Test Line Research Articles

Graphene oxide coatings enhance fluorescence signals in a lateral flow immunoassay for the detection of UCH-L1, a marker for trauma brain injury

We present a lateral flow immunoassay (LFA) for the quantitative, fluorescence-based detection of the trauma brain injury (TBI) biomarker ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) that features a layer of graphene oxide (GO) particles over the test zone of nitrocellulose (NC) strips. The introduction of 80 ng of GO at test lines increased fluorescence signals 2–3-fold. This was attributed to an increase in the immobilization of capture antibodies in the top layer of NC, which increased the density of recognition complexes at the surface and hence the signals reaching the reader used. A linear concentration–response relationship was observed in the 0–200 pg/mL UCH-L1 range with an inter assay CoV of 2.65 %. The LOD and LOQ values of 11.1 pg/mL and 33.5 pg/mL were compatible with threshold levels of UCH-L1. Finally, mock plasma samples with UCH-L1 levels characteristic of TBI patients with negative (60 pg/mL) and positive (130 pg/mL) CT scans were successfully analyzed. Storage stability testing further showed that GO did not affect the biological activity of the capture antibody. In summary, we demonstrate that the use of GO particles in the top layers of the test zone of NC strips improves signal intensity, which could potentially enhance the accuracy and efficiency of LFA-based diagnosis.

Performance of Rice Hope Lines in the Lebak Shallow Swamp Sorong Regency, Southwest Papua

Rice is one of the important food commodities in Indonesia. The extensification of swamp land is expected to play a role in supporting the increase in national rice production. The objective of this study was to evaluate the performance of promising strains of swamp rice produced by BB-Biogen breeding tolerant to Fe poisoning in the shallow swamp area of Sorong-Southwest Papua. The experiment used a randomized block design (RBD) with three replications and 17 genotypes as treatments. The observed characters include the growth and yield component characters. Data analysis included analysis of variance to find out the differences between each test lines and continued with the LSI test to obtain the expected lines that performed well and were superior in the Sorong lebak swamps. The results of the analysis of variance showed that there were significant differences in all the characters tested. The results showed that TT, JAT and JAP showed the best results including the G21 and G11 lines, the G7 line had the highest percentage of filled grain, the G6 line had the heaviest 1000 grain weight. While the G1 and G7 lines had the highest yields per plot equivalent to 6.15 and 5.92 t/ha,

A dual-RPA based lateral flow strip for sensitive, on-site detection of CP4-EPSPS and Cry1Ab/Ac genes in genetically modified crops

Traditional transgenic detection methods require high test conditions and struggle to be both sensitive and efficient. In this study, a one-tube dual recombinase polymerase amplification (RPA) reaction system for CP4-EPSPS and Cry1Ab/Ac was proposed and combined with a lateral flow immunochromatographic assay, named "Dual-RPA-LFD", to visualize the dual detection of genetically modified (GM) crops. In which, the herbicide tolerance gene CP4-EPSPS and the insect resistance gene Cry1Ab/Ac were selected as targets taking into account the current status of the most widespread application of insect resistance and herbicide tolerance traits and their stacked traits. Gradient diluted plasmids, transgenic standards, and actual samples were used as templates to conduct sensitivity, specificity, and practicality assays, respectively. The constructed method achieved the visual detection of plasmid at levels as low as 100 copies, demonstrating its high sensitivity. In addition, good applicability to transgenic samples was observed, with no cross-interference between two test lines and no influence from other genes. In conclusion, this strategy achieved the expected purpose of simultaneous detection of the two popular targets in GM crops within 20 min at 37 ℃ in a rapid, equipment-free field manner, providing a new alternative for rapid screening for transgenic assays in the field.

Triplex-colored nucleic acid lateral flow strip and multiplex polymerase chain reaction coupled method for quantitative identification of beef, pork and chicken

Meat adulteration is one of the worldwide challenges for food quality control. Now, we developed a multiplex PCR and nucleic acid lateral flow strip (mPCR-NALFS) integrated method that employs triplex-color latex microspheres (LMs) as visual labels for simultaneous identification of beef, pork and chicken DNA. First, three primer pairs for beef, pork and chicken DNA are tailed with specific oligonucleotide sequences (anti-recognition/anti-detection/anti-control probes) as swing tags. Then, double-stranded amplicons obtained after mPCR amplification were flanked by tags. Finally, these amplicons can accurately hybridize with the pre-immobilized recognition probes on the conjugate pad, detection probes on the test lines and control probes on the control line, respectively. The LMs and recognition probe modified LMs were characterized by scanning electron microscopy, ultraviolet-visible spectroscopy and Fourier Transform Infrared spectroscopy. This strip can quantitatively detect beef, pork and chicken DNA within 10 min without compromising on accuracy compared to the electrophoresis. Specificity analysis revealed no cross-reactivity with meat derived from beef, pork, chicken, duck, mutton, rabbit, goose, ostrich, camel and horse meat. As low as 0.1 % (wt%) of beef, pork and chicken in the simulated adulterated meats were co-detected. This assay was successfully employed for adulteration identification in 28 commercial beef and lamb products.

