Method For Quantification Research Articles

Preclinical pharmacokinetic studies of villocarine A, an active Uncaria alkaloid.

Villocarine A is a bioactive indole alkaloid isolated from the Uncaria genus. It has demonstrated vasorelaxation activity and potential to protect the central nervous system. To identify the pharmacokinetic properties of villocarine A, a series of in vitro and in vivo studies have been performed. Villocarine A was found to be highly permeable (15.6 ± 1.6*10-6cm/s) across human colorectal adenocarcinoma cell monolayer with high protein binding (>91%) in both rat and human plasma. Hepatic extraction ratio of villocarine A was 0.1 in pooled rat liver and 0.2 in human liver microsomes and was found stable in rat plasma at 37°C. Due to the high permeability and low rate of metabolism properties, villocarine A was initially considered suitable for preclinical development and an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification (linearity: 1-150 ng/ml) in rat plasma was developed and validated for in vivo studies. Essential pharmacokinetic parameters included the volume of distribution and clearance of villocarine A, which were found to be 100.3 ± 15.6L/kg and 8.2 ± 1.1L/h/kg, respectively, after intravenous administration in rats. Following oral dosing, villocarine A exhibited rapid absorption as the maximum plasma concentration (53.2 ± 10.4ng/ml) occurred at 0.3 ± 0.1h, post-dose. The absolute oral bioavailability of villocarine A was 16.8 ± 0.1%. To our knowledge, this was the first pharmacokinetic study of villocarine A, which demonstrated the essential pharmacokinetic properties of villocarine A: large volume distribution, high clearance, and low oral bioavailability in rats.

A Validated Chiral Chromatography Method for Enantiomeric Separation of Pomalidomide in Human Plasma.

In the present work, new chiral stationary phase high-performance liquid chromatography (CSP-HPLC) method was established and validated for the quantification of pomalidomide (PMD) enantiomers in human plasma. The chromatographic enantiomeric separation was achieved on a Daicel-CSP, Chiralpack IA 4.6 × 250mm, 5μm; because of its advantages of high degree of retention, high resolution capacity, better reproducibility, ability to produce lower back pressure and low degree of tailing. The mobile phase was maintained as methanol: glacial acetic acid (499.50ml:50μL). Ultraviolet wavelength for detection was 220nm. PMD enantiomer-I and enantiomer-II were separated at 8.83 and 15.34min, respectively. Limit of detection and limit of quantification for each enantiomer and the calibration curve of standard PMD was linear in range between 10-5,000ngmL-1. The method was validated according to The International Council for Harmonisation of Technical Requirements of Pharmaceuticals for Human Use (ICH(Q2R1)) specific guidelines. We found no interference peak with PMD chromatogram obtained. This is a simple, reliable and specific method for detection and quantification of enantiomer of PMD in human plasma sample.

Oxidative stress induces release of mitochondrial DNA into the extracellular space in human placental villous trophoblast BeWo cells.

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is an indicator of cell death, inflammation, and oxidative stress. ccf-mtDNA differs in pregnancies with placental dysfunction from healthy pregnancies and the direction of this difference depends on gestational age and method of mtDNA quantification. Reactive oxygen species (ROS) trigger release of mtDNA from non-placental cells; yet it is unknown whether trophoblast cells release mtDNA in response to oxidative stress, a common feature of pregnancies with placental pathology. We hypothesized that oxidative stress would induce cell death and release of mtDNA from trophoblast cells. BeWo cells were treated with antimycin A (10-320 mM) or rotenone (0.2-50 mM) to induce oxidative stress. A multiplex real-time quantitative PCR (qPCR) assay was used to quantify mtDNA and nuclear DNA in membrane bound, non-membrane bound, and vesicular-bound forms in cell culture supernatants and cell lysates. Treatment with antimycin A increased ROS (p<0.0001), induced cell necrosis (p=0.0004) but not apoptosis (p=0.6471) and was positively associated with release of membrane-bound and non-membrane bound mtDNA (p<0.0001). Antimycin A increased mtDNA content in exosome-like extracellular vesicles (vesicular-bound form; p=0.0019) and reduced autophagy marker expression (LC3A/B, p=0.0002; p62, p<0.001). Rotenone treatment did not influence mtDNA release or cell death (p>0.05). Oxidative stress induces release of mtDNA into the extracellular space and causes non-apoptotic cell death and a reduction in autophagy markers in BeWo cells, an established in vitro model of human trophoblast cells. Intersection between autophagy and necrosis may mediate the release of mtDNA from the placenta in pregnancies exposed to oxidative stress.

