Paper-based optical sensor arrays for simultaneous detection of multi-targets in aqueous media: A review.

Sensor arrays, which draw inspiration from the mammalian olfactory system, are fundamental concepts in high-throughput analysis based on pattern recognition. Although numerous optical sensor arrays for various targets in aqueous media have demonstrated their diverse applications in a wide range of research fields, practical device platforms for on-site analysis have not been satisfactorily established. The significant limitations of these sensor arrays lie in their solution-based platforms, which require stationary spectrophotometers to record the optical responses in chemical sensing. To address this, this review focuses on paper substrates as device components for solid-state sensor arrays. Paper-based sensor arrays (PSADs) embedded with multiple detection sites having cross-reactivity allow rapid and simultaneous chemical sensing using portable recording apparatuses and powerful data-processing techniques. The applicability of office printing technologies has promoted the realization of PSADs in real-world scenarios, including environmental monitoring, healthcare diagnostics, food safety, and other relevant fields. In this review, we discuss the methodologies of device fabrication and imaging analysis technologies for pattern recognition-driven chemical sensing in aqueous media.

Description of a new quantitative method to assess mitochondrial distribution pattern in mature equine oocytes.

The Mitochondrial distribution pattern or MDP in mammalian oocytes serves as an indicator of their cytoplasmic maturity, with a heterogeneous pattern associated with mature cytoplasm. Currently, MDP assessment involves fluorescent labelling of mitochondria followed by visual evaluation, as no quantitative method exists. Our objective was to develop a quantitative approach to assess MDP in mature equine oocytes. Equine oocytes, obtained by ovum pick up (OPU) were matured in vitro, and only metaphase II oocytes were used in the study (n = 56). Following denudation, oocytes were fixed, stained with MitoTracker™ Red CMXRos (50 nM in TCM-199 with Hank´s salts and 10% FBS) for 15 min at 38 °C, and then incubated with 2.5 µg/ml Hoechst 33342 for 10 min at 38 °C. Confocal microscope images were acquired, and the oocyte's MDP was visually classified as either homogeneous (HoD; n = 17) or heterogeneous (HeD; n = 39). For quantitative analysis, Fiji-ImageJ software was employed. Background subtraction was performed, and a 1-pixel line along the diameter was drawn to calculate the intensity profile. Fluorescence intensities were normalized, and ratios of peripheral to central fluorescence intensity were determined. Student´s t-test was used for comparations; MDP ratio was (mean ± standard error of the mean): 0.8 ± 0.02 for HoD and 0.3 ± 0.02 for HeD (p < 0.001). These results demonstrate concordance between quantitative and qualitative MDP assessment in mature equine oocytes. Our study describes a new approach to quantify mitochondrial distribution pattern and cytoplasmic maturation in mature equine oocytes.

Performance Comparison Between Conventional Fluorescent Spot Test and Quantitative Assay in Detecting G6PD Deficiency in Neonates.

Objectives: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide. The fluorescent spot test (FST) is the conventional method for screening neonates for G6PD. However, it has limitations and quantitative assays such as the CareStart Biosensor 1 are being increasingly recommended. This study aimed to compare FST and CareStart Bioensor 1 in their ability to detect G6PD levels in neonates. Methods: This cross-sectional study involved 455 neonates between June and December 2020. Two milliliters of cord blood were analyzed with CareStart Biosensor 1 and dried cord blood spots with FST. Data was recorded and statistically analyzed. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated to determine the performance of FST at specific G6PD cut-off values; Cohen's kappa analysis assessed the agreement between the two methods. Results: The sensitivity of FST at 30% cut-off G6PD activity level was 91.0%, (95% CI: 57.0-100) and specificity of 97.0% (95% CI: 95.0-98.0). At 60% cut-off, the FST sensitivity sharply declined to 29.0% (95% CI: 19.0-40.0) with a specificity of 100% (95% CI: 98.0-100). The overall prevalence of G6PD deficiency was 5.1% as measured by FST and 17.8% by Biosensor 1 (p < 0.001). Conclusions: In this study, FST missed a significant proportion of cases of intermediate G6PD levels. FST also misclassified several G6PD intermediate individuals as normal, rendering them susceptible to oxidative stress. Biosensor 1 reported a significantly higher prevalence of G6PD deficiency.

