Linear Standard Curve Research Articles

Phytochemical analysis and antioxidant efficacy of Chrysojasminum fruticans (L.) Banfi in Syrian flora

This study investigates the antioxidant properties of Chrysojasminum fruticans (L.) Banfi through a series of assays to measure the total phenolic content (TPC), total flavonoid content (TFC), and free radical scavenging activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, to ensure that it can be used as an antioxidant drug. The TPC, TFC and DPPH assay were performed using spectrophotometric method, and in terms of a linear standard curve for gallic acid, Quercetin, Vitamin C respectively. The.aqueous extract of the flowers exhibited the highest concentration of phenolics, reaching 81.9 mg GAE (Gallic Acid Equivalent) per gram of (Dry Extract) DE, Conversely the ethanolic extract from the fruits demonstrated the lowest phenolic content, with a mere 0.249 (mg GAE/g DE). the aqueous flower extract demonstrated the highest flavonoid concentration, achieving an impressive 113.584 mg quercetin equivalents per gram of DE (mg QUE/g DE). In contrast, the methanolic fruit extract exhibited the lowest flavonoid concentration, measured at a mere 0.695 (mg QUE/g DE). Additionally, the methanolic flower extract demonstrated superior free radical scavenging activity, requiring only 0.348 mg/mL to inhibit 50 % of DPPH radicals, Conversely, the methanolic fruit extract exhibited the least antioxidant activity, as reflected by its highest IC50 value of 1.996 mg/mL.These results underscore Chrysojasminum fruticans (L.) Banfi potent antioxidant capacity and its established role in traditional medicine practices globally, and encourage to be included as a drug contributing to the treatment of many chronic diseases such as diabetes and cardiovascular diseases, after conducting the necessary clinical studies.

Development of an all-in-one real-time PCR assay for simultaneous detection of spotted fever group rickettsiae, severe fever with thrombocytopenia syndrome virus and hantaan virus prevalent in central China.

Central China has been reported to be one of the most important endemic areas of zoonotic infection by spotted fever group rickettsiae (SFGR), severe fever with thrombocytopenia syndrome virus (SFTSV) and hantaan virus (HTNV). Due to similar clinical symptoms, it is challenging to make a definite diagnosis rapidly and accurately in the absence of microbiological tests. In the present study, an all-in-one real-time PCR assay was developed for the simultaneous detection of nucleic acids from SFGR, SFTSV and HTNV. Three linear standard curves for determining SFGR-ompA, SFTSV-L and HTNV-L were obtained within the range of 101-106 copies/μL, with the PCR amplification efficiencies ranging from 93.46% to 96.88% and the regression coefficients R2 of >0.99. The detection limit was 1.108 copies/μL for SFGR-ompA, 1.075 copies/μL for SFTSV-L and 1.006 copies/μL for HTNV-L, respectively. Both the within-run and within-laboratory coefficients of variation on the cycle threshold (Ct) values were within the range of 0.53%-2.15%. It was also found there was no statistical difference in the Ct values between single template and multiple templates (PSFGR-ompA = 0.186, PSFTSV-L = 0.612, PHTNV-L = 0.298). The sensitivity, specificity, positive and negative predictive value were all 100% for determining SFGR-ompA and SFTSV-L, 97%, 100%, 100% and 99.6% for HTNV-L, respectively. Therefore, the all-in-one real-time PCR assay appears to be a reliable, sensitive, rapid, high-throughput and low cost-effective method to diagnose the zoonotic infection by SFGR, SFTSV and HTNV.

A portable microfluidic electrochemical sensor with nonlinear fit strategy for wide-range uric acid detection

In this work, we have developed a portable electrochemical sensing device controlled by a smartphone through Bluetooth, which was applied for regular uric acid (UA) monitoring in prevention and healthcare management. On the core lies a nonlinear fitting model (I = k·(N − exp(−K·cbulk + b))) which is proposed for the first time and given from the differential equation based on the theoretical analysis. We believe this model can reflect the intrinsic relationship between the obtained current and the bulk analyte concentration, therefore leading to an expanding UA detection range (10-fold) compared to the widely used linear standard curve model and reducing the requirements for the modification of the electrode materials. Besides, the integration of the microfluidic chip promoted the removal of UA oxidation products at the electrode surface, yielding excellent sensing stability with a relative standard deviation (RSD) lower than 1 % during 10 consecutive runs. Consequently, we acquired the relationship between Square Wave Voltammetry (SWV) current and UA concentration (I = 15.27*(2.05 − exp(−0.00108*(cbulk − 663.5))), R2 = 0.9999) in the range of 5 to 1000 μM with a limit of detection (LoD) of 2.4 μM using a completely unmodified screen-printed carbon electrode (SPCE). Proof of concept experiments using 25× diluted human urine spiked with UA yielded a recovery rate of 87.5–101.4 % and a satisfactory selectivity result, which is within the value expected for clinical use, indicating the potential of the developed instrument and nonlinear fitting model for urinary UA detection.

