Enzymatic Reaction Research Articles

Quantum Mechanical Cluster Models for Calculations on Enzymatic Reaction Mechanisms: Set-Up and Accuracy.

Enzymes turnover substrates into products with amazing efficiency and selectivity and as such have great potential for use in biotechnology and pharmaceutical applications. However, details of their catalytic cycles and the origins surrounding the regio- and chemoselectivity of enzymatic reaction processes remain unknown, which makes the engineering of enzymes and their use in biotechnology challenging. Computational modelling can assist experimental work in the field and establish the factors that influence the reaction rates and the product distributions. A popular approach in modelling is the use of quantum mechanical cluster models of enzymes that take the first- and second coordination sphere of the enzyme active site into consideration. These QM cluster models are widely applied but often the results are dependent on model choice and selection. Herein, we show that QM cluster models can produce highly accurate results that reproduce experimental product distributions and free energies of activation, regarded that large cluster models with >300 atoms are used. In this tutorial review, we give general guidelines on the set-up and applications of the QM cluster method and discuss its accuracy and reproducibility. Finally, several representative QM cluster model examples on metal-containing enzymes are presented, which highlight the strength of the approach.

Indole Schiff Base Complex: Synthesis and Optical Binding Investigation with Biogenic Amines.

Biogenic amines, produced by bacterial enzymatic reactions in food storage or processing, serve as indicators in food processing industries to assess food quality and freshness. Biogenic amines also often associated with various health problems, including abnormal immune responses and gastrointestinal disease. Previously, salphen base complexes have been reported but still exhibited low fluorescence enhancement upon biogenic amines. This research focused on synthesizing and characterizing new Zn(II) Schiff base complex with indole sidechain to enhance the fluorescence property and exploring their binding behaviour with the biogenic amines, which were phenylethylamine and cadaverine. The Zn(II) indole Schiff base complex's structure was verified by diverse spectroscopic techniques. Then, the binding behaviours between the Zn(II) indole Schiff base complex with the biogenic amines were analyzed using UV-Vis, fluorescence spectroscopy, and Job's plot analysis. UV-Vis binding study results indicated that the synthesized complexes could bind stronger with phenylethylamine than cadaverine, with binding constant, Kb= (8.21 ± 0.58) × 104 M- 1 and (2.506 ± 0.004) × 104 M- 1 respectively. Moreover, Zn(II) indole Schiff base complex-phenylethylamine binding also generated higher fluorescence enhancement than cadaverine, which were 54% and 51% respectively. Based on Job's plot analysis, the complex and biogenic amines were bound in the ratio of 1:1. To conclude, the synthesized complex has promising potential as a sensing material for biogenic amines detection in food. The complex is recommended to be deployed in the development of solid-state fluorescence sensor for biogenic amines detection for monitoring the food spoilage in the food industry in the future.

Development of an amperometric biosensor that can determine the amount of glucose in the blood using the glucose oxidase enzyme: Preparation of polyaniline-polypyrrole-poly(sodium-4-styrenesulfonate) film.

In this study, a new amperometric biosensor was developed for glucose determination. For this purpose, polyaniline-polypyrrole-poly(sodium-4-styrenesulfonate) film was prepared by electropolymerization of aniline and pyrrole with poly(sodium-4-styrenesulfonate) on a platinum plate. The best working conditions of the polyaniline-polypyrrole-poly(sodium-4-styrenesulfonate) film were determined. The glucose oxidase enzyme was immobilized by the entrapment method in polyaniline-polypyrrole-poly(sodium-4-styrenesulfonate) film. Glucose determination was made based on the oxidation of hydrogen peroxide, which is formed as a result of the enzymatic reaction on the surface of the prepared biosensor at +0.40V. The working range for the glucose determination of the biosensor was determined. The effects of pH and temperature on the response of the glucose biosensor were investigated. The reusability and shelf life of the biosensor were determined. The effects of interference in biological environments on the response of the biosensor were investigated. Glucose determination was made in the biological fluid (blood) with the prepared biosensor. This study has a feature that sheds light on biosensor studies to be developed for the detection of substances in the human body, such as glucose, uric acid, and urea. This article will set an example for future scientific research on the development of a sensor for other biological fluids in the human body, such as the sensor developed for blood samples. In addition, this developed sensor provides an innovation that improves the quality of life of patients by allowing them to constantly monitor their glucose levels and intervene when necessary.

