Simultaneous quantitation of neutralizing antibodies against all four dengue virus serotypes using optimized reporter virus particles.

Serum-neutralizing antibody titers are a critical measure of vaccine immunogenicity and are used to determine flavivirus seroprevalence in study populations. An effective dengue virus (DENV) vaccine must confer simultaneous protection against viruses grouped within four antigenic serotypes. Existing flavivirus neutralization assays, including the commonly used plaque/focus reduction neutralization titer (PRNT/FRNT) assay, require an individual assay for each virus, serotype, and strain and easily become a labor-intensive and time-consuming effort for large epidemiological studies or vaccine trials. Here, we describe a multiplex reporter virus particle neutralization titer (TetraPlex RVPNT) assay for DENV that allows simultaneous quantitative measures of antibody-mediated neutralization of infection against all four DENV serotypes in a single low-volume clinical sample and analyzed by flow cytometry. Comparative studies confirm that the neutralization titers of antibodies measured by the TetraPlex RVPNT assay are similar to FRNT/PRNT assay approaches performed separately for each viral strain. The use of this high-throughput approach enables the careful serological study in DENV endemic populations and vaccine recipients required to support the development of a safe and effective tetravalent DENV vaccine. IMPORTANCE: As a mediator of protection against dengue disease and a serological indicator of prior infection, the detection and quantification of neutralizing antibodies against DENV is an important "gold standard" tool. However, execution of traditional neutralizing antibody assays is often cumbersome and requires repeated application for each virus or serotype. The optimized RVPNT assay described here is high-throughput, easily multiplexed across multiple serotypes, and targets reporter viral particles that can be robustly produced for all four DENV serotypes. The use of this transformative RVPNT assay will support the expansion of neutralizing antibody datasets to answer research and public health questions often limited by the more cumbersome neutralizing antibody assays and the need for greater quantities of test serum.

Methods to Discover and Validate Biofluid-Based Biomarkers in Neurodegenerative Dementias.

Neurodegenerative dementias are progressive diseases that cause neuronal network breakdown in different brain regions often because of accumulation of misfolded proteins in the brain extracellular matrix, such as amyloids or inside neurons or other cell types of the brain. Several diagnostic protein biomarkers in body fluids are being used and implemented, such as for Alzheimer's disease. However, there is still a lack of biomarkers for co-pathologies and other causes of dementia. Such biofluid-based biomarkers enable precision medicine approaches for diagnosis and treatment, allow to learn more about underlying disease processes, and facilitate the development of patient inclusion and evaluation tools in clinical trials. When designing studies to discover novel biofluid-based biomarkers, choice of technology is an important starting point. But there are so many technologies to choose among. To address this, we here review the technologies that are currently available in research settings and, in some cases, in clinical laboratory practice. This presents a form of lexicon on each technology addressing its use in research and clinics, its strengths and limitations, and a future perspective.

Challenges of insulin-like growth factor-1 testing.

Insulin-like growth factor 1 (IGF-1), primarily synthesized in the liver, was initially discovered due to its capacity to replicate the metabolic effects of insulin. Subsequently, it emerged as a key regulator of the actions of growth hormone (GH), managing critical processes like cell proliferation, differentiation, and apoptosis. Notably, IGF-1 displays a longer half-life compared to GH, making it less susceptible to factors that may affect GH concentrations. Consequently, the measurement of IGF-1 proves to be more specific and sensitive when diagnosing conditions such as acromegaly or GH deficiency. The recognition of the existence of IGFBPs and their potential to interfere with IGF-1 immunoassays urged the implementation of various techniques to moderate this issue and provide accurate IGF-1 results. Additionally, in response to the limitations associated with IGF-1 immunoassays and the occurrence of discordant IGF-1 results, modern mass spectrometric methods were developed to facilitate the quantification of IGF-1 levels. Taking advantage of their ability to minimize the interference caused by IGF-1 variants, mass spectrometric methods offer the capacity to deliver robust, reliable, and accurate IGF-1 results, relying on the precision of mass measurements. This also enables the potential detection of pathogenic mutations through protein sequence analysis. However, despite the analytical challenges, the discordance in IGF-1 reference intervals can be attributed to a multitude of factors, potentially leading to distinct interpretations of results. The establishment of reference intervals for each assay is a demanding task, and it requires nationwide multicenter collaboration among laboratorians, clinicians, and assay manufacturers to achieve this common goal in a cost-effective and resource-efficient manner. In this comprehensive review, we examine the challenges associated with the standardization of IGF-1 measurement methods, the minimization of pre-analytical factors, and the harmonization of reference intervals. Particular emphasis will be placed on the development of IGF-1 measurement techniques using "top-down" or "bottom-up" mass spectrometric methods.

