Development of an affordable light emitting diode spectrophotometer paired with a Python program for calibration and linearity testing and the measurement of uranium(VI).

Uranium (U) is a radiologically and chemically toxic element that occurs naturally in water, soil, and rock at generally low levels. However, anthropogenic uranium can also leach into groundwater sources due to mining, ore refining, and improper nuclear waste management. Over the last few decades, various methods for measuring uranium have emerged; however, most of these techniques require skilled scientists to run samples on expensive instrumentation for detection or require the pretreatment of samples in complex procedures. In this work, a Schiff base ligand (P1) is used to develop a simple spectrophotometric method for measuring the concentration of uranium (VI) with an accurate and affordable light-emitting diode (LED) spectrophotometer. A test for a higher-order polynomial relationship was used to objectively determine the calibration data's linearity. This test was done with a Python program on a Raspberry Pi computer that captured the spectrophotometer's calibration and sample measurement data.

A machine learning approach for rapid early detection of Campylobacter spp. using absorbance spectra collected from enrichment cultures.

Enumeration of Campylobacter from environmental waters can be difficult due to its low concentrations, which can still pose a significant health risk. Spectrophotometry is an approach commonly used for fast detection of water-borne pollutants in water samples, but it has not been used for pathogen detection, which is commonly done through a laborious and time-consuming culture or qPCR Most Probable Number enumeration methods (i.e., MPN-PCR approaches). In this study, we proposed a new method, MPN-Spectro-ML, that can provide rapid evidence of Campylobacter detection and, hence, water concentrations. After an initial incubation, the samples were analysed using a spectrophotometer, and the spectrum data were used to train three machine learning (ML) models (i.e., supported vector machine - SVM, logistic regression-LR, and random forest-RF). The trained models were used to predict the presence of Campylobacter in the enriched water samples and estimate the most probable number (MPN). Over 100 stormwater, river, and creek samples (including both fresh and brackish water) from rural and urban catchments were collected to test the accuracy of the MPN-Spectro-ML method under various scenarios and compared to a previously standardised MPN-PCR method. Differences in the spectrum were found between positive and negative control samples, with two distinctive absorbance peaks between 540-542nm and 575-576nm for positive samples. Further, the three ML models had similar performance irrespective of the scenario tested with average prediction accuracy (ACC) and false negative rates at 0.763 and 13.8%, respectively. However, the predicted MPN of Campylobacter from the new method varied from the traditional MPN-PCR method, with a maximum Nash-Sutcliffe coefficient of 0.44 for the urban catchment dataset. Nevertheless, the MPN values based on these two methods were still comparable, considering the confidence intervals and large uncertainties associated with MPN estimation. The study reveals the potential of this novel approach for providing interim evidence of the presence and levels of Campylobacter within environmental water bodies. This, in turn, decreases the time from risk detection to management for the benefit of public health.

Application of MTT assay for probing metabolic activity in bacterial biofilm-forming cells on nanofibrous materials.

The quantification of cellular metabolic activity via MTT assay has become a widespread practice in eukaryotic cell studies and is progressively extending to bacterial cell investigations. This study pioneers the application of MTT assay to evaluate the metabolic activity of biofilm-forming cells within bacterial biofilms on nanofibrous materials. The biofilm formation of Staphylococcus aureus and Escherichia coli on nanomaterials electrospun from polycaprolactone (PCL), polylactic acid (PLA), and polyamide (PA) was examined. Various parameters of the MTT assay were systematically investigated, including (i) the dissolution time of the formed formazan, (ii) the addition of glucose, and (iii) the optimal wavelength for spectrophotometric determination. Based on interim findings, a refined protocol suitable for application to nanofibrous materials was devised. We recommend 2 h of the dissolution, the application of glucose, and spectrophotometric measurement at 595 nm to obtain reliable data. Comparative analysis with the reference CFU counting protocol revealed similar trends for both tested bacteria and all tested nanomaterials. The proposed MTT protocol emerges as a suitable method for assessing the metabolic activity of bacterial biofilms on PCL, PLA, and PA nanofibrous materials.

Risk assessment based on spectrophotometric signals used in eco-friendly analytical scenarios for estimation of carvedilol and hydrochlorothiazide in pharmaceutical formulation.

