Microwell fluoride assay screening for enzymatic defluorination.

There is intense interest in removing fluorinated compounds from the environment, environments are most efficiently remediated by microbial enzymes, and defluorinating enzymes are readily monitored by fluoride determination. Fluorine is the most electronegative element. Consequently, all mechanisms of enzymatic C-F bond cleavage produce fluoride anion, F-. Therefore, methods for the determination of fluoride are critical for C-F enzymology and apply to any fluorinated organic compounds, including PFAS, or per- and polyfluorinated alkyl substances. The biodegradation of most PFAS chemicals is rare or unknown. Accordingly, identifying new enzymes, or re-engineering the known defluorinases, will require rapid and sensitive methods for measuring fluoride in aqueous media. Most studies currently use ion chromatography or fluoride specific electrodes which are relatively sensitive but low throughput. The methods here describe refashioning a drinking water test to efficiently determine fluoride in enzyme and cell culture reaction mixtures. The method is based on lanthanum alizarin complexone binding of fluoride. Reworking the method to a microtiter well plate format allows detection of as little as 4 nmol of fluoride in 200 µL of assay buffer. The method is amenable to color imaging, spectrophotometric plate reading and automated liquid handling to expedite assays with thousands of enzymes and/or substrates for discovering and improving enzymatic defluorination.

Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Molecular electronics sensors on a scalable semiconductor chip: A platform for single-molecule measurement of binding kinetics and enzyme activity.

For nearly 50 years, the vision of using single molecules in circuits has been seen as providing the ultimate miniaturization of electronic chips. An advanced example of such a molecular electronics chip is presented here, with the important distinction that the molecular circuit elements play the role of general-purpose single-molecule sensors. The device consists of a semiconductor chip with a scalable array architecture. Each array element contains a synthetic molecular wire assembled to span nanoelectrodes in a current monitoring circuit. A central conjugation site is used to attach a single probe molecule that defines the target of the sensor. The chip digitizes the resulting picoamp-scale current-versus-time readout from each sensor element of the array at a rate of 1,000 frames per second. This provides detailed electrical signatures of the single-molecule interactions between the probe and targets present in a solution-phase test sample. This platform is used to measure the interaction kinetics of single molecules, without the use of labels, in a massively parallel fashion. To demonstrate broad applicability, examples are shown for probe molecule binding, including DNA oligos, aptamers, antibodies, and antigens, and the activity of enzymes relevant to diagnostics and sequencing, including a CRISPR/Cas enzyme binding a target DNA, and a DNA polymerase enzyme incorporating nucleotides as it copies a DNA template. All of these applications are accomplished with high sensitivity and resolution, on a manufacturable, scalable, all-electronic semiconductor chip device, thereby bringing the power of modern chips to these diverse areas of biosensing.

The challenge of determining lysyl oxidase activity: Old methods and novel approaches.

The lysyl oxidase (LOX) family of enzymes catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-linkages, an essential process for extracellular matrix (ECM) maturation. Elevated LOX expression levels leading to increased LOX activity is associated with diverse pathologies including fibrosis, cancer, and cardiovascular diseases. Different protocols have been so far established to detect and quantify LOX activity from tissue samples and cultured cells, all of them showing advantages and drawbacks. This review article presents a critical overview of the main features of currently available methods as well as introduces some recent technologies called to revolutionize our approach to LOX catalysis.

Acquisition of enzymatic progress curves in real time by quenching-free ion exchange chromatography.

We describe a quenching-free, 'online' ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SUBJECT: Enzymatic assays and analysis.

AgCu@CuO aerogels with peroxidase-like activities and photoelectric responses for sensitive biosensing.

Photoelectrochemical (PEC) enzymatic biosensors integrate the excellent selectivity of enzymes and high sensitivity of PEC bioanalysis, but the drawbacks such as high cost, poor stability, and tedious immobilization of natural enzymes on photoelectrodes severely suppress their applications. AgCu@CuO aerogel-based photoelectrode materials with both remarkable enzyme-like activities and outstanding photoelectric properties were innovatively designed and synthesized to evaluate the activity of xanthine oxidase with a wide linear detection range and a low limit of detection.

The Use of Acoustic Mist Ionization Mass Spectrometry for High-Throughput Screening.

