Construction of a dual-signal readout platform for effective glutathione S-transferase sensing based on polyethyleneimine-capped silver nanoclusters and cobalt-manganese oxide nanosheets with oxidase-mimicking activity.

A novel dual-mode fluorometric and colorimetric sensing platform is reported for determining glutathione S-transferase (GST) by utilizing polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) and cobalt-manganese oxide nanosheets (CoMn-ONSs) with oxidase-like activity. Abundant active oxygen species (O2•-) can be produced through the CoMn-ONSs interacting with dissolved oxygen. Afterward, the pink oxDPD was generated through the oxidation of colorless N,N-diethyl-p-phenylenediamine (DPD) by O2•-, and two absorption peaks at 510 and 551 nm could be observed. Simultaneously, oxDPD could quench the fluorescence of PEI-AgNCs at 504 nm via the inner filter effect (IFE). However, in the presence of glutathione (GSH), GSH prevents the oxidation of DPD due to the reducibility of GSH, leading to the absorbance decrease at 510 and 551 nm. Furthermore, the fluorescence at 504 nm was restored due to the quenching effect of oxDPD on decreased PEI-AgNCs. Under the catalysis of GST, GSH and1-chloro-2,4-dinitrobenzo (CDNB) conjugate to generate an adduct, initiating the occurrence of the oxidation of the chromogenic substrate DPD, thereby inducing a distinct colorimetric response again and the significant quenching of PEI-AgNCs. The detection limits for GST determination were 0.04 and 0.21 U/L for fluorometric and colorimetric modes, respectively. The sensing platform illustrated reliable applicability in detecting GST in real samples.

A two-dimensional iron-doped carbon-based nanoenzyme with catalase-like activity for the detection of alkaline phosphatase and ascorbate oxidase.

Herein, we successfully synthesized two-dimensional iron-doped carbon-based nanosheets (Fe-N800 CS) with catalase-like activity through doping Fe into Zn MOF and introducing graphitic C3N4 (g-C3N4). The interaction of the Fe-N800 CS with hydrogen peroxide could generated abundant reactive oxygen species (ROS) and further oxidize o-Phenylenediamine (OPD) to 2,3-diaminophenazine (DAP) which has constant fluorescence at 560 nm. Ascorbic acid (AA) could be generated via the hydrolysis reaction between alkaline phosphatase (ALP) and ascorbic acid 2-phosphate (AAP). AA can be oxidized to dehy-droascorbic acid (DHA) by ROS, and then combined with OPD to generate 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) with fluorescence at 440 nm, which could increase as the concentration of AA enhanced. DHA could also be generated through oxidation of AA by ascorbate oxidase (AAO). Thus, by monitoring the fluorescence ratio (I560/I440), a ratiometric fluorescence biosensing platform for ALP and AAO was established with the linear ranges in 0.2-10 U/L and 1-60 U/L, respectively. The limit of detection for ALP and AAO were 0.12 U/L and 0.59 U/L. Furthermore, the biosensing platform was successfully applied for the detection of ALP and AAO activity in human serum samples. This work provides a potential tool for future biomedical diagnostics.

Hydrogel-involved portable colorimetric sensor based on oxidase mimic Fe/Co-NC for acetylcholinesterase detection and pesticides inhibition assessment.

Herein, we synthesized a novel N-doped carbon layer encapsulated Fe/Co bimetallic nanoparticles (Fe/Co-NC), which exhibited superior oxidase-like activity due to the facilitation of electron penetration and the formation of metal-nitrogen active sites. Fe/Co-NC could catalyze the oxidation of 3,3,5,5-tetramethylbenzidine (TMB) to blue oxTMB. Acetylcholinesterase (AChE) could catalyze the hydrolysis of thioacetylcholine to produce reducing thiocholine, which prevented TMB from oxidation. Thus, a portable hydrogel colorimetric sensor was developed for on-site and visual monitoring of AChE with the detection limit of 0.36 U L-1, and successfully applied to detect AChE in human erythrocyte samples. Furthermore, this platform was used to investigate the inhibition of triazophos on AChE activity.

