Comparison between capillary electrophoresis and fluorescence anisotropy competitive immunoassay for glucagon.

Glucagon plays a crucial role in regulating glucose homeostasis; unfortunately, the mechanisms controlling its release are still unclear. Capillary electrophoresis (CE)- and fluorescence anisotropy (FA)-immunoassays (IA) have been used for online measurements of hormone secretion on microfluidic platforms, although their use in glucagon assays is less common. We set out to compare a glucagon-competitive IA using these two techniques. Theoretical calibration curves were generated for both CE- and FA-IA and results indicated that CE-IA provided higher sensitivity than FA-IA. These results were confirmed in an experiment where both assays showed limits of detection (LOD) of 30 nM, but the CE-IA had ∼300-fold larger sensitivity from 0 to 200 nM glucagon. However, in online experiments where reagents were mixed within the device, the sensitivity of the CE-IA was reduced ∼3-fold resulting in a higher LOD of 70 nM, whereas the FA-IA remained essentially unchanged. This lowered sensitivity in the online CE-IA was likely due to poor sampling by electroosmotic flow from the high salt solution necessary in online experiments, whereas pressure-based sampling used in FA-IA was not affected. We conclude that FA-IA, despite lowered sensitivity, is more suitable for online mixing scenarios due to the ability to use pressure-driven flow and other practical advantages such as the use of larger channels.

A graphene oxide-assisted protein immobilization paper-tip immunosensor with smartphone and naked eye readout for the detection of okadaic acid.

BACKGROUND: Okadaic acid (OA), as a diarrhetic shellfish poisoning, can increase the risk of acute carcinogenic or teratogenic effects for the ingestion of OA contaminated shellfish. At present, much effort has been made to graft immunoassay onto a paper substrate to make paper-based sensors for rapid and simple detection of shellfish toxin. However, the complicated washing steps and low protein fixation efficiency on the paper substrate need to be further addressed. RESULTS: A novel paper-tip immunosensor for detecting OA was developed combined with smartphone and naked eye readout. The trapezoid paper tip was consisted of quantitative and qualitative detection zones. To improve the OA antigen immobilization efficiency on the paper substrate, graphene oxide (GO)-assisted protein immobilization method was introduced. Meanwhile, Au nanoparticles composite probe combined with the lateral flow washing was developed to simplify the washing step. The OA antigen-immobilized zone, as the detection zone Ⅰ, was used for quantitative assay by smartphone imaging. The paper-tip front, as the detection zone Ⅱ, which could qualitatively differentiate OA pollution level within 45 min using the naked eye. The competitive immunoassay on the paper tip exhibited a wide linear range for detecting OA (0.02-50 ng∙mL-1) with low detection limit of 0.02 ng∙mL-1. The recovery of OA in spiked shellfish samples was in the range of 90.3 %-113.%. SIGNIFICANCE: These results demonstrated that the proposed paper-tip immunosensor could provide a simple, low-cost and high-sensitivity test for OA detection without the need for additional large-scale equipment or expertise. We anticipate that this paper-tip immunosensor will be a flexible and versatile tool for on-site detecting the pollution of marine products.

Development of a Phage-Displayed Nanobody-Based Competitive Immunoassay for the Sensitive Detection of Soybean Agglutinin.

Soybean agglutinin (SBA) is a primary antinutritional factor in soybeans that can inhibit the growth of humans and mammals, disrupt the intestinal environment, and cause pathological changes. Therefore, detecting and monitoring SBA in foods is essential for safeguarding human health. In this paper, M13 phage-displayed nanobodies against SBA were isolated from a naive nanobody library. An M13 phage-displayed nanobody-based competitive enzyme-linked immunosorbent assay (P-cELISA) was then established for SBA analysis using biotinylated anti-M13 phage antibody (biotin-anti-M13) and streptavidin poly-HRP conjugate (SA-poly-HRP). The biotin-anti-M13@SA-poly-HRP probe can easily amplify the detection signal without the chemical modifications of phage-displayed nanobodies. The established P-cELISA presented a linear detection range of 0.56-250.23 ng/mL and a limit of detection (LOD) of 0.20 ng/mL, which was 12.6-fold more sensitive than the traditional phage-ELISA. Moreover, the developed method showed good specificity for SBA and acceptable recoveries (78.21-121.11%) in spiked wheat flour, albumen powder, and whole milk powder. This study proposes that P-cELISA based on biotin-anti-M13@SA-poly-HRP may provide a convenient and effective strategy for the sensitive detection of SBA.

