Hydrazone chemistry-mediated CRISPR/Cas12a system for bacterial analysis.

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.

Boronic Ester-Mediated Dual Recognition Coupled with a CRISPR/Cas12a System for Lipopolysaccharide Analysis.

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.

Tetrahedron supported CRISPR/Cas13a cleavage for electrochemical detection of circular RNA in bladder cancer.

As a diagnostic biomarker, the detection of circular RNA (circRNA) is vital for the early screening of bladder cancer. Usually, the low abundance of circRNA in clinic samples results in the necessarily complicated extraction before detection. In this work, a tetrahedron supported CRISPR/Cas13a cleavage has been explored for direct electrochemical detection of circRNA in urine from bladder cancer. CRISPR/Cas13a system has been reasonably designed to recognize the characteristic back-splice junction site of circRNA. The activated CRISPR/Cas13a by circRNA can cleave uracil bases composed of DNA tetrahedron immobilized on the surface of gold electrode, resulting in the breakage of DNA tetrahedron and the release of electrochemical active molecule methylene blue. By virtue of highly catalytic efficiency of CRISPR/Cas13a and rigid structure of tetrahedron, the developed electrochemical biosensor can directly detect circRNA in 25 µL urine sample with the lowest detection limit of 0.089 fM and linear detection range from 10 fM to 50 nM in less than 10 min, so as to avoid complicated extraction process and benefit for on-site detection.

Application of Chemoselective Ligation in Biosensing.

With the advantages of mild reaction condition as well as high stereoselectivity and efficiency, chemoselective ligations including oxime chemistry, hydrazone chemistry, cycloaddition reaction, C-C multiple bond addition reaction, nucleophilic rings opening reaction, and alkynes-based reaction, have been applied in the field of biosensing. In this review, the roles of these chemoselective ligations for the construction of biosensors have been summarized. The ligations can serve for reactant preparation, interface modification, signal probe synthesis, molecular recognition, signal amplification, and output. Meanwhile, their recent applications for detection of various targets including small molecules, metal ions, protein, and nucleic acid so on, have been reviewed. Moreover, the development trend of these ligations in biosensing has also been prospected in this review.

Electrochemical Evaluation of Tumor Development via Cellular Interface Supported CRISPR/Cas Trans-Cleavage.

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.

Tetrahedron supported click ligation initiated by dual recognition for precise bacterial analysis.

For the 16S rRNA gene of bacterial analysis, the current usage of single recognition probe always causes the false positive result. Meanwhile, it is usually impossible for direct ligation of two free DNA strands modified with click ligation groups in the solution. In our work, A DNA tetrahedron supported click ligation has been elaborately designed; thereby a new method has been further developed for bacterial analysis with dual recognition on two target regions of 16S rRNA gene. Compared with free click ligation, DNA tetrahedron supported click ligation exhibits high reaction rate and ligation efficiency as a result of proximity effect on the supporting interface. The designed DNA tetrahedron can simultaneously bind with two target regions of 16S rRNA gene in bacteria, inducing the proximity of reaction groups and efficient occurrence of click ligation. The established method shows the practical applicability in the serum sample. In a word, inspired by high ligation efficiency on the interface, DNA tetrahedron supported click ligation has been firstly developed and served for bacterial analysis through dual recognition with high specificity, high sensitivity and good performance.

Smartphone-assisted colorimetric biosensor for on-site detection of Cr3+ ion analysis.

A simple and low-cost biosensor has been established for the detection of Cr3+ ion by using the image processing function of the smartphone. In the presence of Cr3+ ion, its dependent DNAzyme can specifically cleave the substrate strand, resulting in the release of ligation probe, which will trigger hyperbranched rolling circle amplification (HRCA) to produce hydrogen ions, causing the color change of the solution containing cresol red. A free APP named "Pixel Picker" has been further used to measure the RGB values. It can be well found that the R/G values correlate with Cr3+ ion concentrations with a linear detection range of 0.5 µM-500 µM and a lowest detection limit of 0.12 µM. In addition, the proposed biosensor has been successfully applied to determine Cr3+ ions in various milks. In a word, the constructed smartphone-assisted colorimetric biosensor has the advantages of simplicity, low cost, high specificity and anti-interference ability.

Fabrication of pH-Adjusted Boronic Acid-Aptamer Conjugate for Electrochemical Analysis of Conjugated N-Glycolylneuraminic Acid.

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.