Lateral Flow Immunosensor Research Articles

Integrated Ultrasound-Enrichment and Machine Learning in Colorimetric Lateral Flow Assay for Accurate and Sensitive Clinical Alzheimer's Biomarker Diagnosis.

The colloidal gold nanoparticle (AuNP)-based colorimetric lateral flow assay (LFA) is one of the most promising analytical tools for point-of-care disease diagnosis. However, the low sensitivity and insufficient accuracy still limit its clinical application. In this work, a machine learning (ML)-optimized colorimetric LFA with ultrasound enrichment is developed to achieve the sensitive and accurate detection of tau proteins for early screening of Alzheimer's disease (AD). The LFA device is integrated with a portable ultrasonic actuator to rapidly enrich microparticles using ultrasound, which is essential for sample pre-enrichment to improve the sensitivity, followed by ML algorithms to classify and predict the enhanced colorimetric signals. The results of the undiluted serum sample testing show that the protocol enables efficient classification and accurate quantification of the AD biomarker tau protein concentration with an average classification accuracy of 98.11% and an average prediction accuracy of 99.99%, achieving a limit of detection (LOD) as sensitive as 10.30pgmL-1. Further point-of-care testing (POCT) of human plasma samples demonstrates the potential use of LFA in clinical trials. Such a reliable lateral flow immunosensor with high precision and superb sensing performance is expected to put LFA in perspective as an AD clinical diagnostic platform.

NIR-II fluorescence lateral flow immunosensor based on efficient energy transfer probe for point-of-care testing of tumor biomarkers

Fluorescence lateral flow immunoassay (LFA) has emerged as a powerful tool for rapid screening of various biomarkers owing to its simplicity, sensitivity and flexibility. It is noteworthy that fluorescent probe mainly determines the analytical performance of LFA. Due to the emission and excitation wavelengths are located in the visible region, most fluorophores are inevitably subject to light scattering and background autofluorescence. Herein, we reported a novel LFA sensor based on the second near-infrared (NIR-II) fluorescent probe with excellent anti-interference capability. The designed NIR-II probe was the Nd3+ and Yb3+ doped rare earth nanoparticles (RENPs) by employing Nd3+ as energy donor and Yb3+ as energy acceptor, which of the donor-acceptor energy transfer (ET) efficiency reached up to 80.7 %. Meanwhile, relying on the convenient and effective encapsulation strategy of poly(lactic-co-glycolic acid) (PLGA) microspheres to RENPs, the surface functionalized NIR-II probe (RE@PLGA) was obtained for subsequent bioconjugation. Benefiting from the optical advantages of NIR-II probe, this proposed NIR-II LFA displayed a good linear relationship ranging from 7 ng/mL to 200 ng/mL for the detection of α-fetoprotein (AFP), an important biomarker of hepatocellular carcinoma (HCC). The limit of detection (LOD) was determined as low as 3.0 ng/mL, which was of 8.3 times lower than clinical cutoff value. It is promising that LFA sensor based on this efficient RENPs probe provides new opportunities for high sensitive detection of various biomarkers in biological samples.

Colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor using phage-displayed shark nanobody for the detection of crustacean allergen tropomyosin

Tropomyosin (TM) is the primary allergenic protein responsible for crustacean food allergies, and thus sensitive and rapid methods are required for the screening of crustacean TM in food. In this study, using the phage-displayed shark nanobody (PSN) as a multifunctional biomaterial, we developed a colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor (CM/SERS-LFI) for competitive detection of crustacean TM. The SERS tag AuMBA@AgNPs with the Raman signal molecule 4-mercaptobenzoic acid (4-MBA) was prepared and immobilized on the PSN to construct the immunoprobe AuMBA@Ag-PSN. The probe can identify free TM that competes with TM on the T-line, and the optimized CM/SERS-LFI enables quantitative analysis of TM using the probe with a limit of detection (LOD) of 0.0026 μg/mL (SERS mode) and 0.0057 μg/mL (colorimetric mode), respectively. Additionally, it can implement a qualitative analysis by the naked eye with a visual LOD of 0.01 μg/mL. The CM/SERS-LFI exhibited excellent performance in the tests of selectivity, accuracy, precision, and stability. Moreover, the method’s effectiveness in the analysis of real samples was confirmed by a commercial ELISA kit. Therefore, the developed CM/SERS-LFI was demonstrated to be a powerful and reliable tool for the rapid and sensitive detection of crustacean TM in food.

