Assay Time Research Articles

Protein-bridged size-permeable DNAzyme@PEG hollow nanosphere reactor facilitates direct and rapid monitoring of trace circulating microRNAs in undiluted cancer patient serums

The direct monitoring of low levels of circulating microRNAs (miRNAs) in undiluted human serums is of significant clinical importance for the diagnosis of different diseases in the early stages; however, this is highly challenged by both the short lengths, low abundance and homogeneity nature of miRNAs and the complicated sample matrix of human serums. In this work, we show the synthesis of a new protein-bridged and size-permeable DNAzyme@PEG hollow nanosphere reactor (p-DPHNR) that enables direct and rapid detection of trace circulating microRNAs in undiluted serums from prostate carcinoma patients. The hydrophilic PEG shell of p-DPHNR efficiently reduces protein adsorption; its size-permeability only allows short miRNAs to enter the core cavity and protects the core DNA signal probes from digestion; and the confinement of the NIR signal amplification DNA probes in the nanosphere core accelerates the reaction kinetics and minimizes the serum background fluorescence. Such p-DPHNR exhibits a 1.8 pM detection limit with a short 20 min assay time for miRNA-141 and can realize clear discrimination of prostate carcinoma patients from healthy donors, thereby manifesting its robust clinical applications for the convenient and early diagnosis of various diseases

Low-cost portable sensor for rapid and sensitive detection of Pb2+ ions using capacitance sensing integrated with microfluidic enrichment.

Lead ion (Pb2+) pollution is a critical global issue due to its ability to accumulate in the human body, resulting in severe health problems. Despite extensive research efforts devoted to the detection of Pb2+ contamination, practical, rapid, and field-deployable sensors for Pb2+ is yet to be developed to effectively safeguard the environment and public health. Herein, a label-free affinity-based sensing device is developed based on printed circuit board (PCB) for low-cost, easy-to-use, and real-time on-site detection of Pb2+ ions. The sensors are prepared by forming a self-assembled monolayer of glutathione (GSH) on the surface of gold-plated PCB electrodes, which serves as a molecular probe to recognize Pb2+. Rapid and sensitive detection is achieved by using capacitance sensing integrated with microfluidic enrichment. The sensor's interfacial capacitance is used to indicate specific binding, while the capacitance reading process simultaneously induces alternating current electrothermal (ACET) acceleration of analyte's travel towards the probes. Thus, the enrichment and detection are integrated into a single step, making pre-concentration unnecessary and shortening the assay time to 30s. This Pb2+ sensor has demonstrated one of the lowest limits of detection reported so far (1.85 fM) with a linear range of 0.01-10pM. To evaluate the sensor's specificity, non-target metal ions are tested, all showing negligible responses. Testing of tap water sample also yields reasonable results, validating the sensor's robustness. The above-mentioned features, together with a commercial portable readout, make this sensor well-suited for point-of-use Pb2+ detection at low cost.

NanoString nCounter-Based Assay for Detection of Fusion-Associated Salivary Gland Tumors.

Salivary gland tumors include numerous subtypes that vary from benign to highly aggressive, with many showing overlapping histopathological features that can make diagnosis challenging. Most subtypes express driver fusion genes that are tumor specific, and detection of such fusions is useful for differentiating amongst specific diagnoses, determining appropriate tumor grading, and guiding effective treatment. Currently, fusions can be detected by FISH, RT-PCR or through next-generation sequencing approaches, all of which are highly effective methodologies but can be costly or time consuming. We developed a rapid NanoString nCounter platform-based assay to detect salivary gland tumor fusions using a combination of fusion junction-specific probes and an approach through differential exon expression analysis. The assay includes 68 junction-specific probes and analysis of exon expression across 9 fusion-associated genes in a single multiplex assay. Out of 55 retrospective and 171 prospective cases assayed, we accurately detected the majority of cases of pleomorphic adenoma, adenoid cystic carcinoma, cribriform adenocarcinoma, clear cell carcinoma, secretory carcinoma and NUT-rearranged carcinoma, including cases of these tumor types arising in non-salivary gland sites, with the major drawback being an inability to detect MAML2-containing mucoepidermoid samples. With mucoepidermoid carcinoma excluded, the assay shows an overall sensitivity of 96.1% and specificity of 100%. We show that the majority of salivary gland tumor fusions can be effectively detected with a single rapid NanoString based assay, which can serve as a useful adjunctive tool for routine diagnostic head and neck pathology. The assay is low cost with a rapid turnaround time (30h total assay time per sample batch, with minimal technician input required) compared to alternate detection methods.

Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips.

This work demonstrates a novel, non-fluorescence approach to the length identification of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products, utilizing capillary flow velocities on paper microfluidic chips. It required only a blank paper chip, aminated microspheres, and a smartphone, with a rapid assay time and under ambient lighting. A smartphone evaluated the initial capillary flow velocities on the paper chips for the PCR and RPA products from various bacterial samples, where the pre-loaded aminated microspheres differentiated their flow velocities. Flow velocities were analyzed at different time frames and compared with the instantaneous flow velocities and interfacial tension (γLV) data. Subsequent error analysis justified the use of the initial time frames. A robust linear relationship could be established between the initial flow velocities against the square root of the product lengths, with R2 values of 0.981 for PCR and 0.993 for RPA. The assay seemed not to have a significant dependency on the cycle numbers and initial target concentrations. This novel method can be potentially used with various paper microfluidic methods of nucleic acid amplification tests towards rapid and handheld assays.

Glycan-Evocated Metallization for Amplification-Free Electrochemical Detection of Glycoproteins at Low Concentration Levels.

Glycoproteins have become the most often screened tumor markers in the in vitro diagnostics. Although a large number of electrochemical methods have been proposed to sensitively detect glycoproteins, most of them involve the aid of laborious signal amplification. Herein, we report the use of glycan-evocated metallization (GlyMetal) for the amplification-free electrochemical detection of glycoproteins at low concentration levels. Briefly, the glycoproteins of interest are captured by an aptamer recognition layer, and then the glycans of targets are oxidized by NaIO4 to convert the 1,2-diol sites into aldehyde groups for the silver deposition-based metallization, followed by the electrochemical stripping assay of the deposited metallic silver for glycoprotein quantification via the established solid-state Ag/AgCl voltammetric process. As GlyMetal can enable the deposition of a large amount of metallic silver and a high signal-to-background ratio can be obtained for the solid-state Ag/AgCl voltammetric stripping assay, the developed GlyMetal-based electrochemical method is applicable to the amplification-free detection of glycoproteins. As a proof of concept, a detection limit of 1.65 pg/mL has been achieved for carcinoembryonic antigen (CEA) detection. In addition to the high selectivity, desirable results have been obtained with respect to the use of the method for CEA detection in serum samples. In consideration of the desirable simplicity, short assay time, and cost-effectiveness of the amplification-free approach, the GlyMetal-based electrochemical method shows great promise in the point-of-care detection of glycoproteins.

Metal organic frameworks (MOFs)-based fluorescent CA125 analysis: A comparative study of the quenching effects of MIL-101, Cu-MOF, ZIF-8, UiO-66

Metal-organic frameworks (MOFs) are a class of materials with highly ordered pore structures. Due to their unique physical and chemical properties, they show great potential in the field of biosensors. Some MOFs can adsorb fluorescently labeled nucleic acid aptamers through various interaction mechanisms (such as electrostatic interactions, π-π stacking, hydrogen bonding, etc.). These interactions not only ensure the stable binding of the aptamers but also allow for their controlled release under specific conditions (such as changes in pH, temperature, or the presence of specific molecules). This mechanism provides multiple possibilities for the design of biosensors. Herein, we have systematically compared the quenching effects of widely used MOFs that can bind to aptamers, i.e., Fe-MOF (MIL-101), Cu-MOF, Zn-MOF (ZIF-8) and Zr-MOF (UiO-66). The study on the kinetics, quenching efficiency, and influencing factors such as ionic strength pH and temperature is performed. Interestingly, Cu-MOF exhibits superior quenching abilities to the other three materials in both the quenching efficiency and kinetics. Thus, a Cu-MOF based fluorescent sensor is reported to detect the ovarian cancer marker carbohydrate antigen 125 (CA125), which provides convenient detection performance (assay time about 10 min), and a detection range from 0.1 to 400 ng/mL. Moreover, it is designed in a simple mix-and-detect format and can be directly applied to clinical sample detection. This work may offer guidance for the choice of MOFs and elaborate design of biosensors.

