A simple fluorescence detection of acetylcholinesterase with peroxidase-like catalysis from iodide.

Acetylcholinesterase (AChE) is an important enzyme in the central and peripheral nervous system that regulates the balance of the neurotransmitter acetylcholine. In this work, a simple, selective and sensitive fluorescence assay was developed toward AChE activity. A conventional AChE substrate acetylthiocholine iodide (ATCI) was applied. Instead directly rendering a signaling, it was found that free iodide ions was released during the enzymatic hydrolysis of ATCI. These ions further catalyzed the oxidation of non-emissive o-phenylenediamine (OPD) into a fluorescent product. This gave a response differed from frequently-adopted sulfhydryl- -based signals and thus minimized related interferences. All materials included in this process were directly available and no additional syntheses were required. Due to the extra iodide-based catalysis included, this scheme was capable of providing a sensitive response toward AChE in the range of 0.01-8 U/L, with a limit of detection at 0.006 U/L. This method was further extended onto chlorpyrifos as an exemplary AChE inhibitor, with a detection down to 3 pM.

Simple and sensitive fluorescence detection of trypsin with Cu2+-Bovine serum albumin complex as a peroxidase mimic.

Trypsin is a serine protease playing a key role in regulating pancreatic exocrine function and can be applied as a marker for the diagnosis of pancreatitis. In this work, a convenient and sensitive fluorescent assay was developed toward trypsin. Hydrogen peroxide slowly oxidized a non-fluorescent o-phenylenediamine (OPD) into a fluorescent product 2,3-diaminophenothiazine (DAP) under the catalytic from copper ions. After the introduction of bovine serum albumin (BSA), the combination of BSA with copper ions formed a peroxidase mimic and significantly accelerated the reaction rate. As an efficient protease, trypsin cleaved the lysine and arginine residues in BSA. This destroyed the binding between Cu2+ and BSA, and brought in a reduction of the catalytic effect. The accompanying decrease in fluorescence provided a response to trypsin in the range of 0.01-600 ng/mL, with a detection limit of 0.007 ng/mL. The scheme had a good selectivity and was successfully applied to the detection of real samples.

Novel Electrochemiluminescent Immunosensor Using Dual Amplified Signals from a CoFe Prussian Blue Analogue and Au Nanoparticle for the Detection of Lp-PLA2.

Coronary heart disease (CHD) poses an important threat to human health, and its pathogenesis is the formation of atheromatous plaques in coronary ventricles. Compared to other biomarkers, lipoprotein-associated phospholipase A2 (Lp-PLA2), which is involved in multiple processes of atherosclerosis, is a noticeable inflammatory biomarker related to CHD. Herein, using a multifunctional nanocomposite containing a CoFe Prussian blue analogue (PBA) and Au nanoparticles (AuNPs) (AuNPs@CoFe PBA) as a sensing substrate, an electrochemiluminescent (ECL) immunosensor was developed for the highly sensitive detection of Lp-PLA2. Benefiting from the synergistic effect of the PBA and AuNPs, the nanocomposite exhibits excellent peroxidase-like activity and can catalyze the luminol-ECL reaction, amplifying the ECL signal by ∼29-fold. Meanwhile, the enlarged specific surface area of the nanocomposite and the presence of abundant AuNPs allow the immobilization of more antibody proteins, thereby improving the sensing response of the immunosensor. When the target Lp-PLA2 is captured by the antibody on the sensor surface, the sensor emits a reduced ECL signal because of the increased mass and electron transfer resistance due to the formation of the immune complex. Under optimized conditions, the constructed ECL immunosensor exhibits a broad linear range from 1 to 2200 ng/mL and a low detection limit of 0.21 ng/mL. Additionally, the ECL immunosensor exhibits high specificity, stability, and reproducibility. This work provides a new approach to diagnose CHD and broadened the application of the PBA in the field of ECL sensors.

Colorimetric detections of iodide and mercuric ions based on a regulation of an Enzyme-Like activity from gold nanoclusters.

