Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

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.

Ti3C2@UiO-TCPP Schottky junction photoelectrochemical sensor for detecting alkaline phosphatase through the steric hindrance effect of phosphopeptide.

Alkaline phosphatase (ALP) is a major biomarker for clinical diagnosis, but detection methods of ALP are limited in sensitivity and selectivity. In this paper, a novel method for ALP determination is proposed. A photoelectrochemical (PEC) sensor was prepared by growing UiO-tetratopic tetrakis (4-carbox-yphenyl) porphyrin (TCPP) in situ between layered Ti3C2 through a one-pot hydrothermal method. The obtained Schottky heterojunction photoelectric material Ti3C2@UiO-TCPP not only has a large light absorption range but also greatly improves the efficiency of photogenerated electron hole separation and thereby enhances sensitivity for PEC detection. The phosphate group on the phosphorylated polypeptide was utilized to form a Zr-O-P bond with the zirconium ion on UiO-66, and then photocurrent decreases due to the steric hindrance effect of phosphorylated polypeptides, that is, the hindrance of electron transfer between the photoelectric material and a solution. The specific interaction between ALP and phosphorylated polypeptides shears the bond between phosphate and zirconium ion on UiO-66 in the peptides then weakens the hindrance effect and increases the photocurrent, thus realizing ALP detection. The linear range of ALP is 0.03-10,000 U·L-1, and the detection limit is 0.012 U·L-1. The method is highly sensitive and selective, and has been applied in detection of ALP in serum samples.

Ratiometric fluorometric assay triggered by alkaline phosphatase: Proof-of-concept toward a split-type biosensing strategy for DNA detection.

Herein, a sensitive ratiometric and split-type fluorescent sensing platform has been constructed for DNA detection based on one signal precursor and two fluorescent signal indicators. In this assay, o-phenylenediamine (OPD) was selected as the signal precursor. On one hand, Cu2+ can oxidize OPD to produce 2, 3-diaminophenazine (DAP), which with an emission peak at 555 nm. On the other hand, ascorbic acid (AA) could react with Cu2+ to generate dehydroascorbic acid (DHAA), which could further react with OPD to form 3-(1, 2-dihydroxy ethyl)furo[3, 4-b]quinoxalin-1 (3H)-on (DFQ) with a strong emission peak at 420 nm. As a result, the formation of DAP was inhibited, and leading to the decrease of fluorescence intensity at 555 nm. Alkaline phosphatase (ALP) could catalyze the substrate l-ascorbic acid-2-phosphate (AA2P) to produce AA in situ. Inspired by the successful use of ALP as a biocatalytic marker in bioassay, a split-type ratiometric fluorescent assay has been designed for DNA detection by using H1N1 DNA as the target model. It was realized for ratiometric fluorescent determination of H1N1 in a linear ranging from 50 pM to 1.5 nM with a limit of detection of 10 pM. The novel strategy could reduce the mutual interferences between the biomolecular recognition system and the fluorescence signal conversion system, which improving the accuracy of detection and effectively reducing the background signal. Furthermore, the strategy provided a promising platform for biomarkers detection in the fields of ratiometric fluorescent biosensors and bioanalysis.

The Global ALPL gene variant classification project: Dedicated to deciphering variants.