A sensitive and selective voltammetric method for the detection of pyrogallol in tomato and water samples using platinum electrode modified with alizarin red S film.

In contrast to the hyperactive platinum electrode, ARS modified platinum electrode presents a remarkable inertness toward adsorption and surface processes and lends it for further voltammetric applications. Measuring pyrogallol levels in samples is significant for assessing their antioxidant activity, which is crucial for understanding their potential health benefits and ability to combat oxidative stress. In addition, the excess consumption of pyrogallol can have significant negative effects on human health. A voltammetric sensor has been developed for the determination of pyrogallol using ARS modified platinum electrode. The electrode was prepared by electrodeposition of alizarin red S on a platinum electrode using cyclic voltammetry with a potential scan range of - 0.4 to 1.2V against an Ag/AgCl quasi reference electrode for 60 cycles as optimum number of cycles. The modified electrode was characterized by CV and SEM techniques. This modified alizarin red S platinum electrode showed remarkable electrocatalytic performance and stability, resulting in a significant increase in pyrogallol oxidation current by 11.05% compared to the pyrogallol oxidative current at the unmodified platinum electrode. A well-defined oxidation peak was observed at ~ 0.40V. The sensor exhibited a low limit of detection (LOD) of 0.28µM and a linear standard curve covering the ranges of 1.0-40µM and 0.01-10.0mM pyrogallol. Extensive studies were performed to evaluate possible interferences from various organic and inorganic compounds and yielded satisfactory results that confirm the selectivity of the developed sensor for pyrogallol determination. In addition, the ARS-Pt electrode provided consistently reliable results for the accurate detection of pyrogallol in water and tomato samples.

Formulation and Assessment of Lipid-based Nanoformulation of Luliconazole

The luliconazole preformulation study aimed to determine the best way to administer medicine using SNEDDS. The plain white powder, luciconazole, showed a number of different crystal structures and was very soluble in isopropyl myristate and oleic acid, as confirmed through partition coefficients in n-octanol. With a robust linear standard curve at 295 nm, UV spectroscopy verified its dependability for analysis. Luliconazole and the necessary excipients for SNEDDS formulation were found to be compatible according to fourier-transform infrared spectroscopy (FTIR) spectra. Using a 32-factorial design enhanced the efficacy and stability of SN-7 batches, especially when diluted with water. Thermal studies revealed formulation dynamics-critical thermal behaviors and crystalline structures (DSC and XRD). The end SNEDDS showed a strong suppression of Candida albicans growth and a higher viscosity, allowing for longer surface retention. Ethical approval facilitated comparative evaluations against a commercial formulation. High-performance liquid chromatography (HPLC) analysis validated SN-7’s purity, enabling a benchmark for monitoring luliconazole plasma concentrations post-application and ex-vivo permeation studies, vital for potential fungal infection treatments. A rigorous three-month stability study affirmed SN-7’s unwavering properties across diverse environmental conditions, ensuring sustained effectiveness. An IEAC-approved skin irritation study on albino wistar rats endorsed SN-7’s minimal irritancy, underscoring its safety and comfort, which is pivotal for pharmaceutical acceptance in topical drug delivery.

