Substrate Ascorbic Acid 2-phosphate Research Articles

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

BackgroundAlkaline 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. ResultsHerein, 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 525nm due to fluorescence resonance energy transfer (FRET) and absorption competition quenching (ACQ) effects. In this process, the PDA fluorescence intensity at 325nm 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. SignificanceThis 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.

Inter filter effect between fluorescent copper nanoclusters and Cr(VI) and its application for probing the activity of alkaline phosphatase

Herein, a novel fluorescent sensing strategy for the detection of alkaline phosphatase activity (ALP) was successfully established using highly luminescent glutathione-stabilized copper nanoclusters (GSH-CuNCs) as fluorescent probe. The GSH-CuNCs were synthesized with GSH as stabilizer and ascorbic acid (AA) as reductant. The optical properties of GSH-CuNCs were further investigated using fluorescence and UV–vis spectrophotometry. And its sensing ability for AA and ALP were explored. The fluorescence intensity of GSH-CuNCs was strongly quenched by Cr(VI) due to the inner filter effect (IFE). However, in the presence of AA, the fluorescence of GSH-CuNCs/Cr(VI) probes was restored due to the weakening of the IFE. It also exhibited a good linear range to the concentration of AA in the range of 0.1–500 μM, and the detection limit was 20 nM. In addition, it has been found that the substrate ascorbic acid 2-phosphate (AA2P) had no effect on the fluorescence of the (GSH-CuNCs)-Cr(VI) probe. Since ALP could specifically hydrolyze AA2P into AA, the emission intensity of the probes was restored by an ALP-triggered reaction. As a result, ALP was sensitively and selectively quantified with a wide linear range of 0.1 mU•mL−1 to 200 mU•mL−1 and a low detection limit of 0.02 mU•mL−1. In order to evaluate the practicality of the detection strategy, ALP in real sample analysis was also determined. The results showed that the recoveries and relative standard deviations were acceptable towards the probes. The proposed biosensor could be applied in fields of environmental and biological analysis.

Preparation of fluorescent polydopamine nanoparticles regulated by CoOOH nanosheets for detection of alkaline phosphatase activity

Alkaline phosphatase (ALP) is an important enzyme which is widely distributed in organisms and participate in the vital process of dephosphorylation, often serve as a common biomarker for clinical diagnosis and treatment of human diseases. Herein, we reported a novel and simple strategy for the preparation of polydopamine nanoparticles (PDANPs) by employing CoOOH nanosheets as an oxidant. In the presence of CoOOH nanosheets, dopamine is easy to be oxidized and self-polymerize into fluorescent PDANPs. By employing the synthesized PDANPs as an effective fluorescence indicator, a fluorescence biosensor was established for label-free and sensitive determination of ALP activity based on the redox reaction between CoOOH nanosheets and the ALP hydrolysis product ascorbic acid (AA), and the dependence of CoOOH nanosheets concentration for formation of PDANPs. In the presence of ALP, the substrate ascorbic acid 2-phosphate (Asc-2P) was catalytically hydrolyzed and released free ascorbic acid, which could in situ reduction of Co(III) of CoOOH to Co(II) ions, resulting in the suppression of oxidation ability of CoOOH nanosheets, thus preventing the synthesis of fluorescent PDA. Therefore, the activity of ALP can be feasibly detected according to the changes of fluorescence peak at 490 nm of PDANPs. The sensor displays a linear range from 0.5 U/L to 300 U/L with a detection limit of 0.1 U/L. Moreover, the developed strategy exhibits good selectivity over other potential interference existing in human serum sample. The synthesis process of fluorescence PDANPs was facile and cost-effective, the proposed convenient and label-free fluorescence PDANPs detection platform shows potential applications in clinical diagnosis and provides a new paradigm for biological analysis.

Colorimetric aptasensor for ochratoxin A detection based on enzyme-induced gold nanoparticle aggregation

An innovative colorimetric method based on enzyme-induced gold nanoparticle aggregation was developed to detect the activity of alkaline phosphatase (ALP), and it was further applied to construct an aptasensor to monitor ochratoxin A (OTA) concentrations. In the presence of ALP, the substrate ascorbic acid 2-phosphate was hydrolyzed to generate ascorbic acid (AA). Subsequently, reduction of MnO2 nanosheets by AA produced manganese ions, which mediated gold nanoparticle aggregation. The color of the detection solution changed from brown–red to purple to blue as the ALP concentration increased, and a detection limit of 0.05 U·L−1 was achieved. Furthermore, this strategy was successfully utilized to devise a target-responsive aptasensor for colorimetric detection of an important mycotoxin, OTA, which causes food poisoning and has various toxic effects on humans. The proposed method offers high sensitivity with a detection limit as low as 5.0 nM together with high specificity. When applied to analyze red wine and grape juice samples, no complex sample pretreatment or bulky instruments were required. Overall, a colorimetric platform based on enzyme-induced gold nanoparticle aggregation was successfully established to improve the simplicity and sensitivity of ALP and OTA detection. This platform appears highly promising for mycotoxin-related food safety monitoring.