A simple colorimetric method was developed for sensitive and selective detections of I- and Hg2+. Histidine stabilized gold nanoclusters (His-AuNCs) were synthesized and catalyzed the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to a blue product. As a strong ligand toward gold, iodide (I-) attached to the surface of the His-AuNCs and significantly enhanced the oxidase-like activity of the His-AuNCs. Based on this enhancement, a sensitive colorimetric response toward I- was obtained. Furthermore, the strong interaction between Hg2+ and I- was adopted for an indirect Hg2+ detection. Under the optimal conditions, the platform presented high selectivity for the determinations of I- and Hg2+ in the ranges 0.02-1 µM and 0.05-0.8 µM, with detection limits as 3.3 nM and 8 nM respectively. This colorimetric assay was successfully applied for analysis of real samples.

Ready-to-Use Colorimetric Platform for Versatile Enzyme Assays through Copper Ion-Mediated Catalysis.

A low cost and versatile colorimetric platform is developed for selective detections of various enzymes. Similar to peroxidases, free copper ion catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H2O2 and turns TMB into a blue product. Bindings from ligands toward copper ions inhibit this catalysis. Enzymes catalyze the reactions of related substrates with generation or consumption the ligands for the binding and thus in turn alter the color changes as responses toward the enzymes. With suitable substrates, exemplary enzymes, including trypsin, acid phosphatase, and tyrosinase, can be sensitively measured, with limits of detection of 0.003 µg/mL, 0.004 U/L, and 0.02 U/mL, respectively. This platform is built with directly available reagents, and the signals can be obtained with inexpensive photometers or visual observations. The low cost and convenience make it suitable for cases where complicated instrumentations are not available, such as point-of-care testing.

Sensitive detection of glutathione through inhibiting quenching of copper nanoclusters fluorescence.

A method for a sensitive fluorescence detection of glutathione was established. Glutathione-stabilized copper nanoclusters (CuNCs) were synthesized via a facile process. These CuNCs showed blue fluorescence with a peak around 450 nm. In the presence of p-benzoquinone (PBQ), the electron transfer from the copper nanoclusters to PBQ quenched the fluorescence of the CuNCs. Glutathione (GSH), as a reducing agent, formed a complex with PBQ. This formation inhibited the quenching from PBQ, and a restored fluorescence was obtained. This interaction provided a fluorescence enhancement for the measurement of GSH. Under the optimal condition, linear responses were obtained toward GSH in the ranges of 0.06-6.0 µM, with a limit of detection at 20 nM. This developed assay was easy in operation with high sensitivity and selectivity. The applicability was approved with successful glutathione measurements in real samples.

Clinical effect of Chinese herbal medicine for removing blood stasis combined with acupuncture on sequelae of cerebral infarction.

OBJECTIVE: To assess the clinical efficacy of Chinese herbal medicine for removing blood stasis combined with acupuncture in the treatment of sequelae of cerebral infarction. METHODS: Ninety patients with cerebral infarction admitted to our hospital from April 2018 to April 2020 were enrolled and equally allocated to an experimental group and a control group. The control group was treated with aspirin, and the experimental group was treated with Chinese herbal medicine for removing blood stasis combined with acupuncture. The recovery of the ability of daily living (ADL), recovery of hemiplegic limb function, blood viscosity, total cholesterol (TC), triglyceride (TG), and quality of life were evaluated. RESULTS: After treatment, the ADL of patients in the two groups witnessed a remarkable recovery, with superior results in the experimental group than the control group (P < 0.05). The hemiplegic limb recovery of the experimental group was observed to be significantly improved when compared with the control group (P < 0.05). Remarkably lower blood viscosity-related indexes of TC and TG of the experimental group compared to the control group were identified (P < 0.05). As to the total remission rate (TRR), the experimental group demonstrated a higher level than the control group (P < 0.05). The scores of quality of life of patients in the experimental group after treatment were evidently higher than those of the control group (P < 0.05). CONCLUSION: Chinese herbal medicine for removing blood stasis combined with acupuncture treatment can better improve the hemiplegic limb function and the quality of life, and reduce blood viscosity of patients with sequelae of cerebral infarction.