Multiple heart-cutting two-dimensional liquid chromatography/charged aerosol detector assay of ginsenosides for quality evaluation of ginseng from diverse Chinese patent medicines.

For quality control of Chinese patent medicines (CPMs) containing the same herbal medicine or different herbal medicines that have similar chemical composition, current ″one standard for one species″ research mode leads to poor universality of the analytical approaches unfavorable to discriminate easily confused species. Herein, we were aimed to elaborate a multiple heart-cutting two-dimensional liquid chromatography/charged aerosol detector (MHC-2DLC/CAD) approach to quantitatively assess ginseng from multiple CPMs. Targeting baseline resolution of 16 ginsenosides (noto-R1/Rg1/Re/Rf/Ra2/Rb1/Rc/Ro/Rb2/Rb3/Rd/Rh1/Rg2/Rg3/Rg3(R)/24(R)-p-F11), experiments were conducted to optimize key parameters and validate its performance. A Poroshell 120 EC-C18 column and an XBridge Shield RP18 column were separately utilized in the first-dimensional (1D) and the second-dimensional (2D) chromatography. Eight consecutive cuttings could achieve good separation of 16 ginsenosides within 85 min. The developed MHC-2DLC/CAD method showed good linearity (R2 > 0.999), repeatability (RSD < 6.73%), stability (RSD < 5.63%), inter- and intra-day precision (RSD < 5.57%), recovery (93.76-111.14%), and the limit of detection (LOD) and limit of quantification (LOQ) varied between 0.45-2.37 ng and 0.96-4.71 ng, respectively. We applied it to the content determination of 16 ginsenosides simultaneously from 28 different ginseng-containing CPMs, which unveiled the ginsenoside content difference among the tested CPMs, and gave useful information to discriminate ginseng in the preparation samples, as well. The MHC-2DLC/CAD approach exhibited advantages of high specificity, good separation ability, and relative high analysis efficiency, which also justified the feasibility of our proposed ″Monomethod Characterization of Structure Analogs″ strategy in quality evaluation of diverse CPMs that contained different ginseng.

How to Design and Validate a Clinical Flow Cytometry Assay.

Multiparametric flow cytometry assays are long recognized as an essential diagnostic test for leukemias and lymphomas. Lacking Food and Drug Administration-approved standardized tests, these assays remain laboratory developed tests. The recently published guidelines, CLSI H62, are the most detailed and up-to-date instructions for designing and validating clinical flow cytometry assays. This review provides a historical background for the current situation, summarizes key points from the CLSI guidelines, and lists practical points for assay development gained from personal experience.

Comparative Characterization of the Ginsenosides from Six Panax Herbal Extracts and Their In Vitro Rat Gut Microbial Metabolites by Advanced Liquid Chromatography-Mass Spectrometry Approaches.

Ginseng extracts are extensively used as raw materials for food supplements and herbal medicines. This study aimed to characterize ginsenosides obtained from six Panax plant extracts (Panax ginseng, red ginseng, Panax quinquefolius, Panax notoginseng, Panax japonicus, and Panax japonicus var. major) and compared them with their in vitro metabolic profiles mediated by rat intestinal microbiota. Ultrahigh-performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) with scheduled multiple reaction monitoring (sMRM) quantitation methods were developed to characterize and compare the ginsenoside composition of the different extracts. After in vitro incubation, 248 ginsenosides/metabolites were identified by UHPLC/IM-QTOF-MS in six biotransformed samples. Deglycosylation was determined to be the main metabolic pathway of ginsenosides, and protopanaxadiol-type and oleanolic acid-type saponins were easier to be easily metabolized. Compared with the ginsenosides in plant extracts, those remaining in six biotransformed samples were considerably fewer after biotransformation for 8 h. However, the compositional differences in four subtypes of the ginsenosides among the six Panax plants became more distinct.