An Electrochemical Bioplatform Based on Acrylamide‐Induced Inhibition of DNA‐RNA Hybridization for the Determination of Acrylamide

AbstractThis study reports the development of an innovative electrochemical bioplatform for the indirect determination of acrylamide (AM) based on its inhibitory effect on the hybridization of nucleic acids through the formation of AM‐guanine single‐stranded (ssDNA) adducts. The method combines the advantages of the formation and selective immunorecognition of DNA/RNA heteroduplex, with the use of MBs and amperometric transduction using disposable SPCEs. Increasing concentrations of acrylamide prevented the efficient hybridization of a synthetic biotinylated DNA probe immobilized on streptavidin‐coated magnetic beads with its complementary RNA target and the as‐formed heteroduplex recognition with a specific antibody and a secondary HRP‐labeled antibody used for electrochemical readout. The resulting magnetic bioconjugates were magnetically trapped on the surface of the working electrode of a SPCE and the amperometric transduction was carried out in the presence of the H2O2/HQ system, recording a cathodic response inversely proportional to the AM concentration. The developed bioplatform is more competitive with other reported methods in terms of simplicity and assay time, allowing the selective detection of 1 nM AM in 60 min. In addition, the bioplatform was applied to the analysis of potato crisp water extracts.

Streptococcus pneumoniae in Medical Districts of Burkina Faso: Serotype Distribution and Antibiotic Susceptibility during 2010 to 2012

Introduction: Streptococcus pneumoniae is a highly virulent pathogen. Despite the recent introduction of vaccines in the program of prevention in Burkina Faso, treatment by antibiotics has remained in use. The aim of this study was to determine the serotypes implied in invasive pneumococcal infections and their resistance to antibiotics used for routine treatment in Burkina Faso. Methods: From March 2010 to December 2012, a total of 6,102 cerebrospinal fluid (CSF) and pleural fluid (PF) specimens were collected in Burkina Faso. The specimens were analyzed by latex agglutination assay, culture, and real time polymerase chain reaction (rt-PCR) for species identification. Serotyping was performed by rt-PCR and minimum inhibitory concentrations were determined by the E-test. Results: Among 562 S. pneumoniae strains identified from 6,102 specimens, 440 isolates were serotyped by rt-PCR. Serotypes 1 and 5 were the most common (50%); however, 70 (15.9%) strains were not detected by rt-PCR methodology. Among patients of less than 20 years of age, serotype 1 was the most frequent strain identified (9.3% to 13.2%). Ampicillin and ceftriaxone were the most active antibiotics in vitro against the strains identified. The rate of S. pneumoniae strains susceptible to chloramphenicol was 83%. Serotypes 1 (7/49) and 5 (5/7) were non-susceptible to chloramphenicol. Conclusion: Serotypes 1 and 5 were the common serotypes identified. Conjugate vaccines introduced in Burkina Faso should take in account these valences, to decrease the incidence of pneumococcal infections efficiently.

