Steady-state kinetic analysis of reversible enzyme inhibitors: A case study on calf intestine alkaline phosphatase.

Enzymes are important drug targets and inhibition of enzymatic activity is an important therapeutic strategy. Enzyme assays measuring catalytic activity are utilized in both the discovery and development of new drugs. Colorimetric assays based on the release of 4-nitrophenol from substrates are commonly used. 4-Nitrophenol is only partly ionized to 4-nitrophenolate under typical assay conditions (pH 7-9) leading to under-estimation of product formation rates due to the much lower extinction coefficient of 4-nitrophenol compared to 4-nitrophenolate. Determination of 4-nitrophenol pKa values based on absorbance at 405 nm as a function of experimental pH values is reported, allowing for calculation of a corrected extinction coefficient at the assay pH. Characterization of inhibitor properties using steady-state enzyme kinetics is demonstrated using calf intestine alkaline phosphatase and 4-nitrophenyl phosphate as substrate at pH ∼8.2. The following kinetic parameters were determined: Km= 40±3 µM; Vmax= 72.8±1.2 µmolmin-1mg protein-1; kcat= 9.70±0.16 s-1; kcat/Km= 2.44±0.16 × 105 M-1s-1 (mean± SEM, N = 4). Sodium orthovanadate and EDTA were used as model inhibitors and the following pIC50 values were measured using dose-response curves: 6.61±0.08 and 3.07±0.03 (mean±SEM, N = 4). Rapid dilution experiments determined that inhibition was reversible for sodium orthovanadate and irreversible for EDTA. A Ki value for orthovanadate of 51±8 nM (mean±SEM, N = 3) was determined. Finally, data analysis and statistical design of experiments are discussed.

Tetrazolium-based colorimetric assays underestimat the direct antitumor effects of anti-VEGF agent bevacizumab.

The direct antitumor effect of bevacizumab (BEV) has long been debated. Assessment of the direct cytotoxic activities of drugs is usually conducted via in vitro experiments, of which tetrazolium-based colorimetric assays are widely employed to measure the direct antitumor activity of BEV. This study aimed to investigate whether tetrazolium-based colorimetric assays are applicable when evaluating the cytotoxicity of BEV against tumor cells. Our results showed that BEV significantly augmented tumor-cell mitochondrial metabolism. Enhanced mitochondrial metabolism caused changes in cellular oxidation-and-reduction environment and upregulated succinate dehydrogenase, which in turn promoted the reduction of tetrazolium to produce formazan. Increased formazan formation resulted in underestimation of the in vitro direct antitumor effect of BEV. Furthermore, inhibition of mitochondrial hypermetabolism partially corrected the underestimation of colorimetric assays in evaluating the direct antitumor activity of BEV. Our findings suggest that tetrazolium-based colorimetric assays are unsuitable for accurately assessing the in vitro cytotoxicity of anti-VEGF drugs and may be the methodological reason for the controversial direct antitumor effect of BEV.

Colorimetric Determination of Adenylation Domain Activity in Nonribosomal Peptide Synthetases by Using Chrome Azurol S.

Adenylation domains are the main contributor to structural complexity among nonribosomal peptides due to their varied but stringent substrate selection. Several in vitro assays to determine the substrate specificity of these dedicated biocatalysts have been implemented, but high sensitivity is often accompanied by the cost of laborious procedures, expensive reagents or the requirement for auxiliary enzymes. Here, we describe a simple protocol that is based on the removal of ferric iron from a preformed chromogenic complex between ferric iron and Chrome Azurol S. Adenylation activity can be rapidly followed by a decrease in absorbance at 630 nm, visualized by a prominent color change from blue to orange.

Addressing artifacts of colorimetric anticancer assays for plant-based drug development.

Cancer has become the silent killer in less-developed countries and the most significant cause of morbidity worldwide. The accessible and frequently used treatments include surgery, radiotherapy, chemotherapy, and immunotherapy. Chemotherapeutic drugs traditionally involve using plant-based medications either in the form of isolated compounds or as scaffolds for synthetic drugs. To launch a drug in the market, it has to pass through several intricate steps. The multidrug resistance in cancers calls for novel drug discovery and development. Every year anticancer potential of several plant-based compounds and extracts is reported but only a few advances to clinical trials. The false-positive or negative results impact the progress of the cell-based anticancer assays. There are several cell-based assays but the widely used include MTT, MTS, and XTT. In this article, we have discussed various pitfalls and workable solutions.