The design of multifunctional fluorescent probe for detecting Zn2+/ ClO−/H2O and application in high-security level information encryption

In this article, a Schiff base-structured compound (TNB) was synthesized using 3-hydroxy-2-naphthaldehyde hydrazone and 2-hydroxy-5-tert-butylbenzaldehyde as starting materials. The structure of TNB was characterized using NMR spectroscopy, high-resolution MS, and IR spectroscopy. Studies showed that TNB can selectively recognize Zn2+/ClO− in solvent system of H2O/DMSO. Fluorescence and UV–vis spectrophotometry were used to investigate the spectral properties of TNB. TNB displayed a significant fluorescence enhancement effect when bound to Zn2+, while binding to ClO− resulted in fluorescence quenching. Job’s plot and NMR titration were used to study the binding mechanism of TNB with Zn2+/ClO−. Linear standard curves were fitted for different Zn2+/ClO− concentrations corresponding to fluorescence intensity, and detection limits (LOD) were determined as 5.0300 × 10−9 mol/L (Zn2+) and 3.1700 × 10−9 mol/L (ClO−). Furthermore, TNB was used to determine trace water in organic solvents. The fluorescence intensity changes with water content were measured in three organic solvents (THF, DMSO, and acetone). Detection limits (LOD) were calculated as 1.4 × 10−4 % (THF), 8.3 × 10−4 % (DMSO), and 1.2 × 10−3 % (acetone). Using the characteristic reactions of TNB with Zn2+, a two-dimensional code information encryption technology was developed and can be used for high-security information encryption and transmission.

Adaptation of a Commercial Qualitative BAX® Real-Time PCR Assay to Quantify Campylobacter spp. in Whole Bird Carcass Rinses.

Poultry is the primary reservoir of Campylobacter, a leading cause of gastroenteritis in the United States. Currently, the selective plating methodology using selective agars, Campy Cefex and Modified Charcoal Cefoperazone Deoxycholate agar, is preferentially used for the quantification of Campylobacter spp. among poultry products. Due to the specific nature of Campylobacter, this methodology is not sensitive, which can lead to skewed detection and quantification results. Therefore, Campylobacter detection and quantification methods are urgently needed. The objective was to develop a shortened enrichment-based quantification method for Campylobacter (CampyQuant™) in post-chill poultry rinsates using the BAX® System Real-Time PCR assay for Campylobacter. The specificity and sensitivity for the detection of C. jejuni, C. coli, and C. lari in pure culture were determined. The BAX® System Real-Time PCR assay consistently detected and identified each species 100% of the time with an enumeration range of 4.00 to 9.00 Log10 CFU/mL. Enrichment time parameters for low-level concentrations (0.00, 1.00, and 2.00 Log10 CFU/mL) of Campylobacter using the BAX® System Real-Time PCR assay were elucidated. It was determined that an enrichment time of 20 h was needed to detect at least 1.00 Log10 CFU/mL of Campylobacter spp. Using the BAX® System Real-Time PCR assay for Campylobacter. As a result, time of detection, detection limits, and enrichment parameters were used to develop the CampyQuant™ linear standard curve using the detected samples from the BAX® System Real-Time PCR assay to quantify the levels in post-chill poultry rinsates. A linear fit equation was generated for each Campylobacter species using the cycle threshold from the BAX® System Real-Time PCR assay to estimate a pre-enrichment of 1.00 to 4.00 Log10 CFU/mL of rinsates detected. The statistical analyses of each equation yielded an R2 of 0.93, 0.76, and 0.94 with a Log10 RMSE of 0.64, 1.09, and 0.81 from C. jejuni, C. coli, and C. lari, respectively. The study suggests that the BAX® System Real-Time PCR assay for Campylobacter is a more rapid, accurate, and efficient alternative method for Campylobacter enumeration.

Optimization of Automated Dimethylmethylene Blue Assay for Quantification of Pediatric Urine Samples.

