A one-tube dual-readout biosensor for detection of nucleic acids and non-nucleic acids using CRISPR-ALP tandem assay.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have been considered a next-generation molecular diagnosis tool. Single-readout mode has been extensively employed in massive CRISPR/Cas12a-based biosensors. In this work, we propose a one-tube dual-readout biosensor (CRISAT) for the first time for the detection of ultrasensitive nucleic acids and non-nucleic acids developed by harnessing CRISPR-ALP tandem assay. In the presence of a target, Cas12a is activated to randomly cut the single-stranded hyDNA sequence of MB@hyDNA-cALP, thus releasing abundant alkaline phosphatase (ALP) in the supernatant solution. By using 4-aminophenol phosphate as the substrate of ALP, p-aminophenol is produced, which then reacts with N-[3-(trimethoxysilyl)propyl]ethylenediamine or diethylenetriamine to generate silicon-containing polymer carbon dots (Si PCDs) or polymer carbon dots (PCDs) in situ, which can be observed by the naked eye or detected using a fluorescent device in the same solution. Using this strategy, a fluorescence and colorimetry dual-readout nanoplatform for CRISPR-based biosensors can be rationally developed. We ascertain the applicability of CRISAT by detecting the SARS-CoV-2 pseudovirus, achieving superior sensitivity and specificity. With simple modification of crRNAs, the CRISAT platform can also be employed to detect monkeypox virus (MPXV) and non-nucleic acids of adenosine triphosphate (ATP). This work shows great potential for the detection of nucleic acids and non-nucleic acids.

Manganese-doped iron coordination polymer nanoparticles with enhanced peroxidase-like activity for colorimetric detection of antioxidants.

A convenient and sensitive antioxidant assay with high performance is essential for assessing food quality and monitoring the oxidative stress level of biological matrices. Although coordination polymer nanoparticles (CPNs)-based nanozymes have emerged as candidates in the analytical field, strategies to improve the catalytic activity of CPNs have been scarcely revealed and studied. Herein, we demonstrate a manganese (Mn) doping strategy to enhance the peroxidase-mimetic activity of Fe-based CPNs. By tuning the Mn doping amounts and selecting 2,5-dihydroxyterephthalic acid (H4DHTP) as ligands, the produced nanozymes in amorphous state followed the catalytic activity order of Fe5Mn-DHTP > Fe8Mn-DHTP > Fe2Mn-DHTP > Fe-DHTP > Mn-DHTP. Ulteriorly, benefitting from the best catalytic performance and definite catalytic mechanism of Fe5Mn-DHTP, versatile colorimetric assays for ultrasensitive detection of one exogenous antioxidant (ascorbic acid, AA) and two endogenous antioxidants (glutathione, GSH; cysteine, Cys) have been deftly devised based on the inhibition of the 3,3',5,5'-tetramethylbenzidine chromogenic reaction in presence of H2O2. It was found that mercaptan (GSH and Cys) and AA exhibited different inhibition mechanisms. Practically, such a colorimetric assay was viable to determine the total antioxidant capacity of drugs and foods with desirable results. This work proposes a feasible strategy for embellishing CPN nanozymes used for designing sensitive and convenient assays for various antioxidants based on an explicit detection mechanism.

Ultrathin PdCu alloy nanosheet-assembled 3D nanoflowers with high peroxidase-like activity toward colorimetric glucose detection.

Enzyme-mimetic properties of nanomaterials can be efficiently tuned by controlling their size, composition, and structure. Here, ultrathin PdCu alloy nanosheet-assembled three-dimensional (3D) nanoflowers (Pd1Cux NAFs) with tunable surface composition are obtained via a generalized strategy. In presence of H2O2, the as-synthesized Pd1Cux NAFs can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to the oxidized form of TMB (oxTMB) with a characteristic absorption peak at 652 nm. Interestingly, Pd1Cux NAFs show obviously composition-dependent peroxidase-like catalytic activities because of the synergistic interaction of nanoalloy. Additionally, different from 2D Pd nanosheets, the distinctive 3D superstructures are featured with rich approachable sites and proper layer spacing, which are in favor of fast mass transport and electron transfers during the catalytic process. Among the studied Pd1Cux NAFs, the Pd1Cu1.7 NAFs show the highest enzyme-like activities and can be successfully applied for the colorimetric detection of glucose with a low detection limit of 2.93 ± 0.53 µM. This work provides an efficient avenue to fabricate PdCu NAF nanozymes in biosensing toward glucose detection. Two-dimensional (2D) PdCu ultrathin nanosheet-assembled 3D nanoflowers (Pd1Cux NAFs) with tunable surface composition exhibit substantially enhanced intrinsic peroxidase-like catalytic activities. The Pd1Cu1.7 NAFs are successfully used as peroxidase mimic catalyst for the colorimetric detection of glucose with low detection limit of 2.93 µM.

