Biomarker detection via lab-on-a-chip integrated immunoaffinity approach for fluorescence and mass spectrometry readout

Abstract: Popular Abstract in English The human plasma proteome containing disease-correlated protein biomarkers reflects the pathological and physiological changes relating to diseases. These biomarkers are of interest to aid in diagnosis, prognosis and monitoring of diseases such as cancer. Because of the interests in the disease-correlated biomarkers, plasma, although is a complex biological sample, spanning over 10 orders of magnitude in protein concentrations, is the most widely used clinical specimen for analysis. Detecting these biomarkers represents biological and technical challenges in clinical diagnostics. Lab-on-a-chip approach could provide insights for a simplified and improved detection platform, eliminating the laborious sample preparations compulsory in most conventional methods. With regards to protein biomarker detection for use in point-of-care settings, recent advancements, paved by the microfluidic technology have been demonstrated. Microfluidic devices, manipulating fluids in minute volumes in the range of micro to nanoliter or less, offer insights into enabling a fully integrated, high throughput, cost-effective, rapid ‘sample to answer’, miniaturized immunoassay systems. Microfluidic whole-blood immunoassays hold potential in point-of-care diagnostics application, which has brought us to the exploration of combining the microfluidic technology and antibody microarrays described in this thesis work. At the Department of Electrical Measurements, we have earlier developed a sandwich antibody microarray on 3D structured porous silicon surfaces. Using this method, in this thesis, the focus in the beginning was on detecting the prostate cancer biomarker, Prostate Specific Antigen (PSA) in plasma samples. We have also shown a signal enhancement step utilizing europium nanoparticles to improve the detection limit of our microarray platform for future development of a multiplex prostate cancer biomarker detection platform. Along the line, microfluidic technology specifically utilizing ultrasonic standing wave forces present in the acoustic field has been extensively explored at the department. Since plasma is highly demanded in clinical diagnostics, we designed an acoustophoresis-based microchip capable of separating plasma from undiluted whole blood. We demonstrated the potential application of the acoustic plasmapheresis microchip by linking it to a subsequent off line detection of PSA on our porous silicon antibody microarray. We then moved forward with the idea of combining on-chip plasma separation and a microarray immunoassay, in which a manifold encompassing both platforms in an integrated manner was designed. We showed the capability of rapid immunoaffinity-capturing of PSA directly from whole blood samples, which hold promise of establishing a ‘whole blood sample to answer’ assay. Another interesting lab-on-a-chip application is in the field of mass spectrometrybased plasma proteomics which focuses on reducing the laborious sample preparation step prior to mass spectrometry analysis. Generally, the chip-based capillary electrophoresis separation, on-chip protein digestion and chip for direct infusion i.e. for ESI/MS interface are among the much earlier explored applications in the field of proteomics. In clinical proteomics, where target proteins/ peptides are often known, mass spectrometry analysis in combination with a pre step of immunoaffinity separation, could eliminate the need for extensive separation methods, offering a simplified quantitative analysis path for detection of protein biomarkers. At the Department of Electrical Measurements, we use lab-on-a-chip integrated immunoaffinity approach in combination with mass spectrometry that could offer insights in specifically detecting protein/peptide biomarkers from complex biological samples. We realized the need for integrated platforms that include reduction in sample complexity, minimize sample transfers and the importance to be able to enrich the targeted protein of interest prior to mass spectrometry analysis. Using our microfabricated Integrated Selective Enrichment Target which is compatible for use in any MALDI instrument, we developed immunoaffinity, on-target protein biomarker digestion and sample cleanup protocols to be able to show detection of a protein biomarker, PSA on an integrated platform. This opens potential application for use in the development of SRM analysis for quantitative measurements of proteins. For detecting peptide from complex samples, we developed a porous silicon array-based immunoMALDI protocol demonstrated by the detection of Angiotensin I peptide from plasma samples.

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