Mn Single-Atom Nanozymes with Superior Loading Capability and Superb Superoxide Dismutase-like Activity for Bioassay.

Single-atom nanozymes (SANs) with highly exposed active sites and remarkable catalytic activity have shown noteworthy practicability in heterogeneous catalysis-based bioassay. Nevertheless, most of them were reported with peroxidase-like activity and ordinary loading capability. It is still a challenge to prepare high-loading SANs with desirable superoxide dismutase (SOD)-like activity. In this work, Mn SAN was successfully confined in the frameworks of Prussian blue analogues formed on Ti3C2 MXene sheets with the assistance of massive surfactants, which show a superior loading efficiency of 13.5 wt % (typically <2.0 wt %). The prepared Mn SAN exhibits desirable superoxide radical anion elimination capability because of its SOD-like activity. Moreover, due to the wide-spectrum absorption behavior of the carriers, Mn SAN shows a synergistically quenching efficiency up to 98.89% on the emission of the reactive oxygen species-mediated chemiluminescent (CL) system. Inspired by these features, a CL quenching method was developed on a lateral flow test strip platform by utilizing Mn SAN as a signal quencher and acetamiprid as a model analyte. The method for detecting acetamiprid shows a detection range of 1.0-10,000 pg mL-1 and a limit of detection of 0.3 pg mL-1. Its accuracy has been validated by detecting acetamiprid in medicinal herbs with acceptable recoveries. This work opens an avenue for preparing SANs with a surfactant-assisted protocol and pioneers the study of SANs with SOD-like activity in bioassay.

Synergetic Dual-Site Atomic Catalysts for Sensitive Chemiluminescent Immunochromatographic Test Strips.

In view of the outstanding catalytic efficiency, single-atom catalysts (SACs) have shown great promise for the construction of sensitive chemiluminescent (CL) platforms. However, the low loading amount of active sites dramatically obstructs the improved catalytic activity of these metal SACs. Benefiting from the exceedingly unique catalytic properties of the metal-metal bonds, atomic clusters may give rise to enhancing the catalytic properties of SACs based on the synergistic effects of dual atomic-scale sites. Inspired by this, atomic Co3N clusters-assisted Co SACs (Co3N@Co SACs) were synthesized through a facile doping method. Through X-ray absorption spectroscopy, the active metal sites in the synergetic dual-site atomic catalysts of Co3N@Co SACs were confirmed to be Co-O4 and Co3-N moieties. Co3N@Co SACs served as a superior co-reactant to remarkably enhance the luminol CL signal by 2155.0 times, which was prominently superior to the boosting effect of the pure Co SACs (98.4 times). The synergetic dual-site atomic catalysts contributed to accelerating the decomposition of H2O2 into singlet oxygen as well as superoxide radical anions to display superb catalytic performances. For a concept employment, Co3N@Co SACs were attempted to utilize as CL probes for establishing a sensitive immunochromatographic assay to quantitate pesticide residues, in which imidacloprid was adopted as the model analyte. The quantitative range of imidacloprid was 0.05-10 ng mL-1 with a detection limit of 1.7 pg mL-1 (3σ). Furthermore, the satisfactory recovery values in mock herbal medicine samples demonstrated the effectiveness of the proposed Co3N@Co SAC-based CL platform. In the proof-of-concept work, synergetic dual-site atomic catalysts show great perspectives on trace analysis and luminescent biosensing.

Water dispersible cobalt single-atom catalysts with efficient Chemiluminescence enhancement for sensitive bioassay.

