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In this study, we described a novel impedance biosensor combining immunomagnetic separation with urease catalysis for sensitive detection of foodborne bacteria using Listeria monocytogenes as model and an immobilization-free microelectrode as detector. The monoclonal antibodies (MAbs) were immobilized on the surface of the magnetic nanoparticles (MNPs) with the diameter of 180 nm by biotin–streptavidin system for specifically and efficiently separating Listeria cells from sample background. The polyclonal antibodies (PAbs) and the urease were modified onto the surface of the gold nanoparticles (AuNPs) with the diameter of 20 nm and the modified AuNPs were used to react with Listera to form the MNP–MAb– Listeria –PAb–AuNP–urease sandwich complexes. The urease in the complexes could catalyze the hydrolysis of the urea into ammonium carbonate and this led to an increase in the ionic strength of the media, which could be detected by the microelectrode. The magnetic separation efficiencies for L. monocytogenes at the concentrations ranging from 3.0×10 1 to 3.0×10 4 CFU/mL were over 95% for the pure cultures and over 85% for the spiked lettuce samples. The lower detection limit of this biosensor for L. monocytogenes was found to be 300 CFU/mL in both the pure cultures and the spiked lettuce samples. The microelectrode was demonstrated to be reusable for over 50 times with thorough cleaning by deionized water. This biosensor showed its potential to provide a simple, low-cost and sensitive method for rapid screening of foodborne pathogens and could be extended for detection of other biological or chemical targets. [ABSTRACT FROM AUTHOR]

Early screening of suspected cases is the key to control the spread of avian influenza (AI) H5N1. In our previous studies, an impedance biosensor with an interdigitated array microelectrode based biochip was developed and validated with pure AI H5 virus, but had limitations in cost and reliability of the biochip, specificity of the antibody against Asian in-field H5N1 virus and detection of H5N1 virus in real samples. The purpose of this study is to develop a low-cost impedance immunosensor for rapid detection of Asian in-field AI H5N1 virus in chicken swabs within 1 h and validate it with the H5N1 virus. Specific monoclonal antibodies against AI H5N1 virus were developed by fusion of mouse myeloma cells with spleen cells isolated from an H5N1-virus-immunized mouse. Dot-ELISA analysis demonstrated that the developed antibodies had good affinity and specificity with the H5N1 virus. The microelectrodes were redesigned with compact size, fabricated using an improved wet-etching micro-fabrication process with a higher qualified production rate of 70–80%, and modified with the antibodies by the Protein A method. Equivalent circuit analysis indicated that electron transfer resistor was effective with the increase in impedance after capturing of the H5N1 viruses. Linear relationship between impedance change and logarithmic value of H5N1 virus at the concentrations from 2 −1 to 2 4 HAU/50 μl was found and the lower limit of detection was 2 −1 HAU/50 μl. No obvious interferences from non-target viruses such as H6N2, H9N2, Newcastle disease virus, and infectious bronchitis virus were found. Chicken swab tests showed that the impedance immunosensor had a comparable accuracy with real-time RT-PCR compared to viral isolation. [ABSTRACT FROM AUTHOR]

