Single‐Molecule Analysis Determines Isozymes of Human Alkaline Phosphatase in Serum

Alkaline phosphatase (ALP) is an important biomarker, as high levels of ALP in blood can indicate liver disease or bone disorders. However, current clinical blood tests only measure the total concentration of ALP but are unable to distinguish enzyme isotypes. Here, we demonstrate a novel and rapid approach to profile various ALP isozymes in blood via a single‐molecule‐analysis platform. The microarray platform provides enzyme kinetics of hundreds of individual molecules at high throughput. Using these single molecule kinetics, we characterize the different activity profiles of ALP isotypes. By analyzing both healthy and disease samples, we found the single molecule activity distribution of ALP in serum reflects the health status of patients. This result demonstrates the potential utility of the method for improving the conventional ALP test, as well as for analyzing other enzymatic biomarkers, including enzyme isotypes.     

An Activatable AIEgen Probe for High‐Fidelity Monitoring of Overexpressed Tumor Enzyme Activity and Its Application to Surgical Tumor Excision

Monitoring fluctuations in enzyme overexpression facilitates early tumor detection and excision. An AIEgen probe (DQM‐ALP) for the imaging of alkaline phosphatase (ALP) activity was synthesized. The probe consists of a quinoline‐malononitrile (QM) core decorated with hydrophilic phosphate groups as ALP‐recognition units. The rapid liberation of DQM‐OH aggregates in the presence of ALP resulted in aggregation‐induced fluorescence. The up‐regulation of ALP expression in tumor cells was imaged using DQM‐ALP. The probe permeated into 3D cervical and liver tumor spheroids for imaging spatially heterogeneous ALP activity with high spatial resolution on a two‐photon microscopy platform, providing the fluorescence‐guided recognition of sub‐millimeter tumorigenesis. DQM‐ALP enabled differentiation between tumor and normal tissue ex vivo and in vivo, suggesting that the probe may serve as a powerful tool to assist surgeons during tumor resection.     

An Activatable AIEgen Probe for High-Fidelity Monitoring of Overexpressed Tumor Enzyme Activity and Its Application to Surgical Tumor Excision

Monitoring fluctuations in enzyme overexpression facilitates early tumor detection and excision. An AIEgen probe (DQM-ALP) for the imaging of alkaline phosphatase (ALP) activity was synthesized. The probe consists of a quinoline-malononitrile (QM) core decorated with hydrophilic phosphate groups as ALP-recognition units. The rapid liberation of DQM-OH aggregates in the presence of ALP resulted in aggregation-induced fluorescence. The up-regulation of ALP expression in tumor cells was imaged using DQM-ALP. The probe permeated into 3D cervical and liver tumor spheroids for imaging spatially heterogeneous ALP activity with high spatial resolution on a two-photon microscopy platform, providing the fluorescence-guided recognition of sub-millimeter tumorigenesis. DQM-ALP enabled differentiation between tumor and normal tissue ex vivo and in vivo, suggesting that the probe may serve as a powerful tool to assist surgeons during tumor resection.     

Platform for Highly Sensitive Alkaline Phosphatase‐Based Immunosensors Using 1‐Naphthyl Phosphate and an Avidin‐Modified Indium Tin Oxide Electrode

We report a versatile platform for highly sensitive alkaline phosphatase (ALP)‐based electrochemical biosensors that uses an avidin‐modified indium tin oxide (ITO) electrode as a sensing electrode and 1‐naphthyl phosphate (NPP) as an ALP substrate. Almost no electrocatalytic activity of NPP and good electrocatalytic activity of 1‐naphthol (ALP product) on the ITO electrodes allow a high signal‐to‐background ratio. The effective surface covering of avidin on the ITO electrodes allows very low levels of nonspecific binding of proteins to the sensing electrodes. The platform technology is used to detect mouse IgG with a detection limit of 1.0 pg/mL.     