SARS-CoV-2 N-Antigen Quantification in Respiratory Tract, Plasma and Urine: Kinetics and Association with RT-qPCR Results.

Qualitative SARS-CoV-2 antigen assays based on immunochromatography are useful for mass diagnosis of COVID-19, even though their sensitivity is poor in comparison with RT-PCR assays. In addition, quantitative assays could improve antigenic test performance and allow testing with different specimens. Using quantitative assays, we tested 26 patients for viral RNA and N-antigen in respiratory samples, plasma and urine. This allowed us to compare the kinetics between the three compartments and to compare RNA and antigen concentrations in each. Our results showed the presence of N-antigen in respiratory (15/15, 100%), plasma (26/59, 44%) and urine (14/54, 28.9%) samples, whereas RNA was only detected in respiratory (15/15, 100%) and plasma (12/60, 20%) samples. We detected N-antigen in urine and plasma samples until the day 9 and day 13 post-inclusion, respectively. The antigen concentration was found to correlate with RNA levels in respiratory (p < 0.001) and plasma samples (p < 0.001). Finally, urinary antigen levels correlated with plasma levels (p < 0.001). Urine N-antigen detection could be part of the strategy for the late diagnosis and prognostic evaluation of COVID-19, given the ease and painlessness of sampling and the duration of antigen excretion in this biological compartment.

Porous Cellulose Substrate Study to Improve the Performance of Diffusion-Based Ionic Strength Sensors.

Microfluidic paper-based analytical devices (µPADs) are leading the field of low-cost, quantitative in-situ assays. However, understanding the flow behavior in cellulose-based membranes to achieve an accurate and rapid response has remained a challenge. Previous studies focused on commercial filter papers, and one of their problems was the time required to perform the test. This work studies the effect of different cellulose substrates on diffusion-based sensor performance. A diffusion-based sensor was laser cut on different cellulose fibers (Whatman and lab-made Sisal papers) with different structure characteristics, such as basis weight, density, pore size, fiber diameter, and length. Better sensitivity and faster response are found in papers with bigger pore sizes and lower basis weights. The designed sensor has been successfully used to quantify the ionic concentration of commercial wines with a 13.6 mM limit of detection in 30 s. The developed µPAD can be used in quantitative assays for agri-food applications without the need for any external equipment or trained personnel.

Development of an efficient reproducible cell-cell transmission assay for rapid quantification of SARS-CoV-2 spike interaction with hACE2.

Efficient quantitative assays for measurement of viral replication and infectivity are indispensable for future endeavors to develop prophylactic or therapeutic antiviral drugs or vaccines against SARS-CoV-2. We developed a SARS-CoV-2 cell-cell transmission assay that provides a rapid and quantitative readout to assess SARS-CoV-2 spike hACE2 interaction in the absence of pseudotyped particles or live virus. We established two well-behaved stable cell lines, which demonstrated a remarkable correlation with standard cell-free viral pseudotyping for inhibition by convalescent sera, small-molecule drugs, and murine anti-spike monoclonal antibodies. The assay is rapid, reliable, and highly reproducible, without a requirement for any specialized research reagents or laboratory equipment and should be easy to adapt for use in most investigative and clinical settings. It can be effectively used or modified for high-throughput screening for compounds and biologics that interfere with virus-cell binding and entry to complement other neutralization assays currently in use.

A Simple Quantitative Assay for Measuring ß-Galactosidase Activity Using X-Gal in Yeast-Based Interaction Analyses.

Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme ß-galactosidase (ß-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-ß-d-galactopyranoside) is an inexpensive and sensitive substrate of ß-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5'-dibromo-4,4'-dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (ortho-nitrophenyl-ß-galactopyranoside) as a substrate for ß-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent yeast cells and transformation Alternate Protocol 1: In-house preparation of yeast competent cells for use in lithium acetate (LiAc)-mediated yeast transformation Support Protocol: Long-term storage and revival of frozen yeast strain stocks Basic Protocol 2: Measuring ß-galactosidase activity via the quantitative X-gal assay Alternate Protocol 2: Quantification of interaction strength using liquid ONPG assay.

