Enhancing the Sensitivity of Lateral Flow Assay with Europium Nanoparticles for Accurate Human IgG Quantification.

Accurate quantification of immunoglobulin G (IgG) levels is vital for understanding immune status and diagnosing various medical conditions. Lateral flow assays (LFAs) offer rapid and convenient diagnostic tools, but their sensitivity has been a limitation. Our research introduces a refined method incorporating europium nanoparticles, enhancing both sensitivity and accuracy of LFAs in human IgG measurement. Utilizing a unique sandwich format, carboxylate-modified polystyrene Eu (III) chelate microparticles (CM-EUs) acted as the primary reporters. The concentrations of both detection and capture antibodies on the strip were optimized to bolster the LFA's quantitative performance. The subsequent calibration curve between the IgG concentration and the measured intensity ratio (VR) established the linearity and analytical sensitivity of our method with a high correlation coefficient (r = 0.99) and an impressively low limit of detection (LoD = 0.04 ng/mL). Our precision assessment, segmented into intra-assay and inter-assay evaluations, showcases the method's consistency and reproducibility. The LFA assay's stability was established by demonstrating its resistance to degradation and affirming its potential for extended storage without a dip in performance. The study's findings underscore the potential of this method to contribute to diagnostic medicine and improve patient care.

A Lateral Flow Assay for the Detection of Leptospira lipL32 Gene Using CRISPR Technology.

The clinical manifestation of leptospirosis is often misdiagnosed as other febrile illnesses such as dengue. Therefore, there is an urgent need for a precise diagnostic tool at the field level to detect the pathogenic Leptospira lipL32 gene at the molecular level for prompt therapeutic decisions. Quantitative polymerase chain reaction (qPCR) is widely used as the primary diagnostic tool, but its applicability is limited by high equipment cost and the lack of availability in every hospital, especially in rural areas where leptospirosis mainly occurs. Here, we report the development of a CRISPR dFnCas9-based quantitative lateral flow immunoassay to detect the lipL32 gene. The developed assay showed superior performance regarding the lowest detectable limit of 1 fg/mL. The test is highly sensitive and selective, showing that leptospirosis diagnosis can be achieved with a low-cost lateral flow device.

Exploring carbohydrate binding module fusions and Fab fragments in a cellulose-based lateral flow immunoassay for detection of cystatin C.

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the kidney biomarker cystatin C that features conjugates of capture antibodies and fusions of carbohydrate binding modules (CBM) with ZZ domains anchored on cellulose deposited over nitrocellulose (NC). The ZZ-CBM3 fusion provides a biomolecular interface between the cellulose layer and the Fc portion of the capture antibodies. By resorting to detection Fab fragments that lack the Fc portion we overcome the observed interference of full-length detection antibodies with the ZZ-CBM3 fusion at the test lines. Using the new LFA architecture, a linear concentration-response relationship was observed in the 0-10 ng/mL cystatin C concentration range, which is compatible with the clinically normal (5-120 ng/mL) and abnormal (> 250 ng/mL) levels of cystatin C, as long as proper dilutions are made. An inter assay CoV of 0.72% was obtained. Finally, mock urine samples characteristic of normal (100 ng/mL) and kidney tubular disease (4000 ng/mL) patients were successfully analyzed. Overall, we demonstrate an innovative LFA architecture that combines NC strips with layered cellulose, ZZ-CBM3 fusions and fluorescently labeled Fab fragments.

Development and Evaluation of Europium-Based Quantitative Lateral Flow Immunoassay for the Chronic Kidney Disease Marker Cystatin-C.

This study aimed to establish a Europium label time-resolved fluorescence immunoassay (TRFIA) to detect the chronic kidney disease (CKD) biomarker Cystatin-C. An Europium based Time resolved fluorescence immunoassay was developed to detect the concentration of Cystatin-C in a urine sample to increase the sensitivity with captured anti-Cystatin-C antibodies immobilized on nitrocellulose membrane and then bonded with detection anti-Cystatin-C labelled with CM-EU, followed by fluorescence measurement using time-resolved fluorometry in 15 min. The performance of this TRFIA was evaluated using the clinical urine serum and compared with the ELISA assays. The linear calibration range was 0.015-32 µg/ml, and the limit of detection (LOD) quantified was 0.0001 µg/ml. This current work has improved the LOD of our previous work from 0.013 µg/ml to 0.001 µg/ml. These results indicated that the CM-EU nanoparticle-based LFIA is rapid, more sensitive, reliable, and reproducible for point-of-care testing of Cys-C concentrations in urine.