Integrating UPLC-QTOF-MS/MS and UPLC-DAD to evaluate the influence of sulfur-fumigated Paeoniae Radix Alba on the overall quality of three Si-Wu-Tang formulations.

Sulfur-fumigation of Paeoniae Radix Alba (PRA) could induce the chemical transformation of its bioactive component paeoniflorin into a sulfur-containing derivative paeoniflorin sulfite, and thus alter the quality, bioactivities, pharmacokinetics, and toxicities of PRA. However, how sulfur-fumigated PRA (S-PRA) affects the quality of PRA-containing complex preparations has not been intensively evaluated. We intend to evaluate the influence of S-PRA on the overall quality of three kinds of Si-Wu-Tang (SWT) formulations, i.e., decoction (SWT-D), granule (SWT-G), and mixture (SWT-M). AnUPLC-DAD multi-components quantification method was used to compare the transfer rates of paeoniflorin sulfite and other 10 bioactive components between S-PRA-containing and NS-PRA-containing SWT formulations. An UPLC-QTOF-MS/MS-based target metabolomics approach was applied to explore the differential sulfur-containing derivatives in S-PRA-containing SWT formulations. The transfer rates of paeoniflorin sulfite in three S-PRA-containing SWT formulations were all higher than 100%. Moreover, S-PRA also increased the transfer rate of 5-hydroxymethylfurfural, 1,2,3,4,6-O-pentagalloylglucose, whereas decreased that of paeoniflorin, albiflorin, and ferulic acid in three SWT formulations. Six pinane monoterpene glucoside sulfites originally identified in S-PRA, were also detectable in three S-PRA-containing SWT formulations. In addition, seven phenolic acid sulfites including (3Z)-6-sulfite-ligustilide, (3E)-6-sulfite-ligustilide, 6,8-disulfite-ligustilide, ferulic acid sulfite, neochlorogenic acid sulfite, chlorogenic acid sulfite, and angelicide sulfite (or isomer) were newly identified in these three S-PRA-containing formulations. S-PRA could differentially affect the transfer rate of paeoniflorin sulfite and other bioactive components during the preparation of three SWT formulations and subsequently the overall quality thereof.

Smartphone microscopic method for imaging and quantification of microplastics in drinking water.

Analysis of microplastics in drinking water is often challenging due to smaller particle size and low particle count. In this study, we used a low cost and an easy to assemble smartphone microscopic system for imaging and quantitating microplastic particles as small as 20 μm. The system consisted of a spherical sapphire ball lens of 4 mm diameter attached to a smartphone camera as a major imaging component. It also involved pre-concentration of the sample using ZnCl2 solution. The spike recovery and limit of detection of the method in filtered distilled and deionized water samples (n = 9) were 55.6% ± 9.7% and 34 particles/L, respectively. Imaging performance of the microscopic system was similar to a commercial bright field microscopic system. The method was further implemented to examine microplastic particles in commercial bottled and jar water samples (n = 20). The particles count in bottled and jar water samples ranged from 0-91 particles/L to 0-130 particles/L, respectively. In both sample types, particles of diverse shape and size were observed. The particles collected from water samples were further confirmed by FTIR spectra (n = 36), which found 97% of the particles tested were made of plastic material. These findings suggested that the smartphone microscopic system can be implemented as a low-cost alternative for preliminary screening of microplastic in drinking water samples. RESEARCH HIGHLIGHTS: Ball lens based smartphone microscopic method was used for microplastic analysis. Particles of diverse shape and size were found in bottle and jar water samples.

Preparation of surface molecularly‐imprinted magnetic kaolin for selective separation and determination of thiabendazole

AbstractIn this study, a selective sorbent was fabricated by combining a surface molecularly‐imprinted polymer and magnetic kaolin for reliable quantification of thiabendazole (TBZ) coupled with spectrofluorimetry. Several surface characterization methods were conducted to confirm the synthesis of TBZ‐imprinted magnetic kaolin with a polymer thickness of ~48 nm. The molecularly‐imprinted magnetic kaolin (MIP@MKLN) showed high adsorption capacity (8.7 mg/g), remarkable selectivity with an imprinting factor of 3.71 and excellent regeneration ability for up to 10 uses without significant change of its initial adsorption capacity for TBZ. The proposed method had linear range from 5 to 500 μg/mL TBZ concentration with a correlation coefficient of 0.995 and the limit of detection was 1.4 μg/mL. MIP@MKLN was used for selective separation and quantification of TBZ in orange and lemon samples. There were high recovery values ranging from 96.4% to 100.2% with lower relative standard deviations (less than 5%) in the spiked samples under optimized conditions. Overall, the proposed method based on MIP@MKLN as an excellent sorbent could produce a feasible, convenient, and promising method for rapid, selective, and sensitive quantification of TBZ in real samples.

