PubMed · 1947737
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H U Bergmeyer. 1991. Immunoassay standardization.. https://doi.org/10.3109/00365519109104596
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Single-molecule immunoassays (SMIs) overcome the sensitivity limitations of conventional bulk measurements by enabling a paradigm shift from analog to digital signal readouts, thereby facilitating highly sensitive quantification of ultra-low-abundance biomarkers for precision diagnostics. This review provides a systematic overview of recent advances in SMI technologies and the conceptual framework underlying their evolution. First, discretization strategies for single-molecule counting are classified into hard discretization, based on physical confinement, and soft discretization, based on spatiotemporal isolation, within heterogeneous and homogeneous assay systems, respectively. The fundamental mechanisms by which these strategies mitigate diffusion limitations and enhance signal-to-noise ratios are discussed. Second, the integration of SMIs with CRISPR-based diagnostic systems (CRISPR-dx) is examined, with particular emphasis on their complementary roles in target recognition and signal amplification. Finally, recent applications of SMIs in the diagnosis of oncological, neurological, infectious, and cardiovascular diseases are summarized, along with a critical discussion of current engineering challenges and future directions toward clinical translation.
BACKGROUND: We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes. RESULTS: Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 microg/ml and 1.6 microg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. CONCLUSIONS: These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.
Herein we describe a new class of microfluidic immunoassays based upon solid supported lipid bilayers. Two-dimensionally fluid bilayer material, which can accommodate multivalent binding between surface-bound ligands and aqueous receptors, was coated on the surface of poly(dimethylsiloxane) microchannels. The bilayers contained dinitrophenyl (DNP)-conjugated lipids for binding with bivalent anti-DNP antibodies. Twelve independent data points of surface coverage versus bulk protein concentration could be made simultaneously by forming a linear array of channels and flowing fluorescently labeled antibodies into them. This enabled an entire binding curve to be obtained in a single experiment. The measured apparent binding constant for the DNP/anti-DNP system was 1.8 microM. The methodology for performing heterogeneous assays developed here not only produces rapid results but also requires much less protein than traditional procedures and eliminates some standard sources of experimental error.