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Labeling DNA Probes

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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Expanding High-Fidelity Multiplexing in Ultrasensitive Single-Molecule Protein Detection via Proximity Barcoding.

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    We developed PRO-MOSAIX, a novel digital immunoassay for ultrasensitive protein detection. This platform achieves high accuracy and multiplexing, overcoming limitations of current methods for biofluid analysis.

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Analytical Chemistry

    Background:

    • The human proteome is rich in information but challenging to analyze due to low protein abundance in biofluids.
    • Existing ultrasensitive assays like digital ELISA struggle with accurate, high-throughput multiplexing due to cross-reactivity and limited signal channels.

    Purpose of the Study:

    • To introduce PRO-MOSAIX (PROximity-barcoded Molecular On-bead Signal Amplification for Individual MultipleXing), a novel platform for high-accuracy, ultrasensitive multiplex digital immunoassays.
    • To overcome limitations in multiplexing accuracy, sensitivity, and throughput for protein detection in biofluids.

    Main Methods:

    • PRO-MOSAIX integrates single-molecule protein detection with DNA barcode-based proximity ligation.
    • It generates specific "ON" signals from proximal, matched affinity reagents, minimizing false positives.
    • A single signal readout channel and DNA barcoding circumvent spectral overlap limitations, with further mitigation of DNA amplification false positives.

    Main Results:

    • A 15-plex PRO-MOSAIX assay was established and validated in human plasma.
    • The assay demonstrated low femtomolar sensitivity and high measurement accuracy.
    • PRO-MOSAIX successfully bridges analytical sensitivity and high-order multiplexing capabilities.

    Conclusions:

    • PRO-MOSAIX is a modular, broadly accessible platform utilizing standard lab instrumentation and high-throughput flow cytometry.
    • It offers a significant advancement for analyzing low-abundance proteins in complex biological samples.
    • This technology enhances diagnostics and basic research by enabling precise, ultrasensitive multiplexed protein quantification.