The molecular basis of pharmacological chaperoning in human α-galactosidase

Fabry disease patients show a deficiency in the activity of the lysosomal enzyme α-galactosidase (α-GAL or α-Gal A). One proposed treatment for Fabry disease is pharmacological chaperone therapy, where a small molecule stabilizes the α-GAL protein, leading to increased enzymatic activity. Using enzyme kinetics, tryptophan fluorescence, circular dichroism, and proteolysis assays, we show that the pharmacological chaperones 1-deoxygalactonojirimycin (DGJ) and galactose stabilize the human α-GAL glycoprotein. Crystal structures of complexes of α-GAL and chaperones explain the molecular basis for the higher potency of DGJ over galactose. Using site-directed mutagenesis, we show the higher potency of DGJ results from an ionic interaction with D170. We propose that protonation of D170 in acidic conditions leads to weaker binding of DGJ. The results establish a biochemical basis for pharmacological chaperone therapy applicable to other protein misfolding diseases.     

Fluorescence immunoassay based on alkaline phosphatase-induced in situ generation of fluorescent non-conjugated polymer dots

Alkaline phosphatase (ALP) activity assay is not only significant to the clinical diagnosis of some related disease, but also momentous to the construction of ALP-based enzyme-linked immunosorbent assay (ELISA). Herein, for the first time, we have discovered that ascorbic acid (AA) can specially react with N-methylethylenediamine (N-MEDA) to generate fluorescent non-conjugated polymer dots (NCPDs) under mild conditions. On the basis of the AA-responsive emission and ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AA2P) to AA, we have exploited a fluorometric ALP activity assay with high sensitivity and selectivity. Furthermore, by means of conventional ALP-based ELISA platform, a conceptual fluorescent ELISA has been constructed and applied in the potential clinical diagnosis, during which cardiac troponin I (cTnI), a well-established biomarker of acute myocardial infarction, has been chosen as the model target. We envision that such original fluorescent NCPDs generation-enabled ELISA could become a versatile tool in biochemical sensing and medical diagnosis in the future.     

Monitoring Cardiac Biomarkers with Aptamer-Based Molecular Pendulum Sensors

Reagent-free electronic biosensors capable of analyzing disease markers directly in unprocessed body fluids will enable the development of simple & affordable devices for personalized healthcare monitoring. Here we report a powerful and versatile nucleic acid-based reagent-free electronic sensing system. The signal transduction is based on the kinetics of an electrode-tethered molecular pendulum—a rigid double stranded DNA with one of the strands displaying an analyte-binding aptamer and the other featuring a redox probe—that exhibits field-induced transport modulated by receptor occupancy. Using chronoamperometry, which enables the sensor to circumvent the conventional Debye length limitation, the binding of an analyte can be monitored as these species increase the hydrodynamic drag. The sensing platform demonstrates a low femtomolar quantification limit and minimal cross-reactivity in analyzing cardiac biomarkers in whole blood collected from patients with chronic heart failure.     

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)₂^2? (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)₂^2? into Cu(BCDS)₂^3?, 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.     

Biofunctionalization of aqueous dispersed, alumina membrane-templated polymer nanorods for use in enzymatic chemiluminescence assays

The noncovalent immobilization of alkaline phosphatase (ALP) onto aqueous dispersed nylon 6 nanorods ( approximately 310 nm mean diameter; approximately 6 microm mean length) prepared by anodic aluminum oxide (AAO) membrane templating was studied. Using multi-stacked layer-by-layer (LBL) assembly with the cationic quaternary ammonium polymer Sapphire II , the amount of ALP enzyme loaded onto the polymer nanostructures was found to be 115+/-7 microg mg(-1) nanorod. The biofunctionalized nanorods were also characterized for their chemiluminescent activity with the dioxetane substrate, CSPD . The results indicate that the kinetic parameters, K(m) and V(max), for the catalytic activity of the nanostructure-bound ALP enzyme are different from those of soluble ('free') ALP. While the K(m) value was measured to be 156 microM for free ALP, the apparent K(m) value determined for the LBL-immobilized ALP is approximately 20% lower (122 microM). Furthermore, despite the relatively high enzyme loading capacity of the nanorods, the specific activity of the bound ALP enzyme was found to be almost nine times lower than that measured for free ALP. Finally, additional experiments revealed that the catalytic activities of both free ALP and nanorod-conjugated ALP are affected similarly by changes in pH, with optimal performance levels occurring under conditions of pH 9.5. To the best of our knowledge, this study represents the first report examining the preparation of aqueous dispersed, AAO-templated polymer nanorods for potential application as enzyme scaffolds in chemiluminescent-based assay systems.     