Detection of silver through amplified quenching of fluorescence from polyvinyl pyrrolidone-stabilized copper nanoclusters.

Silver ion detection with ultra-high sensitivity was established. We synthesized copper nanoclusters (CuNCs) with blue fluorescence through a one-pot process. Instead of a direct quencher toward the CuNCs, silver ions activated the strong oxidation from persulfate and subsequently converted divalent manganese ion into manganese dioxide (MnO2). The surface charges of MnO2 and the CuNCs brought them together and quenched the fluorescence from the latter. Due to silver ions' role as the catalyst in the process, it cycled and even a small amount leads to a significant fluorescence change. This signaling provided the determination of  silver ions in the range 5 pM~1 nM, with a detection limit of  1.2 pM. The method is selective, and its applicability was validated through practical water sample analyses.

Turn-on fluorescence measurement of acid phosphatase activity through an aggregation-induced emission of thiolate-protected gold nanoclusters.

A fluorescence method was developed for a turn-on measurement of acid phosphatase (ACP) activity. It was found that cerous ion (Ce3+) could lead to an enhancement of glutathione protected gold nanocluster fluorescence through an aggregation-induced-emission (AIE) process, while its higher valent counterpart ceric ion (Ce4+) could not. In a weakly acidic environment, ACP catalyzed the dephosphorylation of a phosphate ester of ascorbic acid, with a generation of ascorbic acid (AA). AA reduced Ce4+ into Ce3+, which subsequently enhanced the nanocluster fluorescence. This kind of turn-on fluorescence linearly related to the ACP activity in the range of 0.005-2.4 U/L, with a limit of detection as 0.001 U/L. Human serum samples were measured after a trichloroacetic acid treatment and a simple dilution. The whole analyses were accomplished in 1.5 h with results in good accordance with a reference method.

Determination of dopamine based on its enhancement of gold-silver nanocluster fluorescence.

Dopamine (DA) is one of the most important neurotransmitters in human bodies and its sensitive detection remains a challenge. Herein, protein stabilized gold-silver nanoclusters (Au-AgNCs) were synthesized at first. It was found that the introduction of dopamine lead to a significant enhancement of the fluorescence from the nanoclusters, together with a red-shift of the peak. Through related spectroscopic and electrochemical studies, the fluorescence enhancement was attributed to the reduction of the nanoclusters by dopamine. This enhancement was then adopted for quantitative measurements, and linear responses toward dopamine in the ranges 0.01-1.7 µM and 1.7-10 µM were constructed. A limit of detection was obtained at 6.9 nM. The present study provided a facile and efficient method for the determination of dopamine, and the method was successfully applied for related measurements in serum samples.

Fluorescence detections of hydrogen peroxide and glucose with polyethyleneimine-capped silver nanoclusters.

Detection of hydrogen peroxide is of significant importance for biological assays, and fluorescence methods are intensively reported for this purpose. Due to the highly oxidative property of this species, usually fluorescence quenching is obtained during the interactions and decreased signals are rendered. In this report, this oxidative property was adopted for an increased fluorescence signaling. Photoluminescent silver nanoclusters (AgNCs) were synthesized with polyethyleneimine as the stabilizer. This fluorescence from these nanoclusters could be quenched by reduced glutathione (GSH) through an interaction from its thiol group. As an oxidant, hydrogen peroxide converted GSH into an oxidized form (GSSG) with an elimination of the free thiols, and inhibited the quenching. This interaction presented an increased response toward hydrogen peroxide in the range of 0.1-20 µM with a detection limit of 35 nM. The scheme was further coupled with glucose oxidase for a glucose analysis down to 0.11 µM. This method was selective and was successfully applied for glucose measurement in human serum samples.

Sensitive detection of molybdenum through its catalysis and quenching of gold nanocluster fluorescence.