State-of-the-art colloidal particles and unique interfaces-based SARS-CoV-2 detection methods and COVID-19 diagnosis.

In March 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-based infections were declared 'COVID-19 pandemic' by the World Health Organization. Pandemic raised the necessity to design and develop genuine and sensitive tests for precise specific SARS-CoV-2 infections detection. Nanotechnological methods offer new ways to fight COVID-19. Nanomaterials are ideal for unique sensor platforms because of their chemically versatile properties and they are easy to manufacture. In this context, selected examples for integrating nanomaterials and distinct biosensor platforms are given to detect SARS-CoV-2 biological materials and COVID-19 biomarkers, giving researchers and scientists more goals and a better forecast to design more relevant and novel sensor arrays for COVID-19 diagnosis.

Development and Evaluation of a Quantitative Fluorescent Lateral Flow Immunoassay for Cystatin-C, a Renal Dysfunction Biomarker.

The diagnosis, prognosis, and control of chronic kidney disease rely on an understanding of the glomerular filtration rate (GFR). The renal clearance of the cystatin-C is closely associated with the GFR. Cystatin-C is a more suitable GFR marker than the commonly used creatinine. General techniques for cystatin-C calculation, such as particle-enhanced turbidimetric and nephelometric assay, are time-consuming and tedious. Here, we propose a rapid, quantitative immunoassay for the detection of cystatin-C. A fluorescence-based lateral-flow kit was developed in a sandwich format by using a monoclonal antibody. A Linear calibration was obtained over the clinical diagnostic range of 0.023-32 µg/mL and the limit of detection (LOD) was 0.023 µg/mL and the limit of quantification (LOQ) was 0.029 µg/mL. Average recoveries from spiked urine samples ranged from 96-100% and the coefficient of variation was less than 4% for both intra and inter-day assays with excellent repeatability. With the comparison with an ELISA kit, the developed kit is highly sensitive, performs well over the detection range, provides repeatable results in a short time, and can easily be used at point-of-care (POC), making it an ideal candidate for rapid testing in early detection, community screening for renal function disorders.

Potential of brain mast cells for therapeutic application in the immune response to bacterial and viral infections.

A wide range of microorganisms can infect the central nervous system (CNS). The immune response of the CNS provides limited protection against microbes penetrating the blood-brain barrier. This results in a neurological deficit and sometimes leads to high morbidity and mortality rates despite advanced therapies. For the last two decades, different studies have expanded our understanding of the molecular basis of human neuroinfectious diseases, especially concerning the contributions of mast cell interactions with other central nervous system compartments. Brain mast cells are multifunctional cells derived from the bone marrow and reside in the brain. Their proximity to blood vessels, their role as "first responders" their unique receptors systems and their ability to rapidly release pathogen responsive mediators enable them to exert a crucial defensive role in the host-defense system. This review describes key biological and physiological functions of mast cells, concerning their ability to recognize pathogens via various receptor systems, followed by a coordinated and selective mediator release upon specific interactions with pathogenic stimulating factors. The goal of this review is to direct attention to the possibilities for therapeutic applications of mast cells against bacterial and viral related infections. We also focus on opportunities for future research activating mast cells via adjuvants.

A Cellulose Paper-Based Fluorescent Lateral Flow Immunoassay for the Quantitative Detection of Cardiac Troponin I.

