Fluorescence Readings Research Articles

Development and Validation of a Highly Sensitive RT-qLAMP Assay for Rapid Detection of SARS-CoV-2: Methodological Aspects.

Specific and reliable diagnostic methods are becoming increasingly essential to identify patients in light of the high transmission rate and the recent appearance of the new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For the specific detection of SARS-CoV-2, our quantitative reverse transcription loop-mediated isothermal amplification (RT-qLAMP) assay implementation demonstrates how flexible it can be with two readouts: visualized colorimetric and real-time fluorescence. Different factors were optimized to improve the reaction conditions, including temperature (60°C), assay runtime (60min), primers, MgSO4 (6mM), dNTPs (1mM), LAMP Buffer (1.2mM Tris-HCl), KCl (50mM), pH (8), and phenol red (10mM) concentrations. Regarding analytical sensitivity, the colorimetric RT-LAMP method detected samples with Ct values up to 29, while the RT-qLAMP assay identified up to Ct = 31. RT-qLAMP was evaluated on 40 clinical samples (25 positives and 15 negatives) for viral RNA detection. All negative samples were found negative through fluorescent reading in RT-qLAMP and quantitative reverse transcription PCR (RT-qPCR) assays. Twenty-three clinically positive samples demonstrated a positive RT-qLAMP reaction (up to Ct ≤ 31) with 92% clinical sensitivity, 100% clinical specificity, 100% positive predictive value (PPV), 88.24% negative predictive values (NPV), and 95% accuracy.

A Rapid Detection Method for H3 Avian Influenza Viruses Based on RT-RAA.

The continued evolution of H3 subtype avian influenza virus (AIV)-which crosses the interspecific barrier to infect humans-and the potential risk of genetic recombination with other subtypes pose serious threats to the poultry industry and human health. Therefore, rapid and accurate detection of H3 virus is highly important for preventing its spread. In this study, a method based on real-time reverse transcription recombinase-aided isothermal amplification (RT-RAA) was successfully developed for the rapid detection of H3 AIV. Specific primers and probes were designed to target the hemagglutinin (HA) gene of H3 AIV, ensuring highly specific detection of H3 AIV without cross-reactivity with other important avian respiratory viruses. The results showed that the detection limit of the RT-RAA fluorescence reading method was 224 copies/response within the 95% confidence interval, while the detection limit of the RT-RAA visualization method was 1527 copies/response within the same confidence interval. In addition, 68 clinical samples were examined and the results were compared with those of real-time quantitative PCR (RT-qPCR). The results showed that the real-time fluorescence RT-RAA and RT-qPCR results were completely consistent, and the kappa value reached 1, indicating excellent correlation. For visual detection, the sensitivity was 91.43%, the specificity was 100%, and the kappa value was 0.91, which also indicated good correlation. In addition, the amplified products of RT-RAA can be visualized with a portable blue light instrument, which enables rapid detection of H3 AIV even in resource-constrained environments. The H3 AIV RT-RAA rapid detection method established in this study can meet the requirements of basic laboratories and provide a valuable reference for the early diagnosis of H3 AIV.

AB067. New drug development for the use of PARP1-E2F1 transcriptional inhibitors in the treatment of glioblastoma.