Sample pretreatment is a key to rapid screening of pathogens for prevention and control of foodborne diseases. Magnetic immunoseparation is a specific method based on antibody–antigen reaction to capture the target bacteria and concentrate them in a smaller-volume buffer. The use of nano-sized magnetic particles could improve the separation efficiency of bacteria but require much higher gradient and strength magnetic field. In this study, a strong magnetic bioseparator with a mean field strength of 1.35 T and a mean gradient of 90 T/m was developed with the use of the 30 nm and 180 nm magnetic particles to specifically separate and efficiently concentrate foodborne bacterial pathogens using Escherichia coli O157:H7 as a model bacterium. The polyclonal antibodies against E. coli were evaluated using Dot ELISA analysis for their good affinity with the target bacteria and then used to modify the surface of the magnetic nanoparticles by 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) method and streptavidin-biotin binding. The magnetic particle concentrations were optimized to be 40 µg/ml and 100 µg/ml for the 30 nm and 180 nm particles, respectively, the immunoreaction time was optimized to be 45 min for both sizes of particles, and the separation times were optimized to be 60 min and 2 min for the 30 nm and 180 nm particles, respectively. The total magnetic separation time was 2 h and 1 h for the 30 nm and 180 nm particles, respectively. The experimental results demonstrated that the bioseparator with the use of either 30 nm or 180 nm immunomagnetic particles could achieve a separation efficiency of >90% for E. coli O157:H7 at the concentrations ranging from 10 2 to 10 5 cfu/ml. No obvious interferences from non-target foodborne pathogens, such as Salmonella Typhimurium and Listeria innocua , were found. For overall consideration of the consuming time, the cost, and the separation efficiency, the 180 nm magnetic particles are practical for rapid screening applications; however the 30 nm magnetic particles are preferable for specific detection applications. This immunomagnetic bioseparator can be integrated with either conventional culture methods or some rapid detection methods, such as biosensors and PCR, for more sensitive detection of foodborne pathogens. [ABSTRACT FROM AUTHOR]

Abstract: Current methods for detection of avian influenza virus (AIV) based on virus culture and RT-PCR are well established, but they are either time consuming or require specialized laboratory facilities and highly trained technicians. A simple, rapid, robust, and reliable test, suitable for use in the field or at the patient''s bedside, is urgently needed. In this study, the performance of a newly developed portable impedance biosensor was evaluated by comparison with real-time reverse transcriptase PCR (rRT-PCR) and virus culture for detection of AIV in tracheal and cloacal swab samples collected from experimentally H5N2 AIV infected chickens. The impedance biosensor system was based on a combination of magnetic nanobeads, which were coated with AIV subtype-specific antibody for capture (separation and concentration) of a target virus, and a microfluidic chip with an interdigitated array microelectrode for transfer and detection of target virus, and impedance measurement of the bio-nanobeads and AI virus complexes in a buffer solution. A comparison of results obtained from 59 swab samples using virus culture, impedance biosensor and rRT-PCR methods showed that the impedance biosensor technique was comparable in sensitivity and specificity to rRT-PCR. Detection time for the impedance biosensor is less than 1h. [Copyright &y& Elsevier]

Conventional soil nitrate and potassium detection methods are reliable, but time-consuming and expensive. Rapid lower-cost detection methods for soil nitrate and potassium are urgently needed. In this study, a temperature-calibrated soil nitrate and potassium rapid simultaneous detection system was developed based on two ion-selective electrodes (ISEs). At room temperature, the potentiometric experiments were conducted to evaluate the performance of both ISEs and simple linear regression (SLR) was used for modelling. The lower detection limits of both ISEs were 1 x 10-5 mol/litre with regression coefficients of 0.994 and 0.997, respectively. Under different temperatures, the potentiometric experiments were conducted and multiple SLRs were used for temperature-calibrated modelling with a maximum relative error of c. 6% and c. 10%, respectively. An ISE signal acquisition system was developed based on a microprocessor, including an ISE signal conditioning unit and a temperature measurement unit using a thermal resistor. This study might be suitable for in-field soil nitrate and potassium rapid detection application. [ABSTRACT FROM AUTHOR]

A colorimetric biosensor was elaboratively designed for fast, sensitive and multiplex bacterial detection on a single microfluidic chip using immune magnetic nanobeads for specific bacterial separation, immune gold@platinum palladium nanoparticles for specific bacterial labeling, a finger-actuated mixer for efficient immunoreaction and two coaxial rotatable magnetic fields for magnetic nanobead capture (outer one) and magnet-actuated valve control (inner one). First, preloaded bacteria, nanobeads and nanozymes were mixed through a finger actuator to form nanobead-bacteria-nanozyme conjugates, which were captured by the outer magnetic field. After the inner magnetic field was rotated to successively wash the conjugates and push the H2O2-TMB substrate for resuspending these conjugates, colorless TMB was catalyzed into blue TMBox products, followed by color analysis using ImageJ software for bacterial determination. This simple biosensor enabled multiplex Salmonella detection as low as 9 CFU per sample in 45 min. [ABSTRACT FROM AUTHOR]