Optimization and Validation of Procyanidins Extraction and Phytochemical Profiling of Seven Herbal Matrices of Nutraceutical Interest

Several medicinal herbal plants are extensively used as sources of bioactive compounds with beneficial effects on human health. This study assessed the procyanidin and polyphenol profiles together with the antioxidant potential of seven herbal medical matrices. To achieve this aim, procyanidin extraction from grape pomace was optimized and validated by monitoring monomeric-trimeric procyanidins. The proposed quantification method was applied to the seven medical herbs, and it proved to be a very efficient protocol for procyanidin-rich extracts analysis. In addition, the Paullinia cupana Kunth. seed was identified as a very rich source of procyanidins (about 5 mg/g dry matrix of each dimeric and about 3 mg/g dry matrix trimeric) with high antioxidant properties. The polyphenolic profile was assessed by HPLC-HESI-MS/MS analysis. The in vitro antioxidant activity was evaluated by DPPH assay to explore the antioxidant properties of the extracts, which were substantially higher in Peumus boldus Molina leaves extracts (935.23 ± 169 μmol of Trolox equivalent/g of dry weight) concerning the other matrices. Moreover, a high Pearson coefficient value was observed between the total flavonoid content (TFC) and DPPH in comparison with the total polyphenol content (TPC) and DPPH, indicating flavonoids as the principal bioactive with antioxidant activity in the extracts.

Transcriptomics coupled with proteomics reveals osimertinib-induced myocardial mitochondrial dysfunction

Osimertinib, an irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) used for cancer treatment, can cause significant cardiac toxicity. However, the specific mechanism of osimertinib-induced cardiotoxicity is not fully understood. In this study, we administered osimertinib to mice and neonatal rat ventricular myocytes (NRVMs). We observed significant structural and functional damage to the hearts of these mice, along with a marked increase in cardiac injury biomarkers and accompanying ultrastructural damage to mitochondria. We integrated 4D label-free protein quantification and RNA-Seq methods to analyze the sequencing data of NRVMs under osimertinib treatment (0 and 2.5 μM). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis evidenced that differentially expressed genes (DEGs)and differentially expressed proteins (DEPs) were distinctly enriched for oxidative phosphorylation (OXPHOs). Simultaneously, osimertinib primarily affected the contents of adenosine triphosphate (ATP). Further investigations revealed that osimertinib disrupts the functions of the ATP synthase (complex V), leading to a reduction in ATP production. Taken together, our data demonstrated that osimertinib causes mitochondrial dysfunction, which in turn leads to the onset of cardiac toxicity.

Mammalian and avian species quantification in homogenized foods: real time PCR and digital PCR as tools for label compliance controls

Currently food fraud and authenticity of products composition are topics of great concern; ingredients quantification could allow to identify small amounts of contaminats or voluntary addition of improper components. Many molecular methods are available for species identification in foodstuffs but, for a better application, they should not be affected by the interference of other ingredients. The main purpose of this work was to verify the Real Time PCR and the Digital PCR (dPCR) quantification performances on baby food samples, specifically selected for their high miscibility to limit variability; chicken was selected as target to verify the performance of quantification of methods after having spiked the same quantity in different baby foods. The other aims were: (1) to verify a constant genome copies ratio existence between mammalian and avian species (2) to verify the dPCR performance, set up on housekeeping, to quantify mammalian and avian species in commercial products. Digital PCR showed fewer differences respect to Real Time PCR, at the same 15% w/w chicken spiking level. Despite the constant difference between mammalian and avian genome copies, in samples with the same spiking weight, the confidence intervals increasing towards the extreme values, made impossible to use genome copies ratio as a sort of correction factor between species. Finally, the dPCR system using the myostatin housekeeping gene to determine the chicken content seemed reliable to verify the labelling compliance in meat-based commercial products.

