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

Periodic Reporting for period 2 - RAIS (Scalable, point-of-care and label free microarray platform for rapid detection of Sepsis)

Reporting period: 2016-07-01 to 2018-03-31

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.

Microarrays are a powerful set of technologies which are widely used for the detection of pathogenic micro-organisms, proteins and DNA sequencing, among others. Most commercial equipment relies on large readers and labelled detection, whereas most of the advanced research-level, label-free systems suffer from one of more drawbacks, including insufficient sensitivity, high cost 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 project also aims to assess the technology both in the laboratory and real settings (hospital) with human samples.
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.
Testing of different classes of biomarkers (i.e. proteins, microRNAs, and bacteria) allowed the diagnostic value of the RAIS prototype to be demonstrated. RAIS results have demonstrated that is possible to detect E. coli down to 10 100 CFU/mL, which represents an advance beyond the state of the art. Moreover, the assay is very fast as it can deliver results in minutes.

The detection sensitivity of the reader was enhanced through plasmonic nanostructures, as mentioned below. However, the reader on its own is a significant advance in microscopy to detect tiny quantities of material. For comparison, the RAIS reader has the same axial resolution (1 Angstrom) as that of the Carl Zeiss DIC microscope, currently the most advanced system to detect transparent material. Yet, the reader has a much lower cost (almost two orders of magnitude). However, the most remarkable advantage is the large field of view of reader, three orders of magnitude larger than Carl Zeiss DIC microscope. All these features make the RAIS reader a powerful technology not only for biomarker detection but for a wide range of applications.

Finally, a new label-free plasmonic phase-sensitive detection platform for measuring protein biomarker microarrays in a high-throughput manner was investigated for the first time. This platform allows detection of atomically thick (angstrom-level) topographical changes in a microarray setting, allowing potentially millions of protein biomarker spots to be imaged simultaneously.

Before RAIS, there was no efficient way to exploit the high sensitivity of phase-enhanced interferometric effects, due to the need for bulky and complex optical equipment. For the first time, we exploit the sharp phase transitions at the resonant spectral position of plasmonic gold nanohole arrays instead of the traditional amplitude-sensitive approach for rapid and high-throughput biosensing applications. The compact phase contrast imaging technology, together with large-area nanoplasmonic microarray chip, show the potential to be a powerful tool in PoC applications. Moreover, far-field phase-interrogation is a disruptive technology that will inspire the development of advanced nanophotonic designs.

Blood culture analysis remains the gold standard methodology for sepsis diagnosis. Thus, the time to diagnosis is still too long to effectively impact patient management. E. coli has been reported as the most frequently detected bacterial pathogen in causing sepsis and we have proven the ability of the RAIS instrument to rapidly discriminate SIRS from sepsis patients, i.e non-infectious from infectious processes.