Recent Advance in Single-Molecule Fluorescent Biosensors for Tumor Biomarker Detection

Abstract

The construction of biosensors for specific, sensitive, and rapid detection of tumor biomarkers significantly contributes to biomedical research and early cancer diagnosis. However, conventional assays often involve large sample consumption and poor sensitivity, limiting their further application in real samples. In recent years, single-molecule biosensing has emerged as a robust tool for detecting and characterizing biomarkers due to its unique advantages including simplicity, low sample consumption, ultra-high sensitivity, and rapid assay time. This review summarizes the recent advances in the construction of single-molecule biosensors for the measurement of various tumor biomarkers, including DNAs, DNA modifications, RNAs, and enzymes. We give a comprehensive review about the working principles and practical applications of these single-molecule biosensors. Additionally, we discuss the challenges and limitations of current single-molecule biosensors, and highlight the future directions.

Keywords: DNA/RNA sensor; biomarker; biosensor; enzymatic biosensor; fluorescence microscopy; single-molecule biosensor.

Figure 1

(A) CRISPR/Cas12a-integrated single-microbead sensing platform for amplification-free measurement of DNA [26]. (B) RNase H-based single-molecule biosensor for simultaneously detecting multiple HTLV DNAs [22]. (C) Ligation–transcription circuit-based single-molecule biosensor for simultaneous determination of multiple SNPs [59].

Figure 2

(A) Methylation-sensitive transcription-based single-molecule biosensor for accurate analysis of DNA methylation [65]. (B) Silver-coordinated Watson–Crick pairing-based single-molecule biosensor for simultaneously detecting genomic 4mdC and 6mdA [71]. (C) Bsu DNA polymerase-mediated single-molecule biosensor for rapid detection of OG in telomeres of cancer cells [81].

Figure 3

(A) QD-based single-molecule biosensor for monitoring lncRNAs by coupling PER with isothermal circular strand-displacement polymerization reaction [102]. (B) Single-molecule biosensor for the simultaneous quantification of multiple lncRNAs by utilizing magnetic separation techniques and enzyme-free strand displacement reaction [89]. (C) Single-molecule biosensor for quantitative analysis of miRNA assay by integrating CRISPR/Cas13a with DNA-PAINT [100]. (D) Single-molecule biosensor for one-step detection of miRNAs in lung cancer tissues by utilizing PER and CRISPR-Cas12a [101].

Figure 4

(A) DSN-driven QD biosensor for one-step measurement of hTR at the single-molecule level [106]. (B) QD biosensor based on multi-cycle ligation amplification for sensitive detection of piRNA [111]. (C) QD biosensor for highly specific detection of circRNA based on TWJ skeleton and EXP-RCA [120].

Figure 5

(A) Multiple DNAzyme-powered biosensor for single-molecule monitoring of FTO [127]. (B) Bidirectional strand displacement-driven single-molecule biosensor for simultaneously measuring hAAG and hSMUG1 [128]. (C) Single-molecule biosensor for the measurement and imaging of UDG activity based on CHA-induced FRET [138]. (D) AuNP-based single-molecule biosensor for monitoring APOBEC3A activity by utilizing ERA reaction [144].

Figure 6

(A) Enzyme-free entropy-driven QD biosensor for the imaging of intracellular telomerase activity [145]. (B) HCR-based biosensor for single-molecule monitoring of ALP [156]. (C) AuNP-based nanomachine for the quantification of caspase-8 and caspase-9 by combining Exo III-mediated signal amplification with single-molecule detection [166]. (D) RCA-integrated CRISPR/Cas12a biosensor for isothermal single-molecule measurement of FEN1 in breast cancer tissues [173].