Direct Determination of Absolute Radical Quantum Yields in Hydroxyl and Sulfate Radical-Based Treatment Processes

Direct Determination of Absolute Radical Quantum Yields in Hydroxyl and Sulfate Radical-Based Treatment Processes

Determination of Fe3O4 Content and Total Nonhydraulic Minerals in Steel Slag

The nonhydraulic minerals (Fe3O4, RO phase, Fe) in slag are important indicators for evaluating the pozzolanic activity and detecting the quality of the slag activation processing technology. Fe3O4 is an important characteristic mineral among the nonhydraulic minerals. In order to accurately assess the pozzolanic activity of steel slag powder and to monitor the quality of the activation process of steel slag powder for separate nonhydraulic minerals, it is imperative to precisely determine the nonhydraulic mineral content within the steel slag. Further refinement and enhancement are required for both the HNO3 dissolution method used in determining Fe3O4 content in steel slag, as well as for the EDTA-DEA-TEA (ethylenediamine tetraacetate sodium-diethylamine-triethanolamine) dissolution method employed in determining total nonhydraulic minerals, due to potential deviations caused by challenging impurity separations. The results show that the content of Fe3O4 is determined by 10%HNO3-20%NaOH-chemical analysis method, which solves the problem that the impurities of refractory materials (quartz, corundum, mullite) and amorphous phase affects the content determination in HNO3 dissolution method. The total amount of nonhydraulic minerals (Fe3O4, RO phase, Fe) was determined by the EDTA-NaOH-TEA dissolution method, which solved the problem that the incomplete dissolution of C2F in the EDTA-DEA-TEA dissolution method affected the content determination. The maximum error between the content determination value and the theoretical calculation value of the two methods is less than 0.50%. The improved Fe3O4 and total nonhydraulic mineral quantification methods are feasible and reliable.

Exploring polyphosphates in soil: presence, extractability, and contribution to microbial biomass phosphorus

AbstractPolyphosphates (Poly-P) are known to fulfil several important physiological functions. Many microorganisms can accumulate large amounts of Poly-P in their biomass. Regardless of these facts, systematic research on Poly-P in soil is missing, probably due to the absence of any method of direct Poly-P quantification. In this study, we attempted to unequivocally prove the presence of Poly-P in the biomass of soil microorganisms and quantify their extractability and contribution to microbial biomass phosphorus. To do so, we combined several approaches that can indicate Poly-P presence in soil microbial biomass indirectly, i.e. growth of soil inoculum on media without phosphorus, associated with measurement of changes in the microbial biomass stoichiometry, and the colour of the microbial suspension stained by the Neisser method. All soil microbial communities exhibited growth on media without phosphorus. As the growth on this media depleted Poly-P content, the biomass carbon to phosphorus and nitrogen to phosphorus ratio increased and the colour of the microbial suspension stained by the Neisser method changed predictively. The associated Poly-P addition experiment indicated that the recovery of added Poly-P from soil in form of soluble reactive phosphorus in sodium bicarbonate extract may reach up to 93% mainly due to abiotic depolymerization. Using a simple stoichiometric model applied to measured data, we calculated that the Poly-P content of microbial biomass in our soils may be up to 45 or 70% of total microbial biomass phosphorus depending on the assumptions applied regarding parameter values. We discuss the magnitude of error associated with the measurement of soil microbial phosphorus due to the high extractability of Poly-P.

Advances in contezolid: novel oxazolidinone antibacterial in Gram-positive treatment.

The principal objective of this project was to review and thoroughly examine the chemical characteristics, pharmacological activity, and quantification methods associated with contezolid. The article was based on published and ongoing preclinical and clinical studies on the application of contezolid. These studies included experiments on the physicochemical properties of contezolid, in vitro antimicrobial research, in vivo antimicrobial research, and clinical trials in various phases. There were no date restrictions on these studies. In June 2021, contezolid was approved for treating complicated skin and soft tissue infections. The structural modification of contezolid has resulted in better efficacy compared to linezolid. It inhibits bacterial growth by preventing the production of the functional 70S initiation complex required to translate bacterial proteins. The current evidence has indicated a substantial decline in myelosuppression and monoamine oxidase inhibition without impairing its antibacterial properties. Contezolid was found to have a more significant safety profile and to be metabolised by flavin monooxygenase 5, reducing the risk of harmful effects due to drug-drug interactions. Adjusting doses is unnecessary for patients with mild to moderate renal or hepatic insufficiency. As an oral oxazolidinone antimicrobial agent, contezolid is effective against multi-drug resistant Gram-positive bacteria. The introduction of contezolid provided a new clinical option.