Engineering DNA tetrahedron as a sensing surface of lateral flow test strips and ratiometric visual detection of exosomal microRNA-150–5p

We here report that DNA tetrahedral probe-based sensing surfaces are capable of efficiently capturing gold nanoparticles (AuNPs) on lateral flow test strips, and we have developed a ratiometric biosensor for exosomal microRNAs by integrating a tetrahedral probe and a catalytic hairpin assembly (CHA). DNA tetrahedrons were devised with barcodes and labeled with biotin as capture probes on the test and control lines of the strip. The CHA system consists of two hairpin substrates that can form stable duplexes via a programmable assembly reaction triggered by exosomal miRNAs. The duplexes hybridized with barcoded tetrahedra via complementary sequences and bound to streptavidin-modified AuNPs (SA-AuNPs) via biotin. Thus, a red line was observed on the test line owing to the immobilized SA-AuNPs. The remaining SA-AuNPs were captured by using a biotinylated tetrahedron on the control line. Because the barcoded and biotinylated tetrahedra consume SA-AuNPs simultaneously, a stronger signal in the test line results in a weaker signal in the control line, thus forming a ratiometric result. Tetrahedral probes avoid movement along with sample flow on sensing zones like single-stranded probes. The strip can have a detection limit of 58.90 fM with high selectivity due to the use of ratiometric color changing response, CHA signal amplification and tetrahedral capture probes. This system is able to rapidly detect exosomal microRNA-150–5p and may have a potential utility in point-of-care diagnosis of incipient diabetic nephropathy.

Heterosis for yield and its component traits in Indian mustard (Brassica juncea L., Czern & Coss)

Four lines were crossed with eleven testers in line x tester mating design for estimation of combining ability and heterosis for yield and its component characters in mustard (Brassica juncea). Lines PM-25 and PM-27 were foundto be superior general combiners for length of siliqua and seed yield. PM-25 was also found to be a very good general combiner for number of seeds/siliqua and 1000 seed weight. The cross Varuna x Pusa Tarak showed high sca for seed yield followed by Varuna × Pusa LES-39 while for 1000 seed weight trait, Varuna x PM-28 and Varuna x PM-25 were found to be better. Varuna x Pusa Tarak also showed high sca for total number of siliqua/plant.Among the 44 hybrids, Varuna × Pusa LES-39 recorded highest heterosis for seed yield /plant over mid parent and better parent followed by Varuna × PM-27, Varuna × PM-26, Kranti× PM-27 and Varuna × Pusa Tarak. The resultsof this study suggest that the cross Varuna x Pusa Les 39 is best for high seed yield due to high siliqua length, number of siliqua at main axis, total number of siliqua/plant and number of seeds/siliqua.

Joint analysis of phenotype-effect-generation identifies loci associated with grain quality traits in rice hybrids

Genetic improvement of grain quality is more challenging in hybrid rice than in inbred rice due to additional nonadditive effects such as dominance. Here, we describe a pipeline developed for joint analysis of phenotypes, effects, and generations (JPEG). As a demonstration, we analyze 12 grain quality traits of 113 inbred lines (male parents), five tester lines (female parents), and 565 (113×5) of their hybrids. We sequence the parents for single nucleotide polymorphisms calling and infer the genotypes of the hybrids. Genome-wide association studies with JPEG identify 128 loci associated with at least one of the 12 traits, including 44, 97, and 13 loci with additive effects, dominant effects, and both additive and dominant effects, respectively. These loci together explain more than 30% of the genetic variation in hybrid performance for each of the traits. The JEPG statistical pipeline can help to identify superior crosses for breeding rice hybrids with improved grain quality.