A simplified lateral flow immunosensor for the assay of carcinoembryonic antigen in low-resource settings.

Carcinoembryonic antigen (CEA) is a glycoprotein widely used as a tumor marker. In this work, a colorimetric lateral flow immunosensor is developed for rapid and low-cost quantification of CEA in human blood serum. The immunosensor consists of a glass fiber sample/conjugation pad, a nitrocellulose detection pad and a cellulose absorption pad. The detection is based on a sandwich immunoreaction: the sample/conjugation pad is modified with gold nanoparticles (GNPs)-labeled anti-CEA conjugate probes which bind to the CEA target molecules in the sample and the complexes are captured at capture anti-CEA immobilized at the test line. The color intensity of the test line, measured from a scanned image of the strip, is related to the CEA concentration in the sample. The different assay parameters are studied in detail. The linearity holds from 1.25 to 640 ng mL-1 of CEA, the instrumental and visual limits of detection are 0.45 and 0.63 ng mL-1, respectively, and the total assay time is 15 min. The specificity of the immunoassay versus other cancer biomarkers is satisfactory. The recovery in samples of human serum spiked with CEA is in the range of 81-118% and the coefficient of variation of the method is ≤10%. Results obtained with the lateral flow immunosensor correlated well with a reference radioimmunoassay method (R2 = 0.99). This immunosensor can be readily applied to CEA monitoring at the point-of-care (POC) or in resource-limited settings thanks to its low-cost and simplicity.

Detection of carbofuran in fruits and vegetables by Raman spectroscopy combined with immunochromatography.

It has long been desired to develop rapid methods for the rapid identification and quantification of pesticides and their metabolites. Carbofuran, a representative pesticide of the carbamate group, is highly systemic and is used on vegetables, fruits and grains, which has led many countries to test for residues in food and the environment. In this study, gold and silver composite core-shell (Au@Ag) nanoparticles were used to label the carbofuran antibody and Raman molecule 5,5-dithiobis-2-nitrobenzoic acid (DTNB) to synthesize Raman immune probes. The signal value of DTNB was read using a Raman spectrometer, and the quantitative detection technology of carbofuran was established based on lateral flow immunochromatographic assay (ICA) combined with surface-enhanced Raman spectroscopy (SERS). SERS-ICA is a rapid, quantitative and ultrasensitive test for the determination of carbofuran in fruits and vegetables with a sensitivity of 0.1 pg mL-1. Consequently, the results demonstrate that the SERS-based lateral flow immunosensor developed in this study has the advantages of excellent assay sensitivity and remarkable multiplexing capability, and thus it will have great application potential in food safety monitoring.

Dosage-sensitive and simultaneous detection of multiple small-molecule pollutants in environmental water and agriproducts using portable SERS-based lateral flow immunosensor

