Min Incubation Time Research Articles

Fast and on-site detection of fenthion in rice using core-shell Au@Ag nanoparticles and a portable Raman spectrometer

The residue of pesticide fenthion is a common issue in food safety, and how to conveniently detect it is a challenge in the food industry. Therefore, combined with a portable Raman spectrometer, a surface enhanced Raman spectroscopy (SERS) method based on a high-performance substrate Au@AgNPs had been designed for the rapid and sensitive detection of fenthion. Au@AgNPs can be induced to generate more hot spots to improve SERS signals because of their core-shell structure. The quantitative detection of fenthion was achieved under the optimal conditions: pH 7.0, 10−3 M CaCl2, 70 % Au@AgNPs concentration, and 4 min of incubation time. The results showed that the method had a wider detection range of 0.1–100 mg/L, a low detection limit (LOD) of 0.036 mg/L, excellent anti-interference ability, and reproducibility. In addition, the method was successfully validated with the actual sample rice. Therefore, the method had the advantages of being fast, on-site, and easy to use, which is useful for promoting its application.

Non-enzymatic sensor based on doped polyindole/multi-walled carbon nanotube for detecting neurotransmitter acetylcholine

A facile non-enzymatic acetylcholine sensor composed of doped-polyindole/multi-walled carbon nanotube (dPIn-MWCNT) composite was designed, fabricated, and presented here. The negative current response toward acetylcholine concentration was first found and reported here. The confirmation of acetylcholine detection mechanism was elucidated by XPS, where the acetylcholine interaction resulted in a decrease in the % (dPIn doping level and an increase of the dPIn structural defect. The response optimizations with dPIn concentrations, applied potentials, and incubation times were systematically investigated. The 45dPIn-15MWCNT operated at + 0.6 V vs. Ag/AgCl with 5 min of incubation time was the optimum condition to obtain the highest acetylcholine current response. Chronoamperometric acetylcholine detection revealed two distinct current regimes from the acetylcholine surface interaction and the de-doping of dPIn. Regime I was related to the diffusion-controlled process as observed in the low concentration range of 10−8–10−5 M. Regime II corresponded to the mixed diffusion-controlled and adsorption-controlled process as observed in the concentration range of 10−5–10−2 M. For regime I, the calculated LOD was as low as 1.27 nM with a very high acetylcholine response sensitivity. Regime I is suggested for the point-of-care utility, where the detectable concentration range covered the acetylcholine level normally found in the healthy human blood. Regime II can be applicable and useful in the treatment monitoring of neurological diseases.

Evaluation of a Revised Point-of-Care Test for the Detection of Feline Leukaemia p27 Antigen and Anti-p15E Antibodies in Cats.

The first point-of-care (PoC) test (v-RetroFel®; modified version 2021) determining the presence of FeLV p27 antigen and FeLV anti-p15E antibodies has become recently commercially available to identify different feline leukaemia virus (FeLV) infection outcomes. This study aimed to assess this PoC test's performance concerning FeLV p27 antigen and FeLV anti-p15E antibody detection. Sensitivity, specificity, positive and negative predictive values (PPV, NPV) were assessed after ten minutes (recommended) and 20 min (prolonged) incubation times. The test results were evaluated as either positive or negative. Serum samples from 934 cats were included, originating from Italy (n = 269), Portugal (n = 240), Germany (n = 318), and France (n = 107). FeLV p27 antigen and anti-p15E antibodies were measured by reference standard ELISAs and compared to the PoC test results. The PoC test was easy to perform and the results easy to interpret. Sensitivity and specificity for FeLV p27 antigen were 82.8% (PPV: 57.8%) and 96.0% (NPV: 98.8%) after both, ten and 20 minues of incubation time. Sensitivity and specificity for anti-p15E antibodies were 31.4% (PPV: 71.6%) and 96.9% (NPV: 85.1%) after ten minutes incubation time; sensitivity was improved by a prolonged incubation time (20 min) to 40.0% (PPV: 76.3%), while specificity remained the same (96.9%, NPV: 86.7%). Despite the improved sensitivity using the prolonged incubation time, lower than ideal sensitivities for both p27 antigen and especially anti-p15E antibodies were found, indicating that the PoC test in its current version needs further improvement prior to application in the field.