Amplification-free detection of Ascochyta blight in chickpea using a simple molecular beacon assay

Ascochyta blight is a major biotic stress that limits chickpea production globally. Fungicide application remains one of the effective control measures for the endemic spread. Due to the serious threat that synthetic fungicides pose to crop quality, early diagnosis of the pathogen is imperative. Whilst there have previously been several conventional lab-based diagnostic methods developed for early detection of Ascochyta rabiei, they require long assay times, specialised equipment and facilities, and trained personnel to process the samples. To overcome this challenge, a rapid amplification-free detection assay using a molecular beacon probe was developed. The method consists of a simple assembly assay that accurately detects pathogen within 30 min. The developed assay is species-specific and has a similar sensitivity level as conventional amplification-based methods. Although it is still a lab-based technique, considering the simplicity of the assay, it has a great potential to be developed further as a reliable in-field diagnostic device for early detection and quantification of fungal pathogen spores.

A gold nanoparticle-enhanced dCas9-mediated fluorescence resonance energy transfer for nucleic acid detection

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas proteins coupled with pre-amplification have shown great potential in molecular diagnoses. However, the current CRISPR-based methods require additional reporters and time-consuming process. Herein, a gold nanoparticle (AuNP)-enhanced CRISPR/dCas9-mediated fluorescence resonance energy transfer (FRET) termed Au-CFRET platform was proposed for rapid, sensitive, and specific detection of nucleic acid for the first time. In the Au-CFRET sensing platform, AuNP was functionalized with dCas9 and used as nanoprobe. Target DNA was amplified with FAM-labeled primers and then precisely bound with AuNP-dCas9. The formed complex rendered the distance between AuNP acceptor and FAM donor to be short enough for the occurrence of FRET, thus resulting in fluorescence quenching. Moreover, AuNPs were demonstrated to enhance binding efficiency of dCas9 to target DNA in Au-CFRET system. The key factors regarding the FRET efficiency were analyzed and characterized in detail, including the length of donor/acceptor and the size of AuNPs. Under the optimal conditions, Au-CFRET could determinate CaMV35S promoter of genetically modified rice as low as 21 copies μL−1. Moreover, Au-CFRET sensing system coupled with one-step extraction and recombinase polymerase amplification can identify the genuine plant seeds within 30 min from sampling to results at room/body temperature without expensive equipment or technical expertise, and requires no additional exogenous reporters. Therefore, the proposed sensing platform significantly simplified the system and shortened the assay time for nucleic acid diagnoses.

A-324 Performance and Turnaround Time of Plasma Lactate Assay on POCT, Blood Gas Instrument, and Chemistry Analyzer

Abstract Background Time is a critical factor of plasma lactate testing in the management of patients with sepsis. To best serve these patients, we must optimize testing methods to provide a turnaround time (TAT) that guides efficient patient care. Point of care tests (POCTs) provide rapid results for emergency department and medical intensive care unit but are not feasible for all hospital settings. Laboratory testing methods on an automation line provide greater efficiency but result slowly due to need of specimen preparation and longer analytical time. The goal of this study is to provide detailed TATs of plasma lactate testing among the methods of POCT, chemistry analyzer in an automation line, and blood gas analyzer. Methods Plasma lactate testing was performed by three methods, GEM Premier ChemSTAT (POCT in ER only), Beckman AU5800 (chemistry analyzer), and Radiometer ABL800 (blood gas instrument). The TAT of lactate for each method was measured and include the time from specimen collection to laboratory receipt (except for POCT method), and time from specimen receipt to resulting (including specimen preparation for Beckman AU5800). Results The average TATs of lactate testing for each method in all hospital sites are detailed with respect to each event and subcategorized (Table 1). The average TAT of 12.0 min on Radiometer ABL800 is higher than the TAT of 2.5 on GEM Premier ChemSTAT method but is greatly reduced compared to the TAT of 35.4 min on Beckman AU5800. Conclusions Radiometer ABL800 method could be the optimal laboratory method for plasma lactate testing fulfilling the TAT (average 12 min), with less test cancellation due to specimen stability issue, and combines testing with blood gases. Using three Radiometer ABL800 instruments, we manage the performance of around 70 plasma lactate tests daily.