The co-contamination of pesticide residues and mycotoxins in agricultural products is a global concern, with the potential for cumulative and synergistic damaging effects, imposing substantial health and economic burdens to the public. The dosage-sensitive and simultaneous detection of multiple pollutants, with a heightened sensitivity in real samples, poses a significant demand and challenge. Herein, we propose a portable detection method integrating surface-enhanced Raman scattering (SERS)-with lateral flow immunoassay (LFIA), offering high sensitivity and multiplex analysis capabilities. This approach enables the simultaneous detection of imidacloprid (IMI), pyraclostrobin (PYR) and aflatoxin B1 (AFB1) through a single test strip. Utilizing the immune-specific binding between antigen and antibodies, we immobilised antibody- conjugated SERS nanotags on three test lines of the strips to generate Raman signal amplification in the proposed biosensor. Accurate quantitative analysis was performed by measuring the SERS signal intensity on the test lines. The limits of detection were 8.6 pg/mL for IMI, 97.4 pg/mL for PYR and 8.9 pg/mL for AFB1, exhibiting sensitivities 12-fold, 102-fold and11-fold higher than the colorimetric signals, respectively. Importantly, the SERS-LFIA immunosensor demonstrated robust performance when applied to real samples, yielding recoveries ranging from 86.16 % to 115.0 %, with relative standard deviation values below 8.67 %. These results underscore the excellent stability, high selectivity and reliability the proposed SERS-LFIA immunosensor. Consequently, it holds promise for the detection of multiple pesticides and mycotoxins in both environmental and agricultural samples.

UiOL@AIEgens-assisted lateral flow immunosensor for the ultrasensitive dual-modal point-of-care detection of aflatoxin B1

Aflatoxin B1 (AFB1) contamination in food has attracted worldwide attention. The sensitive detection of AFB1 is vital for ensuring food quality and safety. This study developed an ultrasensitive signal-enhanced lateral flow immunosensor (LFIS) based on the functionalized zirconium metal-organic framework (MOF) of a UiO linker enriched with abundant aggregation-induced emission luminogen (UiOL@AIEgens) probes for the rapid dual-modal point-of-care (POC) determination of AFB1. Using UiO MOFs with numerous active sites as the carrier facilitated abundant AIEgens enrichment on the surface. After coupling with enough anti-AFB1 monoclonal antibodies (mAbs), the green-emissive UiOL@AIEgens-mAbs probes with high specificity and remarkably-enhanced fluorescence responses were obtained to competitively capture target AFB1 in the standard or sample solution and AFB1 antigen immobilized on the test (T) line of the POC LFIS. Under optimum conditions, the LFIS was capable of visual qualitative and smartphone-assisted dual-modal determination of target AFB1 within 7 min. Detection occurred in a range of 0.01–5 ng/mL at an ultra-low detection limit of 0.003 ng/mL, which was 300- and 600-fold lower than traditional immunoassays and the maximum limit set by the European Union, respectively. Moreover, the feasibility and robustness of the LFIS platform were assessed by detecting AFB1 in maize and lotus seed samples with average recoveries of 94.3–109.0%. The developed UiOL@AIEgens-based POC LFIS can be used for ultrasensitive, reliable, on-site detection in food. This study provides a new method for the real-time monitoring of AFB1 and other harmful contaminants in food and more complex matrices.

Electrochemical lateral flow immunosensor with enhanced reproducibility for milk allergen detection

Electrochemical lateral flow platforms are applied for many applications due to their advantages of higher sensitivity in electrochemical detection than in conventional test strips. However, these devices have low reproducibility since the interface between the test strips and the electrode is difficult to replicate. Herein, for the first time, dual-working electrodes on a lateral flow immunoassay (dWE-LFIA) were used as an internal current on a single device, in which the current of both working electrodes was calculated as a normalized current to increase the device-to-device reproducibility. In this work, the detection of β-lactoglobulin, an allergen found in milk, was used as a model for evaluating the efficacy of dWE-LFIA. A study of reproducibility using this device showed a surprising 15-fold %RSD improvement compared to a single electrode on LFIA. Moreover, the analytical performance of this sensor for detecting β-lactoglobulin exhibited a wide linearity range of 1–1000 ng/mL and a detection resolution of 0.31 ng/mL. Therefore, our design of dWE-LFIA exhibited outstanding performance of high reproducibility, had high sensitivity detection, and could be applied and further developed for commercial biosensors in the future.

