Final Report Summary - PARCIVAL (Partner Network for a Clinically Validated Multi-Analyte Lab-on-a-Chip Platform)
Executive Summary:
Project acronym: PARCIVAL
Project title: Partner Network for a Clinically Validated Multi-Analyte Lab-on-a-Chip Platform
Grant agreement No.: 278090
Project start: 01 January 2012
Project end: 31 December 2014
Website: http://www.parcival-project.eu
Participants:
Participant no. Participant name Country
1 (Coordinator) Erasmus MC Netherlands
2 HSG-IMIT Germany
3 National Institute for Research and Development in Microtechnologies
IMT Bucharest Romania
4 Dr. Stein und Kollegen Labora¬toriums¬medizin, Mikrobiologie, Infektionsepidemiologie, Virologie, Transfusionsmedizin und Human genetik GbR Germany
5 Pathofinder BV Netherlands
6 Rohrer AG Switzerland
7 ASKION GmbH Germany
8 Agrobiogen GmbH Biotechnology Germany
9 EADS Deutschland GmbH, Innovation Works Germany
Introduction
The objective of PARCIVAL is to develop an integrated and automated multi-analyte lab-on-a-disk platform for the fast and reliable sample in answer out diagnosis of highly infectious respiratory tract pathogens, antibiotic resistance patterns and biomarkers for individual severity of the infection. The platform format will be based on completely integrated microfluidic foil-disposables with no external connections except one rotational axis of the centrifugal processing device and the sample inlet ports. The disposables will feature different panels (selection of pathogens, antibiotic resistance patterns, biomarkers), which can be freely selected and combined to allow a comprehensive choice of diagnostic parameters by the clinician at the point-of-care, vastly increasing the access to diagnostic information for doctors in everyday situations and emergencies. The integrated platform with pre-packaged liquid and dry reagents on each test carrier will automate all processing steps from sample preparation, over assay processing up to result reporting.
This will allow evidence-based therapeutic decisions, allow antibiotic prescriptions which are tailored to the individual patient and thus offer the prospect to greatly improve therapeutic outcomes. Also, an air-sampling add-on module for monitoring of clinical ventilation systems will be developed and interfaced to the platform. By acting as early warning system that is able to identify airborne pathogens including resistances.
The consortium consisted of 9 partners (6 enterprises (4 SMEs), 3 institutes). The project was heavily based on the research capabilities of innovative SMEs. SMEs leaded the innovation by implementing diagnostic tests (PathoFinder BV), sample preparation (Agrobiogen GmbH), a processing device (ASKION GmbH), and a production process for the disposables (Rohrer AG). HSG-IMIT was involved with the microfluidic integration of the sample preparation and the diagnostic test on the discs. IMT Bucharest delivered the software needed for the interpretation of the results and EADS Deutschland GmbH developed a system to sample the environment for airborne pathogens. Two clinical partners Erasmus MC and Dr. Stein collected reference samples in order to perform a clinical validation of the system. By the end of the project all elements are nearly ready to be integrated into a full functioning prototype of the system. Unfortunately a clinical validation of the system could not be performed within the timeframe of the project.
Project Context and Objectives:
A summary description of project context and objectives
The objective of PARCIVAL is to develop an integrated and automated multi-analyte detection platform, consisting of the low-cost centrifugal microfluidic disposable “PARCIVAL disk” and the processing device “PARCIVAL player” for the fast and re-liable sample-in answer-out analysis of highly infectious respiratory pathogens, resistance patterns and biomarkers for diagnosis of infections. As an advantage com-pared to non-centrifugal microfluidic platforms, no connections to external actuators / pressure sources is required what tremendously reduces the risk for cross contamination. Fluid actuation is a result of defined centrifugal forces only. The PARCIVAL disk will feature different panels (selection of respiratory pathogens, resistance pat-terns, biomarkers), which can be freely selected and combined to allow a comprehensive choice of diagnostic parameters by the clinician at the point-of-care, vastly increasing the access to diagnostic information for doctors in everyday situations and emergencies. The cartridge will contain pre-packaged liquid and dry reagents and automates all processing steps from sample preparation, over assay processing up to result reporting thereby reducing the hands on time and the need for highly trained stuff.
The developed PARCIVAL platform will allow evidence-based therapeutic decisions, allow antibiotic prescriptions which are tailored to the individual patient and thus offer the prospect to greatly improve therapeutic outcomes. Also, an air-sampling add-on module for monitoring of clinical ventilation systems will be developed and interfaced to the platform. By acting as early warning system that is able to identify airborne pathogens including resistances.
Figure 1: Sample in > Answer out
The project is SME-targeted and is designed to encourage SME efforts towards research and innovation.
The consortium consisted of 9 partners (6 enterprises (4 SMEs), 3 institutes).
Expected impacts listed in the work programme in relation to the topic
“The availability of multi-analyte diagnostic tests is expected to allow for antibiotic prescription that takes both the type of infection of the patient, and the presence of resistant pathogens in the clinical setting into account.”
The objective of PARCIVAL is to implement a multi-analyte diagnostic test based on a lab-on-a-disk system with the capability of running several panel tests at the same time. This either permits screening of e.g. 1-4 patients with a specific panel (e.g. in case of emerging epidemics) or to screen a single patient for an extended panel plus information about primary immune responses (e.g. in critical infections with unknown cause). The initial panels were chosen to allow the identification of common respiratory tract pathogens, antibiotic resistance patterns and biomarkers which offer insight into the state of the suspected infection. Thus, the prescribed medication can be tailored exactly to the cause of infection and the presence of antibiotic resistance. This allows the clinician an unprecedented information depth and a case-specific, evidence-based medication. PARCIVAL could therefore, due to the very fast access of comprehensive diagnostic information at the point-of-care, have a substantial impact on clinical practice in terms of diagnostics and treatment. In summary, in critical cases as well as in routine diagnostics the physician could select one or several diagnostic panel(s), take a patient sample, put it into the point-of-care platform, and receive the analysis result within 1-2 hours. Then, treatment with a specific therapy fitting to patient and specific pathogen can start immediately. The amount of information about pathogens, biomarkers and resistances that will be available in this short timeframe is unprecedented in the state-of-the-art diagnostics. Additionally, by adapting and expanding the panels with new assays, the platform’s scope can be expanded easily to other pressing and emerging health threats.
“Such improved prescription should speed up patient recovery and retard the development of multi-drug resistance.”
By allowing the precise identification of pathogen(s), resistances, and immune response in an automated platform at the point-of-care, PARCIVAL will enable the clinician to immediately select the best-fitting treatment and medication the state-of-the-art has to offer to the individual patient. This will greatly reduce prescription of ineffective antibiotics and thus greatly reduce the risk of generating new resistant strains.
“SME-targeted research is designed to encourage SME efforts towards research and innovation”
The PARCIVAL consortium consists of 3 academic and 6 industrial partners of whom 5 are SMEs. The project is partly coordinated by an SME on scientific aspects ensuring that the outcome of the project satisfies the need of highly innovative European SMEs. SME innovation plays a key-role in the project, since the diagnostic assay, the production technology and the sample preparation unit is based on SME research. Therefore, SMEs will be the main beneficiaries of the project’s exploitation.
Expected impact listed in the area Anti-microbial drug resistance
“The strategic objective of this area is to confront the increasing emergence and spread of antimicrobial drug resistant pathogens in Europe and in a multi-disciplinary approach through the development of effective infection prevention and control strategies.“
The chosen multi-disciplinary approach integrates diagnostic partners with technological cutting-edge R&D and ensures sustainable research by integration of production- and end-user know-how. The platform’s capability to identify a large spectrum of pathogens is perfectly complemented by integration of an add-on module for air sampling and, by interfacing it to the lab-on-a-disk-platform, for monitoring of clinics, transport hubs and critical infrastructures. In case of emerging epidemics, this will provide earliest warning of the pathogenic spread and allow effective prevention and control strategies to be implemented.
Expected impacts listed in the work programme HEALTH 2011
“Improving the health of European citizens and increasing the competitiveness and boosting the innovative capacity of European health-related industries while businesses and addressing global health issues including emerging epidemics”
Automated diagnostics with an affordable device could be a breakthrough for routine diagnostics that allow tailored patient medication, since it enables fast and accurate diagnosis in almost every setting. This personalized medication has the potential of greatly increasing health, comfort and prognosis of the patient.
Competitiveness
The aim of PARCIVAL is to provide innovative European SMEs with a platform that addresses current shortcomings of labs-on-a-chip concerning handling, sample preparation, and production process and allows the establishment of a leading role for Europe in lab-on-a-chip applications by instituting a supply chain for this technology.
Emerging epidemics
The impact of the recent outbreaks of for example the Influenza H1N1 strain in various parts of the world on our health care system and on our society in general immediately stresses the importance a very fast and adequate diagnostic and surveillance system for monitoring respiratory tract infections. Monitoring respiratory tract infections directly by the local physician using a point of care device will be the first step in controlling the outbreak, since fast and specific treatments and/or quarantine measures will help to slow down disease spreading.
Routine diagnostics
In routine diagnostics, this platform has also a great economic and social impact, since knowledge about the pathogen responsible for an acute case of respiratory disease will help to specifically treat the case, thus reducing recovery time. The successful development of a platform with a fast adaptability to new assays will also lead to other systems to identify infectious diseases such as sexually transmitted diseases which could be developed and marketed by the same consortium.
On-line monitoring of air for biological contaminations
The possibility of detecting biological contaminations in the environment is a key capability required during emerging epidemics and other situations. A few devices exist for monitoring the pathogen load in air; however, detection usually relies on a cultivation step. These cultivation procedures are time consuming and require trained personnel. So far, there is no possibility of monitoring air “on-line”, i.e. at short time intervals, high sensitivity, quickly and at acceptable long-term costs (maintenance, reagents, etc.). The impacts of the targeted system will be several fold, for example
i) Better hygiene monitoring in hospitals: Air conditioning systems in hospitals have to be checked for biological contaminations once a year. The testing is done in the ducts of the air conditioning system, not in the rooms themselves. Regarding the high risk of nosocomial infections that exist in hospitals; on-line pathogen detection in hospitals could highly improve the hygienic situation.
ii) Improved safety of infrastructures: The fast spreading of diseases like SARS or swine flu have shown that today the risk of epidemics or pandemics is very high due to the high mobility of people. The monitoring of infrastructures like airports together with an early warning system would greatly increase the safety of the population.
iii) Improvement in pharmaceutical production: Pharmaceuticals are produced and packaged in clean room environments. Testing intervals are usually about 1 month or more with about 1 week for analysis. An on-line detection method would reduce the risk of drugs being produced under conditions which would not comply with the regulations.
Steps that will be needed to bring about these impacts
The validation and integration of the lab-on-a-disk platform in PARCIVAL into a clinical setting will be initialized and tested during the course of the project by Erasmus MC and Labor Stein. This will allow integrating end-user feedback into the research in an early stage. The consortium is heavily based on industrial partners (6/9) and does of course aim for a post-project product development commercialization phase. This combination of early end-user integration and strong product orientation has the high likelihood of resulting in a system with the potential for very good acceptance in clinical settings and a sustainable supply by a partner network for the platform.
Project Results:
In the PARCIVAL project four phases were identified: Specification, development of the components, integration of the components, clinical validation and feedback. The results for these phases are summarized below:
1. Specification
The integration of the components required a careful specification phase in the beginning. Here, interfaces, communication channels, requirements from the individual applications etc. are collected in a specifications document. The specifications were delivered in the first reporting period and guided the next steps.
2. Development of the components
After the first phase, the first versions of the components were developed independently by the respective partners according to the set specifications. Developments in this period were performed on individual test stands / measurement setups.
In total 27 assays were designed by PathoFinder targeting atypical pathogens, 8 targeting typical pathogens, 9 resistance markers and 1 internal control. The NA extraction method has been designed by Agrobiogen.
EADS developed a sampling device.
HSG-IMIT delivered the design of the required disposables for microfluidic tests. Rohrer worked on the implementation of the replication technology.
3. Integration of the components
The individual development phase ended in month 18 of the project with the assembly of the first processing device prototype by Askion. A total of 5 prototypes were created during the project and placed at PathoFinder, HSG-IMIT, ASKION, Erasmus and Labor Stein. Software for the presentation of the results was designed by IMT.
4. Clinical validation and feedback
It was planned that at project month 30, the demonstration of microfluidic assay integration would be achieved. This however was to optimistically planned. The delivery of the prototypes by ASKION was delayed, but more importantly the implementation of the replication could not completely by established within the timeframe of the project. Therefore the planned validation phase targeted at testing of the system with surplus patient samples and comparison to state-of-the art systems could not be performed.
Work packages
The work in Parcival was divided in 8 work packages:
• Work package 1 Management
• Work package 2 System specifications
• Work package 3 Assay development
• Work package 5 Production technology
• Work package 6 Sample preparation
• Work package 7 Microfluidic integration
• Work package 8 Clinical validation and testing
Figure 2: The PARCIVAL microfluidic platform is based on a centrifugal microfluidic disposable test carrier, which microfluidically integrates sample preparation and assay, including pre-storage of all reagents. The test carrier will be handled by processing device including analysis software. The platform will be clinically validated during the project. An add-on air sampling module will allow monitoring of airborne pathogens with little extra research effort. Components are shown in ellipses, work packages as boxes and involved partners are written in boldface. WP1 and WP 2 are project management and specification, respectively, and are not shown in this figure.
Work performed in the different work packages except Work package 1 (Management)
Work package 2 System specifications
Results:
The rather complex system integration approach requires careful specifications. The specification document describes in detail all requirements needed to develop an integrated and automated multi-analyte lab-on-a-disk platform. The document describes end-user specifications in terms of handling, process time, price of device and disposables but also details about the operating conditions, sample taking, sample analyses, the pathogens to be detected. The document can be seen as a summary of the results within all workpackages within the project.
Some highlights of the specification document are:
• The aim is Simple handling and fast time to result.
