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Scalable, point-of-care and label free microarray platform for rapid detection of Sepsis

Periodic Reporting for period 1 - RAIS (Scalable, point-of-care and label free microarray platform for rapid detection of Sepsis)

Reporting period: 2015-01-01 to 2016-06-30

Sepsis is a potentially fatal whole-body inflammatory reaction caused by severe infection and, with a mortality of 35%, responsible for ∼20,000 deaths per day worldwide. The cost of Sepsis is high – and rising. In 2008, more than €10 billion was spent on hospitalizations for Sepsis in Europe and the USA. Between 1997 and 2008, the total costs for treating patients hospitalized for Sepsis increased by an average of 11.9% per year, adjusted for inflation. The costs related to long-term damage resulting from Sepsis are unknown. In Germany, the cost of a typical episode of Sepsis has more than doubled over the past decade, from approximately €25,000 to €55,000 per patient, and the human cost of Sepsis is incalculable.

Microarrays are a powerful set of technologies which are widely used for the detection of pathogenic micro-organisms, proteins and Deoxyribonucleic acid (DNA) sequencing, among others. Most commercial equipment relies on large readers (also called scanners) and labelled detection, whereas most of the advanced research-level portable and label-free designs suffer from one of more drawbacks, including insufficient sensitivity and specificity, high cost and sophisticated implementation and reduced scalability (number of spots).

The overall objective of the RAIS project is to develop a new, point-of-care, label-free microarray platform based on a proprietary, interferometric, lens-less microscopy design and validate it for quantifying levels of specific Sepsis biomarkers within 30 minutes. The rapid detection of Sepsis, essential to increase the survival rate of the patient, is an ideal proof-of-concept to demonstrate the disruptive capability of the new proposed tool. However, it could also be extended to perform other types of disease screening or multiple simultaneous diagnoses, especially those requiring to rapidly screen a large number of biochemical targets on a single microarray.

The specific objectives of the RAIS project include the development of:
• An optical microarray reader, based on a novel design combining interferometric lens-free microscopy and proximity large-area charge-coupled device (CCD) or complementary metal-oxide-semiconductor (CMOS) image sensing, for high-throughput, label-free, rapid and sensitive detection of nucleic acids, proteins and pathogenic microorganisms. The proposed technology will have a small form factor and manufacturing cost less than €1000, very suitable for point-of-care applications.
• A microarray plate consisting of a transparent slide with a novel nano-structured surface geometry to increase the optical interaction (i.e. detection sensitivity) and covered by specific receptors to capture the biomarkers. Once combined with the new reader, the engineered substrate will allow detection of biomarker levels of a few pg/mL, or even below.
• A disposable cartridge combining the nano-structured microarray plate and microfluidics delivery, capable of detecting a number of Sepsis biomarkers using a few microliters of blood or serum samples.
• The integration of the microarray reader and cartridge in a portable (point-of-care) instrument which can provide a result within 30 minutes, at a cost per patient of less than €50.

The project also aims to assess the technology both in the laboratory and real settings (hospital) with human samples and develop specific testing and management protocols for the utilisation of the test results in the treatment of Sepsis. The collected data will form the basis for the subsequent clinical trials and CE marking validation of the platform, also in accordance to the European Committee on Antimicrobial Susceptibility Testing (European Medicines Agency) protocols and guidelines for medical diagnostic method development.

The project is clearly driven by end-user needs and in this context the consortium aims at exploiting the technology through the existing industrial partners as well as possible spin-off activities by
At the beginning of the project, the system requirements were defined from the perspective of the end-user. From this, a full set of details system specifications were agreed by all partners.

The most suitable biomarkers for detecting Sepsis in patient samples were selected at the beginning of the project. Selected biomarkers include four proteins (C-reactive protein, interleukin-6, MR-proadrenomedullin, and procalcitonin), two bacteria (Escherichia coli and Staphylococcus aureus), and four micro-RNAs (miRNA146a, miRNA223, miRNA15a and miRNA16). The role in sepsis of these kinds of biomarkers is relatively new, and none of them are included in routine clinical diagnosis so far.

Antibodies for the identification of four proteins and two bacteria were identified and tested using standard techniques (ELISA and CLIA). Experiments demonstrated the compatibility of these antibodies with conjugation techniques to be used in the microarray. An immunoassay protocol was set up for every protein demonstrating the feasibility of detecting these biomarkers in the concentration range requested to discriminate sepsis patients from healthy subjects. E. coli and S. aureus were not detected in the requested range because of the limitation of using ELISA methods. Therefore, these antibodies will be used with RAIS technology to evaluate their diagnostic potential.

Appropriate biofunctionalization protocols have been developed, based on the formation of arrays of receptors compatible with the RAIS plate reader. Surface modification and immobilization of receptors has been carried out with glass-based substrates and also with gold-based substrates, mimicking in this latter case the material used for the nanostructured substrates for optical signal enhancement.