Ultrasensitive and selective detection of sulfamethazine in milk via a Janus-labeled Au nanoparticle-based surface-enhanced Raman scattering-immunochromatographic assay

Sulfamethazine (SM2) is an antibacterial drug，which has been extensively used in human and veterinary medicine, long-term consumption of which may lead to the accumulation of sulfonamides in the body. Detection of sulfonamides often uses microbiological approaches, mass spectrometry and chromatography, which are expensive and time-consuming. Surface-enhanced Raman scattering-based immunochromatographic assay (SERS-ICA) has been recently applied in the detection. Herein, a Janus-labeled Au nanoparticle with subnanosized SiO2-monoclonal antibody and SERS reporter (DTNB) modified simultaneously (mAbAuNpDTNB) has been developed in a SERS-based lateral flow immunosensor, which can be used for rapid, quantitative and ultrasensitive detection of sulfamethazine residue in milk. The mAbAuNpDTNB exhibits a specific array on a paper stripe, which not only identifies sulfamethazine but also straightforwardly exposes the Raman reporter between the AuNps via self-assembly. The detection sensitivity of SERS-ICA for sulfamethazine reached 0.1 pg/mL, which was far below the previously published value by ELISA and the maximum residue limit set by the European Union. The entire SERS-ICA detection for sulfamethazine was completed within 15 min. Furthermore, high accuracy for this assay was exhibited in the spiking experiment with a recovery percentage of 88.1%–112.7%. The results demonstrated that this SERS-ICA can potentially be applied in point-of-care testing as an ultrasensitive and quantitative to semi-quantitative analytical method.

Ultrasensitive detection of SARS-CoV-2 antigen using surface-enhanced Raman spectroscopy-based lateral flow immunosensor.

The fast spread and transmission of the coronavirus 2019 (COVID-19) has become one of serious global public health problems. Herein, a surface enhanced Raman spectroscopy-based lateral flow immunoassay (LFA) was developed for the detection of SARS-CoV-2 antigen. Using uniquely designed core-shell nanoparticle with embedded Raman probe molecules as the indicator to reveal the concentration of target protein, excellent quantitative performance with a limit of detection (LOD) of 0.03 ng/mL and detection range of 10-1000 ng/mL can be achieved within 15 min. Besides, the detection of spiked virus protein in human saliva was also performed with a portable Raman spectrometer, proposing the feasibility of the method in practical applications. This easy-to-use, rapid and accurate method would provide a point-of-care testing way as the ideal alternative for current detection requirement of virus-related biomarkers.

Resistance-Based Lateral Flow Immunosensor with a NFC-Enabled Smartphone for Rapid Diagnosis of Leptospirosis in Clinical Samples.

Leptospirosis is one of the most life-threatening tropical diseases caused by pathogenic Leptospira. To date, a diagnostic device that offers rapid and sensitive detection of leptospires has been still in demand for proper treatment to reduce the mortality rate. Herein, we create a resistance-based lateral flow immunosensor diagnosis device (R-LFI) that integrates near-field communication (NFC) with a portable smartphone for leptospiral detection in clinical samples. A specific monoclonal antibody against the pathogen was coated on a nitrocellulose membrane (NCM) where the test line was collocated. Two electrodes with a sandwich-like configuration were installed employing a conductive double-sided adhesive tape and connected with a NFC smartphone-based detection system. A half-sandwich immunocomplex formation induced high proton conduction, resulting in a considerable decrement in resistive response. The performance of the R-LFI sensor was evaluated using recombinant LipL32 (rLipL32), Leptospira interrogans, and clinical samples. The R-LFI device exhibited linear responses toward rLipL32 protein in phosphate buffer and L. interrogans-spiked healthy human serum samples within the concentration ranging from 1 to 1000 ng mL-1 (limit of detection (LOD): 0.29 ng mL-1) and from 104 to 106 cell mL-1 (LOD: 4.89 × 103 cell mL-1), respectively. Our R-LFI sensor successfully detected L. interrogans-positive clinical samples as confirmed by polymerase chain reaction (PCR). This platform offers high specificity, selectivity, simplicity, miniscule sample volume, and no labeling element requirement. These desirable features make it particularly suitable for countries where medical facilities and resources are limited.