Comparison of the Antioxidant Properties of Green Macroalgae from Diverse European Water Habitats by Use of Several Semi-Quantitative Assays.

Nowadays, algae are becoming more and more popular as a food group rich in nutrients, cosmetic raw materials full of antioxidants or valuable dietary supplements. They are of interest for the industry because they are found almost all over the world, in all climatic zones, both in fresh and salt waters. The aim of this study was to take a broad look at green algae (Chlorophyta) and to show how large the variability of the content of active compounds may depend on the species and the place and time of sample collection. Particular attention was paid to compounds with antioxidant activity, whose simplified profiles were created on the basis of complementary, semi-quantitative methods. Additionally, time-yield extraction optimizations were performed. Three different specimens of Ulva lactuca were compared: from the coastal zone of the Baltic Sea, from the open Baltic Sea area around Bornholm and Ulva spiralis (Ulva lactuca polymorph) from the Atlantic Ocean. The studied algae of the Cladophora genera were three different species of freshwater algae from various habitats: a lake (Cladophora glomerata), a river (Cladophora rivularis) and aquarium farming (Cladophora aegagropila, syn. Aegagropila linnaei). The content of antioxidants and the extraction efficiency varied significantly depending on the species.

Nitrogen-Enriched Conjugated Polymer Enabled Metal-Free Carbon Nanozymes with Efficient Oxidase-Like Activity.

Metal-free carbon nanozymes could be promising with the unique features of intrinsic catalytic ability, structure diversity, and strong tolerance to acidic/alkaline media. However, to date, the study of metal-free carbon nanozymes fell far behind metal-based nanomaterials, in which, the majority reported much more peroxidase-like activity than other enzyme-mimicking behavior (e.g., oxidase). Thus, the exploit of high-performance carbon nanozymes is of importance but challenging. In this work, the nitrogen-rich conjugated polymer (Aza-CPs) with rigid network structure is utilized as precursor to yield N-doped carbon material QAU-Z1 in high yield via a direct pyrolysis method. Surprisingly, QAU-Z1 stood out in oxidase-like behavior, which significantly outperformed the control materials GNC-900 and QAU-Z2 with nucleobase or conjugated small molecule as precursor, respectively. More importantly, it is a crucial revelation that the catalytic performance is closely related to the change of zeta potential for carbon nanozyme during the substrate 3,3',5,5'-tetramethylbenzidine oxidation process, as well as its catalytical capacity to O2 , which could be insightful to understand the inherent mechanism. This work not only presents the potential of conjugated polymers in constructing highly efficient carbon nanozyme, but also reveals the vital role of interaction mode between the nanozyme and substrate in the catalytic performance.

Detection of Lysyl Oxidase Activity in Tumor Extracellular Matrix Using Peptide-Functionalized Gold Nanoprobes.

High LOX levels in the tumor microenvironment causes the cross-linking of extracellular matrix components and increases the stiffness of tumor tissue. Thus, LOX plays an important role in tumorigenesis and in lowering the tumor response to anticancer drugs. Despite comprehensive efforts to identify the roles of LOX in the tumor microenvironment, sensitive and accurate detection methods have not yet been established. Here, we suggest the use of gold nanoparticles functionalized with LOX-sensitive peptides (LS-AuNPs) that aggregate upon exposure to LOX, resulting in a visual color change. LOX-sensitive peptides (LS-peptides) contain lysine residues that are converted to allysine in the presence of LOX, which is highly reactive and binds to adjacent allysine, resulting in the aggregation of the AuNPs. We demonstrated that the synthesized LS-AuNPs are capable of detecting LOX sensitively, specifically both in vitro and in the tissue extract. Moreover, the suggested LS-AuNP-based assay is more sensitive than commonly employed assays or commercially available kits. Therefore, the LS-AuNPs developed in this study can be used to detect LOX levels and can be further used to predict the stiffness or the anticancer drug resistance of the tumor.

Nanozyme Applications: A Glimpse of Insight in Food Safety.

Nanozymes own striking merits, including high enzyme-mimicking activity, good stability, and low cost. Due to the powerful and distinguished functions, nanozymes exhibit widespread applications in the field of biosensing and immunoassay, attracting researchers in various fields to design and engineer nanozymes. Recently, nanozymes have been innovatively used to bridge nanotechnology with analytical techniques to achieve the high sensitivity, specificity, and reproducibility. However, the applications of nanozymes in food applications are seldom reviewed. In this review, we summarize several typical nanozymes and provide a comprehensive description of the history, principles, designs, and applications of nanozyme-based analytical techniques in food contaminants detection. Based on engineering and modification of nanozymes, the food contaminants are classified and then discussed in detail via discriminating the roles of nanozymes in various analytical methods, including fluorescence, colorimetric and electrochemical assay, surface-enhanced Raman scattering, magnetic relaxing sensing, and electrochemiluminescence. Further, representative examples of nanozymes-based methods are highlighted for contaminants analysis and inhibition. Finally, the current challenges and prospects of nanozymes are discussed.

