Molecular Detection of Multidrug Resistance and Characterizations of Mutations in Mycobacterium Tuberculosis Using Polycarbonate Track-Etched Membrane Based DNA Bio-Chip.

With the widespread use of rifampicin (RMP) and isoniazid (INH), multidrug resistance (MDR) in Mycobacterium tuberculosis (M.tb) poses a threat to the success of tuberculosis (TB) control programs. We have developed a new polycarbonate track-etched membranes (PC-TEM) based DNA bio-chip designed for rapid detection of mutations conferring MDR in M.tb culture isolates. Bio-chips were designed to contain 14 specific probes for wild type and mutated allele of selected codons within 80 bp rifampicin resistance determining region of rpoB gene, katG gene and mabA-inhA regulatory region. RMP-resistance-associated gene mutation points rpoB 516, 526, 531 and 533, and the INH-resistance-associated gene mutation points katG315 and inhA-15 were targeted. Bio-chip signal was detected using enhanced chemiluminescence. A total of 50 culture isolates that were sensitive or resistant to RMP and/or INH were analyzed by bio-chip. The results of culture-based drug susceptibility testing (DST) were used as the gold standard and gene sequencing was performed to resolve the discordance. Amongst 50 culture isolates, we have detected 18 MDR, 9 RMP mono-resistant, 6 INH mono-resistant, and 17 fully susceptible isolates. The developed DNA bio-chip has a sensitivity of 90% for RMP and MDR and 100% for INH resistance. The bio-chip has a specificity of 100% for RMP and MDR and 88.8% for INH detection. The identification of mutations using the DNA bio-chip was 100% concordant with the sequencing data for the probes covered by the bio-chip. The detection of rpoB, katG and inhA gene mutation points by a DNA bio-chip may be used as a rapid, accurate, and economical, clinical detection method for MDR detection in M.tb. This is very valuable for the control of TB epidemics.

Development of DNA Bio-chip for Detection of Mutations of rpoB, embB and inhA Genes in Drug-Resistant Mycobacterium Tuberculosis.

Drug-resistant (DR) tuberculosis (TB) is a global threat to health security and TB control programs. Since conventional drug susceptibility testing (DST) takes several weeks, we have developed a molecular method for the rapid identification of DR strains of Mycobacterium Tuberculosis (M.tb) utilizing DNA bio-chips. DNA bio-chips were prepared by immobilizing oligonucleotides (probes) on highly microporous polycarbonate track-etched membranes (PC-TEM) as novel support. Bio-chip was designed to contain 15 specific probes to detect mutations in three genes (rpoB, embB, and inhA). A sensitive and specific chemiluminescence based bio-chip assay was developed based on multiplex PCR followed by hybridization on bio-chip. Fifty culture isolates were used to evaluate the ability of in-house developed bio-chip to detect the mutations. Bio-chip analysis shows that 37.7% of samples show wild type sequences, 53.3% of samples were monoresistance showing resistance to either rifampicin (RMP), isoniazid (INH), or ethambutol (EMB). 4.4% of samples were polydrug resistant showing mutations in both the rpoB gene and embB gene while 4.4% of samples were multidrug-resistant (MDR), harboring mutations in the rpoB and inhA genes. The results were compared with DST and sequencing. Compared to sequencing, bio-chip assay shows a sensitivity of 96.5% and specificity of 100% for RMP resistance. For EMB and INH, the results were in complete agreement with sequencing. This study demonstrates the first-time use of PC-TEMs for developing DNA bio-chip for the detection of mutations associated with drug resistance in M.tb. Developed DNA bio-chip accurately detected different mutations present in culture isolates and thus provides detailed and reliable data for clinical diagnosis.

A Non-Instrumental Green Analytical Method Based on Surfactant-Assisted Dispersive Liquid-Liquid Microextraction-Thin-Layer Chromatography-Smartphone-Based Digital Image Colorimetry(SA-DLLME-TLC-SDIC) for Determining Favipiravir in Biological Samples.

