A rapid
and sensitive isothermal method is crucial for point-of-care
(POC) nucleic acid testing. Recently, RNA-guided CRISPR/Cas12a proteins
were discovered to exhibit target-triggered nonspecific single-stranded
deoxyribonuclease (ssDNase) activity. Herein, the ssDNase cleavage
capacity of the CRISPR/Cas12a system for interfacial hairpin DNA (hpDNA)
and linear DNA was investigated in detailed. A novel electrochemical
DNA biosensor was then developed via target-induced Cas12a cleaving
interfacial hpDNA. In this strategy, the RNA-guided target DNA binding
activates the robust Cas12a ssDNase activity. The immobilized hpDNA
electrochemical reporters with a low surface coverage and incompact
morphological structure present accessible substrates for highly efficient
Cas12a cleavage, leading to a highly sensitive electrochemical DNA
biosensor. Under the optimal conditions, as low as 30 pM target DNA
was detected in about 60 min with 3.5 orders of magnitude dynamic
range from 50 pM to 100 nM. Furthermore, the practical application
ability of the established sensing method for detecting the target
in complex matrices was also demonstrated. The proposed strategy enables
rapid and sensitive DNA determination, providing a potential tool
for POC molecular diagnostics.
A novel electrochemical cytosensing strategy was designed based on the specific recognition of integrin receptors on a cell surface to arginine-glycine-aspartic acid-serine (RGDS)-functionalized single-walled carbon nanotubes (SWNTs). The covalent conjugation of the RGDS tetrapeptide to SWNTs was proved with Raman and FT-IR spectra. The conjugated RGDS showed a predominant ability to capture cells on the electrode surface by the specific combination of RGD domains with integrin receptors. With the use of BGC-823 human gastric carcinoma cells (BGC cells) as a model, the cell surface mannosyl groups could specifically bind with horseradish peroxidase labeled concanavalin A, producing an electrochemical cytosensor. On the basis of the dual signal amplification of SWNTs and enzymatic catalysis, the cytosensor could respond down to 620 cells mL (-1) of BGC cells with a linear calibration range from 1.0 x 10 (3) to 1.0 x 10 (7) cells mL (-1), showing very high sensitivity. The dual signal amplification could be further used to evaluate the mannosyl groups on the cell surface, and the mannosyl groups on a single living intact BGC cell were detected to correspond to 5.3 x 10 (7) molecules of mannose. This strategy presents a promising platform for highly sensitive cytosensing and convenient evaluation of surface carbohydrates on living cells.
The study of glycobiology has been seriously hampered due to lack of an ideal assay tool. This work proposes a robust carbohydrate monolayer platform to solve the problems of active site inaccessibility and lectin denaturation associated with protein arrays reported for detection of cell surface carbohydrates and develops a convenient method for monitoring cell surface carbohydrate sites of interest, with high sensitivity, acceptable rapidity, low cost, and excellent extensibility. It utilizes the competitive binding of solid-surface-confined and cell-surface-residing carbohydrates to quantum dot labeled carbohydrate recognition protein and subsequent voltammetric quantification of the metal signature. The mannan monolayer strategy exhibited sensitive response to K562 cells and possessed potential specificity due to the specific interaction between lectin and corresponding carbohydrate. By comparing the competitive binding of K562 cells with mannan in solutions, the average Con A binding capacity of a single K562 cell could be estimated to correspond to 6.9 pg or 2.3 x 10(10) mannose moieties. This strategy integrates the advantages of surface assembly, nanotechnology, bioconjugate techniques, and electrochemical detection and can be expanded for profiling cell surface carbohydrates and high-throughput multiple detection by simultaneously using more pairs of lectin and carbohydrate owing to the multiple coding capability of QDs, which provides an important protocol for the quantitative evaluation of cell surface carbohydrate sites.
Sugar and spice: The title method for the simultaneous multiplex analysis of intact cell‐surface glycans (see picture) shows excellent performance in sensitivity, stability, and practicality. The strategy can be used to analyze the dynamic variation of the cell‐surface glycome and to decipher cellular pathophysiological processes.
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