Glioblastoma (GBM) is the most malignant brain tumor and ranks among the most lethal of all human cancers, without improvements in survival over the last 30 years. Data obtained in our group suggest that PARP1, a well-known DNA-repairing protein, could also play a key role in the regulation of cell cycle through its interaction with the transcription factor E2F1. Therefore, considering that most oncogenic processes are associated with cell cycle deregulation, we hypothesized that disruption of PARP1-E2F1 interaction would provide a novel therapeutic approach to different types of cancer. The identification of novel compounds disrupting PARP1-E2F1 interaction was carried out by combining in silico and in vitro screening, using a rational drug design. The virtual screen was performed using a molecular library of several million compounds at the selected target site, using AtomNet® (Atomwise, San Francisco, CA, USA), the first deep learning neural network for structure-based drug design and discovery. Since there is no complete structural information of the PARP1-E2F1 protein-protein interaction, a homologous structure of the BRCT domain of BRCA1 complex with the phospho-peptide (PDBID: 1T2V) was used to identify the potential binding interface of BRCT domain of PARP-1 (PDBID: 2COK) and the E2F1 protein. Top scoring compounds were clustered and filtered to obtain a final subset of 83 compounds that were incorporated to our in vitro screening, which included both transcriptional E2F1 activity and survival studies. Complete culture medium supplemented with the compounds selected in the in silico screening (10 μM) were added and incubated for 24 hours. E2F1 activity was observed by measuring luminescence. For the viability assay, the fluorescence reading was performed (excitation 544 nm and emission 590 nm). The in silico and in vitro screening resulted in 12 compounds that inhibited E2F1 transcriptional activity and significantly reduced cell number. The highest inhibition of both E2F1 transcriptional activity and cell growth was observed with compound 3797, which was selected for further studies. Both in silico and in vitro results indicate that inhibition of PARP1-E2F1 transcriptional activity may provide a new rationale for designing novel therapeutic approaches for the treatment of GBM.

Evaluation of the remineralization potential of self-assembling peptide P11-4 with fluoride compared to fluoride varnish in the management of incipient carious lesions: a randomized controlled clinical trial

ObjectivesThe present trial’s aim was to compare the remineralization potential of self-assembling peptide P11-4 combined with fluoride to that of fluoride varnish.Materials and methodsTwenty-eight participants with 58 incipient carious lesions were enrolled in the present trial. Participants were randomly divided into two groups with 14 participants and 29 incipient lesions in each group. Patients were assigned either to self-assembling peptide combined with fluoride (Curodont Repair Fluoride Plus™) or sodium fluoride varnish (NaF, Bifluorid 10) groups. Both agents were applied according to the manufacturer’s instructions on non-cavitated incipient carious lesions. Lesions were assessed by two calibrated and blinded assessors at baseline, and after one-, three- and six-months using a laser fluorescence device (DIAGNOdent).ResultsAlthough laser fluorescence scores significantly improved in both groups over time (p < 0.05), no notable differences were evident between both groups at one-month (p > 0.05). Yet, at three- and six-months statistically lower laser fluorescence readings were evident in the self-assembling peptide combined with fluoride group in comparison to the fluoride alone group (p < 0.05). There was 60% less risk for caries progression for Curodont Repair Fluoride Plus™ when compared to NaF varnish after six months. Self-assembling peptide combined with fluoride was able to change 65.5% of non-cavitated carious lesions from DIAGNOdent score 3 (11–20) to score 1 (0–4). Fluoride varnish was able to change 13.8% of the lesions from score 3 to score 1 after six months.ConclusionsThe self-assembling peptide combined with fluoride varnish showed higher remineralization potential than fluoride varnish alone for incipient carious lesions over a six-months follow up.Clinical relevanceThe combination of self-assembling peptide P11-4 and fluoride could offer a new tool in managing incipient carious lesions.

Water-soluble vitamin-E for enhancing fluorescence diagnosis in infected human dentine treated with NaOCl

