Portable electrochemical assay system for on-chip quantitative estimation of bladder cancer biomarkers in real samples

Project Information

BlaC E-assay

Grant agreement ID: 655176

DOI

10.3030/655176

Project closed

EC signature date 13 March 2015

Start date 5 October 2015

End date 4 October 2017

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 195 454,80

EU contribution

€ 195 454,80

Coordinated by

UNIVERSITY OF BATH
United Kingdom

Periodic Reporting for period 1 - BlaC E-assay (Portable electrochemical assay system for on-chip quantitative estimation of bladder cancer biomarkers in real samples)

Reporting period: 2015-10-05 to 2017-10-04

Summary of the context and overall objectives of the project

• Overall objective
The objective of this project is to develop a microfluidic based electrochemical platform focused on the development of inexpensive, accurate, and reliable POC diagnostic devices for bladder specific cancer biomarker detection with multiplexing capabilities and bring them to a pre-commercial stage. Electrochemical sandwich ELISA, branch of electrochemical immunoassay have been increasingly attractive for use in biosensing application due to its highly sensitive, label-free, and real-time multiplexed detection of biomolecules.

• Importance for society
Cancer is a life-threatening disease and its various types may affect almost any organ in human. Among various cancer types, Bladder cancer (BCa) is one of major cancer and affect over 70,000 people every year in the United States alone. One of the best approaches to improve cancer survival rate is to diagnose it at an early stage. In spite of the rapid explosion of new technology platforms, traditional optical ELISA is used in clinical practice, which is laborious, time-consuming, require large sample volume and suffer from low sensitivity. On the other hand, recently proposed electrochemical sensors, which promise easy operation, accuracy, high sensitivity, low cost and compact size mainly suffer from background signals, non-specificity and selectivity in clinical samples.

• Problem being addressed
In this project attempts were made to find solutions for key issues of non-specificity, sensitivity and selectivity in clinical samples for sensitive detection. Attempts were made to solve the key issues via separation of assay chamber and detection chamber with innovative surface chemistry and microfluidic design with electrochemical detection techniques. The project started with the fabrication of hair comb structured gold electrode followed by its characterization. The scope of the project includes development of biosensor platform via separation of assay chamber and detection chamber with innovative surface chemistry, microfluidic design and electrochemical detection techniques, Further it include immobilization of antibodies on the biosensor matrix, evaluation of sensor detection range, specificity study, and prototype development.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The work was focused around four work packages (WP) with a set of milestones (MS) accomplished:
WP1. Sensor Chip and assay chamber fabrication and characterization
MS1. Design, fabrication & characterization of sensor chip
MS2. Assay matrix selection and surface chemistry

WP2. Fluidic system and bioassay
MS3. Fluidic setup and characterization for reagents processing/fluidic networking to conduct all steps of the bio-assay
MS4. Bioassay development and optimization of chemistry and fluidic parameters for selective and sensitive estimation of biomarkers

WP3. Detection of BlaC biomarkers
MS5.Validation and standard curve generation for BlaC specific biomarker estimation in standard solutions
MS6. BlaC specific biomarker estimation in spiked serum/urine sample
MS7. Multiplex detection of BlaC specific biomarkers

WP4. System automation and prototype
MS8. Prototype development and automation of bioassay
MS9. Prototype system validation using standard and spiked samples

Off-site matrix with on-chip detection platform
Figure 1 shows the general schematic for FNAB based matrix modification and antibody binding along with optical images of 3D laser engraved PMMA and 2D PC matrices at various stages of FNAB modification. The sensor was assembled onto glass via fluidic tubings, providing off site sensor for on-chip detection. Figures 2 and 3 show the general schematic sensor setup assembly along with the schematic for prototype concept. As immunoassay steps occur on matrix and only final electroactive product was moved to sensor chip, it resulted in better sensitivity and improved reproducibility.

Sensor response with urine samples
Both the NUMA1 and the CFHR1 sensors were tested via differential pulse voltammetry for different protein concentrations in 10% urine samples. With increasing concentration, the response current was found to be increasing, suggesting successful immunoassay. Both sensors work well in the range 1-100 ng/ml. The detection limits were found to be 1.29 ng/ml and 0.97 ng/ml for NUMA1 and CFHR1, respectively.
Results suggest that measurements for NUMA1 and CFHR1 in urine samples using off site matrix connected to sensor chip via fluidic line based system may provide new and better biosensor platform for measuring of protein markers in urine. The sensors do not show significant interference and are specific for their respective antigen. Further, the electrodes were tested for 3 weeks shelf life at 4 C and found to show more than 95% response.

Prototype response
Using developed procedure and platform, prototype was designed and software was developed in such way that it can control all steps automatically as programmed on display and can run one chip in continuous flow manner or can run multiple chips simultaneously in pulse mode i.e. it will pull solution from reagent bay and deliver to matrix with predefined speed and time and then move to 2nd chip for given instruction or wait till incubation period is over before doing 2nd step of assay. Prototype can be run in manual or automatic mode and can be operated directly from its screen or after connected to computer via USB cable and developed software. Further, it can be connected to an electrochemical potentiostat to run measurement at last step automatically via trigger control. During assay solution after each incubation and washing solution goes directly to waste chamber bypassing sensor chip and only in the last step solution from matrix goes to sensor chip following given time and speed parameter and trigger the connected electrochemical system for signal measurement. Initial results for NUMA1 suggest that developed prototype is working and measurement system can be made automated and multiple sensor testing can be programmed for simultaneous assay.

In summary, fluidic connected off-site matrix based biosensor platform has been developed and prototype has been made showing electrochemical ELISA for multiple chips, which can be automated and performed easily. In validation of the developed off-site matrix based platform, antibody-based detection of bladder cancer biomarkers NUMA1 and CFHR1 was achieved in urine samples.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Project has made the desired impact. The applicant with his experience and skills has created a new platform of biosensing techniques with an increased sensitivity and specificity. Spending time in the UK in a young but highly reputed team has provided him best chances for the career development and he has gained experience in interdisciplinary and industrial collaborations and benefit from an advanced training on topics at the forefront of the research in the field. As a result of his research in Bath and Industrial collaboration he has diversified his methodological skills toolbox, broaden the topics portfolio and added a new interdisciplinary dimension to his research, making him well-prepared for leading new research initiatives. Further secondment at GWENT provided him hands on experience of commercially viable healthcare product development and as a result he got advancement in career and got development manager job in industry in UK itself.

Schematic of prototype concept

Schematic of PC and PMMA based matrix assembly

Image of prototype developed