B-033 Real World Assessment of the Impact of Infectious Disease Assays on Workflow Capabilities of Siemens Atellica IM Analyzers

Abstract Background Instrument immunoassay throughputs publicized in the industry are often theoretical, performed in relatively smaller scale, and/or in the best case generated only with fast assays, not representing the typical assay mix of routine activity. Infectious disease (ID) serology assays typically have long analytical times and have the potential to influence throughput capabilities and turnaround time (TAT) of other assays with shorter analytical times. The objective of this study is to utilize real world evidence across a large fleet of Atellica® IM 1300 and IM 1600 analyzers representing variously sized laboratories, using variations of ID assay mixes, to assess the impact on TAT on select STAT immunoassays. Methods Assay mix, test volumes and TAT data was mined from the Atellica® Smart Remote Services (SRS), a Siemens Healthineers proprietary remote connectivity platform over 3 distinct 14-day time windows. Real world data from >1800 Atellica IM and >8 million tests were queried per time window for the following ID assays: HIV, Hepatitis B, Hepatitis C, TORCH, Syphilis (long analytical time) and a selection of non-ID immunoassays: hs-troponin I (TNIH), Thyroid Stimulating Hormone (TSH3UL), total HCG (ThCG), and B-Type Natriuretic Peptide (BNP) (short analytical time). Median TAT for short assay was analyzed with 6 variations of ID assay mix (0%, <10%, <20%, <30%, <40%, <50%) in the run representing increasing percentages of assays requiring longer incubation times. TAT was represented as barcode to result and aspiration to result. Results The median TAT for TNIH remained consistent at 10.1 minutes across increasing % of ID assays (N=100961). The median TAT for TNIH for platforms not running ID assays (83237) remained consistent at 10.03 minutes. The median TAT for TSH remained consistent at 14.02 minutes across increasing % of ID assays (N=1183344). The median TAT for TSH for platforms not running ID assays (N=480219) remained consistent at 13.98 minutes. The median TAT for ThCG remained consistent at 10.28 minutes across increasing % of ID assays (N=24677). The median TAT for ThCG for platforms not running ID assays (N=79275) remained consistent at 10.27 minutes. The median TAT for BNP remained consistent at 10.1 minutes across increasing % of ID assays (N=42949). The median TAT for BNP for platforms not running ID assays (N=14990) remained consistent at 10.03 minutes. Conclusions Throughput and TAT on the Atellica IM Analyzer is relatively unaffected by mix of longer-incubation and shorter-incubation assays. The dual incubation rings allow more flexibility in the mix of incubation times with predictable and consistent TAT for all assays including STAT.

A-052 Clinical usefulness of ADAMS A1c HA-8190V automatic switching mode for the HbA1c measurement in Hb variants

Abstract Background High-performance liquid chromatography (HPLC) is widely used as a HbA1c routine assay. However, quantification of glycated hemoglobin (HbA1c) is a challenge in patients with hemoglobin (Hb) variants. In this study, we evaluated the clinical utility and analytical performance of new developed ARKRAY ADMAS HA-8190V assay using autoswitch mode, that automatically switches between variant and fast mode depending on patient Hb characteristics, in patients with Hb variant suspected in Korea. Methods The measuring time for HbA1c assay was compared between the HA-8180V fast mode (existing model) and the autoswitch mode of HA-8190V (new model) in 60 normal Hb samples. And the HbA1c measured values was compared bewteen the HA-8180V fast mode and the HA-8190V autoswitch mode in 30 low Hb samples. In addition, flag detection for the abnormal Hb and measured values for 21 Hb variant samples were analyzed using the HA-8180V fast mode and the HA-8190V autoswitch mode. Results It took 50.8 minutes and 39.1 minute to measure 60 normal Hb samples by HA-8180V fast mode and HA-8190V autoswitching mode, respectively. The values could not be measured at the HA-8180V fast mode for 7 of 30 low Hb samples, whereas all samples could be measured at HA-8190V autoswitching mode. For 21 Hb variant samples, HA-8180V fast mode showed abnormal flag detections for only 14 samples, but HA-8190V autoswitch mode reported abnormal variant flags for 20 samples. In the result accuracy evaluation (% difference) based on capillary electrophoresis HbA1c assay (Sebia, Lisses, France) in the 21 Hb variant samples, the % difference of HA-8190 V autoswitch mode was 3.9%, which was superior to HA-8180V fast mode of -11.65%. Conclusions Autoswitch mode of The HA-8190V (automatically switch between variant and fast mode based on patient Hb characteristics) showed more accurate flag detection and HbA1c measurements in patients with Hb variants, along with shorter test time and manual reduction.