A Compact Detection Platform Based on Gradient Guided-Mode Resonance for Colorimetric and Fluorescence Liquid Assay Detection.

This paper presents a compact spectral detection system for common fluorescent and colorimetric assays. This system includes a gradient grating period guided-mode resonance (GGP-GMR) filter and charge-coupled device. In its current form, the GGP-GMR filter, which has a size of less than 2.5 mm, can achieve a spectral detection range of 500-700 nm. Through the direct measurement of the fluorescence emission, the proposed system was demonstrated to detect both the peak wavelength and its corresponding intensity. One fluorescent assay (albumin) and two colorimetric assays (albumin and creatinine) were performed to demonstrate the practical application of the proposed system for quantifying common liquid assays. The results of our system exhibited suitable agreement with those of a commercial spectrometer in terms of the assay sensitivity and limit of detection (LOD). With the proposed system, the fluorescent albumin, colorimetric albumin, and colorimetric creatinine assays achieved LODs of 40.99 and 398 and 25.49 mg/L, respectively. For a wide selection of biomolecules in point-of-care applications, the spectral detection range achieved by the GGP-GMR filter can be further extended and the simple and compact optical path configuration can be integrated with a lab-on-a-chip system.

A Colorimetric Method for Quantifying Cis and Trans Alkenes Using an Indicator Displacement Assay.

A colorimetric indicator displacement assay (IDA) amenable to high-throughput experimentation was developed to determine the percentage of cis and trans alkenes. Using 96-well plates two steps are performed: a reaction plate for dihydroxylation of the alkenes followed by an IDA screening plate consisting of an indicator and a boronic acid. The dihydroxylation generates either erythro or threo vicinal diols from cis or trans alkenes, depending upon their syn- or anti-addition mechanisms. Threo diols preferentially associate with the boronic acid due to the creation of more stable boronate esters, thus displacing the indicator to a greater extent. The generality of the protocol was demonstrated using seven sets of cis and trans alkenes. Blind mixtures of cis and trans alkenes were made, resulting in an average error of ±2 % in the percentage of cis or trans alkenes, and implementing E2 and Wittig reactions gave errors of ±3 %. Furthermore, we developed variants of the IDA for which the color may be tuned to optimize the response for the human eye.

A review on metal nanozyme-based sensing of heavy metal ions: Challenges and future perspectives.

Large scale mining, manufacturing industries, exploitation of underground water, depletion of groundwater level, and uncontrolled discharge of industrial wastes have caused severe heavy metal ion pollution to the environment throughout the world. Therefore, the rapid detection of such toxic metal ions is inevitable. However, conventional methods require sophisticated instruments and skilled manpower and are difficult to operate in on-field conditions. Recently, metal nanozyme-based assays have been found to have the potential as an alternative to conventional methods due to their portability, simplicity, and high sensitivity to detect metal ion concentration to as low as parts per trillion (ppt). Metal nanozyme-based systems for heavy metal ions enable rapid and cheap screening on the spot with a very simple instrument such as a UV-vis absorption spectrophotometer and therefore, are convenient for use in field operations, especially in remote parts of the world. The sensing mechanism of a nanozyme-based sensor is highly dependent on its surface properties and specific interactions with particular metal ion species. Such method often encounters selectivity issues, unlike natural enzyme-based assays. Therefore, in this review, we mainly focus our discussion on different types of target recognition and inhibition/enhancement mechanisms, and their responses toward the catalytic activity in the sensing of target metal ions, design strategies, challenges, and future perspectives.

Assays for the identification and quantification of sialic acids: Challenges, opportunities and future perspectives.