The metachromatic dye dimethylmethylene blue is used to quantify total glycosaminoglycans in urine. Understanding the interaction of dimethylmethylene blue with glycosaminoglycans is pertinent to optimize the assay procedure depending on the type of sample and interpret the findings meaningfully. The present spectrophotometric study determined the optimum sample-to-dye ratio, primary wavelength for measuring absorbance, after studying the interaction of two different chondroitin sulfate species (unfractionated chondroitin sulfate from bovine trachea vs. chondroitin sulfate oligosaccharide with degree of polymerization of 12, from shark cartilage) with dimethylmethylene blue. Respective dye-glycosaminoglycan complexes of the two chondroitin sulfate species showed significantly different absorbance maxima, while that of the chondroitin sulfate oligosaccharide was closer to absorbance maxima of urine glycosaminoglycans. The chondroitin sulfate oligosaccharide showed relatively less stable absorbance readings at higher concentrations in the reaction volume. Furthermore, the chondroitin sulfate reference materials exhibited differences in the linearity of standard curves and hence parallelism. Based on the findings, the method was semiautomated on Beckman Coulter D✕C 700 biochemistry analyzer using the chondroitin sulfate oligosaccharide as the standard. The urine glycosaminoglycan concentration obtained was slightly lower but reasonably close to that obtained through the External Quality Assurance (EQA) scheme administrated by ERNDIM (European Research Network, Inherited Disorders of Metabolism). The findings of the present study can be used to guide the dimethylmethylene blue assay optimization, redevelopment efforts, and harmonization across laboratories. The chondroitin sulfate oligosaccharide is better than the unfractionated chondroitin sulfate from bovine trachea due to its absorbance maxima closer to urine glycosaminoglycans. On the other hand, unfractionated chondroitin sulfate exhibit poor parallelism leading to falsely lower urine glycosaminoglycan levels.

Levofloxacin impregnation and extended release: Concentration model for insulin catheters

Increasing the lifetime of percutaneous insulin delivery catheters to minimize painful needle sticks increases the risk of infection. To combat surface-adherent bacteria and potential biofilm formation, we developed a method to impregnate levofloxacin antibiotic into the outer polymer of the catheter so that it can leach into the external medium. Complementarily, we developed a method to quantify the concentration of antibiotic to ensure the minimum inhibitory concentration (MIC) is maintained throughout the duration of catheter use. Additionally, we have investigated the extent of antibiotic resistance after a zwitterionic nonfouling coating treatment to ensure suitable release after further surface modification. Levofloxacin was incorporated into the catheter through the swelling of the outer polyurethane of the bilayer catheter and subsequent solvent evaporation. The zone of inhibition was measured over time with S. epidermidis on LB agar plates. The release of levofloxacin was measured using optical absorbance spectroscopy in water and quantified against a linear standard curve at 292 nm. We developed a model using Fick's second law of diffusion to calculate the concentration around the catheter. Visible inhibition of bacterial growth and biofilm formation was shown for up to 38 days. A total of 8–45 µg of antibiotic was released over 26 days and a concentration of 820,000 µg/mL in the 0.17 µL control volume was reached at day 26 for the minimally-loaded samples, an amount many orders of magnitude greater than the MIC. After the nonfouling treatment, the lowest concentration reached was 16 µg/mL after 26 days. In conclusion, we have demonstrated a suitable method to induce antibacterial activity for insulin catheters, maintain the MIC, and sustain antibacterial character following further surface coatings.

Automated and fast online method for simultaneously determining a broad spectrum of per- and polyfluoroalkyl substances in a small volume of cerebrospinal fluid.

Per- and polyfluoroalkyl substances (PFASs) are potentially neurotoxic compounds. Levels of PFASs in cerebrospinal fluid (CSF) could directly reflect their potential harm to the central nervous system. Because of the variety of PFASs and the rarity of CSF, there is an urgent need to establish a rapid online method to detect a broad spectrum of PFASs accurately and simultaneously by consuming a small amount of CSF. In this study, we developed a fast and automated method to analyze 52 PFASs in human CSF samples using online TurboFlow ultra-high-performance liquid chromatography-tandem mass spectrometry. Our method offered excellent matrix-matched standard curve linearity (correlation coefficient > 0.99), good limits of quantitation (MLOQs) (0.01 to 0.08 ng mL-1), satisfactory accuracy (recoveries of 74.6%-119.1%) and precision (relative standard deviations of 1.4%-13.2%), small sample amount consumption (50 μL), and fast analysis time (18 min per sample) without complex sample pretreatment procedures. These are advantageous for the high throughput screening of PFASs in environmental epidemiology studies. Repeated freeze-thaw experiments showed that it was better to perform the analytical process soon as possible after sample collection. The established method was used to analyze PFASs in 60 people. Short-chain PFASs, perfluorobutanoic acid (PFBA), perfluoropentanoic acid (PFPeA), and novel PFASs [sodium 2-(N-ethylperfluorooctane-1-sulfonamido)ethyl phosphate (SAmPAP), perfluoroethylcyclohexanesulfonate (PFECHS), and perfluoro-3, 7-dimethyloctanoic acid (P37DMOA)] were reported in CSF for the first time. PFBA and PFPeA were detected in all samples with mean concentrations of 0.24 and 0.22 ng mL-1, respectively. We also calculated the blood-brain barrier transmission efficiency of PFASs (RPFAS), and the mean RPFBA value was above 1, which indicated that PFBA might transfer from serum to CSF.