BACKGROUND: Hypophosphatasia (HPP) is an inherited multisystem disorder predominantly affecting the mineralization of bones and teeth. HPP is caused by pathogenic variants in ALPL, which encodes tissue non-specific alkaline phosphatase (TNSALP). Variants of uncertain significance (VUS) cause diagnostic delay and uncertainty amongst patients and health care providers. RESULTS: The ALPL gene variant database (https://alplmutationdatabase.jku.at/) is an open-access archive for interpretation of the clinical significance of variants reported in ALPL. The database contains coding and non-coding variants, including single nucleotide variants, insertions/deletions and structural variants affecting coding or non-coding sequences of ALPL. Each variant in the database is displayed with details explaining the corresponding pathogenicity, and all reported genotypes and phenotypes, including references. In 2021, the ALPL gene variant classification project was established to reclassify VUS and continuously assess and update genetic, phenotypic, and functional variant information in the database. For this purpose, the database provides a unique submission system for clinicians, geneticists, genetic counselors, and researchers to submit VUS within ALPL for classification. An international, multidisciplinary consortium of HPP experts has been established to reclassify the submitted VUS using a multi-step process adhering to the stringent ACMG/AMP variant classification guidelines. These steps include a clinical phenotype assessment, deep literature research including artificial intelligence technology, molecular genetic assessment, and in-vitro functional testing of variants in a co-transfection model to measure ALP residual activity. CONCLUSION: This classification project and the ALPL gene variant database will serve the global medical community, widen the genotypic and phenotypic HPP spectrum by reporting and characterizing new ALPL variants based on ACMG/AMP criteria and thus facilitate improved genetic counseling and medical decision-making for affected patients and families. The project may also serve as a gold standard framework for multidisciplinary collaboration for variant interpretation in other rare diseases.

SERS Immunoassay Based on an Enzyme-Catalyzed Cascade Reaction and Metal-Organic Framework/Alkaline Phosphatase for Ultrasensitive Detection of Adenosine Triphosphate.

Herein, an adenosine triphosphate (ATP)-induced enzyme-catalyzed cascade reaction system based on metal-organic framework/alkaline phosphatase (MOF/ALP) nanocomposites was designed to establish a surface-enhanced Raman spectroscopy (SERS) biosensor for use in rapid, sensitive ATP detection. Numerous ALP molecules were first encapsulated using ZIF-90 to temporarily deactivate the enzyme activity, similar to a lock. Au nanostars (AuNSs), as SERS-enhancing substrates, were combined with o-phenylenediamine (OPD) to form AuNSs@OPD, which could significantly improve the Raman signal of OPD. When the target ATP interacted with the MOF/ALP nanocomposites, ATP could act as a key to open the MOF structure, releasing ALP, which should further catalyze the conversion of OPD to oxOPD with the aid of ascorbic acid 2-phosphate. Therefore, with the increasing concentrations of ATP, more ALP was released to catalyze the conversion of OPD, resulting in the reduced intensity of the Raman peak at 1262 cm-1, corresponding to the level of OPD. Based on this principle, the ATP-induced enzyme-catalyzed cascade reaction SERS biosensor enabled the ultrasensitive detection of ATP, with a low detection limit of 0.075 pM. Consequently, this study provides a novel strategy for use in the ultrasensitive, rapid detection of ATP, which displays considerable potential for application in the fields of biomedicine and disease diagnosis.

Capsulation of EBTAC into ZIF-8 for the development of a signal-on fluorescent biosensor to detect alkaline phosphatase.

Diseases such as liver cancer, extrahepatic biliary obstruction and osteocarcinoma are closely associated with the abnormal level of alkaline phosphatase (ALP). Hence, it is essential to develop a convenient assay to detect ALP activity. Herein, a novel signal-on fluorescent biosensor on account of the fluorescence signal of the aggregation-induced emission (AIE) fluorochrome 2,2',2'',2'''-((ethene-1,1,2,2-tetrayltetrakis(benzene-4,1-diyl))tetrakis(oxy))tetraacetic acid (EBTAC) encapsulated zeolitic imidazolate framework-8 (ZIF-8@EBTAC) was designed to monitor ALP. Due to the aggregation-induced emission of EBTAC, the synthetic ZIF-8@EBTAC shows robust fluorescence. Once pyrophosphate (ppi) was added, its complexation with Zn2+ in ZIF-8 triggered the collapse of the ZIF-8 framework, releasing encapsulated EBTAC molecules and restoring to free state, leading to the dramatical decrease in fluorescence. ALP could catalyze the hydrolysis of ppi to phosphate (pi), which is difficult to bind to Zn2+ and has little effect on the fluorescence of ZIF-8@EBTAC. Therefore, with the assistance of the substrate ppi, the ultimate fluorescence of ZIF-8@EBTAC was positively related with ALP activity. The constructed biosensor was able to monitor the ALP activity well from 0.01 to 100 U L-1, and a detection limit of 0.01 U L-1 was achieved. Based on the ability of EBTAC serving as a fluorescent probe with aggregation-induced luminescence properties, this proposed design can be applied to diverse targets and provide new ideas for the establishment of fluorescent biosensors.