Plasma Glucose Concentrations in Different Sampling Tubes Measured on Different Glucose Analysers.

The German Diabetes Association recommends using sampling tubes with citrate and fluoride additives to diagnose diabetes by oral glucose tolerance test to inhibit glycolysis. The effect of different tubes on measurement results was assessed. In a first study, an oral glucose tolerance test was performed on 41 participants without anamnestically known diabetes. Venous blood was sampled in two different tubes with citrate/fluoride additives from different manufacturers and one with only lithium-heparin additive. A second study with 42 participants was performed to verify the initial results with an adapted design, in which a third tube with citrate buffer was used, and glucose measurements were performed on two additional devices of another analyser model. Samples were centrifuged either immediately (<5 min incubation time) or after 20 min or 4 h. All glucose measurements were performed in plasma. Glucose concentrations in lithium-heparin tubes with<5 min incubation time served as baseline concentrations. In the first study, glucose concentrations in one of the citrate/fluoride tubes were similar to the baseline. In the other citrate/fluoride tube, markedly lower concentrations (approximately - 5 mg/dL (- 0.28 mmol/L)) were measured. This was reproduced in the verification study for the same analyser, but not with the other analyser model. Lithium-heparin tubes centrifuged after 20 and 240 min showed systematically lower glucose concentrations. The results confirm that glycolysis can be effectively inhibited in citrate/fluoride-containing sampling tubes. However, glucose measurement results of one analyser showed a relevant negative bias in tubes containing liquid citrate buffer.

Visualizing anions by color changing response: Exploitation of pyrimidine-based probe for simultaneous detection of CN− and F− ions

An easily scalable pyrimidine-based charge-transfer probe has been synthesized that can be utilized for simultaneous detection of both CN− and F− ions. The orange-colored solution of compound turns into yellow upon addition of F− ion, while the spiking of CN- ion leads to a colorless solution. Such color-changes have also been noticed on solid-supported materials, such as modified paper strips. Interestingly, the interaction with F- is found to be rapid as well as reversible (checked against water), while for cyanide ion, the scenario is completely opposite, the interaction is rather slow (∼10 min incubation time), and irreversible. The detailed spectroscopic investigation indicated that F−, being basic in nature, participates in the hydrogen bonding interaction. However, CN− ion, due to its highly nucleophilic character, formed Michael addition reaction product. Both these interactions substantially curtail the extent of intramolecular charge transfer and thereby resulting distinct changes in visible color. Detection of multiple anions via distinct naked-eye color changing response is not very common in the literature and therefore will be beneficial for future development of new sensory systems.

Colorimetric sensing of hydrogen peroxide using capped Morus nigra-sawdust deposited zinc oxide nanoparticles via Trigonella foenum extract.

Hydrogen peroxide (H2O2) is one of the main byproducts of most enzymatic reactions, and its detection is very important in disease conditions. Due to its essential role in healthcare, the food industry, and environmental research, accurate H2O2 determination is a prerequisite. In the present work, Morus nigra sawdust deposited zinc oxide (ZnO) nanoparticles (NPs) were synthesized by the use of Trigonella foenum extract via a hydrothermal process. The synthesized platform was characterized by various techniques, including UV-Vis, FTIR, XRD, SEM, EDX, etc. FTIR confirmed the presence of a Zn‒O characteristic peak, and XRD showed the hexagonal phase of ZnO NPs with a 35nm particle size. The EDX analysis confirmed the presence of Zn and O. SEM images showed that the as-prepared nanoparticles are distributed uniformly on the surface of sawdust. The proposed platform (acetic acid-capped ZnO NPs deposited sawdust) functions as a mimic enzyme for the detection of H2O2 in the presence of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetrically. To get the best results, many key parameters, such as the amount of sawdust-deposited nanoparticles, TMB concentration, pH, and incubation time were optimized. With a linear range of 0.001-0.360μM and an R2 value of 0.999, the proposed biosensor's 0.81nM limit of quantification (LOQ) and 0.24nM limit of detection (LOD) were predicted, respectively. The best response for the proposed biosensor was observed at pH 7, room temperature, and 5min of incubation time. The acetic acid-capped sawdust deposited ZnO NPs biosensor was also used to detect H2O2 in blood serum samples of diabetic patients and suggest a suitable candidate for in vitro diagnostics and commercial purposes.