Simple Determination of Affinity Constants of Antibodies by Competitive Immunoassays.

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody-antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols.

Electrochemical bioplatform to manage alpha-gal syndrome by tracking the carbohydrate allergen in meat.

This work presents the first bioplatform described to date for the determination of galactose-α-1,3-galactose (α-Gal), a non-primate mammalian oligosaccharide responsible for almost all cases of red meat allergy. The bioplatform is based on the implementation of an indirect competitive immunoassay and enzymatic labeling with the enzyme horseradish peroxidase (HRP) built on the surface of magnetic microparticles (MBs) and amperometric transduction on screen-printed carbon electrodes (SPCEs) using the H2O2/hydroquinone (HQ) system. The target α-Gal competed with biotinylated α-Gal immobilized on the surface of neutravidin-modified MBs for the limited immunorecognition sites of a detection antibody enzymatically labeled with an HRP-conjugated secondary antibody. The resulting magnetic immunoconjugates were trapped on the surface of the SPCE working electrode and amperometric transduction was performed, providing a cathodic current variation inversely proportional to the concentration of α-Gal in the analyzed sample. The developed biotool was optimized, characterized and applied with satisfactory results to the determination of the target allergen in different samples of raw and processed meats.

Competitive Immunoassay in a Microfluidic Biochip for In-Field Detection of Abscisic Acid in Grapes.

Viticulture and associated products are an important part of the economy in many countries. However, biotic and abiotic stresses impact negatively the production of grapes and wine. Climate change is in many aspects increasing both these stresses. Routine sample retrievals and analysis tend to be time-consuming and require expensive equipment and skilled personnel to operate. These challenges could be overcome through the development of a miniaturized analytic device for early detection of grapevine stresses in the field. Abscisic acid is involved in several plant processes, including the onset of fruit ripening and tolerance mechanisms against drought stress. This hormone can be detected through a competitive immunoassay and is found in plants in concentrations up to 10-1 mg/mL. A microfluidic platform is developed in this work which can detect a minimum of 10-11 mg/mL of abscisic acid in buffer. Grape samples were tested using the microfluidic system alongside benchmark techniques such as high-performance liquid chromatography. The microfluidic system could detect the increase to 10-5 mg/mL of abscisic acid present in real berry samples at the veraison stage of ripening.

Flexible nanoplasmonic sensor for multiplexed and rapid quantitative food safety analysis with a thousand-times sensitivity improvement.

The accumulation of trace amounts of certain small molecules in food poses considerable human health challenges, including the potential for carcinogenesis and mutagenesis. Here, an ultrasensitive gold-platinum nanoflower-coupled metasurface plasmon resonance (MetaSPR) (APNMSPR) biosensor, based on a competitive immunoassay, was developed for the multiplexed and rapid quantitative analysis of trace small molecules in eggs, offering timely monitoring of food safety. This one-step biosensor can be integrated into either a newly designed detachable high-throughput MetaSPR chip-strip plate device or a standard 96-well plate for multiplexed small-molecule detection within a single egg. The limits of detection were 0.81, 1.12, and 1.74 ppt for florfenicol, fipronil, and enrofloxacin, respectively, demonstrating up to 1000-fold increased sensitivity and a 15-fold reduction in analysis time compared with those of traditional methods. The results obtained using the APNMSPR biosensor showed a strong correlation with those obtained using liquid chromatography-tandem mass spectrometry. The APNMSPR biosensor holds immense promise for the multiplexed, highly sensitive, and rapid quantitative analysis of small molecules for applications in food safety control, early diagnosis, and environmental monitoring.

Establishment of an indirect competitive immunoassay for the detection of dicamba based on a highly specific nanobody.