Analytical Methods for Brassinosteroid Analysis: Recent Advances and Applications.

Brassinosteroids (BRs) are plant steroidal hormones that play crucial roles in plant growth and development. Accurate quantification of BRs in plant tissues is essential for understanding their biological functions. This study presents a comprehensive overview of the latest methods used for the quantification of BRs in plants. We discuss the principles, advantages, and limitations of various analytical techniques, including immunoassays, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS) that are used for the detection and quantification of BRs from complex plant matrices. We also explore the use of isotopically labeled internal standards to improve the accuracy and reliability of BR quantification.

Improved efficiency using sequential automated immunoassays for syphilis screening in blood donors.

Using sequential immunoassays for the screening of blood donors is well described for viral serology testing but not for the screening of syphilis. In this study, we report the evaluation results and 2-year sequential testing data using two highly sensitive automated serology assays, the Alinity s Syphilis chemiluminescent immunoassay for screening, with all repeatedly reactive samples then tested on the Elecsys Syphilis electrochemiluminescence immunoassay. We screened 1,767,782 blood donor samples between 7 July 2021 and 6 July 2023 and found the Alinity false-positive rate to be low at 0.08% (1,456/1,767,782). The common false-positive rate between the two assays was also low (3.83%, 58/1,514). Concordantly reactive samples were further tested using a Treponema pallidum particle agglutination test, a rapid plasma reagin test, and a fluorescent treponemal antibody absorption test. There were 262/1,376 concordantly reactive Alinity and Elecsys blood donor samples with reactivity on one or more of the confirmatory tests. A total of 26/1,376 donors had a current syphilis infection, 152/1,376 reported a past history of syphilis and had been treated, and 84/1,376 did not report a past history of syphilis. We suggest that future studies could explore the use of sequential immunoassays to aid in the serodiagnosis for syphilis. IMPORTANCE: The serodiagnosis for syphilis usually follows two methodologies-a "traditional" algorithm using a non-treponemal test followed by confirmation using a treponemal test, or a "reverse" algorithm using a treponemal test followed by a non-treponemal test. There are limited reports in the literature of using a modified reverse algorithm (treponemal test followed by a second treponemal test), and to the best of knowledge, there are currently no published articles using two highly sensitive automated immunoassays to aid the serodiagnosis of syphilis. In addition, the Treponema pallidum particle agglutination (TPPA) assay is commonly used as a confirmatory test for the diagnosis of syphilis. With the withdrawal of the TPPA assay from Australia and presumably from the global market also, alternative testing algorithms are now required. This study provides proof of concept for using sequential immunoassays in the diagnosis of syphilis.

Immunological Quantitation of the Glycation Site Lysine-414 in Serum Albumin in Human Plasma Samples by Indirect ELISA Using Highly Specific Monoclonal Antibodies.