Effects of compositional uncertainties in cracked NH3/biosyngas fuel blends on the combustion characteristics and performance of a combined-cycle gas turbine: A numerical thermokinetic study

Blending of partially cracked ammonia with biosyngas is an attractive strategy for improving NH3 combustion. In practice, products of biomass gasification and those of thermo-catalytic cracking of NH3 are subject to some compositional uncertainties. Despite their practical importance, so far, the effects of such uncertainties on combustion systems remained largely unexplored. Hence, this paper quantifies the effects of small compositional uncertainties of reactants upon combustion of partially cracked NH3/syngas/air mixtures. An uncertainty quantification method, based on polynomial chaos expansion and a data-driven model, is utilised to investigate the effects of uncertainty in fuel composition on the laminar flame speed (SL) and adiabatic flame temperature (Tad) at different inlet pressures (Pi). The analysis is then extended to the power output of a combined-cycle gas turbine fuelled by the reactants. It is found that 1.5% fuel compositional uncertainty can cause 12–21% of SL uncertainty depending on the inlet pressure. Furthermore, the effect of compositional uncertainty on Tad increases at higher ratios of H2 to NH3. Sensitivity analysis reveals that the uncertainty of CO contribution to SL uncertainty is higher than that of NH3, while the trend is reversed for the Tad uncertainty. In addition, the power output from the combined-cycle gas turbine system varies between 4 and 6% with 1.5% of fuel compositional uncertainty. This become more noticeable at elevated Pi [5–10 atm], particularly when the fuel mixture contains high H2 which is the main contributor to Tad variability.

U-Plume: automated algorithm for plume detection and source quantification by satellite point-source imagers

Abstract. Current methods for detecting atmospheric plumes and inferring point-source rates from high-resolution satellite imagery are labor-intensive and not scalable with regard to the growing satellite dataset available for methane point sources. Here, we present a two-step algorithm called U-Plume for automated detection and quantification of point sources from satellite imagery. The first step delivers plume detection and delineation (masking) with a U-Net machine learning architecture for image segmentation. The second step quantifies the point-source rate from the masked plume using wind speed information and either a convolutional neural network (CNN) or a physics-based integrated mass enhancement (IME) method. The algorithm can process 62 images (each measuring 128 pixels × 128 pixels) per second on a single 2.6 GHz Intel Core i7-9750H CPU. We train the algorithm using large-eddy simulations of methane plumes superimposed on noisy and variable methane background scenes from the GHGSat-C1 satellite instrument. We introduce the concept of point-source observability, Ops=Q/(UWΔB), as a single dimensionless number to predict plume detectability and source rate quantification error from an instrument as a function of source rate Q, wind speed U, instrument pixel size W, and instrument-dependent background noise ΔB. We show that Ops can powerfully diagnose the ability of an imaging instrument to observe point sources of a certain magnitude under given conditions. U-Plume successfully detects and masks plumes from sources as small as 100 kg h−1 in GHGSat-C1 images over surfaces with low background noise and successfully handles larger point sources over surfaces with substantial background noise. We find that the IME method for source quantification is unbiased over the full range of source rates, while the CNN method is biased towards the mean of its training range. The total error in source rate quantification is dominated by wind speed at low wind speeds and by the masking algorithm at high wind speeds. A wind speed of 2–4 m s−1 is optimal for detection and quantification of point sources from satellite data.

Evaluating lumbar disc degeneration by MRI quantitative metabolic indicators: the perspective of factor analysis

PurposeThis study aimed to investigate an early diagnostic method for lumbar disc degeneration (LDD) and improve its diagnostic accuracy.MethodsQuantitative biomarkers of the lumbar body (LB) and lumbar discs (LDs) were obtained using nuclear magnetic resonance (NMR) detection technology. The diagnostic weights of each biological metabolism indicator were screened using the factor analysis method.ResultsThrough factor analysis, common factors such as the LB fat fraction, fat content, and T2* value of LDs were identified as covariates for the diagnostic model for the evaluation of LDD. This model can optimize the accuracy and reliability of LDD diagnosis.ConclusionThe application of biomarker quantification methods based on NMR detection technology combined with factor analysis provides an effective means for the early diagnosis of LDD, thereby improving diagnostic accuracy and reliability.