It is clear from the analysis of the distribution of approved drug targets that enzymes continue to be a major target class for the pharmaceutical industry. The application of high-throughput screens designed to monitor the activity of these enzyme targets, and the ability of test compounds to modulate this activity, is still the predominant hit finding approach in the industry. The widespread use of enzyme activity-based screens has led to the development of several useful guidelines for the development and validation of robust and reliable assays. Key learnings for the development, validation, and implementation of acoustic mist ionization mass spectrometry for high-throughput enzyme assays are described.

A portable personal glucose meter method for enzyme activity detection and inhibitory activity evaluation based on alkaline phosphatase-mediated reaction.

In this study, an effective and portable method for enzyme activity detection and inhibitory activity evaluation was developed based on the alkaline phosphatase (ALP)-mediated reaction in a personal glucose meter (PGM). In this method, ALP catalyzes the hydrolysis of substrate amifostine (WR-2721) to produce ethanethiol (WR-1065), which can trigger the reduction of ferricyanide (K3[Fe(CN)6]), an electron transfer mediator in glucose test strips, to ferrocyanide ([K4Fe(CN)6]) and generate a PGM-detectable signal. Thus, WR-1065 can be directly quantified by a PGM as simply as detecting glucose in blood. After being systematically optimized, the method was applied to evaluate the inhibitory activity of ten small-molecule compounds and six Cordyceps sinensis (CS) extracts on ALP. The results showed that adenosine-5-monophosphate and theophylline had high inhibitory activity, but two CS extracts have promotion potency on ALP with the values of -20.7 ± 1.3% and -46.6 ± 2.1%, respectively. Moreover, the binding sites and modes of small-molecule compounds to ALP were investigated by molecular docking, while a new substrate competitor with theoretically good inhibitory activity against ALP was designed by scaffold hopping. Finally, the accuracy of the PGM method for enzyme activity detection was assessed by detecting ALP from milk samples, and the recovery ranged from 87.7% to 116.9%. These results indicate that it is feasible to evaluate enzyme activity and the inhibitory activity of small-molecule compounds and CS extracts on ALP using a PGM based on ALP-mediated reaction. Graphical abstract.

Synthesis and evaluation of sensitive coumarin-based fluorogenic substrates for discovery of α-N-acetyl galactosaminidases through droplet-based screening.

As part of a search for a substrate for droplet-based microfluidic screening assay of α-N-acetylgalactosaminidases, spectral and physical characteristics of a series of coumarin derivatives were measured. From among these a new coumarin-based fluorophore, Jericho Blue, was selected as having optimal characteristics for our screen. A reliable method for the challenging synthesis of coumarin glycosides of α-GalNAc was then developed and demonstrated with nine examples. The α-GalNAc glycoside of Jericho Blue prepared in this way was shown to function well under screening conditions.

Comparison of a new enzymatic assay for serum homocysteine on Toshiba TBA-c16000 against an immunoassay on Abbott Architect.

Homocysteine, a risk factor for cardiovascular disease, is commonly analyzed using enzymatic measurements and immunoassays. We compared the results of a new enzymatic assay with those of an immunoassay, using new reagents for homocysteine. The 87 serum samples were analyzed using the Abbott Architect i2000sr (immunoassay) and Toshiba TBA-c16000 (enzymatic assay), and the results obtained from the two assays were compared for precision, correlation, linearity, sample carryover, and reference range verification according to the Clinical and Laboratory Standards Institute guidelines. Repeatability and total imprecision were within the desirable range (Westgard QC, 4.15%). Correlation analysis revealed a strong correlation with a slope ranging from 0.9887 to 1.052, a correlation coefficient (R 2) of 0.9886 [95% confidence interval (CI) of 0.9899-0.9968], and a y-intercept from -0.5741 to 0.6252. Linearity was acceptable (R 2 = 0.9993), and the recovery rate was within ±10% of the expected value. The enzymatic assay showed an acceptable carryover rate (-0.15%) and a shorter turnaround time (10-12 min) compared with that of the immunoassay (30 min). Our new enzymatic assay for the measurement of homocysteine showed an acceptable performance in terms of precision, correlation, linearity, carryover test, cost-effectiveness, and speed.

Digital Microfluidics-Enabled Analysis of Individual Variation in Liver Cytochrome P450 Activity.