Magnetic nanobeads have been widely used for magnetic separation, target labeling and signal amplification in the development of various biosensors. This review summarized (1) the basic knowledge of magnetic nanobeads, including physical structure, chemical characteristics and surface modification; (2) the development and applications of magnetic separation, from the earliest conventional magnetic separation, to high gradient magnetic separation, then to magnetophoretic separation and magnetic continuous-flow separation, finally to the latest magnetic grid separation; and (3) recent advances and applications of magnetic nanobead based biosensors, where the magnetic nanobeads work as either detection signal for electromagnetic biosensors, magnetic lateral flow biosensors and magnetic relaxation switching biosensors, or signal amplifying tag for electrochemical biosensors, QCM biosensors and SPR biosensors, or both. Besides, the important issues and future trends for magnetic nanobead based separation and biosensor were also discussed and summarized. • Basic knowledge of magnetic nanobeads were summarized. • Recent advances on MNBs for magnetic separation were reviewed. • Recent advances on MNBs for target label were reviewed. • Recent advances on MNBs for signal amplification were reviewed. [ABSTRACT FROM AUTHOR]

Background: Automatic signal-feature extraction algorithms are crucial for profile processing in bioinformatics. Both baseline drift and noise seriously affect the position and peak area of signals. An efficient algorithm named the derivative passing accumulation (DPA) method for simultaneous baseline correction and signal extraction is presented in this article. It is an efficient method using only the first-order derivatives which are obtained through taking the simple differences. Results: We developed a new signal feature extracting procedure. The vector representing the discrete first-order derivative was divided into negative and positive parts and then accumulated to build a signal descriptor. The signals and background fluctuations are easily separated according to this descriptor via thresholding. In addition, the signal peaks are simultaneously located by checking the corresponding intervals in the descriptor. Therefore, the eternal issues of parsing the 1-dimensional output of detectors in biological instruments are solved together. Thereby, the baseline is corrected, and the signal peaks are extracted. Conclusions: We have introduced a new method for signal peak picking, where baseline computation and peak identification are performed jointly. The testing results of both authentic and artificially synthesized data illustrate that the new method is powerful, and it could be a better choice for practical processing. [ABSTRACT FROM AUTHOR]

This study reported a bacterial detection platform using chitosan electrostatic adsorption for nucleic acid purification, RAA-T7-CRISPR/Cas13a system for nucleic acid quantification and centrifugal microfluidic platform for full-automatic operation. First, the nucleic acids in the lysed bacterial sample were captured by chitosan modified in the microfluidic PDMS channel through electrostatic adsorption, followed by washing with sodium acetate buffer solution. Then, these captured nucleic acid templates were eluted using Tris-HCl buffer solution through restoring the electrical neutrality of chitosan. Finally, the eluted nucleic acid templates were transferred into the RAA-T7-CRISPR/Cas13a system and isothermally amplified at 37 °C, followed by real-time detection of fluorescent products. A centrifugal microfluidic chip was elaborately designed with a chitosan modified channel, eight functional chambers and three siphon valves to achieve sequential capture, washing, elution, amplification and detection of nucleic acids. This platform has the capacity of automatically detecting Salmonella within 1 h. The detection limit of this platform was 10 CFU/mL in chicken supernatant under the use of optimal conditions. The average recovery and standard deviation of this platform are 106.9% and 6.4%, respectively. • A new centrifugal microfluidic platform was developed for Salmonella detection. • DNA extraction and purification were achieved by chitosan modified PDMS channel. • RAA-T7-Crispr Cas13a was used for quantification of RAA amplified DNA. • The centrifugal platform enabled Salmonella detection as low as 10 CFU/mL in 1 h. • The mean recovery of Salmonella in spiked chicken supernatant was ∼107%. [ABSTRACT FROM AUTHOR]