Optimizing Molecular Detection for the Hop Downy Mildew Pathogen Pseudoperonospora humuli in Plant Tissue

Downy mildew-free hop plantlets and rhizomes are essential to limit the introduction of this destructive pathogen, Pseudoperonospora humuli, into hopyards. The objective of this research was to determine which DNA-based diagnostic tools are optimal for P. humuli detection in plant tissue. Quantitative real-time PCR (qPCR) assays with TaqMan probes for nuclear (c125015.3e1) and mitochondrial (orf359) DNA loci were developed and tested side by side. A recombinase polymerase amplification (RPA) assay was designed based on the orf359 DNA locus. The mitochondrial qPCR assay had a 10-fold lower limit of detection (100 fg of genomic DNA) and was 60% more effective in detecting P. humuli in asymptomatic stems than the nuclear-based assay. Both qPCR assays had linear standard curves (R2 > 0.99) but lacked the quantitative precision to differentiate leaf infections beyond 1 day postinoculation. A wide range of Cq values (≥4.9) in standardized tests was observed among isolates, suggesting that the number of mitochondria and nuclear DNA targets can vary. The absence of P. humuli DNA in symptomatic rhizomes was explained, in part, by the detection of Phytophthora DNA. However, the Phytophthora-specific atp9-nad9 assay cross-reacted with P. humuli, leading to false positive amplification. Sensitivity in the RPA assay was reduced by crude plant DNA extract. Improvements to the objectivity of calling positive amplifications and determining the onset of amplification from RPA fluorescence data were realized by applying the first and second derivatives, respectively. The orf359 qPCR assay is specific and sensitive, making it well suited for P.humuli diagnostics in plant tissue.

A HPLC-ESI-Q-ToF-MS Method for the Analysis of Monomer Constituents in PHGG, Gum Arabic And Psyllium Husk Prebiotic Dietary Fibre Supplements

ABSTRACT “Soluble” type dietary fibre contains polysaccharides with monomer constituents that play a major role in lowering the glycemic index of food (“low-GI”) and improving colonic health through gut microbiome fermentation. Soluble fibres such as partially hydrolysed guar gum (PHGG), gum Arabic and psyllium husk are of particular interest in gastrointestinal research, exploring their effects in motility disorders such as gastroparesis, constipation, diarrhea and irritable bowel syndrome (IBS) is important. The primary aim of this research was to develop a rapid pre-column derivatized HPLC-ESI-Q-ToF-MS method for the identification and quantification of monomer sugar constituents in commercial dietary fibre supplements of PHGG, gum Arabic and psyllium husk. Polysaccharides in the samples were hydrolysed and derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP). A rapid 21 min HPLC-ESI-Q-ToF-MS method separated nine different PMP-labelled monomer sugars. Five different sugars (i.e., Galactose, Arabinose, Rhamnose, Mannose and Xylose) were identified and quantified. Standard curve linearity was excellent (R2 > 0.999), with good intra-laboratory precision (< ±5% SD for concentrations, ≤ ±0.02 min peak shifting for retention times). The average three-level (50%, 75% and 100%) spiking recoveries for the analytes was acceptable (96.22–109.49%). Positive scanning mode ESI-MS/MS was used to generate [M + H]+ precursor ions and three (m/z) product ion fragments to provide identity confirmation. The concentrations of monomer sugars in gum Arabic and psyllium husk dietary fibres were consistent with literature reports. In PHGG, the concentration of galactose was unexpectedly higher than mannose, indicating that the commercial hydrolysis process of guar gum during PHGG manufacturing affected monomer composition. The method presented here provides the basis for the standardisation and labelling of commercial PHGG, gum Arabic and psyllium husk supplements. Future research will need to explore the monomer variability in other prebiotic soluble fibres and their efficacy in the production of beneficial short-chain fatty acids (SCFA) in the colonic environment.

Discrimination of mucinous pancreatic cysts using an enzymatic turnover assay to improve clinical diagnostic accuracy.