IntroductionBacterial fluorescence methods are of interest in endodontics for informing endpoints for debridement. This study explored potential fluorescence quenching reversal effects of a water-soluble vitamin E conjugate (d-α-Tocopherol polyethylene glycol 1000 succinate, TPGS) when applied to polymicrobial biofilms grown on dentine that had been exposed to sodium hypochlorite (NaOCl) to cause quenching. MethodExtracted human teeth were debrided, embedded in transparent acrylic resin and sectioned. After smear layer removal, tooth dentine sections were inoculated with a polymicrobial inoculum, and cultured for 7 days to create biofilms. Samples (n = 8 per group) were exposed to 1 % or 4 % NaOCl for 2 or 4 min, and then treated with TPGS. Bacterial fluorescence readings under laser excitation at 655 nm were assessed over 10 min using a calibrated DIAGNOdent device. All data were assessed for normality (Kolmogorov-Smirnov test) and analysed with ANOVA followed by Bonferroni post-hoc tests. ResultsNaOCl at both concentrations quenched fluorescence readings of biofilms grown on dentine samples, with a maximal reduction of 40.4 % at 5 min after 4 % NaOCl. Treatment with TPGS gave faster recovery of fluorescence readings compared to the control at 5 and 10 min. ConclusionThe water-soluble antioxidant TPGS partially reversed fluorescence quenching caused by NaOCl. This agent may have value clinically for reducing the time needed for fluorescence readings to recover when NaOCl is used as an irrigant. This will facilitate more accurate assessment of endpoints for canal debridement.

High resolution chlorophyll- a in-situ fluorescence sensors versus in-vitro chlorophyll- a measurements in mesocosms with contrasting nutrient and temperature treatments.

Harmful algal blooms (HABs) are a significant threat to freshwater ecosystems, and monitoring for changes in biomass is therefore important. Fluorescence in-situ sensors enable rapid and high frequency real-time data collection and have been widely used to determine chlorophyll- a (Chla) concentrations that are used as an indicator of the total algal biomass. However, conversion of fluorescence to equivalent Chla concentrations is often complicated due to biofouling, phytoplankton composition and the type of equipment used. Here, we validated measurements from 24 Chla and 12 phycocyanin (cyanobacteria indicator) fluorescence in-situ sensors (Cyclops-7F, Turner Designs) against spectrophotometrically (in-vitro) determined Chla and tested a data-cleaning procedure for eliminating data errors and impacts of non-photochemical quenching. The test was done across a range of freshwater plankton communities in 24 mesocosms (i.e. experimental tanks) with a 2x3 (high and low nutrient x ambient, IPCC-A2 and IPCC-A2+50% temperature scenarios) factorial design. For most mesocosms (tanks), we found accurate (r 2 ≥ 0.7) calibration of in-situ Chla fluorescence data using simple linear regression. An exception was tanks with high in-situ phycocyanin fluorescence, for which multiple regressions were employed, which increased the explained variance by >16%. Another exception was the low Chla concentration tanks (r 2 < 0.3). Our results also show that the high frequency in-situ fluorescence data recorded the timing of sudden Chla variations, while less frequent in-vitro sampling sometimes missed these or, when recorded, the duration of changes was inaccurately determined. Fluorescence in-situ sensors are particularly useful to detect and quantify sudden phytoplankton biomass variations through high frequency measurements, especially when using appropriate data-cleaning methods and accounting for factors that can impact the fluorescence readings.

Abstract 3188: Chitosan-glycerol injectable gel for intratumoral retention of immunotherapeutics