• Target fabrication cost for one PARCIVAL disk is < 10 € at mass production including disposable and biochemistry and a retail price of approximately 50 € per PARCIVAL disk.
• Price of the processing device (PARCIVAL player) should be below 5000 €, a favourable price would be 2000€.
• Two panels will be tested: a Respiratory pathogen panel (Panel A) and a panel with resistance patterns and some typical pathogens (Panel B). It has been decided not to include the biomarkers C-reactive protein and Procalcitonin (see WP3).
• Operating conditions should comply with standard laboratory environment.
• Three types of pretreatment will be used for three types of clinical samples: Sputum, Broncho-Alveolar lavage (BAL) and Throat swab.
• To facilitate sample pre-treatment and sample loading to the PARCIVAL disk, dedicated sample-transfer modules will be developed for swabs and sputum.
• A workflow for air sampling will be included.
• Sequence of sample preparation, including lysis and nucleic acid purification based on magnetic bead based extraction protocol. The sample preparation protocol has to be automated by the PARCIVAL disk.
• To be able to detect relevant pathogens, first priority is to use real-time PCR (PCR) assays in PARCIVAL. A quantitative result could offer additional benefits, but is considered as optional.
• For the integration of the real-time PCR assay into the PARCIVAL disk, all reagents will be dryly prestored in the amplification chambers.
• A specific thermocycling protocol has to be adopted by the PARCIVAL player for PCR on the PARCIVAL disk.
• Fluidic unit operations / fluidic structures are described in detail
• The processing device is described in detail.
• The requirements of the cartridge (disc) are described in detail.
• The system must comply to directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices.
A first version of the specification document was delivered on 27.03.2012.
Work package 3 Assay development
Results:
Task 3.1 Development of validation targets
PathoFinder and the Erasmus Medical Centre made an inventory of all relevant a-typical and typical respiratory pathogen as well as all important antibiotic resistance markers. Based upon this selection, primers and probes were designed and a verification and validation plan was made. Validation was started using oligo sequences flanking the primer and probe region and for the extended validation experiments cloned constructs were made. In this task all construct were designed and cloned using ´gene synthesis service´ and T7 RNA transcripts were made of all targets with a RNA origin. Every single construct contains a maximum of 8 targets which makes it easy to use due to its equimolar ratio and suitable for quality control experiments.
Task 3.2 Development of new assays
The composition of the assay was split into two different panels; Panel A targeting 22 atypical viruses (Influenza A, RS, Rhino, Boca etc.) and bacteria (M.pneumoniae L.pneumophila etc.) and one internal RNA control (MS2 phage). Panel B consists of 23 typical bacteria (S.pneumoniae E.coli A.baumanii etc.), resistance markers (Mec A (MRSA), KPC, OXA-48 etc.) and 1 internal DNA control. All assays were designed in a way that they can be performed in one tube using four different fluorescent channels. In this matter only 7 qPCR assays are needed to target a large panel of pathogens. The assays have been designed, optimised and validated in strict compliance with the MIQE and CE-IVD guidelines. Verification of the first panel, targeting 21 respiratory viruses and atypical bacteria, was performed and resulted in similar sensitivities in comparison with commercial qPCR assays.
It was decided that the integration of an assay for biomarkers is not relevant. This decision was made in collaboration with our clinical partners (Erasmus MC and Labor Stein). Based on their experience as well as research in current literature, we found that the use of such markers in our diagnostic package has no relevant value. Our goal is to develop a Point-of-Care (POC) assay which can be used for the screening of patients for the presence of microorganisms that cause respiratory tract infections and to differentiate either bacterial- or viral pathogens, leading to a logical empirical treatment regimen. Patients can be divided in 2 groups: community-acquired (CA) patients who visit the general practitioner having respiratory tract problems and patients with healthcare-associated (HA) lower-tract respiratory tract infections (LWI).
This POC test can be used by the general practitioner to diagnose the CA infection for a targeted treatment. The decision to screen HA patients with a suspected LWI is mostly based on a clinical suspicion of the infection that includes physical parameters, such as fever and so forth. Our POC is an excellent test to determine the pathogen in this stage which will be used for targeted treatment. The use of biomarkers like C-reactive protein (CRP) and neopterin are only relevant in cases where there is severe progress in the infection which can results in bacteremia or bacterial sepsis. In the case that the focus of the infection is known, our test can be used for pathogen identification. Nevertheless, the biomarker procalcitonin has been shown to be heightened specifically in bacterial infections, and its levels can be used to establish the duration of antibiotic treatment, but not the type of antibiotic treatment. Furthermore a disappointing diagnostic accuracy has been shown in the patient group where our POC is intended for (trans r soc trop med hyg 2012; annals of emergency medicine 50(1):34-31); sensitivity of 76% and specificity of 70%, even in bacteremia patients means false results, and possibly a negative value of its determination in a significant amount of performed tests. Moreover, the decision of not implementing a protein marker in a RNA/DNA chip resulted in a more straight forward design of the disc because these biomarkers should be tested from blood and this requires a different approach in sample treatment in comparison to samples from the respiratory tract (sputa, bronchoalveolar lavage etc.). This will result in the development of a more cost effective final product and will simplify the market introduction because this platform can better compete with other POC devices. Overall, the decision to not include protein biomarkers is beneficial for the total project.
Task 3.3 Uniforming of reaction conditions / Task 3.4 (Time-)Optimization of reactions
The assay conditions of all assays were adapted and modified for some assays to create one uniform protocol for all sets within this panel. Within this task several enzyme mixes for combined reverse transcription and amplification were tested with the aim to obtain similar results for all different assays. Pre-clinical validation was performed with External Quality Assessment (EQA) programmes from QCMD. The EQA panels were tested from the years 2009 to 2012 and fulfilled all requirements. These assays will used in the next period for the integration on the lab-on-a-disk by HSG-IMIT. Several sets within Panel B are designed and further optimized within the next report period.
Task 3.5 Coating against unspecific adsorption of DNA
The aim of the task was to prevent nucleic acid and/or protein loss in microfluidic channels with a high surface-to-volume ratio. The specific objective was to evaluate different blocking agents within this period. First the assays for validation experiments to set-up a golden standard in the Parcival project were selected. These validation experiments were performed at the facility of PathoFinder and shipped to partner HSG-IMIT. The most important selection criteria for the validation experiments were sensitivity and reproducibility. HSG-IMIT has achieved the same sensitivity with all validation experiments performed with the Labdisk. Because HSG-IMIT met the requirements of the gold standard, there was no reason to start the use of a coating. In conclusion, no loss of specific RNA and DNA was observed, resulted in equal sensitivities obtained with the diagnostics assays of PathoFinder in combination with the newly developed Labdisk by HSG-IMIT.
Summary
In total 27 assays were designed by PathoFinder targeting atypical pathogens, 8 targeting typical pathogens, 9 resistance markers and 1 internal control. We developed 7 control constructs compromising a maximum number of 8 targets per construct. The assays and controls were tested for functionality and used in validation experiments in 2014
Three different objectives were pursued in this study, we tested:
1. Repeatability/reproducibility (inter-/intra-laboratory study) by PathoFinder, Agrobiogen, Dr. Stein and Erasmus;
2. Sensitivity and robustness of the assay;
Using different types of real-time cyclers by multiple technicians and other laboratory variations, on 2 different days using reference NA extraction methods and real-time PCR.
And, 3. The utility was determined by comparison of Cycle Threshold (Ct) values.
It was concluded that the assays are ready for integration on the discs.
Work package 4 Processing device
Objectives
Hard- and software design and engineering of the processing device. Data interpretation and acquisition of the results and the user interface will be provided.
Results:
Task 4.1 Design and construction of the device
Task 4.2 Engineering of control units
The task of ASKION is to develop the hardware platform for the project with the help of the other partners. During feasibility phase all physical and technical systems have been checked in the lab of the ASKION GmbH. This resulted in an approved concept for the first prototypes of processing device.
In order to automate the processes of the centrifugal micro fluidic platform, a processing device is required consisting of the following main hardware modules:
- a spinning drive
- a mechanical cartridge support
- a magnet module,
- a thermal module,
- an optical module
- a control unit.
Task 4.3 Software programming
Control software and an experimental graphical user interface (GUI) will be designed by ASKION in collaboration with IMT while software for data analysis and interpretation will be designed by IMT only.
The report D4.1 contains the following issues:
- Technical drawings of processing device
- Electronic control units of processing device
- Software packages for controlling the spinning drive, the magnet module, the thermal module (including thermo sensors), the optical module, safety features and a GUI.
Task 4.4 Mechanical and electrical assembly, initial operation and testing
Task 4.5 Design review and optimization (hard- and software)
In August 2013 the first processing device was delivered to HSG IMIT in Freiburg (MS8). It was necessary to improve the microfluidic technology. Askion used a remained device for further development and improvement of the functions. Especially the positioning system and the heater concept needed to be optimized. In the first quarter of 2014 we made sure that all required functions have been reached. In February 2014 the first PCR result was running on a player. This result was the basis to set up the next machines for the Parcival consortium. In March 2014 Askion was able to deliver an optimized processing device to HSG IMIT which fulfilled all required functions (D4.2).
From April 2014 until the summertime many tests were performed. Thereby the chemical kits of PathoFinder were used. Parallel to these improvements many stable tests have been performed. These tests made sure to have reproducible results on all 4 devices.
In May 2014 the current results have been presented at a project meeting by EADS in Munich. Together with the colleagues from HSG agreements concerning the controlling system have been made.
All these activities were completed by a ceremonial handover in Gera with all partners who got a device.
Some pictures of the device
Work package 5 Production technology
Objectives
The implementation of the replication technology for the microfluidic disposables and the realization of a physical barrier in the fluidic network which can be removed by the liquid exerting centrifugal pressure at high centrifugal frequencies, acting as a valve.
Results:
Task 5.1 Conceptual design and layout of the disk forming and sealing
The manufacturing process has been proved to be undertaken in the following stages: cutting of the foil from a coil, micro-thermoforming of the cut foil, precutting of the disc, cutting of the sealing foil and sealing onto the disc, final cutting of the disc.
Tests done so far demonstrate that it is possible to form microfluidic structures with the necessary sharp edges. However limits are given by the design of the disc e.g. depth of the cavities or distances from each other.
For example it requires a surface temperature at the PDMS master of about 180 [°C]. With the current tool set-up with a cover ring to separate master and tool environment the foil still most frequently tears at an approximate temperature of 160 [°C]between ring and tool frame.
Another issue is the slow heating up of the PDMS master. We observe that shallow features are formed much better than massive cavities. The mentioned surface temperature is therefore an important process parameter. The lower tool temperature is measured inside the heating plate. In the course of production PDMS masters tend to shrink. It obviously reduces its dimension already over the very first forming cycles. Furthermore, the first couple of disks formed exhibit a bubble structure in the foil that refers to the release of humidity from the PDMS master with the effect of bristling the material and easy breaking the foil.
Design of experiments (DOE) capabilities provides a method for simultaneously investigating the effects of multiple variables on an output variable (response). These experiments consist of runs or tests, in which purposeful changes are made to input variables or factors, and data are collected at each run. The goal is to identify the process conditions and product components that influence quality and then determine the input variable (factor) settings that maximize results.
Task 5.2 Implement prototyping line for sealing and forming procedures
Among other features Minitab, the tool of our choice, offers the factorial design of experiments. Due to the poor forming quality no statistical optimization procedure has so far been applied. A stable process on the actual system can still not be executed and DOE makes little sense so far.
Thermoforming with PDMS master tool
At the beginning of similar projects it has been decided to start with PDMS master tools. Due to lots of design changes on the micro-fluidic design of the disc it is easy to prepare new tools with this technology. The manufacture is quite inexpensive and quick, therefore lots of designs can be tested.
The disadvantage of this technology is the limited tool life. After about 20 cycles the material becomes brittle and tends to break outs of small geometries. Furthermore the measures of the tool are not stable but decrease dimensions with increasing number of cycles.
Based on the experience we made with PDMS masters it has been decided to work with a metal master tool for the final version right from the beginning of the project. Work on PDMS masters tools has therefore not been started.
Thermoforming with Aluminium master tool
Compared to PDMS tools the use of metal tools for commercial productions requires some considerable changes for the tool manufacturing. Whereas deforming of the disc from PDMS tools is not a problem due to the flexibility of the tool it became a severe issue with the aluminium tool. The high vacuum between the foil and the tool during the forming phase is responsible for the strong adherence of the disc to the tool after cooling. A flimsy removal of the disc without any damages is no longer possible.
Coatings and surface treatments
Several coatings and surface treatments which should alleviate the deforming and improve the roughness of the tool have therefore been tested.
Different tool manufacturing technologies were tried out to find the most suitable solution for making the micro thermoforming tools. The focus was on sharp edges, precise dimensions and surfaces with a low roughness.
Conventional machined tool
Conventional machined tool
Furthermore severe problems with the deforming of the disc came up. Because of creating a vacuum for the forming process there is no air between the disc and the tool and therefore the plastic material sticks to the metal.
The manufacturing of the tool is quite complex and expensive. As benefit it could be proved that the processed discs were stable in dimensions.
3D-Plot master tool
As an alternative to the conventional manufacturing, 3D-plotting has been tried out. (Pictures 14 & 15). Reason for the trial was the technology, which builds up the tool layer by layer with micro drops which consist of a blend of aluminium and plastic. This technology should make sharp edges possible. Unfortunately it was proven that the dimensions of the whole plot are not precise enough for the required purposes. The surface furthermore is rougher than grinded surfaces. A later grinding of the tool after plotting was not successful as the surface of the material tends to smear.
During processing tests it was also detected that the processing time increases caused by a bad heat transformance of the material. Longer cycle times were the result. Also with this kind of tool the deforming was a severe challenge based on the a.m. problem.
Although the manufacturing of the tool with this technology is quick and inexpensive 3D plotting was not further traced.