Molybdenum (Mo) is an essential nutrient for the proper functioning of some enzymes in living organisms as human beings. Conventional methods for its detection require complicated instrumentations as atomic absorption or mass spectrometers. In this work, a sensitive kinetic fluorescence was developed as an alternative. Gold nanoclusters (AuNCs) with red fluorescence emission were synthesized, and this fluorescence was effectively quenched by iodine through an etching process. It was found that the presence of Mo significantly speeded up a reaction for the generation of iodine, and thus enhanced the quenching. This effect was then adopted for the development of a sensitive fluorescent measurement toward Mo. The method was capable of detecting Mo down to 0.2 nM and was successfully applied for the analyses of mung bean and tea leaf samples.

An experimental study on the treatment of diabetes-induced cognitive disorder mice model with exosomes deriving from mesenchymal stem cells (MSCs).

The occurrence rate of senile dementia among diabetes patients is high, but there is still no effective therapeutic method. This study aimed to explore curative effect of exosomes deriving from MSCs treating diabetes-induced cognition impairment. After BM-MSCs culture was purified, Western Blot and electron microscope were used to identify exosomes which were injected into diabetes mouse through intracranial injection. Purified exosomes in Western blot analysis and electron microscope observation were verified. After therapy with exosomes deriving BM-MSC, mouse cognition impairment and histologic abnormity were recovered. Exosomes deriving from BM-MSCs can be a new preparation treating cognition with application prospect.

Fluorometric determination of nitrite through its catalytic effect on the oxidation of iodide and subsequent etching of gold nanoclusters by free iodine.

A method for sensitive detection of nitrite is presented. It is found that the red fluorescence of gold nanoclusters (with excitation/emission maxima at 365/635 nm) is quenched by traces of iodine via etching. Free iodide is formed by oxidation of iodide by bromate anion under the catalytic effect of nitrite. This catalytic process provides a sensitive means for nitrite detection. Under the optimal conditions, fluorescence linearly dropos in the 10 nM to 0.8 µM nitrite concentration range. The limit of detection is 1.1 nM. This is a few orders of magnitude lower than the maximum concentration allowed by authorities. Graphical abstract Schematic representation of a method for detection of nitrite via a redox reaction. Iodine was produced in the reaction and subsequently quenched the fluorescence from gold nanoclusters by etching their metallic cores, and a sensitive assay for nitrite down to 1.1 nM was developed.

Determination of alkaline phosphatase activity based on enzyme-triggered generation of a thiol and the fluorescence quenching of silver nanoclusters.

A fluorimetric method is described for the determination of alkaline phosphatase (ALP) activity. It is based on the use of polyethyleneimine-coated silver nanoclusters (AgNCs), which display an intense blue fluorescence peaking at 450 nm (under 375 nm excitation). ALP catalyzes the dephosphorylation of the thiophosphate amifostine to generate a thiol that binds to the AgNCs and causes its fluorescence to be quenched. Under the optimal experimental conditions, fluorescence linearly drops in the 0.08-2.0 U L-1 ALP activity range, and the limit of detection is 0.02 U L-1. The method was successfully applied to the determination of ALP activity in spiked human serum samples. Graphical abstract Alkaline phosphatase (ALP) catalyzes the degradation of amifostine with a generation a thiol product. The thiol quenches the fluorescence of silver nanoclusters, and a method for the detection of ALP down to 0.02 U L-1 was developed.

Fluorescence red-shift of gold-silver nanoclusters upon interaction with cysteine and its application.

In this work, gold-silver alloy nanoclusters (AuAg NCs) were demonstrated as a novel probe for fluorescent detection of cysteine (Cys). The alloy nanoclusters were fabricated by bovine serum albumin as a template and NaBH4 as a reducer. They showed a red emission at 650 nm. The interaction between AuAg NCs and Cys was investigated. The thiol group in Cys molecules has strong affinity on the surface of metals, which results in variation of fluorescence peak wavelength. It was further demonstrated that this red-shift of fluorescence had a good linear relationship with the concentration of Cys in the range of 2-100 µM. The method was successfully applied for human plasma analysis with satisfactory results. This novel strategy was expected to provide a potential opportunity for extending the application of novel metal nanoclusters in fluorescence.

Copper ion detection with improved sensitivity through catalytic quenching of gold nanocluster fluorescence.