Abstract A novel, injectable chitosan-glycerol hydrogel was developed and characterized. Upon injection with co-formulated therapeutics, the gel vastly improved molecule retention at the injection site, as compared to molecule delivery in less viscous media. Methods. The hydrogel was formed through a centrifugation process. 70% deacetylated, medium viscosity chitosan was dissolved in 0.1M HCl. Glycerol was then added, followed by vortexing. Next, 1x PBS and 1M NaOH were added to neutralize the mixture, which induced gelation. After hydrogel centrifugation at 10,000 rpm for 5 minutes, a hydrogel was separated from precipitate. Several formulation parameters were tested, including chitosan:glycerol volume ratio, chitosan concentration, chitosan degree of deacetylation, and chitosan molecular weight. The retention of model therapeutics, formulated in hydrogel or saline vehicles, following injection in agar-based tumor phantoms was quantified via absorbance and fluorescence readings. To assess biocompatibility, a non-contact cell viability assay was performed utilizing 3T3 fibroblasts seeded in the bottom of transmembrane wells with hydrogels added to the upper chamber. After 48 hours, cells were collected and counted utilizing trypan blue. New Data. A hydrogel for intratumoral therapeutic delivery was constructed with several desirable qualities, including biocompatibility and injectability through a 25-gauge needle. Different relative glycerol to chitosan volumes, from 5 to 95 w/v % glycerol, were tested to determine a range of gelation conditions. Glycerol volume most significantly impacted gelation conditions independent of chitosan concentration, with gelation occurring between 70-85% relative volume glycerol. Increasing chitosan solution concentration and chitosan molecular weight increased the range of chitosan:glycerol ratios resulting in gelation. The hydrogel improved load retention at the agar tumor phantom injection site for various sizes of model therapeutics compared to less viscous solutions. Cytotoxicity testing of the hydrogel showed a statistically insignificant difference between the live:dead cell ratios of the experimental and negative control groups. Conclusion. An injectable, biocompatible hydrogel was created through the neutralization and centrifugation of a chitosan-glycerol solution. The hydrogel displays physiological pH, injectability through a 25-gauge needle, biocompatibility, and vastly improved therapeutic load retention at the injection site compared to less-viscous delivery media. Chitosan-glycerol hydrogels may be useful for intratumoral delivery and retention of immunomodulators within the tumor microenvironment. Citation Format: Robert L. Kobrin, Siena M. Mantooth, David A. Zaharoff. Chitosan-glycerol injectable gel for intratumoral retention of immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3188.

A simple, robust, broadly applicable insertion mutagenesis method to create random fluorescent protein: target protein fusions.

A simple, broadly applicable method was developed using an in vitro transposition reaction followed by transformation into Escherichia coli and screening plates for fluorescent colonies. The transposition reaction catalyzes the random insertion of a fluorescent protein open reading frame into a target gene on a plasmid. The transposition reaction is employed directly in an E. coli transformation with no further procedures. Plating at high colony density yields fluorescent colonies. Plasmids purified from fluorescent colonies contain random, in-frame fusion proteins into the target gene. The plate screen also results in expressed, stable proteins. A large library of chimeric proteins was produced, which was useful for downstream research. The effect of using different fluorescent proteins was investigated as well as the dependence of the linker sequence between the target and fluorescent protein open reading frames. The utility and simplicity of the method were demonstrated by the fact that it has been employed in an undergraduate biology laboratory class without failure over dozens of class sections. This suggests that the method will be useful in high-impact research at small liberal arts colleges with limited resources. However, in-frame fusion proteins were obtained from 8 different targets suggesting that the method is broadly applicable in any research setting.

Quantitative analysis of Δ9-tetrahydrocannabinol (Δ9-THC) in cannabis plants using the Fast Blue BB (FBBB) and 4-aminophenol (4-AP) colorimetric tests

The Fast Blue BB (FBBB) and 4-aminophenol (4-AP) colorimetric tests have been reportedly used for the qualitative determination of Δ9-THC in plants and for the differentiation between marijuana and hemp-type cannabis. We report the miniaturization of the FBBB colorimetric reaction on a silicone treated filter paper substrate and the analytical figures of merit for a quantitative determination of Δ9-THC for the first time. The reaction between Δ9-THC and FBBB forms a red chromophore that fluoresces when irradiated with visible (480 nm) or UV (365 nm) light, providing a 3-fold increase in sensitivity. Portable instruments are introduced for the objective color determination for both tests and for the fluorescence reading of the THC + FBBB complex. We report a fluorescence signal with Δ9-THC, Δ8-THC, and CBN. The limit of detection (LOD) was determined to be 1.6 ng/μL with precision ∼12 % RSD for standard Δ9-THC solutions ranging between 5 and 20 ng/μL. The linear dynamic range for this test is reported between 1.6 ng/μL and 20 ng/μL for the portable fluorescence detector. The miniaturization of both colorimetric tests and the increased sensitivity of the FBBB test using fluorescence analysis, coupled to portable instruments allows for limited quantitative analysis of cannabis plants in the field.

