Click chemical ligation-enabled digital particle counting for multiplexed microRNA analysis.

Digital counting assays, that quantify targets by counting individual signal entities, provide a promising way for the sensitive analysis of biomarkers even at the single-molecule level. Considering the requirements of complex enzyme-catalyzed amplification techniques and specialized instruments in traditional digital counting biosensors, herein, a simple digital counting platform for microRNA (miRNA) analysis is developed by employing the miRNA-templated click chemical ligation to hinge ultrabright quantum dot-doped nanoparticles (QDNPs) on the bottom of microplate well. Compared with the traditional short miRNA-mediated sandwich hybridization mechanism, the click chemistry-mediated ligation featured enhanced stability, achieving higher sensitivity by directly counting the number of QDNPs with a common wide-field fluorescence microscope. Furthermore, enzyme-free cycling click ligation strategy is adopted to push the detection limit of miRNA down to a low level of 8 fM. What is more, taking advantages of the tunable emission wavelength and narrow emission spectra of fluorescent nanoparticles, the platform enables simultaneous detection of multiplex miRNA targets without cross interference. Benefiting from the simple operation, high sensitivity, and good generality, miRNA analysis in complex samples is successfully achieved. This method not only pioneers a new route for digital counting assays but also holds great potential in miRNA-related biological researches.

Augmented Fluorescence Signaling on a Single BaTiO3 Microbead Optical Booster toward High-Sensitive Biosensing.

In this work, we report a new generation of single microbead bioassay that employs a single BaTiO3 microbead as an optical booster for target biomarker enrichment and optical enhancement toward protein and nucleic acid analysis. The single BaTiO3 microbead can not only concentrate the target molecules by nearly 104-fold but also act as an optical booster to prominently enhance the target-induced fluorescence signal by the whispering gallery mode for improving the excitation efficiency and the microlens effect for promoting the signal collecting efficiency, respectively. Compared with using a conventional single microbead, this optical booster exhibits nearly 2 orders of magnitude higher sensitivity without the assistance of any signal amplification techniques or costly instruments. Moreover, this single microbead optical booster is capable of detecting different kinds of protein and nucleic acid biomarkers in a simple mix-and-read manner, holding great potential for early clinical diagnosis.

Digital-like Enzyme Inhibition Mechanism-Based Strategy for the Digital Sensing of Heparin-Specific Biomarkers.

A new microbead (MB)-based digital flow cytometric sensing system is proposed for the sensitive detection of heparin-specific biomarkers, including heparin-binding protein (HBP) and heparinase. This strategy takes advantage of the inherent space-confined enzymatic behavior of T4 polynucleotide kinase phosphatase (T4 PNKP) around a single MB and the heparin's digital-like inhibitory effect on T4 PNKP. By integrating with an on-bead terminal deoxynucleotidyl transferase (TdT)-catalyzed fluorescence signal amplification technology, the concentration of HBP and heparinase can be digitally determined by the number of fluorescence-positive/-negative MBs which can be easily counted by flow cytometry. This is not only the first test to expand the application scenario of T4 PNKP to the digital detection of different biomarkers but also pioneers a new direction for fabricating digital biosensing platforms based on the enzyme inhibition mechanism.

Recent Progress of Perovskite Nanocrystals in Chem/Bio Sensing.

Perovskite nanocrystals (PNCs) are endowed with extraordinary photophysical properties such as wide absorption spectra, high quantum yield, and narrow emission bands. However, the inherent shortcomings, especially the instability in polar solvents and water incompatibility, have hindered their application as probes in chem/bio sensing. In this review, we give a fundamental understanding of the challenges when using PNCs for chem/bio sensing and summarize recent progress in this area, including the application of PNCs in various sensors and the corresponding strategies to maintain their structural integrity. Finally, we provide perspectives to promote the future development of PNCs for chem/bio sensing applications.

Microchamber-Free Digital Flow Cytometric Analysis of T4 Polynucleotide Kinase Phosphatase Based on Single-Enzyme-to-Single-Bead Space-Confined Reaction.

Digital bioassays have attracted extensive attention in biomedical applications due to their ultrahigh sensitivity. However, traditional digital bioassays require numerous microchambers such as droplets or microwells, which restricts their application scope. Herein, we propose a microchamber-free flow cytometric method for the digital quantification of T4 polynucleotide kinase phosphatase (T4 PNKP) based on an unprecedented phenomenon that each T4 PNKP molecule-catalyzed reaction can be spatially self-confined on a single microbead, which ultimately enables the one-target-to-one-fluorescence-positive microbead digital signal transduction. The digital signal-readout mode can clearly detect T4 PNKP concentrations as low as 1.28 × 10-10 U/µL, making it most sensitive method to date. Significantly, T4 PNKP can be specifically distinguished from other phosphatases and nucleases in complex samples by digitally counting the fluorescence-positive microbeads, which cannot be realized by traditional bulk measurement-based methods. Taking advantage of the novel space-confined enzymatic feature of T4 PNKP, this digital mechanism can use T4 PNKP as the enzyme label to fabricate digital sensing systems toward various biomolecules such as digital enzyme-linked immunosorbent assay (ELISA). Therefore, this work not only enlarges the toolbox for high-sensitivity biomolecule detection but also opens new gates to fabricate next-generation digital assays.

Amplification-Free and Mix-and-Read Analysis of Multiplexed MicroRNAs on a Single Plasmonic Microbead.

In this work, a single microbead covered with a plasmonic layer is employed as the microreactor for the multiplexed miRNA analysis without nucleic acid amplification. On the plasmonic layer, the S9.6 antibody is adopted as the universal module for binding DNA/miRNA duplexes regardless of the sequence. Meanwhile, there is also a SERS reporter gold nanoparticle (GNP) pool, in which each group of GNPs is labeled with both a Raman coding molecule and a DNA probe for recognizing a given miRNA of interest. The target miRNAs will lead to the specific capture of the corresponding SERS reporter GNPs onto the plasmonic layer, which will enormously enhance the target miRNA-induced SERS signals. Finally, the enhanced SERS signals concentrated on the microbead will be mapped out by a confocal Raman microscope. The proposed method achieves the high-precision sensing of sub-pM target miRNA in a simple mix-and-read format and possesses multiplexed assay capability.