A promising tool for clinical diagnostics: Dual-emissive carbonized polymer dots based cross-linking enhanced emission for sensitive detection of alkaline phosphatase and butyrylcholinesterase.

Compared with single signal readout, dual-signal readout commendably corrects the impact of systematic or background error, achieving more accurate results for the diagnosis of many diseases. This work aimed to design and prepare dual-emissive fluorescent probes for the construction of ratiometric fluorescence biosensors to detect liver disease biomarkers. Sodium alginate (SA) with numerous potential sub-fluorophores and active sites and 4,4',4'',4'''-(porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) (TCPP) with macrocyclic conjugated structures were introduced to prepare the carbonized polymer dots (CPDs) with red/blue dual emission based on the cross-linking enhanced emission (CEE) effect and the luminescence of macrocyclic conjugated structures. The ratiometric fluorescence sensing systems were constructed by integrating the specific response of CPDs to Cu2+ and the affinity difference of Cu2+ to substrates or products of enzymes. The sensing systems, CPDs/Cu2+/PPi and CPDs/Cu2+/BTCh, were designed to detect liver disease biomarkers, alkaline phosphatase (ALP) and butyrylcholinesterase (BChE), respectively. The limit of detection for ALP and BChE was 0.35 U/L and 0.19 U/L, respectively. The proposed sensors were successfully applied to human serum samples from different health stages with satisfactory recoveries. These results demonstrate the successful design of a novel dual-emissive fluorescent probe and provide a feasible strategy for clinical detection.

Synergistic effect-triggered fluorescence quenching enables rapid and sensitive detection of alkaline phosphatase.

The development of biosensors mediated by synergistic quenching effect is of great significance for rapid and accurate clinical diagnosis. Hence, we prepared a cyan-emitting fluorescent Si dots for alkaline phosphatase (ALP) detection through the synergistic quenching effect of inner filter effect (IFE) and photo-induced electron transfer (PET). Si dots were prepared by microwave-assisted method, which displayed high quantum yield (28.7%), as well as good physiochemical properties, such as photo-stability, pH stability, and chemical stability. As the hydrolysate of 4-nitrophenyl phosphate disodium salt hexahydrate catalyzed by ALP, both IFE and PET of 4-nitrophenyl to Si dots were used for the turn-off mode detection of ALP. The linear relationships were established between the change of fluorescence intensity and ALP concentration in the range of 0.05 U L-1 to 5.0 U L-1, and 5.0 U L-1 to 80.0 U L-1, respectively. The detection limit was 0.01 U L-1. The synergistic quenching effect caused the turn-off mode detection to be more sensitive, and it can also be used for the accurate detection of ALP in human serum, thereby showing great anti-interference ability in complex environments.

Colorimetric Sensing Strategy through the Coordination Chemistry between Ascorbic Acid 2-Phosphate and Copper Ions.

The coordination chemistry between phosphorylated molecules and metal ions has been reported, while few studies focus on its sensing capability. Herein, we report a colorimetric sensing strategy through the coordination chemistry between ascorbic acid 2-phosphate (AAP) and copper ions. The phosphate group-containing AAP can coordinate with copper ions to induce a visible color change from blue to green in a rapid way, which can be easily read by the naked eye or a smartphone based on the blue-to-green (B/G) ratio. This coordination chemistry provides a facile and convenient strategy for designing colorimetric assays. Alkaline phosphatase can catalyze the hydrolysis of AAP to ascorbic acid (AA), thus modulating the AAP/AA transformation and the AAP-mediated coordination, offering a straightforward way for monitoring the enzymatic activity. This colorimetric sensing strategy shows good performances in stability, sensitivity, cost, and scale-up production, holding great promise as a point-of-care technique for diagnostic applications.