A multiplex recombinase polymerase amplification assay combined with CRISPR/Cas12a for the detection of respiratory syncytial virus and respiratory adenovirus.

Respiratory syncytial virus (RSV) and respiratory adenovirus (ADV) are two common pathogens that cause acute respiratory tract infections in children. We aimed to develop a rapid method for detecting both pathogens simultaneously. The recombinase polymerase isothermal amplification (RPA) method was combined with the CRISPR/Cas detection system. The assay's specificity and sensitivity were explored by designing RPA primers and CRISPR RNAs (crRNAs) through multi-sequence comparisons, optimizing the reaction conditions, and using a fluorescent reading device. The consistency of the test results of 160 clinical pharyngeal swab samples was studied using quantitative polymerase chain reaction (qPCR) results as a comparative control. RSV and ADV could be detected at levels as low as 104 copies/mL and 103 copies/mL, respectively, within 50 minutes with no cross-reactivity with other similar pathogens. For the clinical samples, compared with the qPCR method, the sensitivities for RSV and ADV were 98.1% and 91.4%, respectively, and the detection specificities were both 100%. The Kappa values were greater than 0.95, suggesting a high degree of consistency. This method for detecting RSV and ADV is rapid, sensitive, and specific. It can accurately detect mixed infections in a timely manner, making it suitable for use in areas with scarce healthcare resources.

Understanding Netosis Priming and Induction through Nucleosome Changes and Real-Time Monitoring in Isolated Neutrophils and in an Ex-Vivo Model

Sepsis is a leading cause of death, affecting 1.7 million adults annually in the US, where it is implicated in ~350,000 deaths (1). Rapid recognition and intervention are required to avoid progression to septic shock and death. Sepsis results from a dysregulated host response to infection (2). We aimed to study part of this dysregulated response by analyzing NETosis in neutrophils. Neutrophils are integral to the innate immune response. They can undergo a unique type of programmed cell death, NETosis, where nuclear chromatin is extruded out of the cell to capture and deactivate pathogens and prevent dissemination. These neutrophil extracellular traps (NETs) contain nucleosomes, DNA and histone proteins. NETs help to trap invading pathogens but in excess, lead to endothelial damage, excessive activation of platelets and promote microthrombosis in distal organs leading to organ failure. Our understanding of NETosis largely stems from mouse models, immortalized cell lines, and isolated neutrophils. However, these don't entirely mirror the complexity of human immune response. Here we present a model of ex vivo NETosis induction in whole blood, which can be used to provide greater clarity on the complex signaling and chromatin changes associated with NETosis. We developed an ex vivo Synthetic Sepsis TM model for observing induced NETosis in whole blood. Whole blood from healthy volunteers was induced with Phorbol 12-myristate 13-acetate (PMA), Calcium Ionophore (CI) or Lipopolysaccharides (LPS) and NETosis was monitored using H3.1 Nu.Q® levels. In parallel we adapted a SpectraMax plate reader for whole-blood fluorescent readings via Sytox Green interacalation to measure DNA release associated with NETosis. Neutrophils were isolated using the MACSxpress® Whole Blood Neutrophil Isolation Kit and NETosis of isolated neutrophils was visualized using the Incucyte® system from Sartorius. A myeloperoxidase (MPO) inhibitor, ABAH, was used to assess NETosis inhibition in PMA-induced isolated neutrophils, which allowed us to detect a reduction in both nucleosome levels using our H3.1 Nu.Q® assay and the measured DNA release via real-time fluorescence techniques. We next compared the NETosis profiles in our ex vivo Synthetic Sepsis TM model with isolated neutrophils to assess the impact of other blood cells and circulating proteins. Further, Methylation analysis post-ONT sequencing facilitated cell of origin analysis. H3.1 Nu.Q® levels increased with NETosis induction, consistent with DNA release as measured by Sytox green. NETosis was detected 1.5 hours after PMA induction using H3.1 Nu.Q® and Sytox assays. DNA Methylation analysis confirmed neutrophil cell of origin post induction. MPO inhibition resulted delayed onset of NETosis, supporting NET release rather than other forms of cell death. Netosis visualization using microscopy confirmed NETosis. Importantly, we found that although PMA successfully induced NETosis in both contexts, CI failed to elicit a response in the ex vivo whole bloodsystem, highlighting the importance of a full blood model for unbiased NETosis profiling. Our findings demonstrate that H3.1 nucleosome levels are elevated in response to NETosis-inducing treatments, with a time course that is consistent with NETosis and correlates with other established NETosis markers, validating its utility as a reliable indicator of NETosis. The successful induction of NETosis in an ex vivo whole blood model provides a valuable tool for studying disease-associated NETosis within a biologically relevant context, which will likely shed additional insight which is not possible using isolated neutrophils. Utilizing these approaches provides a platform to scrutinize a wide array of both synthetic and naturally occurring NETosis regulators. It facilitates insight into the signalling mechanisms at the core of NETosis and the influence of chromatin-associated changes within an in vivo like microenvironment of whole blood. By unravelling the intricate dynamics of neutrophil biology and its interplay with the microenvironment, we can develop tailored interventions to modulate NETosis in sepsis and other inflammatory conditions. 1. https://www.cdc.gov/sepsis/index.html. 2. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016 Feb 23;315(8):801.

