Optimal Assay Conditions Research Articles

Screening and application of aptamers as fluorescent biosensors for selective and sensitive detection of hepatocellular carcinoma and in vivo targeted delivery studies.

The incidence of primary hepatocellular carcinoma (HCC) has recently ranked fifth in the world, and the incidence rate is increasing year by year worldwide. Therefore, early diagnosis is the highest priority in the treatment of HCC. In this paper, four anti-HCC aptamers were obtained using magnetic bead SELEX technology. Among them, Apt-1 had the smallest Kd value(5.9nM) and the highest affinity. Flow cytometry results showed that the FITC-aptamers only specifically recognized HCC serum. Circular dichronism (CD) spectral characterization showed a positive peak near 275nm and a negative peak near 250nm for all aptamers, elucidating that the secondary structure formed by the candidate aptamers was a stem-loop B-DNA structure. In addition, molecular docking simulations showed that the binding of the HCC target to the candidate aptamer sequences was mainly dominated by hydrogen bonding. The results of the aptamer sensing performance analysis showed that under the optimized assay conditions, a linear relationship (ranging from 1nM to 1µM) was achieved, with a limit of detection (LOD) down to 0.75nM and a LOQ of 2.32nM. This was further validated in clinical samples, with a positive detection rate of more than 90%. Furthermore, aptamer-mediated in vivo delivery of luciferase mRNA showed that Apt-1-luciferase mRNA could be targeted to the liver and hepatic luciferase expression was significantly increased. These results demonstrate that the aptamer paves the way for clinical application, evidencing significant potential to offer reference information for early diagnosis.

Thg1 family 3'-5' RNA polymerases as tools for targeted RNA synthesis.

Members of the 3'-5' RNA polymerase family, comprised of tRNAHis guanylyltransferase (Thg1) and Thg1-like proteins (TLPs), catalyze templated synthesis of RNA in the reverse direction to all other known 5'-3' RNA and DNA polymerases. The discovery of enzymes capable of this reaction raised the possibility of exploiting 3'-5' polymerases for posttranscriptional incorporation of nucleotides to the 5'-end of nucleic acids without ligation, and instead by templated polymerase addition. To date, studies of these enzymes have focused on nucleotide addition to highly structured RNAs, such as tRNA and other noncoding RNAs. Consequently, general principles of RNA substrate recognition and nucleotide preferences that might enable broader application of 3'-5' polymerases have not been elucidated. Here, we investigated the feasibility of using Thg1 or TLPs for multiple nucleotide incorporation to the 5'-end of a short duplex RNA substrate, using a templating RNA oligonucleotide provided in trans to guide 5'-end addition of specific sequences. Using optimized assay conditions, we demonstrated a remarkable capacity of certain TLPs to accommodate short RNA substrate-template duplexes of varying lengths with significantly high affinity, resulting in the ability to incorporate a desired nucleotide sequence of up to eight bases to 5'-ends of the model RNA substrates in a template-dependent manner. This work has further advanced our goals to develop this atypical enzyme family as a versatile nucleic acid 5'-end labeling tool.

Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

Molecularly Imprinted Viral Protein Integrated Zn-Cu-In-Se-P Quantum Dots Superlattice for Quantitative Ratiometric Electrochemical Detection of SARS-CoV-2 Spike Protein in Saliva.

Solution-processable colloidal quantum dots (QDs) are promising materials for the development of rapid and low-cost, next-generation quantum-sensing diagnostic systems. In this study, we report on the synthesis of multinary Zn-Cu-In-Se-P (ZCISeP) QDs and the application of the QDs-modified electrode (QDs/SPCE) as a solid superlattice transducer interface for the ratiometric electrochemical detection of the SARS-CoV-2-S1 protein in saliva. The ZCISeP QDs were synthesized through the formation of In(Zn)PSe QDs from InP QDs, followed by the incorporation of Cu cations into the crystal lattice via cation exchange processes. A viral-protein-imprinted polymer film was deposited onto the QDs/SPCE for the specific binding of SARS-CoV-2. Molecular imprinting of the virus protein was achieved using a surface imprinting electropolymerization strategy to create the MIP@QDs/SPCE nanosensor. Characterization through spectroscopic, microscopic, and electrochemical techniques confirmed the structural properties and electronic-band state of the ZCISeP QDs. Cyclic voltammetry studies of the QDs/SPCE superlattice confirmed efficient electron transport properties and revealed an intraband gap energy state with redox peaks attributed to the Cu1+/2+ defects. Binding of SARS-CoV-2-S1 to the MIP@QDs/SPCE cavities induced a gating effect that modulated the Fe(CN)6 3-/4- and Cu1+/2+ redox processes at the nanosensor interface, producing dual off/on ratiometric electrical current signals. Under optimal assay conditions, the nanosensor exhibited a wide linear detection range (0.001-100 pg/mL) and a low detection limit (0.34 pg/mL, 4.6 fM) for quantitative detection of SARS-CoV-2-S1 in saliva. The MIP@QDs/SPCE nanosensor demonstrated excellent selectivity against nonspecific protein targets, and the integration with a smartphone-based potentiostat confirmed the potential for point-of-care applications.