Favipiravir (FAV) has become a promising antiviral agent for the treatment of COVID-19. Herein, a green, fast, high-sample-throughput, non-instrumental, and affordable analytical method is proposed based on surfactant-assisted dispersive liquid-liquid microextraction (SA-DLLME) combined with thin-layer chromatography-digital image colourimetry (TLC-DIC) for determining favipiravir in biological and pharmaceutical samples. Triton X-100 and dichloromethane (DCM) were used as the disperser and extraction solvents, respectively. The extract obtained after DLLME procedure was spotted on a TLC plate and allowed to develop with a mobile phase of chloroform:methanol (8:2, v/v). The developed plate was photographed using a smartphone under UV irradiation at 254 nm. The quantification of FAV was performed by analysing the digital images' spots with open-source ImageJ software. Multivariate optimisation using Plackett-Burman design (PBD) and central composite design (CCD) was performed for the screening and optimisation of significant factors. Under the optimised conditions, the method was found to be linear, ranging from 5 to 100 µg/spot, with a correlation coefficient (R2) ranging from 0.991 to 0.994. The limit of detection (LOD) and limit of quantification (LOQ) were in the ranges of 1.2-1.5 µg/spot and 3.96-4.29 µg/spot, respectively. The developed approach was successfully applied for the determination of FAV in biological (i.e., human urine and plasma) and pharmaceutical samples. The results obtained using the proposed methodology were compared to those obtained using HPLC-UV analysis and found to be in close agreement with one another. Additionally, the green character of the developed method with previously reported protocols was evaluated using the ComplexGAPI, AGREE, and Eco-Scale greenness assessment tools. The proposed method is green in nature and does not require any sophisticated high-end analytical instruments, and it can therefore be routinely applied for the analysis of FAV in various resource-limited laboratories during the COVID-19 pandemic.

Cellulose paper sorptive extraction (CPSE): A simple and affordable microextraction method for analysis of basic drugs in blood as a proof of concept.

Aiming towards simplifying sample preparation procedure, the present work explores use of unmodified laboratory filter paper as sorbent for extraction of nine basic drugs (five antidepressants, four benzodiazepines, and ketamine) from human blood samples and their analysis by gas chromatography-mass spectrometry (GC-MS). The procedure termed as cellulose paper sorptive extraction (CPSE) is straightforward. It involves adsorption of target analytes from deproteinized diluted blood samples on the unmodified cellulose paper followed by elution into 2 mL of methanol. Multivariate optimization, consisting of Placket-Burman design (PBD) and central composite design (CCD), was used to screen and optimize significant factors for CPSE. The proposed method follows the principles of green analytical chemistry (GAC), as the unmodified filter paper used as the sorbent is inexpensive and biodegradable. The technique is easy to perform and requires only 2 mL of MeOH during the entire extraction procedure. Under the optimized conditions, the limit of detection and quantification for the target analytes were estimated to be in the range of 0.003-0.035 and 0.010-0.117 µg mL-1, respectively. In contrast, the relative standard deviations were consistently below 10 %. The calibration curves were linear in the range of 0.015-2 µg mL-1 with a coefficient of determination (R2) in the range of 0.995-0.999.Satisfactory recoveries ranging from 87 to 99 % was achieved. As proof of concept, the analysis of nine drugs in blood samples from the patients was performed to demonstrate the potential application of the proposed method.

Simple determination of dichlorvos in cases of fatal intoxication by gas Chromatography-Mass spectrometry.

Dichlorvos (DDVP) is an organophosphorous insecticide which is classified as "highly hazardous" Class 1B chemical by World Health Organization (WHO) and largely misused for the purpose of self-poisoning in developing countries. Forensic toxicology laboratories are routinely encountering cases of pesticide poisoning due to their fatal intoxication. Herein; a method is described based on vortex-assisted dispersive liquid-liquid microextraction (VA-DLLME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) for the determination of an organophosphorous insecticide; dichlorvos (DDVP) in human autopsy samples (blood, stomach content and liver). Under the optimum conditions, the method was found to be linear in the range of 0.5-10 µg mL-1 and 1.5-10 µg g-1 for blood and tissue samples, respectively. Limit of quantification was set at 0.55 µg mL-1 and 1.1 µg g-1 for blood and tissue samples, respectively. Intraday and inter-day precisions were less than 8 and 12 %, respectively. Good recoveries in the range of 86-95 % were obtained for the proposed procedure. The method has been satisfactorily applied for the determination of DDVP in autopsy samples from two different cases received in our laboratory. In comparison to previous methods; the proposed method is relatively short, high sample throughput, inexpensive and adheres to the principles of green analytical chemistry (GAC) for determination of DDVP in human autopsy samples. The method can be adopted in forensic toxicological laboratories for analysis of DDVP in autopsy samples. In addition, the green character of the proposed method was evaluated using ComplexGAPI procedure.

Rapid Determination of Non-Steroidal Anti-Inflammatory Drugs in Urine Samples after In-Matrix Derivatization and Fabric Phase Sorptive Extraction-Gas Chromatography-Mass Spectrometry Analysis.