Discovery of potent and selective activity-based probes (ABPs) for the deubiquitinating enzyme USP30.

Ubiquitin-specific protease 30 (USP30) is a deubiquitinating enzyme (DUB) localized at the mitochondrial outer membrane and involved in PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKβ-USP30-ACLY-regulated lipogenesis/tumorigenesis. A USP30 inhibitor, MTX652, has recently entered clinical trials as a potential treatment for mitochondrial dysfunction. Small molecule activity-based probes (ABPs) for DUBs have recently emerged as powerful tools for in-cell inhibitor screening and DUB activity analysis, and here, we report the first small molecule ABPs (IMP-2587 and IMP-2586) which can profile USP30 activity in cells. Target engagement studies demonstrate that IMP-2587 and IMP-2586 engage active USP30 at nanomolar concentration after only 10 min incubation time in intact cells, dependent on the presence of the USP30 catalytic cysteine. Interestingly, proteomics analyses revealed that DESI1 and DESI2, small ubiquitin-related modifier (SUMO) proteases, can also be engaged by these probes, further suggesting a novel approach to develop DESI ABPs.

Fluorescence Naphthalene Cationic Schiff Base Reusable Paper as a Sensitive and Selective for Heavy Metals Cations Sensor: RSM, Optimization, and DFT Modelling.

Heavy metals are particularly damaging contaminants in the environment, and even trace concentrations represent a risk to human health due to their toxicity. To detect the heavy metals of Mn2+ and Co2+ ions, a novel selective reusable paper-based Fluorescence naked-eye sensor based on naphthalene cationic Schiff base (NCSB) was synthesized and confirmed using FT-IR, 1H-NMR, and MS tools. Based on a blue to colorless color change in the aqueous solution, the NCSB sensor is utilized to Mn2+ and Co2+ cations selectively among other metal ions (Fe2+, Cu2+, Mg2+, Ni2+, Zn2+, Cd2+, Hg2+, Pb2+, Sn2+ and Cr3+). In the aqueous medium, the NCSB sensor displayed high sensitivity, with limits of detection (LOD) values of 0.014 µM (14.08 nM) and 0.041 µM (41.47 nM) for Mn2+ and Co2+ cations, respectively. The paper-based sensor naked-eye detected Mn2+ and Co2+ cations in water at concentrations as low as 0.65 µM (65 nM) and 0.086 µM (86 nM), respectively. It was discovered that 5min of incubation time and a pH range of 7 to 11 were optimal for the complexation reaction between the Mn2+ and Co2+ ions and the NCSB sensor. Through a static quenching process, the interaction of the different metal ions with the Schiff base group in the NCSB molecule results in the development of a ground-state non-fluorescent complex. NCSB sensor was also successfully applied in analysis of Mn2+ and Co2+ in environmental water with good recoveries of 94.8-105.9%. The theoretical calculations based on density functional theory (DFT) studies are in support of experimental interpretations. The links between the input factors and the anticipated response were evaluated using the quadratic model of the response surface methodology (RSM) modeling.