The optical microarray reader has been developed. Initially, an alpha prototype instrument was developed. This has been used to test the technology and for initial experiments by some partners. A key achievement with this instrument was the optical detection of ultra-thin (2 nm) dielectric patterns and single protein (BSA) layers.
Different designs of the disposable cartridge combining the nano-structured microarray plate and microfluidics delivery, have been developed and prototypes for testing by the partners produced. In parallel, a holder for the disposable cartridge has been designed which is compatible with the reader. This simplifies the handling of the cartridge for the user.

Towards the end of the period, a beta prototype reader was developed with all necessary functionality including both reader improvements and advanced system integration. Improvements include a reduction in volume of 25 times and a significant reduction in the cost of materials. The size reduction has also been important to drastically improve the optical performance by minimizing the optical path in the reader.

Software development is on-going but most of the required actions are implemented in the beta reader, although an improvement in efficiency of the read-out is still required. Post-processing software has been developed to simplify analysis by the partners. This will be the basis of the automated analysis software in the next period.

In addition to the technology development detailed above, the RAIS project will need to assess the system in a real, clinical setting. For this reason, blood samples collected from patients with sepsis will be indispensable for this validation step. Moreover, such samples will be required for the evaluation of the diagnostic performance of the assay in WP3 and the optimization of the biofunctionalization protocol for each biomarker in human samples in WP5.

In order to have available a representative number of blood samples and to obtain them in advance for proper shipment to the respective partners, ICS have created a Sepsis biobank. This is composed of blood samples from patients diagnosed with sepsis, and controls (samples from patients with non-infectious Systemic In
One Unique Selling Point of the RAIS reader is the time to obtain a measurement result, which is targeted at less than 30 minutes. The microarray reader development work performed in this period has further pushed the state of the art performance of lens-free microscopes. In particular, the functionality has been successfully demonstrated with the detection of monolayers of BSA proteins.

With respect to the microfluidic cartridge, the latest design of the chip has shown promising performance: it took only ca. 10-15 seconds to fill the measurement chamber (water based ink) by driving the liquid through capillary forces, i.e. there are no pumps and metering sensors required, which helps to keep the reader system small and compact. Also there no valves integrated on the disposable cartridge which helps to keep the disposable costs low and maximize the future impact.

As result of the RAIS R&D activities, DIESSE will commercialize the reagents and microarray plates which, together with the microfluidic cartridge from microTEC, are the main components of the disposable diagnostic test for Sepsis. The development of diagnostic kits for the biomarkers will result in new products to be added in DIESSE pipeline and will allow the entrance of DIESSE in a serology market segment different from the ones dedicated to the diagnosis of infectious and autoimmune diseases.

The knowledge acquired by CSIC includes the development of biofunctionalization protocols using a spotting-based approach in order to obtain microarrayed substrates, the development of each individual assay for each biomarker and the final combination in a multiplexed format. This is highly relevant in the development of biosensors, microarrays and in the field of in vitro diagnostics. It involves valuable information capable of being exploited in these areas. The know-how related to the microarray spotting and covalent biofunctionalization of the bioreceptors can be further exploited for other clinical scenarios using the same reader platform developed in RAIS which requires the use of antibodies or DNA probes as capture receptors. The surface modification needed to avoid interferences coming from the serum is usually highly complex and succeeding in this aspect can represent an important milestone for the final goal of the project. The results obtained can also be further applied to other optical devices which also operate under label-free conditions. Moreover, especially exploitable is the combination of both microarray-spotted substrates and antifouling conditions for the direct detection in serum and plasma or in other complex environments (as urine or saliva). The achievement of this particular goal expands the potential for multiplexed analysis which is currently highly demanded in diagnostics. Overall the know-how related to these aspects will be thoroughly used by CSIC in establishing new collaborations with clinical and industrial partners by combining it either with associates developing biosensors or with our own proprietary optical devices.

A major goal that ICS have achieved is the creation of the Sepsis Biobank. This is important to ensure proper handling and storage of the samples to be tested in the present study as well as availability when required. The major impact of this action is to have a collection of samples taken from sepsis patients, non-infectious SIRS patients and healthy controls, for further studies to be developed in the future, not only from our group but also from any other research group or company focused on this area.

RAIS will have a significant impact in the healthcare arena and its outcomes will benefit European industries, making them more competitive in the point-of-care medical market for detection of Sepsis and other infectious diseases, as well as microarrays for DNA sequencing and proteins. Sepsis affects over 18 million people each year and is one of the 10 leading causes of death worldwide. Each hour of delayed treatment i
RAIS Optical Reader Technology
RAIS Platform Concept