4151 Background: One impact of medical imaging technology has been an approximately 3-fold increase in the incidental detection of pancreatic cysts during routine clinical examinations. To reduce burden on the healthcare system and patients, clinicians desire accurate classification of pancreatic cysts into benign non-mucinous or potentially malignant, mucinous populations. Using EUS-FNA, fluid from these cysts can provide molecular biomarkers of predictive value. However, current in vitro diagnostics lack the desired sensitivity and specificity for clinicians to accurately stratify patients for risk of pre-malignant cancers. This situation can be improved by introducing new biomarkers and novel assay platforms. An enzymatic biomarker, pepsin C, has shown high accuracy for diagnosing mucinous cysts. The use of enzymatic activity assays is applicable to clinical workflows without disruption of current standards of care. Methods: A pepsin C activity assay using a magnetic bead-based platform was developed, with both fluorescent and surface-enhanced Raman spectroscopy (SERS) readouts. The assay platform utilizes selective peptide substrates, and a dimeric Rhodamine-6G-based dye, which allows ultrasensitive detection and significantly decreases the sample volume requirement for analysis, down to 1 µL of cyst fluid. The dye-labeled substrate is immobilized on magnetic beads and reacted with enzyme-containing samples to produce a quantitative assay signal that is standardized and expressed in true enzyme activity units. Results: While both readouts were quantitative and produced linear standard curves, SERS-based analysis was more robust against established biological matrix effects than fluorescence. Nevertheless, both assay modes successfully differentiated mucinous and non-mucinous cysts in a retrospective cohort of 69 cyst fluid samples. Compared with the standard of care CEA assay, this activity-based assay displays much improved sensitivity in diagnosing mucinous pancreatic cysts (Table). Conclusions: This pepsin c activity assay differentiates between mucinous and non-mucinous cysts better than the CEA assay and provides a quantifiable standardized readout. This work establishes a path to a true rule-out assay enabling clinicians to better stratify patients into low risk vs. potential malignancy thus impacting treatment and monitoring plans. [Table: see text]

Evaluation of a Quantitative Taqman Real-Time PCR Assay to Measure Proviral load from Human Immunodeficiency Virus Type 1 individuals

Human Immunodeficiency Virus (HIV) is a virus belonging to the family Retroviridae. HIV – 1 is found to be predominant in India and many parts of Africa. The intention of this study was to quantify the HIV Proviral Deoxyribonucleic Acid (DNA) from newly infected HIV-1 individuals. Fifty patients who were tested positive for HIV were included in this study. Proviral Ribo Nucleic Acid (RNA) was extracted by QIAmp® RNA Mini Kit (QIAGEN, Germany) method. Complementary Deoxyribo Nucleic Acid (cDNA) was synthesized by using Invitrogen Superscript III cDNA synthesis Kit (USA). This cDNA was subjected to Polymerase Chain Reaction (PCR) and Gene cloning by transformation method. The quantification of Real time PCR was done by Applied Bio-System (ABI)-Prism 7700. A linear standard curve was obtained 10 copies to 106 copies per reaction. The assay had good analytic sensitivity and linear dynamic range greater than 6 logs. From the results obtained in this study, It was concluded that Taqman Real-Time PCR Assay plays a major role in monitoring the HIV infected patients in routine diagnostics and clinical practice.

Solvothermal production of tea residue derived carbon dots by the pretreatment of choline chloride/urea and its application for cadmium detection

Cadmium contamination is a serious environmental issue and its detection is important. In this study, tea residue after pretreatment by the deep eutectic solvent of choline chloride/urea was used for one-pot solvothermal synthesis of carbon dots (CDs) applied in cadmium detection for the first time. The effect of choline chloride/urea molar ratio, reaction period, and reaction temperature on the CDs synthesis was learned and a promising synthesis system (choline chloride/urea molar ratio of 1:2, reaction at 200 °C for 8 h) was built. Under this reaction condition, the CDs yield and the quantum yield were 2.25% and 16.99%, respectively. The optical properties and structure of CDs were learned by fluorescence spectrophotometer, UV–vis spectrophotometer, transmission electron microscope (TEM), field emission scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray diffractometer (XRD), and X-ray photoelectron spectrometer (XPS). According to the characterization, the CDs had a strong peak at 410 nm with the corresponding excitation wavelength of 330 nm and contained various functional groups including hydroxyl, amino, and carbonyl. The CDs were suitable for cadmium detection by a linear standard curve with a high correlation coefficient (R2 =0.99). Overall, this study offers a promising method for utilization of waste biomass (tea residue) for fluorescent materials (CDs) production.