Disulfide Bonds Are Not Necessary for Intrinsic TNSALP Activity.

Recent developments in single-molecule enzymology (SME) have allowed for the observation of subpopulations present in enzyme ensembles. Tissue-nonspecific alkaline phosphatase (TNSALP), a homodimeric monophosphate esterase central to bone metabolism, has become a model enzyme for SME studies. TNSALP contains two internal disulfide bonds that are critical for its effective dimerization; mutations in its disulfide bonding framework have been reported in patients with hypophosphatasia, a rare disease characterized by impaired bone and tooth mineralization. In this paper, we present the kinetics of these mutants and show that these disulfide bonds are not crucial for TNSALP enzymatic function. This surprising result reveals that the enzyme's active conformation does not rely on its disulfide bonds. We posit that the signs and symptoms seen in hypophosphatasia are likely not primarily due to impaired enzyme function, but rather decreased enzyme expression and trafficking.

Self-assembly of protein-DNA superstructures for alkaline phosphatase detection in blood.

We designed a paper-based analytical device by integrating horseradish peroxidase (HRP)-encapsulated 3D DNA for visual detection of alkaline phosphatase (ALP). This device allows on-paper sample pre-treatment, target recognition and signal readout, enabling simple (without additional pre-treatment of blood samples) and rapid (within 23 min) determination of ALP in clinical samples.

Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.

Alkaline Phosphatase-Mediated Bioetching of CoOOH/BiVO4 for Signal-On Organic Photoelectrochemical Transistor Bioanalysis.

Organic photoelectrochemical transistor (OPECT) bioanalytics has recently appeared as a promising route for biological measurements, which has major implications in both next-generation photoelectrochemical (PEC) bioanalysis and futuristic biorelated implementations. Via biological dissociation of materials, bioetching is a useful technique for bio-manufacturing and bioanalysis. The intersection of these two domains is expected to be a possible way to achieve innovative OPECT bioanalytics. Herein, we validate such a possibility, which is exemplified by alkaline phosphatase (ALP)-mediated bioetching of a CoOOH/BiVO4 gate for a signal-on OPECT immunoassay of human immunoglobulin G (HIgG) as the model target. Specifically, target-dependent bioetching of the upper CoOOH layer could result into an enhanced electrolyte contact and light accessibility to BiVO4, leading to the modulated response of the polymeric poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) channel that could be monitored by the channel current. The introduced biosensor achieves sensitive detection of HIgG with high selectivity and sensitivity. This work features bioetching-enabled high-efficacy OPECT bioanalysis and is anticipated to serve as a generic protocol, considering the diverse bioetching routes.

Dual-product synergistically enhanced ratiometric fluorescence assay for alkaline phosphatase activity using core-shell lanthanide-based nanoprobe.

We developed a new core-shell ratiometric fluorescent nanoprobe simply prepared by a seeded growth method. The strong luminescence arising from the shell formed by coordination self-assembly of an alkaline phosphatase (ALP) substrate, l-ascorbic acid 2-phosphate (AAP), and Tb3+, together with the weak fluorescence of the core-a metal-organic framework, UiO-66-NH2, constitutes an ideal dual-emission characteristic. Since AAP can be specifically cleaved by ALP, the well-formulated core-shell nanostructure was destroyed upon exposure to ALP. In this case, the luminescence of Tb3+ was quenched due to the inefficient antenna effect, while the fluorescence of UiO-66-NH2 was strengthened by the synergistical enhancement of dual hydrolysates to inhibit the ligand-to-metal charge transfer (LMCT) process. Using the dual signal response, this nanoprobe was employed for ratiometric fluorescence detection of ALP activity in the range of 0.05-0.6 U mL-1 with a detection limit of 0.018 U mL-1, accompanied by a discernible fluorescence color evolution from turquoise to blue. In virtue of good properties in accuracy, sensitivity, selectivity and simplicity, this assay enabled quantitative detection of ALP activity in human serum and efficient screening of ALP inhibitors. More Importantly, a smartphone-assisted paper-based sensing platform was designed for on-site visual analysis of ALP activity in human serum, which may be very promising as a facile, portable, and robust format to facilitate relevant biological and clinical applications.