N-Acetyl neuraminic acid (sialic acid) is a monosaccharide generally found as the terminating unit on glycans, which in turn are found on the surface of cells and glycoproteins. These glycans aid in a variety of biological functions such as cell interactions and immune response. Sialic acid has been identified as a biomarker for cardiovascular disease, diabetes and a range of other inflammatory and degenerative conditions. It has also been identified as a marker for different types of cancer. Sialic acid levels vary depending on the level of inflammation present during the course of an inflammatory disease and it is overexpressed by tumours as a shield against the immune system. Since the discovery of sialic acid, numerous assays have been developed for the identification and quantification of different sialic acid derivative monosaccharides and these assays fall into four main groups: colorimetric, fluorometric, enzymatic and chromatographic/mass spectrometric, with much overlap between these. Given the importance of sialic acids in biological pathways, this review article critically appraises assays that are used to detect and quantify sialic acid and its derivatives. Thus it details the method, sensitivity, specificity and wider scope of a range of assays, and concludes by suggesting some future directions for assay development and application. In this way, insight is provided into assays that allow for the accurate quantitation of sialic acid in biological samples, which may facilitate identification of the roles of sialic acid in healthy and disease pathways.

Cerium metal organic framework mediated molecular threading for point-of-care colorimetric assays.

This work prepared a Cerium metal organic framework (Ce-MOF), which exhibits colorimetric and fluorescent response for H2O2 simultaneously. The Ce-MOF was integrated in the origami paper SlipChip to directly analyze glucose and uric acid level in serum without sample handling or extra analytical equipment except for a smartphone. Its excellent stability in wide pH and temperature, which ranges from pH 1 to 12 and from 20 to 90 °C respectively, provides wider application prospects than conventional colorimetric strategy in the horseradish peroxidase (HRP) based detection. In colorimetric assays, the "coffee ring effect" is a fatal problem which refers to the instable and inhomogeneous color rendering, and the coating of color rendering zones by dried serum proteins also seriously hinders color reading. Herein, the anchored Ce-MOF not only solves the "coffee ring effect" by modulating the mass transport, but also constructs an ingenious bio-like barrier for selectively blocking proteins in serum. To achieve that, the externally actuated strategy and Ce-MOF mediating molecular threading-dependent transport strategy were employed. Relatively good performance of our device has been demonstrated whatever in color stability and uniformity, serum protein filtration or the direct clinical sample diagnosis.

Single and multiple detections of foodborne pathogens by gold nanoparticle assays.

A late detection of pathogenic microorganisms in food and drinking water has a high potential to cause adverse health impacts in those who have ingested the pathogens. For this reason there is intense interest in developing precise, rapid and sensitive assays that can detect multiple foodborne pathogens. Such assays would be valuable components in the campaign to minimize foodborne illness. Here, we discuss the emerging types of assays based on gold nanoparticles (GNPs) for rapidly diagnosing single or multiple foodborne pathogen infections. Colorimetric and lateral flow assays based on GNPs may be read by the human eye. Refractometric sensors based on a shift in the position of a plasmon resonance absorption peak can be read by the new generation of inexpensive optical spectrometers. Surface-enhanced Raman spectroscopy and the quartz microbalance require slightly more sophisticated equipment but can be very sensitive. A wide range of electrochemical techniques are also under development. Given the range of options provided by GNPs, we confidently expect that some, or all, of these technologies will eventually enter routine use for detecting pathogens in food. This article is categorized under: Diagnostic Tools > Biosensing.

Smartphone controlled interactive portable device for theranostics in vitro.

In this work, a smartphone controlled interactive theranostic device has been developed to perform in vitro photodynamic therapy (PDT) and diagnostic assays for treatment assessment on a single platform. Further, silver nanorod (Ag NR) was identified as a photosensitizer and its effect was studied in three different cell lines. PDT was achieved with Ag NRs using low irradiation (1.4 mW/cm2 at 632 nm) from light emitting diodes (LEDs) in the device. Specifically, PDT in conjugation with widely used chemotherapeutic drug doxorubicin (Dox) proved effective in killing of HeLa cancer cells and multicellular tumor spheroids at a minimum dose of Ag (2.5 µg/mL). The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) assays performed with the device indicated the therapeutic success of the delivered PDT. The device is portable and can be adapted for different wavelength irradiations and radiation doses. Additionally, wireless operation using a custom designed smartphone application makes it convenient to use in complex environments without much of human intervention.

Identification of Several Toxic Metal Ions Using a Colorimetric Sensor Array.

Water pollution by toxic metal ions is a worldwide environmental and health problem, and therefore monitoring of toxic metal ions in water resources is highly desired. In this chapter, we describe a simple colorimetric sensor array for simultaneous detection of multiple toxic heavy metal ions (Hg2+, Cd2+, Fe3+, Pb2+, Al3+, Cu2+, and Cr3+) in water. This assay is produced by using 11-mercaptoundecanoic acid (MUA)-capped gold nanoparticles (AuNPs) and five amino acids (lysine, cysteine, histidine, tyrosine, and arginine). The presence of amino acids can enhance or diminish the aggregation MUA-capped AuNPs with metal ions. The color change of the sensor array after aggregation in some of the channels creates unique response patterns for each metal ion.