Plasma microrna quantification protocol.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.

Adjusting the Fitting of Fluorescence-Based Dose-Response Kinase Inhibition Assay to Account for Fluorescent Inhibitors.

Fluorescence is routinely used to monitor kinase inhibition in commercial assays. Occasionally fluorescent compounds can interfere with the fluorescent reading. To address this issue, the problematic data are usually truncated to improve the fit, however, this approach raises ethical and reproducibility concerns. Instead, it is suggested to adjust the fitting formula, to account for the autofluorescence of the compounds and improve the fit of the data compared with a naive approach. Finally, it was noticed that truncating the data can result in a small underestimation of the IC50 values and should therefore be used carefully.

Carbon dots-based fluorescent molecularly imprinted photonic crystal hydrogel strip: Portable and efficient strategy for selective detection of tetracycline in foods of animal origin

Rapid, portable, and sensitive detection of tetracycline (TC) is crucial for the environment and human health. In this study, we developed carbon dots (CDs)-based fluorescent molecularly imprinted photonic crystal hydrogel (FMIPH) strips for TC detection in animal-derived foods. CDs emit fluorescent signals, and molecularly imprinted polymers provide specific recognition sites for TC. Inverse opal photonic crystals afford stable 3D macroporous mass transfer channels that considerably reduce binding time between TC and the strips. The portable FMIPH strip exhibited a linear fluorescence response to TC in the concentration range of 0.1–50 μg mL−1, with a detection limit of 0.067 μg mL−1. Good recoveries of TC (93.86–112.59%) were observed in TC-spiked commercially available pork, eggs, and milk. A combination of FMIPH strips with a portable fluorescent reading device could achieve sensitive, on-site, and real-time detection of TC in animal-derived foods.

Point-of-Care Platform Based on Solid-Phase Fluorescence Filter Effect for Urinary Iodine Testing in Children and Pregnant Women.