Special attention is given to the pharmacological treatment of combined medication of Carvedilol and hydrochlorothiazide which is the most effective and the most beneficial therapy for hypertensive patients with diabetes and various metabolic comorbidities. This work represents spectrophotometric platform scenarios based on factorized spectrum (FS) using interpoint data difference resolution scenarios (IDDRS) coupled with spectrum subtraction method (SS) for the concurrent quantification of carvedilol (CAR) and hydrochlorothiazide (HCT) when present together in a combination without the need for any initial physical separation steps. This IDD resolution scenario based on manipulating the zero-order spectra (D0) of both drugs in the mixture with various spectral features at different wavelength regions (200-400 nm), region I (220-250 nm), region II (240-300 nm) and region III (270-320 nm) via absorbance resolution (AR) and induced absorbance resolution (IAR) methods coupled with corresponding spectrum subtraction (SS). The calibration curves were established across the linearity ranges of 2.0-12.0 µg/mL at 242.50 nm and 4.0-40.0 µg/mL at 285.5 nm for CAR and 1.0-11.0 µg/mL at 226.10 nm and 2.0-20.0 µg/mL at 270.5 nm for HCT. Moreover, methods' validation was confirmed via ICH guidelines. A Multicenter comparison between sensitivity, specificity in respect resolution sequence were applied using different wavelength regions with various concentration ranges was applied and finally spectral resolution recommendation is issued and cumulative validation score (CVS) is calculated as an indicator in the risk analysis. In quality control laboratories, the studied approaches are applicable for conducting analysis on the mentioned drugs. In addition, the selection of spectrophotometry aligns with the principles of green analytical chemistry, an approach that resonates with the overarching theme of minimizing environmental impact. Via four metric tools named: analytical greenness (AGREE), green analytical procedure index (GAPI), analytical eco-scale, and national environmental method index (NEMI), methods' greenness profile was guaranteed.

Anatomical characteristics of different tooth groups. Effect on in-office bleaching treatment

AIM: The objective of the present study was to investigate the association between the anatomical characteristics of different tooth groups and the diffusion and bleaching effect of hydrogen peroxide (H2O2). MATERIALS AND METHODS: Computed tomography (CT) images from five patients were used to assess the hard tissue thickness and pulp volume (PV) of four tooth groups: lower (mandibular) incisors (LI), upper (maxillary) incisors (UI), canines (C), and premolars (PM). Additionally, 80 bovine tooth disks were divided into four groups (n = 20) to match the thickness of each tooth group studied. All the specimens were exposed to a 35% H2O2 bleaching gel, with 50 µL applied for 45 min during the first, second, and third sessions. Diffusion was evaluated using the peroxidase enzyme method. Color change analyses (∆E, ∆E00, and ∆WID) were performed after the three application sessions and 7 days after the bleaching treatment using a spectrophotometer. RESULTS: The PM group showed greater thickness and PV, followed by the C, UI, and LI groups (P 0.001). The LI group had six times greater H2O2 diffusion compared with the PM group (P 0.001), while the PM group exhibited a PV nine times larger than the LI group. Furthermore, the LI and UI groups achieved color saturation with one fewer session than the C and PM groups. CONCLUSIONS: Specific tooth groups have anatomical characteristics that interfere with bleaching treatment in terms of the diffusion and whitening effect of H2O2. Furthermore, the diffusion capacity of H2O2 was inversely proportional to the thickness of the tooth groups.

Evaluation of the Reliability of the CCM-300 Chlorophyll Content Meter in Measuring Chlorophyll Content for Various Plant Functional Types.