Single-atom catalysts (SACs) have been applied in various fields as they display extremely high utilization efficiency of catalytic sites. A majority of SACs prepared by high-temperature calcination suffer from poor water dispersion and lose of labelling groups. Herein cobalt SACs (CSACs) were synthesized with a solvothermal method by adopting hybridized MOFs Fe2O3/MIL-100(Fe) as the carriers to load cobalt atoms. Compared with original MOFs MIL-100(Fe), the carriers possess superior loading capacity, and the loading amount of cobalt element is up to 4.69 wt%. The implantation of cobalt atoms in hybridized MOFs Fe2O3/MIL-100(Fe) vastly improved the specific surface of the carriers for 68 times. CSACs at 1.0 µg mL-1 can catalyze H2O2 to generate numerous reactive oxygen species and enormously boost the chemiluminescent emission of luminol-H2O2 system up to 2297 times. The CSACs also exhibit satisfactory dispersion in aqueous medium. Benefiting from these attracting features, the CSACs were applied as sensitive signal probes for detecting carbendazim in Chinese medicinal herbs with a chemiluminescent immunoassay method. The dynamic range is 10 pg mL-1 - 50 ng mL-1 and the limit of detection is 1.8 pg mL-1. The proof-of-principle work paves a pathway to the exploitation of SACs as sensitive probes for tracing biological recognition events.

Antidepressant-like Effect of 3-n-Butylphthalide in Rats Exposed to Chronic Unpredictable Mild Stress: Modulation of Brain-Derived Neurotrophic Factor Level and mTOR Activation in Cortex.

3-n-Butylphthalide (NBP), an extract from seeds of Apium graveolens Linn. (Chinese celery), has been demonstrated to have antidepressant effects in suspension chronic-stressed rats by our group. The purpose of this study was to investigate the possible involvement of brain-derived neurotrophic factor (BDNF) and mammalian target of rapamycin (mTOR) in the antidepressant mechanism of NBP. Chronic unpredictable mild stress (CUMS) was applied for 6 weeks to induced a depressive-like behavior, characterized by decreased locomotor activity, sucrose preference and the NE, DA and 5-HT levels in cortex. Oral treatment with NBP (30 or 100 mg/kg, p.o.), similarly to fluoxetine (2 mg/kg, p.o.), can prevention of these alterations. The NBP (30 or 100 mg/kg, p.o.) reversed the decrease in the BDNF, p-ERK, mTOR and synapsin-1 protein levels in rat cortex caused by CUMS. And rapamycin, an mTOR inhibitor, completely inhibited the antidepressant-like activity of NBP in vivo. In conclusion, these findings indicate that NBP treatment attenuated the depression-like behaviors through the modulation of serotonergic system and BDNF-ERK-mTOR signaling in rat.

Pressure/colorimetric dual-readout immunochromatographic test strip for point-of-care testing of aflatoxin B1.

Immunochromatographic test strip (ITS) for point-of-care testing (POCT) has attracted prominent attention due to the advantages including rapid response, low cost and good portability. Here, we developed a sensitive ITS for detecting aflatoxin B1 (AFB1) by using dendritic platinum nanoparticles (DPNs) as novel pressure/colorimetric dual-readout probes. DPNs-labeled antibody of AFB1 were used as the signal tracer of the immunochromatographic process. After 10-min competitive immunoreaction, black color appeared on the test line of ITS due to the accumulation of DPNs, which was observed visually as a colorimetric readout for qualitation purpose. Furthermore, DPNs with peroxidase-like activity caused decomposition of hydrogen peroxide aqueous solution to produce pressure change signal in vials, which was detected by a hand-held pressure meter for quantitation purpose. With the pressure readout mode, the detection range was 0.05-10 ng mL-1, and the detection limit was 0.03 ng mL-1 (S/N = 3) for AFB1. The proposed ITS was successfully utilized for detecting AFB1 in herbal medicine samples, and the acceptable recoveries of 93.77-114.09% indicated the reliability for real sample detection. It provides a new avenue for POCT with great application potential in various area including drug and food quality control, pollutants monitoring as well as medical diagnosis.

Novel cobalt-based metal-organic frameworks with superior catalytic performance on N-(4-aminobutyl)-N-ethylisoluminol chemiluminescent reaction.