Novel fluorometric protocol for assessing myeloperoxidase activity

Neutrophil myeloperoxidase (MPO) is an essential enzyme for the innate immune system. Measuring MPO activity is vital for understanding neutrophil characteristics and functions in various diseases. MPO activity can be measured using several methods, including spectrophotometric and fluorometric protocols. This paper introduces a fluorometric method for specifically quantifying MPO activity based on the H2O2-dependent oxidation of thiamine. We optimized this new method using the robust statistical approach response surface methodology (RSM) and Box Benken Design (BBD). We extensively examined the effects of several experimental parameters using the RSM methodology and determined the best conditions for accurate and sensitive MPO activity measurement. The optimal conditions were determined using the analysis of variance (ANOVA) for second-order polynomial equations. The resulting F-value (4.86) indicated that the model was significant. However, the lack-of-fitness F-value (1.79) suggested it did not differ significantly from the corresponding p-value. The greatest MPO activity (30 ± 2 U L−1) was obtained under optimum conditions, which were 1000 µM of H2O2, 10 min incubation time, and 1000 µM of thiamine. Our results suggest that this advanced fluorometric method has significant accuracy, sensitivity, and linearity up to 60 IU. The new and standard colorimetric methods also showed a good correlation. These results indicate that the new fluorometric method can be dependable and efficient for assessing MPO activity. The new method is characterized by excellent accuracy, sensitivity, and linearity, making it a valuable protocol for researchers and clinicians interested in assessing MPO activity.

The Dual-Response-Single-Amplification Fluorescent Nanomachine for Tumor Imaging and Gastric Cancer Diagnosis.

Gastric cancer (GC) is one of the most common tumors worldwide and is the leading cause of tumor-related mortality. Traditional biomarkers and screening methods cannot meet the clinical demands. There is an urgent need for highly sensitive diagnostic markers as well as accurate quantification methods for early gastric cancer (EGC) screening. Here a dual-target cooperatively responsive fluorescent nanomachine by the innovative application of two targets─responsive strand migration system with a single-amplification-cycle element was developed for the simultaneous detection of GC biomarkers miR-5585-5p and PLS3 mRNA, which were selected by next-generation sequencing and RT-qPCR. It was also an RNA extraction-free, PCR-free, and nonenzymatic biosensor to achieve tumor cell imaging and serum diagnosis. Requiring only a 20 μL serum sample and 20 min incubation time, the nanomachine achieved an ultrasensitive detection limit of fM level with a broad linear range from fM to nM. More importantly, a higher AUC value (0.884) compared to the clinically used biomarker CA 72-4 was obtained by the nanomachine to distinguish GC patients successfully. Notably, for the key concerns of diagnosis of EGC patients, the nanomachine also achieved a satisfactory AUC value of 0.859. Taken together, this work has screened and obtained multiple biomarkers and developed a fluorescent nanomachine for combination diagnosis of GC, providing an ingenious design of a functionalized DNA nanomachine and a feasible strategy for the transformation of serum biomarkers into clinical diagnosis.

Coffee mucilage clarification: A promising raw material for the food industry

Coffee mucilage is a by-product of interest for its potential for producing antioxidant beverages. This study examined the effect of operating conditions on coffee mucilage clarification to improve the process and transform it in a raw material. A 24 Full Factorial Design (FD) with a central point was used to evaluate the incubation times (6–140 h), clarifying agent concentrations (0.1–1.5 mg/mL), pH (4.0–5.5), and temperatures (21–60 °C) for gelatin and enzymatic treatment on mucilage turbidity, total content of phenolic compounds (substances having an aromatic ring with one or more hydroxyl groups), protein, and carbohydrate content. The statistical analysis of the results was conducted by means of a variance analysis (ANOVA) with 95 % reliability. Gelatin treatment reduced coffee mucilage turbidity by 46–57 % but presented a 39 % reduction in total phenolic content. Statistical analysis showed the enzymatic treatment (pectinase) as the best evaluated clarifying agent, with a 63.7 % decrease in turbidity (0.744 ± 0.021) without significantly affecting the total phenolic content. Pectinase concentration and pH were the factors that most affected the process, suggesting operating conditions of pH 4, 1.1 % pectinase concentration, process temperature 21 °C, and 30 min incubation time.

Quantitative Evaluation of Interleukin-4 by Immunowall Devices Made of Gelatin Methacryloyl Hydrogel.