New corn resistant lines to stalk rot disease (Dickeya zeae) in Indonesia

Abstract. Suriani, Patandjengi B, Muis A, Junaid M, Mirsam H, Azrai M, Efendi R, Sebayang A. 2023. New corn resistant lines to stalk rot disease (Dickeya zeae) in Indonesia. Biodiveritas 24: 3190-3200. Stalk rot disease caused by Dickeya zeae is one of the important diseases of corn in Indonesia. Host resistance cultivars are an effective and sustainable control measure of the disease. Therefore, the present study aimed to evaluate the resistance of 15 S1 hybrid maize lines to stalk rot disease. The research was conducted in two seasons (DS and WS) using a randomized block design with 3 replications. The D. zeae suspension with 108cfu/mL concentration was inoculated into the plant test 45 Days After Planting (DAP). Disease incidence and severity were observed during the two seasons. The results showed that all tested lines were infected with stalk rot disease but had various resistance reactions. Disease incidence and severity in the dry season were higher than in the rainy season. In the rainy season all test lines followed the 3 models of disease development, but in the dry season, all lines followed the monomolecular model. Further analysis showed that 3 lines of hybrid maize had the lowest AUDPC value with a protection index of more than 50% in two growing seasons. Stalk lignin content had negative correlation with a disease incidence of -0.60877, so it can be used as a parameter of plant resistance to disease. Tested lines that show resistance to the disease could potentially be useful as new varieties of maize.

Efficiency of ferromagnetic shielding of superconducting coils of high-speed maglev crew

Background: Magnetic levitation transport with combined traction, suspension and guidance systems based on superconducting coils (SCC) allows reaching speeds of up to 500600 km/h with a very significant (up to 150200 mm) air gap, which is an important factor in ensuring the safety of high-speed transportation. However, SCC are a source of strong external magnetic fields, which, in conditions of limited crew dimensions, can have a harmful effect on both passengers and on-board auxiliary equipment.
 Aim: to analyze the external magnetic fields of the SCC systems for traction, suspension and direction of maglev vehicles and the effectiveness of ferromagnetic screens as a means of ensuring the electromagnetic safety of passengers and the electromagnetic compatibility of the SCC with other onboard equipment.
 Materials and methods: to achieve this goal, methods of analytical and numerical modeling of the magnetic fields of the SCC using modern software packages were used. As a prototype of the combined magnetic system of traction, suspension and direction, the system of the MLX-L0 maglev vehicle, which is undergoing pilot commercial operation at the Yamanashi test line (Japan), was adopted.
 Results: it is shown that the value of the external magnetic fields of the SCC of the traction and guiding systems in the passenger cabin of the prototype crew exceeds the maximum permissible levels established by domestic and foreign regulatory documents, both without shielding and with passive shielding with flat steel sheets.
 Conclusion: passive shielding of the SCC with steel sheets, including multilayer ones, does not provide the required reduction in the level of external magnetic fields for a given vehicle dimensions.

Mining the Australian Grains Gene Bank for Rust Resistance in Barley.

Global barley production is threatened by plant pathogens, especially the rusts. In this study we used a targeted genotype-by-sequencing (GBS) assisted GWAS approach to identify rust resistance alleles in a collection of 287 genetically distinct diverse barley landraces and historical cultivars available in the Australian Grains Genebank (AGG) and originally sourced from Eastern Europe. The accessions were challenged with seven US-derived cereal rust pathogen races including Puccinia hordei (Ph-leaf rust) race 17VA12C, P. coronata var. hordei (Pch-crown rust) race 91NE9305 and five pathogenically diverse races of P. striiformis f. sp. hordei (Psh-stripe rust) (PSH-33, PSH-48, PSH-54, PSH-72 and PSH-100) and phenotyped quantitatively at the seedling stage. Novel resistance factors were identified on chromosomes 1H, 2H, 4H and 5H in response to Pch, whereas a race-specific QTL on 7HS was identified that was effective only to Psh isolates PSH-72 and PSH-100. A major effect QTL on chromosome 5HL conferred resistance to all Psh races including PSH-72, which is virulent on all 12 stripe rust differential tester lines. The same major effect QTL was also identified in response to leaf rust (17VA12C) suggesting this locus contains several pathogen specific rust resistance genes or the same gene is responsible for both leaf rust and stripe rust resistance. Twelve accessions were highly resistant to both leaf and stripe rust diseases and also carried the 5HL QTL. We subsequently surveyed the physical region at the 5HL locus for across the barley pan genome variation in the presence of known resistance gene candidates and identified a rich source of high confidence protein kinase and antifungal genes in the QTL region.

Study on the Integration Strategy of Online EOL Testing of Pure Electric Vehicle Power Battery.