The improved purification technique for isolation of the novel CGTase from the alkaliphilc strain Caldalkalibacillus mannanilyticus IB-OR17-B1

Cyclodextrin-glucanotransferase (CGTase, EC 2.4.1.19) is a multifunctional enzyme that catalyzes many enzymatic reactions including cyclization, binding, disproportionation and hydrolysis reactions, playing an important role in the enzymatic synthesis of compounds that are widely used in agriculture, pharmaceuticals, food, chemical and biotechnology industries. The present research is aimed to optimize the purification protocol for the extracellular CGTase of alkaliphilc bacterial strain Caldalkalibacillus mannanilyticus IB-OR17-B1 guaranteeing the enzyme homogeneity and its high yield. The improved combination of ultrafiltration and corn-starch (5% w/v) affinity sorption techniques resulted to mild and rapid isolation of electrophoritically homogenic enzyme at 18 × increase of its specific activity and yield 56%. The developed two-step procedure instead the practiced tree-step one using commonly ion-exchange chromatography as final purification technique highly contributes in advance of cost-effectiveness for industrial production and isolation of valuable CGTases.

Encapsulation of HRP-Immobilized Silica Particles into Hollow-Type Spherical Bacterial Cellulose Gel: A Novel Approach for Enzyme Reactions within Cellulose Gel Capsules

We revealed that the encapsulation of enzyme-immobilized silica particles in hollow-type spherical bacterial cellulose (HSBC) gels enables the use of the inside of HSBC gels as a reaction field. The encapsulation of horseradish peroxidase (HRP)-immobilized silica particles (Si-HRPs, particle size: 40–50 μm) within HSBC gels was performed by using a BC gelatinous membrane produced at the interface between Komagataeibacter xylinus suspension attached onto an alginate gel containing Si-HRPs and silicone oil. After the biosynthesis of the BC gelatinous membrane, formed from cellulose nanofiber networks, the alginate gel was removed via immersion in a phosphate-buffered solution. Si-HRP encapsulated HSBC gels were reproducibly produced using our method with a yield of over 90%. The pore size of the network structure of the BC gelatinous membrane was less than 1 μm, which is significantly smaller than the encapsulated Si-HRPs. Consequently, the encapsulated Si-HRPs could neither pass through the BC gelatinous membrane nor leak from the interior cavity of the HSBC gel. The activity of the encapsulated HRPs was detected using the 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system, demonstrating that this method can encapsulate the enzyme without inactivation. Since HSBC gels are composed of a network structure of biocompatible cellulose nanofibers, immune cells cannot enter the hollow interior, thus, the enzyme-immobilized particles encapsulated inside the HSBC gel are protected from immune-cell attacks. The encapsulation technique demonstrated in this study is expected to facilitate the delivery of enzymes and catalysts that are not originally present in the in vivo environment.

Differing temperature dependencies of functional homologs zebrafish Abcb4 and human ABCB1

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C–40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

Enhanced Saccharification Yield of Alkali Pretreated Sugarcane Bagasse Utilizing Customized Cellulase Cocktail from Trichoderma harzianum and Trichoderma viride

The experiment was conducted during 2023 at Bioenergy Laboratory, Department of RBEE, College of Agricultural Engineering and Technology, CCSHAU, Hisar, Haryana, India. Compositional changes in sugarcane bagasse subjected to varying concentrations of sodium hydroxide (0.3% to 1.2%), revealing significant increases in glucan content (from 37.13% to 53.81%) alongside decreases in xylan, acid-insoluble lignin, acid-soluble lignin, ash, and other extractives. These changes were validated using microscopic technique SEM, confirming the efficacy of the pretreatment process. The utilization of a customized cellulase cocktail derived from Trichoderma harzianum and Trichoderma viride holds significant promise in enhancing the saccharification from alkali-pretreated sugarcane bagasse. This study investigates the synergistic effects of cellulase enzymes produced by these fungi on the hydrolysis of lignocellulosic biomass. The enzymatic hydrolysis process is optimized by varying enzyme dosages, reaction conditions, and incubation times to maximize the release of fermentable sugars. Results indicate a substantial improvement in saccharification efficiency with the customized cellulase cocktail, highlighting its potential for sustainable biofuel production. The pretreated sugarcane bagasse, when saccharified with Trichoderma harzianum and Trichoderma viride individually, released 254.43 mg g-1 and 325.53 mg g-1 of reducing sugars, respectively, after 40 h of incubation. In contrast, the combined enzymatic cocktail achieved a substantial increase in glucose yields (345.12 mg g-1) at 40 h, showcasing the synergistic effect of the combined enzymatic activity. This research contributes to advancing bioconversion technologies for utilizing lignocellulosic biomass resources efficiently and economically, thus addressing key challenges in sustainable energy production.