This paper presents a lateral flow assay (LFA) for the quantitative, fluorescence-based detection of the cardiac biomarker troponin I (cTnI) that features an analytical strip made of cellulose filter paper. The results show that the wicking and test time are comparable to those obtained with conventional nitrocellulose (NC)-based LFAs. Further, the cellulose paper provides an excellent background with no auto-fluorescence that is very adequate in detecting fluorescent lines. While fluorescence that was generated with cellulose strips was lower when compared to that generated in NC strips, signals could be improved by layering carbon nanofibers (CNF) on the cellulose. A nonlinear behavior of the concentration-response relationship was observed for the LFA architectures with NC, cellulose, and cellulose-CNF in the 0 to 200 ng/mL cTnI concentration range. The measurements were consistent and characterized by coefficients of variation lower than 2.5%. Detection and quantitation limits that were in the range 1.28-1.40 ng/mL and 2.10-2.75 ng/mL were obtained for LFA with cellulose and cellulose CNF strips that are equivalent to the limits obtained with the standard NC LFA. Overall, we showed that commercially available filter paper can be used in the analytical strip of LFA.

A paper microfluidics-based fluorescent lateral flow immunoassay for point-of-care diagnostics of non-communicable diseases.

In the emergency diagnosis of patients, acute myocardial infarction (AMI) is always time-consuming to diagnose, and the process requires multiple laboratory procedures, expensive equipment and skilled workers. Herein, we developed an easy-to-use, low-cost and portable fluorescent lateral flow immunoassay based on paper microfluidics for the point-of-care diagnostics of non-communicable diseases. The fluorescent lateral flow immunoassay can produce results in less than 10 minutes, and the limit of detection (LOD) is 0.019 ng ml-1. The slope was linear from 0 to 100 ng ml-1; the equation is y = 0.0342e2.1181x and R2 = 0.9618, which are distinctive features that ensure maximum amplification of the signal and recording of quantitative values by an analyser. The detection sensitivity showed an exceptional increase to 0.01 ng ml-1. Compared with conventional bioassay readers, our analyser shows some advantages to easily, clearly and effectively read data. The present point-of-care test for cardiac troponin I decreases the turnaround time and has a high coefficient of variation even at lower concentrations of troponin. So, the development of lateral flow assay-based point-of-care assays with higher analytical performance for real world samples can decrease the rule-out time for AMI in emergency departments and other fields.

Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 µmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities.

Water soluble and efficient amino acid Schiff base receptor for reversible fluorescence turn-on detection of Zn²âº ions: Quantum chemical calculations and detection of bacteria.

An amino acid Schiff base (R) capable of recognizing Zn(2+) ions selectively and sensitively in an aqueous medium was prepared and characterized. Upon addition of Zn(2+) ions, the receptor exhibits fluorescence intensity enhancements (~40 fold) at 460 nm (quantum yield, Φ=0.05 for R and Φ=0.18 for R-Zn(2+)) and can be detected by naked eye under UV light. The receptor can recognize the Zn(2+) (1.04×10(-8) M) selectively for other metal ions in the pH range of 7.5-11. The Zn(2+) chelation with R decreases the loss of energy through non-radiative transition and leads to fluorescence enhancement. The binding mode of the receptor with Zn(2+) was investigated by (1)H NMR titration and further validated by ESI-MS. The elemental color mapping and SEM/EDS analysis were also used to study the binding of R with Zn(2+). Density functional theory calculations were carried out to understand the binding mechanism. The receptor was applied as a microbial sensor for Escherichia coli and Staphylococcus aureus.

Rapid in vitro protein synthesis pipeline: a promising tool for cost-effective protein array design.

Several protein expression systems for construction of protein arrays have been established in recent years. However, current protocols for protein synthesis are still time consuming, laborious and expensive. This study has established an alternative workflow that covers rapid construction of expression cassettes, in-tube and on-membrane synthesis of recombinant proteins, and straightforward screening of synthesized proteins. Eighteen membrane associated eukaryotic proteins and two secretory complement regulators (C1 inhibitor and vitronectin) were included in the study. To generate hybrid genes, double-overlap extension PCR was employed to fuse the 5' fragment (consisting of a T7 promoter and a species independent translation sequence), ORFs of the target proteins, and the 3' fragment (encompassing GFP fusion, Myc-tag and stop codon). OE-PCR generated fragments were directly mixed with the Leishmania torentolae lysate (translation mix) for protein synthesis. In order to establish a cheap and user-friendly alternative to existing cell-free protein array techniques, PCR products were spotted on the hydrophobic substrate (PVDF membrane), air-dried and covered with only 2 µL of translation mix. All synthesized proteins were spontaneously immobilized on the membrane due to the hydrophobic interaction between C-terminally fused GFP and PVDF. Synthesis and immobilization of proteins were confirmed simply by assessing the GFP chromophore under a laser scanner or a fluorescent microscope.