A novel self-assembled dual-emissive ratiometric fluorescent nanoprobe for alkaline phosphatase sensing.

BACKGROUND: Alkaline phosphatase (ALP) is widely found in various organs and tissues of the human body which could assist in the verification of the presence of various diseases through its content in the blood. In the past few years, many analytical methods for ALP activity assays have been explored. However, a simple and economical method with high sensitivity and specificity also remains great challenge. Therefore, the development of sensitive and efficient approach for ALP analysis is of great significance in biomedical studies. RESULTS: Herein, we constructed a highly sensitive and label-free ratiometric fluorometric biosensing platform for the determination of ALP activity, which utilizing lysozyme(Ly)-functionalized 5-methyl-2-thiouracil(MTU)-modified gold nanoclusters (MTU-Ly@Au NC) and poly-dopamine (PDA) as signal indicators. Dopamine (DA) can self-polymerizes to form PDA under alkaline conditions that can further quenched the fluorescence of MTU-Ly@Au NC at 525 nm due to fluorescence resonance energy transfer (FRET) and absorption competition quenching (ACQ) effects. In this process, the PDA fluorescence intensity at 325 nm was nearly unchanged. After the addition of ALP, ascorbic acid (AA) which can alleviate the self-polymerization process of DA was generated from the substrate ascorbic acid 2-phosphate (AAP), thus changing ratiometric fluorescence intensity of I525/I325. Hence, by monitoring the fluorescence ratio (I525/I325), a ratiometric fluorescence biosensing platform for ALP was established with the linear calibration in the range of 0.5-8 U L-1 and the limit of detection of 0.157 U L-1. SIGNIFICANCE: This work not only synthesized a novel fluorescence probe with simple preparation and low cost for ALP which has excellent anti-interference properties and selectivity. Furthermore, this biosensing platform was successfully applied for the determination of ALP activity in human serum samples. This work provided a potential tool for biomedical diagnostics in the future.

Construction of a Label-Free Ratiometric Biosensor Based on Target Recycling Amplification and Hg-ZnSe QDs for Assay of BChE and OPs.

Herein, we constructed a label-free ratiometric fluorescence biosensing strategy for the determination of butyrylcholinesterase (BChE) activity and organophosphorus (OPs) concentration. BChE promoted the hydrolysis of iodized s-butyrylthiocholine (BTCh) into a reducing substance thiocholine, which can decompose CoOOH nanosheets (CoOOH NSs) to Co2+. Subsequently, the single-stranded DNA (ssDNA) on the surface of CoOOH NSs was released. Then, ssDNA hybridized with hairpin DNA (h-DNA) and triggered the target recycling amplification process, producing large amounts of G-quadruplex. After adding thioflavin T (ThT), the target BChE was converted into activatable G-quadruplex/ThT with an amplified yellow fluorescence signal. The addition of OPs could significantly inhibit the hydrolysis of BTCh by BChE and thus unable to produce the yellow fluorescence G-quadruplex/ThT complex. Throughout the entire process, the fluorescence intensity of Hg-ZnSe QDs as a reference signal remained unchanged at 630 nm. Furthermore, this work provided an effective approach for detecting the BChE activity in serum samples and OPs in fruits and vegetables.

A sensing platform for on-site detection of glutathione S-transferase using oxidized Pi@Ce-doped Zr-based metal-organic frameworks(MOFs).