Immunoassays, which use antigen-antibody reactions, are the primary techniques used to selectively quantify specific disease markers in blood. Conventional immunoassays, such as the microplate-based enzyme-linked immunosorbent assay (ELISA) and paper-based immunochromatography, are widely used, but they have advantages and disadvantages in terms of sensitivity and operating time. Therefore, in recent years, microfluidic-chip-based immunoassay devices with high sensitivity, rapidity and simplicity, which are compatible with whole blood assays and multiplex assays, have been actively investigated. In this study, we developed a microfluidic device using gelatin methacryloyl (GelMA) hydrogel to form a wall-like structure in a microfluidic channel and perform immunoassays inside the wall-like structure, which can be used for rapid and highly sensitive multiplex assays with extremely small sample amounts of ~1 μL. The characteristics of GelMA hydrogel, such as swelling rate, optical absorption and fluorescence spectra, and morphology, were carefully studied to adapt the iImmunowall device and immunoassay. Using this device, a quantitative analysis of interleukin-4 (IL-4), a biomarker of chronic inflammatory diseases, was performed and a limit of detection (LOD) of 0.98 ng/mL was achieved with only 1 μL sample and 25 min incubation time. The superior optical transparency over a wide range of wavelengths and lack of autofluorescence will help to expand the application field of the iImmunowall device, such as to a simultaneous multiple assay in a single microfluidic channel, and provide a fast and cost-effective immunoassay method.

UV–vis Study on β‐Cyclodextrin as Dual Function for Synthesis AgNPs and Antibacterial Application

AbstractThe current study reveals the synthesis of silver nanoparticles (AgNPs) with β‐cyclodextrin (β‐CDs) as a dual function, reducing, and stabilizing agent. UV–vis study is carried out to monitor the optimum condition of AgNPs formation. It is observed that setting pH at 12, 1:33 concentration ratio of AgNO3 to β‐CDs, and 5 min of incubation time are the optimum condition to synthesize AgNPs. The stability of AgNPs 4 weeks after synthesized is confirmed by the UV–vis spectra. Fourier transform infrared (FTIR) characterization reveals there is a peak appearing assigned to C=O which proves a redox reaction occurs between AgNO3 and β‐CDs. Gram‐positive (Bacillus subtilis and Staphylococcus aureus) and Gram‐negative bacteria (Escherichia coli and Pseudomonas aeruginosa) are tested to investigate the antibacterial activity of AgNPs. This study shows that AgNPs exhibit inhibition of bacterial growth.

Enzyme assisted juice extraction from Dacryodes macrophylla as a potential bio-resource for wine production

Atom fruit (Dacryodes macrophylla) is a Non-timber Forest Product (NTFP) that comprises a large seed, thick pulp, and a thin hard outer covering. The structural component of its cell wall and thick pulp make it difficult in extracting the juice. Also, Dacryodes macrophylla fruit is greatly underutilized, therefore the need to process and transform it into other value-added products. This work aims to enzymatically extract juice from Dacryodes macrophylla fruit with the aid of pectinase, ferment and test the acceptability of wine produced from this extract. The enzyme and non-enzyme treatments were carried out under the same conditions and their physicochemical properties such as pH, juice yield, total soluble solids, and Vitamin C were compared. A central composite design was used for the optimization of the processing factors for the enzyme extraction process. Enzyme treatment had a great impact on the juice yield (%) and Total soluble solids (TSS) (0Brix) of samples as it was as high as 81 ± 0.7% and 10.6 ± 0.02 0Brix whereas, that of the non-enzyme treatments were 46 ± 0.7% and 9.5 ± 0.02 0Brix respectively. However, the Vitamin C content of enzyme-treated juice decreased to 11.32 ± 0.13 mg/ml as compared to that of the non-enzyme-treated juice sample (15.7 ± 0.04 mg/ml). The optimum processing condition in the extraction of juice from the atom fruit was 1.84% enzyme concentration, 49.02 ֯C Incubation temperature, and 43.58 min Incubation time. During wine processing within 14 days of primary fermentation, the pH of the must decreased from 3.42 ± 0.07 to 3.26 ± 0.07 whereas the Titratable acidity (TA) increased from 0.16 ± 0.05 to 0.51 ± 0.0. The wine produced from Dacryodes macrophylla fruit showed promising results as its sensorial scores for all attributes including color, clarity, flavor, mouthfeel, alcoholic burn after taste and overall acceptability were all above 5. Thus, enzymes can be used to improve the juice yield of Dacryodes macrophylla fruit and hence, can be a potential bioresource for wine production.

