Cyano-Hydrol green derivatives: Expanding the 9-cyanopyronin-based resonance Raman vibrational palette.

9-cyanopyronin is a promising scaffold that exploits resonance Raman enhancement to enable sensitive, highly multiplexed biological imaging. Here, we developed cyano-Hydrol Green (CN-HG) derivatives as resonance Raman scaffolds to expand the color palette of 9-cyanopyronins. CN-HG derivatives exhibit sufficiently long wavelength absorption to produce strong resonance Raman enhancement for near-infrared (NIR) excitation, and their nitrile peaks are shifted to a lower frequency than those of 9-cyanopyronins. The fluorescence of CN-HG derivatives is strongly quenched due to the lack of the 10th atom, unlike pyronin derivatives, and this enabled us to detect spontaneous Raman spectra with high signal-to-noise ratios. CN-HG derivatives are powerful candidates for high performance vibrational imaging.

Establishment of a multi-line immunochromatography based on magnetic nanoparticles for simultaneous screening of multiple biomarkers.

Biomarkers screening is a benefit approach for early diagnosis of major diseases. In this study, magnetic nanoparticles (MNPs) have been utilized as labels to establish a multi-line immunochromatography (MNP-MLIC) for simultaneous detection of carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA 19-9), and alpha-fetoprotein (AFP) in a single serum sample. Under the optimal parameters, the three biomarkers can be rapidly and simultaneously qualitative screening within 15 min by naked eye. As for quantitative detection, the MNP-MLIC test strips were precisely positioned and captured by a smartphone, and signals on the test and control lines were extracted by ImageJ software. The signal ratio of test and control lines has been calculated and used to plot quantitative standard curves with the logarithmic concentration, of which the correlation coefficients are more than 0.99, and the limit of detection for CEA, CA 19-9, and AFP were 0.60 ng/mL, 1.21 U/mL, and 0.93 ng/mL, respectively. The recoveries of blank serum were 75.0 ~ 112.5% with the relative standard deviation ranging from 2.5 to 15.3%, and the specificity investigation demonstrated that the MNP-MLIC is highly specific to the three biomarkers. In conclusion, the developed MNP-MLIC offers a rapid, simple, accurate, and highly specific method for simultaneously detecting multiple biomarkers in serum samples, which provides an efficient and accurate approach for the early diagnosis of diseases.

A fluorescent sensor array based on antibiotic-stabilized metal nanoclusters for the multiplex detection of bacteria.

To address the need for facile, rapid detection of pathogens in water supplies, a fluorescent sensing array platform based on antibiotic-stabilized metal nanoclusters was developed for the multiplex detection of pathogens. Using five common antibiotics, eight different nanoclusters (NCs) were synthesized including ampicillin stabilized copper NCs, cefepime stabilized gold and copper NCs, kanamycin stabilized gold and copper NCs, lysozyme stabilized gold NCs, and vancomycin stabilized gold/silver and copper NCs. Based on the different interaction of each NC with the bacteria strains, unique patterns were generated. Various machine learning algorithms were employed for pattern discernment, among which the artificial neural networks proved to have the highest performance, with an accuracy of 100%. The developed prediction model performed well on an independent test dataset and on real samples gathered from drinking water, tap water and the Anzali Lagoon water, with prediction accuracy of 96.88% and 95.14%, respectively. This work demonstrates how generic antibiotics can be implemented for NC synthesis and used as recognition elements for pathogen detection. Furthermore, it displays how merging machine learning techniques can elevate sensitivity of analytical devices.

Progress and Challenge of Sensors for Dairy Food Safety Monitoring.

