Development Of Assay Research Articles

Development of an Automated Workflow for Screening the Assembly and Host-Guest Behavior of Metal-Organic Cages Towards Accelerated Discovery.

Metal-organic cages (MOCs) are a class of self-assembled materials with promising applications in chemical purifications, sensing, and catalysis. Their potential is, however, hampered by challenges in the targeted design of MOCs with desirable properties. MOC discovery is thus often reliant on trial-and-error approaches and brute-force manual screening, which are time-consuming, costly, and material-intensive. Translating the synthesis and property screening of MOCs to an automated workflow is therefore attractive, to both accelerate discovery and provide the datasets crucial for data-led approaches to accelerate MOC discovery and to realize their targeted properties for specific applications. Here, an automated workflow for the streamlined assembly and property screening of MOCs was developed, incorporating automated high-throughput screening of variables pertinent to MOC synthesis, data curation and automated analysis, and development of a host-guest assay to rapidly assess binding behavior. Computational modelling supplemented this automated experimental workflow for post priori rationalization of experimental outcomes. This study lays the groundwork for future large-scale MOC screening: from a relatively modest screen of 24 precursor combinations under one set of reaction conditions, 3 clean MOC species were identified, and subsequent screening of their host-guest behavior highlighted trends in binding and the identification of potential applications in molecular separations.

The development of a high-throughput acoustic droplet ejection mass-spectrometry assay and a solid-supported membrane (SSM)-based electrophysiological assay to study the pharmacological inhibition of SLC1-A3, -A2 and -A1 in a drug discovery program

IntroductionThe solute carrier (SLC) family comprises a diverse group of membrane proteins essential for transporting a variety of substrates across cellular membranes. These transporters play crucial roles in cellular homeostasis, nutrient uptake, and neurotransmitter clearance. The SLC1 subfamily, specifically SLC1A3 (EAAT1), SLC1A2 (EAAT2), and SLC1A1 (EAAT3), are excitatory amino acid transporters that regulate glutamate concentrations in the synaptic cleft, making them important targets for neurological disorder therapeutics. Despite their significance, drug discovery efforts targeting these transporters have been hampered by limitations in available screening methodologies.MethodsWe are utilizing advanced methodologies such as Acoustic Droplet Ejection Mass Spectrometry (ADE‐MS) and Solid Supported Membrane (SSM)-based electrophysiology to develop assays for the SLC1 family members: SLC1A3 (EAAT1), SLC1A2 (EAAT2), and SLC1A1 (EAAT3).Results and DiscussionIn this manuscript, we present the successful development of novel assays specifically designed for drug discovery applications targeting the SLC1 family members. Our Acoustic Droplet Ejection Mass Spectrometry (ADE‐MS) platform demonstrated high sensitivity and reproducibility in detecting substrate transport activity across all three transporters. The complementary Solid Supported Membrane (SSM)‐based electrophysiology assay provided real-time kinetic measurements of transporter function with minimal background interference. These assays exhibited Z’ factors exceeding 0.7, indicating their robustness for high-throughput screening campaigns. Initial validation using known inhibitors confirmed the assays’ ability to identify compounds with varying potencies and mechanisms of action against SLC1A3, SLC1A2, and SLC1A1.ConclusionWe endeavor to establish robust assays that can facilitate future drug discovery campaigns.

Structure-affinity relationship analysis and affinity maturation of a calprotectin-binding peptide.

Peptide 3 is an 18-amino acid linear peptide binding with sub-micromolar affinity to the inflammation marker calprotectin. Its application in point-of-care diagnostic assays has shown promising results, yet improving its affinity for calprotectin could facilitate the development of sensitive and robust assays. Herein we report a detailed structure-activity relationship analysis of Peptide 3 to better understand the importance of each individual amino acid for the binding to calprotectin. Moreover, we have followed two different approaches, one based on prolonging the peptide with random sequences and phage display selection, and one on screening chemically synthesized peptide variants containing non-canonical amino acids, to increase its binding affinity. Combining several mutations that enhance affinity by small factors yielded Peptide 4 binding human calprotectin with a KD of 39 ± 5 nM measured by surface plasmon resonance spectroscopy, which is a 5-fold improvement compared to the previously reported Peptide 3.

Centrifugal Microfluidic Lateral Flow Assay Enables High Sensitivity Interleukin-6 Detection and Ultrafast Readout of Elevated Analyte Levels.