Performance assessment of the new Xpert® HIV-1 viral load XC assay for quantification of HIV-1 viral loads.

BACKGROUND: HIV-1 RNA quantification is a key component of treatment monitoring. OBJECTIVES: To assess the performance of a redesigned HIV-1 RNA quantitative assay that uses a dual-target approach: Xpert® HIV-1 Viral Load (VL) XC. STUDY DESIGN: Fresh and frozen samples (N = 533) from HIV-1 positive patients tested with Abbott HIV-1 assays (Alinity m and RealTime [m2000]) were retested using the new Xpert XC assay. Three samples with known underquantification using the previous single-target Xpert assay were retested. RESULTS: The Xpert XC assay yielded valid results in 98.5% (N = 528/536) of cases and showed high sensitivity in 80 fresh samples that had undetectable VLs or ≤1.7 log copies/mL with Alinity m. Linear regression and Bland-Altman analyses showed high concordance with the Abbott tests for quantified samples over a wide VL range (1.6-6.9 copies/mL), including non-B subtypes (mean difference=-0.1±0.23 log copies/mL). Mutations associated with integrase resistance did not impact the results. Very good linearity and reproducibility was shown for the tested subtypes B, CRF06_cpx, and CRF02_AG. Xpert XC VLs in samples that were previously underquantified using the original single-target Xpert assay were similar to those detected by the Abbott assays (±0.11 log copies/mL). CONCLUSIONS: The Xpert XC assay showed excellent correlation with the Abbott assays for all tested HIV-1 subtypes. Sensitivity, linearity and accuracy were high in the therapeutically relevant VL range. With a time to result of only 90 min, this on-demand decentralized assay is a safe, reliable and fast option for VL monitoring in HIV-1-infected patients.

Alg mannosyltransferases: From functional and structural analyses to the lipid-linked oligosaccharide pathway reconstitution.

BACKGROUND: N-glycosylation is initiated from the biosynthesis of lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER), which is catalyzed by a series of Alg (asparagine-linked glycosylation) proteins. SCOPE OF REVIEW: This review summarizes our recent studies on the enzymology of Alg mannosyltransferases (MTases). We also discuss the membrane topology and physiological importance of several ER cytosolic Alg proteins. MAJOR CONCLUSIONS: Utilizing an efficient prokaryotic protein expression system and a new LC-MS quantitative activity assay, we overexpressed all Alg MTases and performed enzymology studies. Moreover, by reconstituting the LLO pathway, the high-yield chemoenzymatic synthesis of high-mannose-type N-glycans was accomplished using recombinant Alg MTases. GENERAL SIGNIFICANCE: The analysis of the enzymology and topology of Alg MTases has provided valuable biochemical information in the LLO biosynthesis pathway. In addition, an efficient chemoenzymatic strategy that could prepare various oligomannose-type N-glycans in sufficient amounts was established for further biological assays.

The clinical value of fluorescence quantitative assay and G6PD/6PGD ratio method in the diagnosis of glucose-6-phosphate dehydrogenase deficiency and gene mutation / 中华检验医学杂志