This paper analyzes the electrical test items of the EOL testing line in automotive manufacturers. On this basis, this paper proposes and designs an integrated and automated testing strategy to deal with the problems of slow testing speed, high dependence on manual labor and low efficiency. This article mainly analyzes the various tests of the two main tests in battery EOL testing: Battery Management System (BMS) testing and electrical testing. We propose an innovative integrated solution based on various testing items, including the reception, transmission, and self-analysis of different UDS protocol messages, a unique automated electrical performance measurement scheme, and a requirement and logic design of an integrated software end based on Python. The experimental results of actual testing show that the implementation of the integrated strategy greatly reduces the complexity of the testing steps, improves the testing efficiency, and reduces errors caused by human operation.

Glowstick-inspired smartphone-readable reporters for sensitive, multiplexed lateral flow immunoassays

The COVID-19 pandemic has increased demand for point-of-care (POC) screening tests such as lateral flow assays (LFAs) and highlighted the need for sensitive and cost-effective POC diagnostic platforms. Here, we demonstrate an LFA platform using standard fluorescent nanoparticle reporters in which optical excitation is replaced by chemical excitation using the peroxyoxalate-based chemistry of inexpensive, shelf-stable glowsticks. The one-step chemi-excitation of fluorescent particles produces visible light readable by an unmodified smartphone, enhancing sensitivity while preserving simplicity and cost-effectiveness. Our Glow LFA detected the common model analyte human chorionic gonadotropin with a limit of detection (LoD) of 39 pg/mL—over ten times more sensitive than standard gold nanoparticles using the same antibodies. We also demonstrate its application to the detection of SARS-CoV-2 nucleoprotein at 100 pg/mL in nasal swab extract. Multiple fluorescent dyes can be chemi-excited by a single reagent, allowing for color multiplexing on a single LFA strip with a smartphone camera. The detection of three analytes on a single LFA test line was demonstrated using red, green, and blue fluorescent reporter particles, making glow LFA a promising platform for multiplexed detection.

Partially miscible droplet microfluidics to enhance interfacial adsorption of hydrophilic nanoparticles for colloidosome synthesis

Colloidosomes are spherical shells composed of closed-packed nanoparticles, which can be prepared through the self-assembly of nanoparticles at the interphase of two immiscible liquids. However, the method leads to colloidosomes of various sizes and limited synthesis due to charge repulsion and nanoparticle wettability. Herein, a microfluidic droplet device and partially miscible liquids are integrated for colloidosome synthesis. Dissolving liquids within droplets enables hydrophilic nanoparticles to close packing at the liquid interface to form colloidosomes. Moreover, the microfluidic droplet system achieved colloidosomes within a narrow and specific range of sizes. The close-packing of nanoparticles is proved by modifying colloidosomes into probes in signal-enhanced lateral flow assays (LFAs). Due to the plasmon resonance effect, the testing lines are black, demonstrating the close packing of nanoparticles and their signal enhancement property compared to traditional LFAs. Accordingly, this approach is a potential bacterial detection platform with a detection time of 10 min, higher sensitivity, and specificity. Moreover, since colloidosomes are hollow spheres, they are capable of drug cargo with an encapsulation rate of 99.76 %. With this proposed partially miscible fluid-based droplet microfluidic system, colloidosomes can be fabricated with versatile properties, extending their application to advanced material fabrication, drug encapsulation, and biosensing.

Luminescent diagnostics and quantitative assessment of malaria based on a lateral flow immunoassay with cdte quantum dots

A lateral flow immunoassay (LFI) is a simple, low-cost and rapid diagnostic tool to identify various diseases by detecting analytes such as antibodies, parasites, or other relevant biomarkers. However, most LFIs can only confirm the presence or absence of a target analyte being applied as qualitative diagnostic tools. In addtion, the conventional LFIs, which require visual inspection of the test line, may demonstrate insufficient sensitivity for the mild form or early detection of infections and for this reason, the result can be falsely negative. This study presents the results of employing thioglycolic acid capped CdTe quantum dots as a probe to enhance luminescence and subsequently detection sensitivity in LFIs for diagnosis of malaria. The chemical route synthesis of thioglycolic acid capped CdTe quantum dots was optimized systematically by probing optical properties of the quantum dots. These optimized quantum dots of thioglycolic acid capped CdTe have been conjugated with the anti-malaria antibodies against HRP2 protein (P. falciparum) and were then incorporated into LFIs. Further, an image processing code has been developed to carry out the quantification of malaria parasites in terms of the ratio on intensities of control and test lines on the LFIs. The results have been compared with those obtained using the standard, colloidal gold based LFIs. It has been demonstrated that sensitivity and lower detection limits of malaria under low parasite concentration increase significantly due to enhanced luminescence of control and test lines under UV light, owing to the presence of thioglycolic acid capped CdTe quantum dots. It has also been shown that the image processing based quantification of malaria will likely minimize the chances of false negative results under low parasite concentration and assist in early diagnosis of malaria.