Copper sulfide nanoparticle-decorated graphene as a catalytic amplification platform for electrochemical detection of alkaline phosphatase activity

Copper sulfide nanoparticle-decorated graphene sheet (CuS/GR) was successfully synthesized and used as a signal amplification platform for electrochemical detection of alkaline phosphatase activity. First, CuS/GR was prepared through a microwave-assisted hydrothermal approach. The CuS/GR nanocomposites exhibited excellent electrocatalytic activity toward the oxidation of ALP hydrolyzed products such as 1-naphthol, which produced a current response. Thus, a catalytic amplification platform based on CuS/GR nanocomposite for electrochemical detection of ALP activity was designed using 1-naphthyl phosphate as a model substrate. The current response increased linearly with ALP concentration from 0.1 to 100 U L⁻¹ with a detection limit of 0.02 U L⁻¹. The assay was applied to estimate ALP activity in human serum samples with satisfactory results. This strategy may find widespread and promising applications in other sensing systems that involves ALP.     

A covalent organic framework (COF)-MnO2 based dual signal sensing platform for sensitive alkaline phosphatase activity detection via dynamic regulating the mimicking oxidase content

It is of great significance to accurately monitor the alkaline phosphatase (ALP) level as it plays an important role in living body activities. Herein, we develop a COF- MnO₂ system for ALP activity detection via the dynamic regulating the MnO₂ nanosheets content. MnO₂ nanosheets with oxidase-mimicking property can oxide the colorless 3,3′,5,5′-Tetramethylbenzidine (TMB) into blue oxidized TMB (oxTMB). The hexagonal structure and ordered mesoporous channels of DMTP-TAPB COF provide excellent space to accommodate the product oxTMB. The confinement of the dye molecules into COF structure leads to enhance color change and obvious fluorescence quench of the sensing system. The fluorescence quenching and color change dependent on the ALP level as it can dynamic regulate the MnO₂ content via the enzymatic hydrolysis of ascorbate-2-phosphate. Therefore, a COF-MnO₂ based dual signal sensing platform is successfully constructed to detect ALP activity, giving detection limit of 0.11 U L^-1 and 0.23 U L^-1 for fluorescence and colorimetric procedures, respectively. The practical application of the designed sensing platform is verified through the detection of ALP activity in serum samples, and satisfactory results are obtained.     

Fluorescent Dye Conjugates of Rabbit Arylsulfatase A as a Biological Tracer for Protein Endocytosis

Fluorescent dye conjugates of arylsulfatase A (ASA) from rabbit liver were prepared at pH 9.0 in 0.1 M sodium bicarbonate buffer. The modification of amino or sulphadryl groups by dichlorotriazinylamino-fluorescein or Lucifer yellow fluorescent dyes did not alter the characteristic features of the enzyme molecule such as enzyme activity, dimerization of the protein molecule at pH 4.5 and anomalous kinetics of the native enzyme. The fluorescence intensity of the Lucifer yellow enzyme conjugates were quenched when the pH of the protein solution was changed from pH 7.5 to 4.5. Therefore, the Lucifer yellow enzyme conjugate can be used to study the kinetics of pH-dependent association and dissociation of the ASA. Availability of such fluorescent dyes conjugates of ASA or other lysosomal enzyme may be used as a biological tracer to study the receptor dependent endocytosis of enzyme molecules.     