Effect of processing variables on characterization of ofloxacin loaded lipospheres prepared by melt dispersion technique.

A melt dispersion technique was employed to prepare ofloxacin lipospheres, by using cetyl alcohol (polar lipid). Effects of various process parameters such as selection of surfactants (gelatin, Tween 40 and poly vinyl alcohol) and selection of stirring speed were studied. Lipospheres were evaluated for morphology, drug entrapment and in vitro drug release profiles. The optimized liposphere batch was selected and formulated as tablets and evaluated the in vitro drug release profile. These studies showed that ofloxacin loaded lipospheres were able to control the drug release for a period of 16h.

NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus.

The development of novel therapeutic agents is essential for combating the increasing number of cases of dengue fever in endemic countries and among a large number of travelers from non-endemic countries. The dengue virus has three structural proteins and seven non-structural (NS) proteins. NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. The serine protease domain of NS3 plays a central role in the replicative cycle of dengue virus. This review discusses the recent structural and biological studies on the NS2B-NS3 protease-helicase and considers the prospects for the development of small molecules as antiviral drugs to target this fascinating, multifunctional protein.

NS3 protease from flavivirus as a target for designing antiviral inhibitors against dengue virus

The development of novel therapeutic agents is essential for combating the increasing number of cases of dengue fever in endemic countries and among a large number of travelers from non-endemic countries. The dengue virus has three structural proteins and seven non-structural (NS) proteins. NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) that is responsible for proteolytic processing of the viral polyprotein, and a C-terminal region that contains an RNA triphosphatase, RNA helicase and RNA-stimulated NTPase domain that are essential for RNA replication. The serine protease domain of NS3 plays a central role in the replicative cycle of dengue virus. This review discusses the recent structural and biological studies on the NS2B-NS3 protease-helicase and considers the prospects for the development of small molecules as antiviral drugs to target this fascinating, multifunctional protein.

Excision of selectable marker genes from transgenic crops as a concern for environmental biosafety.

The main task in the development of transgenic plants is the capability to distinguish between plant cells with an integrated transgene and the bulk of non-transformed cells. Selectable marker genes are required to achieve this goal within the transgene, and to select for their expression. These selectable markers are mostly based on genes conferring antibiotic or herbicide resistance. The presence of the marker gene will lead to unpredictable environmental hazards, so on the basis of economic incentives and safety concerns, several methods, such as site-specific recombination, homologous recombination and co-transformation, have been developed to eliminate these genes from the genome after successful transformation has been achieved. Gene transfer without the incorporation of an antibiotic-resistance marker or herbicide-resistance marker in the host genome should convince the public with regard to the field release of transgenic organisms. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host. Copyright © 2007 Society of Chemical Industry.

Biological and biomedical aspects of genetically modified food.

Genetically modified (GM) foods are the product of one of the most progressive fields of science-biotechnology. There are major concerns about GM foods in the public; some of them are reasonable, some of them are not. Biomedical risks of GM foods include problems regarding the potential allergenicity, horizontal gene transfer, but environmental side effects on biodiversity must also be recognized. Numerous methods have been developed to assess the potential risk of every GM food type. Benefits of the first generation of GM foods were oriented towards the production process and companies, the second generation of GM foods offers, on contrary, various advantages and added value for the consumer. This includes improved nutritional composition or even therapeutic effects. Recombinant probiotics and the principle of alternative gene therapy represent the latest approach of using GM organisms for biomedical applications. This article tries to summarize and to explain the problematic topic of GM food.