Diabetes mellitus, a metabolic disorder that is characterized by elevated blood glucose levels, is common throughout the world and its prevalence is steadily increasing. Early diagnosis and treatment are important to prevent acute complications and life-threatening long-term organ damage. Glycation sites in human serum albumin (HSA) are considered to be promising biomarkers of systemic glycemic status. This work aimed to develop a sensitive and clinically applicable ELISA for the quantification of glycation site Lys414 in HSA (HSAK414 ). The monoclonal antibodies (mAbs) were generated by immunizing mice with a glycated peptide. The established indirect ELISA based on mAb 50D8 (IgG1 isotype) yielded a limit of detection of 0.39 nmol/g HSA for HSAK414 with a linear dynamic range from 0.50 to 6.25 nmol/g glycated HSA. The inter- and intra-day assays with coefficients of variation less than 20 % indicated good assay performance and precision. Assay evaluation was based on plasma samples from diabetic and non-diabetic subjects with known HSAK414 glycation levels previously determined by LC-MS. Both data sets correlated very well. In conclusion, the generated mAb 50D8 and the established ELISA could be a valuable tool for the rapid quantitation of glycation site HSAK414 in plasma samples to evaluate its clinical relevance.

A breakthrough of immunoassay format for hapten: recent insights into noncompetitive immunoassays to detect small molecules.

Immunoassay based on the antibodies specific for targets has advantages of high sensitivity, simplicity and low cost, therefore it has received more attention in recent years, especially for the rapid detection of small molecule chemicals present in foods, diagnostics and environments. However, limited by low molecular weight and only one antigenic determinant existed, immunoassays for these small molecule chemicals, namely hapten substances, were commonly performed in a competitive immunoassay format, whose sensitivities were obviously lower than the sandwich enzyme-linked immunosorbent assay generally adaptable for the protein targets. In order to break through the bottleneck of detection format, researchers have designed and established several novel noncompetitive immunoassays for the haptens in the past few years. In this review, we focused on the four representative types of noncompetitive immunoassay formats and described their characteristics and applications in rapid detection of small molecules. Meanwhile, a systematic discussion on the current technologies challenges and the possible solutions were also summarized. This review aims to provide an updated overview of the current state-of-the-art in noncompetitive immunoassay for small molecules, and inspire the development of novel designs for small molecule detection.

Clinical specificity of two assays for immunoglobulin kappa and lambda free light chains.

OBJECTIVES: Free light chain (FLC) assays and the ratio of κ/λ are recommended for diagnosis, prognosis and monitoring of plasma cell dyscrasias (PCD). Limited data exists on FLC clinical specificity in patients diagnosed with other conditions. METHODS: We assessed the κ, λ, and κ/λ FLC ratio using the FreeLite assay and the Sebia FLC ELISA assay in 176 patients with clinical presentations of fatigue, anemia, polyclonal hypergammaglobulinemia, joint disorders, kidney disease and non PCD-cancers with no monoclonal protein observed on serum protein electrophoresis or MASS-FIX immunoglobulin isotyping. Manufacturer defined reference intervals (RI) and glomerular filtration rate (GFR) specific RI (renal RI) were utilized. RESULTS: For the κ/λ ratio, 68.7 % (121/176) of specimens on the FreeLite and 87.5 % (154/176) of specimens on the Sebia assay were within RI. For κ, 68.2 % (120/176) and 72.2 % (127/176) of results were outside RI for FreeLite and Sebia respectively. For λ, 37.5 % (66/176) and 84.1 % (148/176) of FreeLite and Sebia results were outside RI. With FreeLite and Sebia, patients with kidney disease (n=25) had the highest κ/λ ratios. 44 patients (25.0 %) had GFR <60 mL/min/BSA. When renal RI were applied, 13.6 % had a FLCr outside the renal RI with FreeLite, and 4.5 % with Sebia. CONCLUSIONS: In a cohort of patients with signs and symptoms suggestive of PCDs, but ultimately diagnosed with other conditions, Sebia FLC had improved clinical specificity relative to FreeLite, if one was using an abnormal κ/λ ratio as a surrogate for monoclonality.