Chlorophyll fluorescence is a well-established method to estimate chlorophyll content in leaves. A popular fluorescence-based meter, the Opti-Sciences CCM-300 Chlorophyll Content Meter (CCM-300), utilizes the fluorescence ratio F735/F700 and equations derived from experiments using broadleaf species to provide a direct, rapid estimate of chlorophyll content used for many applications. We sought to quantify the performance of the CCM-300 relative to more intensive methods, both across plant functional types and years of use. We linked CCM-300 measurements of broadleaf, conifer, and graminoid samples in 2018 and 2019 to high-performance liquid chromatography (HPLC) and/or spectrophotometric (Spec) analysis of the same leaves. We observed a significant difference between the CCM-300 and HPLC/Spec, but not between HPLC and Spec. In comparison to HPLC, the CCM-300 performed better for broadleaves (r = 0.55, RMSE = 154.76) than conifers (r = 0.52, RMSE = 171.16) and graminoids (r = 0.32, RMSE = 127.12). We observed a slight deterioration in meter performance between years, potentially due to meter calibration. Our results show that the CCM-300 is reliable to demonstrate coarse variations in chlorophyll but may be limited for cross-plant functional type studies and comparisons across years.

Assessment of 5-Hydroxymethylfurfural in Food Matrix by an Innovative Spectrophotometric Assay.

Foods contaminants pose a challenge for food producers and consumers. Due to its spontaneous formation during heating and storage, hydroxymethylfurfural (HMF) is a prevalent contaminant in foods rich in carbohydrates and proteins. Colorimetric assays, such as the Seliwanoff test, offer a rapid and cost-effective method for HMF quantification but require careful optimization to ensure accuracy. We addressed potential interference in the Seliwanoff assay by systematically evaluating parameters like incubation time, temperature, and resorcinol or hydrochloric acid concentration, as well as the presence of interfering carbohydrates. Samples were analyzed using a UV-Vis spectrophotometer in scan mode, and data obtained were validated using HPLC, which also enabled quantification of unreacted HMF for assessing the protocol's accuracy. Incubation time and hydrochloric acid percentage positively influenced the colorimetric assay, while the opposite effect was observed with the increase in resorcinol concentration. Interference from carbohydrates was eliminated by reducing the acid content in the working reagent. HPLC analyses corroborated the spectrophotometer data and confirmed the efficacy of the proposed method. The average HMF content in balsamic vinegar samples was 1.97 ± 0.94 mg/mL. Spectrophotometric approaches demonstrated to efficiently determine HMF in complex food matrices. The HMF levels detected in balsamic vinegars significantly exceeded the maximum limits established for honey. This finding underscores the urgent need for regulations that restrict contaminant levels in various food products.

[Validation of a method for measuring the antielastolytic activity of human circulating alpha1-antitrypsin]. / Validation d'une méthode de mesure de l'activité antiélastasique de l'alpha1-antitrypsine circulante humaine.

The existence of alpha-1 antitrypsin variants with apparently unremarkable phenotypes and serum concentrations, contrasting with a clinical picture suggestive of a severe deficiency, led us to investigate whether in these cases there was a reduction or even suppression of the capacity of alpha-1 antitrypsin to inhibit elastase. To this end, in two different laboratories, we adapted and validated a method for measuring the functional activity of alpha-1 antitrypsin, based on spectrophotometric kinetic analysis of the inhibition by serum alpha-1 antitrypsin of the hydrolytic activity of porcine pancreatic elastase on a chromogenic substrate. This method has proved to be robust, reproducible and transferable and made possible to define, on the basis of an analysis of a hospital population, a functionality index with a confidence interval comprised between 0.87 and 1.2, allowing to identify subjects likely to have a functional deficiency of alpha-1 antitrypsin, whether this deficiency being of a genetic origin without any quantitative or phenotypic translation, or whether being acquired under the effect of external agents (cigarette smoke or viruses).

Smart Chemometric-Assisted Spectrophotometric Approaches for Simultaneous Quantification of Tertiary Combination Recommended for COVID-19 Supportive Treatments With Their Greenness Assessment.