Alkaline phosphatase assay using a near-infrared fluorescent substrate merocyanine 700 phosphate

Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that is commonly used as a conjugating enzyme in biological research. A wide variety of substrates have been developed to assay its activity. In this study, we developed an ALP assay method utilizing merocyanine 700 (MC700) based substrate MC700 phosphate (MC700p). MC700 is a near-infrared fluorescent merocyanine dye, and has excitation/emission maxima at 686 nm/722 nm in ALP assay buffer. Upon hydrolysis by ALP, MC700p is converted to MC700. The fluorescence of MC700 is dependent on the pH and detergent concentration in the buffer. The fluorescence signal produced by MC700p hydrolysis is linearly related to the ALP amount and substrate concentration. A stop solution containing EDTA could be used to stop the ALP/MC700p reaction. It was also demonstrated that MC700p could substitute pNpp as the ALP substrate in a commercial 17β-Estradiol enzyme immunoassay kit.     

Distinct and long-lived activity states of single enzyme molecules

Individual enzyme molecules have been observed to possess discrete and different turnover rates due to the presence of long-lived activity states. These stable activity states are thought to result from different molecular conformations or post-translational modifications. The distributions in kinetic activity observed in previous studies were obtained from small numbers of single enzyme molecules. Due to this limitation, it has not been possible to fully characterize the different kinetic and equilibrium binding parameters of single enzyme molecules. In this paper, we analyze hundreds of single beta-galactosidase molecules simultaneously; using a high-density array of 50,000 fL-reaction chambers, we confirm the presence of long-lived kinetic states within a population of enzyme molecules. Our analysis has isolated the source of kinetic variability to kcat. The results explain the kinetic variability within enzyme molecule populations and offer a deeper understanding of the unique properties of single enzyme molecules. Gaining a more fundamental understanding of how individual enzyme molecules work within a population should provide insight into how they affect downstream biochemical processes. If the results reported here can be generalized to other enzymes, then the stochastic nature of individual enzyme molecule kinetics should have a substantial impact on the overall metabolic activity within a cell.     

Enzyme Catalysis Induced Polymer Growth in Nanochannels: A New Approach to Regulate Ion Transport and to Study Enzyme Kinetics in Nanospace

Method that could regulate the ion transport in nanochannel in an efficient and rapid manner is still a challenge. Here, we introduced enzyme‐catalysis‐induced polymer growth in nanochannels to develop a new method to regulate the ion transport and evaluate the enzyme catalysis kinetics in nano‐space. As a model enzyme, Horseradish peroxidase (HRP) was immobilized in the nanochannels through a volume‐controlled‐drying method. In the presence of H₂O₂, HRP catalyzed o‐phenylenediamine (o‐PD) to trigger its polymer growth, in turn blocked the ion transport and led to the decrease of the ion current. Taking advantages of the high efficiency of enzyme catalysis and the nano‐confinement of nanochannels, the system readily achieved blocking ratios of ion current even reaching 99.6 % of the initial. Based on above concept, we developed a new method to evaluate the enzyme catalysis kinetics in nano‐confined space. By comparing with those in free state in solution and absorbed on planar surface, HRP confined in nanochannels presented similar apparent Michaelis constant (K_m) values for the substrate H₂O₂ but much higher K_m values for the substrate o‐PD, due to the steric hindrance and diffusion suppression. The enzyme‐catalysis‐induced polymerization in nanochannels might lead to new concept for the nano‐blocking/switching and provide a new platform for single molecule analysis and detection.     