3D-Plot master tool 3D-Plot master tool (detail)
Multi-part tool
A technical breakthrough was achieved with the multi-part tool. With this technology all elevated parts of the structure are made separately. At the base of the forming tool adequate physical dimensions where milled out so that the separately made parts can be pushed through the base from the backside. With this technology there are no more radii at the base of the structure. Instead there are sharp corners finally producing sharp edges at the disc. As the tool has to be produced with highest precision in order to make all parts fitting well also the thermoformed disc is of high precision. Unfortunately this necessary precision makes the tool very expensive.
Achievements
After testing several different tool conceptions it has been found out that the multipart tool is indeed the most expensive one but due to its sharp edges and the high precision it has shown best results in microfluidic properties.
Multi-part tool
The problem of deforming of the disc could be solved by the installation of an overpressure between the ready formed disc and the lower tool part. A slight pressure impulse pushes the disc from the tool.
A challenge for the thermoforming process was the forming of channels with a cross section of just 50µm. Channels of 50µm could be achieved but improvement is necessary regarding the consistency of the dimensions as the dimensions increase towards the base of the forming tool. This is probably due to a too big radius of the milling cutter at its top.
50µm channel 50µm channel
Finally discs were made and tested. As several areas of the disc were identified where design improvements could be made, a new tool has been ordered with the improved design. Transitions from small to big channels were furnished with bigger radii as well as the distance from the stick pack cavities to the bead structure was increased.
Conclusion
After testing several different tool concepts with different materials it has been found out that the a multipart tool made of Aluminium has the best characteristics for the production of discs with sharp edges and the high precision needed to have the necessary microfluidic properties. Problems were caused by the deforming of the discs when they are removed from the forming tool. The problem of deforming of the disc could be solved by the installation of an overpressure between the ready formed disc and the lower tool part. A slight pressure impulse pushes the disc from the tool. Another challenge for the thermoforming process was the forming of channels with a cross section of just 50μm. Channels of 50μm could be achieved but improvement is necessary regarding the consistency of the dimensions as the dimensions increase towards the base of the forming tool.
Finally discs were made and tested. As several areas of the disc were identified where design improvements could be made, a new tool has been ordered with the improved design. With the new tool discs can be made which will most probably meet all set requirements for the microfluidic properties. However this could not be established within time frame of the project which was the main reason that a clinical validation of the system could not be performed.
Work package 6 Sample preparation
Objectives
Integration of sample preparation in microfluidic system.
Development of an automated capturing and separation system based on filtration and magnetic separation to be combined with the centrifugal microfluidic platform in PARCIVAL.
The integration of sample preparation comprises several tasks. The nucleic acid extraction and purification with the Agrobiogen/nexttec system will be adapted for use in the centrifugal microfluidic platform. The add-on module for air-sampling to enable monitoring of airborne pathogens will be developed by EADS and interfaced to the microfluidic detection platform of HSG-IMIT.
Results:
Task 6.1 Sampling microorganisms with filters, especially gelatin or similar filters
A hand-held air sampler device based on gelatin filters was developed with three different applications.
1. respiratory sampling (medical application)
2. wiping sampling
3. air sampling
Common to all three applications is the gelatin filter membrane with a diameter of 13 mm. This membrane was cut out from the original filter membrane and was placed in the air sampler by a magnetic holder. A pump is than connected at the sampling device and air sampling could be started. For medical applications, a nozzle will be contacted at the sampler and patients can blow their breath by a moderate pumping rate at the gelatin membrane. This application is specific for the detection of respiratory diseases. For wiping tests a special wiping head was constructed and connected at the air sample in front of the membrane. This wiping head has specific canals at the surface that the membrane couldn’t stick to the surface.
A)
C)
B)
D)
(A) Hand-held air sampling device for gelatin filters with pump;(B) Air sampler with magnetic holder and cut out gelatin filters with a diameter of 13mm; (C) medical application of air sampler with a nozzle for detecting respiratory diseases; (D) wiping application of air sampler with canals at the surface to avoid sucking of the surface area.
After manufacturing the air sampler device was tested to capture microorganisms. Different sampling applications were tested by the system:
• 1m3 inside
• 1m3 outside
• 1m2 floor
• respiratory sampling
• surface of skin from arm
• surface of computer keyboard
Results show a very high capturing of microorganisms by using gelatin filters. In addition occupied gelatin filters were also tested in the protocol for nucleic acid isolation. As Result a very less inhibition or dysfunction of the protocol could be observed.
Task 6.2 Development of an automated filter handling and liquid handling device
After air sampling two different ways of sample preparation were planned:
1. Unspecific sampling preparations by dissolving the gelatin filters and direct processing of the liquid at centrifugal microfluidic platform.
2. Specific sampling preparation by usage of magnetic beads coated with recombinant G3P bacteriophage protein to capture specific bacteria out of the liquid of dissolved gelatin filters.
Concerning of specific sampling occupied gelatin filters were dissolved in liquid together with magnetic beads. To capture specific bacteria form the solution, magnetic beads coated with a bacteriophage protein were used. As example G3P protein from E. coli M13 phage were cloned in three domains with different length in a T7 expression vector with a 6xHis-Tag. The small domain is N1 (255bp and 85AS) followed by N1L2 (315bp and 104 AS*) and N1L1N2 (705bp and 235AS). Results show that the smallest G3P domain N1 was expressed as an insoluble protein. But both lager domains, N1L2 and N1L1N2, were soluble expressed and could use for coating at magnetic beads for specific sampling of E. coli as model organism.
* Amino acids
A)
B)
C)
Expression of E. coli M13 bacteriophage protein G3P for coating of magnetic beads; (A) schematic drawing of magnetic beads coated with phage protein and captured bacteria; (B) schematic drawing of M13 G3P protein with domains and there differed lengths, N: N-terminal domain, L: Loop, TD: Trans membrane domain at C-terminus, bp: base pairs, AS: amino acids; (C) SDS-PAGE: soluble proteins after Ni-NTA purification of G3P domains N1L1 (approx. 14kDa) and N1L1N2 (approx. 35kDa) shown in characteristic bands; insoluble N1 domain no characteristic band could be observed.
Task 6.3 Nucleic acid purification
First, a real-time PCR system for detection of PCR inhibitors in nucleic acid preparations was developed in order to test their quality. Then, a lysis system for model bacteria (gram-negative bacterial species: E. coli, gram-positive bacterial species: S. epidermidis) was established on the basis of the nexttec™ principal according to the project specifications. In spiking experiments with saliva as sample material a sensitivity of 5 to 15 bacterial genome equivalents per PCR reaction was demonstrated. During optimisation the lysis time was shortened from 40 to 25 min. without remarkable loss in DNA yield.
In cooperation with project partner EADS gelatin filters for air sampling of pathogens were analysed. It was found that 0.5 to 1 filter containing bacteria and viruses can be directly dissolved and used for lysis with the nexttec™ buffer system. The resulting DNA preparation does not inhibit real-time PCR. Gelatin and sample background (dust particles etc.) were effectively removed by nexttec™ cleanColumns.
DNA as well as ribosomal RNA was isolated from lysed bacteria in sample background (e.g. saliva). But, after spiking saliva samples with an RNA bacteriophage (MS-2) according to the specifications (MS-2 as model RNA virus) the phage RNA cannot be detected in the corresponding nucleic acid preparation purified on nexttec™ cleanColumns. We concluded that the nexttec™ buffer system is not able to inhibit RNases sufficiently for subsequent detection of RNA viruses. Therefore, in milestone 5 the decision for a nucleic acid purification system was made in favour of a silica-based magnetic bead system.
A lysis and washing buffer system for nucleic acid purification on magnetic silica beads was developed in parallel to the nexttec™ system. After optimisation of the method DNA and RNA from bacteria and viruses could be isolated. The system was also adapted and tested for the isolation of nucleic acids from gelatin filters after air sampling. The silica magnetic bead system is now introduced together with partners HSG-IMIT and Rohrer to the centrifugal lab-disc.
Summary
During the first period of the project a nucleic acid isolation method on the basis of the nexttec™ 1step system was adapted to the conditions of the PARCIVAL microfluidic disk. But it was found that RNA could not be efficiently stabilized for sensitive detection. Therefore, the decision for a nucleic acid purification system was made in favour of a silica-based magnetic bead system. A basic method was developed and tested. During the second project period the method was optimized for the PARCIVAL goals and tested for the detection of different target organisms in real sample background.
All these optimization works and tests with different types of pathogens and back-ground were the basis for an interlaboratory validation study. The finally developed methods from sample handling to amplification and detection (developed by PathoFinder) were used in four different labs and compared with standard methods. See WP3.
The other part of this Work package included the development of an automated capturing and separation system based on filtration and magnetic separation which could be combined with the centrifugal microfluidic platform in PARCIVAL.
A sampling device has been designed and tested. The tool included three sampling concepts:
1. Respiratory sampling (medical application); 2. Wiping sampling and 3. Air sampling.
Furthermore a Docking station with a filter holder and UV light source was developed. Test were performed on the dissolvent of loaded gelatin membrane filters and the capturing of bacteria with magnetic beads.
Capture proteins (phage proteins) to capture microorganisms were produced and tested.
An existing device was modified for processing of the gelatin filters together with coated magnetic beads.
Because there was no possibility of handling with pathogens in the laboratories of Airbus Group Innovations a validation only with beads concerning total bead loss and precision of pipetting has been performed and showed reasonable results. The estimated capture efficiencies of gelatin filter in combination with magnetic beads was 54%.
Work package 7 Microfluidic integration
Objectives
Development of all required microfluidic components and reagent storage and release concepts. Integration of sample preparation, pathogen test and the biomarker test into the automated microfluidic system. Combination of these tests onto panel test carriers and parallelization.
Results:
Task 7.1 Development, evaluation and selection of microfluidic components
Microfluidic unit operations are required for automation of the assays within the PARCIVAL project. These unit operations have now been selected and described. Unit operations such as sample handling, integration of the sorbent material and switching of fluid flow are being developed to fit the requirements of the project that were previously unavailable. As a next step, all unit operations will be integrated to fabricate the LabDisk with final assay format as defined in the specification document.
Task 7.2 Integration of a nucleic acid purification protocol
Nucleic-Acid purification and extraction module
For magnetic bead based nucleic acid extraction, a microfluidic unit operation for gas-phase transition magnetophoresis was selected and tailored to the requirements of the PARCIVAL project. The microfluidic integration fully automates the following process:
(1) Mixing of 200 µL liquid sample with 10 µL DTT and 200 µL of a lysis buffer containing chaotropic agents
(2) Addition of 200 µL binding buffer; binding of nucleic acids to the magnetic beads
(3) Transport of magnetic beads into 200 µL washing buffer and mix
(4) Transport of magnetic beads into 200 µL of a second washing buffer
(5) Transport of magnetic beads into 200 µL elution buffer to release purified DNA from the bead-surface.
After nucleic acid purification, the elution buffer, containing the purified DNA is transferred to the nucleic acid amplification module. The microfluidic process flow, that is integrated in the PARCIVAL disk demonstrator, is depicted in the following picture sequence [Fig 2 a – f). Results are described in Report on deliverable D7.3.
Fig 2a: CAD Design of PARCIAL Disk for nucleic acid analysis Fig. 2b: 200 µL Sample (1) and 200 µL lysis buffer (2) are mixed in lysis chamber.
Fig 2c: 200 µL binding buffer (3) dissolve 10 µL DTT (1M) and magnetic beads (4) and are transferred into lysate. Fig. 2d: 200 µL washing buffer 1 (5) and 200 µL washing buffer 2 (70% Ethanol) (6) are transferred into washing chambers. Washing buffer 3 (7) is optional. Magnetic beads are transported from lysis chamber into washing chambers and finally into elution chamber (white arrows)
Fig. 2e: 200 µL elution buffer (8) are transferred into elution chamber Fig. 2f: After elution, elution buffer is transferred (9) into aliquoting structure.
Nucleic acid amplification
The feasibility of RT-PCR has been demonstrated on a demonstrator with microfluidic test structures (Fig .3). The demonstrator automatically aliquots a mixture of elution buffer and PCR polymerase into 8 x 20 µL, each aliquot is then transferred into a fluidically separated amplification chamber where it is brought in contact with dryly prestored primers and probes. Thereby, geometric multiplexing, targeting multiple analytes in parallel, could be achieved. The fluidic test structures were designed for being processed in a commercially available PCR thermocycler. Thereby, fluidic structures for aliquoting and feasibility of RT-PCR could be investigated and operating condition could be optimized while the PARCIVAL processing device is being developed in parallel by project partner 7 ASKION GmbH (D4.2)
Fig. 3: Graphical depiction of demonstrator for functional testing of aliquoting and (RT-) PCR in commercially available PCR thermocycler. The validated unit-operation for aliquoting will be integrated into the final PARCIVAL disk.
On-Disk reagent prestorage
The PARCIVAL demonstrator disk included pre-packed liquid reagents for nucleic acid purification (Fig. 1). For packaging of liquid reagents, aluminium stick packs were chosen. The stick packs were fabricated from a polymer-aluminium composite foil with excellent vapour barrier properties. The applied stick pack technology enables long term storage of liquid reagents what has been demonstrated before by van Oordt et al. for water (loss of 0.6 % after 42 days at 70°C) and alcohol containing buffers (loss of 0.8 % after 21 days at 70°C) and will be repeated for the final nucleic acid purification reagents in PARCIVAL. All reagents are released from the stick packs at defined rotational speeds via a peelable seam at the radial outer end. The final PARCIVAL demonstrator will include 5 stick packs containing the following reagents and volumes.
Reagent Volume
Lysis Buffer 200 µL
Binding Buffer 200 µL
Washing Buffer 1 200 µL
Washing Buffer 2 200 µL
Elution Buffer 200 µL
Conclusions and outlook
All microfluidic unit operations for nucleic acid analysis in the PARCIVAL disk have been selected and the microfluidic and bio-chemical feasibility of the single components has been proven. A demonstrator of the PARCIVAL disk for nucleic acid purification including prestored liquid reagents has been fabricated and functionality has been demonstrated successfully. Results of nucleic acid purification are described in Report on deliverable 7.3. Microfluidic unit operations for aliquoting have been selected and fluidic test structures for validation of the (RT-) PCR based amplification in commercially available real-time PCR thermocycler have been fabricated as the laboratory set-up for the PARCIVAL processing device will be finalized in parallel.