BACKGROUND: There is an increasing interest of the scientific community in developing innovative methodologies for their analysis needs within a green analytical chemistry framework. UV spectrophotometry is one of the most promising eco-friendly methods, which is integrated with advanced chemometric tools to enhance the selectivity of the analysis of complex mixtures with severe overlapped signals. OBJECTIVE: Simultaneous determination of a triple-combination of pseudoephedrine hydrochloride (PSE), carbinoxamine maleate (CRX), and paracetamol (PAR) using an artificial intelligence system and multivariate calibration methods. This combination has been recently recommended for COVID-19 home-treated patients as part of a symptomatic treatment. METHODS: Namely, the suggested models are artificial neural networks, partial least-squares, and principal component regression. The proposed algorithms were optimized and developed with the aid of a five-level, three-factor experimental design. RESULTS: The investigated methods were applied over the concentration range of 100-180 µg/mL, 18-16 µg/mL, and 4-12 µg/mL for PSE, CRX, and PAR, respectively. The models' validation results demonstrated excellent recoveries (around 98 to 102%), signaling the approaches' outstanding resolution capacity for the cited compounds in the presence of common excipients. The outcomes of the studied methods were statistically compared to the official approaches, and no significant difference was found. CONCLUSIONS: The suggested models were efficiently employed to determine the selected drugs in their combined tablets without any initial separation steps. The impact of these methods on the environment was evaluated via greenness tools: namely, the National Environmental Method Index, Raynie and Driver's green assessment method, Analytical Eco-Scale, Green Analytical Procedure Index, and Analytical Greenness Metric. HIGHLIGHTS: Green chemometric quality assessment of PSE, CRX, and PAR in their pure and pharmaceutical dosage forms. The established approaches are innovative, sustainable, smart, fast, selective, and cost-effective. These models are potential green nominees for routine analysis of the investigated mixture in quality control laboratories.

A Novel and Simple Method for a Differentiation of Alcohol Types.

BACKGROUND: Alcohol poisoning is a significant global problem that has become an epidemic. The determination of the alcohol type is hereby essential as it may affect the course of the treatment; however, there is no routine laboratory diagnostic method for alcohol types other than for ethanol. In this study, we aimed to define a simple method for alcohol type differentiation by utilizing a combination of breathalyzer and spectrophotometrically measured serum ethanol results. METHODS: A breathalyzer and spectrophotometry were used to measure four different types of alcohol: ethanol, isopropanol, methanol, and ethylene glycol. To conduct serum alcohol analysis, four serum pools were created, each containing a different type of alcohol. The pools were analyzed using the spectrophotometric method with an enzymatic ethanol test kit. An experiment was conducted to measure the different types of alcohol using impreg-nated cotton and a balloon, simulating a breathalyzer test. An algorithm was created based on the measurements. RESULTS: Based on the results, the substance consumed could be methanol or isopropanol if the breathalyzer test indicates a positive reading and if the blood ethanol measurement is negative. If both the breathalyzer and the blood measurements are negative, the substance in question may be ethylene glycol. CONCLUSIONS: This simple method may determine methanol or isopropanol intake. This straightforward and innovative approach could assist healthcare professionals in different fields with diagnosing alcohol intoxication and, more precisely, help reducing related morbidity and mortality.

The Effect of Resin Infiltration Technique on the Calculated Color Change of Demineralized Lesions of Different Severities.

OBJECTIVE: To assess the color change of demineralized enamel lesions of different severities after resin infiltration using both clinical spectrophotometry and digital photography. METHODS AND MATERIALS: Sixty sound human premolars were randomly divided into 3 groups according to the demineralization level. All the teeth were immersed in a demineralizing solution of a pH adjusted to 4.4 at 37°C. Three levels of demineralization were obtained (D1 shallow, D2 moderate, D3 deep) according to the demineralization time. The demineralized area was then infiltrated by low-viscosity resin (ICON, DMG, Germany). Two instrumental methods were utilized to assess the color difference, a clinical spectrophotometer and digital photography at three time points (sound, demineralized, and infiltrated enamel) to calculate the color difference between sound and demineralized enamel (ΔE1) and between sound and infiltrated enamel (ΔE2). Statistical analysis was performed by ANOVA, followed by Tukey's post hoc test. The correlation was analyzed using linear regression. RESULTS: Two-way ANOVA showed statistically significant differences for both levels of the study (p≤0.05). The color change (ΔE1) and (ΔE2) for different demineralization levels showed statistically significant differences between all groups. For both clinical spectrophotometry and digital photography, D3 showed the highest difference followed by D2 and then D1. As for (ΔE1) calculations, digital photography had a significantly higher difference than spectrophotometry for the D1 group (5.47±0.93 vs 2.78±0.58). As for (ΔE2) digital photography had a statistically significantly lower difference than spectrophotometry (5.55±1.05 vs 6.48±0.76) for the D3 group. CONCLUSIONS: Color correction after resin infiltration is affected by the demineralization level of enamel. Clinical spectrophotometry and digital photography can detect similarly the color change of demineralized enamel after resin infiltration in shallow and moderate demineralization. However, in deep demineralization clinical spectrophotometry tends to exaggerate the color change compared to digital photography.