Iodine is an essential element that is used to make thyroid hormones. However, people usually ignore their iodine nutrition level, thus leading to a series of thyroid diseases, particularly in areas where medical resources are scarce. Thus, development of a portable, economical, and simple method for the detection of urinary iodine is of significant importance. Herein, a solid-phase fluorescence filter effect (SPFFE) induced by iodine was used to develop an SPFFE-based point-of-care testing (POCT) platform for the detection of urinary iodine by coupling with headspace sample introduction. This method can not only alleviate the matrix interference that occurred in the conventional inner filter effect (IFE) but also achieve high sensitivity. Furthermore, the urinary iodine (UI) POCT platform was developed through the integration of a sample pretreatment and fluorescence readout. This whole system costs less than US $20 and provides accurate temperature control and a portable fluorescence reading within 15-20 min. Compared to the traditional IFE-based assay, the SPFFE-based POCT platform allows the selective detection of iodine as low as 10 nM and has a linear range of 0.05-4 μM. In addition, it provides notable visualization from blue-violet to orange-red in the presence of iodine, which tends to indicate the iodine nutritional status of the human body. Eventually, the clinical applicability and feasibility of the UIPOCT platform as an early diagnostic test kit were confirmed by determining the iodine in urine samples from children and pregnant women.

Laser fluorescence is a predictor of lesion depth in non-cavitated root carious lesions - an in vitro study.

This in vitro study was designed to compare the assessment of carious root surfaces using laser fluorescence or visual assessment and µCT. A total of 107 extracted human teeth were included in the study. For the subsequent assessment, a clinical evaluation according to Ekstrand and Nyvad criteria, laser fluorescence measurements (DIAGNOdent-Pen©), and µCT imaging (Bruker Skyscan 1172) of the included root surfaces were performed. For cavitated lesions, the surface was reconstructed on µCT. Target parameters were: clinical appearance, laser fluorescence reading (0-99), and lesion depth in µm (µCT). The statistical analysis included chi² and Spearman-Rho tests. Laser fluorescence values were significantly higher (p<0.01) in leathery non-cavitated (median [IQR]: 73.50 [48.00]) and cavitated (88.50 [29.00]) surfaces than in hard intact surfaces (7.00 [35.00]). In addition, laser fluorescence values showed a significant correlation with lesion depth for non-cavitated surfaces (0.750; p<0.01). Further statistically significant correlations were not detectable. Regression analysis showed that the laser fluorescence values increased with increasing demineralization depth (β: 0.648, CI95: 0.631-1.053; p<0.01). Laser fluorescence measured by DIAGNOdent© is a predictor for the depth of demineralization in carious root caries lesions with intact surfaces. Considering the limitations of this in vitro study, laser fluorescence is suitable for assessing the depth extent of (non-cavitated) root caries lesions and is a tool for clinical diagnosing and assessing the course of lesion development.

Design and development of a lanthanide-labeled immunochromatographic strip for simultaneous detection of morphine, methamphetamine and ketamine in hair.

Colloidal gold immunoassay is the most widely used method in the field of drug detection. However, this method often has poor quantitative identification ability and low analytical sensitivity, which is not suitable for the analysis of hair poisoning ingredients. In order to solve these limitations, we developed an immunochromatographic test strip for simultaneously screening multiple drugs in this study. This hand-held test strip used fluorescent nanoparticles loaded with lanthanide chelates as the signal carrier of fluorescence reading, and conducted quantitative testing of various drugs based on the competitive immune reaction between the analyte and antigen. Under the optimal conditions, the competition curves of morphine (MOP), methamphetamine (MET) and ketamine (KET) were obtained on a single band. The detection limit (LOD) of this analytical method was 100-1000 times lower than that of colloidal gold test strips. The detection limits of MOP, MET and KET were 0.06 ng mL-1, 0.1 ng mL-1 and 1.0 ng mL-1, respectively. No cross-reaction was observed when morphine, methamphetamine and ketamine were tested simultaneously with this method. And 184 hair samples were tested simultaneously, and the detected amount was very close to the results of LC-MS. The immunochromatographic strip showed good stability in repeated tests, and the coefficient of variation was less than 15%. Fluorescence immunochromatography strips and handheld strip readers have the characteristics of portability, speed, ease of operation and high sensitivity, and may become powerful tools for screening drug abuse in hair in forensic medicine.