A longitudinal study of endocrinology and foraging ecology of subadult gray whales prior to death based on baleen analysis.

Individual-level assessments of wild animal health, vital rates, and foraging ecology are critical for understanding population-wide impacts of exposure to stressors. Large whales face multiple stressors, including, but not limited to, ocean noise, pollution, and ship strikes. Because baleen is a continuously growing keratinized structure, serial extraction, and quantification of hormones and stable isotopes along the length of baleen provide a historical record of whale physiology and foraging ecology. Furthermore, baleen analysis enables the investigation of dead specimens, even decades later, allowing comparisons between historic and modern populations. Here, we examined baleen of five sub-adult gray whales and observed distinct patterns of oscillations in δ15N values along the length of their baleen plates which enabled estimation of baleen growth rates and differentiation of isotopic niche widths of the whales during wintering and summer foraging. In contrast, no regular patterns were apparent in δ13C values. Prolonged elevation of cortisol in four individuals before death indicates that chronic stress may have impacted their health and survival. Triiodothyronine (T3) increased over months in the whales with unknown causes of death, simultaneous with elevations in cortisol, but both hormones remained stable in the one case of acute death attributed to killer whale predation. This parallel elevation of cortisol and T3 challenges the classic understanding of their interaction and might relate to increased energetic demands during exposure to stressors. Reproductive hormone profiles in subadults did not show cyclical trends, suggesting they had not yet reached sexual maturity. This study highlights the potential of baleen analysis to retrospectively assess gray whales' physiological status, exposure to stressors, reproductive status, and foraging ecology in the months or years leading up to their death, which can be a useful tool for conservation diagnostics to mitigate unusual mortality events.

Prevalence, incidence, risk factors and residual risk associated with viral infections among eligible Brazilian blood donors.

Knowledge regarding the profile of eligible blood donors presenting positive results in laboratory screening is essential for reducing transfusion-transmitted human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV). Our study aimed to evaluate the prevalence, incidence, predictor variables and residual risk (RR) of HIV/HBV/HCV in blood bags donated in Minas Gerais, Brazil. This study analysed data retrieved from the records of a large blood bank relating to donations collected at multiple centres within the period 2012-2018, during which 1 991 120 blood bags were screened using immunoassays and nucleic acid tests (NATs). Multilevel modelling was used to investigate the association between sex, civil status and age group with HIV/HBV/HCV. RR was estimated from the incidence values (restricted to negative and positive tests within the study period) and window periods for infections. The prevalence in first time donors, incidence and RR of HCV (223.73 cases per 100 000; 54.84 per 100 000 persons-year and 1.6527 per 100 000, respectively) were higher than those of HIV (172.65 cases per 100 000; 28.25 per 100 000 persons-year and 0.8514 per 100 000) and HBV (168.17 cases per 100 000; 18.54 per 100 000 persons-year and 0.5588 per 100 000). The odds of acquiring infection were greater in male, single and older donors. Sixteen donors were identified as seronegative and NATs+ during the 7-year span of the study. Our study has clarified some spatiotemporal trends regarding HIV/HBV/HCV infections in donated blood in Brazil. The results will contribute to the formulation of directives addressed to high-risk donors.

Improving diagnostic performance of coronavirus disease 2019 rapid antigen testing through computer-based feedback training using open-source experimental psychology software.