DNAzyme-regulated CRISPR/Cas12a based fluorescent biosensor for sensitive detection of alkaline phosphatase activity and inhibition.

Alkaline phosphatase (ALP) is regarded as an emerging biomarker and therapeutic target for various diseases. Herein, we developed a DNAzyme-regulated CRISPR/Cas12a cascade signal amplification strategy for sensitive and selective detection of ALP activity and inhibition. In this assay, the substrate strand of Cu2+-specific DNAzyme was designed as the DNA activator for Cas12a. The substrate strand would be cleaved into two fragments when ALP-catalyzed hydrolysis reaction disabled the complexation between Cu2+ and pyrophosphate (PPi). In this case, the cleaved sequences could not further bind to the Cas12a-crRNA complex to activate the trans-cleavage activity of CRISPR/Cas12a, which would hamper the cleavage of doubly labeled single-stranded DNA (ssDNA) fluorescence reporter, and thus a turn-off fluorescence signal. The cascade signal amplification strategy greatly improved the detection sensitivity for ALP activity, with a detection limit as low as 0.04 U/L. Moreover, the proposed method was successfully applied for ALP activity detection in serum samples and ALP inhibitory capability evaluation. This method overcomes the shortcoming of conventional methods that show unsatisfactory sensitivity and are susceptible to interfering substances, which might hold great promise in clinical diagnosis and biomedical research.

Structural Characterization of Functionally Important Chloride Binding Sites in the Marine Vibrio Alkaline Phosphatase.

Enzyme stability and function can be affected by various environmental factors, such as temperature, pH, and ionic strength. Enzymes that are located outside the relatively unchanging environment of the cytosol, such as those residing in the periplasmic space of bacteria or extracellularly secreted, are challenged by more fluctuations in the aqueous medium. Bacterial alkaline phosphatases (APs) are generally affected by ionic strength of the medium, but this varies substantially between species. An AP from the marine bacterium Vibrio splendidus (VAP) shows complex pH-dependent activation and stabilization in the 0-1.0 M range of halogen salts and has been hypothesized to specifically bind chloride anions. Here, using X-ray crystallography and anomalous scattering, we have located two chloride binding sites in the structure of VAP, one in the active site and another one at a peripheral site. Further characterization of the binding sites using site-directed mutagenesis and small-angle X-ray scattering showed that upon binding of chloride to the peripheral site, structural dynamics decreased locally, resulting in thermal stabilization of the VAP active conformation. Binding of the chloride ion in the active site did not displace the bound inorganic phosphate product, but it may promote product release by facilitating rotational stabilization of the substrate-binding Arg129. Overall, these results reveal the complex nature and dynamics of chloride binding to enzymes through long-range modulation of electronic potential in the vicinity of the active site, resulting in increased catalytic efficiency and stability.

Nanobody-based fluorescent immunoassay using carbon dots anchored cobalt oxyhydroxide composite for the sensitive detection of fenitrothion.

Fenitrothion (FN) residue in food is a serious threat to public health. Consequently, a sensitive, cost-effective, and convenient immunoassay for FN urgently needs to be fabricated to safeguard human health. Herein, a nanobody-alkaline phosphatase fusion protein (Nb-ALP)-based fluorescent ELISA using red emissive carbon dots (r-CDs) anchored cobalt oxyhydroxide nanosheet (CoOOH NS) composite was developed for detecting FN. Briefly, a Nb-ALP was obtained by autoinduction expression and employed as a recognition, signal transduction, and amplification element. As the fluorescence signal source, r-CDs were assembled with CoOOH NS to yield the r-CDs@CoOOH NS composite, leading to the fluorescence quenching of r-CDs via Förster resonance energy transfer (FRET). After competitive immunoreaction, the Nb-ALP bounded to the immobilized antigen can mediate the production of ascorbic acid, which can reduce the CoOOH NS to Co2+, breaking the FRET between r-CDs and CoOOH NS, accompanied by the fluorescence recovery of r-CDs. This fluorescent ELISA is highly sensitive to FN with a detection limit of 0.14 ng mL-1, which is 25-fold lower than that of conventional colorimetric ELISAs. The recovery test of food samples and the validation by GC-MS/MS further demonstrated the proposed assay was an ideal tool for detecting FN.