Colloidal gold and fluorescent immunochromatographic test strips for canine parvovirus detection.

Canine parvovirus (CPV) is an acute and highly infectious virus causing disease in puppies and, thus, affecting the global dog industry. The current CPV detection methods are limited by their sensitivity and specificity. Hence, the current study sought to develop a rapid, sensitive, simple, and accurate immunochromatographic (ICS) test to detect and control the spread and prevalence of CPV infection. More specifically, 6A8, a monoclonal antibody (mAb) with high specificity and sensitivity, was obtained by preliminary screening. The 6A8 antibody was labelled with colloidal gold particles. Subsequently, 6A8 and goat anti-mouse antibodies were coated onto a nitrocellulose membrane (NC) as the test and control lines, respectively. Furthermore, 6A8 and rabbit IgG antibodies were labelled with fluorescent microspheres and evenly sprayed onto a glass fibre membrane. Both strips could be prepared in 15min with no noticeable cross-reactivity with other common canine intestinal pathogens. The strips were simultaneously used to detect CPV in 60 clinical samples using real-time quantitative PCR, hemagglutination, and hemagglutination inhibition assays. The colloidal gold (fluorescent) ICS test strip was stable for 6 (7) and 4 (5) months at 4 °C and room temperature (18-25 °C). Both test strips were easy to prepare and rapidly detected CPV with high sensitivity and specificity. Moreover, the results were easily interpretable. This study establishes a simple method for two CPV diseases, colloidal gold and fluorescent immunochromatographic (ICS) test strips. KEY POINTS: • CPV test strips do not exhibit cross-reactivity with other canine intestinal pathogens. • The strips are stable for months at 4 °C and at room temperature (18-25 °C). • These strips are a promising approach for the timely diagnosis and treatment of CPV.

Lateral Flow Assay for Hepatitis B Detection: A Review of Current and New Assays.

From acute to chronic hepatitis, cirrhosis, and hepatocellular cancer, hepatitis B infection causes a broad spectrum of liver diseases. Molecular and serological tests have been used to diagnose hepatitis B-related illnesses. Due to technology limitations, it is challenging to identify hepatitis B infection cases at an early stage, particularly in a low- and middle-income country with constrained resources. Generally, the gold-standard methods to detect hepatitis B virus (HBV) infection requires dedicated personnel, bulky, expensive equipment and reagents, and long processing times which delay the diagnosis of HBV. Thus, lateral flow assay (LFA), which is inexpensive, straightforward, portable, and operates reliably, has dominated point-of-care diagnostics. LFA consists of four parts: a sample pad where samples are dropped; a conjugate pad where labeled tags and biomarker components are combined; a nitrocellulose membrane with test and control lines for target DNA-probe DNA hybridization or antigen-antibody interaction; and a wicking pad where waste is stored. By modifying the pre-treatment during the sample preparation process or enhancing the signal of the biomarker probes on the membrane pad, the accuracy of the LFA for qualitative and quantitative analysis can be improved. In this review, we assembled the most recent developments in LFA technologies for the progress of hepatitis B infection detection. Prospects for ongoing development in this area are also covered.

Operation Control Method for High-Speed Maglev Based on Fractional-Order Sliding Mode Adaptive and Diagonal Recurrent Neural Network

The speed profile tracking calculation of high-speed maglev trains is mainly affected by running resistance. In order to reduce the adverse effects and improve tracking accuracy, this paper presents a maglev train operation control method based on a fractional-order sliding mode adaptive and diagonal recurrent neural network (FSMA-DRNN). First, the kinematic resistance equation is established due to the three types of resistance that occur during the actual operation of a train: air resistance, guide eddy current resistance, and suspension frame generator coil resistance. Then, the FSMA-DRNN control law and parameter update law are designed, and a FSMA-DRNN operation controller is composed of three parts: speed feed forward, fractional-order sliding mode adaptive equivalent control, and diagonal recurrent neural network resistance compensation. Furthermore, by using the designed operation controller, it is proven effective by the Lyapunov theory for the stability of the closed-loop control system. Apart from the proposed theoretical analysis, the proposed approaches are verified by experiments on the high-speed maglev hardware-in-the-loop simulation platform Rt-Lab, in line with the 29.86 km test line and a five-car train from the Shanghai maglev, showing the effectiveness and superiority for operation optimization.