One of the most consumed foods is milk and milk products, and guaranteeing the suitability of these products is one of the major concerns in our society. This has led to the development of numerous sensors to enhance quality controls in the food chain. However, this is not a simple task, because it is necessary to establish the parameters to be analyzed and often, not only one compound is responsible for food contamination or degradation. To attempt to address this problem, a multiplex analysis together with a non-directed (e.g., general parameters such as pH) analysis are the most relevant alternatives to identifying the safety of dairy food. In recent years, the use of new technologies in the development of devices/platforms with optical or electrochemical signals has accelerated and intensified the pursuit of systems that provide a simple, rapid, cost-effective, and/or multiparametric response to the presence of contaminants, markers of various diseases, and/or indicators of safety levels. However, achieving the simultaneous determination of two or more analytes in situ, in a single measurement, and in real time, using only one working 'real sensor', remains one of the most daunting challenges, primarily due to the complexity of the sample matrix. To address these requirements, different approaches have been explored. The state of the art on food safety sensors will be summarized in this review including optical, electrochemical, and other sensor-based detection methods such as magnetoelastic or mass-based sensors.

Mesophilic Argonaute-Based Single Polystyrene Sphere Aptamer Fluorescence Platform for the Multiplexed and Ultrasensitive Detection of Non-Nucleic Acid Targets.

The rapid, simultaneous, and accurate identification of multiple non-nucleic acid targets in clinical or food samples at room temperature is essential for public health. Argonautes (Agos) are guided, programmable, target-activated, next-generation nucleic acid endonucleases that could realize one-pot and multiplexed detection using a single enzyme, which cannot be achieved with CRISPR/Cas. However, currently reported thermophilic Ago-based multi-detection sensors are mainly employed in the detection of nucleic acids. Herein, this work proposes a Mesophilic Argonaute Report-based single millimeter Polystyrene Sphere (MARPS) multiplex detection platform for the simultaneous analysis of non-nucleic acid targets. The aptamer is utilized as the recognition element, and a single millimeter-sized polystyrene sphere (PSmm ) with a large concentration of guide DNA on the surface served as the microreactor. These are combined with precise Clostridium butyricum Ago (CbAgo) cleavage and exonuclease I (Exo I) signal amplification to achieve the efficient and sensitive recognition of non-nucleic acid targets, such as mycotoxins (<60 pg mL-1 ) and pathogenic bacteria (<102 cfu mL-1 ). The novel MARPS platform is the first to use mesophilic Agos for the multiplex detection of non-nucleic acid targets, overcoming the limitations of CRISPR/Cas in this regard and representing a major advancement in non-nucleic acid target detection using a gene-editing-based system.

Paper-based multiplex biosensors for inexpensive healthcare diagnostics: a comprehensive review.

Multiplex detection is a smart and an emerging approach in point-of-care testing as it reduces analysis time and testing cost by detecting multiple analytes or biomarkers simultaneously which are crucial for disease detection at an early stage. Application of inexpensive substrate such as paper has immense potential and matter of research interest in the area of point of care testing for multiplexed analysis as it possesses several unique advantages. This study presents the use of paper, strategies adopted to refine the design created on paper and lateral flow strips to enhance the signal, increase the sensitivity and specificity of multiplexed biosensors. An overview of different multiplexed detection studies performed using biological samples has also been reviewed along with the challenges and advantages offered by multiplexed analysis.

Multiplex detection of clinical pathogen nucleic acids via a three-way junction structure-based nucleic acid circuit.

Nucleic acid testing technology has made considerable progress in the last few years. However, there are still many challenges in the clinical application of multiple nucleic acid assays, such as how to ensure accurate results, increase speed and decrease cost. Herein, a three-way junction structure has been introduced to specifically translate analytes of loop-mediated isothermal amplification to a catalytic hairpin assembly. For different analyses, a well-optimized nucleic acid circuit can be directly applied to detection, through only one-component replacement, which only not avoids duplicate sequence design but also saves detection cost. Thanks to this design, multiple and logical analysis can be easily realized in a single reaction with ultra-high sensitivity and selectivity. In this paper, Mycoplasma pneumoniae and Streptococcus pneumoniae can be clearly distinguished from the clinical mixed sample with negative control or one analyte in one tube single fluorescence channel. The fair experimental results of actual clinical samples provide a strong support for the possibility of clinical application of this methodology.