Fluorometric turn-on determination of the activity of alkaline phosphatase by using WS2 quantum dots and enzymatic cleavage of ascorbic acid 2-phosphate.

A method is described for the determination of the activity of alkaline phosphatase (ALP). It is based on the reversible modulation of the fluorescence of WS2 quantum dots (QDs). The fluorescence of the QDs is quenched by Cr(VI) but restored by free ascorbic acid (AA). The detection scheme relies on the fact that ALP hydrolyzes the substrate ascorbic acid 2-phosphate to produce AA, and that enzymatically generated AA can restore the fluorescence of the QDs. The signal (best measured at excitation/emission peak wavelengths of 365/440 nm) increases linearly in the 0.5 to 10U·L-1 ALP activity range, with a detection limit of 0.2U·L-1. The method was applied to the determination of ALP activity in human serum samples and demonstrated satisfactory results. Graphical abstract The fluorescence of chromate-loaded tungsten disulfide quantum dots (QDs) is quenched but restored after reaction with ascorbic acid that is formed by the catalytic action of alkaline phosphatase (ALP) on ascorbic acid 2-phosphate (AAP). The increase in fluorescence can be related to the activity of ALP.

Metal-to-Ligand Charge-Transfer-based Visual Detection of Alkaline Phosphatase Activity

The ability to directly detect alkaline phosphatase (ALP) activity in undiluted serum samples is of great importance for clinical diagnosis. In this work, we report the use of the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of the Cu(BCA)2+ (BCA = bicinchoninic acid) reporter for the visual detection of ALP activity. In the presence of ALP, the substrate ascorbic acid 2-phosphate (AAP) can be enzymatically hydrolyzed to release ascorbic acid (AA), which in turn reduces Cu2+ to Cu+. Subsequently, the complexation of Cu+ with the BCA ligand generates the chromogenic Cu(BCA)2+ reporter, accompanied by a color change of colorless-to-purple of the solution with a sharp absorption band at 562 nm. The underlying MLCT-based mechanism has been demonstrated on the basis of density functional theory (DFT) calculations. Needless of any sequential multistep operations and elaborately designed colorimetric probe, the proposed MLCT-based method allows for a fast and sensitive visual detection of ALP activity within a broad linear range of 20 - 200 mU mL-1 (R2 = 0.999), with a detection limit of 1.25 mU mL-1. The results also indicate that it is highly selective and has great potential for the screening of ALP inhibitors in drug discovery. More importantly, it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum samples. Owing to the prominent simplicity and practicability, it is reasonable to conclude that the proposed MLCT-based method has a high application prospect in clinical diagnosis.

FRET Effect between Fluorescent Polydopamine Nanoparticles and MnO2 Nanosheets and Its Application for Sensitive Sensing of Alkaline Phosphatase.

As an essential and universal hydrolase, alkaline phosphatase (ALP) has been identified as a crucial indicator of various diseases. Herein, we, for the first time, expanded the application of fluorescent polydopamine (F-PDA) nanoparticles to nanoquencher-based biosensing system, as well as discovered the reversible quenching effect of manganese dioxide (MnO2) nanosheets on the fluorescence of F-PDA nanoparticles and intensively confirmed the quenching mechanism of Förster resonance energy transfer by using transmission electron microscopy, UV-vis, Fourier transform infrared spectroscopy, and fluorescence lifetime experiments. By means of the ALP-triggered generation of ascorbic acid (AA) from the substrate ascorbic acid 2-phosphate, the AA-triggered reduction of MnO2 nanosheets to Mn2+, as well as the clear quenching mechanism of F-PDA nanoparticles by MnO2 nanosheets, we have developed a label-free, low-cost, visual, and facile synthetic fluorescent biosensor for convenient assay of ALP activity. The fluorescent bioassay shows a good linear relationship from 1 to 80 mU/mL (R2 = 0.999), with a low detection limit of 0.34 mU/mL, and the excellent applicability in human serum samples demonstrates potential applications in clinical diagnosis and biomedical research.

Determination of the activity of alkaline phosphatase by using nanoclusters composed of flower-like cobalt oxyhydroxide and copper nanoclusters as fluorescent probes.