Balancing sensitivity and time to result is always a challenging part of the development of analytical tools and is of particular importance in clinical and point-of-care diagnostics. In this study, a highly sensitive fluorescence interleukin-6 lateral flow assay (LFA) was developed using centrifugal microfluidics. The sample flow rate through the lateral flow membrane is determined by centrifugal force, which can be precisely controlled with a processing device. Using this precise flow control, an ultrafast early readout after 30 s with a sensitivity of 78.3 pg/mL and a quantitative measurement up to 2000 pg/mL was achieved. Afterward, the flow rate was reduced, and thus, the incubation time increased to achieve a maximum sensitivity of 1.2 pg/mL within 13 min of run time. This high-performance LFA is intended to help particularly vulnerable patient groups, such as pregnant women and neonates, where a rapid and highly sensitive diagnosis of inflammatory biomarkers can make a life-saving difference. In addition to medical applications, the presented system can also be used for the analysis of binding kinetics directly on the lateral flow strip. This enables the development of lateral flow assays with the highest possible sensitivity in the shortest time. Therefore, this advancement leads to a new era of point-of-care testing with future prospects for fully automated centrifugal cassettes with enhanced performance.

Developing a serological test for the diagnosis of toxocarasis using a novel recombinant multi-epitope.

Developing a serological test for the diagnosis of toxocarasis using a novel recombinant multi-epitope.

Development of a fluorescence-based assay for screening of urate transporter 1 inhibitors (II): Optimization of fluorescent substrates and structure-activity relationships analysis.

Development of a fluorescence-based assay for screening of urate transporter 1 inhibitors (II): Optimization of fluorescent substrates and structure-activity relationships analysis.

Development of an RT-RPA assay for La Crosse virus detection provides insights into age-dependent neuroinvasion in mice

BackgroundLa Crosse virus (LACV) is a mosquito-borne arbovirus responsible for pediatric encephalitis in North America, predominantly affecting children under 16 years. Early and accurate diagnosis is critical to reducing morbidity in this vulnerable population. However, existing molecular and serological methods are limited in sensitivity, specificity, and accessibility.MethodsTo address these limitations, we developed a reverse transcription recombinase polymerase amplification (RT-RPA) assay for LACV detection. Primers targeting the divergent M segment of the LACV genome were designed and screened for optimal performance. The assay’s analytical sensitivity was evaluated through serial dilutions of LACV RNA prior to reverse transcription, while specificity was assessed using reverse transcribed RNA from related or geographically relevant arboviruses. We further adapted the RT-RPA test into a lateral flow assay (LFA) format for potential point-of-care use. Additionally, we employed a murine model to explore the age-dependent dynamics of LACV neuroinvasion and clearance, with the virus detected using RT-RPA and reverse transcription quantitative polymerase change reaction (RT-qPCR).ResultsPrimer screening identified an optimal primer pair that amplified LACV cDNA within 20 min at 39 °C, with a limit of detection (LOD) of 100 copies. The assay demonstrated high specificity, with no amplification of related or other geographically relevant arboviruses. Integration of the RT-RPA test into an LFA format preserved the LOD and specificity, enabling visual detection via test strips. In the murine model, weanling mice exhibited LACV neuroinvasion as early as 4 days post-infection (dpi), with sustained detection between 5 and 7 dpi. In adult mice, neuroinvasion was first detected at 5 dpi, plateauing between 6 and 10 dpi, and cleared entirely by 20 dpi in surviving animals.ConclusionsThis study establishes the RT-RPA assay as an efficient, specific, and sensitive diagnostic platform for LACV, with potential for adaptation into field-deployable LFA tests. Moreover, our findings provide valuable insights into the age-dependent dynamics of LACV neuroinvasion and clearance, informing future diagnostic and therapeutic strategies.

β-Galactosidase-Mediated, Mn2+-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim.

The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of p-aminophenyl β-D-galactopyranoside to generate p-aminophenol. This compound subsequently facilitates the decomposition of MnO2 nanosheets, leading to the generation of Mn2+ ions. The Mn2+ ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn2+-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.

Development of a Visual Assay for Detection of Viable Cronobacter sakazakii Using RT-PSR and Hydroxynaphthol Blue Indicator

Cronobacter sakazakii is a foodborne pathogen in powdered infant formula, which poses a significant risk to susceptible populations such as infants and the elderly. This study aims to develop a visual detection method for viable C. sakazakii using the reverse transcription-polymerase spiral reaction and hydroxynaphthol blue indicator. Under the optimized conditions, the detection process could be completed within 55 min with low equipment dependence. It was evaluated to have high specificity and sensitivity with the detection limit low to 1.2 × 101 CFU/mL. The assay also showed 100% accuracy in artificially contaminated samples.