Novel cobalt-based metal-organic frameworks (Co MOFs) were synthesized by a facile "controlled synthesis" strategy. The MOFs displayed superior catalytic performance on the chemiluminescent (CL) reaction between N-(4-aminobutyl)-N-ethylisoluminol (ABEI) and H2O2. UV-vis absorption, CL spectrum, ESR, and radical scavenger experiments were conducted for clarifying the catalytic mechanism of Co MOFs. All results revealed that Co MOFs can accelerate decomposition of H2O2 and production of OH•, O2•-as well as 1O2 radicals. The rapid reaction between these reactive oxygen species and ABEI resulted in the generation of ABEI-ox∗. The excited-state oxidation product emitted a very intensive CL signal with a maximal emission wavelength of 430 nm as it returned to the ground state. To explore their application potential in CL assay, Co MOFs were used as powerful CL reaction catalyst for establishing a very sensitive method for immunoassay of aflatoxin B1. The detection range was 0.05-60 ng mL-1, and the limit of detection was 4.3 pg mL-1. The result for detecting herbal medicine samples demonstrates the acceptable reliability of the Co MOFs-based CL immunoassay. The proof-of-principle work verifies the application potential of Co MOFs on boosting intensive CL signal, and meets the demand for high sensitivity in various bioassay fields.

Novel chemiluminescent immunochromatographic assay using a dual-readout signal probe for multiplexed detection of pesticide residues.

A novel immunochromatographic assay (ICA) using a dual-readout signal probe was developed for multiplexed detection of pesticide residues by adopting methyl parathion and fenpropathrin as model analytes. Luminol-reduced Au nanoparticles (LRAuNPs) were synthesized and utilized in the proposed ICA platform as a colorimetric/chemiluminescent (CL) dual-readout probe. The methyl parathion antibody and fenpropathrin antibody were tagged with the prepared LRAuNPs to conduct spatially-resolved multiplexed detection. After the occurrence of two immunoreactions on the test strip, the probes were captured by the immobilized antigens on the two test zones. The red color resulting from the accumulation of captured LRAuNPs was adopted as the visual and semi-quantitative readout. For the sensitive quantitative detection of the analytes, the CL signals caused by the luminophore in the LRAuNPs were collected after triggering the luminol-H2O2 CL reaction. Under the optimal conditions, the detection limits for methyl parathion and fenpropathrin were 0.17 ng mL-1 and 0.10 ng mL-1 (S/N = 3), respectively. The whole procedure for ICA was completed within 15 min. The present ICA protocol was successfully applied for detection of pesticide residues in spiked traditional Chinese medicine samples. This dual-readout ICA platform showed merits such as low cost, time efficiency, easy operation and high sensitivity. Its application potential has been demonstrated in the rapid screening and field detection of multiple pesticide residues.

Dual-Readout Immunochromatographic Assay by Utilizing MnO2 Nanoflowers as the Unique Colorimetric/Chemiluminescent Probe.

Manganese dioxide nanoflowers (MnO2 NFs) were synthesized and used as a dual readout probe to develop a novel immunochromatographic test strip (ITS) for detecting pesticide residues using chlorpyrifos as the model analyte. MnO2 NFs-labeled antibody for chlorpyrifos was employed as the signal tracer for conducting the ITS. After 10 min competitive immunoreaction, the tracer antibody was captured by the immobilized immunogen in the test strip, resulting in the captured MnO2 NFs on test line. The captured MnO2 NFs led to the appearance of brown color on the test line, which could be easily observed by the naked eye as a qualitative readout. Due to the very slight colorimetric difference of chlorpyrifos at trace concentrations, the semiquantitative readout by naked eyes could not meet the demand of quantitative analysis. MnO2 NFs showed a significant effect on the luminol-H2O2 chemiluminescent (CL) system, and the CL signal driven by MnO2 NFs were used to detect the trace concentration of chlorpyrifos quantitatively. 1,3-Diphenylisobenzofuran quenching studies and TMB-H2O2 coloration assays were conducted for studying the enhancing mechanism of MnO2 NFs, which was based on the oxidant activity to decompose H2O2 for forming reactive oxygen species. Under optimal conditions, the linear range of chlorpyrifos was 0.1-50 ng/mL with a low detection limit of 0.033 ng/mL (S/N = 3). The reliability of the dual-readout ITS was successfully demonstrated by the application on traditional Chinese medicine and environmental water samples. Due to the simultaneous rapid-qualitative and sensitive-quantitative detection, the dual-readout protocol provides a promising strategy for rapid screening and field assay on various areas such as environmental monitoring and food safety.