Interfacially crystallized Enzyme@MOFs biocatalytic membranes for highly efficient synthesis of D-amino acids via continuously recirculating stereoinversion

D-Amino acids (D-AAs) constitute essential building blocks found in many pharmaceuticals and fine chemicals. We herein presented the coating of polyvinylidene fluoride (PVDF) hollow fiber membrane with L-amino acid oxidase (LAAO) embedded zeolitic imidazolate framework-8 (ZIF-8) layer by a single-step interfacial biomineralization strategy for efficient synthesis of D-AAs. The ZIF-8 skeleton and PVDF membrane structure not only guaranteed free enzyme activity and stability, but also reinforced the applicability of this bioreactor for continuous enzymatic reactions. By placing the prepared biocatalytic membrane in the constructed circulation device, only the L-AAs can be specifically oxidized. The D-AAs are accumulated and synthesized after multiple rounds of continuously recirculating stereoinversion. Impressively, using tryptophan (Trp) as the model molecule, the thus-designed bioreactor exhibited an excellent conversion rate (92.10%) and ee% (99.41%) of D-Trp with a shorter time under constant flow conditions. This study provides an unprecedented protocol for highly efficient synthesis of D-AAs, and immensely advances the promising application field of membrane bioreactors.

Efficient derivatization-free monitoring of glycosyltransferase reactions via flow injection analysis-mass spectrometry for rapid sugar analytics.

The widespread application of enzymes in industrial chemical synthesis requires efficient process control to maintain high yields and purity. Flow injection analysis-electrospray ionization-mass spectrometry (FIA-ESI-MS) offers a promising solution for real-time monitoring of these enzymatic processes, particularly when handling challenging compounds like sugars and glycans, which are difficult to quickly analyze using liquid chromatography-mass spectrometry due to their physical properties or the requirement for a derivatization step beforehand. This study compares the performance of FIA-MS with traditional hydrophilic interaction liquidchromatography (HILIC)-ultra high-performance liquid chromatography (UHPLC)-mass spectrometry (MS) setups for the monitoring of the enzymatic synthesis of N-acetyllactosamine (LacNAc) using beta-1,4-galactosyltransferase. Our results show that FIA-MS, without prior chromatographic separation or derivatization, can quickly generate accurate mass spectrometric data within minutes, contrasting with the lengthy separations required by LC-MS methods. The rapid data acquisition of FIA-MS enables effective real-time monitoring and adjustment of the enzymatic reactions. Furthermore, by eliminating the derivatization step, this method offers the possibility of being directly coupled to a continuously operated reactor, thus providing a rapid on-line methodology for glycan synthesis as well.

Interpenetrating Polymer Network Capturing FRET-Sensitive Polymers Available for an Enzyme Assay.

Fluorogenic glycomonomers have been used for biological evaluations, and water-soluble and Förster resonance energy transfer (FRET)-sensitive glycopolymers have also been reported. A FRET-sensitive polymer was conveniently prepared from a fluorogenic donor monomer and a fluorogenic acceptor monomer by means of simple radical polymerization in high yield. Continuous fluorospectroscopic monitoring of the polymer in the presence of an enzyme was performed, and the results showed the possible application of the FRET-sensitive glycopolymer for practical use. In addition to the use of aqueous solution phase, the water-soluble and FRET-sensitive glycopolymer was completely captured into an interpenetrating polymer network (IPN) by means of radical polymerization with a combination of acrylamide and bis-acrylamide as used for the cross-linking reagent system. The IPN including the FRET-sensitive glycopolymer was allowed to react with amylases in an aqueous buffer solution at 37 °C, and the enzymatic reaction was continuously and conveniently monitored by means of fluorometric spectroscopy.

Photoelectrochemical/Colorimetric Dual-Mode Specific Detection of Staphylococcus aureus Based on the Enzymatic Reaction Triggered by Catalase from Lysed Bacteria.