Dicamba, a traditional highly effective and low toxicity herbicide, has gained new life with the development of dicamba-tolerant transgenic crops in recent years. However, dicamba is highly volatile and therefore easy to cause drift damage to sensitive crops. The development of efficient and sensitive detection methods is essential for monitoring of trace dicamba in the environment. Nanobody-based immunoassay plays an important role in on-site detection of pesticides. However, now rapid and sensitive immunoassay methods based on nanobody for dicamba detection were lacking. In this study, the nanobodies specifically recognizing dicamba were successfully obtained by immunising camels and phage display library construction, and then an indirect competitive immunoassay based on Nb-242 was constructed with IC50 of 0.93 µg/mL and a linear range of 0.11-8.01 µg/mL. Nb-242 had good specificity with no cross-reactivities against the dicamba analogs other than 2,3,6-trichlorobenzoic acid and the developed immnoassay had a good correlation with the standard HPLC in the spike-recovery studies. Finally, the key amino acid Ala 123, Tyr 55, Tyr 59 and Arg 72 of Nb-242 that specifically recognizing and binding with dicamba were identified by homologous modeling and molecular docking, laying an important foundation for further structural modification of Nb-242. This study has important guiding significance for constructing immunoassay method of dicamba based on nanobody and provides a sensitive, specific, and reliable detection method that is suitable for the detection of dicamba in the environment.

Palladium nanoclusters as a label to determine GFAP in human serum from donors with stroke by bimodal detection: inductively coupled plasma-mass spectrometry and linear sweep voltammetry.

Water-soluble, stable, and monodisperse palladium nanoclusters (PdNCs) were synthesized using NaBH4 as a reductant and lipoic acid as a ligand. PdNCs, measured by high-resolution transmission electron microscopy, showed a round shape and a diameter of 2.49 ± 0.02 nm. It was found that each PdNC contains 550 Pd atoms on average. These PdNCs offer high amplification as a label of biochemical reactions when inductively coupled plasma-mass spectrometry (ICP-MS) is used as a detector. In addition, PdNCs have catalytic activity on electrochemical reactions, allowing detection by linear sweep voltammetry (LSV). As a proof of applicability, a competitive immunoassay based on PdNC labels was developed for the determination of glial fibrillary acidic protein (GFAP) in human serum, comparing ICP-MS and LSV detection. GFAP is a biomarker for differentiating between patients with ischemic stroke (IS) and hemorrhagic stroke (HS). The limit of detection (LoD), corresponding to IC10 (4-parameter logistic curve), was 0.03 pM of GFAP, both by ICP-MS and LSV, being lower than the 0.31 pM LoD provided by the ELISA commercial kit. Using the error profile method, 0.03 pM and 0.11 pM LoDs were obtained respectively by ICP-MS and LSV: LoD is lower by ICP-MS due to the better precision of the measurements. The analyses of human serum samples from IS, HS, and control (CT) donors using PdNC labels and detection by ICP-MS and LSV were validated with a commercial ELISA kit (for CT donors only ICP-MS provided enough sensitivity). Results point out toward the future use of PdNCs as a label in other immunoprobes for the determination of specific proteins requiring very low LoDs as well as the development of electrochemical decentralized methodologies.

MIP-based immunoassays: A critical review.

Molecularly imprinted polymers, MIPs, are man-made receptors mimicking the thermodynamic and kinetic binding behaviour of natural antibodies. Therefore, it is not surprising that many researchers have thought about MIPs as artificial receptors in immunoassay-like analytical applications, where the general machinery of the assay is maintained, but the molecular recognition is no longer assured by an antibody but by an artificial receptor. However, the number of papers devoted explicitly to applications of MIPs in the immunoassay field is quite limited if compared to the huge number of papers covering the multifaceted molecular imprinting technology. For this reason, this critical review wants to give a general view of MIP-based immunoassays, trying to highlight the critical points that have so far prevented a wider application of molecular imprinting technology in the immunoassay field and, possibly, try to suggest strategies to overcome them.

A Metasurface Plasmonic Analysis Platform Combined with Gold Nanoparticles for Ultrasensitive Quantitative Detection of Small Molecules.