Electrochemical detection of Oxaliplatin induced DNA damage in G-quadruplex structures

Oxaliplatin (OXP) is a platinum-based chemotherapeutic agent that induces DNA damage by forming intra- and interstrand crosslinks, mainly at the N7s of adenine (A) and guanine (G) bases. In addition to double-stranded DNA, G-rich G-quadruplex (G4)-forming sequences can also be targeted by OXP. However, high doses of OXP can lead to drug resistance and cause serious adverse effects during treatment. To better understand the targeting of G4 structures by OXP, their interactions as well as the molecular mechanisms underlying OXP resistance and adverse effects, there is a need for a rapid, quantitative, and cost-effective method to detect OXP and the damage it causes. In this study, we successfully fabricated a graphite electrode biosensor modified with gold nanoparticles (AuNPs) to investigate the interactions between OXP and the G4-forming promoter region (Pu22) of Vascular endothelial growth factor (VEGF). The overexpression of VEGF is known to be associated with tumor progression and the stabilization of VEGF G4 by small molecules is shown to suppresses VEGF transcription in different cancer cell lines. Differential pulse voltammetry (DPV) was used to investigate the interactions between OXP and Pu22-G4 DNA by monitoring the decrease in the oxidation signal of guanine with increasing OXP concentration. Under the optimized conditions (37 °C, 1:2 v/v AuNPs/water as electrode surface modifier, and 180 min incubation time) the developed probe showed a linear dynamic range of 1.0–10.0 μM with a detection limit of 0.88 μM and limit of quantification of 2.92 μM. Fluorescence spectroscopy was also used to support the electrochemical studies. We observed a decrease in the fluorescence emission of Thioflavin T in the presence of Pu22 upon addition of OXP. To our knowledge, this is the first electrochemical sensor developed to study OXP-induced damage to G4 DNA structures. Our findings provide new insights into the interactions between VEGF G4 and OXP, which could aid in targeting VEGF G4 structures and the development of new strategies to overcome OXP resistance.

Ascorbic Acid-Caused Quenching Effect of Protein Clusteroluminescence Probe: The Fast Fluorescent Detection of Ascorbic Acid in Vegetables.

It is interesting and meaningful to explore fluorescent probes for novel rapid detection methods. In this study, we discovered a natural fluorescence probe, bovine serum albumin (BSA), for the assay of ascorbic acid (AA). Due to clusterization-triggered emission (CTE), BSA has the character of clusteroluminescence. AA shows an obvious fluorescence quenching effect on BSA, and the quenching effect increases with increasing concentrations of AA. After optimization, a method for the rapid detection of AA is established by the AA-caused fluorescence quenching effect. The fluorescence quenching effect reaches saturation after 5 min of incubation time and the fluorescence is stable within more than one hour, suggesting a rapid and stable fluorescence response. Moreover, the proposed assay method shows good selectivity and a wide linear range. To further study the mechanisms of AA-caused fluorescence quenching effect, some thermodynamic parameters are calculated. The main intermolecular force between BSA and AA is electrostatic, presumably leading to the inhibiting CTE process of BSA. This method also shows acceptable reliability for the real vegetable sample assay. In summary, this work will not only provide an assay strategy for AA, but also open an avenue for the application expansion of CTE effect of natural biomacromolecules.