Label-Free, Non-Optical Readout of Bead-Based Immunoassays with and without Electrokinetic Preconcentration.

In this article, we report a microfluidic bead-based lateral flow immunoassay (LFIA) with a novel sensing mechanism for label-free, non-optical detection of protein binding. This device comprises two packed beds of microbeads: first, bioconjugated microbeads that serve as a test line, and second, a three-dimensional (3D) electrode for sensing. As the protein target binds the bioconjugated microbeads, a shift in ionic conductivity across the bioconjugated beads is produced and can be directly measured at the surface of the 3D electrode by obtaining current-voltage curves before and after incubation of the analyte. We use a model antigen, rabbit IgG, for quantitative evaluation of this sensor, obtaining a limit of detection (LOD) of 50 nM for the LFIA. We demonstrate that this device can be used to measure binding kinetics, exhibiting a rapid (<3 min) increase in the signal after the introduction of the analyte and an exponential decay in the signal after replacing the sample with buffer only. To improve the LOD of our system, we implement an electrokinetic preconcentration technique, faradaic ion concentration polarization (fICP), to increase the local concentration of antigen available during binding as well as the time the antigen interacts with the test line. Our results indicate that this enrichment-enhanced assay (fICP-LFIA) has an LOD of 370 pM, an 135-fold improvement over the LFIA and a 7-fold improvement in sensitivity. We anticipate that this device can be readily adapted for point-of-care diagnostics and translated to any desired protein target by simply modifying the biorecognition agent on these off-the-shelf microbeads.

Field observations of wet snow accretion on overhead transmission lines at the Kushiro test line

Wet snow accretion on overhead lines often causes large-scale snow damage. The Kushiro test line, Japan, was constructed in 2013 for field observations of wet snow accretion in conductors and insulators as well as galloping resulting from snow accretion in overhead lines. Several cases of noticeable wet snow accretion have been observed at this site, especially in the winter of 2014. In the most notorious case, more than 2 kg/m of snow was found to be accreted on the ACSR240 single conductor, which has a diameter of 22.4 mm. In the case of single conductors without countermeasures, snow accretion developed to form a cylindrical sleeve with wire rotation. However, in some cases, the cylindrical sleeve of snow accretion also occurred in four-bundled conductors, where line spacers prevent the wire from rotating. This indicates that the accreted wet snow slides along the strands of the wire to form a cylindrical sleeve. Accordingly, the effectiveness of the snow resistance ring, which is the most commonly used anti-snow-damage device in Japan, in preventing snow accretion was confirmed. As the ring prevents the accreted snow from sliding, accretion areas tend to split, causing the snow to be shed from the conductor. More heat transfer from the air to snow and heat generated by the electric current facilitated the sliding of snow along the strand, making the snow resistance ring more effective.

Au@SiO2 SERS nanotags based lateral flow immunoassay for simultaneous detection of aflatoxin B1 and ochratoxin A.

Agricultural products are frequently contaminated by mycotoxins. Multiplex, ultrasensitive, and rapid determination of mycotoxins is still a challenging problem, which is of great significance to food safety and public health. Herein, a surface-enhanced Raman scattering (SERS) based lateral flow immunoassay (LFA) for the simultaneous on-site determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) on the same test line (T line) was developed, in this study. In practice, two kinds of Raman reporters 4-mercaptobenzoic acid (4-MBA), and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) encoded silica-encapsulated gold nanotags (Au4-MBA@SiO2 and AuDNTB@SiO2) were used as detection markers to identify the two different mycotoxins. Through systematic optimization of the experimental conditions, this biosensor has high sensitivity and multiplexing with the limits of detection (LODs) at 0.24pgmL-1 for AFB1 and 0.37pgmL-1 for OTA. These are far below the regulatory limits set by the European Commission, in which the minimum LODs for AFB1 and OTA are 2.0 and 3.0μgkg-1. In the spiked experiment, the food matrix are corn, rice, and wheat, and the mean recoveries of the two mycotoxins ranged from 91.0%±6.3%-104.8%±5.6% for AFB1 and 87.0%±4.2%-112.0%±3.3% for OTA. These results demonstrate that the developed immunoassay has good stability, selectivity, and reliability, which can be used for routine monitoring of mycotoxin contamination.