A New Strategy for Measuring Salivary Alpha‐Amylase Activity Using Glucometer

AbstractAlpha‐amylase is one of the most important enzymes in the saliva. It is an important biomarker for many health conditions. Saliva sampling is a non‐invasive method and is the preferred choice for sampling at point of the care and home testing. Measurement of salivary alpha‐amylase activity as a rapid test using test strips with Flavin‐dependent glucose dehydrogenases (FAD‐GDH) as glucose is the aim of the current research. FAD‐GDH is an oxygen‐independent enzyme and has a narrow substrate specificity. For this goal, starches from various sources are tested as a substrate for alpha‐amylase and chose MS‐101 starch as the best substrate, between six different starch types. Under optimal assay conditions, when hydrolyzed starch (in 20 min, 37 °C, pH = 7) reacts with FAD‐GDH enzymes that exist in test strips for 5 s glucometer can show the amount of glucose produced in samples from patient samples. This glucose value is directly correlated with the salivary alpha‐amylase activity (p‐value <0.01).

Protocol improvement and multisite validation of a digital soft agar colony formation assay for tumorigenic transformed cells intermingled in cell therapy products

Background aimsThe administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs. Methods: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells. Results: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314–570 wells) based on the laboratory-specific CFE (42–76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone. ConclusionsThe D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.

Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to β-naphthoflavone (β-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to β-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

A versatile kinase mobility shift assay (KiMSA) for PKA analysis and cyclic AMP detection in sperm physiology (and beyond).

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-32P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

Development of a colloidal gold immunochromatographic strip for rapid detection of ribavirin in pig urine

ABSTRACT A specific and sensitive monoclonal antibody (mAb) for ribavirin (RBV) was generated by immunizing BALB/c mice with RBV – bovine serum albumin. Using the mAb, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic (CGI) test strip were developed for detecting RBV in pig urine. Under optimum assay conditions, IC50 was 0.08 ng/mL in 0.05 M phosphate-buffered saline and the recovery rate in real-world samples ranged between 77.3% and 99%. The cut-off limit of the CGI test strip for RBV was 5 ng/mL in pig urine. The cross-reactivity rate of the test strip to the tested antiviral drugs including acyclovir and moroxydine hydrochloride and the metabolites 1,2,4-triazole-3-carboxamide and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxylic acid was below 0.1%. The results indicate that ic-ELISA and the CGI test strip have high sensitivity and specificity and that these methods are suitable for the rapid detection of RBV in pig urine.

Magnetic DNAzyme nanomachine fluorescent biosensor for Pb(Ⅱ) detection

The lead ion (Pb2+) is harmful to human. Detecting low content of Pb2+ in the environment requires high sensitive and specific detection technology. In this study, we developed a magnetic DNAzyme nanomachine fluorescent biosensor for rapid and ultra-sensitive detection of Pb2+. We focused on using bovine serum albumin (BSA) as a bridge to connect magnetic beads and DNAzyme in the magnetic nanomachine, aiming to improve the detection sensitivity. In the presence of Pb2+, the enzyme cleavage activity was activated, leading to the substrate chains cleaved and released. Meanwhile, Pb2+ participated in the cyclic process of recognition and release, which contributed to signal amplification. The cleavage products and stem-loop chains were assembled and amplified using the double primer realtime fluorescence quantitative reaction (qPCR) method. By applying such triple amplifications of the signal, Pb2+ was quantitatively detected by measuring the change in the Ct value (ΔCt) before and after cleavage of the DNAzyme. Under optimized assay conditions, a linear relationship ranging from 2 nM to 150 nM was observed with a limit of detection (LOD) of 3.7 nM for Pb2+. In conclusion, our approach offers a potentially useful method for simple, rapid, and sensitive Pb2+ detection.

Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes.

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

The Optimization of Assay Conditions and Characterization of the Succinic Semialdehyde Dehydrogenase Enzyme of Germinated Tartary Buckwheat.

In this study, the conditions for optimizing the determination of succinic semialdehyde dehydrogenase (SSADH, EC 1.2.1.79) activity in germinated Tartary buckwheat were investigated. Based on a single-factor test, the effects of temperature, pH, and succinic semialdehyde (SSA) concentration on the enzyme activity of germinated buckwheat SSADH were investigated by using the response surface method, and optimal conditions were used to study the enzymatic properties of germinated buckwheat SSADH. The results revealed that the optimum conditions for determining SSADH enzyme activity are as follows: temperature-30.8 °C, pH-8.7, and SSA concentration-0.3 mmol/L. Under these conditions, SSADH enzyme activity was measured as 346 ± 9.61 nmol/min. Furthermore, the thermal stability of SSADH was found to be superior at 25 °C, and its pH stability remained comparable at pH levels of 7.6, 8.1, and 8.6 in germinated Tartary buckwheat samples; however, a decline in stability was observed at pH 9.1. Cu2+, Co2+, and Ni2+ exhibited an activating effect on SSADH activity in germinating Tartary buckwheat, with Cu2+ having the greatest influence (p < 0.05), which was 1.21 times higher than that of the control group. Zn2+, Mn2+, and Na+ inhibited SSADH activity in germinating Tartary buckwheat, with Zn2+ showing the strongest inhibitory effect (p < 0.05). On the other hand, the Km and Vmax of SSADH for SSA in germinated Tartary buckwheat were 0.24 mmol/L and 583.24 nmol/min. The Km and Vmax of SSADH for NAD+ in germinated Tartary buckwheat were 0.64 mmol/L and 454.55 nmol/min.

Fluorometric assay for phenol oxidase activity in soils and its controlling variables

Phenolic compounds constitute the major component of recalcitrant carbon pools in soils and undergo degradation by enzymes such as phenol oxidases. Despite their significance, a common, appropriate assay to determine its activity is yet to be found. Until recently, only colorimetric assays, using substrates such as L-DOPA, pyrogallol, and ABTS, have been dominantly employed to measure phenol oxidase activity, despite their drawbacks, such as interference of soil background color, unavailability of a commercial product, and low sensitivity. Therefore, this study aimed to identify the optimal conditions for the novel fluorometric Amplex Red assay, compare the effectiveness and reproducibility of this assay with the conventional colorimetric L-DOPA assay, and reveal the key controlling variables of phenol oxidase activity by applying it to various soil types. Results showed that the optimal phenol oxidase activity can be determined at a substrate concentration of 500 μM and when the soil suspension is prepared with sonication only, using deionized water as a solvent. The comparison of the Amplex Red and L-DOPA assay indicated that the fluorometric assay has greater reproducibility, with a significantly smaller coefficient of variation (19.9 % and 107 %, respectively). For the L-DOPA assay, there was substantial variability between replicates and strong dependence on soil suspension preparation methods. Although this study reveals that the Amplex Red assay is more suitable for measuring phenol oxidase activity, additional research on other soil types is important to confirm its applicability, and adjustments to incubation conditions may be necessary for specific soil types. The application of the Amplex Red assay on various soil types uncovered soil pH and organic matter as the two most important factors controlling phenol oxidase activity, which aligned with previous findings. A positive linear correlation between phenol oxidase activity and soil pH (R = 0.734, P ≤ 0.000) but a negative exponential correlation with soil organic matter content (R = −0.547, P ≤ 0.01).

Developing DPYD Genotyping Method for Personalized Fluoropyrimidines Therapy.

Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy. A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129-5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing. From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129-5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129-5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results. A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.

Study of the Electrochemical Impedance Immunosensor

Colloidal gold has been extensively studied for its unique properties in various fields, including diagnostics and therapeutics. The colloidal gold electrochemical impedance immunosensor is a promising tool in antigen detection. It has high sensitivity, specificity, and rapid results. However, it also faces certain challenges, such as the stability of colloidal gold particles and optimization of assay conditions. To address these challenges, this paper suggests several recommendations. First, advancements in colloidal gold synthesis and surface modification techniques can enhance the stability and reproducibility of the particles. Second, optimizing assay conditions, including sample pre-treatment and antigen binding conditions, can improve the overall performance of the electrochemical impedance immunosensor. Furthermore, research is needed to develop the full potential of this technique in different fields and expand its practical applications. In conclusion, the colloidal gold electrochemical impedance immunosensor shows great promise in antigen detection. By comparing it with other methods and proposing solutions to its limitations, this review paper aims to contribute to the advancement of this technique and its applications in various industries. Continued research and development in this field will undoubtedly lead to further improvements and broaden the scope of this powerful tool.

