The most suitable biomarkers for detecting Sepsis in patient samples were selected. These included four proteins (CRP, IL-6, MR-proadrenomedullin and PCT), two bacteria (E. coli and S. aureus), and four micro-RNAs. However, it was decided to focus on measuring two protein biomarkers (CRP and PCT), one bacteria (E. coli) and one miRNA (miRNA-16), as representative examples.
Antibodies for the identification of the four proteins and two bacteria were identified and tested. An immunoassay protocol was set up for every protein demonstrating the feasibility of detecting these biomarkers in the required concentration range. Appropriate biofunctionalization protocols were developed, using both glass-based and gold-based substrates, mimicking in this latter case the material used for the nanostructured substrates for optical signal enhancement.
Two major designs of the microfluidic chip were developed:
• Chip design based on capillary filling of the chambers (compact and easy to automate).
• Chip design with the liquid driven by a pump (enables faster and more flexible testing of all RAIS platform elements).
The optical microarray reader has been developed. Initially, an alpha prototype instrument was produced for first experiments. A beta prototype reader was then developed with all necessary functionality. Improvements include a reduction in volume of 25 times along with a significant reduction in the cost of materials, and also improved, automated post-processing software and Graphical User Interface to simplify the measurements and analysis by the partners.
A nanostructured substrate was developed which utilizes plasmonic local field enhancements to increase the detection sensitivity of the interferometric lens-free optical reader. The use of the plasmonic chips as microarray plates covered by specific receptors to capture the biomarkers was demonstrated.
A nanoparticle-enhanced bioassay was also developed to amplify the signal from each captured biomarker on the sensor surface, without extending the total turnaround time of the bioassay. The implementation of the NP-enhanced bioassay finally enabled the detection of PCT, one of the most challenging biomarkers of Sepsis due to its small molecular weight and low clinical concentration range.
A Sepsis Biobank was created, composed of blood samples from patients diagnosed with sepsis, and controls (samples from patients with non-infectious SIRS and from healthy volunteers). Various samples were selected and the biomarkers initially proposed were determined using standard laboratory techniques.
Finally, the detection and quantification of CRP and E. coli in real patient samples was attempeted with the RAIS reader. E. coli was successfully detected and it was possible to discriminate between samples which came from sepsis patients and those from SIRS patients and healthy controls. However, due to issues with non-specific adsorption, it was not possible to detect CRP with sufficient sensitivity in patient samples. Nevertheless, the result with E. coli is a key achievement, and with further work on the biochemistry, it should be possible to detect both CRP and PCT in patient samples, based on the LOD achieved in laboratory experiments.
Dissemination activities have been continued throughout the project and have intensified in the last 6 months, as interesting results have been generated. Two workshops have been successfully organised and a press interview was conducted.