Objective:To investigate the diagnostic value of fluorescence quantitative method and G6PD/6PGD ratio method in glucose-6-phosphate dehydrogenase (G6PD) deficiency and the type of gene mutation.Methods:A total of 1 201 patients (711 males and 490 females) with suspected G6PD deficiency in Shanghai Children′s Hospital were collected from June 2018 to March 2021. Fluorescence quantification method, G6PD/6PGD ratio method and multicolor melting curve were used to detects enzyme activity, ratio and gene mutation type. Comparison of each index and evaluation of its diagnostic efficiency were performed.Results:Among 1 201 suspicious samples, 163 cases (135 males and 28 females) were finally diagnosed. 156 cases were diagnosed by fluorescence quantitative method with a detection rate of 95.71%, and 140 cases were diagnosed by G6PD/6PGD ratio method with a detection rate of 85.89%. enzymatic activity of G6PD and ratio of G6PD/6PGD in male were significantly lower than female, and the differences were statistically significant ( U=642.5, 734.5, P<0.001). 112 cases received G6PD gene mutation detection and 92 cases were diagnosed, 74 were hemizygous mutations, 1 were homozygous mutations, 15 were heterozygous mutations, and 2 were compound heterozygous mutations. Among 15 cases of heterozygous mutations, 11 cases were diagnosed by fluorescence quantitative method, the diagnosed rate was 73.33%, 4 cases were diagnosed by G6PD/6PGD ratio method, and the diagnosed rate was 26.67%. A total of 7 mutation sites were detected and the proportions were c.1388G>A (32.22%), c.1376G>T (30.00%), c.871G>A (13.33%), c.1024C>T (11.11%). c.95A>G (7.78%), c.487G>A (4.44%), c.392G>T (1.11%). The enzymatic activities of c.1376G>T and c.1024C>T, c.487G>A were statistically significant ( P<0.001,0.015); the G6PD/6PGD ratios of c.1024C>T and c.1388G>A, c.1376G>T were statistically significant ( P=0.017,0.002,0.011,0.013). Fluorescence quantitative method had sensitivity of 100%, specificity of 95.65%, and the area under the curve (AUC) is 0.972. The sensitivity of the G6PD/6PGD ratio method was 100%, the specificity was 94.57%, and the AUC was 0.979. The sensitivity of fluorescence quantitative method combined with G6PD/6PGD ratio was 96.7%, the specificity was 100%, and the AUC was 0.992. Conclusions:Compared with fluorescence quantification, the G6PD/6PGD ratio method might not be able to diagnose female heterozygotes effectively; The panel of G6PD fluorescence quantification and G6PD/6PGD ratio was helpful to reduce the missed diagnosis. Combined with gene mutation analysis, it could improve the diagnosis rate of G6PD deficiency in the children.

Performance characteristics of the light-initiated chemiluminescent assay for quantitative determination of progesterone.

BACKGROUND: To study the performance of quantitative determination of progesterone by light-initiated chemiluminescent assay (LICA). METHODS: Clinical samples of serum were used for detection of progesterone by LICA. The precision study was performed according to Clinical and Laboratory Standards Institute (CLSI) EP15-A3, the linear range validation was performed according to CLSI EP06-A, accuracy was evaluated according to CLSI EP9-A3, and the performance of detection capability was confirmed according to CLSI EP17-A2. All data were analyzed using SPSS software. Function regression analysis was performed by OriginPro software. RESULTS: The LICA-800 system exhibited low coefficients of variation (CVs) and high reproducibility, and the calculated synthetic CV was 2.16%. The access progesterone assay showed excellent linearity in the assay measuring range (0.37-40 ng/mL) using the polynomial regression method in accordance with CLSI EP06-A. Bias assessment was used to verify accuracy, and the percentage deviation met the quality requirements of the laboratory's allowable deviation of 10.00%. In terms of the detection capability of LICA, the calculated limit of blank (LoB) was 0.046 ng/mL, limit of detection (LoD) was 0.057 ng/mL, and the limit of quantitation (LoQ) value was 0.161 ng/mL. CONCLUSIONS: The competitive LICA provided a highly sensitive, accurate and precise method for measuring serum progesterone level.

Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

Fragile X syndrome results from the absence of the FMR1 gene product-Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

Simultaneous quantitative assays of 15 ginsenosides from 119 batches of ginseng samples representing 12 traditional Chinese medicines by ultra-high performance liquid chromatography coupled with charged aerosol detector.