Foodborne pathogen contamination is a main source of food safety issues. Rapid screening of pathogens in foods is imperative for preventing the outbreaks of foodborne diseases, which have posed serious threats to human health and economic development. This review aimed at recent progress of the microfluidic separators and biosensors for rapid and sensitive detection of foodborne pathogens. The key technologies for developing lab-on-chip separators and biosensors were illustrated. In particular, we systematically discussed the strategy of lab-on-chip magnetic separation for continuous-flow isolation of pathogenic bacteria. Challenges and future perspectives for practical applications of lab-on-chip technologies in foods were also discussed. Lab-on-chip devices are a promising tool to integrate separation, enrichment and detection in one single chip to facilitate "sample-in-answer-out" testing of pathogens in agri-food. [Display omitted] • Recent advances on microfluidic separators for isolation and enrichment of foodborne pathogens are summarized. • The strategy of microfluidic magnetic separation in continuous-flow isolation of pathogens are systematically discussed. • Recent progress of microfluidic electrochemical and optical biosensors for rapid detection of pathogens are summarized. • Challenges and future perspectives of practical application of lab-on-chip technology in agri-food are discussed. [ABSTRACT FROM AUTHOR]

Astaxanthin with high antioxidant activity is of great practical value and Haematococcus pluvialis is recognized as the best natural astaxanthin producer. The yield of Haematococcus pluvialis was often affected by the ciliate during its production, however, the use of biochemical pesticides might have a great impact on Haematococcus pluvialis. Therefore, a simple microfluidic chip with the spiral microchannel was developed for continuous‐flow physical separation of ∼10 µm ciliate from ∼30 µm Haematococcus pluvialis since their different sizes resulted in different equilibrium positions in the channel due to the Dean‐coupled inertial migration. First, a spiral microchannel with a width of 700 µm and a height of 130 µm in the microfluidic chip was developed using three‐dimensional printing and verified to completely separate polystyrene particles of 10 µm from those of 30 µm. Then, this microfluidic chip was used to separate the actual sample, and experimental results showed that ∼80% of ciliate was continuously separated from Haematococcus pluvialis at a flow rate of 2.8 ml/min. More importantly, no additional biochemical reagents were used and the activity of Haematococcus pluvialis was not affected. This microfluidic chip featured with simple design, automatic operation, and small size is promising for purification and breeding of Haematococcus pluvialis. [ABSTRACT FROM AUTHOR]

This study reported an automatic centrifugal system for rapid quantification of foodborne pathogenic bacteria based on immunomagnetic separation (IMS) for target bacteria enrichment and recombinase aided amplification (RAA) for nucleic acid detection. First, target bacteria were captured by immune magnetic nanoparticles (MNPs) to form magnetic bacteria, which were purified and enriched by magnetic separation. Then, nucleic acid extraction buffer was used to extract genomic DNA of magnetic bacteria and dissolve lyophilized RAA reagent. Finally, isothermal amplification and fluorescent detection were conducted for bacteria quantification. Bacteria magnetic separation, nucleic acid extraction and fluorescent RAA detection were elaborately achieved in a centrifugal disc with unique functional chambers and multistage siphon channels. A supporting device was developed to automatically and successively perform the programmed centrifugal protocol, including temperature control for isothermal amplification and fluorescence detection for real-time RAA analysis. Under optimal conditions, this centrifugal system enabled Salmonella detection as low as 10 CFU mL−1 in spiked chicken samples in 1 h with average recovery of 105.6% and average standard deviation of 8.4%. It has been demonstrated as an alternative for rapid detection of Salmonella. [ABSTRACT FROM AUTHOR]

