Prostate cancer (PCa) is a significant global health concern, and current monitoring methods using prostate-specific antigen (PSA) lack sufficient specificity and sensitivity. Liquid biopsies, offering access to circulating biomarkers, present a promising alternative. MicroRNAs (miRNAs), short non-coding RNAs involved in gene regulation, are dysregulated in various cancers, including PCa. miR-141-3p and miR-375-3p, specifically, show overexpression in PCa patients' blood and are implicated in tumorigenesis and metastasis. However, translating miRNA biomarkers into clinical practice faces challenges due to limitations of conventional technologies. RNA extraction is labor-intensive and introduces variability. PCR amplification introduces bias and hinders absolute quantification. Existing technologies, including bead- or electrode-based capture probes and microfluidics, often necessitate expensive consumables or large sample volumes. While single-molecule methods like fluorescence microscopy offer high sensitivity, their multiplexing capabilities are limited. Nanopore sensors, although label-free and sensitive, have lacked the multiplexing capacity and selectivity needed for detecting clinically relevant miRNAs directly from complex biological fluids. This paper introduces a novel electro-optical sensing platform that overcomes these limitations, aiming to provide a highly sensitive, amplification-free, multiplexed method for miRNA detection directly from unprocessed serum samples, potentially revolutionizing PCa diagnosis and monitoring.

The literature highlights the need for improved prostate cancer diagnostic tools beyond PSA. Liquid biopsies, utilizing circulating biomarkers like miRNAs, offer a potentially more sensitive and specific approach. Studies have demonstrated the association between dysregulated miRNA expression, specifically miR-141-3p and miR-375-3p, and prostate cancer progression. The limitations of current miRNA detection methods, namely RNA extraction and PCR amplification, are extensively discussed. These conventional techniques are laborious, prone to bias, and often require large sample volumes, hindering their clinical translation. Existing single-molecule and nanopore technologies show promise, but lack the necessary multiplexing capacity and sensitivity for direct detection in complex biological fluids like serum.

The researchers developed a novel electro-optical sensing platform combining single-molecule nanopore sensing with fluorescence microscopy for label-free, amplification-free detection of miRNAs. The key innovation lies in the design of custom molecular probes. These probes comprise a long DNA carrier (with varying lengths for multiplexing) and a molecular beacon (MB). The MB is a stem-loop structured oligonucleotide labeled with a fluorophore and a quencher. Upon target miRNA binding, the stem-loop opens, separating the quencher from the fluorophore and thus generating a fluorescent signal. The length of the DNA carrier serves as an electrical barcode, allowing for multiplexed detection. The experimental setup involves aligning a nanopipette tip with a confocal detection volume. Lambda-phage DNA was enzymatically digested to produce DNA carriers of three different lengths (5.6, 10, and 38.5 kbp). Custom MB sequences complementary to target miRNAs (miR-141-3p, miR-375-3p, let-7a, and miR-21) were hybridized to these carriers. Electro-optical traces were recorded, with electrical signals reflecting the DNA carrier length and optical signals indicating miRNA binding. The sensitivity and selectivity were evaluated using synthetic miRNAs. Finally, the platform was validated using serum samples from prostate cancer patients with active disease and those in remission, and also used for stage classification using three different miRNAs. The entire process is amplification-free and requires only 0.1 µl of serum.

The developed electro-optical nanopore sensing platform demonstrated femtomolar sensitivity (8 and 5 fM for miR-375-3p and miR-141-3p, respectively) and single-base mismatch selectivity. This sensitivity surpasses that of conventional confocal microscopy and bulk fluorescence measurements by almost six orders of magnitude. The platform successfully distinguished between prostate cancer patients with active disease and those in remission based on miR-141-3p and miR-375-3p levels directly from unprocessed serum. The average relative expression levels of miR-141-3p and miR-375-3p were 12.5-fold and 4.2-fold higher, respectively, in the active cancer cohort compared to the remission cohort, with 100% sensitivity and specificity. The technique also showed a significant improvement over detection using serum RNA extracts or RT-qPCR, which exhibited lower sensitivity and significant variability. Analysis of three miRNAs (miR-141-3p, miR-375-3p, and let-7b) demonstrated potential for classifying cancer stages (remission, localized, metastatic), with significant differences (P<0.05) observed in miRNA expression levels between stages, suggesting potential use in disease staging.

The study's findings address the critical need for sensitive and specific diagnostic tools for prostate cancer. The developed electro-optical nanopore platform provides a significant advancement, offering high sensitivity and multiplexing capabilities that surpass current technologies. The ability to detect miRNAs directly from unprocessed serum eliminates the biases and variability associated with RNA extraction and amplification, improving the reliability of results. The femtomolar sensitivity achieved is particularly important for detecting low-abundance miRNAs in clinical samples. The successful differentiation of prostate cancer patients based on miRNA profiles highlights the platform's potential as a minimally invasive diagnostic tool. The ability to classify cancer stages by using multiplexed miRNA analysis further expands the potential applications of this technology. The results suggest this platform could be adapted for a wide range of cancers and other diseases, potentially changing the approach to minimally invasive cancer diagnostics.

This research presents a novel electro-optical nanopore platform for highly sensitive, amplification-free, and multiplexed detection of miRNAs directly from serum. The platform's femtomolar sensitivity, single-base mismatch selectivity, and ability to distinguish between prostate cancer stages directly from unprocessed serum represents a major advancement in liquid biopsy technology. The technology is cost-effective, requires minimal sample volume, and has a fast turnaround time. Future research could focus on expanding the multiplexing capabilities, testing the platform in larger clinical trials with whole blood samples, and exploring its application for other biomarkers and diseases.

The study's sample size for the clinical validation was relatively small, requiring further validation with larger cohorts of patients to confirm the generalizability of the findings. While the platform demonstrates superior sensitivity to existing methods, further optimization could improve the detection limits. The current setup requires specialized equipment, and future efforts should focus on developing a more user-friendly and portable device for clinical use. The impact of exosomes and protein-bound miRNAs on the measured miRNA concentrations warrants further investigation.