A rapid and highly sensitive multiple detection of human adenovirus type 3, type 7 and respiratory syncytial virus by recombinase-aided reverse transcription PCR.

BACKGROUND: Polymerase chain reaction (PCR) has been widely used for many pathogen detection. However, PCR technology still suffers from long detection time and insufficient sensitivity. Recombinase-aided amplification (RAA) is a powerful nucleic acid detection tool with high sensitivity and amplification efficiency, but its complex probes and inability of multiplex detection hinder the further application of this technology. METHODS: In this study, we developed and validated the multiplex reverse transcription recombinase-aided PCR (multiplex RT-RAP) assay for human adenovirus 3 (HADV3), human adenovirus 7 (HADV7), and human respiratory syncytial virus (HRSV) within 1 h with Human RNaseP protein as a reference gene to monitor the whole process. RESULTS: Using recombinant plasmids, the sensitivity of multiplex RT-RAP for the detection of HADV3, HADV7, and HRSV was 18, 3, and 18 copies per reaction, respectively. The multiplex RT-RAP showed no cross-reactivity with other respiratory viruses, demonstrating its good specificity. A total of 252 clinical specimens were tested by multiplex RT-RAP and the results were found to be consistent with those of corresponding RT-qPCR assays. After testing serial dilutions of selected positive specimens, the detection sensitivity of multiplex RT-RAP was two to eightfold higher than that of corresponding RT-qPCR. CONCLUSION: We conclude the multiplex RT-RAP is a robust, rapid, highly sensitive, and specific assay with the potential to be used in the screening of clinical samples with low viral load.

Multiplex Identification of Post-Translational Modifications at Point-of-Care by Deep Learning-Assisted Hydrogel Sensors.

Multiplex detection of protein post-translational modifications (PTMs), especially at point-of-care, is of great significance in cancer diagnosis. Herein, we report a machine learning-assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely-related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real-time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early-stage diagnosis of protein-related diseases.

Multiplex Detection of Antibody Landscapes to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/Influenza/Common Human Coronaviruses Following Vaccination or Infection With SARS-CoV-2 and Influenza.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses continue to co-circulate, representing 2 major public health threats from respiratory infections with similar clinical presentations. SARS-CoV-2 and influenza vaccines can also now be co-administered. However, data on antibody responses to SARS-CoV-2 and influenza coinfection and vaccine co-administration remain limited. METHODS: We developed a 41-plex antibody immunity assay that can simultaneously characterize antibody landscapes to SARS-CoV-2/influenza/common human coronaviruses. We analyzed sera from 840 individuals (11-93 years), including sera from reverse transcription-polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2-positive (n = 218) and -negative (n = 120) cases, paired sera from SARS-CoV-2 vaccination (n = 29) and infection (n = 11), and paired sera from influenza vaccination (n = 56) and RT-PCR-confirmed influenza infection (n = 158) cases. Last, we analyzed sera collected from 377 individuals who exhibited acute respiratory illness (ARI) in 2020. RESULTS: This 41-plex assay has high sensitivity and specificity in detecting SARS-CoV-2 infections. It differentiated SARS-CoV-2 vaccination (antibody responses only to spike protein) from infection (antibody responses to both spike and nucleoprotein). No cross-reactive antibodies were induced to SARS-CoV-2 from influenza vaccination and infection, and vice versa, suggesting no interaction between SARS-CoV-2 and influenza antibody responses. However, cross-reactive antibodies were detected between spike proteins of SARS-CoV-2 and common human coronaviruses that were removed by serum adsorption. Among 377 individuals who exhibited ARI in 2020, 129 were influenza positive; none had serological evidence of SARS-CoV-2/influenza coinfections. CONCLUSIONS: Multiplex detection of antibody landscapes can provide in-depth analysis of the antibody protective immunity to SARS-CoV-2 in the context of other respiratory viruses, including influenza.