The superfamily of hepatic cytochrome P450 (CYP) enzymes is responsible for the intrinsic clearance of the majority of therapeutic drugs in humans. However, the kinetics of drug clearance via CYPs varies significantly among individuals due to both genetic and external factors, and the enzyme amount and function are also largely impacted by many liver diseases. In this study, we developed a new methodology, based on digital microfluidics (DMF), for rapid determination of individual alterations in CYP activity from human-derived liver samples in biopsy-scale. The assay employs human liver microsomes (HLMs), immobilized on magnetic beads to facilitate determination of the activity of microsomal CYP enzymes in a parallelized system at physiological temperature. The thermal control is achieved with the help of a custom-designed, inkjet-printed microheater array modularly integrated with the DMF platform. The CYP activities are determined with the help of prefluorescent, enzyme-selective model compounds by quantifying the respective fluorescent metabolites based on optical readout in situ. The selectivity and sensitivity of the assay was established for four different CYP model reactions, and the diagnostic concept was validated by determining the interindividual variation in one of the four model reaction activities, that is, ethoxyresorufin O-deethylation (CYP1A1/2), between five donors. Overall, the developed protocol consumes only about 15 µg microsomal protein per assay. It is thus technically adaptable to screening of individual differences in CYP enzyme function from biopsy-scale liver samples in an automated fashion, so as to support tailoring of medical therapies, for example, in the context of liver disease diagnosis.

Novel "turn on-off" paper sensor based on nonionic conjugated polythiophene-coated CdTe QDs for efficient visual detection of cholinesterase activity.

An increasing number of patients are living with Alzheimer's disease (AD); thus, the need for a method to detect AD early and sensitively has become urgent, and the demand for an intelligent analytical platform is growing year by year. Abnormal levels of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to be indicative of AD. In this work, a novel conjugated polythiophene (CP) compound was successfully combined with CdTe quantum dots (QDs) to improve their selectivity and sensitivity. The QDs successfully enabled the detection of low concentrations of AChE by turning on the fluorescence of the CdTe/CP via the interaction between CP and thiocholine produced by ATCh hydrolysis and aggregation induced emission enhancement (AIEE). Under optimal conditions, we reached a low detection limit of 0.14 U L-1, which is 7.9 times lower than that of pristine QDs. More importantly, an efficient, inexpensive, and disposable paper-based platform, which allows the efficient visual detection of AChE activity via the color variation of CdTe/CP, was designed. Moreover, the accuracy of the method was demonstrated by conducting a recovery test in human serum, in which the recoveries reached 107% and 110%, proving that CdTe/CP has considerable potential to be used for analyzing real biological samples. The advantages of this method are its simplicity, fast detection capability, affordability, and the fact that it can be used for on-site detection of AChE activity. Furthermore, it has certain guiding significance for detecting AD.

Temporal Sampling of Enzymes from Live Cells by Localized Electroporation and Quantification of Activity by SAMDI Mass Spectrometry.

Measuring changes in enzymatic activity over time from small numbers of cells remains a significant technical challenge. In this work, a method for sampling the cytoplasm of cells is introduced to extract enzymes and measure their activity at multiple time points. A microfluidic device, termed the live cell analysis device (LCAD), is designed, where cells are cultured in microwell arrays fabricated on polymer membranes containing nanochannels. Localized electroporation of the cells opens transient pores in the cell membrane at the interface with the nanochannels, enabling extraction of enzymes into nanoliter-volume chambers. In the extraction chambers, the enzymes modify immobilized substrates, and their activity is quantified by self-assembled monolayers for matrix-assisted laser desorption/ionization (SAMDI) mass spectrometry. By employing the LCAD-SAMDI platform, protein delivery into cells is demonstrated. Next, it is shown that enzymes can be extracted, and their activity measured without a loss in viability. Lastly, cells are sampled at multiple time points to study changes in phosphatase activity in response to oxidation by hydrogen peroxide. With this unique sampling device and label-free assay format, the LCAD with SAMDI enables a powerful new method for monitoring the dynamics of cellular activity from small populations of cells.

Clinical and Analytical Impact of Moving from Jaffe to Enzymatic Serum Creatinine Methodology.