Integration and parallelization of the automated assay on one disposable
A novel concept for handling clinical samples (Swab and Sputum) and for transferring pathogens to the LabDisk has been developed and 3D printed prototypes were fabricated and tested for their liquid handling qualities. Biological tests with real-world samples will be conducted in the near future.
Task 7.3 Optimized nucleic acid purification protocol integrated in microfluidic disk.
In order to provide DNA and RNA in sufficient quantity, a microfluidic module for nucleic acid purification was integrated on the PARCIVAL disk prior to multiplex (RT-) PCR based amplification. Ease-of-use is increased by prestorage of all liquid reagents on the disk.
Nucleic-Acid bench-top purification protocol
As described in the report on deliverable 6.3 the adaptation of Agrobiogen nexttec™ 1-step DNA isolation system to co-purify DNA and RNA was not successful due to insufficient RNase inhibition especially in the first lysis step (see fig 2 & 3).
Fig 1: RT real time PCR with RNA isolated from MS2 phage using the Agrobiogen nexttec™ 1-step DNA isolation system. The MS2 phage was spiked in different sample backgrounds. No RNA was isolated and detected in blood and saliva background. 103 phages in lysis approximately correspond to 35 phages in PCR.
--complete nexttec lysis-- M ----2nd lysis step only----
--lysate-- --eluate-- --lysate-- --eluate--
Fig 2: 1% Agarose gel with DNA and RNA isolated from 200 µl saliva with the Agrobiogen nexttec™ 1-step DNA isolation system. If just the 2nd lysis step is performed, RNA can be isolated.But in the first enzymatic lysis step no Rnase inhibitor or destroying agent is resent. Thus even ribosomal RNA is degraded. M – 1 kb DNA ladder.
Therefore, the consortium decided to integrate a magnetic bead based nucleic acid purification protocol as the fall back solution.
The nucleic acid purification protocol developed and tested under work package 6.3 was further adapted and simplified for integration into the Lab Disk.
During the development usual laboratory steps like vortexing or pipetting up and down for mixing or washing the silica magnetic beads were integrated. In order to resemble the situation in the Lab Disk during binding and washing steps the magnetic bead/ buffer suspension was mixed by slowly overhead rotation. Isolated DNA using the standard mixing (pipetting up and down or vortexing) or “smooth” overhead rotation was compared in a real time PCR (Fig 4).
Fig 3: Real time PCR with DNA isolated from S. epidermidis using the magnetic bead based nucleic acid purification protocol. When comparing the two mixing methods pipetting up and down and mixing overhead, no differences can be seen. 104 bacteria cells in lysis approximately correspond to 500 genomes in PCR.
It could be shown that both methods for mixing give the same result. Real time PCR plots are identical in shape and Ct value.
Further the number of wash steps was reduced from three to two. Often the standard methods for DNA isolation on silica surfaces require different washing steps, which are repeated. First, contaminants like proteins are washed away under high salt conditions. Then, second, salts (Guanidinium) are removed with ethanol keeping the nucleic acid precipitated on the silica surface. In order to save time and space we omitted the wash repeats (Fig 5). Both DNA isolated from bacteria with or without sample background (blood) was analysed in a real time PCR.
Fig 4: Real time PCR with DNA isolated from S. epidermidis using the magnetic bead based nucleic acid purification protocol. One washing step is as effective as two washing steps both in peptone-NaCl and in blood.
Samples processed with one washing step gave the same PCR plot as with two washing steps. Consequently, the procedure was simplified.
For a better binding of nucleic acids from lysates to the beads an alcohol (ethanol or isopropanol) was added to the lysates. We found that isopropanol is best suited and added a pure (100%) solution. But during storage of 100% isopropanol in the Lab Disk the risk of delamination of sealed disks halves is too high. Therefore, different volumes and concentrations of isopropanol were tested. The results in Fig 6 show that 80% isopropanol, which does not cause delamination, can also be used both in the same volume or a slightly higher volume. The best variant was to store the beads in 80% isopropanol. But it is rather difficult to keep the magnetic beads in suspension. For this reason a separate storage was preferred.
Fig 5: RT real time PCR with RNA isolated from MS2 phage using the magnetic bead based nucleic acid purification protocol. Different concentrations and volumes of isopropanol were tested for binding of RNA to the silica beads. The plots show that addition of 80% isopropanol (150 or 200 µl) instead of 100% for binding gives almost the same results. To each sample 103 phages were added (spiked) which approximately corresponds to 50 phages per RT-PCR.
Finally, the optimised procedure for lysis and purification of DNA and RNA has been provided for integration to HSG.
No. Process step
(1) Mix 10 μl DTT (1M) with 200 μl of liquid sample
(2) Add 200 μl lysis buffer (containing GuSCN), invert and incubate 10 min at RT
(3) add 150 μl isopropanol (80%) and 50 μl magnetic bead suspension, incubate 5 min at RT under mixing
(4) collect the beads with a magnet, discard supernatant
(5) add 200 μl wash solution, mix
(6) collect the beads with a magnet, discard supernatant
(7) add 200 μl ethanol (70%), mix
(8) collect the beads with a magnet, discard supernatant
(9) dry the beads (15 min at RT)
(10) add 200 μl pure water, resuspend beads and incubate at 50°C, 5 min
(11) collect the beads with a magnet, transfer supernatant to a new reaction tube
Translation of bench top protocol into microfluidic layout
The provided bench top nucleic acid purification protocol has been translated into a sequence of microfluidic unit operations. The selected unit operations have been integrated into the PARCIVAL disk demonstrator. The process flow is described in report on deliverable e 7.2 and in Fig 7a – f.
Fig 7a: CAD Design of PARCIAL Disk for nucleic acid analysis Fig. 7b: 200 µL Sample (1) and 200 µL lysis buffer (2) are mixed in lysis chamber.
Fig 7c: 200 µL binding buffer (3) dissolve 10 µL DTT (1M) and magnetic beads (4) and are transferred into lysate. Fig. 7d: 200 µL washing buffer 1 (5) and 200 µL washing buffer 2 (70% Ethanol) (6) are transferred into washing chambers. Washing buffer 3 (7) is optional. Magnetic beads are transported from lysis chamber into washing chambers and finally into elution chamber (white arrows)
Fig. 7e: 200 µL elution buffer (8) are transferred into elution chamber Fig. 7f: After elution, elution buffer is transferred (9) into aliquoting structure.
3.3. Results of nucleic acid purification
For the proof-of-principle, DNA has been extracted on the PARCIVAL disk demonstrator from non-pathogenic E. coli cultures. For the experiments, all required liquid reagents were prestored on the PARCIVAL disk. The entire process of nucleic acid purification and release of prestored liquid reagents was controlled by a predefined rotational protocol. On-disk extracted E. coli DNA was quantified by real-time PCR and compared to manual reference extractions conducted according to the provided bench top protocol. The experiments were conducted in duplicates (Fig 8a). To evaluate the presence of inhibitors, the extracted DNA was diluted 1:10 and quantified by real-time PCR again (Fig. 8b). The measured Ct values of real-time PCR are depicted in Table 1:
Fig. 8a: Real-time PCR plots of DNA extraction from E. coli on PARCIVAL disks 1 and 2 and comparison to manual reference extraction.
Fig. 8b: Real-time PCR plots of 1:10 diluted extracted DNA on PARCIVAL disks 1 and 2 and comparison to 1:10 diluted manual reference extraction.
Table 1: Real-time PCR results of DNA extraction (Disk 1, 2 and manual reference) and 1:10 diluted DNA (Disk 1,2 and manual reference). All real-time PCRs were conducted in triplicates.
Conclusions and outlook
Microfluidically integrated nucleic acid purification with prestored reagents has been demonstrated successfully. The delay between manual reference extraction and on-disk extraction was 1 – 1.5 Ct values when quantified with real-time PCR. In a next step, the feasibility of the system will be demonstrated for RNA model organisms what was not possible to date due to the change in nucleic acid purification protocol (month 18). Afterwards, the nucleic acid purification protocol will be further optimized with respect to yield and eluate purity. The yield of extracted nucleic acids will be increased by enhancing the mixing of magnetic beads within the sample, typically improving the binding of nucleic acids. For characterization of the eluate quality, further means for measurement of inhibitors (Ethanol, salt, proteins) in the eluate will be implemented.
Task 7.4 Validated demonstrator
Due to delays in the workplan caused by the technological challenges, it was not possible to perform the validation studies on the prototype of the processing device and discs. First of all there was a delay in the delivery of the 4 prototypes of the centrifugal processing device of about six months, see D4.2 But the most critical issue on the end was the development of the production process of the discs, see D5.2 During the final meeting is was discussed to perform the validation studies after the end of the project period. No final decision was made on this issue, but it was decided to follow up on this issue in the first quarter of 2015.
Summary
In this work package, the laboratory process of pathogen- and biomarker testing has been integrated into an automated microfluidic test carrier. The liquid handling has been realized by microfluidic unit operations on a rotating disk with micro channels and chambers. The fluidics design with pre-storage of liquid and air-dried reagents on the centrifugal microfluidic Disk has been successfully proofed.
The first detected pathogen so far was a MS2 phage (internal control in PathoFinder respiratory panel)
Open task: Demonstration of Parcival Disk for analysis of clinical samples.
Work package 8 Clinical validation and testing
Objectives
The validation and integration of the lab-on-a-disk platform in PARCIVAL into a clinical setting will be initialized and tested during the course of the project by Erasmus MC and Labor Stein. This will allow integrating end-user feedback into the research in an early stage.
Results:
WP8 is targeted at the clinical evaluation of the prototype PARCIVAL platform in order to get a proof of concept. The applicability as a clinical tool will be tested and as such a prediction will be made on the potential for post-project commercialization as a device for routine use in diagnostics. The aim of D8.1 is to collect reference samples that can be used to test the system as a whole and to validate it on normal clinical material. The material has been tested using the nucleic acid extraction method and PCR assays in the project in an ex disc setup (see also D3.3). It was also planned to do this on the prototype of the PARCIVAL platform and discs but due to delays in the workplan caused by the technological challenges, it was not possible to perform the validation studies on the prototype of the processing device and discs.
Clinical specimens
The objective was to have at least 5 positive samples per target. Which might be difficult for C. pneumonia and L. pneumophila (spiked material).
Erasmus collected samples containing bacteria that are also screened for antibiotic resistance. Labor Stein collected samples containing viruses and bacteria. Erasmus MC specimens are screened with a-typical set (Panel A) using MagNA Pure 96 DNA and Viral NA Small Volume Kit. Labor Stein specimens screened with resistance set (Panel B) using QiaSymphony/ MagNA Pure Kit
Deliverable D8.1 has been finalized and the following clinical specimens are available for the evaluation of the system:
PANEL A
PANEL B
D8.2 Report on (clinical) validation of automated respiratory disease detection.
D8.3 Report on laboratory validation of automated airborne pathogen detection.
Due to delays in the workplan caused by the technological challenges, it was not possible to perform the validation studies on the prototype of the processing device and discs.
First of all there was a delay in the delivery of the 4 prototypes of the centrifugal processing device of about six months, see D4.2
But the most critical issue on the end was the development of the production process of the discs, see D5.2
During the final meeting is was discussed to perform the validation studies after the end of the project period. No final decision was made on this issue, but it was decided to follow up on this issue in the first quarter of 2015.
Summary
Due to a delay in work package 4 and more critical a delay in the production of enough discs (work package 5) we were not able to perform a clinical validation study with the Parcival system within the time frame of the project. However by the end of the project period all elements are ready. A validation study has been performed in an in tube format (see work package 3).
Potential Impact:
The objective of PARCIVAL is to develop an integrated and automated multi-analyte detection platform, consisting of the low-cost centrifugal microfluidic disposable “PARCIVAL disk” and the processing device “PARCIVAL player” for the fast and reliable sample-in answer-out analysis of highly infectious respiratory pathogens, resistance patterns and biomarkers for diagnosis of infections. As an advantage compared to non-centrifugal microfluidic platforms, no connections to external actuators / pressure sources is required what tremendously reduces the risk for cross contamination. Fluid actuation is a result of defined centrifugal forces only. The PARCIVAL disk will feature different panels (selection of respiratory pathogens, resistance patterns, biomarkers), which can be freely selected and combined to allow a comprehensive choice of diagnostic parameters by the clinician at the point-of-care, vastly increasing the access to diagnostic information for doctors in everyday situations and emergencies. The cartridge will contain pre-packaged liquid and dry reagents and automates all processing steps from sample preparation, over assay processing up to result reporting thereby reducing the hands on time and the need for highly trained stuff.
The developed PARCIVAL platform will allow evidence-based therapeutic decisions, allow antibiotic prescriptions which are tailored to the individual patient and thus offer the prospect to greatly improve therapeutic outcomes. Also, an air-sampling add-on module for monitoring of clinical ventilation systems will be developed and interfaced to the platform. By acting as early warning system that is able to identify airborne pathogens including resistances.
General requirements and end-user specification
Simple handling and fast time to result
• Reduced number of manual handling and processing steps by process automation
• Easy interpretation of results by providing yes or no answer.
• The process time (including sample preparation and amplification / detection) of the cycler should not exceed 90 – 120 minutes.
Market size (rough estimate)
As labor Stein is part of the Limbach group, we could estimate the market size for the whole group. The Limbach group receives clinical samples from around 30.000 resident doctors and 600 hospitals which could be potential customers for the PARCIVAL platform.
Breakdown of expected selling price (rough estimate)
Target fabrication cost for one PARCIVAL disk is < 10 € at mass production
including disposable and biochemistry and a retail price of approximately 50€ per PARCIVAL disk.
Price of the processing device (PARCIVAL player) should be below 5000€, a favourable price would be 2000€.