Food safety related to drug residues in food has become a widespread public concern. Small-molecule drug residue analysis often relies on mass spectrometry, thin-layer chromatography, or enzyme-linked immunosorbent assays (ELISA). Some of these techniques have limited sensitivity and accuracy, while others are time-consuming, costly, and rely on specialized equipment that requires skilled operation. Therefore, the development of a sensitive, fast, and easy-to-operate biosensor could provide an accessible alternative to conventional small-molecule analysis. Here, we developed a nanocup array-enhanced metasurface plasmon resonance (MetaSPR) chip coupled with gold nanoparticles (AuNPs) (MSPRAN) to detect small molecules. As sulfamethazine drug residues in poultry eggs may cause health issues, we selected this as a model to evaluate the feasibility of using MSPRAN for small-molecule detection. The MSPRAN biosensor employed competitive immunoassay technology for sulfamethazine detection. The limit of detection was calculated as 73 pg/mL, with sensitivity approximately twice that of previously reported detection methods. Additionally, the recovery rate of the biosensor, tested in egg samples, was similar to that measured using ELISA. Overall, this newly developed MSPRAN biosensor platform for small-molecule detection provides fast and reliable results, facile operation, and is relatively cost-effective for application in food safety testing, environmental monitoring, or clinical diagnostics.

Establishment and analytical performance of light-initiated chemiluminescence assay method for detecting thyrotropin receptor antibody.

Aim: To evaluate double-antibody competitive light-initiated chemiluminescence assay method for detecting the thyrotropin receptor antibody. Materials & methods: The optimal working concentrations of competitive antibody and rTSHR were confirmed by checkerboard titration. Assay performance was assessed by precision, linearity, accuracy, limit of blank and clinical evaluation. Results: The coefficient of variation for repeatability and intermediate precision was 3.9-5.9 and 0.9-1.3%, respectively. The correlation coefficient was 0.999 by least squares linear fitting in linearity evaluation. The relative deviation ranged from -5.9 to 4.1%, and the limit of blank of the method was 0.13 IU/l. Compared with the Roche cobas system (Roche Diagnostics, Mannheim, Germany), the relationship between the two assays was shown to be significantly correlative. Conclusion: The light-initiated chemiluminescence assay method for detecting thyrotropin receptor antibody is a rapid, novel and accurate method for thyrotropin receptor antibody measurement.

Graves' disease is common in daily life. Patients often experience rapid heartbeat and weight loss. Blood tests (especially serum autoantibody levels) are meaningful in diagnosis and treatment. In this study, we used a new and useful approach to test blood markers. Compared with other methods, this new technique could perform better in terms of time and expense. Based on a full assessment, we believe that this new method will play an important role in clinical application.

High-performance green-emitting AIE nanoparticles for lateral flow immunoassay applications.

Ultrabright green-emissive AIE nanoparticles (AIENPs) were used as signal-amplification probes to enhance the detectability of lateral flow immunoassay (LFIA). The detection performances of the green-emissive AIENP probes in both sandwich and competitive LFIA formats were systematically evaluated. Benefiting from its remarkable fluorescent brightness, the developed AIENP-LFIA showed versatile applicability for the detection of small molecules and macromolecules by using ochratoxin A (OTA) and procalcitonin (PCT) as model analytes, respectively. Under the optimum conditions, the detection limits (LODs) of the fabricated AIENP-LFIA for OTA and PCT were 0.043 ng mL-1 and 0.019 ng mL-1, respectively. These LOD values are significantly lower than those of conventional LFIA methods using gold nanoparticles as signal reporters. In addition, we demonstrated the practical application potential of AIENP-LFIA for the detection of OTA in real maize samples and PCT in real serum samples. These results indicated that the ultrabright green-emissive AIENPs were promising as signal output materials for building high-performance LFIA platform and broadening the application scenarios of LFIA.

Automated detection of neutralizing SARS-CoV-2 antibodies in minutes using a competitive chemiluminescence immunoassay.