Neurotrophic factor biomarkers for ischemic stroke diagnosis and mechanistic insights

Ischemic stroke (IS), a multifactorial disease resulting from the complex interplay of various environmental and genetic risk factors. Neurotrophic factors (NTFs) have a potential role in IS, but the exact mechanisms are unknown. The aim of this study was to identify biomarkers associated with the occurrence and development of NTFs and to analyze their potential mechanisms of action. In this study, we selected the intersection of neurotrophic factor genes, differentially expressed genes (DEGs) and key genes in the IS module based on IS-related datasets (GSE16561 and GSE58294). Machine learning screened out 5 biomarkers for IS diagnosis (MMP9, MARCKS, IGF2R, HECW2 and CYBRD1). GSEA results showed that different signaling pathways were activated in IS samples with high expression of different diagnostic genes. Furthermore, an immunological analysis was carried out, which demonstrated significant differences in the levels of activated B cells, neutrophils, and activated CD8 T cells between IS patients and normal samples. RT-qPCR results showed that there were significant differences in the expression of CYBRD1, MARCKS and MMP9 between IS and control patients. In conclusion, we identified 5 diagnostic markers that may be involved in the progression of IS, including MMP9, MARCKS, IGF2R, HECW2 and CYBRD1. Finally, differential expression of MMP9, MARCKS, and CYBRD1 was detected in peripheral blood samples from 15 IS and 5 normal cases. Our analysis could serve as a foundation for enhancing comprehension of the underlying molecular mechanisms governing the pathogenesis and progression of IS. The identified biomarkers might serve as targets for the development of novel diagnostic assays, enabling earlier detection of IS and potentially leading to more timely and effective treatment interventions.

Single-B-cell cloning and recombinant antibodies generation to analyze the antigenicity of porcine reproductive and respiratory syndrome virus nonstructural protein 12.

Single-B-cell cloning and recombinant antibodies generation to analyze the antigenicity of porcine reproductive and respiratory syndrome virus nonstructural protein 12.

Development of a quantitative PCR assay to analyze the infection of Tenacibaculum sp. strain Pbs‑1, which is the causative agent of black-spot shell disease

Development of a quantitative PCR assay to analyze the infection of Tenacibaculum sp. strain Pbs‑1, which is the causative agent of black-spot shell disease

Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA

Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R2 values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States.

Discovery of MAGL Inhibition by Lophine Derivatives: An Unexpected Finding from Chemiluminescent Assay Development

The inhibitory effects of two novel lophine derivatives were unexpectedly discovered during the development of a chemiluminescent monoacylglycerol lipase (MAGL) assay. The proposed lophine derivatives were found to exhibit concentration-dependent inhibitory effects on MAGL with the octanoic and palmitic acid esters of 2-(4-hydroxyphenyl)-4,5-diphenylimidazole showing the strongest activity. Reversibility assays using a fluorometric method confirmed that these compounds interact with MAGL in a stable, irreversible manner. To further investigate their mode of interaction, docking studies were performed, supporting the hypothesis that compounds 3 and 4 may act as competitive and irreversible inhibitors. Lophine derivatives were initially designed and synthesized as potential chemiluminescence pro-enhancers. However, assay optimization revealed no signal production upon MAGL hydrolysis, precluding their use as chemiluminescent probes. These findings suggest that lophine is a promising candidate for the development of MAGL inhibitors, although further optimization is needed to enhance binding affinity and selectivity.

A Laboratory-Developed Assay for the Simultaneous Detection of Aspergillus fumigatus and Pneumocystis jirovecii Pulmonary Pathogens

Invasive fungal diseases are a significant threat in immunocompromised patients, underscoring the need for rapid and accurate diagnostics. This study describes the development and validation of a real-time PCR-based laboratory-developed assay (LDA) on the Panther Fusion system for the simultaneous detection of Aspergillus fumigatus (AF) and Pneumocystis jirovecii (PJ) in bronchoalveolar lavage fluid (BALF) samples. The assay was evaluated using 239 clinical BALF samples, including cases confirmed positive for AF or PJ by reference mycological methods. Rigorous optimization ensured compatibility with the automated workflow of the Panther Fusion system, which addresses challenges such as BALF viscosity and fungal DNA recovery. No cross-reactivity with non-target fungal species was observed, and the assay demonstrated high analytical sensitivity and specificity. Only two false-negative results were reported, which could plausibly be reclassified as true negatives when interpreted alongside the serum beta-d-glucan and galactomannan assay results. For PJ detection, the assay showed excellent concordance with the OLM PneumID assay, supporting its reliability in clinical settings. The dual-target approach facilitates the simultaneous detection of both pathogens within a single workflow, improving diagnostic efficiency. The AF/PJ LDA represents a robust and scalable alternative to existing molecular assays, with the potential to enhance routine diagnostics for pulmonary fungal infections.

