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.
MicroRNAs (miRNAs) have been considered as promising cancer biomarkers. However, the simple but sensitive detection of low levels of miRNAs in biological samples still remains challenging. Herein, we wish to report an entirely enzyme-free, simple, and highly sensitive miRNA assay based on the counting of cycling click chemical ligation (3CL)-illuminated fluorescent magnetic nanoparticles (MNPs) with a total internal reflection fluorescence microscopy (TIRFM). In this strategy, each miRNA molecule can trigger many cycles of click chemical ligation reactions to produce plentiful ligated oligonucleotides (ODNs) with both 5′-biotin and 3′-fluorophore, resulting in efficient signal amplification. It is worth noting that only the ligated ODNs can bring fluorophores onto streptavidin-functionalized MNPs (STV-MNPs). Notably, merely 10 fluorescent molecules on each 50 nm MNP can make it bright enough to be clearly visualized by the TIRFM, which can significantly improve the detection sensitivity for miRNA. Through fluorescence counting of individual MNPs and integrating their fluorescence intensities, the amount of target miRNA can be quantitatively determined. This miRNA assay can be accomplished in a mix-and-read manner just by simply mixing the enzyme-free 3CL reaction system with the MNPs before TIRFM imaging, which avoids tedious immobilization, washing, and purification steps. Despite the extremely simple operation, this strategy exhibits high sensitivity with a quite low detection limit of 50 fM target miRNA as well as high specificity to well discriminate miRNA sequences with a single-base variation. Furthermore, the applicability of this method in real biological samples is also verified through the accurate detection of the miRNA target in cancer cells.
As generally acknowledged, terminal
deoxynucleotidyl transferase
(TdT) can only elongate DNA substrates from their 3′-OH ends.
Herein, for the first time, we report that TdT-catalyzed DNA polymerization
can directly proceed on the exosome membrane without the mediation
of any nucleic acids. We prove that both the glycosyl and phenolic
hydroxyl groups on the membrane proteins can initiate the DNA polymerization.
Accordingly, we have developed powerful strategies for high-sensitive
exosome profiling based on a conventional flow cytometer and an emerging
CRISPR/Cas system. By using our strategy, the featured membrane protein
distributions of different cancer cell-derived exosomes can be figured
out, which can clearly distinguish plasma samples of breast cancer
patients from those of healthy people. This work paves new ways for
exosome profiling and liquid biopsy and expands the understanding
of TdT, holding great significance in developing TdT-based sensing
systems as well as establishing protein/nucleic acid hybrid biomaterials.
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