List of Websites:
http://www.parcival-project.eu
Project acronym: PARCIVAL
Project title: Partner Network for a Clinically Validated Multi-Analyte Lab-on-a-Chip Platform
Grant agreement No.: 278090
Project start: 01 January 2012
Project end: 31 December 2014
Website: http://www.parcival-project.eu
Participants:
Participant no. Participant name Country
1 (Coordinator) Erasmus MC Netherlands
2 HSG-IMIT Germany
3 National Institute for Research and Development in Microtechnologies
IMT Bucharest Romania
4 Dr. Stein und Kollegen Labora¬toriums¬medizin, Mikrobiologie, Infektionsepidemiologie, Virologie, Transfusionsmedizin und Human genetik GbR Germany
5 Pathofinder BV Netherlands
6 Rohrer AG Switzerland
7 ASKION GmbH Germany
8 Agrobiogen GmbH Biotechnology Germany
9 EADS Deutschland GmbH, Innovation Works Germany
Introduction
The objective of PARCIVAL is to develop an integrated and automated multi-analyte lab-on-a-disk platform for the fast and reliable sample in answer out diagnosis of highly infectious respiratory tract pathogens, antibiotic resistance patterns and biomarkers for individual severity of the infection. The platform format will be based on completely integrated microfluidic foil-disposables with no external connections except one rotational axis of the centrifugal processing device and the sample inlet ports. The disposables will feature different panels (selection of pathogens, antibiotic resistance patterns, biomarkers), which can be freely selected and combined to allow a comprehensive choice of diagnostic parameters by the clinician at the point-of-care, vastly increasing the access to diagnostic information for doctors in everyday situations and emergencies. The integrated platform with pre-packaged liquid and dry reagents on each test carrier will automate all processing steps from sample preparation, over assay processing up to result reporting.
This will allow evidence-based therapeutic decisions, allow antibiotic prescriptions which are tailored to the individual patient and thus offer the prospect to greatly improve therapeutic outcomes. Also, an air-sampling add-on module for monitoring of clinical ventilation systems will be developed and interfaced to the platform. By acting as early warning system that is able to identify airborne pathogens including resistances.
The consortium consisted of 9 partners (6 enterprises (4 SMEs), 3 institutes). The project was heavily based on the research capabilities of innovative SMEs. SMEs leaded the innovation by implementing diagnostic tests (PathoFinder BV), sample preparation (Agrobiogen GmbH), a processing device (ASKION GmbH), and a production process for the disposables (Rohrer AG). HSG-IMIT was involved with the microfluidic integration of the sample preparation and the diagnostic test on the discs. IMT Bucharest delivered the software needed for the interpretation of the results and EADS Deutschland GmbH developed a system to sample the environment for airborne pathogens. Two clinical partners Erasmus MC and Dr. Stein collected reference samples in order to perform a clinical validation of the system. By the end of the project all elements are nearly ready to be integrated into a full functioning prototype of the system. Unfortunately a clinical validation of the system could not be performed within the timeframe of the project.
Project Context and Objectives:
A summary description of project context and objectives
The objective of PARCIVAL is to develop an integrated and automated multi-analyte detection platform, consisting of the low-cost centrifugal microfluidic disposable “PARCIVAL disk” and the processing device “PARCIVAL player” for the fast and re-liable sample-in answer-out analysis of highly infectious respiratory pathogens, resistance patterns and biomarkers for diagnosis of infections. As an advantage com-pared to non-centrifugal microfluidic platforms, no connections to external actuators / pressure sources is required what tremendously reduces the risk for cross contamination. Fluid actuation is a result of defined centrifugal forces only. The PARCIVAL disk will feature different panels (selection of respiratory pathogens, resistance pat-terns, biomarkers), which can be freely selected and combined to allow a comprehensive choice of diagnostic parameters by the clinician at the point-of-care, vastly increasing the access to diagnostic information for doctors in everyday situations and emergencies. The cartridge will contain pre-packaged liquid and dry reagents and automates all processing steps from sample preparation, over assay processing up to result reporting thereby reducing the hands on time and the need for highly trained stuff.
The developed PARCIVAL platform will allow evidence-based therapeutic decisions, allow antibiotic prescriptions which are tailored to the individual patient and thus offer the prospect to greatly improve therapeutic outcomes. Also, an air-sampling add-on module for monitoring of clinical ventilation systems will be developed and interfaced to the platform. By acting as early warning system that is able to identify airborne pathogens including resistances.
Figure 1: Sample in > Answer out
The project is SME-targeted and is designed to encourage SME efforts towards research and innovation.
The consortium consisted of 9 partners (6 enterprises (4 SMEs), 3 institutes).
Expected impacts listed in the work programme in relation to the topic
“The availability of multi-analyte diagnostic tests is expected to allow for antibiotic prescription that takes both the type of infection of the patient, and the presence of resistant pathogens in the clinical setting into account.”
The objective of PARCIVAL is to implement a multi-analyte diagnostic test based on a lab-on-a-disk system with the capability of running several panel tests at the same time. This either permits screening of e.g. 1-4 patients with a specific panel (e.g. in case of emerging epidemics) or to screen a single patient for an extended panel plus information about primary immune responses (e.g. in critical infections with unknown cause). The initial panels were chosen to allow the identification of common respiratory tract pathogens, antibiotic resistance patterns and biomarkers which offer insight into the state of the suspected infection. Thus, the prescribed medication can be tailored exactly to the cause of infection and the presence of antibiotic resistance. This allows the clinician an unprecedented information depth and a case-specific, evidence-based medication. PARCIVAL could therefore, due to the very fast access of comprehensive diagnostic information at the point-of-care, have a substantial impact on clinical practice in terms of diagnostics and treatment. In summary, in critical cases as well as in routine diagnostics the physician could select one or several diagnostic panel(s), take a patient sample, put it into the point-of-care platform, and receive the analysis result within 1-2 hours. Then, treatment with a specific therapy fitting to patient and specific pathogen can start immediately. The amount of information about pathogens, biomarkers and resistances that will be available in this short timeframe is unprecedented in the state-of-the-art diagnostics. Additionally, by adapting and expanding the panels with new assays, the platform’s scope can be expanded easily to other pressing and emerging health threats.
“Such improved prescription should speed up patient recovery and retard the development of multi-drug resistance.”
By allowing the precise identification of pathogen(s), resistances, and immune response in an automated platform at the point-of-care, PARCIVAL will enable the clinician to immediately select the best-fitting treatment and medication the state-of-the-art has to offer to the individual patient. This will greatly reduce prescription of ineffective antibiotics and thus greatly reduce the risk of generating new resistant strains.
“SME-targeted research is designed to encourage SME efforts towards research and innovation”
The PARCIVAL consortium consists of 3 academic and 6 industrial partners of whom 5 are SMEs. The project is partly coordinated by an SME on scientific aspects ensuring that the outcome of the project satisfies the need of highly innovative European SMEs. SME innovation plays a key-role in the project, since the diagnostic assay, the production technology and the sample preparation unit is based on SME research. Therefore, SMEs will be the main beneficiaries of the project’s exploitation.
Expected impact listed in the area Anti-microbial drug resistance
“The strategic objective of this area is to confront the increasing emergence and spread of antimicrobial drug resistant pathogens in Europe and in a multi-disciplinary approach through the development of effective infection prevention and control strategies.“
The chosen multi-disciplinary approach integrates diagnostic partners with technological cutting-edge R&D and ensures sustainable research by integration of production- and end-user know-how. The platform’s capability to identify a large spectrum of pathogens is perfectly complemented by integration of an add-on module for air sampling and, by interfacing it to the lab-on-a-disk-platform, for monitoring of clinics, transport hubs and critical infrastructures. In case of emerging epidemics, this will provide earliest warning of the pathogenic spread and allow effective prevention and control strategies to be implemented.
Expected impacts listed in the work programme HEALTH 2011
“Improving the health of European citizens and increasing the competitiveness and boosting the innovative capacity of European health-related industries while businesses and addressing global health issues including emerging epidemics”
Automated diagnostics with an affordable device could be a breakthrough for routine diagnostics that allow tailored patient medication, since it enables fast and accurate diagnosis in almost every setting. This personalized medication has the potential of greatly increasing health, comfort and prognosis of the patient.
Competitiveness
The aim of PARCIVAL is to provide innovative European SMEs with a platform that addresses current shortcomings of labs-on-a-chip concerning handling, sample preparation, and production process and allows the establishment of a leading role for Europe in lab-on-a-chip applications by instituting a supply chain for this technology.
Emerging epidemics
The impact of the recent outbreaks of for example the Influenza H1N1 strain in various parts of the world on our health care system and on our society in general immediately stresses the importance a very fast and adequate diagnostic and surveillance system for monitoring respiratory tract infections. Monitoring respiratory tract infections directly by the local physician using a point of care device will be the first step in controlling the outbreak, since fast and specific treatments and/or quarantine measures will help to slow down disease spreading.
Routine diagnostics
In routine diagnostics, this platform has also a great economic and social impact, since knowledge about the pathogen responsible for an acute case of respiratory disease will help to specifically treat the case, thus reducing recovery time. The successful development of a platform with a fast adaptability to new assays will also lead to other systems to identify infectious diseases such as sexually transmitted diseases which could be developed and marketed by the same consortium.
On-line monitoring of air for biological contaminations
The possibility of detecting biological contaminations in the environment is a key capability required during emerging epidemics and other situations. A few devices exist for monitoring the pathogen load in air; however, detection usually relies on a cultivation step. These cultivation procedures are time consuming and require trained personnel. So far, there is no possibility of monitoring air “on-line”, i.e. at short time intervals, high sensitivity, quickly and at acceptable long-term costs (maintenance, reagents, etc.). The impacts of the targeted system will be several fold, for example
i) Better hygiene monitoring in hospitals: Air conditioning systems in hospitals have to be checked for biological contaminations once a year. The testing is done in the ducts of the air conditioning system, not in the rooms themselves. Regarding the high risk of nosocomial infections that exist in hospitals; on-line pathogen detection in hospitals could highly improve the hygienic situation.
ii) Improved safety of infrastructures: The fast spreading of diseases like SARS or swine flu have shown that today the risk of epidemics or pandemics is very high due to the high mobility of people. The monitoring of infrastructures like airports together with an early warning system would greatly increase the safety of the population.
iii) Improvement in pharmaceutical production: Pharmaceuticals are produced and packaged in clean room environments. Testing intervals are usually about 1 month or more with about 1 week for analysis. An on-line detection method would reduce the risk of drugs being produced under conditions which would not comply with the regulations.
Steps that will be needed to bring about these impacts
The validation and integration of the lab-on-a-disk platform in PARCIVAL into a clinical setting will be initialized and tested during the course of the project by Erasmus MC and Labor Stein. This will allow integrating end-user feedback into the research in an early stage. The consortium is heavily based on industrial partners (6/9) and does of course aim for a post-project product development commercialization phase. This combination of early end-user integration and strong product orientation has the high likelihood of resulting in a system with the potential for very good acceptance in clinical settings and a sustainable supply by a partner network for the platform.
Project Results:
In the PARCIVAL project four phases were identified: Specification, development of the components, integration of the components, clinical validation and feedback. The results for these phases are summarized below:
1. Specification
The integration of the components required a careful specification phase in the beginning. Here, interfaces, communication channels, requirements from the individual applications etc. are collected in a specifications document. The specifications were delivered in the first reporting period and guided the next steps.
2. Development of the components
After the first phase, the first versions of the components were developed independently by the respective partners according to the set specifications. Developments in this period were performed on individual test stands / measurement setups.
In total 27 assays were designed by PathoFinder targeting atypical pathogens, 8 targeting typical pathogens, 9 resistance markers and 1 internal control. The NA extraction method has been designed by Agrobiogen.
EADS developed a sampling device.
HSG-IMIT delivered the design of the required disposables for microfluidic tests. Rohrer worked on the implementation of the replication technology.
3. Integration of the components
The individual development phase ended in month 18 of the project with the assembly of the first processing device prototype by Askion. A total of 5 prototypes were created during the project and placed at PathoFinder, HSG-IMIT, ASKION, Erasmus and Labor Stein. Software for the presentation of the results was designed by IMT.
4. Clinical validation and feedback
It was planned that at project month 30, the demonstration of microfluidic assay integration would be achieved. This however was to optimistically planned. The delivery of the prototypes by ASKION was delayed, but more importantly the implementation of the replication could not completely by established within the timeframe of the project. Therefore the planned validation phase targeted at testing of the system with surplus patient samples and comparison to state-of-the art systems could not be performed.
Work packages
The work in Parcival was divided in 8 work packages:
• Work package 1 Management
• Work package 2 System specifications
• Work package 3 Assay development
• Work package 5 Production technology
• Work package 6 Sample preparation
• Work package 7 Microfluidic integration
• Work package 8 Clinical validation and testing
Figure 2: The PARCIVAL microfluidic platform is based on a centrifugal microfluidic disposable test carrier, which microfluidically integrates sample preparation and assay, including pre-storage of all reagents. The test carrier will be handled by processing device including analysis software. The platform will be clinically validated during the project. An add-on air sampling module will allow monitoring of airborne pathogens with little extra research effort. Components are shown in ellipses, work packages as boxes and involved partners are written in boldface. WP1 and WP 2 are project management and specification, respectively, and are not shown in this figure.
Work performed in the different work packages except Work package 1 (Management)
Work package 2 System specifications
Results:
The rather complex system integration approach requires careful specifications. The specification document describes in detail all requirements needed to develop an integrated and automated multi-analyte lab-on-a-disk platform. The document describes end-user specifications in terms of handling, process time, price of device and disposables but also details about the operating conditions, sample taking, sample analyses, the pathogens to be detected. The document can be seen as a summary of the results within all workpackages within the project.
Some highlights of the specification document are:
• The aim is Simple handling and fast time to result.
• Target fabrication cost for one PARCIVAL disk is < 10 € at mass production including disposable and biochemistry and a retail price of approximately 50 € per PARCIVAL disk.