Advanced Uncertainty Quantification for Flood Inundation Modelling

Flood hazards present a significant risk to the UK, with homes, businesses and critical infrastructure exposed to a mixture of fluvial, surface water and coastal flooding. Climate change is expected to influence river flows, changing the frequency and magnitude of future flood events. Flood hazard assessments are used by decision-makers to implement policies and engineering interventions to reduce the impacts of these flood events. Probabilistic flood modelling can explore input and parameter uncertainties in flood models to fully quantify inundation uncertainty. However, probabilistic methods require large computational costs—limiting their application. This paper investigates a range of advanced uncertainty quantification methods (traditional Monte Carlo (FMC), Kriging and multi-fidelity Monte Carlo (MFMC)) to reduce the dichotomy between accuracy and costs. Results suggest that Kriging can reduce computational costs by 99.9% over FMC. The significantly increased efficiency has the potential to improve future policy and engineering decisions, reducing the impacts of future flood events.

Development and Validation of the HPLC Method for Simultaneous Quantification of Glucose and Xylose in Sugar Cane Bagasse Extract.

A validated rapid high-performance liquid chromatography (HPLC)-refractive index (RI) method was developed for the identification and quantification of glucose and xylose in hydrolyzed bagasse extract. The separation of compounds was achieved on Eurokat® H column (300 × 8mm, 10μm) at 75°C, using 0.01N sulfuric acid solution as mobile phase and 0.6mL/min as flow rate. The method was validated based on accuracy, precision, linearity, robustness, uncertainty, limit of detection (LOD) and limit of quantification (LOQ). Total chromatographic analysis time per sample was ~11min. Calibration plots were linear over the concentration ranges 11-100μg/100μL for glucose and xylose. The LOD was 0.8ppm and LOQ was 2.5ppm. The high recovery and low relative standard deviation confirm the suitability of the method for determination of glucose and xylose in bagasse extract. The proposed HPLC-RI method was accurate, fast and robust and required less run time due to less analytes retention times and allowed optimal energy consumption owing to lower column oven temperature.

Development and Validation of Fast and Sensitive RP-HPLC Stability-Indicating Method for Quantification of Piroxicam in Bulk Drug.

A simple, rapid, sensitive, and cost-effective green solvent-assisted reverse-phase high-performance liquid chromatographic technique, coupled with a photodiode array detector, was developed and validated for the estimation of piroxicam (PRXM). The chromatographic separation was achieved by using a C-18 (250 × 4.6) mm, 5-μm stationary phase and a mobile phase consisting of methanol and 0.1% ortho-phosphoric acid in water in a ratio of (80:20) v/v at a flow rate of 1ml/min. The detection was carried out at a wavelength of 254nm with a constant injection volume of 10μL throughout the analysis. The calibration curve was observed to be linear over the optimum concentration range of 50-300μgmL-1, with an R2 value of 0.9995. The developed method was validated as per the International Council for Harmonisation (ICH) Q2 (R1) guideline. Various parameters like selectivity/specificity, accuracy/recovery, linearity, precision, detection limit, quantitation limit, robustness and stability of analyte in solution were performed for the method validation. The PRXM was evaluated under stressed conditions, including acidic, basic, oxidative, thermal and photolytic, as per ICH Q1 (R2) guidelines. Significant degradation was observed in acidic and basic degradation conditions. Conversely, the drug substance showed stability when exposed to oxidative, photolytic and thermal degradation conditions.

Evaluation of quantification methods to determine photodynamic action on mono- and dual-species bacterial biofilms.

The effect of photodynamic inactivation (PDI) sensitized by 5,10,15,20-tetra(4-N,N,N-trimethylammoniophenyl)porphyrin (TMAP4+) on different components of mono- and dual-species biofilms of Staphylococcus aureus and Escherichia coli was determined by different methods. First, the plate count technique showed that TMAP4+-PDI was more effective on S. aureus than E. coli biofilm. However, crystal violet staining revealed no significant differences between before and after PDI biofilms of both bacteria. On the other hand, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method indicated a reduction in viable cells as the light exposure time increases in both, mono- and dual-species biofilms. Furthermore, it was determined that as the irradiation time increases, the amount of extracellular polymeric substances present in the biofilms decreased. This effect was presented in both strains and in the mixed biofilm, being more evident in S. aureus mono-specie biofilm. Finally, scanning electron microscopy analysis showed a decrease in the number of cells forming the biofilm after photosensitization treatments. This information makes it possible to determine whether the photodynamic action is based on damage to metabolic activity, extracellular matrix and/or biomass, which may be useful in establishing a fully effective PDI protocol for the treatment of microorganisms growing as biofilms.