The authors describe a sensitive fluorometric method for the determination of the activity of alkaline phosphatase (ALP). It is based on the use of a composite prepared consisting of flower-like cobalt oxyhydroxide (CoOOH) and copper nanoclusters (CuNCs). On formation of the CuNC-CoOOH aggregates, the fluorescence of the CuNCs is quenched by the CoOOH sheets. If, however, the CoOOH sheets are reduced to Co(II) ions in the presence of ascorbic acid (AA), fluorescence recovers. AA is formed in-situ by hydrolysis of the substrate ascorbic acid 2-phosphate (AA2P) as catalyzed by ALP. Thus, the ALP activity can be detected indirectly by kinetic monitoring of the increase in fluorescence, best at excitation/emission wavelengths of 335/410nm. The assay allows ALP to be determined in 0.5 to 150mU·mL-1 activity range and with a 0.1mU·mL-1 detection limit. The method was successfully applied to the determination of ALP activity in (spiked) human serum samples. The assay has attractive features in being of the off-on type and immune against false positive results. Graphical Abstract A fluorescent bioassay is reported for the determination of the activity of alkaline phosphatase (ALP). It is exploiting the ascorbic acid (AA)-induced decomposition of nanoclusters composed of flower-like cobalt oxyhydroxide and copper nanoclusters. ALP catalyzes hydrolysis of ascorbic acid 2-phosphate (AA2P) and dephosphorylation to form AA.

Fluorescence quenching based alkaline phosphatase activity detection

Simple and fast detection of alkaline phosphatase (ALP) activity is of great importance for diagnostic and analytical applications. In this work, we report a turn-off approach for the real-time detection of ALP activity on the basis of the charge transfer induced fluorescence quenching of the Cu(BCDS)22- (BCDS = bathocuproine disulfonate) probe. Initially, ALP can enzymatically hydrolyze the substrate ascorbic acid 2-phosphate to release ascorbic acid (AA). Subsequently, the AA-mediated reduction of the Cu(BCDS)22- probe, which displays an intense photoluminescence band at the wavelength of 402nm, leads to the static quenching of fluorescence of the probe as a result of charge transfer. The underlying mechanism of the fluorescence quenching was demonstrated by quantum mechanical calculations. The Cu(BCDS)22- probe features a large Stokes shift (86nm) and is highly immune to photo bleaching. In addition, this approach is free of elaborately designed fluorescent probes and allows the detection of ALP activity in a real-time manner. Under optimal conditions, it provides a fast and sensitive detection of ALP activity within the dynamic range of 0–220mUmL−1, with a detection limit down to 0.27mUmL−1. Results demonstrate that it is highly selective, and applicable to the screening of ALP inhibitors in drug discovery. More importantly, it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum and even whole blood samples. Therefore, the proposed charge transfer based approach has great potential in diagnostic and analytical applications.

Facile colorimetric assay of alkaline phosphatase activity using Fe(II)-phenanthroline reporter

We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe3+ to Fe2+. The complexation of Fe2+ with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS)34− reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0–220 mU mL−1 with a detection limit of 0.94 mU mL−1. Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL−1 being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing.

Sensitive and selective colorimetric assay of alkaline phosphatase activity with Cu(II)-phenanthroline complex

Alkaline phosphatase (ALP) plays a vital role in dephosphorylation- and phosphorylation-related cellular regulation and signaling processes. Accordingly, the development of efficient methods for ALP activity assay is of significant importance in clinical diagnosis. In this work, a simple and practical method is reported for the first time for the sensitive and selective colorimetric assay of ALP activity by exploiting a water-soluble Cu(II)-phenanthroline complex as the probe, on the basis of the distinctive metal-to-ligand charge-transfer (MLCT) properties. This method is simply built on a two-step chromogenic reaction: the enzymatic hydrolysis of the substrate ascorbic acid 2-phosphate to ascorbic acid (AA), followed by the reduction of the colorimetric probe Cu(BPDS)22− (BPDS=bathophenanthroline disulfonate) by AA to its cuprous form. The latter process triggers a turn-on spectral absorption at 424nm and a striking color change of the solution from colorless to blackish-green. Needless of complicated protocols and instrumentation, this method allows a sensitive readout of ALP activity within a wide linear range of 0–200mUmL−1, with a detection limit down to 1.25mUmL−1. Results also reveal that it is highly selective and holds great potential in ALP inhibitor efficiency evaluation. In addition, quantitative analysis of ALP activity in spiked serum samples has been realized successfully in the linear range of 0–200mUmL−1, with a detection limit of 1.75mUmL−1. Advantages of simplicity, wide linear range, high sensitivity and selectivity, low cost, and little background interference render this method great potential in practical applications.