B-059 Development and Validation of Multiplex Quantitative Allergy Tests based on Automated Flow Fluorescence Immunoassay Analyzer

Abstract Background Serological allergy tests measuring allergen-specific IgE (sIgE) are simpler and safer than skin prick tests, and the results are not influenced by skin condition or human factors. Quantitative detection of sIgE can also aid in evaluating patient allergies and aiding subsequent treatment. Dymind has researched and developed novel quantitative allergy tests based on the automated flow fluorescence immunoassay analyzer system (MF1280), enabling quantitative detection of multiple sIgE in just one test with one sample. Here, we present the performance of tIgE and two specific IgE test kits using the Dymind MF1280 system. Methods This study evaluated the performance of total IgE (tIgE) and two allergen-specific IgE (sIgE) panels of inhalant and food allergens, inhalant allergen panel including d1 (Dermatophagoides pteronyssinus), d2 (Dermatophagoides farina), i6 (Cockroach), e1 (Cat epithelium), e5 (Dog epithelium), w1(Common ragweed), and food allergen panel including f1 (Egg white), f2 (Cow’s milk), f3 (cod), f23 (Crab), 24 (Shrimp), and f27 (beef). Evaluation criteria included precision, linearity, limit of detection (LOD) and method comparison. As clinical performance of 425 clinical samples collected from 2 hospital laboratory in China was evaluated by both of Dymind allergen IgE MF1280 system and Phadia ImmunoCAP. Results The validated linearity of total IgE and sIgE were 2.0-5000.0 IU/mL and 0.1-100.0 IU/mL respectively. The LOD of total IgE and sIgE determined to be 2 IU/mL and 0.1 IU/mL respectively. The precision was evaluated by assaying a low and a high samples of every sIgE assay and the result of d1, d2, i6, e1, e5, w1, f1, f2, f3, f23, f24, f27 were 3.73% / 1.34% (low sample / high sample), 3.26% / 1.95%, 2.18% / 0.87%, 1.31% / 1.40%, 3.63% / 1.13%, 2.84% / 4.76%, 4.50% / 1.53%, 5.97% / 1.64%, 3.46% / 1.11%, 1.58% / 1.73%, 3.77% / 1.28%, 1.78% / 0.96% respectively. The total mean coefficient of measurements was below 7% to ensure high precision in multiplex quantitative analysis. Method comparison of 425 clinical samples between Phadia ImmunoCAP and Dymind allergen IgE showed total concordance rate was over 85% except f2 (over 80%). Conclusions The Dymind Allergen-specific IgE test kits on the automated flow fluorescence immunoassay analyzer system provide fast and accurate in vitro diagnostic detection of tIgE and sIgE for aiding diagnostics specific allergens sensitization and treatment for allergy patients, with good precision and excellent agreement compared to ImmunoCAP. The MF1280 system has advantages including random access, fully automated, multiplex quantitative detection of allergen sIgE with reducing assay time, sample consumption and lower costs.