The development of point-of-care testing (POCT) for glutathione S-transferase (GST) is an effective way to establish the mechanism of targeted monitoring of cancer chemotherapy drug metabolism. Assays for GST with high sensitivity as well as on-site screening have been urgently required to monitor this process. Herein, we synthesized oxidized Pi@Ce-doped Zr-based metal-organic frameworks (MOFs) by electrostatic self-assembly between phosphate and oxidized Ce-doped Zr-based MOFs. It was found that the oxidase-like activity of oxidized Pi@Ce-doped Zr-based MOFs was substantially increased after phosphate ion (Pi) assembly. And a stimulus-responsive hydrogel-based kit was constructed by embedding oxidized Pi@Ce-doped Zr-based MOFs into a PVA (polyvinyl alcohol) hydrogel system, we integrated a portable hydrogel kit with a smartphone for real-time monitoring of GST for quantitative and accurate analysis. The color reaction was triggered based on oxidized Pi@Ce-doped Zr-based MOFs with 3,3',5,5'-tetramethylbenzidine (TMB). However, in the presence of glutathione (GSH), the above color reaction was hindered due to the reducibility of GSH. Catalyzed by GST, GSH can react with 1-chloro-2,4-dinitrobenzo (CDNB) to form an adduct, which caused the color reaction to occur again, resulting in the color response of the kit. In combination with ImageJ software, the kit image information acquired by smartphone could be converted into hue intensity, providing a direct quantitative tool for the detection of GST with a detection limit of 0.19mU·L-1. Based on the advantages of simple operation and cost-effectiveness, the introduction of the POCT miniaturized biosensor platform will meet the requirements of on-site quantitative analysis of GST.

A dual-signal fluorometric and colorimetric sensing platform based on gold-platinum bimetallic nanoclusters for the determination of ß-galactosidase activity.

Herein, a novel dual-signal sensing system for the determination of ß-galactosidase (ß-Gal) activity was established, which was based on a dual-emission probe assembled from gold-platinum bimetallic nanoclusters (Au-Pt NCs) and rhodamine B. Under the catalysis of ß-Gal, 4-nitrophenyl ß-D-galactopyranoside (PNPG) was rapidly hydrolyzed to generate p-nitrophenol (PNP), which has an obvious UV absorption peak at 400 nm. The hydrolyzed product PNP can quench the fluorescence of Au-Pt NCs effectively by inner filter effect (IFE), and PNP had no impact on the fluorescence of rhodamine B, which will change the emission intensity ratio of Au-Pt NCs and rhodamine B. Therefore, the ratiometric fluorescent and colorimetric dual-signal sensor based on Au-Pt NCs and rhodamine B was successfully constructed for sensitive detection of ß-Gal activity. The linear detection range for the ratiometric fluorescence and colorimetric methods were 2.5-25 U/L and 15-55 U/L with detection limits of 1.2 U/L and 5.2 U/L, respectively. The developed assay method has been used for quantitative detection of ß-Gal in spiked serum samples and showed good performance. And the detection platform has high reliability and excellent selectivity, which opens a new avenue for the further application of Au-Pt NCs in chemical sensing and biological analysis.

Cascade reaction biosensor based on gold nanocluster decorated iron-cobalt oxide nanosheets as a superior peroxidase mimic for dual-mode detection of α-glucosidase and its inhibitor.

Herein, we have synthesized a novel kind of gold nanoclusters decorated iron-cobalt oxide nanosheets (His-AuNCs@FeCo-ONSs) assembled by electrostatic interaction, which possessed both outstanding peroxidase-like activity and fluorescence property. Taking advantage of our bifunctional hybrid nanozyme and enzyme cascade reactions, a sensitive dual-mode (colorimetric/fluorescent) detection method for α-glucosidase was constructed. The detection limits for α-glucosidase were 2.2 U/L and 3.3 U/L in fluorometric and colorimetric mode, respectively. This method not only provides high sensitivity, but also can correct itself to improve the accuracy of analysis due to the dual-response signals. Furthermore, it was employed for α-glucosidase determination in real samples and screening of α-glucosidase inhibitors.

A ratiometric fluorescence method based on nitrogen-doped carbon quantum dots for the determination of the activity of alkaline phosphatase.