Targeted quantitation of CFTR protein expression in vivo using immunoprecipitation &amp; parallel reaction monitoring tandem mass spectrometry

Abstract Background The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a low-abundance membrane protein. The dysfunction of CFTR protein is the fundamental cause of cystic fibrosis (CF), a fatal genetic disease. In recent years, the novel messenger RNA (mRNA)-based therapy shows high potential to treat CF disease, by delivering CFTR mRNA into lung epithelial cells to generate fully functional CFTR replacement protein. To evaluate mRNA drug efficacy, a targeted quantitative proteomics method is needed to estimate the expression level of mRNA encoded CFTR protein. Methods In this paper, a method combining membrane protein extraction, immunoprecipitation (IP), and nanoLC-MS/MS for quantifying CFTR in lung tissue samples was reported for the first time. Absolute quantification was performed by constructing a standard curve by spiking recombinant human CFTR protein in mouse lung tissue matrix. Results This method was qualified, with good linearity of standard curve and lower limit of quantification of human CFTR at 1.4 pg per mg tissue. The coefficient of variation of back calculated concentration of all standards and their back-calculation errors were &lt; 20%. The CFTR expression level in mouse lungs dosed with mRNA encapsulated lipid nanoparticle (LNP), and the endogenous level in wild type human lungs were measured successfully. Conclusions The result demonstrated high sensitivity, precision and accuracy of this proteomics method for quantifying low-abundance CFTR protein in lung tissue sample.

Rapid Detection and Differentiation of Legionella pneumophila and Non-Legionella pneumophila Species by Using Recombinase Polymerase Amplification Combined With EuNPs-Based Lateral Flow Immunochromatography.

Legionella, a waterborne pathogen, is the main cause of Legionnaires’ disease. Therefore, timely and accurate detection and differentiation of Legionella pneumophila and non-Legionella pneumophila species is crucial. In this study, we develop an easy and rapid recombinase polymerase amplification assay combined with EuNPs-based lateral flow immunochromatography (EuNPs-LFIC-RPA) to specifically distinguish Legionella pneumophila and non-Legionella pneumophila. We designed primers based on the mip gene of Legionella pneumophila and the 5S rRNA gene of non-Legionella pneumophila. The recombinase polymerase amplification reaction could go to completion in 10 min at 37°C, and the amplification products could be detected within 5 min with EuNPs-LFIC strips. Using a florescent test strip reader, the quantitative results were achieved by reading the colored signal intensities on the strips. The sensitivity was 1.6 × 101 CFU/ml, and a linear standard linear curve plotted from the test strip reader had a correlation coefficient for the determination of Legionella pneumophila (R 2 = 0.9516). Completed concordance for the presence or absence of Legionella pneumophila by EuNPs-LFIC-RPA and qPCR was 97.32% (κ = 0.79, 95% CI), according to an analysis of practical water samples (n = 112). In short, this work shows the feasibility of EuNPs-LFIC-RPA for efficient and rapid monitoring of Legionella pneumophila and non-Legionella pneumophila in water samples.

Survey and risk assessment on residual pesticides and heavy metals in coffee beans distributed in Daejeon

The content of harmful materials was investigated for coffee beans sold in Daejeon. Total 79 samples were analysed and 213 residual pesticides and 2 heavy metals were analysed simultaneously by GC-MS/MS, GC-NPD, GC-ECD, LC-MS/MS and ICP-MS. The instrumental method was validated with limit of detection (LOD), limit of quantification (LOQ), the linearity of standard curves. LOD of the residual pesticides was between 0.0003 to 0.0021 mg/kg, LOQ of the residual pesticides was between 0.0008 to 0.0064 mg/kg. LOD of the heavy metals was between 0.0077 to 0.0079 μg/kg, LOQ of the heavy metals was between 0.0233 to 0.0239 μg/kg. The linearity correlation coefficient for the calibration curve was between 0.9929 to 0.9999 and the recovery rate was between 95.4% to 106.1%. According to the monitoring of residual pesticides and heavy metals, no pesticide was detected in all coffee bean samples. 88.6% (70 samples) of analysed total 79 coffee beans contained at least 1 heavy metal but there was no sample which exceeded the maximum residual limit. Risk assessment was also carried out based on the content of heavy metals detected in coffee beans. The carcinogenic risk assessment to heavy metals showed that all cancer-risk (CR) values were below 10–6 and it meant that the CR due to heavy metals intake was evaluated as safe. The non-carcinogenic risk assessment to heavy metals showed that all hazard index (HI) were below 1, which was considered acceptable at the current level of exposure. The %PTWI values of lead and cadmium for 55 roasted coffee bean samples were 0.09% and 0.04% respectively, compared with the reference values. This results indicate that there is almost no health risk from heavy metal intake through the consumption of coffee beans in circulation in Daejeon.