Single step and mask-free 3D wax printing of microfluidic paper-based analytical devices for glucose and nitrite assays.

Microfluidic paper-based analytical devices (µPADs) have recently emerged as a simple, portable, user-friendly, and affordable alternative to more instrument-intensive analytical approaches for point-of-care testing (POCT), food safety analysis, and environmental monitoring. However, most of the existing methods for the fabrication of µPADs still face a great challenge because of different trade-offs among cost, convenience, and the pattern resolution. In this work, we report a facile one-step approach to prepare a µPAD using an affordable, easy-to-build 3D printer to generate patterns of solid wax on laboratory filter paper. The presented wax printing method did not require the use of predesigned masks and an external heat source to form complete hydrophobic wax barrier through the use of a custom-made extruder. The results revealed a strong linear relationship (R2 = 0.985) between the nominal and the printed widths of the wax barriers. The achievable resolution of the wax barrier printed on filter paper was 468 ±â€¯72 µm, which was lower than previously reported minimum barrier feature sizes achieved by wax printing and other wax patterning techniques, such as stamping and screen-printing. The analytical utility of the fabricated µPADs was evaluated for colorimetric nitrite and glucose detection in artificial solutions. It was found that the fabricated µPADs provided adequate accuracy and reproducibility for quantitative determination of nitrite and glucose within concentration ranges relevant to the disease detection in human saliva and urine. The wax printing approach reported here provides a simple, rapid, and cost-effective fabrication method for paper-based microfluidics and may bring benefits to medical diagnostics in the developing world.

Colorimetric High-Throughput Screening Assays for the Directed Evolution of Fungal Laccases.

In this chapter we describe several high-throughput screening assays for the evaluation of mutant libraries for the directed evolution of fungal laccases in the yeast Saccharomyces cerevisiae. The assays are based on the direct oxidation of three syringyl-type phenols derived from lignin (sinapic acid, acetosyringone, and syringaldehyde), an artificial laccase mediator (violuric acid), and three organic synthetic dyes (Methyl Orange, Evans Blue, and Remazol Brilliant Blue). While the assays with the natural phenols can be used for laccases with low redox potential, the rest are exclusive for high-redox potential laccases. In fact, the violuric acid assay is devised as a method to ascertain that the high-redox potential of laccase is not lost during directed evolution.

Improved LFIAs for highly sensitive detection of BNP at point-of-care.

Heart failure (HF) has become a major cause of morbidity and mortality with a significant global economic burden. Although well-established clinical tests could provide early diagnosis, access to these tests is limited in developing countries, where a relatively higher incidence of HF is present. This has prompted an urgent need for developing a cost-effective, rapid and robust diagnostic tool for point-of-care (POC) detection of HF. Lateral flow immunoassay (LFIA) has found widespread applications in POC diagnostics. However, the low sensitivity of LFIA limits its ability to detect important HF biomarkers (e.g., brain natriuretic peptide [BNP]) that are normally present in low concentration in blood. To address this issue, we developed an improved LFIA by optimizing the gold nanoparticle (GNP)-antibody conjugate conditions (e.g., the conjugate pH and the amount of added antibody), the diameter of GNP and the concentration of antibody embedded on the test line and modifying the structure of test strip. Through these improvements, the proposed test strip enabled the detection of BNP down to 0.1 ng/mL within 10-15 min, presenting ~15-fold sensitivity enhancement over conventional lateral flow assay. We also successfully applied our LFIA in the analysis of BNP in human serum samples, highlighting its potential use for clinical assessment of HF. The developed LFIA for BNP could rapidly rule out HF with the naked eye, offering tremendous potential for POC test and personalized medicine.

Colorimetric Detection of Acetyl Xylan Esterase Activities.

Colorimetric detection of reaction products is typically preferred for initial surveys of acetyl xylan esterase (AcXE) activity. This chapter will describe common colorimetric methods, and variations thereof, for measuring AcXE activities on commercial, synthesized, and natural substrates. Whereas assays using pNP-acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate (4MUA) are emphasized, common methods used to measure AcXE activity towards carbohydrate analogs (e.g., acetylated p-nitrophenyl ß-D-xylopyranosides) and various acetylated xylans are also described. Strengths and limitations of the colorimetric assays are highlighted.