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Due to the recent pandemic caused by coronavirus disease 2019 (COVID-19), the lateral flow immunoassay used for its rapid antigen test is more popular than ever before. However, the history of the lateral flow immunoassay is about 60 years old, and its original purpose of use, such as a COVID-19 rapid antigen test or a pregnancy test, was the qualitative detection of a target analyte. Recently, the demand for quantitative analysis of lateral flow immunoassays is increasing in various fields. Lateral flow immunoassays for quantitative detection using various materials and sensor technologies are being introduced, and readers for analyzing them are being developed. Quantitative analysis readers are highly anticipated for their future development in line with technological advancements such as optical, magnetic field, photothermal, and electrochemical sensors and trends such as weight reduction, miniaturization, and cost reduction of systems. In addition, the sensing, processing, and communication functions of portable personal devices such as smartphones can be used as tools for the quantitative analysis of lateral flow immunoassays. As a result, lateral flow immunoassays can efficiently achieve the goal of rapid diagnosis by point-of-care testing. Readers used for the quantification of lateral flow immunoassays were classified according to the adopted sensor technology, and the research trends in each were reviewed in this paper. The development of a quantitative analysis system was often carried out in the assay aspect, so not only the readers but also the assay development cases were reviewed if necessary. In addition, systems for quantitative analysis of COVID-19, which have recently been gaining importance, were introduced as a separate section.

Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.

A high-throughput 96-microwell plate fluorometric method was developed and validated to determine omeprazole (OMZ) in its dosage forms. The method was based on the charge-transfer (CT) sensitized fluorescence reaction of OMZ with 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone (DDQ). This fluorescence reaction provided a new approach for simple, sensitive and selective determinations of OMZ in pharmaceutical preparations. In the present method, the fluorescence reaction was carried out in 96-microwell plates as reaction vessels in order to increase the automation of the methodology and the efficiency of its use in quality control laboratories. All factors affecting the fluorescence reaction were carefully studied and the conditions were optimized. The stoichiometry of the fluorescence reaction between OMZ and DDQ was determined and the reaction mechanism was suggested. Under the optimum conditions, the linear range was 100–6000 ng/ml with the lowest LOD of 33 ng/ml. Analytical performance of the proposed assay, in terms of accuracy and precision, was statistically validated and the results were satisfactory; RSD was <2.6 % and the accuracy was 98.6–101.6 %. The method was successfully applied to the analysis of OMZ in its dosage forms; the recovery values were 98.26–99.60 ± 0.95–2.22 %. The developed methodology may provide a safer, automated and economic tool for the analysis of OMZ in quality control laboratories.

Background Flavonols are plant specialized metabolites that regulate plant growth, development, and stress responses. Due to their antioxidant activity, they also confer nutritional benefits to human health. Quantification of flavonols in plant tissues typically relies on chromatographic methods such as HPLC or LC-MS, or on microscopy-based approaches using diphenylboric acid 2-aminoethyl ester (DPBA) staining to visualize flavonols in plant tissues. These methods are time-consuming and resource intensive. Here, we present a rapid, high-throughput, fluorescence-based microplate reader assay for flavonol quantification, in which the flavonol-specific dye DPBA is added to plant extracts to form fluorescent complexes. Results The assay was optimized for extraction efficiency and validated for sensitivity, accuracy, and reproducibility. It also shows consistency with HPLC measurements. We demonstrate its utility by quantifying flavonol levels in different tomato tissues, across different cultivars, and even between plant species. Our assay showed that reproductive tissue in tomato plants has higher flavonol levels than vegetative tissue. Also, we found variation in flavonol levels in tomato fruit skin across different laboratory and commercial cultivars, suggesting that our approach shows promise for use in genome-wide association studies to identify genetic factors underlying variation in flavonol levels. Lastly, we measured flavonol levels in dry leaves of different plants used for brewed beverages. Conclusion In conclusion, the assay represents a simple, robust, and scalable flavonol screening tool for studies in plant metabolism, environmental physiology, breeding, and metabolic engineering.