Survey and risk assessment on residual pesticides and heavy metals in coffee beans distributed in Daejeon

The content of harmful materials was investigated for coffee beans sold in Daejeon. Total 79 samples were analysed and 213 residual pesticides and 2 heavy metals were analysed simultaneously by GC-MS/MS, GC-NPD, GC-ECD, LC-MS/MS and ICP-MS. The instrumental method was validated with limit of detection (LOD), limit of quantification (LOQ), the linearity of standard curves. LOD of the residual pesticides was between 0.0003 to 0.0021 mg/kg, LOQ of the residual pesticides was between 0.0008 to 0.0064 mg/kg. LOD of the heavy metals was between 0.0077 to 0.0079 μg/kg, LOQ of the heavy metals was between 0.0233 to 0.0239 μg/kg. The linearity correlation coefficient for the calibration curve was between 0.9929 to 0.9999 and the recovery rate was between 95.4% to 106.1%. According to the monitoring of residual pesticides and heavy metals, no pesticide was detected in all coffee bean samples. 88.6% (70 samples) of analysed total 79 coffee beans contained at least 1 heavy metal but there was no sample which exceeded the maximum residual limit. Risk assessment was also carried out based on the content of heavy metals detected in coffee beans. The carcinogenic risk assessment to heavy metals showed that all cancer-risk (CR) values were below 10–6 and it meant that the CR due to heavy metals intake was evaluated as safe. The non-carcinogenic risk assessment to heavy metals showed that all hazard index (HI) were below 1, which was considered acceptable at the current level of exposure. The %PTWI values of lead and cadmium for 55 roasted coffee bean samples were 0.09% and 0.04% respectively, compared with the reference values. This results indicate that there is almost no health risk from heavy metal intake through the consumption of coffee beans in circulation in Daejeon.

Simultaneous determination of three antituberculosis drugs in the serum of patients with spinal tuberculosis by capillary electrophoresis.

The pharmacokinetic variations of a single drug in an antituberculosis regimen are associated with acquired drug resistance and therapy failure. This study aimed to develop a simple and effective method for monitoring the serum levels of isoniazid (INH), rifampicin (RFP), and pyrazinamide (PZA), three antibiotics used in patients with spinal tuberculosis using capillary electrophoresis (CE). A standard solution of INH, RFP, and PZA was prepared and mixed with serum to prepare the standard curve. The detection limit, quantification limit, precision, stability, repeatability, and sample recovery were determined. Then, INH, RFP, and PZA were measured from the leftover serum samples of all patients with spinal tuberculosis who were treated with 2SHRZ/2.5H2R2Z2 combined with surgery in a tertiary hospital in Qinghai from October 2015 to September 2017. A total of 107 patients with spinal tuberculosis treated using the 2SHRZ/2.5H2R2Z2 regimen combined with surgery were included in this study. All three antibiotics had linear standard curves with high correlation coefficients (R2 = 0.9997, 0.9994, and 0.9986). The recovery rates were 98.1% for INH, 96.5% for PZA, and 97.2% for RFP. The results from the serum samples showed that the plasma concentrations of INH (4.989 ± 1.692 μg mL-1) and RFP (9.400 ± 1.711 μg mL-1) reached effective therapeutic concentrations in all patients, but not PZA (33.860 ± 1.830 μg mL-1). The CE method for measuring INH, RFP, and PZA simultaneously in serum samples of patients with spinal tuberculosis is simple, rapid, and sensitive. This method is suitable for the routine monitoring of INH, RFP, and PZA concentrations in the serum of patients with spinal tuberculosis.