Coumarin-imidazopyridine hybrids and their first-in-class ZnII metal complexes as potent dual entry and replication inhibitors of Zika viral infection

In this study, we synthesized and characterized a series of coumarin-imidazopyridine hybrid ligands (HL1-HL4) and their corresponding Zn(II) complexes (C1-C4). The ligands were synthesized via a two-step process in 56–90 % yields. The resulting ligands, were utilized to form Zn(II) complexes, characterized by conductivity measurements, HRMS, IR, 1H NMR spectroscopy and X-ray diffractions. Biological evaluations revealed that these compounds exhibited potent antiviral activity against Zika virus (ZIKV), with EC50 values ranging from 0.55 to 4.8 µM and SI of up to 1490. Notably, the complexes (the first-in-class Zn(II) anti-ZIKV complexes) generally displayed enhanced activity compared to their respective ligands, with some compounds outperforming the reference antiviral, ribavirin. The Time of Addition assay suggested that while some compounds interfere with both viral entry (with a virucidal component) and replication phases, other only acted in replication phases. These results together with molecular modeling studies on ZIKV Envelope protein and ZIKV NS2B-NS3 offered insights for their mode of actions and further optimizations.

Exploring the antiviral activities of the FDA-approved drug sulfadoxine and its derivatives against Chikungunya virus.

Currently, there is no antiviral licensed to treat chikungunya fever, a disease caused by the infection with Alphavirus chikungunya (CHIKV). Treatment is based on analgesic and anti-inflammatory drugs to relieve symptoms. Our study aimed to evaluate the antiviral activity of sulfadoxine (SFX), an FDA-approved drug, and its derivatives complexed with silver(I) (AgSFX), salicylaldehyde Schiff base (SFX-SL), and with both Ag and SL (AgSFX-SL) against CHIKV. The anti-CHIKV activity of SFX and its derivatives was investigated using BHK-21 cells infected with CHIKV-nanoluc, a marker virus-carrying nanoluciferase reporter. Dose-response and time of drug-addition assays were performed in order to assess the antiviral effects of the compounds, as well as in silico data and ATR-FTIR analysis for insights on their mechanisms of action. The SFX inhibited 34% of CHIKV replication, while AgSFX, SFX-SL, and AgSFX-SL enhanced anti-CHIKV activity to 84%, 89%, and 95%, respectively. AgSFX, SFX-SL, and AgSFX-SL significantly decreased viral entry and post-entry to host cells, and the latter also protected cells against infection. Additionally, molecular docking calculations and ATR-FTIR analysis demonstrated interactions of SFX-SL, AgSFX, and AgSFX-SL with CHIKV. Collectively, our findings suggest that the addition of metal ions and/or Schiff base to SFX improved its antiviral activity against CHIKV.

Quantification measurement of hemoglobin with large dynamic range and low detection limit via an optical fiber optofluidic laser with enzyme-catalyzed reaction

Quantitative measurement of hemoglobin (Hb) levels is an essential part of routine medical examinations, disease diagnosis and health status monitoring. In this study, we introduced optical fiber optofluidic laser (FOFL) technology combined with catalytic oxidation reaction to design a laser sensor for sensitive Hb detection. For the H2O2-rhodamine B (RhB) oxidation system, radially emitting FOFL was achieved with a thin-walled hollow optical fiber (HOF) as an optical microcavity and amplified the concentration change of RhB during oxidation by H2O2. Hb was employed as peroxide-mimicking enzyme to catalyze the oxidation system, which sped up the reaction, resulting in an earlier laser extinction time. With the laser extinction time as sensing signal, the Hb sensor achieved a dynamic range of five orders of magnitude and a limit of detection (LOD) of 46.0 pM at an assay time of 40 min. The developed method provides ideas for the exploitation of FOFL biosensing based on catalytic oxidation reactions.

Effect of endothelium on the anticoagulant activity of a covalent antithrombin-heparin complex.

We developed a covalent antithrombin-heparin complex (ATH) with superior In vivo anticoagulant efficacy compared to non-covalent antithrombin (AT) + unfractionated heparin (H). Previous in vitro studies of ATH, investigating the mechanisms behind its efficacy, were done in the absence of endothelium. Since the endothelial surface modulates hemostasis, we investigated its impact on the in vitro anticoagulant properties of ATH and AT+H. Discontinuous second order rate constant enzyme inhibition assays, fibrin formation, and plasma clot generation were performed in the presence of ATH or AT+H, with and without endothelium present. ATH had an increased rate of direct inhibition of IIa and Xa, and increased inhibition of IIa-induced fibrin formation, compared to AT+H. When compared at equal anti-Xa levels, ATH was less effective than AT+H at catalyzing inhibition of plasma clot generation. These results were found in both the presence and absence of endothelium. Endothelium decreased the rate of IIa inhibition, and reduced clot time in IIa-induced fibrin formation and plasma clot generation assays, for both ATH and AT+H. Endothelium did not impact the activity of ATH differently to AT+H. This supports the growing body of evidence suggesting ATH may be a beneficial anticoagulant for potential clinical use.