INTRODUCTION: Rapid antigen testing (RAT) results are visually read as whether colored line is present or absent. The subjective interpretation potentially misses detecting weak lines due to lower analyte concentration in samples tested, requiring training. Although routine test experience has improved the result readout skills, it consumes time and resources. Therefore, we created a computer-based feedback training method using open-source experimental psychology software, wherein participants accumulate RAT result readout experience by repeatedly responding positive/negative to randomly presented pictures showing RAT results; then, they receive feedback on their answers as correct or incorrect and are asked to stare at the pictures again with the knowledge of correct answer. This study aimed to examine the training effects in improving the skills, using coronavirus disease 2019 (COVID-19) RAT. METHODS: Twenty-two medical technologists were randomly divided into two groups: the feedback-training and test-experience groups. Using several pictures showing positive and negative results of COVID-19 RAT, after examination of their initial result readout skills, feedback-training group received the feedback training, whereas test-experience group performed an equal number of tests without feedback to accumulate test experience, and their skills were examined again. The ratio of "positive" answers to the pictures showing positive results (i.e., hit rate) was statistically analyzed. RESULTS: The feedback-training group showed a significantly higher hit rate after their training, whereas the test-experience group did not. The feedback training effects were manifested in weak line detection. CONCLUSIONS: This computer-based feedback training method can be an effective tool for improving RAT result readout skills.

Direct single-molecule imaging for diagnostic and blood screening assays.

Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10-18 M) for model assay systems and to the low femtomolar (10-16 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10-18 to 10-5 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.

Dual Functional Ultrasensitive Point-of-Care Clinical Diagnosis Using Metal-Organic Frameworks-Based Immunobeads.

Sepsis is an acute systemic infectious syndrome with high fatality. Fast and accurate diagnosis, monitoring, and medication of sepsis are essential. We exploited the fluorescent metal-AIEgen frameworks (MAFs) and demonstrated the dual functions of protein detection and bacteria identification: (i) ultrasensitive point-of-care (POC) detection of sepsis biomarkers (100 times enhanced sensitivity); (ii) rapid POC identification of Gram-negative/positive bacteria (selective aggregation within 20 min). Fluorescent lateral flow immunoassays (LFAs) are convenient and inexpensive for POC tests. MAFs possess a large surface area, excellent photostability, high quantum yield (∼80%), and multiple active sites serving as protein binding domains for ultrasensitive detection of sepsis biomarkers (IL-6/PCT) on LFAs. The limit of detection (LOD) for IL-6/PCT is 0.252/0.333 pg/mL. Rapid appraisal of infectious bacteria is vital to guide the use of medicines. The dual-functional fluorescent MAFs have great potential in POC tests for the clinical diagnosis of bacterial infections.

Decoupling the Characteristics of Magnetic Nanoparticles for Ultrahigh Sensitivity.

Immunoassays exploiting magnetization dynamics of magnetic nanoparticles are highly promising for mix-and-measure, quantitative, and point-of-care diagnostics. However, how single-core magnetic nanoparticles can be employed to reduce particle concentration and concomitantly maximize assay sensitivity is not fully understood. Here, we design monodisperse Néel and Brownian relaxing magnetic nanocubes (MNCs) of different sizes and compositions. We provide insights into how to decouple physical properties of these MNCs to achieve ultrahigh sensitivity. We find that tricomponent-based Zn0.06Co0.80Fe2.14O4 particles, with out-of-phase to initial magnetic susceptibility χ″/χ0 ratio of 0.47 out of 0.50 for magnetically blocked ideal particles, show the ultrahigh magnetic sensitivity by providing a rich magnetic particle spectroscopy (MPS) harmonics spectrum despite bearing lower saturation magnetization than dicomponent Zn0.1Fe2.9O4 having high saturation magnetization. The Zn0.06Co0.80Fe2.14O4 MNCs, coated with catechol-based poly(ethylene glycol) ligands, measured by our benchtop MPS show 3 orders of magnitude better particle LOD than that of commercial nanoparticles of comparable size.

Optimizing cutpoints for clinical interpretation of brain amyloid status using plasma p-tau217 immunoassays.