Fluorescence assay for alkaline phosphatase based on ATP hydrolysis-triggered dissociation of cerium coordination polymer nanoparticles.

Alkaline phosphatase (ALP) is a significant biomarker for diagnostics. Simple, selective and sensitive detection of ALP activity is thus of critical importance. In this study, an artful fluorescence assay for ALP is proposed based on adenosine triphosphate (ATP) hydrolysis-triggered disassociation and fluorescence quenching of cerium coordination polymer nanoparticles (CPNs). ATP, a recognized natural substrate of phosphatase, can serve as a superb "antenna" to sensitize the luminescence of Ce3+ with the aid of tris(hydroxymethyl) aminomethane (Tris), forming Ce3+-ATP-Tris CPNs. In the presence of ALP, ATP will be catalytically converted into adenosine and inorganic orthophosphate, however neither of them can sensitize Ce3+ in alkaline media. As a result, the obtained CPNs are disassociated, inducing the quenching of the fluorescence. On this basis, a straightforward fluorescence assay for ALP activity is rationally developed. The fluorescence quenching efficiency shows a linear relationship for ALP within the activity range from 0.1 to 10 mU mL-1 with a detection limit of 0.09 mU mL-1 under the optimal experimental conditions. Moreover, this facile yet effective fluorescence method featured simplicity, cost-effectiveness, high sensitivity and high selectivity and can be successfully utilized for the quantitative detection of ALP in human serum samples.

CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout.

Most multiplex nucleic acids detection methods require numerous reagents and high-priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point-of-care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR-RDB (CRISPR-based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost-effectiveness, instrument-free, ease to use, and visualization is reported. CRISPR-RDB integrates the trans-cleavage activity of CRISPR-Cas12a with a commercial RDB technique. It utilizes different Cas12a-crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific-oligonucleotide probes. It has demonstrated that the versatility of CRISPR-RDB by constructing a four-channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2. With a simple modification of crRNAs, the CRISPR-RDB can be modified to detect human papillomavirus, saving two-thirds of the time compared to a commercial PCR-RDB kit. Further, a user-friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR-RDB represents a desirable option for multiplexed biosensing and on-site diagnosis.

Malathion detection based on polydopamine enhanced oxidase-mimetic activity of palladium nanocubes.

Nowadays, fabricating simple and efficient pesticide detection methods become a research focus due to the great threat pesticide residues posed to human health and environment. Herein, we constructed a high-efficiency and sensitive colorimetric detection platform for malathion detection based on polydopamine-dressed Pd nanocubes (PDA-Pd/NCs). The Pd/NCs coated with PDA exhibited excellent oxidase-like activity, which was attributed to the substrates accumulation and accelerated electron transfer induced by PDA. What's more, we successfully achieved sensitive detection of acid phosphatase (ACP) using 3,3',5,5'-tetramethylbenzidine (TMB) as the chromogenic substrate, relying on the satisfactory oxidase activity from PDA-Pd/NCs. However, the addition of malathion could inhibit the activity of ACP and limit the production of medium AA. Therefore, we constructed a colorimetric assay for malathion based on PDA-Pd/NCs + TMB + ACP system. The wide linear range (0-8 µM) and low detection limit (0.023 µM) indicate excellent analytical performance, which is superior to most malathion analysis methods previously reported. This work not only provides a new idea for dopamine coated nano-enzyme to improve its catalytic activity, but also creates a new tactics for the detection of pesticides such as malathion.

An anti-fouling aptasensor for detection of thrombin by dual polarization interferometry.

An anti-fouling surface was designed to effectively resist nonspecific protein adsorption using dual polarization interferometry, based on which the aptasensor for detection of thrombin was fabricated according to the specific interaction between thrombin and its 15-mer aptamer.