The development of eco-friendly analytical methods for the detection of foodborne pathogens is of great importance for protecting human health and alleviating environmental pressure. Here, we demonstrate that a material (CaCO 3 microparticles) prepared from discarded eggshells can be used as signal probes for micro-orifice resistance assay (MORA) and can be combined with ligation-mediated branching hybridization chain reaction (LB-HCR)-based signal-amplification technique (e-LMORA) for high sensitivity detection of Listeria monocytogenes. This strategy used LB-HCR-induced aggregation of streptavidin-modified CaCO 3 microparticles, which were recognized by the particle counter for quantitative analysis of the target DNA. Compared with traditional MORA using polystyrene microspheres as signal probes, CaCO 3 microparticles prepared from waste eggshells are more environmentally friendly. The combination of DNA-recognition probe, signal probes, and enzyme-free signal-amplification strategy not only reduces the cost synergistically, but also enables the biosensor to attain high sensitivity (limit of detection = 21 CFU/mL) due to its efficient signal amplification. • A micro-orifice resistance assay based on CaCO 3 microparticles and LB-HCR was proposed. • This assay enables highly sensitive detection of Listeria monocytogenes. • Eco-friendly probes made from discarded eggshells can turn waste into treasure. • A more efficient signal amplification strategy results in high detection sensitivity. [ABSTRACT FROM AUTHOR]

[Display omitted] • The next-generation digital platform based on novel Ago gene editing tools. • Ultrafast polydisperse droplet preparation system for quick digital detection. • Machine learning system based on droplet area and intensity dual parameters. • True sense one step/one pot multiplex nucleic acid detection by mesophilic Ago. • Asymmetric RPA enhances the cleavage effect of CbAgo at low temperatures. Foodborne pathogens endanger human health. Therefore, sensitive and accurate detection assays are key for their prevention and control. Microdroplet digital nucleic acid platforms can achieve single-molecule sensitivity in the presence of specialized instrumentation for generating homogenized monodisperse droplets, expensive thermal cyclers, high temperature-resistant reagents, and time-consuming multistep operations in the lab. Argonaute (Ago), an emerging next-generation gene editing tool, can be used for the detection of pathogen genetic information. Current reports mainly adopt the high temperature-dependent thermophilic Ago in stepwise nucleic acid detection, which hinders its applications in ultrasensitive digital detection. Here, we expressed and purified the mesophilic Clostridium butyricum Argonaute (CbAgo) and developed a polydisperse micro d roplet reactors-assist asymmetric r ecombinase polymerase amplification driven Cb Ago report digital detectio n platform (DRAGON). It realized the signal-enhanced nucleic acid amplification and CbAgo report compatibility in novel low-cost and ultrafast-prepared polydisperse microdroplet reactors. The microdroplet reactor area and fluorescence intensity were combined to establish a dual parameter-based random forest machine learning model, which achieved high sensitivity (1 CFU/mL) and efficiency (<45 min) for detecting pathogens. This is the first time that mesophilic Ago was used to achieve true sensitive one-step and one-pot low-temperature digital nucleic acid detection. Our system accomplished multiplex detection in one-step and one-pot with a single nuclease which is impossible for CRISPR/Cas system and thermophilic Ago-based stepwise platforms. DRAGON represents the next-generation platform for pathogen detection based on gene editing tools and intelligent digital nucleic acid detection system. [ABSTRACT FROM AUTHOR]

Culture plating is worldwide accepted as the gold standard for quantifying viable foodborne pathogens. However, it is time-consuming (1–2 days) and requires specialized laboratory and personnel. This study reported a deep learning enhanced digital microfluidic platform for multiplex detection of viable foodborne pathogens. The new method used a Time-Lapse images driven EfficientNet-Transformer Network (TLENTNet) to type and quantify the bacteria through spatiotemporal features of bacterial growth and digital enumeration of bacterial culture. First, the bacterial sample was prepared with LB medium and injected into a pre-vacuumed microfluidic chip with an array of 800 microwells to encapsulate at most one bacterium in each well. Then, a programmed sliding microscopic platform was used to scan all microwells every 15 min, capturing time-lapse images of bacterial growth within each microwell. Finally, the TLENTNet was used to facilitate bacterial typing and quantification. Under optimal conditions, this platform was able to detect four bacterial species (S. typhimurium, E. coli O157:H7 , S. aureus and B. cereus) with an average accuracy of 97.72% and a detection limit of 63 CFU/mL in 7 h. [ABSTRACT FROM AUTHOR]