Rapid and sensitive detection of zearalenone in corn using SERS-based lateral flow immunosensor

Zearalenone (ZEN) is a universal mycotoxin contaminant in corn and its products. A surface-enhanced Raman scattering (SERS) based test strip was proposed for the detection of ZEN, which had the advantages of simplicity, rapidity, and high sensitivity. Core-shell Au@AgNPs with embedded reporter molecules (4-MBA) were synthesized as SERS nanoprobe, which exhibited excellent SERS signals and high stability. The detection range of ZEN for corn samples was 10–1000 μg/kg with the limit of detection (LOD) of 3.6 μg/kg, which is far below the recommended tolerable level (60 μg/kg). More importantly, the SERS method was verified by HPLC in the application on corn samples contaminated with ZEN, and the coincidence rates were in the range of 86.06%–111.23%, suggesting a high accuracy of the SERS assay. Therefore, the SERS-based test strip with an analysis time of less than 15 min is a promising tool for accurate and rapid detection of ZEN-field contamination.

Based lateral flow immunosensor for ultrasensitive and selective surface-enhanced Raman spectroscopy stroke biomarkers detection

Despite optimistic trends in mortality and incidence, stroke is still a major cause of disability and death in populations. The application of blood-based biomarkers is known as a promising field for predicting the disease severity and prognosis to further guide therapy strategy. However, proposing a method that can accurately and sensitively identify patients by biomarkers detection remains a notable challenge due to the complexity of stroke pathogenesis and low abundance of target molecules. In this experiment, we designed a target antibody modified surface-enhanced Raman scattering (SERS) active nanospheres (GNPs) combined with probe-immobilized lateral flow strip as nanostructure to assess multiple-biomarker in blood samples of stroke patients simultaneously. Then the sensitive and quantitative measures toward stroke-related matrix metalloproteinase-9 (MMP-9), S100 calcium-binding protein B protein (S100-β), and neuro-specific enolase (NSE) were implemented in a wide range from 0.0001 ng/mL to 1000 ng/mL and a limit of detection of 0.01 pg/mL. Furthermore, the accuracy of SERS imaging was detected by enzyme linked immunosorbent assay (ELISA); the outcome obtained through SERS imaging was in accord with those obtained by ELISA. Therefore, this method can provide a reliable strategy for stroke diagnosis and prognosis evaluation with showing great prospect for clinical applications through further quantitative analysis of molecules in blood samples.

Electrochemiluminescence-Incorporated Lateral Flow Immunosensors Using Ru(bpy)32+-Labeled Gold Nanoparticles for the Full-Range Detection of Physiological C-Reactive Protein Levels.

C-reactive protein (CRP) is used as a general biomarker for inflammation and infection. During stroke and myocardial infarction, CRP increases and is present in a broad concentration range of 1-500 μg/mL. Therefore, full-range CRP detection is crucial to identify patients who need close follow-up or intensive treatment after a heart attack. Here, we report the first attempt to develop an electrochemiluminescent lateral flow immunosensor (ECL-LFI) that allows full-range CRP detection. Ru(bpy)32+-labeled gold nanoparticles (AuNPs) are used as a CRP-targeting probe and a signal generator; they form sandwich immunocomplexes at the test line of the strip and generate strong ECL emission via a Ru(bpy)32+/tripropylamine system. The ECL-LFI shows high sensitivity in detecting CRP in spiked serum, with a limit of detection of 4.6 pg/mL within 15 min, and a broad detection range of 0.01-1000 ng/mL, which is 2 orders of magnitude broader than that of conventional colorimetric LFI. The clinical usability of the ECL-LFI was evaluated using 30 clinical serum samples (200 ng/mL to 5 mg/mL), which showed a good linear correlation (R2 = 0.9896), with a clinical chemistry analyzer. The results suggest that the ECL-LFI holds great potential for CRP detection in point-of-care diagnostics.