Potentiometric flow-injection system for determination of alkaline phosphatase in human serum

A flow-injection system for detection of alkaline phosphatase (ALP) activity in human serum samples has been developed. As a specific and inexpensive ALP substrate for this kinetic assay monofluorophosphate (MFP) was applied. For detection of fluoride ions, generated in the course of the biocatalytic hydrolysis of MFP, conventional fluoride ion-selective electrode based on LaF₃-crystalline membrane was applied. After optimization the system allows analysis of human serum with high selectivity and relatively short time of analysis (5–6 samples h⁻¹). Volume of serum required for analysis is 0.05 mL. The system is useful for determination of the enzyme activity in human serum samples at physiological and pathological levels as well as for detection of isoenzymatic forms of ALP.     

A Rapidly Reversible Chemical Dimerizer System to Study Lipid Signaling in Living Cells

Chemical dimerizers are powerful tools for non‐invasive manipulation of enzyme activities in intact cells. Here we introduce the first rapidly reversible small‐molecule‐based dimerization system and demonstrate a sufficiently fast switch‐off to determine kinetics of lipid metabolizing enzymes in living cells. We applied this new method to induce and stop phosphatidylinositol 3‐kinase (PI3K) activity, allowing us to quantitatively measure the turnover of phosphatidylinositol 3,4,5‐trisphosphate (PIP₃) and its downstream effectors by confocal fluorescence microscopy as well as standard biochemical methods.     

Exploring the trifluoromenadione core as a template to design antimalarial redox-active agents interacting with glutathione reductase

Menadione is the 2-methyl-1,4-naphthoquinone core used to design potent antimalarial redox-cyclers to affect the redox equilibrium of Plasmodium-infected red blood cells. Exploring the reactivity of fluoromethyl-1,4-naphthoquinones, in particular trifluoromenadione, under quasi-physiological conditions in NADPH-dependent glutathione reductase reactions, is discussed in terms of chemical synthesis, electrochemistry, enzyme kinetics, and antimalarial activities. Multitarget-directed drug discovery is an emerging approach to the design of new antimalarial drugs. Combining in one single 1,4-naphthoquinone molecule, the trifluoromenadione core with the alkyl chain at C-3 of the known antimalarial drug atovaquone, revealed a mechanism for CF(3) as a leaving group. The resulting trifluoromethyl derivative 5 showed a potent antimalarial activity per se against malarial parasites in culture.     

Metallic-Nanostructure-Enhanced Fluorescence of Single Flavin Cofactor and Single Flavoenzyme Molecules

The enzyme cofactors are intrinsically fluorescent and participate directly in the single molecule enzymology studies. Due to photobleaching, one cannot follow kinetics continuously by cofactor fluorescence for more than several minutes typically. Modification of spectral properties of fluorophores, such as the amplification of emission intensity, can be achieved through coupling with surface plasmons in close proximity to metallic nanostructures. This process, referred to as metal-enhanced fluorescence, offers promise for a range of applications, including bioassays, sensor technology, microarrays, and single-molecule studies. Here, we demonstrated up to a 100-fold increase in the emission of the single cofactors and flavoenzymes near silver nanostructures. Amplified fluorescence of different types of flavins and flavoenzymes has been interpreted by using time-resolved single molecule fluorescence data. The results show considerable promise for the studies of enzyme kinetics using the intrinsic fluorescence from the cofactors.     

2-Carboxy-1-naphthyl phosphate as a substrate for the fluorimetric determination of alkaline phosphatase

A new substrate, 2-carboxy-1-naphthyl phosphate (CNP), was developed for the fluorimetric determination of alkaline phosphatase (ALP) activity. The product of the enzyme reaction is 1-hydroxy-2-naphthoic acid (HNA), which is a strong fluorescent product. The amount of HNA generated is proportional to ALP activity. Optimal conditions for the determination of ALP were investigated. The linear range and detection limit for the determination of ALP are 0.01-4.8 U/L and 7.44 mU/L, respectively. This method is simple, practical and can be successfully applied to assess ALP in human serum with good accuracy and precision. The results were evaluated by comparison with a standard colorimetric assay using p-nitrophenyl phosphate as ALP substrate.