Evaluation of a MALDI-TOF MS cephalosporinase assay for blood culture isolates

Objectives: Rapid detection of beta-lactamases has the potential to improve antimicrobial stewardship and sepsis outcomes. The aim of this study was to evaluate a recently TGA-approved cephalosporinase detection method utilising matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) on blood culture isolates. This was compared to standard antimicrobial susceptibility testing and phenotypic cephalosporinase detection. Methods: A total of 114 gram negative blood culture isolates were retrospectively analysed. Isolates were selected to either be ceftriaxone non-susceptible by Vitek 2 semi-automated system (version 08.01; bioMerieux, USA, AST-N246 card) (n=31) and/or demonstrate an inducible cephalosporinase or ESBL on disc approximation testing (n=32). This cohort was compared to an equal number of ceftriaxone-susceptible isolates by Vitek 2 (n=57). The range of organisms included Escherichia coli (n=62), followed by Serratia marcescens (n=12), Pseudomonas aeruginosa (n=11), Enterobacter cloacae complex (n=10), Klebsiella aerogenes (n=5), Klebsiella pneumoniae (n=3), Klebsiella oxytoca (n=3), Morganella morganii (n=3), Acinetobacter calcoaceticus-A. baumannii complex (n=3), and Citrobacter freundii (n=2). Sub-cultured colonies were prepared with the MBT STAR-Cepha IVD Kit and third-generation cephalosporin hydrolysis was detected using the MBT STAR-BL IVD Module (Bruker Daltronics) on MALDI-TOF MS after 30 min incubation time. Results: The MALDI-TOF MS assay sensitivity and specificity for third-generation cephalosporinase activity was 61.3% and 77.1% respectively. The assay detected 11/11 (100%) ESBL producers and 5/6 (83.3%) AmpC type third-generation cephalosporinase producers. Additionally, third generation cephalosporinase activity was detected by the MALDI-TOF MS assay in 17 of 26 inducible cephalosporinase positive isolates (65.4%) that tested susceptible to ceftriaxone by Vitek 2. Of all species included, Pseudomonas aeruginosa had the lowest concordance of MALDI-TOF MS and Vitek 2 results, with only 1/11 isolates (9%) positive by MALDI-TOF MS. Conclusions: Overall the MALDI-TOF MS cephalosporinase assay lacked sensitivity in detection of third generation cephalosporinases compared to antimicrobial susceptibility testing by Vitek 2. The risk for false negative results in screening for resistance in blood culture isolates is too high to consider its implementation in the standard workflow, although there may be possible benefit in detecting ESBL and ampC type cephalosporinase activity following species identification as an alternative to standard disc approximation testing.

Addition of exogenous γ-glutamyl hydrolase eliminates the need for lengthy incubation of whole blood lysate for quantitation of folate vitamers by high performance liquid chromatography-tandem mass spectrometry

BackgroundMeasurement of folate monoglutamates by HPLC-MS/MS in whole blood lysate (WBL) requires lengthy incubation prior to analysis, risking degradation of labile folate vitamers. ObjectiveWe explored whether addition of a commercially available recombinant exogenous γ-glutamyl hydrolase (exoGGH) enzyme reduced the required incubation time of WBL for measurement of folate as monoglutamates. MethodsFor conventional deglutamylation of polyglutamates, WBL was incubated 4 h at 37°C. Alternatively, we added exoGGH to WBL at varying concentrations (1–10 µg/mL) and incubation times (0–90 min). We also investigated modifications to the sample diluent (pH, ascorbic acid vs. sodium ascorbate, and ascorbate concentration). Finally, we tested the effect of the enzyme in different sample types: WBL from frozen whole blood vs. frozen WBL or vs. frozen washed red blood cells (RBC). Samples (n ≤ 15 per experiment) were analyzed by HPLC-MS/MS for 6 folate monoglutamates and 5-methyltetrahydrofolate diglutamate. ResultsOptimal deconjugation of folate polyglutamates was achieved using 1% ascorbic acid and 5 µg enzyme/mL WBL, requiring ≤ 30 min incubation time to achieve complete folate recovery as monoglutamates. This treatment resulted in similar folate concentrations as conventional deglutamylation (4 h at 37°C). The exoGGH enzyme was effective in samples stored frozen as whole blood and as WBL. However, extended thaw time of whole blood resulted in 5-methyltetrahydrofolate loss and unacceptable changes to non-methyl folate concentration. Total folate (with exoGGH) measured in washed RBC was ∼15% lower than RBC folate calculated from WBL concentrations (conventional deglutamylation). ConclusionsUse of exoGGH minimized incubation time and thus may avoid degradative losses of labile folate forms during sample preparation. The lower folate results in washed RBC may be due to inadequate packing of RBC among other unidentified factors. A larger study is required to confirm lack of differences in folate concentrations determined with and without the use of exoGGH.