Colorimetric and chemiluminescent dual-readout immunochromatographic assay for detection of pesticide residues utilizing g-C3N4/BiFeO3 nanocomposites.

Graphitic carbon nitride/bismuth ferrite nanocomposites (g-C3N4/BiFeO3 NCs) were synthesized by a facile one step sol-gel combustion method and employed as a peroxidase-like catalyst. Based on the catalytical activity on the luminol-H2O2 reaction, the nanocomposites were utilized as a colorimetric/chemiluminescent dual-readout immunochromatographic assay (ICA) for the multiplexed detection of pesticide residues by utilizing chlorpyrifos and carbaryl as the model analytes. In the proposed protocol, chlorpyrifos antibody and carbaryl antibody were tagged to g-C3N4/BiFeO3 NCs for developing the spatially-resolved multianalyte ICA. After two competitive immunoreactions completed on the ICA test strip, the tracer antibodies were captured by the immobilized antigens on two test lines. The accumulation of g-C3N4/BiFeO3 NCs led to the appearance of brown color, which were observed as a colorimetric and semi-quantitative signal. Furthermore, the g-C3N4/BiFeO3 NCs-driven generation of CL signal was collected as a sensitively quantitative signal after initiating the luminol-H2O2 reaction on the test lines. Under the optimal conditions, the limits of detection of chlorpyrifos and carbaryl were both 0.033ng/mL. The dual-readout ICA was successfully used to detect chlorpyrifos and carbaryl spiked in environmental water and traditional Chinese medicine samples with acceptable recovery values of 80-119% and 90-118%. Due to many advantages including low cost, time efficiency, high sensitivity and good portability, the novel ICA showed great potential in many areas such as drug safety, environmental monitoring and clinical diagnosis.

Multiplexed immunochromatographic test strip for time-resolved chemiluminescent detection of pesticide residues using a bifunctional antibody.

A novel bifunctional antibody (BfAb) that could recognize methyl parathion and imidacloprid simultaneously was prepared by a hybrid hybridomas technique. Using the BfAb as the sole recognition reagent, a multiplexed immunochromatographic test strip based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of pesticide residues. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were used as the CL probes to label the haptens of methyl parathion and imidacloprid, respectively. After the labeled haptens competed with methyl parathion and imidacloprid to bind with the BfAb immobilized on the test strip, the two CL reactions catalyzed by the enzymes were triggered simultaneously by coreactants injection. Due to the distinct CL kinetics characteristics of HRP and ALP, the signals for methyl parathion and imidacloprid detections were collected at 2.5s and 300s, respectively. The linear ranges for methyl parathion and imidacloprid were both 0.1-250ngmL-1, with detection limits of 0.058ngmL-1 (S/N=3). The whole assay process could be accomplished within 22min. The detection results for spiked traditional Chinese medicine samples demonstrated its application potential. The proposed method provided a low-cost, facile and rapid tool for multiplexed screening of pesticide residues using single antibody.

An ultra-facile and label-free immunoassay strategy for detection of copper (II) utilizing chemiluminescence self-enhancement of Cu (II)-ethylenediaminetetraacetate chelate.

The establishment of facile, rapid, sensitive and cost-effective protocols for the detection of heavy metals is of great significance for human health and environmental monitoring. Hereby, an ultra-facile and label-free immunoassay strategy was designed for detecting heavy metal ion by using Cu (II) as the model analyte. Cu (II) reacted previously with ethylenediaminetetraacetate (EDTA) was captured by immobilized monoclonal antibody for Cu (II)-EDTA chelate. Then Cu (II) was detected based on the self-enhancing effect of Cu (II)-EDTA chelate to luminol-H2O2 chemiluminescence reaction. The CL intensity is linear relative with Cu (II) concentration in a very wide range of 1.0-1000ng/mL, with a detection limit of 0.33ng/mL (S/N=3). Since the specificity of this proposed strategy relied on both the specificity of monoclonal antibody and the specificity of luminol-H2O2 system, it could avoid interference from most common ions. The proposed method was used successfully to detect Cu (II) in traditional Chinese medicine and environmental water samples with acceptable recovery values of 82-113%. This proof-of-principle work demonstrated the feasibility of the label-free immunoassay for heavy metal ions, and opened a new avenue for rapid screening and field assay for drug safety, environmental monitoring and clinical diagnosis.