Staphylococcus aureus (S. aureus) is abundant in nature and frequently leads to various health issues. Bacteriophages as obligate intracellular parasites of bacteria have the ability to specifically identify and infect S. aureus, causing bacterial lysis and the release of endogenous catalase (CAT). The released CAT triggers the conversion of H2O2 into O2 and H2O, resulting in a notable decrease in UV absorption at 570 nm and a concurrent surge in photocurrent. On the basis of this, a photoelectrochemical/colorimetric dual-mode biosensor for the detection of S. aureus was developed. In the photoelectric detection mode, the reactions involving endogenous enzymes occur directly in the solution, requiring only the simple drop-coating of TiO2@CdS onto the indium tin oxide (ITO) electrode surface. There was no need for immobilizing additional biomolecules, thereby significantly minimizing nonspecific adsorption and improving the biosensor's stability and reproducibility. For colorimetry, we utilized a cost-effective and operationally simple approach based on KI and starch. Remarkably, this photoelectrochemical/colorimetry exhibited a linear range of 102-109 CFU/mL for S. aureus, achieving detection limits of 7 and 10 CFU/mL, respectively. Herein, phage identification ensures the specific detection of live S. aureus, thereby effectively mitigating the potential for false signals. The dual-signal readout mode improves the detection accuracy and reliability. In conclusion, this present method offers numerous advantages, including simplicity, time-efficiency, cost-effectiveness, high specificity, and therefore excellent accuracy.

Dynamics of HSD17B3 expression in human fetal testis: implications for the role of Sertoli cells in fetal testosterone biosynthesis

Introduction: Androgens play a pivotal role in shaping male sexual characteristics, with testosterone being an essential hormone in orchestrating various developmental processes. Testosterone biosynthesis involves a series of enzymatic reactions, among which the 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) holds significance. While its role in adult Leydig cells is well established, its localization and importance during the fetal period remain less known, especially in humans. This study aims to delineate the dynamics of HSD17B3 expression in human fetal testes to clarify the contribution of specific cell types to testosterone biosynthesis.Methods: Using immunofluorescence staining, we investigated the expression pattern of HSD17B3 in human fetal and adult testicular tissues.Results and discussion: The findings of this study revealed a distinct temporal and cellular expression pattern of HSD17B3 protein in the fetal period. We detected its expression exclusively in Sertoli cells, the highest during the second trimester. This unique localization suggests the inclusion of fetal Sertoli cells in testosterone production during the critical masculinization-programming window. Furthermore, we demonstrated a shift in HSD17B3 expression from Sertoli cells to Leydig cells in adulthood, corroborating findings from rodent studies. This study sheds light on the intricate, still underexplored regulation of steroidogenesis during fetal development, whose disturbance might lead to testicular dysgenesis. Further research is warranted to elucidate the regulatory pathways governing the expression of HSD17B3 and its transition between Sertoli and Leydig cells, potentially paving the way for novel therapeutic interventions in disorders of sexual development.

Quantum Cascade Laser Infrared Spectroscopy for Glycan Analysis of Glycoprotein Solutions.

Glycans are oligosaccharides attached to proteins or lipids and affect their functions, such as drug efficacy, structural contribution, metabolism, immunogenicity, and molecular recognition. Conventional glycosylation analysis has relied on destructive, slow, system-sensitive methods, including enzymatic reactions, chromatography, fluorescence labeling, and mass spectrometry. Herein, we propose quantum cascade laser (QCL) infrared (IR) spectroscopy as a rapid, nondestructive method to quantify glycans and their monosaccharide composition. Previously, we demonstrated high-sensitivity IR spectroscopy of protein solution using solvent absorption compensation (SAC) and double-beam modulation (DBM) techniques. However, the SAC-DBM approach suffered a limited frequency scanning range (<400 cm-1) due to the light dispersion by acousto-optic modulators (AOMs). Here, we implemented a mirror-based double-pass AOM in the SAC-DBM scheme and successfully extended the frequency range to (970 to 1840 cm-1), which encompasses the vibrational fingerprint of biomolecules. The extended frequency range allowed the simultaneous observation of monosaccharide ring bands (1000 to 1200 cm-1) and protein amide bands (1500 to 1700 cm-1). We compared the IR spectra of six glycoproteins and two nonglycosylated proteins with the results from intact mass spectrometry. The IR absorbance ratios of the ring band to the amide band of glycoproteins in solutions showed a linear correlation with the ratios of glycan to protein backbone masses. Furthermore, a multivariate analysis produced monosaccharide compositions consistent with the reported database for the glycoproteins, and the monosaccharide compositions were used to improve the predictability of the glycan-protein mass ratio from the IR-absorbance ratio. This nondestructive, high-sensitivity QCL-IR spectroscopy could be used as a standard method to monitor batch-to-batch comparability during drug manufacturing and quantify the glycosylation and monosaccharide composition of new glycoproteins and other glycosylated biosystems.