Droplet digital PCR (ddPCR) is accurate in nucleic acid quantification owing to its linearity and high sensitivity. Amplification of nucleic acid in droplets, however, is limited by the stability of droplets against thermal cycling. While the use of fluorinated oil or supplementation of surfactant could improve the stability of droplets, this process has also greatly increased the cost of ddPCR and limited post-PCR analysis. Here, we report a novel method known as gel capsule-based digital PCR (gc-dPCR) which includes a method to prepare hydrogel capsules encapsulating the PCR reaction mix, conducting PCR reaction, and readout by either quantitative PCR (qPCR) system or fluorescence microplate reader. We have compared the developed method to vortex ddPCR. Our approach results in higher fluorescence intensity compared to ddPCR suggesting higher sensitivity of the system. As hydrogel capsules are more stable than droplets in fluorinated oil throughout thermal cycling, all partitions can be quantified, thus preventing loss of information from low-concentration samples. The new approach should extend to all droplet-based PCR methods. It has greatly improved ddPCR by increasing droplets stability and sensitivity, and reducing the cost of ddPCR, which help to remove the barrier of ddPCR in settings with limited resources. Graphical Abstract

Cardiac toxicity represents one of the leading causes of drug failure along different stages of drug development. Multiple very successful pharmaceuticals had to be pulled from the market or labeled with strict usage warnings due to adverse cardiac effects. In order to protect clinical trial participants and patients, the International Conference on Harmonization published guidelines to recommend that all new drugs to be tested preclinically for hERG (K v 11.1) channel sensitivity before submitting for regulatory reviews. However, extensive studies have demonstrated that measurement of hERG activity has limitations due to the multiple molecular targets of drug compound through which it may mitigate or abolish a potential arrhythmia, and therefore, a model measuring multiple ion channel effects is likely to be more predictive. Several phenotypic rapid-throughput methods have been developed to predict the potential cardiac toxic compounds in the early stages of drug development using embryonic stem cells- or human induced pluripotent stem cell-derived cardiomyocytes. These rapid-throughput methods include microelectrode array-based field potential assay, impedance-based or Ca 2+ dynamics-based cardiomyocytes contractility assays. This review aims to discuss advantages and limitations of these phenotypic assays for cardiac toxicity assessment.

We evaluated the ability of different fluorescent indicators by various analytical instruments, including a laser scanning confocal microscope (LSCM), fluorescence plate reader, and flow cytometer (FCM), to measure the mitochondrial membrane potential (ΔΨm) of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury. The mitochondrial oxygen consumption rate and a transmission electron microscope were used to detect changes in mitochondrial functions and morphology, respectively. Cardiac H9c2 cells were exposed to H2O2 (500, 750, 1000, and 1250 μM) to induce mitochondrial oxidative stress injury, and fluorescent indicators including tetramethyl rhodamine ethyl ester (TMRE), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1), and rhodamine 123 (R123) were used to detect changes in ΔΨm using an LSCM, fluorescence plate reader, and FCM. The decrease in ΔΨm caused by H2O2 was determined by endpoint and dynamic analyses after staining with JC-1 or TMRE. With the R123 probe, the LSCM could only detect the change in ΔΨm caused by 1000 μM H2O2. Moreover, R123 was less effective than JC-1 and TMRE for measurement of ΔΨm by the LSCM. Our data indicated that an LSCM is the most suitable instrument to detect dynamic changes in ΔΨm, whereas all three instruments can detect ΔΨm at the endpoint.

This chapter contains sections titled: Introduction Standard Methods for Biomarker Purification, Enrichment, and Detection Smart Reagents for Biomarker Purification and Processing Sample‐Processing Modules for Smart Conjugate Bioassays Devices for Use in Smart Conjugate Bioassays Conclusions References

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8 , in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

Background: Membrane-active antimicrobial peptides (AMPs) are interesting candidates for the development of novel antimicrobials. Although their effects were extensively investigated in model membrane systems, interactions of AMPs with living microbial membranes are less known due to their complexity. The aim of the present study was to develop a rapid fluorescence-based microplate assay to analyze the membrane effects of AMPs in whole Staphylococcus aureus and Staphylococcus epidermidis. Methods: Bacteria were exposed to bactericidal and sub-inhibitory concentrations of two membrane-active AMPs in the presence of the potential-sensitive dye 3,3′-dipropylthiadicarbocyanine iodide (diSC3(5)) and the DNA staining dye propidium iodide (PI), to simultaneously monitor and possibly distinguish membrane depolarization and membrane permeabilization. Results: The ion channel-forming gramicidin D induced a rapid increase of diSC3(5), but not PI fluorescence, with slower kinetics at descending peptide concentrations, confirming killing due to membrane depolarization. The pore-forming melittin, at sub-MIC and bactericidal concentrations, caused, respectively, an increase of PI fluorescence in one or both dyes simultaneously, suggesting membrane permeabilization as a key event. Conclusions: This assay allowed the distinction between specific membrane effects, and it could be applied in the mode of action studies as well as in the screening of novel membrane-active AMPs.