Switching from Multiplex to Multimodal Colorimetric Lateral Flow Immunosensor.

Multiplex lateral flow immunoassay (LFIA) is largely used for point-of-care testing to detect different pathogens or biomarkers in a single device. The increasing demand for multitargeting diagnostics requires multi-informative single tests. In this study, we demonstrated three strategies to upgrade standard multiplex LFIA to multimodal capacity. As a proof-of-concept, we applied the strategies to the differential diagnosis of Human Immunodeficiency Virus (HIV) infection, a widespread pathogen, for which conventional multiplex LFIA testing is well-established. In the new two-parameter LFIA (x2LFIA), we exploited color encoding, in which the binding of multiple targets occurs in one reactive band and the color of the probe reveals which one is present in the sample. By combining the sequential alignment of several reactive zones along the membrane of the LFIA strip and gold nanoparticles and gold nanostars for the differential visualization, in this demonstration, the x2LFIA can furnish information on HIV serotype and stage of infection in a single device. Three immunosensors were designed. The use of bioreagents as the capturing ligand anchored onto the membrane or as the detection ligand labelled with gold nanomaterials affected the performance of the x2LFIA. Higher detectability was achieved by the format involving the HIV-specific antigens as capturing agent and labelled secondary bioligands (anti-human immunoglobulins M and protein G) as the probes.

Duplex Surface Enhanced Raman Scattering-Based Lateral Flow Immunosensor for the Low-Level Detection of Antibiotic Residues in Milk.

A duplex surface enhanced Raman scattering (SERS)-based lateral flow immunosensor was established for the simultaneous detection of two common antibiotic residues including tetracycline and penicillin in milk. The newly synthesized Au@Ag nanoparticles were labeled with different Raman molecules including 5,5-dithiobis-2-nitrobenzoic acid (DTNB) or 4-mercaptobenzoic acid (MBA), followed by the conjugation of anti-tetracycline monoclonal antibody or anti-penicillin receptor, forming two kinds of SERS nanoprobes. The two nanoprobes can recognize tetracycline-BSA and ampicillin-BSA, respectively, which facilitates the simultaneous detection of the two types of antibiotics on a single test line. After optimization, detection limits of tetracycline and penicillin as low as 0.015 ng/mL and 0.010 ng/mL, respectively, were achieved. These values were far below those of most of other documented bio-analytical approaches. Moreover, the spiking test demonstrates an excellent assay accuracy with recoveries of 88.8% to 111.3%, and satisfactory assay precision with relative standard deviation below 16%. Consequently, the results demonstrate that the SERS-based lateral flow immunosensor developed in this study has the advantages of excellent assay sensitivity and remarkable multiplexing capability, thus it will have great application potential in food safety monitoring.

Lateral Flow Immunosensor for Ferritin Based on Dual Signal-Amplified Strategy by Rhodium Nanoparticles.

Lateral flow immunoassay (LFIA) is one of the most widely used tools for analysis and field measurement and has the advantages of high efficiency, simple operation, portability, and low cost. Therefore, in this study, we designed a proof-of-concept of LFIA based on rhodium nanoparticles and investigated its improvement by further introducing the tetramethyl benzidine and H2O2 mixture as the substrate to trigger the color reaction. The proposed methods were qualitative research by the naked eye and quantitative measurement by a smartphone and software. Under the optimal condition, the detection of ferritin was successfully established with the limit of detection of 0.3 ng/mL. The lowest visually detectable amount was 0.05 ng/mL. To verify the performance of the RhNPs-LFIA, three spiked serum samples were tested, and the recovery rate increased from 88.9 to 129.9%, revealing that the proposed methods were applicable and practically reliable for testing serum samples. The developed RhNP-based LFIA is highly sensitive and convenient, which provides a promising technology for accurate, rapid, high sensitivity, and high screening detection of ferritin in clinical diagnosis.

Ru(bpy)32+ -Loaded Mesoporous Silica Nanoparticles as Electrochemiluminescent Probes of a Lateral Flow Immunosensor for Highly Sensitive and Quantitative Detection of Troponin I.