Turn‐On Colorimetric Platform for Dual Activity Detection of Acid and Alkaline Phosphatase in Human Whole Blood

The activity detection of acid phosphatase (ACP) and alkaline phosphatase (ALP) is of great importance to the diagnosis and prognosis of related diseases. In this work, we report for the first time a turn-on colorimetric platform for the activity detection of ACP and ALP, by exploiting Cu(BCDS)22- (BCDS=bathocuproinedisulfonate) as the probe. The presence of ACP or ALP dephosphorylates the substrate ascorbic acid 2-phosphate to produce ascorbic acid, which then reduces Cu(BCDS)22- into Cu(BCDS)23- , leading to a turn-on spectral absorption at 484 nm and a dramatic color change of the solution from colorless to orange-red. The underlying metal-to-ligand charge-transfer mechanism has been demonstrated by quantum mechanical computations. This platform allows a rapid, sensitive readout of ACP and ALP activities within the dynamic range from 0 to 220 mU ml-1 . In addition, it is highly immune to false-positive results and also highly selective. More importantly, it is applicable in the presence of human serum and even whole blood samples. These results demonstrate that our platform holds great potential in clinical practices and in the point-of-care analysis.

Fluorescence Immunoassay System via Enzyme-Enabled in Situ Synthesis of Fluorescent Silicon Nanoparticles.

The emergence of fluorescent nanomaterials with excellent performances has triggered the development of fluorescence analysis technique, which possesses several advantages in the research and clinical applications. However, current strategies for fluorescence immunoassay usually involve the routine fluorophore-labeled antibody and/or awkward signal generation procedure that may not be available in conventional enzyme-linked immunosorbent assay (ELISA) systems. Herein, we circumvent this problem by imparting an exquisite signal generation mechanism to commercially available alkaline phosphatase (ALP)-based ELISA platform and putting forward a conceptual fluorescent ELISA system based on an original ALP-enabled in situ synthesis of fluorescent nanomaterials. After adding target antigen, the presence of ALP labeled on antibody catalyzes the transformation of the substrate ascorbic acid 2-phosphate into ascorbic acid. Then the resultant ascorbic acid (i.e., ascorbate) interacts with amine-containing silane molecules (no fluorescence) to produce intense cyan fluorescent silicon nanoparticles. For the proof-of-concept, alpha-fetoprotein and human immunoglobulin G are chosen as the model antigen targets, and our proposed immunoassay (designated as the nanoparticles generation-based fluorescent ELISA) enables the detection with either fluorescence spectroscopy or naked-eye readout under the ultraviolet lamp. The convincing recognition mechanism and assay performance ensure fluorescent ELISA to quantitatively evaluate the alpha-fetoprotein level in serologic test and potentially apply in the clinic diagnosis of hepatocellular carcinoma.

A fluorescent ELISA based on the enzyme-triggered synthesis of poly(thymine)-templated copper nanoparticles.

Inspired by the ALP-triggered hydrolysis of the substrate ascorbic acid 2-phosphate as well as the product ascorbate-controlled generation of polyT-CuNPs with bright red fluorescence, we have developed a novel, selective and sensitive fluorescence turn-on assay for ALP activity sensing. Based on the conventional ELISA platform and commercially available antibody-ALP conjugates, an unconventional fluorescent ELISA system has been rationally developed and successfully applied in the quantitative measurement of model antigen proteins using fluorescence spectroscopy and a naked-eye readout under ultraviolet light.

Use of Fe₃O₄ Nanoparticles for Enhancement of Biosensor Response to the Herbicide 2,4-Dichlorophenoxyacetic Acid.

Magnetic nanoparticles of Fe3O4 were synthesized and characterized using transmission electron microscopy and X-ray diffraction. The Fe3O4 nanoparticles were found to have an average diameter of 5.48 ±1.37 nm. An electrochemical biosensor based on immobilized alkaline phosphatase (ALP) and Fe3O4 nanoparticles was studied. The amperometric biosensor was based on the reaction of ALP with the substrate ascorbic acid 2-phosphate (AA2P). The incorporation of the Fe3O4 nanoparticles together with ALP into a sol gel/chitosan biosensor membrane has led to the enhancement of the biosensor response, with an improved linear response range to the substrate AA2P (5-120 μM) and increased sensitivity. Using the inhibition property of the ALP, the biosensor was applied to the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The use of Fe3O4 nanoparticles gives a two-fold improvement in the sensitivity towards 2,4-D, with a linear response range of 0.5-30 μgL-1. Exposure of the biosensor to other toxicants such as heavy metals demonstrated only slight interference from metals such as Hg2+, Cu2+, Ag2+ and Pb2+. The biosensor was shown to be useful for the determination of the herbicide 2, 4-D because good recovery of 95-100 percent was obtained, even though the analysis was performed in water samples with a complex matrix. Furthermore, the results from the analysis of 2,4-D in water samples using the biosensor correlated well with a HPLC method.