ChlorTox scale assessment, greenness, and whiteness evaluation of selective spectrophotometric analysis of dimenhydrinate and cinnarizine.

Nausea and vomiting are considered common series side effects induced by chemotherapy treatment in cancer patients. This annoying side effect can impair the patient's compliance to cancer treatment and affect their quality of life. Dimenhydrinate and cinnarizine in combined pharmaceutical dosage form is used to control chemotherapy induced nausea and vomiting in cancer patients. For safety, selective spectrophotometric methods based on novel dual resolution strategies were introduced to estimate dimenhydrinate and cinnarizine in presence of their harmful impurities namely benzophenone and 1- (diphenylmethyl)piperazine, respectively. These methods namely, dual ratio difference (DRD), dual ratio extraction (DRE) and dual absorbance extraction coupled with dual ratio extraction (DAE-DRE) were successfully performed to simultaneously analyze the drug of interests dimenhydrinate and cinnarizine in their pure form, synthetic mixtures and in market dosage form. Linearity ranges were 6.0-60.0 µg/mL and 3.0-30.0 µg/mL for dimenhydrinate and cinnarizine, respectively with good recovery% of Mean ± SD for all the proposed methods 99.82 ± 0.48, 99.79 ± 0.40, 100.14 ± 0.82, 100.03 ± 0.69, respectively. ICH guidelines were adhered in accordance with confirming validation of the proposed methods where fulfilling results were accomplished. Various unified greenness and whiteness assessment tools, such as the chlorTox scale, greenness index via spider chart, AGREE (The Analytical Greenness Metric), green certificate, and the RGB12 algorithm were employed in this research to assess the greenness and sustainability of the introduced UV-spectrophotometric methods in comparison to the reported HPLC method. As a result, these methods hold significant potential for utilization in the quality control department of pharmaceutical companies, contributing to enhanced pharmaceutical product analysis and overall sustainability practices.

Quantification of arsenic in real samples using a spectrophotometric cloud point extraction of the formed ion pair with astrazon orange G.

A novel cloud-point extraction (CPE) procedure for the determination of ultra-trace amounts of arsenic species in real samples, purchased from the local market by spectrophotometer was developed. Inorganic arsenic species analysis in water, beverages, and foods has become increasingly important in recent years, as arsenic species are considered carcinogenic and are assessed at significant levels in samples. The technique is established on a selective ternary complex of As(V) with astrazon orange G (AOG+) in the presence of tartaric acid and polyethylene glycol tertoctylphenyl ether (Triton X-114) at pH 4.0. The calibration curve developed within range 3.0-160 ng/mL with a correlation coefficient of 0.9988 for As(V) provided a preconcentration factor of 200 and a limit of detection (3S blank/m) of 0.88 ng/mL under optimum investigation conditions. The results of molar absorptivity and Sandell sensitivity are calculated and found to be 4.38 × 105 L/mol cm and 0.018 ng cm-2, respectively. The statistical treatment of data obtained from the proposed and GF-AAS procedures are compared in terms of Student's t-tests and variance ratio F-tests has revealed no significant differences. The methodology has been effectively confirmed by assessing real samples and comparing it to the GF-AAS method statistically.

Spectrophotometric and chromatographic analysis of creatine:creatinine crystals in urine.