The lateral flow immunosensor (LFI) is a widely used diagnostic tool for biomarker detection; however, its sensitivity is often insufficient for analyzing targets at low concentrations. Here, an electrochemiluminescent LFI (ECL-LFI) is developed for highly sensitive detection of troponin I (TnI) using Ru(bpy)32+ -loaded mesoporous silica nanoparticles (RMSNs). A large amount of Ru(bpy)32+ is successfully loaded into the mesoporous silica nanoparticles with excellent loading capacity and shows strong ECL signals in reaction to tripropylamine. Antibody-immobilized RMSNs are applied to detect TnI by fluorescence and ECL analysis after a sandwich immunoassay on the ECL-LFI strip. The ECL-LFI enables the highly sensitive detection of TnI-spiked human serum within 20min at femtomolar levels (≈0.81 pg mL-1 ) and with a wide dynamic range (0.001-100ng mL-1 ), significantly outperforming conventional fluorescence detection (>3 orders of magnitude). Furthermore, TnI concentrations in 35 clinical serum samples across a low range (0.01-48.31ng mL-1 ) are successfully quantified with an excellent linear correlation (R2 = 0.9915) using a clinical immunoassay analyzer. These results demonstrate the efficacy of this system as a high-performance sensing strategy capable of capitalizing on future point-of-care testing markets for biomolecule detection.

Dual near-infrared fluorescence-based lateral flow immunosensor for the detection of zearalenone and deoxynivalenol in maize

A novel dual near-infrared fluorescence-based lateral flow immunosensor was developed to determine zearalenone and deoxynivalenol in maize. Two near-infrared dyes with distinct fluorescence characteristics were utilized to separately label the anti-zearalenone and anti-deoxynivalenol antibodies as detection reagents. The capture antigens zearalenone-BSA and deoxynivalenol-BSA were mixed and immobilized on the same test line of nitrocellulose membrane. This assay format facilitates simultaneous detection of the two mycotoxins on a single test line. After optimizing experimental parameters, the limits of detection for zearalenone and deoxynivalenol were as low as 0.55 μg/kg and 3.8 μg/kg in maize, respectively. The spiking experiment yielded recovery ratios ranging from 81.7% to 107.3% with coefficients of variation less than 14% demonstrating high assay accuracy and precision. Moreover, the actual sample analysis produced consistent results between this method and instrumental method. Therefore, the developed immunosensor can serve as an accurate and efficient approach for monitoring mycotoxins in agricultural products.

Surface enhanced Raman scattering-based lateral flow immunosensor for sensitive detection of aflatoxin M1 in urine

Aflatoxin M1 (AFM1) is generally used as a biomarker in urine for the assessment of aflatoxin exposure in humans and animals. However, there is no approach for the rapid and on-site monitoring of AFM1 level in urine. Here, we report a surface enhanced Raman scattering (SERS)-based lateral flow immunosensor built for such a purpose. Raman molecule 5,5-dithiobis-2-nitrobenzoic acid and anti-AFM1 monoclonal antibody were conjugated with Au (core)@Ag (shell) nanoparticle to serve as SERS nanoprobe. AFM1-bovine serum albumin was conjugated with gold nanoparticle and then applied onto nitrocellulose membrane as a visible “GOLD” test line. Quantitation of AFM1 was performed by the readout of Raman signal from the SERS nanoprobes captured on the test line. After optimizing experimental parameters, the detection limit of this immunosensor can achieve as low as 1.7 pg/mL of AFM1 in urine, which is far below the recommended tolerable level (30 pg/mL) of AFM1 in urine. The spiking experiment yielded 93.8%–111.3% recovery with coefficients of variation below 17% demonstrating high assay accuracy and precision. Moreover, this immunosensing assay is fast with an assay time below 20 min. Therefore, the developed immunosensor is a promising tool for the rapid assessment of aflatoxin exposure in the field.