Rapid screening of foodborne pathogens is key to preventing food poisoning. In this study, a slidable centrifugal disc was developed for automatic and multi-channel detection of Salmonella typhimurium using Flinders Technology Associates (FTA) cards for nucleic acid extraction and recombinase aided amplification (RAA) for nucleic acid detection. The slidable FTA switching and centrifugal fluidic control were elaborately combined to achieve fully automatic operations, including centrifugation of the bacterial sample to obtain the concentrated bacteria, heating and drying of the FTA card to extract the nucleic acids, washing of the FTA card to remove the impurities, and RAA detection of the extracted DNA to determine the concentration. Under the optimal conditions, this slidable centrifugal disc was able to detect 10 CFU mL−1 in a spiked chicken meat supernatant in 1 h with an average recovery of 101.8% and an average standard deviation of 6.5%. This disc has been demonstrated as an alternative for sample-in-result-out detection of Salmonella and has shown potential for simultaneous detection of multiple bacteria. [ABSTRACT FROM AUTHOR]

The development of a low-cost, high-sensitivity, portable bioassay for detecting the highly toxic and thermally stable aflatoxin B 1 (AFB 1) is of significant importance to food safety and human health. A pipette–smartphone integrated biosensor based on a super "four-in-one" DNA sea-urchin-like composite catalyst (FSCC) was constructed for the efficient analysis of AFB 1. Detection is possible via a self-made poly(methyl methacrylate) pipette, combined with a smartphone to further increase the biosensor's portability. The combination of the two greatly simplifies experimental operations, reduces testing costs, and can minimize matrix interferences through sample pre-treatment operations. And the smartphone-APP signal readout strategy can avoid the need for a reading instrument, making it more suitable for field testing. The FSCC was developed by fully utilizing the properties of a Fe-Zr-metal–organic framework (Fe-Zr-MOF), which exhibits high catalytic activity and enables the encapsulation of abundant horseradish peroxidase in its pores, the in-situ reduction and enrichment of Pt nanoparticles on its surface, and the coordination of biotinylated DNA to achieve a high adsorption capacity of streptavidin-horseradish peroxidase. This ensured detection sensitivity, with a limit of detection = 1.57 pg/mL. The developed biosensor displays notable application potential, especially in resource-poor areas due to the advantages of low costs, simple operation, ultra-high sensitivity, portability, etc. [Display omitted] • The "four-in-one" composite catalyst is constructed for effective signal amplification. • The composite catalyst is achieved by full characteristics of Fe-Zr-MOF (Fe-PCN-224). • The biosensor for AFB 1 detection is based on self-made PMMA pipet with pipette. • The pipette-smartphone integrated biosensor is low cost, ultra-sensitive and portable. [ABSTRACT FROM AUTHOR]

Rapid screening of foodborne pathogens is key to prevent food poisoning. In this study, a lab-on-chip device was developed for rapid, automatic and sensitive detection of viable Salmonella typhimurium using loop-mediated isothermal amplification (LAMP) and smartphone real-time turbidity monitoring. First, magnetic nanoparticles (MNPs) coated with anti-Salmonella capture antibodies in propidium monoazide (PMA) were fully mixed with bacterial samples using two active magnetic stirring mixers at reverse rotating directions, and incubated in the serpentine channel with 470 nm blue light exposure, allowing specific formation of magnetic bacteria and sufficient PMA pretreatment of the DNA of dead bacteria. Then, the PMA-treated magnetic bacteria were separated in the separation chamber using the magnetic field and their genomic DNA templates were extracted using lysis buffer at 70 °C. Finally, the viable bacteria's DNA was amplified using LAMP in the detection chamber preloaded with the lyophilized LAMP reagents at 67.5 °C after blocking with paraffin oil to avoid aerosol cross contamination. Finally, the turbidity of the LAMP reaction system was monitored in a real-time manner for the quantitative detection of viable bacteria. The experimental results demonstrated that this device was able to automatically detect viable Salmonella as low as 14 CFU mL−1 in spiked chicken meat supernatants within 1.5 h. This device is very promising to provide a sample-in-result-out solution for the in-field detection of Salmonella and could be easily extended for other foodborne pathogens. [ABSTRACT FROM AUTHOR]