In vitro and in vivo efficacy of zinc oxide green nanoparticles against multidrug-resistant Salmonella Typhi.

Antibiotic resistance is an increasing threat, requiring novel therapeutic solutions. Metal nanoparticles e.g., zinc oxide nanoparticles (ZnO NPs) exhibited the potential against many bacterial pathogens. Strains of Salmonella enterica serovar Typhi resistant to ceftriaxone were reported first from Pakistan in 2016. Since then, S. Typhi is a pathogen of concern globally owing to its rapidly emerging resistance potential against many last resort antibiotics. In the present study, in vitro and in vivo antimicrobial activity of ZnO NPs against multidrug resistant (MDR) and extensively drug resistant (XDR) Salmonella Typhi strains from Pakistan was evaluated. Zinc oxide green nanoparticles (ZnO GNPs), synthesized from Aloe vera, were characterized by SEM, XRD, UV-vis and Raman spectroscopy. In vitro antibacterial activity of two different concentrations of ZnO GNPs (7 and 15%) was checked using agar well diffusion method. Further, broth microdilution and time kill assays were performed using the ZnO GNPs. In vivo assays were conducted in BALB/c mice sepsis models. In all the three methods, agar well diffusion assay broth microdilution and time kill assay, different zinc oxide dihydrate precursor concentrations had shown the antibacterial activity. The minimum inhibitory concentration (MIC) of ZnO GNPs nanoparticles against MDR and XDR S. Typhi strains was found as 16 to 64µg/ml. In vivo experiment has shown a significant decrease in CFU/ml in the mice treated with ZnO GNPs as compared to the control group. Our findings have revealed that ZnO GNPs have significant antibacterial activity against MDR and XDR S. Typhi, both in vitro and in vivo.

Universal All-In-One Lateral Flow Immunoassay with Triple Signal Amplification for Ultrasensitive and Simple Self-Testing of Treponema pallidum Antibodies.

Lateral flow immunoassay (LFIA) is valued for its simplicity and rapidity for on-site screening, however, it experienced false negatives in real sample analysis due to low sensitivity. Although many signal amplification techniques can improve the sensitivity, they usually require additional complicated steps. To address these issues, taking Treponema pallidum (T. pallidum) antibodies as a model detecting target, herein, we report an all-in-one LFIA (AIO-LFIA) with triple-step signal amplification to significantly improve sensitivity while maintaining simplicity. This LFIA utilizes a biotin-streptavidin system for initial signal amplification, followed by introducing a release controller with a specific imprinted structure for timed multicomponent release, which avoids the extra steps when adding components in traditional LFIA. Particularly, a 3D-printed programmed metal in situ growth (MISG) device is integrated to localize signal enhancement at specific sites, overcoming limitations of traditional MISG and substantially reducing reagent usage and assay time, and the nitrocellulose membrane surface was much cleaner than the conventional approach, which facilitates signal readout. After optimization, the proposed AIO-LFIA is capable of visual detection down to 1 pg/mLT. pallidum antibodies in 15 min, 1000-fold lower than the gold nanoparticle-based LFIA. In clinical testing of 152 samples, the AIO-LFIA can distinguish all positive samples, outperforming commercial LFIA which missed those positive samples with relatively low antibody levels. Thus, this study presents a universal ultrasensitive and reliable AIO-LFIA strategy for infectious diseases self-testing, providing an effective promising prospect to address the challenge over emerging infectious diseases in the future.