Rapid determination of the antimicrobial properties of surfaces using an enzymatic activity surrogate

AbstractTypical approaches for assessing the antimicrobial activity of metals‐based surfaces involve the contact of a bacterial culture with the surface for a period of time, followed by culturing on agar plates to assess the decrease in microbial viability versus controls. This is a time‐consuming methodology requiring at least 24 h to produce a set of results, which can be a bottleneck for productivity in novel materials development. An enzyme‐based method was shown to be a satisfactory and much more rapid surrogate test for this application. A β‐galactosidase solution was applied to copper, silver, and zinc‐based antimicrobial surfaces for up to 1 h, and then the rate of colour development at 578 nm was monitored for a few minutes after addition of the chromogenic enzyme substrate chlorophenol red‐β‐d‐galactopyranoside (CPRG). Highly active antimicrobial surfaces were detected by a lack of colour development, due to enzyme inhibition by the metals. The enzymatic reaction rates were quantified and compared, demonstrating that the copper sample showed the greatest inhibition effect followed by the silver and zinc samples. The antimicrobial activity was quantified using bacteria and the plate count method, and the results correlated well with this enzyme assay, demonstrating that the metals‐based antimicrobial activities of both hard and soft (textile) surfaces could be quickly assessed with this enzyme‐based methodology.

Enzymatically Mediated In Situ Generation of Z-Scheme Bi2S3/Bi2MoO6 Heterojunction-Based Organic Photoelectrochemical Transistor for METTL3/METTL14 Detection.

The OPECT biosensing platform, which connects optoelectronics and biological systems, offers significant amplification and more possibilities for research in biological applications. In this work, a homogeneous organic photoelectrochemical transistor (OPECT) biosensor based on a Bi2S3/Bi2MoO6 heterojunction was constructed to detect METTL3/METTL14 protein activity. The METTL3/METTL14 complex enzyme was used to catalyze adenine (A) on an RNA strand to m6A, protecting m6A-RNA from being cleaved by an E. coli toxin (MazF). Alkaline phosphatase (ALP) catalyzed the conversion of Na3SPO3 to H2S through an enzymatic reaction. Due to the adoption of the strategy of no fixation on the electrode, the generated H2S was easy to diffuse to the surface of the ITO electrode. The Bi2S3/Bi2MoO6 heterojunction was formed in situ through a chemical replacement reaction with Bi2MoO6, improving photoelectric conversion efficiency and realizing signal amplification. Based on this "signal on" mode, METTL3/METTL14 exhibited a wide linear range (0.00001-25 ng/μL) between protein concentration and photocurrent intensity with a limit of detection (LOD) of 7.8 fg/μL under optimal experimental conditions. The applicability of the developed method was evaluated by investigating the effect of four plasticizers on the activity of the METTL3/METTL14 protein, and the molecular modeling technique was employed to investigate the interaction between plasticizers and the protein.

Insight into the role of droplets in quinoa starch-based Pickering high internal phase emulsion for enzymatic hydrolysis reaction

Pickering high internal phase emulsion has emerged as potential platforms for biocatalytic reactions. Herein, native quinoa starch was used as the emulsifier to form a stable Pickering HIPE with an internal phase up to 80 %. The catalytic performance of lipase loaded in HIPE was evaluated for the enzymatic hydrolysis of hexyl hexanoate. Compared with the typical Pickering emulsion, biphasic, and monophasic systems, the Pickering HIPE system displayed higher conversion (83.54 % at 1440 min) and specific enzyme activity (1.41 U/mg), thus emphasizing the crucial role of microdroplets and large interface area created by both the starch particles and high internal phase volume. Subsequently, HIPEs with different starch concentrations were fabricated to further understand the catalysis behavior happening at compartmentalized microdroplets and interfaces. The microstructural, interfacial, and rheological properties of the HIPEs were systematically determined to explain the correlation between emulsion properties and catalytic efficiency. High starch concentration reduced the droplet size and increased the interfacial area, thus shortening the mass transfer distance and enlarging the reaction area. Meanwhile, the increased surface coverage and viscosity led to poor accessibility and limited mass movement. This trade-off made the maximum conversion be obtained at 2 wt% concentration. The study provides novel information about the role of emulsion droplets in the catalytic performance of lipase in starch-based HIPE.