INTRODUCTION: We aimed to evaluate clinical interpretation cutpoints for two plasma phosphorylated tau (p-tau)217 assays (ALZpath and Lumipulse) as predictors of amyloid status for implementation in clinical practice. METHODS: Clinical performance of plasma p-tau217 against amyloid positron emission tomography status was evaluated in participants with mild cognitive impairment or mild dementia (n = 427). RESULTS: Using a one-cutpoint approach (negative/positive), neither assay achieved ≥ 90% in both sensitivity and specificity. A two-cutpoint approach yielding 92% sensitivity and 96% specificity provided the desired balance of false positives and false negatives, while categorizing 20% and 39% of results as indeterminate for the Lumipulse and ALZpath assays, respectively. DISCUSSION: This study provides a systematic framework for selection of assay-specific cutpoints for clinical use of plasma p-tau217 for determination of amyloid status. Our findings suggest that a two-cutpoint approach may have advantages in optimizing diagnostic accuracy while minimizing potential harm from false positive results. HIGHLIGHTS: Phosphorylated tau (p-tau)217 cutpoints for detection of amyloid pathology were established. A two-cutpoint approach exhibited the best performance for clinical laboratory use. p-tau217 assays differed in the percentage of results categorized as intermediate.

Immunoassays: Analytical and Clinical Performance, Challenges, and Perspectives of SERS Detection in Comparison with Fluorescent Spectroscopic Detection.

Immunoassays (IAs) with fluorescence-based detection are already well-established commercialized biosensing methods, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA). Immunoassays with surface-enhanced Raman spectroscopy (SERS) detection have received significant attention from the research community for at least two decades, but so far they still lack a wide clinical commercial application. This review, unlike any other review that we have seen, performs a three-dimensional performance comparison of SERS IAs vs. fluorescence IAs. First, we compared the limit of detection (LOD) as a key performance parameter for 30 fluorescence and 30 SERS-based immunoassays reported in the literature. We also compared the clinical performances of a smaller number of available reports for SERS vs. fluorescence immunoassays (FIAs). We found that the median and geometric average LODs are about 1.5-2 orders of magnitude lower for SERS-based immunoassays in comparison to fluorescence-based immunoassays. For instance, the median LOD for SERS IA is 4.3 × 10-13 M, whereas for FIA, it is 1.5 × 10-11 M. However, there is no significant difference in average relative standard deviation (RSD)-both are about 5-6%. The analysis of sensitivity, selectivity, and accuracy reported for a limited number of the published clinical studies with SERS IA and FIA demonstrates an advantage of SERS IA over FIA, at least in terms of the median value for all three of those parameters. We discussed common and specific challenges to the performances of both SERS IA and FIA, while proposing some solutions to mitigate those challenges for both techniques. These challenges include non-specific protein binding, non-specific interactions in the immunoassays, sometimes insufficient reproducibility, relatively long assay times, photobleaching, etc. Overall, this review may be useful for a large number of researchers who would like to use immunoassays, but particularly for those who would like to make improvements and move forward in both SERS-based IAs and fluorescence-based IAs.

An Analysis of the Biotin-(Strept)avidin System in Immunoassays: Interference and Mitigation Strategies.

An immunoassay is an analytical test method in which analyte quantitation is based on signal responses generated as a consequence of an antibody-antigen interaction. They are the method of choice for the measurement of a large panel of diagnostic markers. Not only are they fully automated, allowing for a short turnaround time and high throughput, but offer high sensitivity and specificity with low limits of detection for a wide range of analytes. Many immunoassay manufacturers exploit the extremely high affinity of biotin for streptavidin in their assay design architectures as a means to immobilize and detect analytes of interest. The biotin-(strept)avidin system is, however, vulnerable to interference with high levels of supplemental biotin that may cause elevated or suppressed test results. Since this system is heavily applied in clinical diagnostics, biotin interference has become a serious concern, prompting the FDA to issue a safety report alerting healthcare workers and the public about the potential harm of ingesting high levels of supplemental biotin contributing toward erroneous diagnostic test results. This review includes a general background and historical prospective of immunoassays with a focus on the biotin-streptavidin system, interferences within the system, and what mitigations are applied to minimize false diagnostic results.

An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays.