Rapid One-Pot Microwave Assisted Green Synthesis Nitrogen Doped Carbon Quantum Dots as Fluorescent Precursor for Estimation of Modafinil as Post-Covid Neurological Drug in Human Plasma with Greenness Assessments

The neuro-stimulant anti-narcoleptic drug as modafinil (MOD) is used to treatment neurological conditions caused by COVID-19. MOD was used to treatment narcolepsy, shift-work sleep disorder, and obstructive sleep apnea-related sleepiness. So, an innovative, quick, economical, selective, and ecologically friendly procedure was carried out. A highly sensitive N@CQDs technique was created from green Eruca sativa leaves in about 4 min using microwave synthesis at 700 w. The quantum yield of the synthesized N@CQDs was found to be 41.39%. By increasing the concentration of MOD, the quantum dots' fluorescence intensity was gradually quenched. After being excited at 445 nm, the fluorescence reading was recorded at 515 nm. The linear range was found to be in the range 50 – 700 ng mL−1 with lower limit of quantitation (LOQ) equal to 45.00 ng mL−1. The current method was fully validated and bio analytically according to (US-FDA and ICH) guidelines. Full characterization of the N@CQDs has been conducted by high resolution transmission electron microscope (HRTEM), Zeta potential measurement, fluorescence, UV–VIS, and FTIR spectroscopy. Various experimental variables including pH, QDs concentration and the reaction time were optimized. The proposed study is simply implemented for the therapeutic drug monitoring system (TDMS) and various clinical laboratories for further pharmacokinetic research.

Automotive Diesel Fuel Internal Stability Testing with the Use of UV and Temperature as Degradation Factors

Diesel fuel stability can be considered from many points of view, of which the two considered most important are stability in contact with the environment and internal stability. Fuel stability in touch with the environment is often defined as oxidation stability, of which measurement procedures are well developed. The presented paper shows that fuel's internal stability can also be important. The internal stability of diesel fuel with the local use of thermal and ultraviolet radiation (UV) as degradation factors and fluorescence signals as a probe is presented in this paper. We show that the internal degradation of fuel with temperature use differs from that with UV and simultaneous both factors use. Our study shows that using temperature as a degradation factor introduces significant fluorescence fading. Moreover, the fluorescence signal restores significantly later than the sample stabilizes at room temperature. The novelty proposed based on examination is hybrid degradation and an examination cycle that enables the simultaneous use of degradation factors and fluorescence reading. For this purpose, a dedicated measurement setup of signal control and processing was constructed and programmed. The measurement procedure of the data series for specific wavelength enables calculation of signal shifts that allow the internal stability classification of diesel fuel samples in less than 30 min with the cost of a single disposable capillary probe and one polymer plug. Premium and regular fuel examination results show that internal fuel stability can be related to polycyclic aromatic hydrocarbons (PAH) concentrations and can be modified with dedicated additives.

Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis.

Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings. IMPORTANCE Accurate tests for the diagnosis of SARS-CoV-2, the virus causing coronavirus disease 2019 (COVID-19), are important for timely treatment and infection control decisions. Conventional tests such as real-time reverse transcription-PCR (RT-PCR) require specialized equipment and are expensive. On the other hand, rapid antigen tests suffer from a lack of sensitivity. In this study, we describe a novel assay format for the diagnosis of COVID-19 that is based on principles of loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas chemistry. A major advantage of this assay format is that it does not require expensive equipment to perform, and results can be read visually. This method proved to be fast, easy to perform, and inexpensive. The test compared well against an RT-PCR assay in terms of the ability to detect SARS-CoV-2 RNA in clinical samples. No false-positive test results were observed. The new assay format is ideal for SARS-CoV-2 diagnosis in resource-limited settings.