Metabolic profiling of antioxidants constituents in Artemisia selengensis leaves.

This study aimed to evaluate the antioxidant potential of Artemisia selengensis Turcz (AST) leaves, a byproduct when processing AST stalk, and identify the antioxidant constituents by using HPLC-QTOF-MS(2). The total phenolics content (TPC), total flavonoids content (TFC) and antioxidant abilities of fractions resulted from the successively partition of chloroform, ethyl acetate and n-butanol were compared. Ethyl acetate fraction (EAF) exhibited the highest TFC (65.44 mg QuE/g fraction), n-butanol fraction (nBuF) showed the highest TPC (384.78 mg GAE/g fraction) and the best DPPH scavenging ability, ABTS(+) scavenging ability and reducing power. Totally, 57 compounds were identified or tentatively identified in nBuF and EAF, 40 of them were reported in AST for the first time. The major constituents in EAF were flavonoids, and the major constituents in nBuF were phenolic acids and organic acids. Thus, AST leaves might be a potential low-cost resource of natural antioxidants.

Chemiluminescence reaction kinetics-resolved multianalyte immunoassay strategy using a bispecific monoclonal antibody as the unique recognition reagent.

The multianalyte immunoassay (MIA) has attracted increasing attention due to its high sample throughput, short assay time, low sample consumption, and reduced overall cost. However, up to now, the reported MIA methods commonly require multiple antibodies since each antibody can recognize only one antigen. Herein, a novel bispecific monoclonal antibody (BsMcAb) that could bind methyl parathion and imidacloprid simultaneously was produced by a hybrid hybridomas strategy. A chemiluminescence (CL) reaction kinetics-resolved strategy was designed for MIA of methyl parathion and imidacloprid using the BsMcAb as the unique recognition reagent. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were adopted as the signal probes to tag the haptens of the two pesticides due to their very different CL kinetic characteristics. After competitive immunoreactions, the HRP-tagged methyl parathion hapten and the ALP-tagged imidacloprid hapten were simultaneously bound to the BsMcAb since there were two different antigen-binding sites in it. Then, two CL reactions were simultaneously triggered by adding the CL coreactants, and the signals for methyl parathion and imidacloprid detections were collected at 0.6 and 1000 s, respectively. The linear ranges for methyl parathion and imidacloprid were both 1.0-500 ng/mL, with detection limits of 0.33 ng/mL (S/N = 3). The proposed method was successfully used to detect pesticides spiked in ginseng and American ginseng with acceptable recoveries of 80-118%. This proof-of-principle work demonstrated the feasibility of MIA using only one antibody.

Response surface optimization and physicochemical properties of polysaccharides from Nelumbo nucifera leaves.

Dynamic high pressure microfluidization (DHPM)-assisted extraction (DHPMAE) of lotus (Nelumbo nucifera) leaves polysaccharides (LLPs) was optimized by response surface methodology. The optimal extraction conditions were: liquid/solid ratio of 35:1 (v/m, mL/g), processing pressure of 180 MPa, processed two times, extraction temperature of 76°C, extraction time of 50 min. Under the optimal extraction conditions, DHPMAE produced a higher polysaccharides yield (6.31%) than leaching (2.95%). Scanning electron microscope (SEM) analysis revealed that DHPM could reduce the particles size and make the surface more unconsolidated. The LLPs prepared by both methods showed similar FT-IR spectrum, and were consisted of the same monosaccharides, including rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose. The content of each monosaccharide in extracts, however, was quite different. The average molecular weight of LLPs prepared by DHPMAE is 550 kDa, smaller than 578 kDa obtained by leaching. The LLPs prepared by DHPMAE exhibited stronger DPPH scavenging ability (IC50 value of 0.38 mg/mL), HO scavenging ability (IC50 value of 0.61 mg/mL) and reducing power. Therefore, DHPMAE can be a promising alternative to traditional extraction techniques for polysaccharides from plants, and lotus leaves might be a potential resource of natural antioxidants.