A colorimetric immunosensor was developed for the determination of Salmonella Typhimurium using rotating magnetic separation, gold nanorod (GNR) indication, and click chemistry amplification. The target bacteria were first separated from large-volume sample using a rotating magnetic field and a small amount (50 μg) of immunomagnetic nanoparticles (MNPs), resulting in the forming of magnetic bacteria. Then, the magnetic bacteria were conjugated with catalase (CAT)-labeled antibodies, which were synthesized using trans-cyclooctene/1,2,4,5-tetrazine click chemistry reaction, resulting in the forming of enzymatic bacteria. Then the CATs on the enzymatic bacteria were used to decompose an excessive amount of hydrogen peroxide (H2O2), the remaining H2O2 was mixed with horseradish peroxidase to etch the GNRs, resulting in color change and absorbance peak shift of the GNRs. Finally, the peak shift was measured and analyzed for the quantitative determination of target bacteria. This immunosensor was able to detect Salmonella Typhimurium with a linear range of 101–105 CFU mL−1 in 3 h with a low detection limit of 35 CFU mL−1. The mean recovery for Salmonella Typhimurium in spiked chicken samples was 109%. [ABSTRACT FROM AUTHOR]

Immunoassays such as the enzyme-linked immunosorbent assay (ELISA) are utilized extensively for detecting protein biomarkers and small molecules in healthcare, environmental monitoring, and food analysis. Unfortunately, the current strategies for immunoassays often require sophisticated apparatus such as a microplate reader, which might not be available in resource-limited areas. To mitigate this problem, we designed a compact smartphone based-device and a multicolor response immunosensor. First, we designed a compact and cost-effective 3D-printed attachment, where a light-emitting diode was used as a light excitation source and a smartphone captured the fluorescent emission signals. Second, by combining quantum dots hybrid and chemical redox reaction, multiple color responses were displayed in the presence of the analyte at different concentrations. Third, solutions with distinct tonality could be readily distinguished by the naked eye and they were suitable for quantitative analysis using the hue-saturation-lightness color space based on a smartphone application. The versatility of the proposed sensing system was demonstrated by implementing an indirect competitive ELISA for analyzing trace drug residues in foodstuffs. The multicolor response of this sensing strategy allows us to visually quantify drug residues in foodstuffs. Moreover, the smartphone-based immunosensor can assess the exact concentration of the analyte by using a self-designed mobile application. The proposed assay provides a highly sensitive performance that the limit of detection was 0.37 ng/mL by visual detection and 0.057 ng/mL using the compact device. Due to its advantages in terms of portability, straightforward visual detection, high sensitivity, and cost effectiveness, the proposed immunosensor has great potential for applications in areas without access to laboratories or expensive infrastructure. Image 1 • A ratiometric fluorescent immunosensor based on quantum dots hybrid and smartphone. • A multiple fluorescence color changes enabled highly sensitive visual detection. • A smartphone-based device was designed as a compact and sensitive readout system. • The smartphone-based device was used for on-site analysis of drug residues. [ABSTRACT FROM AUTHOR]

A disposable visual microfluidic immunosensor is described for the determination of foodborne pathogens using immunomagnetic separation, enzymatic catalysis and distance indication. Specifically, a sensor was designed to detect Salmonella typhimurium as a model pathogen. Magnetic nanoparticles (MNPs) were modified with the anti-Salmonella monoclonal antibodies and then used to enrich S. typhimurium from the sample. This is followed by conjugation to polystyrene microspheres modified with anti-Salmonella polyclonal antibodies and catalase to form the MNP-bacteria-polystyrene-catalase sandwich. The catalase on the complexes catalyzes the decomposition of hydrogen peroxide to produce oxygen after passing a micromixer. The generated oxygen gas increases the pressure in the chip and pushes the indicating red dye solution to travel along the channel towards the unsealed outlet. The travel distance of the red dye can be visually read and related to the amount of S. typhimurium using the calibration scale. The sensor can detect as low as 150 CFU·mL−1 within 2 h. [ABSTRACT FROM AUTHOR]