BACKGROUND: Identification and monitoring of chronic kidney disease (CKD) requires accurate quantification of serum creatinine. The poor specificity of Jaffe creatinine methods is well documented, and guidelines recommend enzymatic methodology. We describe our experience of moving from Jaffe to enzymatic creatinine methodology. We present comparison of >5000 paired Jaffe and enzymatic creatinine results, examine interferences, and attempt to assess clinical consequences of changing methodology. METHODS: Overall, 5303 serum samples received for routine creatinine measurement were analyzed using Jaffe and enzymatic methods with an Abbott Architect autoanalyzer. Associated results for glucose, total bilirubin, triglycerides, total protein, and hemolytic, icteric, and lipemic indexes were extracted from the laboratory database. CKD staging was estimated for each sample to assess potential clinical effects. RESULTS: The methods correlated well (r = 0.996) and showed good agreement (Passing-Bablok fit, y = 0.935x + 0.074). Paired analysis, however, showed significant differences (P < 0.001), and approximately 20% of results differed by more than ±10%. Multivariate analysis demonstrated independent associations between difference in creatinine results, glucose (P < 0.0001), and hemolytic index (P = 0.009). Glucose demonstrated positive interference in the Jaffe method, and hemolysis produced negative interference in the enzymatic method. Little or no association was observed with other analytes. CKD staging differed in 4% of samples. CONCLUSIONS: Differences between Jaffe and enzymatic serum creatinine results exceed the recommended 5% target for a significant proportion of samples, particularly at concentrations <1.13 mg/dL (100 µmol/L). Both glucose and hemolysis contribute to the variance in results. Although the clinical impact of these differences seems small, laboratories should continue moving toward enzymatic creatinine estimation to ensure the best estimate of renal function.

Three-dimensional donor-acceptor-type photoactive material/conducting polyaniline hydrogel complex for sensitive photocathodic enzymatic bioanalysis.

Herein, an innovative photocathodic enzymatic biosensor is proposed with poly {4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]-benzo[1,2-b:4,5-b']dithiophene-2,6-diyl-alt-3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophene-4,6-diyl} (PTB7-Th) as donor-acceptor-type photoactive material and three-dimensional (3D) polyaniline hydrogels (PAniHs) as both electron transfer layer and biomolecule carrier. Based on the enhancement effect of PAniHs on the charge separation and electron transfer of PTB7-Th and the competitive consumption of dissolved oxygen (O2) between the xanthine oxidase (XOD)-guanine catalytic reaction and O2-sensitive PTB7-Th/PAniHs, the proposed photocathodic enzymatic biosensor has been demonstrated to detect guanine with the advantages of low limit of detection (0.02 µM), wide linear range (from 0.1 to 80 µM), simple and convenient preparation process, satisfactory stability, and photochemical signal amplification independent of any exogenous electron donor/acceptor or sensitizer. Remarkably, the proposed photocathodic enzymatic biosensor can not only be extended to other aerobic enzymatic bioanalyses, but also pave a horizon for the application of environmentally friendly conductive hydrogel materials in photoelectrochemical bioanalysis.

Mass spectrometric biosensing: Quantitation of multiplex enzymes using single mass probe and fluorous affinity chip.

This work presents a concept of "mass spectrometric biosensing" by using a chip to recognize the targets and mass spectrometry to detect the signals switched by the recognition. The chip is prepared on an ITO slide with a hydrophobic fluorous-tag monolayer to self-assemble the mixture of mass probe and fluorous-tagged cysteine as a spacer through fluorous affinity interaction. The presence of spacer provides suitable conditions for recognition reactions on the chip. By designing a single mass probe as the peptide substrates of corresponding target enzymes, a novel quantitative strategy based on the ratio of signal intensities of different species on the chip is developed for MALDI-MS assay of multiplex enzyme activities. Using caspase-3 and protein kinase A as targets, the reactions with designed mass probe produce three mass shifts to act as two "fingerprint" patterns for obtaining the dual enzyme activities. The proposed biosensing method shows the detectable ranges from 0.05 to 50 µU µL-1 and 0.4-40 µU µL-1 with correlation coefficients of 0.990 and 0.989 for PKA and caspase-3, respectively. The biosensing application has been demonstrated by monitoring these enzymes in cell lysates upon anti-cancer drug treatment, indicating the prospect of the novel biosensing protocol in biomedical study.

Biochemical characterization of RNA-guided ribonuclease activities for CRISPR-Cas9 systems.