• Price of the processing device (PARCIVAL player) should be below 5000 €, a favourable price would be 2000€.
• Two panels will be tested: a Respiratory pathogen panel (Panel A) and a panel with resistance patterns and some typical pathogens (Panel B). It has been decided not to include the biomarkers C-reactive protein and Procalcitonin (see WP3).
• Operating conditions should comply with standard laboratory environment.
• Three types of pretreatment will be used for three types of clinical samples: Sputum, Broncho-Alveolar lavage (BAL) and Throat swab.
• To facilitate sample pre-treatment and sample loading to the PARCIVAL disk, dedicated sample-transfer modules will be developed for swabs and sputum.
• A workflow for air sampling will be included.
• Sequence of sample preparation, including lysis and nucleic acid purification based on magnetic bead based extraction protocol. The sample preparation protocol has to be automated by the PARCIVAL disk.
• To be able to detect relevant pathogens, first priority is to use real-time PCR (PCR) assays in PARCIVAL. A quantitative result could offer additional benefits, but is considered as optional.
• For the integration of the real-time PCR assay into the PARCIVAL disk, all reagents will be dryly prestored in the amplification chambers.
• A specific thermocycling protocol has to be adopted by the PARCIVAL player for PCR on the PARCIVAL disk.
• Fluidic unit operations / fluidic structures are described in detail
• The processing device is described in detail.
• The requirements of the cartridge (disc) are described in detail.
• The system must comply to directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices.
A first version of the specification document was delivered on 27.03.2012.
Work package 3 Assay development
Results:
Task 3.1 Development of validation targets
PathoFinder and the Erasmus Medical Centre made an inventory of all relevant a-typical and typical respiratory pathogen as well as all important antibiotic resistance markers. Based upon this selection, primers and probes were designed and a verification and validation plan was made. Validation was started using oligo sequences flanking the primer and probe region and for the extended validation experiments cloned constructs were made. In this task all construct were designed and cloned using ´gene synthesis service´ and T7 RNA transcripts were made of all targets with a RNA origin. Every single construct contains a maximum of 8 targets which makes it easy to use due to its equimolar ratio and suitable for quality control experiments.
Task 3.2 Development of new assays
The composition of the assay was split into two different panels; Panel A targeting 22 atypical viruses (Influenza A, RS, Rhino, Boca etc.) and bacteria (M.pneumoniae L.pneumophila etc.) and one internal RNA control (MS2 phage). Panel B consists of 23 typical bacteria (S.pneumoniae E.coli A.baumanii etc.), resistance markers (Mec A (MRSA), KPC, OXA-48 etc.) and 1 internal DNA control. All assays were designed in a way that they can be performed in one tube using four different fluorescent channels. In this matter only 7 qPCR assays are needed to target a large panel of pathogens. The assays have been designed, optimised and validated in strict compliance with the MIQE and CE-IVD guidelines. Verification of the first panel, targeting 21 respiratory viruses and atypical bacteria, was performed and resulted in similar sensitivities in comparison with commercial qPCR assays.
It was decided that the integration of an assay for biomarkers is not relevant. This decision was made in collaboration with our clinical partners (Erasmus MC and Labor Stein). Based on their experience as well as research in current literature, we found that the use of such markers in our diagnostic package has no relevant value. Our goal is to develop a Point-of-Care (POC) assay which can be used for the screening of patients for the presence of microorganisms that cause respiratory tract infections and to differentiate either bacterial- or viral pathogens, leading to a logical empirical treatment regimen. Patients can be divided in 2 groups: community-acquired (CA) patients who visit the general practitioner having respiratory tract problems and patients with healthcare-associated (HA) lower-tract respiratory tract infections (LWI).
This POC test can be used by the general practitioner to diagnose the CA infection for a targeted treatment. The decision to screen HA patients with a suspected LWI is mostly based on a clinical suspicion of the infection that includes physical parameters, such as fever and so forth. Our POC is an excellent test to determine the pathogen in this stage which will be used for targeted treatment. The use of biomarkers like C-reactive protein (CRP) and neopterin are only relevant in cases where there is severe progress in the infection which can results in bacteremia or bacterial sepsis. In the case that the focus of the infection is known, our test can be used for pathogen identification. Nevertheless, the biomarker procalcitonin has been shown to be heightened specifically in bacterial infections, and its levels can be used to establish the duration of antibiotic treatment, but not the type of antibiotic treatment. Furthermore a disappointing diagnostic accuracy has been shown in the patient group where our POC is intended for (trans r soc trop med hyg 2012; annals of emergency medicine 50(1):34-31); sensitivity of 76% and specificity of 70%, even in bacteremia patients means false results, and possibly a negative value of its determination in a significant amount of performed tests. Moreover, the decision of not implementing a protein marker in a RNA/DNA chip resulted in a more straight forward design of the disc because these biomarkers should be tested from blood and this requires a different approach in sample treatment in comparison to samples from the respiratory tract (sputa, bronchoalveolar lavage etc.). This will result in the development of a more cost effective final product and will simplify the market introduction because this platform can better compete with other POC devices. Overall, the decision to not include protein biomarkers is beneficial for the total project.
Task 3.3 Uniforming of reaction conditions / Task 3.4 (Time-)Optimization of reactions
The assay conditions of all assays were adapted and modified for some assays to create one uniform protocol for all sets within this panel. Within this task several enzyme mixes for combined reverse transcription and amplification were tested with the aim to obtain similar results for all different assays. Pre-clinical validation was performed with External Quality Assessment (EQA) programmes from QCMD. The EQA panels were tested from the years 2009 to 2012 and fulfilled all requirements. These assays will used in the next period for the integration on the lab-on-a-disk by HSG-IMIT. Several sets within Panel B are designed and further optimized within the next report period.
Task 3.5 Coating against unspecific adsorption of DNA
The aim of the task was to prevent nucleic acid and/or protein loss in microfluidic channels with a high surface-to-volume ratio. The specific objective was to evaluate different blocking agents within this period. First the assays for validation experiments to set-up a golden standard in the Parcival project were selected. These validation experiments were performed at the facility of PathoFinder and shipped to partner HSG-IMIT. The most important selection criteria for the validation experiments were sensitivity and reproducibility. HSG-IMIT has achieved the same sensitivity with all validation experiments performed with the Labdisk. Because HSG-IMIT met the requirements of the gold standard, there was no reason to start the use of a coating. In conclusion, no loss of specific RNA and DNA was observed, resulted in equal sensitivities obtained with the diagnostics assays of PathoFinder in combination with the newly developed Labdisk by HSG-IMIT.
Summary
In total 27 assays were designed by PathoFinder targeting atypical pathogens, 8 targeting typical pathogens, 9 resistance markers and 1 internal control. We developed 7 control constructs compromising a maximum number of 8 targets per construct. The assays and controls were tested for functionality and used in validation experiments in 2014
Three different objectives were pursued in this study, we tested:
1. Repeatability/reproducibility (inter-/intra-laboratory study) by PathoFinder, Agrobiogen, Dr. Stein and Erasmus;
2. Sensitivity and robustness of the assay;
Using different types of real-time cyclers by multiple technicians and other laboratory variations, on 2 different days using reference NA extraction methods and real-time PCR.
And, 3. The utility was determined by comparison of Cycle Threshold (Ct) values.
It was concluded that the assays are ready for integration on the discs.
Work package 4 Processing device
Objectives
Hard- and software design and engineering of the processing device. Data interpretation and acquisition of the results and the user interface will be provided.
Results:
Task 4.1 Design and construction of the device
Task 4.2 Engineering of control units
The task of ASKION is to develop the hardware platform for the project with the help of the other partners. During feasibility phase all physical and technical systems have been checked in the lab of the ASKION GmbH. This resulted in an approved concept for the first prototypes of processing device.
In order to automate the processes of the centrifugal micro fluidic platform, a processing device is required consisting of the following main hardware modules:
- a spinning drive
- a mechanical cartridge support
- a magnet module,
- a thermal module,
- an optical module
- a control unit.
Task 4.3 Software programming
Control software and an experimental graphical user interface (GUI) will be designed by ASKION in collaboration with IMT while software for data analysis and interpretation will be designed by IMT only.
The report D4.1 contains the following issues:
- Technical drawings of processing device
- Electronic control units of processing device
- Software packages for controlling the spinning drive, the magnet module, the thermal module (including thermo sensors), the optical module, safety features and a GUI.
Task 4.4 Mechanical and electrical assembly, initial operation and testing
Task 4.5 Design review and optimization (hard- and software)
In August 2013 the first processing device was delivered to HSG IMIT in Freiburg (MS8). It was necessary to improve the microfluidic technology. Askion used a remained device for further development and improvement of the functions. Especially the positioning system and the heater concept needed to be optimized. In the first quarter of 2014 we made sure that all required functions have been reached. In February 2014 the first PCR result was running on a player. This result was the basis to set up the next machines for the Parcival consortium. In March 2014 Askion was able to deliver an optimized processing device to HSG IMIT which fulfilled all required functions (D4.2).
From April 2014 until the summertime many tests were performed. Thereby the chemical kits of PathoFinder were used. Parallel to these improvements many stable tests have been performed. These tests made sure to have reproducible results on all 4 devices.
In May 2014 the current results have been presented at a project meeting by EADS in Munich. Together with the colleagues from HSG agreements concerning the controlling system have been made.
All these activities were completed by a ceremonial handover in Gera with all partners who got a device.
Some pictures of the device
Work package 5 Production technology
Objectives
The implementation of the replication technology for the microfluidic disposables and the realization of a physical barrier in the fluidic network which can be removed by the liquid exerting centrifugal pressure at high centrifugal frequencies, acting as a valve.
Results:
Task 5.1 Conceptual design and layout of the disk forming and sealing
The manufacturing process has been proved to be undertaken in the following stages: cutting of the foil from a coil, micro-thermoforming of the cut foil, precutting of the disc, cutting of the sealing foil and sealing onto the disc, final cutting of the disc.
Tests done so far demonstrate that it is possible to form microfluidic structures with the necessary sharp edges. However limits are given by the design of the disc e.g. depth of the cavities or distances from each other.
For example it requires a surface temperature at the PDMS master of about 180 [°C]. With the current tool set-up with a cover ring to separate master and tool environment the foil still most frequently tears at an approximate temperature of 160 [°C]between ring and tool frame.
Another issue is the slow heating up of the PDMS master. We observe that shallow features are formed much better than massive cavities. The mentioned surface temperature is therefore an important process parameter. The lower tool temperature is measured inside the heating plate. In the course of production PDMS masters tend to shrink. It obviously reduces its dimension already over the very first forming cycles. Furthermore, the first couple of disks formed exhibit a bubble structure in the foil that refers to the release of humidity from the PDMS master with the effect of bristling the material and easy breaking the foil.
Design of experiments (DOE) capabilities provides a method for simultaneously investigating the effects of multiple variables on an output variable (response). These experiments consist of runs or tests, in which purposeful changes are made to input variables or factors, and data are collected at each run. The goal is to identify the process conditions and product components that influence quality and then determine the input variable (factor) settings that maximize results.
Task 5.2 Implement prototyping line for sealing and forming procedures
Among other features Minitab, the tool of our choice, offers the factorial design of experiments. Due to the poor forming quality no statistical optimization procedure has so far been applied. A stable process on the actual system can still not be executed and DOE makes little sense so far.
Thermoforming with PDMS master tool
At the beginning of similar projects it has been decided to start with PDMS master tools. Due to lots of design changes on the micro-fluidic design of the disc it is easy to prepare new tools with this technology. The manufacture is quite inexpensive and quick, therefore lots of designs can be tested.
The disadvantage of this technology is the limited tool life. After about 20 cycles the material becomes brittle and tends to break outs of small geometries. Furthermore the measures of the tool are not stable but decrease dimensions with increasing number of cycles.
Based on the experience we made with PDMS masters it has been decided to work with a metal master tool for the final version right from the beginning of the project. Work on PDMS masters tools has therefore not been started.
Thermoforming with Aluminium master tool
Compared to PDMS tools the use of metal tools for commercial productions requires some considerable changes for the tool manufacturing. Whereas deforming of the disc from PDMS tools is not a problem due to the flexibility of the tool it became a severe issue with the aluminium tool. The high vacuum between the foil and the tool during the forming phase is responsible for the strong adherence of the disc to the tool after cooling. A flimsy removal of the disc without any damages is no longer possible.
Coatings and surface treatments
Several coatings and surface treatments which should alleviate the deforming and improve the roughness of the tool have therefore been tested.
Different tool manufacturing technologies were tried out to find the most suitable solution for making the micro thermoforming tools. The focus was on sharp edges, precise dimensions and surfaces with a low roughness.
Conventional machined tool
Conventional machined tool
Furthermore severe problems with the deforming of the disc came up. Because of creating a vacuum for the forming process there is no air between the disc and the tool and therefore the plastic material sticks to the metal.
The manufacturing of the tool is quite complex and expensive. As benefit it could be proved that the processed discs were stable in dimensions.
3D-Plot master tool
As an alternative to the conventional manufacturing, 3D-plotting has been tried out. (Pictures 14 & 15). Reason for the trial was the technology, which builds up the tool layer by layer with micro drops which consist of a blend of aluminium and plastic. This technology should make sharp edges possible. Unfortunately it was proven that the dimensions of the whole plot are not precise enough for the required purposes. The surface furthermore is rougher than grinded surfaces. A later grinding of the tool after plotting was not successful as the surface of the material tends to smear.
During processing tests it was also detected that the processing time increases caused by a bad heat transformance of the material. Longer cycle times were the result. Also with this kind of tool the deforming was a severe challenge based on the a.m. problem.
Although the manufacturing of the tool with this technology is quick and inexpensive 3D plotting was not further traced.