The SARS-CoV-2 pandemic has shown the importance of rapid and comprehensive diagnostic tools. While there are numerous rapid antigen tests available, rapid serological assays for the detection of neutralizing antibodies are and will be needed to determine not only the amount of antibodies formed after infection or vaccination but also their neutralizing potential, preventing the cell entry of SARS-CoV-2. Current active-virus neutralization assays require biosafety level 3 facilities, while virus-free surrogate assays are more versatile in applications, but still take typically several hours until results are available. To overcome these disadvantages, we developed a competitive chemiluminescence immunoassay that enables the detection of neutralizing SARS-CoV-2 antibodies within 7 min. The neutralizing antibodies bind to the viral receptor binding domain (RBD) and inhibit the binding to the human angiotensin-converting enzyme 2 (ACE2) receptor. This competitive binding inhibition test was characterized with a set of 80 samples, which could all be classified correctly. The assay results favorably compare to those obtained with a more time-intensive ELISA-based neutralization test and a commercial surrogate neutralization assay. Our test could further be used to detect individuals with a high total IgG antibody titer, but only a low neutralizing titer, as well as for monitoring neutralizing antibodies after vaccinations. This effective performance in SARS-CoV-2 seromonitoring delineates the potential for the test to be adapted to other diseases in the future.

High-performance colorimetric immunoassay for determination of chloramphenicol using metal-organic framework-based hybrid composites with increased peroxidase activity.

Metal-organic frameworks (MOFs) as carriers for high-capacity loading of HRP-IgG and gold nanoparticles are introduced, to prepare MOF hybrids with enhanced peroxidase activity. The prepared MOF hybrids were employed to establish an indirect competitive colorimetric immunoassay for chloramphenicol (CAP) detection, in which the limit of detection for CAP is 0.006 µg·L-1, only one-fifth of that of the conventional ELISA using the same antibodies and antigens. The linear range was 0.008-0.108 µg·L-1, and the recovery of spiked milk samples varied in the range 76.0-106.0% through three independent experiments. Our proposed colorimetric immunoassay using the MOF hybrid immunoprobe provides a novel platform for ultra-sensitive determination of CAP residues, and it also could be used as a signal amplification model for the high-performance colorimetric immunoassay in food safety monitoring.

A Self-Regulated Microfluidic Device with Thermal Bubble Micropumps.

Currently, many microchips must rely on an external force (such as syringe pump, electro-hydrodynamic pump, and peristaltic pump, etc.) to control the solution in the microchannels, which probably adds manual operating errors, affects the accuracy of fluid manipulation, and enlarges the noise of signal. In addition, the reasonable integration of micropump and microchip remain the stumbling block for the commercialization of microfluidic technique. To solve those two problems, we designed and fabricated a thermal bubble micropump based on MEMS (micro-electro-mechanical systems) technique. Many parameters (voltage, pulse time, cycle delay time, etc.) affecting the performance of this micropump were explored in this work. The experimental results showed the flow rate of solution with the assistance of a micropump reached more than 15 µL/min in the optimal condition. Finally, a method about measuring total aflatoxin in Chinese herbs was successfully developed based on the integrated platform contained competitive immunoassay and our micropump-based microfluidics. Additionally, the limit of detection in quantifying total aflatoxin (AF) was 0.0615 pg/mL in this platform. The data indicate this combined technique of biochemical assays and micropump based microchip have huge potential in automatically, rapidly, and sensitively measuring other low concentration of biochemical samples with small volume.

Dual-Mode Electrochemical Competitive Immunosensor Based on Cd2+/Au/Polydopamine/Ti3C2 Composite and Copper-Based Metal-Organic Framework for 17ß-Estradiol Detection.

Herein, a dual-mode electrochemical competitive immunosensor was constructed for the detection of 17ß-estradiol (E2) based on differential pulse voltammetry (DPV) and chronoamperometry (i-t). During the immune recognition process, the E2 antibody (E2-Ab) was immobilized on the Cd2+/Au/polydopamine/Ti3C2 (Cd2+/Au/pDA/Ti3C2) composite-modified electrode; then, the E2-conjugated bovine serum albumin (E2-BSA) was labeled with a copper-based metal-organic framework (Cu-MOF) and competed with E2 in combining the E2-Ab. The Cu-MOF was not only an electroactive species but also possessed good electrocatalytic activity toward H2O2. Thus, E2 could be quantified according to the peak current change of the Cu-MOF in DPV curve or the variation of H2O2 reduction current. For DPV quantification, Cd2+ was introduced as an internal reference in this case, and a highly reproducible ratio readout was obtained. The as-prepared dual-mode E2 electrochemical immunosensor showed good linear relationship in the ranges of 1 pg mL-1-10 ng mL-1 (DPV) and 10 pg mL-1-10 ng mL-1 (i-t), and the detection limits were 0.47 and 5.4 pg mL-1 (S/N = 3), respectively. Furthermore, the dual-mode electrochemical immunosensor exhibited good practicability in real sample analysis.