Despite the extensive consumption of ginseng, precise quality control of different ginseng products is highly challenging due to the containing of ginsenosides in common for different Panax species or different parts (e.g. root, leaf, and flower) of a same species. Herein we performed a comparative investigation of diverse ginseng products by simultaneously assaying 15 saponins (notoginsenoside R1, ginsenosides Rg1, -Re, -Rf, -Ra2, -Rb1, -Rc, -Ro, -Rb2, -Rb3, -Rd, 20(R)-ginsenoside Rg3, 24(R)-pseudoginsenoside F11, chikusetsusaponins IV, and -IVa) using an ultra-high-performance liquid chromatography/charged aerosol detector (UHPLC-CAD) approach. Twelve Panax-derived ginseng products (involving P. ginseng root, P. quinquefolius root, P. notoginseng root, Red ginseng, P. ginseng leaf, P. quinquefolius leaf, P. notoginseng leaf, P. ginseng flower, P. quinquefolius flower, P. notoginseng flower, P. japonicus root, and P. japonicus var. major root) were considered. Benefiting from the condition optimization, the baseline resolution of 15 ginsenosides was achieved on a CORTECS UPLC Shield RP18 column. This method was validated as specific, precise (0.81-1.94% intra-day variation; 0.86-2.35% inter-day variation), and accurate (recovery: 90.73-107.5%), with good linearity (R2 > 0.999), high sensitivity (limit of detection: 0.02-0.21 µg; limit of quantitation: 0.04-0.42 µg) and sample stability (1.49-4.74%). Its application to 119 batches of ginseng samples unveiled vital information enabling the authentication of these different ginseng products. Detection of ginsenosides by CAD exhibited superiority over UV in sensitivity and the ability to monitor chromophore-free structures. Large-scale comparative studies by quantifying multiple markers provide methodological reference to the precise quality control of herbal medicine.

Quantifying SARS-CoV-2 viral load: current status and future prospects.

Introduction: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emerged as a novel viral agent that quickly spread worldwide. SARS-CoV-2 is responsible for the human coronavirus disease 2019 (COVID-19) which has claimed hundreds of thousands of lives and had an immeasurable toll on the economy. Currently, most clinical cases are identified by qualitative molecular testing. However, the need for quantitative assessment is gaining traction.Areas covered: In this review, the current state and future perspective of SARS-CoV-2 viral load quantification is presented.Expert opinion: Viral load quantification is a critical measure that informs clinicians of treatment response, actionable viral load levels, and guidance on patient management. Additionally, for pathogens with epidemiological consequences, viral load can provide information to guide infection control measures and policies. While qualitative detection is sufficient to identify cases and initiate containment and mitigation measures in the vast majority of COVID-19 cases, in certain situations, SARS-CoV-2 quantification is needed to assess viral load trending. However, there are obstacles to quantification, including variability in respiratory specimen collection and the lack of commutable reference material. At the same time, the need for quantification for clinical and epidemiological management is growing, especially concerning individuals with prolonged RNA shedding.

Flow Control in Porous Media: From Numerical Analysis to Quantitative µPAD for Ionic Strength Measurements.

Microfluidic paper-based analytical devices (µPADs) are a promising technology to enable accurate and quantitative in situ assays. Paper's inherent hydrophilicity drives the fluids without the need for external pressure sources. However, controlling the flow in the porous medium has remained a challenge. This study addresses this problem from the nature of the paper substrate and its design. A computational fluid dynamic model has been developed, which couples the characteristics of the porous media (fiber length, fiber diameter and porosity) to the fluidic performance of the diffusion-based µPAD sensor. The numerical results showed that for a given porous membrane, the diffusion, and therefore the sensor performance is affected not only by the substrate nature but also by the inlets' orientation. Given a porous substrate, the optimum performance is achieved by the lowest inlets' angle. A diffusion-based self-referencing colorimetric sensor was built and validated according to the design. The device is able to quantify the hydronium concentration in wines by comparison to 0.1-1.0 M tartaric acid solutions with a 41.3 mM limit of detection. This research showed that by proper adjustments even the simplest µPADs can be used in quantitative assays for agri-food applications.

