In this study, a hydrazone chemistry-mediated clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system has been proposed for the fist time and constructed. In our system, hydrazone chemistry is designed and employed to accelerate the formation of a whole activation strand by taking advantage of the proximity effect induced by complementary base pairing, thus activating the CRISPR/Cas12a system quickly and efficiently. Moreover, the introduction of hydrazone chemistry can improve the specificity of the CRISPR/Cas12a system, allowing it to effectively distinguish single-base mismatches. The established system has been further applied to analyze Pseudomonas aeruginosa by specific recognition of the probe strand with a characteristic fragment in 16S rDNA to release the hydrazine group-modified activation strand. The method shows a wide linear range from 3.8 × 102 colony-forming units (CFU)/ml to 3.8 × 106 CFU/ml, with the lowest detection limit of 24 CFU/ml. Therefore, the introduction of hydrazone chemistry may also broaden the application of the CRISPR/Cas12a system.

In this work, boronic ester-mediated dual recognition has been coupled with a CRISPR/Cas12a system; thus, a new method for highly specific and sensitive detection of lipopolysaccharide (LPS) is proposed via the simultaneous recognition of boronic acid and an LPS aptamer (LPSA) as well as signal amplification by CRISPR/Cas12a. Specifically, boronic acid-modified magnetic beads (MB@APBA) and aptamers are employed for the simultaneous dual recognition of LPS, while polymerase isotherm amplification is further utilized to induce LPS cycling and form a double strand, which can activate the CRISPR/ Cas12a system so as to amplify the signal. Consequently, a linear detection range can be obtained from 0.05 to 5000 ng/mL, with the lowest detection limit of 44.86 pg/mL. The capturing of MB@ APBA on 1, 2-and 1, 3-cis dihydroxyl-containing substances can not only eliminate the interference of other molecules but also enhance the highly specific recognition of LPSA on LPS. Moreover, MB@APBA can be reused by adjusting the pH value of the reaction system. The method can be developed as a universal platform for the analytical detection of other carbohydrates.

In this work, the boronic acid–aptamer conjugate (BAAC) is elaborately designed and explored as a recognition unit. The admirable properties of the pH-dependent boronic acid ester are integrated with the specific capturing capability of the modified aptamer; thus, BAAC can efficiently and selectively bind with the target by adjusting the pH values. An electrochemical biosensor based on pH-adjusted BAAC has been further developed for the analysis of CNeu5Gc, an important biomarker of different kinds of cancer. The boronic acid moiety in BAAC can react with CNeu5Gc to form a BAAC–CNeu5Gc complex under acidic conditions, followed by the release of CNeu5Gc from the complex and subsequent capture by the aptamer moiety with the adjustment of the pH value to alkalinity. With simplicity, high specificity, and efficiency, the biosensor exhibits a wide linear range from 2.816 to 3603.960 ng/mL with a low detection limit of 1.224 ng/mL and can be applied to analyze CNeu5Gc in animal food samples. Besides, this work can also provide a kind of modified aptamer, i.e., the chemical capturing group-modified aptamer, to give a new viewpoint for the exploration of other functionalized aptamers.

Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by Sambucus nigra agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.

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