The majority of bacteria and archaea rely on CRISPR-Cas systems for RNA-guided, adaptive immunity against mobile genetic elements. The Cas9 family of type II CRISPR-associated DNA endonucleases generates programmable double strand breaks in the CRISPR-complementary DNA targets flanked by the PAM motif. Nowadays, CRISPR-Cas9 provides a set of powerful tools for precise genome manipulation in eukaryotes and prokaryotes. Recently, a few Cas9 orthologs have been reported to possess intrinsic CRISPR-guided, sequence-specific ribonuclease activities. These discoveries fundamentally expanded the targeting capability of CRISPR-Cas9 systems, and promise to provide new CRISPR tools to manipulate specific cellular RNA transcripts. Here we present a detailed method for the biochemical characterization of Cas9's RNA-targeting potential.

An enhanced assay to characterize anti-CRISPR proteins using a cell-free transcription-translation system.

The characterization of CRISPR-Cas immune systems in bacteria was quickly followed by the discovery of anti-CRISPR proteins (Acrs) in bacteriophages. These proteins block different steps of CRISPR-based immunity and, as some inhibit Cas nucleases, can offer tight control over CRISPR technologies. While Acrs have been identified against a few CRISPR-Cas systems, likely many more await discovery and application. Here, we report a rapid and scalable method for characterizing putative Acrs against Cas nucleases using an E. coli-derived cell-free transcription-translation system. Using known Acrs against type II Cas9 nucleases as models, we demonstrate how the method can be used to measure the inhibitory activity of individual Acrs in under two days. We also show how the method can overcome non-specific inhibition of gene expression observed for some Acrs. In total, the method should accelerate the interrogation and application of Acrs as CRISPR-Cas inhibitors.

Measurement of Nitrate Reductase Activity in Tomato (Solanum lycopersicum L.) Leaves Under Different Conditions.

Nitrogen is one of the crucial macronutrients essential for plant growth, development, and survival under stress conditions. Depending on cellular requirement, plants can absorb nitrogen mainly in multiple forms such as nitrate (NO3-) or ammonium (NH4+) or combination of both via efficient and highly regulated transport systems in roots. In addition, nitrogen-fixing symbiotic bacteria can fix atmospheric nitrogen in to NH4+ via highly regulated complex enzyme system and supply to the roots in nodules of several species of leguminous plants. If NO3- is a primary source, it is transported from roots and then it is rapidly converted to nitrite (NO2-) by nitrate reductase (NR) (EC 1.6.6.1) which is a critical and very important enzyme for this conversion. This key reaction is mediated by transfer of two electrons from NAD(P)H to NO3-. This occurs via the three redox centers comprised of two prosthetic groups (FAD and heme) and a MoCo cofactor. NR activity is greatly influenced by factors such as developmental stage and various stress conditions such as hypoxia, salinity and pathogen infection etc. In addition, light/dark dynamics plays crucial role in modulating NR activity. NR activity can be easily detected by measuring the conversion of NO3- to NO2- under optimized conditions. Here, we describe a detailed protocol for measuring relative NR enzyme activity of tomato crude extracts. This protocol offers an efficient and straightforward procedure to compare the NR activity of various plants under different conditions.

Droplet-based optofluidic systems for measuring enzyme kinetics.

The study of enzyme kinetics is of high significance in understanding metabolic networks in living cells and using enzymes in industrial applications. To gain insight into the catalytic mechanisms of enzymes, it is necessary to screen an enormous number of reaction conditions, a process that is typically laborious, time-consuming, and costly when using conventional measurement techniques. In recent times, droplet-based microfluidic systems have proved themselves to be of great utility in large-scale biological experimentation, since they consume a minimal sample, operate at high analytical throughput, are characterized by efficient mass and heat transfer, and offer high levels of integration and automation. The primary goal of this review is the introduction of novel microfluidic tools and detection methods for use in high-throughput and sensitive analysis of enzyme kinetics. The first part of this review focuses on introducing basic concepts of enzyme kinetics and describing most common microfluidic approaches, with a particular focus on segmented flow. Herein, the key advantages include accurate control over the flow behavior, efficient mass and heat transfer, multiplexing, and high-level integration with detection modalities. The second part describes the current state-of-the-art platforms for high-throughput and sensitive analysis of enzyme kinetics. In addition to our categorization of recent advances in measuring enzyme kinetics, we have endeavored to critically assess the limitations of each of these detection approaches and propose strategies to improve measurements in droplet-based microfluidics. Graphical abstract.