An integrated fluorescent lateral flow assay for multiplex point-of-care detection of four respiratory viruses.

This work established a highly sensitive and specific quantum dot nanobeads-based lateral flow assay for multiplex detection of four respiratory virus markers at point of care. The respiratory virus antigens were detected by fluorescent lateral flow strips within 20 min. The limits of detection for SARS-CoV-2 antigen, IAV antigen, IBV antigen, and ADV antigen were 0.01 ng/mL, 0.05 ng/mL, 0.31 ng/mL, and 0.40 ng/mL, respectively, which were superior to that of conventional AuNPs-based colorimetric lateral flow assay. The coefficients of variation of the test strip were 6.09%, 2.24%, 7.92%, and 12.43% for these four antigens, which indicated that the proposed method had good repeatability. The specificity of the detection system was verified by different combinations of these four respiratory viruses and several other respiratory pathogens. These results indicated that this method could simultaneously detect SARS-CoV-2, IAV, IBV and ADV in a short assay time, showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

A novel DNA tweezers-based nanobiosensor for multiple detections of circulating exosomal microRNAs in breast cancer.

Breast cancer is the prevalent disease in women, and diagnosis of it in early stage and takes preventive measures is very critical. Recently, circulating microRNAs have emerged as promising early biomarkers of cancer. MiR-21 and miR-155 are two significant biomarkers that act as oncomir in breast cancer. In this study, to detect both microRNAs in one test simultaneously, a novel colorimetric nanobiosensor was developed upon the peroxidation property of a specific G-quadruplex nanostructure. The nanostructure forms a DNA Nano-Tweezers after self-assembly of three DNA oligonucleotides with target sequences, and TMB (2, 2'-azino-bis (3-ethylbenzothiazo-line-6-sulfonic acid)) is used as a reporter to produce color. The high sensitivity of the nanobiosensor was determined (in buffer and blood) using different concentrations of target sequences with a linear response range from 0 to 10 nM, and detection limit of 0.38 nM (R2 = 0.98). The method precisely detected target sequences from non-target sequences in both buffer and blood media. These findings demonstrate, the nanobiosensor is superior to most previous published works due to its simultaneous dual detection, simplicity, low response time, and cost. The analytical data is convenient for accurately use for clinical purposes to detect breast cancer in early stage, more significantly.

Base excision-initiated terminal deoxynucleotide transferase-assisted amplification for simultaneous detection of multiple DNA glycosylases.

Various DNA glycosylases involved in base excision repair may be associated with a wide disease spectrum that includes cancer, myocardial infarction, neurodegenerative disorders, etc. In this paper, we developed a sensitive method for simultaneous detection of multiple DNA glycosylases based on the target-initiated removal of damaged base and terminal deoxynucleotidyl transferase (TdT)-assisted labeling and signal amplification. We designed three specific stem-loop probes which contained specific targeting damaged bases in the stem for uracil DNA glycosylase (UDG), human alkyladenine DNA glycosylase (hAAG), and human 8-oxoguanine DNA glycosylase 1 (hOGG1), respectively. Target DNA glycosylase can initiate the recognition and clearance of damaged base on immobilized 3' blocked stem-loop probe, releasing apurine/apyrimidine (AP) site which can be hydrolyzed by AP endonuclease to produce 3'OH probe fragment for TdT extension. Numerous biotin-modified dUTPs were successively labeled on the 3' terminus of the probe fragments, and then reacted with streptavidin-phycoerythrin (SA-PE) for analysis by using the Luminex xMAP array platform. The amplification strategy based on TdT has been utilized to simultaneously and sensitively detect three different DNA glycosylases with detection limits of 10-3 U/ml. Moreover, it could be applied for analyzing DNA glycosylase activity in complex HeLa cell lysate samples. Therefore, this strategy possesses the advantages of high sensitivity, specificity, and multiplex, holding great potential for DNA glycosylase-related biomedical research.

