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Innovative Electrochemical Multiplex Biosensor for Detection and Quantification of Clinically Relevant Circulating miRNAs

Periodic Reporting for period 1 - INFORM (Innovative Electrochemical Multiplex Biosensor for Detection and Quantification of Clinically Relevant Circulating miRNAs)

Reporting period: 2020-01-01 to 2021-12-31

Analysis of peripheral blood as a source of, tumour-shed, cancer biomarkers has been fostered with the concept of liquid biopsy. Circulating aberrant amounts of non-coding microRNA have been shown to be clinically associated with cancer. Data from clinical studies and meta-analysis demonstrated that altered expression of microRNAs in plasma to be associated with prostate cancer (PCa) aggressiveness, distant metastasis, biochemical recurrence and prognosis. The high chemical stability of miRNAs in body fluids makes them powerful biomarkers present at levels suitable for testing in patient samples.
In INFORM a sandwich assay is used to detect miRNAs that are clinically relevant in PCa. The capture strand is immobilized on a screen-printed gold electrode and the detection strand is labelled with a photosensitiser, which produces singlet oxygen, 1O2, upon excitation with light. The 1O2 produce is quantified using chronoamperometry and it is proportional to the concentration of miRNA in solution. Electrochemiluminescent and photoelectrochemical strategies have been developed t detect miR-141, miR-145 and miR-375 in human fluids with the highlight being the multiplex detection and quantification of the three miRNAs in patient samples.
In INFORM project the work developed focused on the development of i) a detection strategy that could detect and quantify prostate cancer (PCa) relevant miRNAs and ii) a multiplex platform that could simultaneously detect three PCa relevant miRNAs.
The first step was no the development of biosensor, based on a sandwich assay combined with a photoelectrochemical approach, that can selectively and sensitively detect the selected miRNAs
Given the complex matrix of human fluids, the biosensor to be developed would have to be sandwich based in order to be able to capture the target analyte in such complex matrix. The developed assay consisted of streptavidin-functionalised surface, a capture probe functionalised with biotin and a detection probe functionalised with the reporter label.
Before employing the sandwich assay in the photoelectrochemical (PEC) detection of the miRNAs under study, an electrochemiluminescent (ECL) study was performed. In the ECL study, the 96 well plate surface was functionalised with streptavidin, hence the capture probe was immobilised directly on the well surface.
The PEC assay differed from the ECL assay only on the surface in which the capture probe would bind. In this case, the streptavidin functionalised were magnetic beads coated with streptavidin and the reporter probe was a photosensitiser. The working conditions of the assay such as i) the amount of magnetic beads, ii) the amount of capture and detection probes, and iii) the hybridization time were optimised in order to be able to detect the lowest amount possible. The PEC assay showed selective and sensitive detection of the three miRNAs detailed in the Description of Action (DoA).
Given the good results obtained in the developemnt of the detection strategy and sandwich assay, the following step was on the detection and quantification of the selected miRNAs in patient samples
Using the ECL and PEC assays developed above, the detection of miR-141, miR-145 and miR-375 was achieved in three different media: i) in buffer, where the optimisations were performed and the assay tested; ii) in human serum, a complex matrix that allowed the proof-of-concept that the assay could detect circulating miRNAs in human fluids; and iii) human plasma, a more complex matrix which was provided by the collaborators from i3S. These samples were collected from patients that were being followed by a physician at the Centro Hospitalar do Porto.
The final, and most important, step of INFORM was the development of a multiplex platform that allowed the simultaneous detection of multiple miRNAs
Together with our collaborators from the Tumour & Microenvironment Interactions Group from i3S and with partners from the FTI-CoSys Lab and the Flanders Make Strategic Research Centre (University of Antwerp) we have developed a 96 well-plate platform for the detection and quantification of PCa relevant miRNAs in human samples.
In the said platform, a calibration curve is performed for each of the three miRNAs under study and, up to, eight (8) patient plasma samples can be analysed revealing the levels for the three analysed miRNAs.
A commercially available 96 well plate (Dropsens 96X110) was used as surface for the electrochemical process, while the light source was designed and fabricated at the University of Antwerp. The light source is placed over the 96 well plate, which is connected to a multipotentiostat via the plate holder and the readings are performed using the software provided by the potentiostat maker modified by the collaborators from the FTI-CoSys Lab.
The work described above was divided in several manuscripts that have been submitted for publication.
INFORM was a highly innovative project whose highlight was the development of a ELISA like platform (96 well plate) that could simultaneously detect and quantify several miRNAs in patient samples. Before reaching that achievement the research work was first based on i) the development of the detection strategy (electrochemiluminescence and photoelectrochemical assays were developed) and on the amplification of the generated signal, ii) the validation of the sandwich assay approach that would give the necessary sensitivity and selectivity to our biosensor and iii) on the selection of the ideal surface for the photoelectrochemical measurements to be performed.
Given the innovative aspect of the developments mentioned above, several scientific manuscripts have been submitted for publication and are currently under review.
As mentioned before the highlight is the platform that allows the simultaneous detection and quantification of the miRNAs of PCa relevant miRNAs in human samples. Such plat form was developed together with our collaborators from the Tumour & Microenvironment Interactions Group from i3S and with partners from the FTI-CoSys Lab and the Flanders Make Strategic Research Centre (University of Antwerp). The platform has several components including a 96 LED borad, which is what allows illumination of the commercially available electrochemical 96 well plate.
In the said platform, a calibration curve is performed for each of the three miRNAs under study and, up to, eight (8) patient plasma samples can be analysed revealing the levels for the three analysed miRNAs.
A commercially available 96 well plate (Dropsens 96X110) was used as surface for the electrochemical process, while the light source was designed and fabricated at the University of Antwerp. The light source is placed over the 96 well plate, which is connected to a multipotentiostat via the plate holder and the readings are performed using the software provided by the potentiostat maker modified by the collaborators from the FTI-CoSys Lab.
The INFORM project contributes to the ‘Europe’s Beating Cancer Plan’ by having a platform that can simultaneously detect and quantify several miRNAs. This platform can be used by clinicians in order to have faster results and a more complete picture of how the tumour looks like, leading to personalised medicine and better survival rates.
Potential users of the technologies developed in INFORM include hospitals and healthcare providers.
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