| In vitro diagnostics(IVDs)technology is active and important in the medical field and is developing towards two different technical directions.One is fast,accurate and simple point-of-care(POC)testing to meet the clinical rapid detection needs and chronic disease monitoring.Due to complex biological samples,low target concentration,and strong timeliness,the development of simple,affordable,yet highly sensitive and specific POC technologies is a still great challenge in science and engineering today.The other is the development of highly integrated and automated instrumentation to achieve precise diagnosis of the disease with extremely high efficiency and sensitivity.The single-molecule counting digital detection technology enables the detection of ultra-trace levels of analytes.As low signal of single target,and background signal interference,the development of simple,efficient,high-throughput single-molecule counting digital detection technology is the key scientific issues.Pursuing point-of-care testing and precise diagnosis,the major works of the thesis are listed as following:Part 1.Portable Analysis based on Nano-catalytic Gas GenerationWe firstly demonstrate that a very familiar,yet underutilized,physical parameter—gas pressure—can serve as signal readout for highly sensitive bioanalysis.Integration of a catalyzed gas generation reaction with an immune molecular recognition component leads to significant pressure changes,which can be measured with high sensitivity using a low-cost and portable pressure meter.In particular,we have combined the decomposition of H2O2 catalyzed by Pt nanoparticles with an antibody-based sandwich assay for detection of various protein analytes.Introduction of the substrate initiates a rapid catalytic gas-generation reaction,leading to a significant pressure increase in the closed reaction chamber allowing a sensitive readout by using a portable pressure meter.This new signaling strategy opens up a new way for simple,portable,yet highly sensitive biomedical analysis in a variety of settings.To further expand the application of the simple and hand-held pressuremeter,a portable method for the rapid detection of the disease biomarker C-reactive protein(CRP)was developed.The pressure-based method with a hand-held pressuremeter could facilitate CRP measurements in POCT scenarios,which can provide valuable information for identification of bacterial infection and timely administration of antibiotics.Enzyme-linked immunosorbent assay(ELISA)is a popular laboratory technique for detection of disease-specific protein biomarkers.However,ELISA requires labor-intensive and time-consuming procedures,with skilled operators and spectroscopic instrumentation.Simplification of the procedures and miniaturization of the devices are crucial for ELISA-based POC testing in resource-limited settings.Here,we present a fully integrated,instrument-free,low-cost and portable POC platform which integrates the process of ELISA and the distance readout into a single microfluidic chip.Based on manipulations using a permanent magnet,the process is initiated by moving magnetic beads with capture antibody through different aqueous phases containing ELISA reagents,and finally converts the molecular recognition signal into a highly sensitive distance readout with a catalyzed gas generation reaction for visual quantitative bioanalysis.Without additional equipment and complicated operations,our integrated ELISA-Chip with distance readout allows ultrasensitive quantitation of disease biomarkers,which shows great potential for quantitative POCT in resource-limited settings.Part 2.Single Protein Digital Analysis based on Droplet MicrofluidicsWe propose a microchannel design that accomplishes the functions of microbeads trapped efficiently and droplet generation rapidly,and apply it to single-molecule enzyme-linked immunosorbent assay(digital ELISA).The digital ELISA approach detected as few as 10-16 M prostate specific antigen(PSA)much lower than conventional ELISA.Beads are encapsulated in generated droplets to achieve one-bead-to-one-trap in the array,which do not rely on Poisson distribution.Our microfluidic chip design with low reagent consumption,low cost,and high-throughput is an ideal device for single-molecule counting digital detection applications. |