In this report, a sensitive fluorescence detection of copper (Ⅱ) ion was developed. Although itself only a weak quencher toward gold nanocluster fluorescence, this ion functioned as a catalyst that accelerated the oxidation of iodide into iodine by a strong oxidant. The so-produced iodine quenched the nanocluster fluorescence through an efficient etching reaction, which rendered a much improved sensitivity for copper detection. Under the optimal conditions, the extent of quenching was found linear to the amount of copper in the range of 0.8-80 nM, and this strategy was capable of detecting copper ion as low as 0.33 nM. The method was selective and was successfully applied for related measurement in practical samples.

A sensitive electrochemiluminescent biosensor based on AuNP-functionalized ITO for a label-free immunoassay of C-peptide.

The C-peptide is a co-product of pancreatic ß-cells during insulin secretion; its content in body fluid is closely related to diabetes. This paper reports an immune-sensing strategy for a simple and effective assay of C-peptide based on label-free electrochemiluminescent (ECL) signaling, with high sensitivity and specificity. The basal electrode was constructed of an indium tin oxide (ITO) glass as a conductive substrate, which was decorated by Au nanoparticles (AuNPs) with hydrolysed (3-aminopropyl)trimethoxysilane as the linker. The characteristics of the fabricated electrode were investigated by electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. After immobilizing the C-peptide antibody, which takes great advantage of AuNPs' binding capacity, this immunosensor can quantify C-peptide using luminol as the ECL probe. By measuring ECL inhibition, calibration can be established to report the C-peptide concentration between 0.05 ng mL-1 and 100 ng mL-1 with a detection limit of 0.0142 ng mL-1. As a proof of concept, the proposed strategy is a promising and versatile platform for the clinical diagnosis, classification, and research of diabetes.

A label-free electrochemiluminescent immunosensor for glutamate decarboxylase antibody detection on AuNPs supporting interface.

In this paper, we will describe a novel lable-free electrochemiluminescent (ECL) immunosensor for the detection of glutamate decarboxylase antibody (GADA) in which Au nanoparticles (AuNPs) had pre-functionalized the indium tin oxide (ITO) glass to support the sensing interface. The preparation of the basal electrode only need a simple two-step drop coating, a thin polymer of hydrolyzed 3-aminopropyl trimethoxysilane, and the AuNPs gel on the ITO substrate. The AuNPs not only enhanced the ECL signal of luminol, but also acted to immobilize the glutamate decarboxylase (GAD) to build the sensing host. This immunosensor exhibits excellent specificity, reproducibility and stability. On resultant sensor, after the direct immunoreaction, the decreased ECL intensity has a good linear regression toward the logarithm of GADA concentration in the range of 0.30 ng mL-1 to 50 ng mL-1 with a detection limit of 0.10 ng mL-1. The proposed senor has prospective capability for the clinical detection of GADA in human serums, which had important value for diagnosis and precaution of type-1 diabetes or latent autoimmune diabetes in adult (LADA).

Gold nanocluster-based ratiometric fluorescent probes for hydrogen peroxide and enzymatic sensing of uric acid.

A method is described for ratiometric fluorometric assays of H2O2 by using two probes that have distinct response profiles. Under the catalytic action of ferrous ion, the 615 nm emission of protein-stabilized gold nanoclusters (under 365 nm photoexcitation) is quenched by H2O2, while an increased signal is generated with a peak at 450 nm by oxidizing coumarin with the H2O2/Fe(II) system to form a blue emitting fluorophore. These decrease/increase responses give a ratiometric signal. The ratio of the fluorescences at the two peaks are linearly related to the concentration of H2O2 in the range from 0.05 to 10 µM, with a 7.7 nM limit of detection. The detection scheme was further coupled to the urate oxidase catalyzed oxidation of uric acid which proceeds under the formation of H2O2. This method provides an simple and effective means for the construction of ratiometric fluorometric (enzymatic) assays that involve the detection of H2O2. Graphical abstract Under catalysis by ferrous ion, hydrogen peroxide quenches the luminescence of gold nanoclusters (AuNCs) and oxidizes coumarin into a fluorescent derivative, which rendered fluorescence ON and OFF at two distinct wavelengths for ratiometric measurements.