Rapid and Sensitive Detection of the Causal Agents of Postharvest Kiwifruit Rot, Botryosphaeria dothidea and Diaporthe eres, Using a Recombinase Polymerase Amplification Assay.

The occurrence of postharvest kiwifruit rot has caused great economic losses in major kiwifruit-producing countries. Several pathogens are involved in kiwifruit rot, notably Botryosphaeria dothidea, and Diaporthe species. In this study, a recombinase polymerase amplification (RPA) assay was developed for the rapid and sensitive detection of the pathogens responsible for posing significant threats to the kiwifruit industries. The RPA primer pairs tested in this study were highly specific for detection of B. dothidea and D. eres. The detection limits of our RPA assays were approximately two picograms of fungal genomic DNA. The optimal conditions for the RPA assays were determined to be at a temperature of 39°C maintained for a minimum duration of 5 min. We were able to detect the pathogens from kiwifruit samples inoculated with a very small number of conidia. The RPA assays enabled specific, sensitive, and rapid detection of B. dothidea and D. eres, the primary pathogens responsible for kiwifruit rots in South Korea.

Development and Validation of Chemometric Assisted RP-HPLC Method for Simultaneous Estimation of Perindopril Erbumine, Indapamide, and Amlodipine Besylate in Bulk and Pharmaceutical Formulation

This research paper illustrates a latterly developed, optimized and validated gradient RP-HPLC approach for simultaneous analysis ofIndapamide, Perindopril erbumine and Amlodipine besylate in bulk and pharmaceutical formulation with the assistance of quality by design. Qualityis predicated on desired and predetermined specifications. Understanding various factors, dependent variables, and their interconnection effectsby a desired set of experiments on the responses to be analyzed is an important component of QbD. Several operating conditions of variousprocesses optimization, chromatographic separation performance improvement, and high extraction efficiency were attained by using QbD. Thepowerful chromatographic conditions were done using the HypersilC18 column (250mm × 4.6mm, 5μm particle Size). The UV detector wasadjusted to 215nm. Design of experiments (DoE) was applied for multivariate optimization of the experimental conditions of the RP-HPLCmethod. Three independent factors, mobile phase composition, phosphate buffer strength, and flow rate, were used to design mathematicalmodels. Central composite design (CCD) was used to examine the response surface methodology and fully examine the results of theseindependent factors. The desirability function was used to optimize the retention time and resolution of the analytes simultaneously. Theimproved and anticipated data from the contour diagram consisted of methanol and phosphate buffer (pH 2.5, strength 0.05M) in the ratio of65:35, respectively, at a flow rate of 1.1 ml/min. Using these optimum conditions, baseline separation of both drugs with good resolution and runtime of less than 5.0 min was achieved. The novelty of the developed method was time-consuming, cost-effective, and sensitive. The optimizedassay conditions were validated according to ICH guidelines. Under the optimized state, the linearity ranges were found to be 10-40 μg/mL, 32–128 μg/mL, and 40-160 μg/mL for Indapamide, Perindopril erbumine, and amlodipine besylate, respectively, with correlation coefficients (R2) of0.999. The mean accuracy studied ranged from 99.18 to 99.58%. The percentage coefficient variation value for the precision study was lower than1%. The proposed method showed good precision and repeatability. Hence the developed RP-HPLC method using quality by design can be usedas a routine quality control analysis of indapamide, perindopril erbumine, and amlodipine besylate.

Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens.