Utilization of a Novel Immunofluorescence Instrument Prototype for the Determination of the Herbicide Glyphosate.

An enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the quantitative analytical determination of the herbicide active ingredient glyphosate in environmental matrices (surface water, soil, and plant tissues). Glyphosate, as a ubiquitous agricultural pollutant, is a xenobiotic substance with exposure in aquatic and terrestrial ecosystems due its extremely high worldwide application rate. The immunoassay developed in Project Aquafluosense is part of a fluorescence-based instrumentation setup for the in situ determination of several characteristic water quality parameters. The 96-well microplate-based competitive immunoassay method applies fluorescence signal detection in the concentration range of 0-100 ng/mL glyphosate. Application of the fluorescent signal provides a limit of detection of 0.09 ng/mL, which is 2.5-fold lower than that obtained with a visual absorbance signal. Beside the improved limit of detection, determination by fluorescence provided a wider and steeper dynamic range for glyphosate detection. No matrix effect appeared for the undiluted surface water samples, while plant tissues and soil samples required dilution rates of 1:10 and 1:100, respectively. No cross-reaction was determined with the main metabolite of glyphosate, N-aminomethylphosphonic acid, and related compounds.

Comparison of lateral flow immunoassays based on oriented and nonoriented immobilization of antibodies for the detection of aflatoxin B1.

In recent years, some studies have found that oriented immobilization of antibodies to microspheres can fully expose the antigen binding sites of antibodies, which can improve the sensitivity of sandwich immunoassays for the detection of proteins. Can this antibody immobilization strategy also improve the sensitivity of competitive immunoassays for the detection of small molecules? To answer this question, the conjugate MS-SPG-Ab (oriented immobilization of aflatoxin B1 antibody to time-resolved fluorescent microspheres via streptococcal protein G) and the conjugate MS-Ab (nonoriented immobilization of aflatoxin B1 antibody to time-resolved fluorescent microspheres) were prepared, and a lateral flow immunoassay (LFIA) for the detection of aflatoxin B1 (AFB1) was established. The detection performance of the two methods was compared. The results showed that under the condition that the number of "effective" antibodies immobilized on TRF-MS was similar, compared with the nonoriented immobilization strategy (IC50 = 0.21 ng mL-1), the LFIA method established by the oriented immobilization strategy reduced the sensitivity of AFB1 detection (IC50 = 0.37 ng mL-1). However, this method can obtain higher detection precision for AFB1, the CV values were all below 8%. And it has stronger tolerance to the matrix of maize and peanut samples. The bias of LFIAs based on oriented immobilization technology (-14.93%-7.92%) was lower than nonoriented immobilization technology (28.16%-34.19%) for AFB1 detection in the two sample extracts. This study suggests that the LFIA method based on the oriented immobilization of antibodies can improve the accuracy of the detection results when performing rapid screening of small molecules.

Smartphone-based paper microfluidic competitive immunoassay for the detection of α-amanitin from mushrooms.

α-Amanitin is often considered the most poisonous mushroom toxin produced by various mushroom species, which are hard to identify from edible, non-toxic mushrooms. Conventional detection methods require expensive and bulky equipment or fail to meet high analytical sensitivity. We developed a smartphone-based fluorescence microscope platform to detect α-amanitin from dry mushroom tissues. Antibody-nanoparticle conjugates were captured by immobilized antigen-hapten conjugates while competing with the free analytes in the sample. Captured fluorescent nanoparticles were excited at 460 nm and imaged at 500 nm. The pixel numbers of such nanoparticles in the test zone were counted, showing a decreasing trend with increasing analyte concentration. The detection method exhibited a low detection limit (1 pg/mL), high specificity, and selectivity, allowing us to utilize a simple rinsing for toxin extraction and avoiding the need for high-speed centrifugation. In addition, this assay's short response time and portable features enable field detection of α-amanitin from amanitin-producing mushrooms.