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Quantum Dot Encoding for In-Solution Single-Molecule Biomarker Counting in Metastatic Prostate Cancer.

Chia-Wei Kuo, Siva Nalla, Suresh Sarkar

    Medrxiv : the Preprint Server for Health Sciences
    |November 24, 2025
    PubMed
    Summary

    We developed a novel, surface-free assay for precise microRNA (miR) quantification in solution. This method enhances sensitivity and throughput for detecting cancer biomarkers like those in metastatic castration-resistant prostate cancer (mCRPC).

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    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Nanotechnology

    Background:

    • Current digital assays for single-molecule biomarker quantification often rely on surface-based methods, limiting sensitivity and throughput.
    • Accurate quantification of microRNAs (miRs) is crucial for diagnosing and monitoring diseases like metastatic castration-resistant prostate cancer (mCRPC).

    Purpose of the Study:

    • To develop a surface-free, wash-free, in-solution assay for highly sensitive and high-throughput sequence-specific counting of miRs.
    • To enable calibration-free measurements and reliable detection of trace biomarkers for precision medicine.

    Main Methods:

    • Utilized DNA nanoflowers (DNFs) densely functionalized with quantum dots (QDs) for stoichiometric assembly with target miRs in solution.
    • Employed fluorescence microscopy and flow cytometry for detecting QD-DNFs as single events without washing.
    • Applied machine learning for assay optimization to achieve high sensitivity and accuracy.

    Main Results:

    • Achieved a limit of detection of approximately 50 aM and high agreement (0.95) with absolute target counts.
    • Demonstrated multiplexed detection of multiple miR sequences using ratiometric and 5-color QD signatures.
    • Successfully detected exosomal miRs from small plasma volumes in mCRPC patients, showing strong agreement with RT-qPCR and reliable detection of miR-375.

    Conclusions:

    • The developed in-solution assay offers enhanced analytical sensitivity and throughput compared to surface-based methods.
    • This technology enables robust absolute counting of clinical biomarkers in plasma, facilitating the translation of cancer precision medicine.
    • The assay's ability to detect trace biomarkers like miR-375 holds significant prognostic value for mCRPC patients.