Ultrasensitive multiplex SERS immunoassay based on porous Au-Ag alloy nanoparticle-amplified Raman signal probe and encoded photonic crystal beads.

An ultrasensitive multiplex surface-enhanced Raman scattering (SERS) immunoassay was developed using porous Au-Ag alloy nanoparticles (p-AuAg NPs) as Raman signal amplification probe coupling with encoded photonic crystal microsphere. p-AuAg NPs were synthesized and modified with the second antibody (Ab2) and Raman tag (mercaptobenzoic acid, MBA) to prepare a Raman signal-amplified probe. The high porosity of the p-AuAg NPs enables significant coupling of the localized surface plasmon resonance and thus abundant inherent hotspots for Raman signal enhancement. 3D-ordered silver nanoparticles-coated silica photonic crystal beads (Ag/SPCBs) were prepared as encoded SERS substrate for multiplex detection using their reflection peaks. The signal-amplified probe was used for multiplex detection of tumor markers carcinoembryonic antigen (CEA) and alpha fetoprotein (AFP). The wide linear ranges of 10-7-103 ng/mL for CEA and 10-4-103 ng/mL for AFP with detection limits of 1.22 × 10-8 ng/mL and 2.47 × 10-5 ng/mL for CEA and AFP at a signal-to-noise ratio of 3 were obtained. The proposed multiplex SERS immunoassay method displays ultrahigh sensitivity, wide linear range, and excellent specificity, which can be successfully applied to measure clinical serum samples with satisfactory results. The research provides a novel SERS signal enhancement strategy for the multiplex bioassay.

Surface-Enhanced Raman Scattering (SERS) Taster: A Machine-Learning-Driven Multireceptor Platform for Multiplex Profiling of Wine Flavors.

Integrating machine learning with surface-enhanced Raman scattering (SERS) accelerates the development of practical sensing devices. Such integration, in combination with direct detection or indirect analyte capturing strategies, is key to achieving high predictive accuracies even in complex matrices. However, in-depth understanding of spectral variations arising from specific chemical interactions is essential to prevent model overfit. Herein, we design a machine-learning-driven "SERS taster" to simultaneously harness useful vibrational information from multiple receptors for enhanced multiplex profiling of five wine flavor molecules at parts-per-million levels. Our receptors employ numerous noncovalent interactions to capture chemical functionalities within flavor molecules. By strategically combining all receptor-flavor SERS spectra, we construct comprehensive "SERS superprofiles" for predictive analytics using chemometrics. We elucidate crucial molecular-level interactions in flavor identification and further demonstrate the differentiation of primary, secondary, and tertiary alcohol functionalities. Our SERS taster also achieves perfect accuracies in multiplex flavor quantification in an artificial wine matrix.

19 F Barcoding Enables Multiplex Detection of Biomarkers Associated with Organ Injury and Cancer.

Simultaneous detection of multiple biomarkers in complex environments is critical for the in-depth exploration of different biological processes, which is challenging for many current analytical methods due to various limitations. Herein, we report a strategy of 19 F barcoding which takes the advantages of 19 F's high magnetic resonance (MR) sensitivity, prompt signal response to environmental changes, negligible biological background, quantitative signal output, and multiplex capacity. A set of 19 F-barcoded sensors responding to different biomarkers involved in organ injury and cancer are designed, synthesized, and characterized. With these sensors, we accomplish concurrent assessment of different biomarkers in the samples collected from the mice with drug-induced liver/kidney injury or tumor, illustrating the feasibility of this approach for multiplexed detection of different biomarkers in complex environments during various biological processes.

Rapid and ultrasensitive detection of food contaminants using surface-enhanced Raman spectroscopy-based methods.