Creatinine is the end product of the catabolism of creatine and creatine phosphate. Creatine phosphate serves as a reservoir of high-energy phosphate, especially in skeletal and cardiac muscle. Besides typical known changes in serum and urinary creatinine concentrations, rare cases associated with changes in serum and urinary creatine levels have been described in the literature in humans. These cases are mostly linked to an excessive intake of creatine ethyl ester or creatine monohydrate, often resulting in increased urine creatinine concentrations. In addition, it is known that at such elevated creatinine concentrations, creatinine crystallisation may occur in the urine. Analysis of crystals and urinary concrements, often of heterogenous chemical composition, may provide diagnostic and therapeutic hints to the benefit of the patient. The aim of the present work was to analyze urine crystals of unclear composition with microscopic and spectroscopic techniques. On routine microscopic analysis of urine, a preliminary suspicion of uric acid or creatinine crystals was expressed. The crystals were of a cuboid shape and showed polarization effects in microscopy. The dried urine sample was whitish-orange in colour, odourless and dissolved well in water. Protein concentration in dry weight (DW) urine was about 0.3 mg/mg. The measured zinc content in the studied sample was approximately 660 µg/g DW sample and copper content was approximately 64 µg/g DW sample. A lead signal of around 10 µg/g DW sample was also observed. UV-Vis analysis showed a maximum creatine peak around 220 nm, compatible with the spectrum of creatinine with a maximum peak of 230 nm. Using HPLC technique, an extreme high ratio of creatine to creatinine of about 38 was measured, which led to the conclusion of the occurrence of rare creatine crystals in urine.

Introduction of a spectrophotometric method for salivary iodine determination on microplate based on Sandell-Kolthoff reaction.

BACKGROUND: Iodine is an essential element for the synthesis of thyroid hormones. Therefore, a reliable marker of iodine supply is important. Iodine is predominantly excreted via kidneys, but also via salivary glands. Our aim was to introduce a new and simple method for determination of salivary iodine concentration (SLIC). MATERIALS AND METHODS: Self-prepared chemicals and standards for Sandell-Kolthoff reaction on microplate with ammonium peroxydisulfate (AP) in the range 0-400 µg/L were used. Suitability of water-based standards (WBS) and artificial saliva-based standards (ASS) for standard curve were tested. We followed standards for method validation, defined concentration of used AP and compared our results with Inductively Coupled Plasma Mass Spectrometry (ICP-MS). RESULTS: WBS gave more reliable results than ASS as an underestimation of iodine concentration was found for ASS. LoB was 6.5 µg/L, LoD 12.0 µg/L, therefore analytical range was 12-400 µg/L. Intra- and inter-assay imprecisions at iodine concentrations, namely 20, 100, 165, and 350 µg/L were 18.4, 5.1, 5.7, and 2.8%, respectively, and 20.7, 6.7, 5.1, and 4.3%, respectively. Suitable molarity of AP was 1.0 mol/L and showed no difference to 1.5 mol/L (P values for samples with concentration 40, 100, and 150 µg/L, were 0.761, 0.085, and 0.275, respectively), whereas there was a significant change using 0.5 mol/L (P<0.001). Saliva samples could be diluted up to 1:8. There was no interference of thiocyanate and caffeine up to 193.5 mg/L. Our original method was comparable to ICP-MS. Spaerman coefficient was 0.989 (95% CI: 0.984-0.993). CONCLUSIONS: The new method for SLIC determination is in excellent agreement with ICP-MS and easy-to-use.

Green and Versatile High-Throughput Microwell Oxidation-Based Spectrophotometric Methods for Determination of Galidesivir in Capsules.

BACKGROUND: Galidesivir (GDV) is a promising new antiviral drug for the potent and safe treatment of a broad spectrum of viral diseases, including COVID-19. In the literature, no analytical method exists for the determination of GDV in bulk and dosage form. OBJECTIVE: The aim of this study was the development of versatile green and simple microwell spectrophotometric methods (MW-SPMs) for the determination of GDV in its bulk form and capsules. METHODS: Three MW-SPMs were developed involving the oxidation of GDV by ammonium metavanadate (AMV), chromium trioxide (CTO), and potassium iodate (PIO) in an acid medium. The reactions were carried out in 96-well plates at room temperature and the absorbances of chromogenic reaction products were measured by an absorbance microplate reader at 780, 595, and 475 nm for AMV, CTO, and PIO, respectively. Variables influencing the reactions were carefully investigated and optimized. RESULTS: Linear relations with excellent correlation coefficients (0.9991-0.9997) were found between the absorbances and GDV concentrations in the range of 25-500 µg/mL. The LODs were ≥8.3 µg/mL. The accuracy and precision of the three MW-SPMs were confirmed by recovery and replicate analysis, respectively. The recovery values were 98.6-101.2% and the RSDs were ≤1.02%. The proposed MW-SPMs were successfully applied to the analysis of GDV in bulk drug and capsules with high accuracy and precision. The greenness of the MW-SPMs was confirmed by three comprehensive metric tools. CONCLUSION: The proposed MW-SPMs combined the inherent advantages of microwell-based analysis and the use of common laboratory reagents for the reactions involved. These advantages include high-throughput, ready automation, reduced sample/reagent volume, precise measurements, and versatility. The advantages of the use of common laboratory reagents include availability, consistency, compatibility, safety, and cost-effectiveness. HIGHLIGHTS: Overall, the proposed MW-SPMs are versatile valuable tools for the quantitation of GDV during its pharmaceutical manufacturing.