Relación entre género y niveles de proteína C reactiva / Relation among gender and levels of C reactive protein

RESUMEN Fundamento: La proteína C reactiva es uno de los mejores marcadores para la valoración y seguimiento de enfermedades inflamatorias; los valores de referencias recomendados para su concentración en suero no están ajustados según género. Objetivo: Determinar si la concentración de proteína C reactiva difiere según el género. Metodología: Se realizó un estudio exploratorio en 3199 muestras de pacientes procedentes del Hospital General Provincial Camilo Cienfuegos y en un grupo control de 76 muestras de sueros de donantes de sangre para cada género, procedentes del Banco de Sangre de Sancti Spíritus. Los niveles se midieron a través un ensayo semicuantitativo de aglutinación con partículas de látex para la muestra supuestamente enferma y uno cuantitativo inmunoturbidimétrico para la supuestamente sana. Se compararon los niveles medios entre género en cada una mediante la prueba t de Student para muestras independientes. Resultados: La media de los niveles de proteína C reactiva en el género masculino y femenino de la muestra supuestamente enferma fue de 3.49 mg/L y 3.41 mg/L respectivamente. En el grupo control la comparación de medias de los niveles de proteína C reactiva entre género fue para los hombres de 1.38 mg/L y para las mujeres de 1.94 mg/L. Conclusión: No se encontraron diferencias significativas entre género en la muestra supuestamente enferma, ni en el grupo control.

ABSTRACT Background: C-reactive protein is one of the best marker for the assessment and monitoring of inflammatory diseases; the recommended reference values for its serum concentration are not gender-adjusted. Objective: To determine whether C-reactive protein concentration differs by gender. Methodology: An exploratory study was conducted on 3199 patient samples from Camilo Cienfuegos General Provincial Hospital and a control group of 76 serum samples from blood donors of each gender from the Sancti Spíritus Blood Bank. Levels were measured by a semi-quantitative latex particle agglutination assay for the presumed diseased sample and a quantitative immunoturbidimetric assay for the presumed healthy sample. Mean levels were compared between genders in each using Student's t-test for independent samples. Results: The mean of C-reactive protein levels in the male and female gender of the supposedly diseased sample was 3.49 mg/L and 3.41 mg/L respectively. In the control group the mean comparison of C-reactive protein levels between genders was 1.38 mg/L for males and 1.94 mg/L for females. Conclusion: No significant gender differences were found in the presumed ill sample, nor in the control group.

Charged aerosol detection in early and late-stage pharmaceutical development: selection of regressionmodels at optimum power function value.

In recent years, the use of quantitative liquid chromatography (LC) coupled charged aerosol detection (CAD) for poor UV absorbing analytes in multicomponent mixtures has grown exponentially across academic and industrial sectors. The ballpark of previous LC-CAD reports is focused on practical applications, as well as optimization of critical parameters such as: response dependencies on temperature, nebulization process, analyte volatility, and mobile-phase composition. However, straightforward approaches to deal with the characteristic nonlinear response of CAD still scarce. A highly overlooked parameter is the power function value (PFV), whose optimization enables a detection signal that is more linear with higher signal-to-noise ratio (S/N) and lower relative standard deviation (RSD) of area counts. Herein, a systematic investigation of different regression models (log-log, first-and second-degree polynomial) by both interpolation and extrapolation process in conjunction with PFV optimization throughout the development of LC-CAD assays is reported. The accuracy of the results via interpolation is always good (< 5%) when operating in the vicinity of the optimum PFV regardless the regression model choice. On the contrary, extrapolation process only worked when applying log-log regression at the optimum PFV (accuracy <5%). This outcome indicates that a first-order regression via interpolation can be a safe and simple choice for quantitative LC-CAD in highly regulated laboratories (GLP, GMP, etc.). Whereas a straightforward extrapolation combined with log-log regression can enable the deployment of high-throughput LC-CAD assays, especially but not limited to laboratories where the synthetic process route is undergoing rapid change and optimization (medicinal chemistry, discovery, biocatalysis, process chemistry, etc.). This approach is crucial in developing quantitative LC-CAD assays for poor UV absorbing pharmaceuticals that are sensitive, precise, accurate and robust across early and late-stage pharmaceutical development.