A nanoscale electrochemical guanine DNA-biosensor based on a flower-like nanocomposite of Tb-doped ZnO for the sensitive determination of pemetrexed.

Pemetrexed is an antineoplastic drug used in chemotherapeutic treatments, especially in malignant mesothelioma and non-small cell lung carcinoma, but can also cause a variety of complications, like stomach pain, nausea, burning, vomiting, numbness, and tingling, emphasizing the need for an approach to quantify the drug in biological matrices. Herein, a DNA-based biosensor was introduced for pemetrexed determination. A hydrothermal approach was used for synthesizing flower-like nanoparticles (NPs) of zinc oxide (ZnO) doped with Tb (FL-NP Tb3+/ZnO). Moreover, energy dispersive X-ray (EDX), field-emission scanning electron microscopy (FESEM), zeta potential, Brunauer-Emmett-Teller (BET), and X-ray diffraction (XRD) analyses were used for characterizing the as-prepared nanocomposite. According to the impedance analysis, FL-NP Tb3+/ZnO was accompanied by very good electrochemical functions for a simple transfer of electrons. In the case of the immobilization of double-stranded deoxyribonucleic acid (ds-DNA) on the FL-NP Tb3+/ZnO and polypyrrole (PP)-modified pencil graphite electrode (ds-DNA/PP/FL-NP Tb3+/ZnO/PGE), a considerable enhancement was found in the electrochemical oxidation of guanine in ds-DNA residue bases. Since there was an interaction between ds-DNA and pemetrexed, the voltammetric current of guanine over the ds-DNA/PP/FL-NP Tb3+/ZnO/PGE declined in the presence of pemetrexed in the electrolytic solution. Moreover, under optimum conditions (25 mg L-1 of ds-DNA and 10 min incubation time, in acetate buffer at 25 °C), a linear decrease in the guanine signal was observed on the ds-DNA/PP/FL-NP Tb3+/ZnO/PGE as the pemetrexed concentration increased in the range from 0.001 μM to 175.0 μM with a limit of detection of 0.17 nM. Finally, the new DNA-based biosensor was successfully used for determining pemetrexed in real samples, indicating its application potential.

Efficient ultrasound-assisted enzymatic method for extraction of immunostimulant QS-21 from Quillaja saponaria Molina

QS-21 from the bark of Quillaja saponaria Molina is a triterpene glycoside saponin , a new alternative immunoadjuvant. However, its application is limited because its primary source is the bark of adult trees and current extraction methods result in low yields. Therefore, this work aimed at extracting QS-21 from Q. saponaria Molina using a novel and efficient ultrasound-assisted enzymatic extraction (UAEE) method to improve yields and optimize resource utilization. Based on the single-factor and Plackett-Burman design (PBD) experiments, Central Composite design (CCD) optimum extraction conditions for improving QS-21 yield were established (71.084 min incubation time, 275.887 U enzyme dosage, and 20.254 min extraction time, yielding 5.778 mg/g of QS-21). Additionally, compared with ultrasound-assisted extraction (UAE) and enzyme-assisted extraction (EAE) methods, the UAEE method showed a higher QS-21 yield, which was 1.748 and 1.977 times higher than those of the UAE and EAE methods, respectively. Besides, cytotoxicity and immune activation assays indicated that the extract by the UAEE method exhibited a significant inhibitory effect on the activity of bone-marrow-derived dendritic cells and had a high potential for immune activation, respectively. Overall, the UAEE method can successfully improve the extraction yield of QS-21 from Q. saponaria Molina, representing an efficient and resource-saving method to obtain QS-21 for use as an adjuvant in human vaccines. • QS-21 was firstly extracted by ultrasound-assisted enzymatic extraction method. • The extraction conditions were optimized by response surface methodology. • The extract showed a significantly higher yield than other extraction methods. • The extract exhibited high immunostimulant activity.