Fungal phytopathogens are the major agents responsible for causing severe damage to and losses in agricultural crops worldwide. Botrytis cinerea, Colletotrichum acutatum, Fusarium proliferatum, and Magnaporthe grisea are included in the top ten fungal phytopathogens that impose important plant diseases on a broad range of crops. Microbial natural products can be an attractive alternative for the biological control of phytopathogens. The objective of this work was to develop and validate a High-throughput Screening (HTS) platform to evaluate the antifungal potential of chemicals and natural products against these four important plant pathogens. Several experiments were performed to establish the optimal assay conditions that provide the best reproducibility and robustness. For this purpose, we have evaluated two media formulations (SDB and RPMI-1640), several inoculum concentrations (1 × 106, 5 × 105 and 5 × 106 conidia/mL), the germination curves for each strain, each strain's tolerance to dimethyl sulfoxide (DMSO), and the Dose Response Curves (DRC) of the antifungal control (Amphotericin B). The assays were performed in 96-well plate format, where absorbance at 620 nm was measured before and after incubation to evaluate growth inhibition, and fluorescence intensity at 570 nm excitation and 615 nm emission was monitored after resazurin addition for cell viability evaluation. Quality control parameters (RZ' Factors and Signal to Background (S/B) ratios) were determined for each assay batch. The assay conditions were finally validated by titrating 40 known relevant antifungal agents and testing 2400 microbial natural product extracts from the MEDINA Library through both HTS agar-based and HTS microdilution-based set-ups on the four phytopathogens.

Optimization of a rapid and sensitive nucleic acid lateral flow biosensor for hepatitis B virus detection.

The utilization of direct amplification of nucleic acid from lysate has attracted interest in the advancement of straightforward and economical point-of-care assays. Consequently, this study primarily focuses on the development of a rapid, precise, and cost-effective lateral flow biosensor for the convenient detection of HBV nucleic acid at the point-of-care. Furthermore, the study evaluates the effectiveness of the direct amplification method in comparison to purified nucleic acid samples within the context of LAMP-LF biosensing approaches. The experiments conducted in this study utilized clinical serum samples that were confirmed as HBV-positive through real-time PCR assays. Sample preparation involved employing spin column nucleic acid purification and serum heat treatment. To amplify a 250bp fragment of the HBV polymerase gene, three pairs of specific LAMP primers were utilized, which were biotin-labeled and FITC-labeled for detection purposes. Various incubation temperatures (ranging from 64 to 68°C) and durations (30min, 45min, and 1h) were investigated to determine the optimal conditions for the LAMP assay. The results were subsequently assessed through fluorometric analysis, white turbidity measurements, and lateral flow assay. Milenia HybriDetect1 strips, designed for immediate use, were employed to visualize the LAMP amplicons. Furthermore, the performance of the lateral flow biosensor was evaluated using 10-fold serial dilutions of a secondary standard containing a viral load of 108 IU/ml. The optimization of the LAMP reaction was achieved at a temperature of 67°C, resulting in significant turbidity after a 30-minute incubation period. When the spin column purification method was employed, varying test bands were observed for templates ranging from 108 IU/ml to 101 IU/ml viral load. However, when serum samples underwent heat treatment and the resulting supernatant was directly used for LAMP, the lateral flow assay was capable of detecting a minimum viral load of 103 IU/ml. In resource-limited settings, the LAMP-LF assay presents a promising solution for HBV testing. However, it is important to note that direct amplification without DNA purification may diminish the performance of the approach.

Development of colorimetric loop-mediated isothermal amplification (LAMP) assay for detecting the azoxystrobin-resistant isolates in Colletotrichum truncatum

Anthracnose, mainly caused by Colletotrichum truncatum, is a serious threat to soybean production worldwide. Azoxystrobin, a quinone outside inhibitor (QoI) fungicide, has long been frequently used in Thailand. As expected, its performance has decreased as the pathogen has developed fungicide resistance, mainly due to mutations in the target cytochrome b (cytb) gene. The substitution from glycine to alanine at codon 143 (G143A) is related to the azoxystrobin-completely resistant phenotype of C. truncatum. We thus used the G143A mutation as the reaction marker to develop a loop-mediated isothermal amplification (LAMP) method combined with hydroxynaphthol blue (HNB) for visual, simple, and rapid detection of azoxystrobin-resistant isolates of C. truncatum. Among four sets of primers designed, primer sets 1, 2, and 4 distinguished the isolate carrying the G143A mutation from wild-type isolate. Optimum conditions for the LAMP assay using primer set 4 were 63 °C for 80 min with a visible detection limit of 1 ng. Moreover, the LAMP assay was highly specific and accurate in tests of 125 isolates of C. truncatum from 13 fields in northern Thailand. This LAMP assay is highly efficient, simple, accurate for rapid field monitoring of azoxystrobin resistance in C. truncatum and implementing effective control measures.