Signal amplification strategies are widely used for improving the sensitivity of lateral flow immunoassays (LFiAs). Herein, the artificial miniaturized peroxidase Fe(III)-MimochromeVI*a (FeMC6*a), immobilized on gold nanoparticles (AuNPs), is used as a strategy to obtain catalytic signal amplification in sandwich immunoassays on lateral flow strips. The assay scheme uses AuNPs decorated with the mini-peroxidase FeMC6*a and anti-human-IgG as a detection antibody (dAb), for the detection of human-IgG, as a model analyte. Recognition of the analyte by the capture and detection antibodies is first evidenced by the appearance of a red color in the test line (TL), due to the accumulation of AuNPs. Subsequent addition of 3,3',5,5'-tetramethylbenzidine (TMB) induces an increase of the test line color, due to the TMB being converted into an insoluble colored product, catalyzed by FeMC6*a. This work shows that FeMC6*a acts as an efficient catalyst in paper, increasing the sensitivity of an LFiA up to four times with respect to a conventional LFiA. Furthermore, FeMC6*a achieves lower limits of detection that are found in control experiments where it is replaced with horseradish peroxidase (HRP), its natural counterpart. This study represents a significant proof-of-concept for the development of more sensitive LFiAs, for different analytes, based on properly designed artificial metalloenzymes.

Engineered Collaborative Size Regulation and Shape Engineering of Tremella-Like Au-MnOx for Highly Sensitive Bimodal-Type Lateral Flow Immunoassays.

Engineered collaborative size regulation and shape engineering of multi-functional nanomaterials (NPs) offer extraordinary opportunities for improving the analysis performance. It is anticipated to address the difficulty in distinguishing color changes caused by subtle variations in target concentrations, thereby facilitating the highly sensitive analysis of lateral flow immunoassays (LFIAs). Herein, tremella-like gold-manganese oxide (Au-MnOx ) nanoparticles with precise MnCl2 regulation are synthesized as immuno signal tracers via a facile one-step redox reaction in alkaline condition at ambient temperature. Avail of the tunable elemental composition and anisotropy in morphology, black-colored tremella-like Au-MnOx exhibits superb colorimetric signal brightness, enhanced antibody coupling efficiency, marvelous photothermal performance, and unrestricted immunological recognition affinity, all of which facilitate highly sensitive multi-signal transduction patterns. In conjunction with the handheld thermal reader device, a bimodal-type LFIA that combines size-regulation- and shape-engineering-mediated colorimetric-photothermal dual-response assay (coined as the SSCPD assay) with a limit of detection of 0.012 ng mL-1 for ractopamine (RAC) monitoring is achieved by integrating Au-MnOx with the competitive-type immunoreaction. This work illustrates the effectiveness of this strategy for establishing high-performance sensing, and the SSCPD assay may be extended to a wide spectrum of future point-of-care (POC) diagnostic applications.

Programmable Gravity Self-Driven Microfluidic Chip for Point-of-Care Multiplied Immunoassays.

Point-of-care testing (POCT) is experiencing a groundbreaking transformation with microfluidic chips, which offer precise fluid control and manipulation at the microscale. Nevertheless, chip design or operation for existing platforms is rather cumbersome, with some even heavily depending on external drivers or devices, impeding their broader utilization. This study develops a unique programmable gravity self-driven microfluidic chip (PGSMC) capable of simultaneous multi-reagent sequential release, multi-target analysis, and multi-chip operation. All necessary reagents are introduced in a single step, and the process is initiated simply by flipping the PGSMC vertically, eliminating the need for additional steps or devices. Additionally, it demonstrates successful immunoassays in less than 60 min for antinuclear antibodies testing, compared to more than 120 min by traditional methods. Assessment using 25 clinically diagnosed cases showcases remarkable sensitivity (96%), specificity (100%), and accuracy (99%). These outcomes underscored its potential as a promising platform for POCT with high accuracy, speed, and reliability, highlighting its capability for automated fluid control.