With the globalization of food and its complicated networking system, a wide range of food contaminants is introduced into the food system which may happen accidentally, intentionally, or naturally. This situation has made food safety a critical global concern nowadays and urged the need for effective technologies capable of dealing with the detection of food contaminants as efficiently as possible. Hence, Surface-enhanced Raman spectroscopy (SERS) has been taken as one of the primary choices for this case, due to its extremely high sensitivity, rapidity, and fingerprinting interpretation capabilities which account for its competency to detect a molecule up to a single level. Here in this paper, we present a comprehensive review of various SERS-based novel approaches applied for direct and indirect detection of single and multiple chemical and microbial contaminants in food, food products as well as water. The aim of this paper is to arouse the interest of researchers by addressing recent SERS-based, novel achievements and developments related to the investigation of hazardous chemical and microbial contaminants in edible foods and water. The target chemical and microbial contaminants are antibiotics, pesticides, food adulterants, Toxins, bacteria, and viruses. In this paper, different aspects of SERS-based reports have been addressed including synthesis and use of various forms of SERS nanostructures for the detection of a specific analyte, the coupling of SERS with other analytical tools such as chromatographic methods, combining analyte capture and recognition strategies such as molecularly imprinted polymers and aptasensor as well as using multivariate statistical analyses such as principal component analysis (PCA)to distinguish between results. In addition, we also report some strengths and limitations of SERS as well as future viewpoints concerning its application in food safety.

Multiplex detection of blood-borne pathogens on a self-driven microfluidic chip using loop-mediated isothermal amplification.

Detection of blood-borne pathogens such as hepatitis C virus (HCV), hepatitis B virus (HBV) and human immunodeficiency virus (HIV) is essential to ensure the safety of blood transfusion. However, traditional PCR-based pathogen nucleic acid detection methods require relatively high experimental facilities and are difficult to apply in areas with limited resources. In this study, a self-driven microfluidic chip was designed to carry out multiplex detection of HBV, HCV and HIV by using loop-mediated isothermal amplification (LAMP). Benefitting from the air permeability of the polydimethylsiloxane material, the chip could accomplish sample loading within 12 min driven by the pressure difference between the reaction chambers and vacuum chambers in the chip without using pumps or any injection devices. Multiplex detection is achieved by presetting LAMP primers specific to different targets in different reaction chambers. Calcein was used as an indicator to indicate the positive amplification reaction, and the result can be recorded by a smartphone camera. After 50 min of isothermal amplification at 63 °C, 2 copies/µL of HBV, HCV and HIV target nucleic acids could be detected. The results of HBV detection of 20 clinical plasma samples by using the chip are consistent with that of the qPCR-based kit, indicating that the LAMP-based self-driven chip has the clinical application potential for blood-borne pathogen detection, especially in resource-limited areas.

A fully automated microfluidic PCR-array system for rapid detection of multiple respiratory tract infection pathogens.

Rapid and accurate identification of respiratory tract infection pathogens is of utmost importance for clinical diagnosis and treatment, as well as prevention of pathogen transmission. To meet this demand, a microfluidic chip-based PCR-array system, Onestart, was developed. The Onestart system uses a microfluidic chip packaged with all the reagents required, and the waste liquid is also collected and stored on the chip. This ready-to-use system can complete the detection of 21 pathogens in a fully integrated manner, with sample lysis, nucleic acid extraction/purification, and real-time PCR sequentially implemented on the same chip. The entire analysis process is completed within 1.5 h, and the system automatically generates a test report. The lower limit-of-detection (LOD) of the Onestart assay was determined to be 1.0 × 103 copies·mL-1. The inter-batch variation of cycle threshold (Ct) values ranged from 0.08% to 0.69%, and the intra-batch variation ranged from 0.9% to 2.66%. Analytical results of the reference sample mix showed a 100% specificity of the Onestart assay. The analysis of batched clinical samples showed consistency of the Onestart assay with real-time PCR. With its ability to provide rapid, sensitive, and specific detection of respiratory tract infection pathogens, application of the Onestart system will facilitate timely clinical management of respiratory tract infections and effective prevention of pathogen transmission. Onestart, a ready-to-use system, can detect 21 pathogens in a fully integrated manner on a microchip within 1.5 h.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.