3D-Plot master tool 3D-Plot master tool (detail)
Multi-part tool
A technical breakthrough was achieved with the multi-part tool. With this technology all elevated parts of the structure are made separately. At the base of the forming tool adequate physical dimensions where milled out so that the separately made parts can be pushed through the base from the backside. With this technology there are no more radii at the base of the structure. Instead there are sharp corners finally producing sharp edges at the disc. As the tool has to be produced with highest precision in order to make all parts fitting well also the thermoformed disc is of high precision. Unfortunately this necessary precision makes the tool very expensive.
Achievements
After testing several different tool conceptions it has been found out that the multipart tool is indeed the most expensive one but due to its sharp edges and the high precision it has shown best results in microfluidic properties.
Multi-part tool
The problem of deforming of the disc could be solved by the installation of an overpressure between the ready formed disc and the lower tool part. A slight pressure impulse pushes the disc from the tool.
A challenge for the thermoforming process was the forming of channels with a cross section of just 50µm. Channels of 50µm could be achieved but improvement is necessary regarding the consistency of the dimensions as the dimensions increase towards the base of the forming tool. This is probably due to a too big radius of the milling cutter at its top.
50µm channel 50µm channel
Finally discs were made and tested. As several areas of the disc were identified where design improvements could be made, a new tool has been ordered with the improved design. Transitions from small to big channels were furnished with bigger radii as well as the distance from the stick pack cavities to the bead structure was increased.
Conclusion
After testing several different tool concepts with different materials it has been found out that the a multipart tool made of Aluminium has the best characteristics for the production of discs with sharp edges and the high precision needed to have the necessary microfluidic properties. Problems were caused by the deforming of the discs when they are removed from the forming tool. The problem of deforming of the disc could be solved by the installation of an overpressure between the ready formed disc and the lower tool part. A slight pressure impulse pushes the disc from the tool. Another challenge for the thermoforming process was the forming of channels with a cross section of just 50μm. Channels of 50μm could be achieved but improvement is necessary regarding the consistency of the dimensions as the dimensions increase towards the base of the forming tool.
Finally discs were made and tested. As several areas of the disc were identified where design improvements could be made, a new tool has been ordered with the improved design. With the new tool discs can be made which will most probably meet all set requirements for the microfluidic properties. However this could not be established within time frame of the project which was the main reason that a clinical validation of the system could not be performed.
Work package 6 Sample preparation
Objectives
Integration of sample preparation in microfluidic system.
Development of an automated capturing and separation system based on filtration and magnetic separation to be combined with the centrifugal microfluidic platform in PARCIVAL.
The integration of sample preparation comprises several tasks. The nucleic acid extraction and purification with the Agrobiogen/nexttec system will be adapted for use in the centrifugal microfluidic platform. The add-on module for air-sampling to enable monitoring of airborne pathogens will be developed by EADS and interfaced to the microfluidic detection platform of HSG-IMIT.
Results:
Task 6.1 Sampling microorganisms with filters, especially gelatin or similar filters
A hand-held air sampler device based on gelatin filters was developed with three different applications.
1. respiratory sampling (medical application)
2. wiping sampling
3. air sampling
Common to all three applications is the gelatin filter membrane with a diameter of 13 mm. This membrane was cut out from the original filter membrane and was placed in the air sampler by a magnetic holder. A pump is than connected at the sampling device and air sampling could be started. For medical applications, a nozzle will be contacted at the sampler and patients can blow their breath by a moderate pumping rate at the gelatin membrane. This application is specific for the detection of respiratory diseases. For wiping tests a special wiping head was constructed and connected at the air sample in front of the membrane. This wiping head has specific canals at the surface that the membrane couldn’t stick to the surface.
A)
C)
B)
D)
(A) Hand-held air sampling device for gelatin filters with pump;(B) Air sampler with magnetic holder and cut out gelatin filters with a diameter of 13mm; (C) medical application of air sampler with a nozzle for detecting respiratory diseases; (D) wiping application of air sampler with canals at the surface to avoid sucking of the surface area.
After manufacturing the air sampler device was tested to capture microorganisms. Different sampling applications were tested by the system:
• 1m3 inside
• 1m3 outside
• 1m2 floor
• respiratory sampling
• surface of skin from arm
• surface of computer keyboard
Results show a very high capturing of microorganisms by using gelatin filters. In addition occupied gelatin filters were also tested in the protocol for nucleic acid isolation. As Result a very less inhibition or dysfunction of the protocol could be observed.
Task 6.2 Development of an automated filter handling and liquid handling device
After air sampling two different ways of sample preparation were planned:
1. Unspecific sampling preparations by dissolving the gelatin filters and direct processing of the liquid at centrifugal microfluidic platform.
2. Specific sampling preparation by usage of magnetic beads coated with recombinant G3P bacteriophage protein to capture specific bacteria out of the liquid of dissolved gelatin filters.
Concerning of specific sampling occupied gelatin filters were dissolved in liquid together with magnetic beads. To capture specific bacteria form the solution, magnetic beads coated with a bacteriophage protein were used. As example G3P protein from E. coli M13 phage were cloned in three domains with different length in a T7 expression vector with a 6xHis-Tag. The small domain is N1 (255bp and 85AS) followed by N1L2 (315bp and 104 AS*) and N1L1N2 (705bp and 235AS). Results show that the smallest G3P domain N1 was expressed as an insoluble protein. But both lager domains, N1L2 and N1L1N2, were soluble expressed and could use for coating at magnetic beads for specific sampling of E. coli as model organism.
* Amino acids
A)
B)
C)
Expression of E. coli M13 bacteriophage protein G3P for coating of magnetic beads; (A) schematic drawing of magnetic beads coated with phage protein and captured bacteria; (B) schematic drawing of M13 G3P protein with domains and there differed lengths, N: N-terminal domain, L: Loop, TD: Trans membrane domain at C-terminus, bp: base pairs, AS: amino acids; (C) SDS-PAGE: soluble proteins after Ni-NTA purification of G3P domains N1L1 (approx. 14kDa) and N1L1N2 (approx. 35kDa) shown in characteristic bands; insoluble N1 domain no characteristic band could be observed.
Task 6.3 Nucleic acid purification
First, a real-time PCR system for detection of PCR inhibitors in nucleic acid preparations was developed in order to test their quality. Then, a lysis system for model bacteria (gram-negative bacterial species: E. coli, gram-positive bacterial species: S. epidermidis) was established on the basis of the nexttec™ principal according to the project specifications. In spiking experiments with saliva as sample material a sensitivity of 5 to 15 bacterial genome equivalents per PCR reaction was demonstrated. During optimisation the lysis time was shortened from 40 to 25 min. without remarkable loss in DNA yield.
In cooperation with project partner EADS gelatin filters for air sampling of pathogens were analysed. It was found that 0.5 to 1 filter containing bacteria and viruses can be directly dissolved and used for lysis with the nexttec™ buffer system. The resulting DNA preparation does not inhibit real-time PCR. Gelatin and sample background (dust particles etc.) were effectively removed by nexttec™ cleanColumns.
DNA as well as ribosomal RNA was isolated from lysed bacteria in sample background (e.g. saliva). But, after spiking saliva samples with an RNA bacteriophage (MS-2) according to the specifications (MS-2 as model RNA virus) the phage RNA cannot be detected in the corresponding nucleic acid preparation purified on nexttec™ cleanColumns. We concluded that the nexttec™ buffer system is not able to inhibit RNases sufficiently for subsequent detection of RNA viruses. Therefore, in milestone 5 the decision for a nucleic acid purification system was made in favour of a silica-based magnetic bead system.
A lysis and washing buffer system for nucleic acid purification on magnetic silica beads was developed in parallel to the nexttec™ system. After optimisation of the method DNA and RNA from bacteria and viruses could be isolated. The system was also adapted and tested for the isolation of nucleic acids from gelatin filters after air sampling. The silica magnetic bead system is now introduced together with partners HSG-IMIT and Rohrer to the centrifugal lab-disc.
Summary
During the first period of the project a nucleic acid isolation method on the basis of the nexttec™ 1step system was adapted to the conditions of the PARCIVAL microfluidic disk. But it was found that RNA could not be efficiently stabilized for sensitive detection. Therefore, the decision for a nucleic acid purification system was made in favour of a silica-based magnetic bead system. A basic method was developed and tested. During the second project period the method was optimized for the PARCIVAL goals and tested for the detection of different target organisms in real sample background.
All these optimization works and tests with different types of pathogens and back-ground were the basis for an interlaboratory validation study. The finally developed methods from sample handling to amplification and detection (developed by PathoFinder) were used in four different labs and compared with standard methods. See WP3.
The other part of this Work package included the development of an automated capturing and separation system based on filtration and magnetic separation which could be combined with the centrifugal microfluidic platform in PARCIVAL.
A sampling device has been designed and tested. The tool included three sampling concepts:
1. Respiratory sampling (medical application); 2. Wiping sampling and 3. Air sampling.
Furthermore a Docking station with a filter holder and UV light source was developed. Test were performed on the dissolvent of loaded gelatin membrane filters and the capturing of bacteria with magnetic beads.
Capture proteins (phage proteins) to capture microorganisms were produced and tested.
An existing device was modified for processing of the gelatin filters together with coated magnetic beads.
Because there was no possibility of handling with pathogens in the laboratories of Airbus Group Innovations a validation only with beads concerning total bead loss and precision of pipetting has been performed and showed reasonable results. The estimated capture efficiencies of gelatin filter in combination with magnetic beads was 54%.
Work package 7 Microfluidic integration
Objectives
Development of all required microfluidic components and reagent storage and release concepts. Integration of sample preparation, pathogen test and the biomarker test into the automated microfluidic system. Combination of these tests onto panel test carriers and parallelization.
Results:
Task 7.1 Development, evaluation and selection of microfluidic components
Microfluidic unit operations are required for automation of the assays within the PARCIVAL project. These unit operations have now been selected and described. Unit operations such as sample handling, integration of the sorbent material and switching of fluid flow are being developed to fit the requirements of the project that were previously unavailable. As a next step, all unit operations will be integrated to fabricate the LabDisk with final assay format as defined in the specification document.
Task 7.2 Integration of a nucleic acid purification protocol
Nucleic-Acid purification and extraction module
For magnetic bead based nucleic acid extraction, a microfluidic unit operation for gas-phase transition magnetophoresis was selected and tailored to the requirements of the PARCIVAL project. The microfluidic integration fully automates the following process:
(1) Mixing of 200 µL liquid sample with 10 µL DTT and 200 µL of a lysis buffer containing chaotropic agents
(2) Addition of 200 µL binding buffer; binding of nucleic acids to the magnetic beads
(3) Transport of magnetic beads into 200 µL washing buffer and mix
(4) Transport of magnetic beads into 200 µL of a second washing buffer
(5) Transport of magnetic beads into 200 µL elution buffer to release purified DNA from the bead-surface.
After nucleic acid purification, the elution buffer, containing the purified DNA is transferred to the nucleic acid amplification module. The microfluidic process flow, that is integrated in the PARCIVAL disk demonstrator, is depicted in the following picture sequence [Fig 2 a – f). Results are described in Report on deliverable D7.3.
Fig 2a: CAD Design of PARCIAL Disk for nucleic acid analysis Fig. 2b: 200 µL Sample (1) and 200 µL lysis buffer (2) are mixed in lysis chamber.
Fig 2c: 200 µL binding buffer (3) dissolve 10 µL DTT (1M) and magnetic beads (4) and are transferred into lysate. Fig. 2d: 200 µL washing buffer 1 (5) and 200 µL washing buffer 2 (70% Ethanol) (6) are transferred into washing chambers. Washing buffer 3 (7) is optional. Magnetic beads are transported from lysis chamber into washing chambers and finally into elution chamber (white arrows)
Fig. 2e: 200 µL elution buffer (8) are transferred into elution chamber Fig. 2f: After elution, elution buffer is transferred (9) into aliquoting structure.
Nucleic acid amplification
The feasibility of RT-PCR has been demonstrated on a demonstrator with microfluidic test structures (Fig .3). The demonstrator automatically aliquots a mixture of elution buffer and PCR polymerase into 8 x 20 µL, each aliquot is then transferred into a fluidically separated amplification chamber where it is brought in contact with dryly prestored primers and probes. Thereby, geometric multiplexing, targeting multiple analytes in parallel, could be achieved. The fluidic test structures were designed for being processed in a commercially available PCR thermocycler. Thereby, fluidic structures for aliquoting and feasibility of RT-PCR could be investigated and operating condition could be optimized while the PARCIVAL processing device is being developed in parallel by project partner 7 ASKION GmbH (D4.2)
Fig. 3: Graphical depiction of demonstrator for functional testing of aliquoting and (RT-) PCR in commercially available PCR thermocycler. The validated unit-operation for aliquoting will be integrated into the final PARCIVAL disk.
On-Disk reagent prestorage
The PARCIVAL demonstrator disk included pre-packed liquid reagents for nucleic acid purification (Fig. 1). For packaging of liquid reagents, aluminium stick packs were chosen. The stick packs were fabricated from a polymer-aluminium composite foil with excellent vapour barrier properties. The applied stick pack technology enables long term storage of liquid reagents what has been demonstrated before by van Oordt et al. for water (loss of 0.6 % after 42 days at 70°C) and alcohol containing buffers (loss of 0.8 % after 21 days at 70°C) and will be repeated for the final nucleic acid purification reagents in PARCIVAL. All reagents are released from the stick packs at defined rotational speeds via a peelable seam at the radial outer end. The final PARCIVAL demonstrator will include 5 stick packs containing the following reagents and volumes.
Reagent Volume
Lysis Buffer 200 µL
Binding Buffer 200 µL
Washing Buffer 1 200 µL
Washing Buffer 2 200 µL
Elution Buffer 200 µL
Conclusions and outlook
All microfluidic unit operations for nucleic acid analysis in the PARCIVAL disk have been selected and the microfluidic and bio-chemical feasibility of the single components has been proven. A demonstrator of the PARCIVAL disk for nucleic acid purification including prestored liquid reagents has been fabricated and functionality has been demonstrated successfully. Results of nucleic acid purification are described in Report on deliverable 7.3. Microfluidic unit operations for aliquoting have been selected and fluidic test structures for validation of the (RT-) PCR based amplification in commercially available real-time PCR thermocycler have been fabricated as the laboratory set-up for the PARCIVAL processing device will be finalized in parallel.