Modeling and simulation of bi‐continuous jammed emulsion membrane reactors for enhanced biphasic enzymatic reactions

Modeling and simulation of bi‐continuous jammed emulsion membrane reactors for enhanced biphasic enzymatic reactions

Boosting of enzymatic cascades by intermediates: Theoretical analysis and model-based optimization

A recent paper demonstrated that adding intermediates of an enzymatic cascade reaction at the start of the reaction process can increase the space-time yield significantly. This method was coined boosting by intermediates (BBI) and can potentially increase the productivity of enzymatic cascade reaction systems. BBI remains largely unexplored, and its mechanism, potential, and limitations are not fully understood. In this short communication, the theoretical background of the method is revealed by studying four chemical reaction networks that exhibit boosting through intermediates. The dynamics of these networks are studied via the quasi-steady state approximation and numerical solutions. A novel graphical method that can be used to deduce which intermediates boost linear, redox-neutral enzymatic cascade reaction networks is presented. Further, how such a reaction process can be designed using model-based optimization is demonstrated.

A Near-Infrared-II Excitable Pyridinium Probe with 1000-Fold ON/OFF Ratio for γ-Glutamyltranspeptidase and Cancer Detection.

Activity-based detection of γ-Glutamyltranspeptidase (GGT) using near-infrared (NIR) fluorescent probes is a promising strategy for early cancer diagnosis. Although NIR pyridinium probes show high performance in biochemical analysis, the aggregation of both the probes and parental fluorochromes in biological environments is prone to result in a low signal-to-noise ratio (SBR), thus affecting their clinical applications. Here, we develop a GGT-activatable aggregate probe called OTBP-G for two-photon fluorescence imaging in various biological environments under 1040 nm excitation. By rationally tunning the hydrophilicity and donor-acceptor strength, we enable a synergistic effect between twisted intramolecular charge transfer and intersystem crossing processes and realize a perfect dark state for OTBP-G before activation. After the enzymatic reaction, the parental fluorochrome exhibits bright aggregation-induced emission peaking at 670 nm. The fluorochrome-to-probe transformation can induce 1000-fold fluorescence ON/OFF ratio, realizing in vitro GGT detection with an SBR > 900. Activation of OTBP-G occurs within 1 min in vivo, showing an SBR > 400 in mouse ear blood vessels. OTBP-G can further enable the early detection of pulmonary metastasis in breast cancer by topically spraying, outperforming the clinical standard hematoxylin and eosin staining. We anticipate that the in-depth study of OTBP-G can prompt the development of early cancer diagnosis and tumor-related physiological research. Moreover, this work highlights the crucial role of hydrophilicity and donor-acceptor strength in maximizing the ON/OFF ratio of the TICT probes and showcases the potential of OTBP as a versatile platform for activity-based sensing.

Reported Awareness and Use of Pyridoxal-5'-Phosphate Supplementation in Alanine Aminotransferase and Aspartate Aminotransferase Assay Reagents: A Survey by the College of American Pathologists Clinical Chemistry Committee.

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assay reagents are available both with and without supplementation with pyridoxal-5'-phosphate (P5P; the active form of vitamin B6), a catalytic cofactor required for their enzymatic reactions. Nonsupplemented assays may miss ALT or AST elevations in patients with vitamin B6 deficiency. To assess awareness and adoption of ALT and AST reagents that are supplemented with P5P. A 4-question survey about ALT and AST reagent supplementation with P5P was included in the College of American Pathologists General Chemistry and Therapeutic Drugs (C program) proficiency testing 2023 B mailing. Overall, 38% (1651 of 4304) of responding laboratories reported using ALT and/or AST reagent supplemented with P5P. P5P supplementation was more common for nonacademic hospital/medical center laboratories (44%; 713 of 1629) relative to other settings. Of the laboratories that reported not using P5P-supplemented reagents, few (5%; 141 of 2611) cited plans to convert in the future. Despite the availability of P5P-supplemented reagents from several major assay manufacturers, the most common stated barrier for adoption was that the laboratory's reagent manufacturer does not provide P5P-supplemented reagents. There is a lack of awareness of the existence and benefits of P5P-supplemented ALT and AST reagents. There is a need for ALT and AST assay manufacturers to clarify and standardize the P5P status of ALT and AST reagents.