In situ encapsulation of SQDs by zinc ion-induced ZIF-8 growth strategy for fluorescent and colorimetric dual-signal detection of alkaline phosphatase.

Controllable encapsulation of sulfur quantum dots (SQDs) into metal-organic frameworks (ZIF-8) by a surface-bound zinc ion-induced growth strategy, and SQDs@ZIF-8 was successfully prepared for alkaline phosphatase (ALP) detection. The new synthesis procedure involves first binding Zn2+ to the surface of SQDs to form SQDs/Zn, and then via zinc ion-induced in situ ZIF-8 growth to obtain SQDs@ZIF-8, which greatly improved the luminous efficiency of SQDs. The specific process of detecting ALP using pH-triggered fluorescence quenching of SQDs@ZIF-8: firstly ALP hydrolyzes 2-phosphate-l-ascorbic acid trisodium salt (AAP) to ascorbic acid (AA), and then the leakage of SQDs in the SQDs@ZIF-8 leads to a decrease in fluorescence intensity based on the destruction of ZIF-8 skeleton by H+ released by AA. A linear relationship was obtained between the fluorescence intensity and the ALP concentration in the range of 0.15-50 U/L, and the detection limit was 0.044 U/L. Moreover, it was found that free SQDs can be complexed with Fe2+ to produce wine red complexes, and the obtained UV absorbance and ALP concentration have a linear relationship in the range of 10-200 U/L. The detection range of ALP is significantly broadened based on the combination of the above two detection methods. Furthermore, SQDs@ZIF-8 exhibited excellent stability in water and was successfully applied to the fluorescence and colorimetric detection of ALP in human serum.

Homogeneous photoelectrochemical biosensor for sensitive detection of omethoate via ALP-mediated pesticide assay and Bi2S3@Bi2Sn2O7 heterojunction as photoactive material.

A simple homogeneous photoelectrochemical (PEC) sensing platform based on an alkaline phosphatase (ALP)-mediated pesticide assay was established for the sensitive detection of omethoate (OM). The Bi2S3@Bi2Sn2O7 heterojunction was used as a photoactive material to provide stable background photocurrent signals. The inhibition of OM on ALP and PEC determination was carried out in the homogeneous system. In the absence of OM, dephosphorylation of L-ascorbic acid 2-phosphate trisodium salt (AAP) was catalyzed by ALP to produce the enzyme-catalyzed product (L-ascorbic acid, AA). AA, as an electron donor, could capture photogenerated holes on the Bi2S3@Bi2Sn2O7 heterojunction, thus inhibiting the recombination of electron holes to achieve an increase of the photocurrent signal. When the OM was introduced, the enzyme activity of ALP was reduced due to the organophosphorus pesticides (OPs)-based enzyme inhibition, and the AA produced by catalytic hydrolysis was also reduced, thus reducing the photocurrent signal. Compared with the traditional PEC sensor for OPs, this homogeneous PEC sensor avoided immobilization procedures, covalent labeling, separation, and the steric hindrance effect caused by immobilized biomolecules, which achieved high recognition efficiency and caused a reduction in analysis time. Additionally, an ALP-mediated pesticide assay for the determination of OPs with a simplified experimental process further improved the stability and reproducibility of the PEC sensor. The PEC sensor showed high sensitivity to the target OM within a dynamic range of 0.05 ~ 500 ng mL-1, and the detection limit was 0.0146 ng mL-1. Additionally, the PEC biosensing system showed good selectivity and anti-interference ability, and exhibited a satisfactory result in spinach and mustard samples. A homogeneous PEC biosensor based on ALP inhibition strategy was constructed for OM detection in vegetable samples via Bi2S3@Bi2Sn2O7 heterojunction as the photoactive substrate material.