Integration and parallelization of the automated assay on one disposable
A novel concept for handling clinical samples (Swab and Sputum) and for transferring pathogens to the LabDisk has been developed and 3D printed prototypes were fabricated and tested for their liquid handling qualities. Biological tests with real-world samples will be conducted in the near future.
Task 7.3 Optimized nucleic acid purification protocol integrated in microfluidic disk.
In order to provide DNA and RNA in sufficient quantity, a microfluidic module for nucleic acid purification was integrated on the PARCIVAL disk prior to multiplex (RT-) PCR based amplification. Ease-of-use is increased by prestorage of all liquid reagents on the disk.
Nucleic-Acid bench-top purification protocol
As described in the report on deliverable 6.3 the adaptation of Agrobiogen nexttec™ 1-step DNA isolation system to co-purify DNA and RNA was not successful due to insufficient RNase inhibition especially in the first lysis step (see fig 2 & 3).
Fig 1: RT real time PCR with RNA isolated from MS2 phage using the Agrobiogen nexttec™ 1-step DNA isolation system. The MS2 phage was spiked in different sample backgrounds. No RNA was isolated and detected in blood and saliva background. 103 phages in lysis approximately correspond to 35 phages in PCR.
--complete nexttec lysis-- M ----2nd lysis step only----
--lysate-- --eluate-- --lysate-- --eluate--
Fig 2: 1% Agarose gel with DNA and RNA isolated from 200 µl saliva with the Agrobiogen nexttec™ 1-step DNA isolation system. If just the 2nd lysis step is performed, RNA can be isolated.But in the first enzymatic lysis step no Rnase inhibitor or destroying agent is resent. Thus even ribosomal RNA is degraded. M – 1 kb DNA ladder.
Therefore, the consortium decided to integrate a magnetic bead based nucleic acid purification protocol as the fall back solution.
The nucleic acid purification protocol developed and tested under work package 6.3 was further adapted and simplified for integration into the Lab Disk.
During the development usual laboratory steps like vortexing or pipetting up and down for mixing or washing the silica magnetic beads were integrated. In order to resemble the situation in the Lab Disk during binding and washing steps the magnetic bead/ buffer suspension was mixed by slowly overhead rotation. Isolated DNA using the standard mixing (pipetting up and down or vortexing) or “smooth” overhead rotation was compared in a real time PCR (Fig 4).
Fig 3: Real time PCR with DNA isolated from S. epidermidis using the magnetic bead based nucleic acid purification protocol. When comparing the two mixing methods pipetting up and down and mixing overhead, no differences can be seen. 104 bacteria cells in lysis approximately correspond to 500 genomes in PCR.
It could be shown that both methods for mixing give the same result. Real time PCR plots are identical in shape and Ct value.
Further the number of wash steps was reduced from three to two. Often the standard methods for DNA isolation on silica surfaces require different washing steps, which are repeated. First, contaminants like proteins are washed away under high salt conditions. Then, second, salts (Guanidinium) are removed with ethanol keeping the nucleic acid precipitated on the silica surface. In order to save time and space we omitted the wash repeats (Fig 5). Both DNA isolated from bacteria with or without sample background (blood) was analysed in a real time PCR.
Fig 4: Real time PCR with DNA isolated from S. epidermidis using the magnetic bead based nucleic acid purification protocol. One washing step is as effective as two washing steps both in peptone-NaCl and in blood.
Samples processed with one washing step gave the same PCR plot as with two washing steps. Consequently, the procedure was simplified.
For a better binding of nucleic acids from lysates to the beads an alcohol (ethanol or isopropanol) was added to the lysates. We found that isopropanol is best suited and added a pure (100%) solution. But during storage of 100% isopropanol in the Lab Disk the risk of delamination of sealed disks halves is too high. Therefore, different volumes and concentrations of isopropanol were tested. The results in Fig 6 show that 80% isopropanol, which does not cause delamination, can also be used both in the same volume or a slightly higher volume. The best variant was to store the beads in 80% isopropanol. But it is rather difficult to keep the magnetic beads in suspension. For this reason a separate storage was preferred.
Fig 5: RT real time PCR with RNA isolated from MS2 phage using the magnetic bead based nucleic acid purification protocol. Different concentrations and volumes of isopropanol were tested for binding of RNA to the silica beads. The plots show that addition of 80% isopropanol (150 or 200 µl) instead of 100% for binding gives almost the same results. To each sample 103 phages were added (spiked) which approximately corresponds to 50 phages per RT-PCR.
Finally, the optimised procedure for lysis and purification of DNA and RNA has been provided for integration to HSG.
No. Process step
(1) Mix 10 μl DTT (1M) with 200 μl of liquid sample
(2) Add 200 μl lysis buffer (containing GuSCN), invert and incubate 10 min at RT
(3) add 150 μl isopropanol (80%) and 50 μl magnetic bead suspension, incubate 5 min at RT under mixing
(4) collect the beads with a magnet, discard supernatant
(5) add 200 μl wash solution, mix
(6) collect the beads with a magnet, discard supernatant
(7) add 200 μl ethanol (70%), mix
(8) collect the beads with a magnet, discard supernatant
(9) dry the beads (15 min at RT)
(10) add 200 μl pure water, resuspend beads and incubate at 50°C, 5 min
(11) collect the beads with a magnet, transfer supernatant to a new reaction tube
Translation of bench top protocol into microfluidic layout
The provided bench top nucleic acid purification protocol has been translated into a sequence of microfluidic unit operations. The selected unit operations have been integrated into the PARCIVAL disk demonstrator. The process flow is described in report on deliverable e 7.2 and in Fig 7a – f.
Fig 7a: CAD Design of PARCIAL Disk for nucleic acid analysis Fig. 7b: 200 µL Sample (1) and 200 µL lysis buffer (2) are mixed in lysis chamber.
Fig 7c: 200 µL binding buffer (3) dissolve 10 µL DTT (1M) and magnetic beads (4) and are transferred into lysate. Fig. 7d: 200 µL washing buffer 1 (5) and 200 µL washing buffer 2 (70% Ethanol) (6) are transferred into washing chambers. Washing buffer 3 (7) is optional. Magnetic beads are transported from lysis chamber into washing chambers and finally into elution chamber (white arrows)
Fig. 7e: 200 µL elution buffer (8) are transferred into elution chamber Fig. 7f: After elution, elution buffer is transferred (9) into aliquoting structure.
3.3. Results of nucleic acid purification
For the proof-of-principle, DNA has been extracted on the PARCIVAL disk demonstrator from non-pathogenic E. coli cultures. For the experiments, all required liquid reagents were prestored on the PARCIVAL disk. The entire process of nucleic acid purification and release of prestored liquid reagents was controlled by a predefined rotational protocol. On-disk extracted E. coli DNA was quantified by real-time PCR and compared to manual reference extractions conducted according to the provided bench top protocol. The experiments were conducted in duplicates (Fig 8a). To evaluate the presence of inhibitors, the extracted DNA was diluted 1:10 and quantified by real-time PCR again (Fig. 8b). The measured Ct values of real-time PCR are depicted in Table 1:
Fig. 8a: Real-time PCR plots of DNA extraction from E. coli on PARCIVAL disks 1 and 2 and comparison to manual reference extraction.
Fig. 8b: Real-time PCR plots of 1:10 diluted extracted DNA on PARCIVAL disks 1 and 2 and comparison to 1:10 diluted manual reference extraction.
Table 1: Real-time PCR results of DNA extraction (Disk 1, 2 and manual reference) and 1:10 diluted DNA (Disk 1,2 and manual reference). All real-time PCRs were conducted in triplicates.
Conclusions and outlook
Microfluidically integrated nucleic acid purification with prestored reagents has been demonstrated successfully. The delay between manual reference extraction and on-disk extraction was 1 – 1.5 Ct values when quantified with real-time PCR. In a next step, the feasibility of the system will be demonstrated for RNA model organisms what was not possible to date due to the change in nucleic acid purification protocol (month 18). Afterwards, the nucleic acid purification protocol will be further optimized with respect to yield and eluate purity. The yield of extracted nucleic acids will be increased by enhancing the mixing of magnetic beads within the sample, typically improving the binding of nucleic acids. For characterization of the eluate quality, further means for measurement of inhibitors (Ethanol, salt, proteins) in the eluate will be implemented.
Task 7.4 Validated demonstrator
Due to delays in the workplan caused by the technological challenges, it was not possible to perform the validation studies on the prototype of the processing device and discs. First of all there was a delay in the delivery of the 4 prototypes of the centrifugal processing device of about six months, see D4.2 But the most critical issue on the end was the development of the production process of the discs, see D5.2 During the final meeting is was discussed to perform the validation studies after the end of the project period. No final decision was made on this issue, but it was decided to follow up on this issue in the first quarter of 2015.
Summary
In this work package, the laboratory process of pathogen- and biomarker testing has been integrated into an automated microfluidic test carrier. The liquid handling has been realized by microfluidic unit operations on a rotating disk with micro channels and chambers. The fluidics design with pre-storage of liquid and air-dried reagents on the centrifugal microfluidic Disk has been successfully proofed.
The first detected pathogen so far was a MS2 phage (internal control in PathoFinder respiratory panel)
Open task: Demonstration of Parcival Disk for analysis of clinical samples.
Work package 8 Clinical validation and testing
Objectives
The validation and integration of the lab-on-a-disk platform in PARCIVAL into a clinical setting will be initialized and tested during the course of the project by Erasmus MC and Labor Stein. This will allow integrating end-user feedback into the research in an early stage.
Results:
WP8 is targeted at the clinical evaluation of the prototype PARCIVAL platform in order to get a proof of concept. The applicability as a clinical tool will be tested and as such a prediction will be made on the potential for post-project commercialization as a device for routine use in diagnostics. The aim of D8.1 is to collect reference samples that can be used to test the system as a whole and to validate it on normal clinical material. The material has been tested using the nucleic acid extraction method and PCR assays in the project in an ex disc setup (see also D3.3). It was also planned to do this on the prototype of the PARCIVAL platform and discs but due to delays in the workplan caused by the technological challenges, it was not possible to perform the validation studies on the prototype of the processing device and discs.
Clinical specimens
The objective was to have at least 5 positive samples per target. Which might be difficult for C. pneumonia and L. pneumophila (spiked material).
Erasmus collected samples containing bacteria that are also screened for antibiotic resistance. Labor Stein collected samples containing viruses and bacteria. Erasmus MC specimens are screened with a-typical set (Panel A) using MagNA Pure 96 DNA and Viral NA Small Volume Kit. Labor Stein specimens screened with resistance set (Panel B) using QiaSymphony/ MagNA Pure Kit
Deliverable D8.1 has been finalized and the following clinical specimens are available for the evaluation of the system:
PANEL A
PANEL B
D8.2 Report on (clinical) validation of automated respiratory disease detection.
D8.3 Report on laboratory validation of automated airborne pathogen detection.
Due to delays in the workplan caused by the technological challenges, it was not possible to perform the validation studies on the prototype of the processing device and discs.
First of all there was a delay in the delivery of the 4 prototypes of the centrifugal processing device of about six months, see D4.2
But the most critical issue on the end was the development of the production process of the discs, see D5.2
During the final meeting is was discussed to perform the validation studies after the end of the project period. No final decision was made on this issue, but it was decided to follow up on this issue in the first quarter of 2015.
Summary
Due to a delay in work package 4 and more critical a delay in the production of enough discs (work package 5) we were not able to perform a clinical validation study with the Parcival system within the time frame of the project. However by the end of the project period all elements are ready. A validation study has been performed in an in tube format (see work package 3).
Potential Impact:
The objective of PARCIVAL is to develop an integrated and automated multi-analyte detection platform, consisting of the low-cost centrifugal microfluidic disposable “PARCIVAL disk” and the processing device “PARCIVAL player” for the fast and reliable sample-in answer-out analysis of highly infectious respiratory pathogens, resistance patterns and biomarkers for diagnosis of infections. As an advantage compared to non-centrifugal microfluidic platforms, no connections to external actuators / pressure sources is required what tremendously reduces the risk for cross contamination. Fluid actuation is a result of defined centrifugal forces only. The PARCIVAL disk will feature different panels (selection of respiratory pathogens, resistance patterns, biomarkers), which can be freely selected and combined to allow a comprehensive choice of diagnostic parameters by the clinician at the point-of-care, vastly increasing the access to diagnostic information for doctors in everyday situations and emergencies. The cartridge will contain pre-packaged liquid and dry reagents and automates all processing steps from sample preparation, over assay processing up to result reporting thereby reducing the hands on time and the need for highly trained stuff.
The developed PARCIVAL platform will allow evidence-based therapeutic decisions, allow antibiotic prescriptions which are tailored to the individual patient and thus offer the prospect to greatly improve therapeutic outcomes. Also, an air-sampling add-on module for monitoring of clinical ventilation systems will be developed and interfaced to the platform. By acting as early warning system that is able to identify airborne pathogens including resistances.
General requirements and end-user specification
Simple handling and fast time to result
• Reduced number of manual handling and processing steps by process automation
• Easy interpretation of results by providing yes or no answer.
• The process time (including sample preparation and amplification / detection) of the cycler should not exceed 90 – 120 minutes.
Market size (rough estimate)
As labor Stein is part of the Limbach group, we could estimate the market size for the whole group. The Limbach group receives clinical samples from around 30.000 resident doctors and 600 hospitals which could be potential customers for the PARCIVAL platform.
Breakdown of expected selling price (rough estimate)
Target fabrication cost for one PARCIVAL disk is < 10 € at mass production
including disposable and biochemistry and a retail price of approximately 50€ per PARCIVAL disk.
Price of the processing device (PARCIVAL player) should be below 5000€, a favourable price would be 2000€.
List of Websites:
http://www.parcival-project.eu