Development of a neutralization assay and bioluminescent imaging mouse model for Dehong virus (DEHV) using a pseudovirus system.

Dehong virus (DEHV) is an emerging filamentous virus of considerable interest. However, research involving DEHV remains limited, and no suitable models exist to investigate its pathogenicity or transmission. In this study, we developed an in vitro neutralization assay to detect DEHV-neutralizing antibodies, as well as an in vivo bioluminescent imaging mouse model based on a pseudovirus system. Our results confirmed that DEHV utilizes the Niemann-Pick disease, type C1 (NPC1) receptor for cellular entry. Additionally, the neutralization assay demonstrated that DEHV antiserum does not exhibit neutralizing activity against Mengla or Marburg viruses. This pseudovirus-based system provides a valuable platform for studying DEHV biology and evaluating therapeutic interventions.IMPORTANCEBats serve as natural reservoirs for diverse filoviruses across Africa, Europe, and East Asia; numerous strains circulate within these populations. Recently, Chinese researchers identified Dehong virus (DEHV), a novel filovirus carried by bats in China. However, the mechanisms underlying the pathogenicity and transmission of DEHV remain poorly understood. Similar to Ebola virus and Marburg virus (MARV), DEHV uses the Niemann-Pick disease, type C1 (NPC1) receptor for host cell invasion. In this study, we utilized a well-established in vitro neutralization assay to confirm that DEHV antiserum lacks neutralizing activity against Mengla and MARV pseudoviruses. Furthermore, we developed an innovative in vivo bioluminescent imaging mouse model using DEHV pseudovirus, which offers a visually intuitive and efficient platform for evaluating antiviral therapies and vaccine candidates. This model has considerable potential for advancing research into DEHV pathogenesis and treatment strategies.

Development of a ptp2-LAMP assay for the specific and sensitive detection of Nosema apis and its comparison with ptp3-LAMP for the detection of Nosema ceranae, in a region endemic for both microsporidium pathogens of the Western honey bee.

Development of a ptp2-LAMP assay for the specific and sensitive detection of Nosema apis and its comparison with ptp3-LAMP for the detection of Nosema ceranae, in a region endemic for both microsporidium pathogens of the Western honey bee.

Development of a urinary antigen test for BK Polyomavirus to help clinicians in patients' follow-up.

Development of a urinary antigen test for BK Polyomavirus to help clinicians in patients' follow-up.

Identification of anti-resorptive GPCRs by high-content imaging in human osteoclasts.

Osteoporosis diagnoses are increasing in the ageing population and although some treatments exist, these have several disadvantages, highlighting the need to identify new drug targets. G protein-coupled receptors (GPCRs) are transmembrane proteins whose surface expression and extracellular activation make them desirable drug targets. Our previous studies have identified 144 GPCR genes to be expressed in primary human osteoclasts, which could provide novel drug targets. The development of high-throughput assays to assess osteoclast activity would improve the efficiency at which we could assess the effect of GPCR activation on human bone cells and could be utilised for future compound screening. Here we assessed the utility of a high-content imaging (HCI) assay that measured cytoplasmic-to-nuclear translocation of the nuclear factor of activated T cells-1 (NFATc1), a transcription factor that is essential for osteoclast differentiation and resorptive activity. We first demonstrated that the HCI assay detected changes in NFATc1 nuclear translocation in human primary osteoclasts using GIPR as a positive control, then developed an automated analysis platform to assess NFATc1 in nuclei in an efficient and unbiased manner. We assessed six GPCRs simultaneously and identified four receptors (FFAR2, FFAR4, FPR1, GPR35) that reduced osteoclast activity. Bone resorption assays and measurements of TRAP activity verified that activation of these GPCRs reduced osteoclast activity, and that receptor-specific antagonists prevented these effects. These studies demonstrate that HCI of NFATc1 can accurately assess osteoclast activity in human cells, reducing observer bias and increasing efficiency of target detection for future osteoclast-targeted osteoporosis therapies.

ATPase activity profiling of three human DExD/H-box RNA helicases.

ATPase activity profiling of three human DExD/H-box RNA helicases.