Fabric phase sorptive extraction (FPSE) has become a popular sorptive-based microextraction technique for the rapid analysis of a wide variety of analytes in complex matrices. The present study describes a simple and green analytical protocol based on in-matrix methyl chloroformate (MCF) derivatization of non-steroidal anti-inflammatory (NSAID) drugs in urine samples followed by FPSE and gas chromatography-mass spectrometry (GC-MS) analysis. Use of MCF as derivatizing reagent saves substantial amounts of time, reagent and energy, and can be directly performed in aqueous samples without any sample pre-treatment. The derivatized analytes were extracted using sol−gel Carbowax 20M coated FPSE membrane and eluted in 0.5 mL of MeOH for GC-MS analysis. A chemometric design of experiment-based approach was utilized comprising a Placket−Burman design (PBD) and central composite design (CCD) for screening and optimization of significant variables of derivatization and FPSE protocol, respectively. Under optimized conditions, the proposed FPSE-GC-MS method exhibited good linearity in the range of 0.1−10 µg mL−1 with coefficients of determination (R2) in the range of 0.998−0.999. The intra-day and inter-day precisions for the proposed method were lower than <7% and <10%, respectively. The developed method has been successfully applied to the determination of NSAIDs in urine samples of patients under their medication. Finally, the green character of the proposed method was evaluated using ComplexGAPI tool. The proposed method will pave the way for simper analysis of polar drugs by FPSE-GC-MS.

Production, characterization and in-vitro applications of single-domain antibody against thyroglobulin selected from novel T7 phage display library.

Single- domain antibodies (SdAbs) have been deployed in various biomedical applications in the recent past. However, there are no reports of their use in the immunoradiometric assays (IRMA) for thyroglobulin (Tg). Tg is the precursor molecule for the biosynthesis of thyroid hormones: thyroxine and triiodothyronine, which are essential for the regulation of normal metabolism in all vertebrates. Patients with differentiated thyroid cancer (DTC) require periodic monitoring of their serum thyroglobulin levels, as it serves as a prognostic marker for DTC. Here, we report a methodology to produce SdAbs against human-Tg, by a hybrid immunization/directed-evolution approach by displaying the SdAb gene-repertoire derived from a hyperimmune camel in the T7 phage display system. We have demonstrated the immunoreactivity of anti-Tg-SdAb (KT75) in immunoassays for thyroglobulin and measured its affinity by surface plasmon resonance (KD ~ 18 picomolar). Additionally, we have shown the quantitative-binding property of SdAb for the first time in IRMA for thyroglobulin. The serum Tg values obtained from SdAb-Tg-IRMA and in-house assay using murine anti-Tg-monoclonal antibody as tracer significantly correlated, r = 0.81, p < 0.05. Our results highlight the scope of using the T7 phage display system as an alternative for the conventional M13-phage to construct single-domain antibody display libraries.

A Microarray Immunoassay for Serum Thyrotropin and Thyroglobulin Using Antibodies Immobilized on Track-Etched Membranes.

Serum thyroglobulin (Tg) and thyroid stimulating hormone (TSH) measurements have evolved as important analytes for monitoring the prognosis of patients with differentiated thyroid cancer, post-thyroidectomy. Individual analyte immunoassay is the current practice in clinical pathology, but the simultaneous assay for all relevant analytes for a given disease, can reduce assay costs, improve patient compliance and give the clinician more information for an unequivocal diagnosis. Microarray immunoassay (MI) can achieve this goal and, hence, we have developed and validated a immuno-radiometric MI for quantitation of serum TSH and Tg by using highly micro-porous polycarbonate (PC) track-etched membranes (TEM) to immobilize the monoclonal anti-TSH and polyclonal anti-Tg antibodies in ~1 mm diameter spots. Non-competitive immunoassays were performed using mixture of 125I labeled monoclonal anti-TSH and anti-Tg antibodies. Phosphorimager was used to quantify the bound radioactivity. TSH and Tg were detected with detection limit of 0.07 µIU/ml and 0.13 ng/ml respectively, which is lower than the clinically required cut-off level. The assay showed: acceptable intra-assay precision within 20 % and recovery in the range of 76-111.2 %. MI compared well with the established immunoradiometric assay (IRMA) with r = 0.98, p < 0.01 (n = 41). No cross-reactivity was seen between the immobilized antibodies. Although two hormones are addressed in this report, MI using PC TEM and isotopic/non-isotopic tracers has the potential for highly automated multiplexed analysis.

A multi-analyte immunoassay for thyroid related analytes.

We describe the development and validation of multianalyte immunoassays (MAIA) for three analytes, viz., thyroxine (T4), thyroid stimulating hormone (TSH), and thyroglobulin (Tg) essential for assessment of thyroid function but having widely varying molecular weights. Using polycarbonate (PC) track-etched membranes (TEM) as an immobilization support and 125I as the tracer, both competitive assay for T4 and non-competitive assay for TSH and Tg were performed on the same TEM. MAIA was found to be highly sensitive and precise with clinically useful working range and correlated very well with individual analyte immunoassays. While we have demonstrated this assay format with radiotracer, it can be used with non-isotopic tracers equally well.