Herein, a simple and sensitive ratiometric fluorescence sensing platform to detect alkaline phosphatase (ALP) activity is developed on the basis of yellow fluorescent nitrogen-doped carbon quantum dots (YNCDs). The hydrolysis of ascorbic acid 2-phosphate (AAP) into ascorbic acid (AA) is catalyzed by ALP. Then, AA will react with o-phenylenediamine (OPD) to form 3-(1,2-dihydroxyethyl)furo[3,4b]-quinoxaline (QXD) which is a blue fluorescent quinoxaline derivative with emission at 435 nm in the presence of Cu2+. YNCDs have yellow fluorescence emission at 555 nm, and can maintain stable in QXD reaction system. Therefore, by utilizing the fluorescence of YNCDs at 555 nm as reference signal and the fluorescence of QXD at 435 nm as report signal, we can detect the ALP activity by monitoring the fluorescence ratio (F435/F555). The linear range is 0.5-5 U/L, and the limit of detection is 0.14 U/L. An application of this method for the analysis of ALP in human serum has given satisfactory results. A ratiometric fluorescent nanoprobe for ascorbic acid and alkaline phosphatase detection with excellent biocompatible and high sensitivity was successfully constructed based on YNCDs and QXD.

Dual-mode colorimetric and fluorescence sensing system for the detection of captopril based on Fe/NC nanozymes and carbon dots.

Metal nitrogen-doped carbon (MNC) nanozymes have received increasing attention in bio-catalysis filed due to adequate catalytic activity, outstanding stability and reusability. Herein, the Fe/NC nanozymes (Fe/NC NZs) with peroxidase-like activity was successfully synthesized and a fluorescence turn on and colorimetric dual-mode sensing system was developed for quantification of captopril (CP) based on Fe/NC NZs and orange-emitting carbon dots (O-CDs). The Fe/NC NZs as an enzyme mimic can efficiently catalyze the 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic reaction, forming blue-colored oxidized TMB product (oxTMB) with the presence of H2O2, leading to the fluorescence quenching of O-CDs simultaneously via the inner filter effect (IFE). When CP was present, the blue oxTMB was reduced to colorless TMB, resulting in the inhibition of IFE and the recovery of fluorescence of O-CDs. The fluorescence increase of O-CDs and absorbance decrease of oxTMB depended on CP concentration. Good linear relationships of fluorescence and colorimetric sensing towards CP were obtained in the range from 1 to 50 µM, and the detection limits were 0.47 and 0.56 µM, respectively. Moreover, this as-constructed dual-mode sensor was used to detect CP in pharmaceutical products with satisfactory results.

Construction of a Sensing Platform Based on DNA-Encoded Magnetic Beads and Copper Nanoclusters for Viral Gene Analysis with Target Recycling Amplification.

The rapid and accurate monitoring of viral genes plays an important role in the area of disease diagnosis, biomedical research, and food safety. Herein, we successfully designed a sensing system that combined the technologies of target DNA recycling amplification, magnetic separation, and in situ formation of fluorescent copper nanoclusters (CuNCs) for viral DNA analysis. In the presence of target viral DNA (tDNA), a large quantity of output DNA (oDNA) was produced from hairpin DNA (hDNA) through an exonuclease III-assisted target recycling amplification strategy. Magnetic beads (MBs) labeled with capture DNA (cDNA) were hybridized with oDNA, and the partially complementary oDNA served as a bridge that could link AT-rich dsDNA on the surface of MBs, which led to a decrease of AT-rich dsDNA in solution after magnetic separation. On account of the lack of AT-rich dsDNA as a template in solution, in situ formation of fluorescent CuNCs was blocked, which resulted in a decrease in the fluorescence intensity at 590 nm. Therefore, taking advantage of one-step magnetic separation and in situ formation of CuNCs, the target viral DNA was sensitively and specifically detected in a linear range from 5 pM to 5 nM with a detection limit of 1 pM. The MB-based platform was not only reusable but also achieved magnetic separation, which could eliminate interferences in complex samples. The assay combining the MB-based probe with fluorescent CuNCs provided a universal, label-free, and reusable platform for viral DNA detection.