Comparison of DeNovix, NanoDrop and Qubit for DNA quantification and impurity detection of bacterial DNA extracts.

Accurate DNA quantification is key for downstream application including library preparations for whole genome sequencing (WGS) and the quantification of standards for quantitative PCR. Two commonly used technologies for nucleic acid quantification are based on spectrometry, such as NanoDrop, and fluorometry, such as Qubit. The DS-11+ Series spectrophotometer/fluorometer (DeNovix) is a UV spectrophotometry-based instrument and is a relatively new spectrophotometric method but has not yet been compared to established platforms. Here, we compared three DNA quantification platforms, including two UV spectrophotometry-based techniques (DeNovix and NanoDrop) and one fluorometry-based approach (Qubit). We used genomic prokaryotic DNA extracted from Streptococcus pneumoniae using a Roche DNA extraction kit. We also evaluated purity assessment and effect of a single freeze-thaw cycle. Spectrophotometry-based methods reported 3 to 4-fold higher mean DNA concentrations compared to Qubit, both before and after freezing. The ratio of DNA concentrations assessed by spectrophotometry on the one hand, and Qubit on the other hand, was function of the A260/280. In case DNA was pure (A260/280 between 1.7 and 2.0), the ratio DeNovix or Nanodrop vs. Qubit was close or equal to 2, while this ratio showed an incline for DNA with increasing A260/280 values > 2.0. The A260/280 and A260/230 purity ratios exhibited negligible variation across spectrophotometric methods and freezing conditions. The comparison of DNA concentrations from before and after freezing revealed no statistically significant disparities for each technique. DeNovix exhibited the highest Spearman correlation coefficient (0.999), followed by NanoDrop (0.81), and Qubit (0.77). In summary, there is no difference between DeNovix and NanoDrop in estimated gDNA concentrations of S. pneumoniae, and the spectrophotometry methods estimated close or equal to 2 times higher concentrations compared to Qubit for pure DNA.

Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

Determination of Phosphoglycolate Phosphatase Activity via a Coupled Reaction Using Recombinant Glycolate Oxidase.

Phosphoglycolate phosphatase (PGLP) dephosphorylates 2-phosphoglycolate to glycolate that can be further metabolized to glyoxylate by glycolate oxidase (GOX) via an oxidative reaction that uses O2 and releases H2O2. The oxidation of o-dianisidine by H2O2 catalyzed by a peroxidase can be followed in real time by an absorbance change at 440 nm. Based on these reactions, a spectrophotometric method for measuring PGLP activity using a coupled reaction with recombinant Arabidopsis thaliana GOX is described. This protocol has been used successfully with either purified PGLP or total soluble proteins extracted from Arabidopsis rosette leaves.

Spectrophotometric Assays for Measuring Hydroxypyruvate Reductase Activity.

Hydroxypyruvate reductase (HPR; EC 1.1.1.81) activity is integral to the photorespiratory pathway. Within photorespiration, HPR catalyzes the reduction of hydroxypyruvate, a product of the serine:glyoxylate aminotransferase reaction to glycerate, a substrate for glycerate kinase, using NADH as cofactor. Here we detail a spectrophotometric assay for measuring HPR activity in vitro by following the consumption of NADH at 340 nm.