The key to prevent and control the spread of foodborne diseases is rapid screening and early warning of pathogenic bacteria in foods. In this study, a microfluidic biosensor using magnetic nanoparticles (MNPs) and catalases was developed for rapid and sensitive detection of Salmonella typhimurium. First, the MNPs modified with anti- Salmonella monoclonal antibodies were used to separate and enrich target bacteria from sample background to form magnetic bacteria, which were reacted with polystyrene microspheres (PSs) modified with anti- Salmonella polyclonal antibodies and catalases to form enzymatic bacteria. Then, the enzymatic bacteria were injected into the capillary in microfluidic chip and captured by high gradient magnetic field. After washing to remove unbound PSs, hydrogen peroxide was finally injected and catalyzed by the catalases on enzymatic bacteria to produce oxygen gap in the capillary, leading to electrical signal-off. The change of electrical voltage was found to have a good linear relationship with the concentration of target bacteria from 3.7 × 101 to 3.7 × 106 CFU/mL. The proposed biosensor was able to detect Salmonella as low as 33 CFU/mL within 2 h, and could be further combined with microfluidics to develop a lab-on-a-chip system for in-field detection of foodborne bacteria to ensure food safety. • The signal-off strategy was successfully used to monitor electrical voltage change. • The catalases were used with capillary to greatly amplify the biological signal. • The biosensor was able to detect Salmonella as low as 33 CFU/mL within 2 h. • The mean recovery of Salmonella in the spiked milk was 104.7%. [ABSTRACT FROM AUTHOR]

Point-of-care testing (POCT) of pathogens is important for avoiding food poisoning. Here, dual-functional manganese dioxide nanoclusters (MnO 2 NCs) were elaboratively used as the fluidic driver and signal amplifier for exploring a new power-free biosensor for POCT of Salmonella. First, H 2 O 2 in three chambers of a microfluidic chip was finger driven simultaneously to dissolve MnO 2 NCs, resulting in the production of O 2 to respectively push preloaded immune magnetic nanobeads (IMNBs), bacterial sample and immune MnO 2 NCs (IMONCs) into a sinusoidal convergence-divergence micromixer. After they were efficiently mixed and incubated in a serpentine channel, IMNB-bacteria-IMONC conjugates were obtained and magnetically separated in a multifunctional chamber. Then, two washing solution were successively driven to rinse these conjugates. Finally, colorless H 2 O 2 -TMB substrate was driven to the multifunctional chamber and catalyzed by the IMONCs on the conjugates into blue catalysate, which was analyzed by developed smartphone APP to determinate bacteria amount. The biosensor enabled quantitative detection of 63 CFU/mL Salmonella within 30 min. The complete bacteria detection procedure was achieved on a single microfluidic chip without any peripheral equipment, and it has great potential for POCT of bacteria. [Display omitted] • Dual-functional MnO 2 nanoclusters were elaborately used as fluidic driver and signal amplifier. • The whole bacterial detection procedure was implemented free-of-power in a microfluidic chip. • The sinusoidal convergence-divergence micromixer was verified with good mixing effect. • Smartphone App was self-developed to accurately measure color change of catalysate. • This microfluidic biosensor was able to detect Salmonella as low as 63 CFU/mL in 30 min. [ABSTRACT FROM AUTHOR]

Aflatoxin B 1 is a carcinogenic contaminant in food or feed, and it poses a serious health risk to humans. Herein, a computer vision-assisted smartphone microscope imaging digital (SMID) immunosensor based on the click chemistry-mediated microsphere counting technology was designed for the detection of aflatoxin B 1 in peanuts. In this SMID immunosensor, the modified polystyrene (PS) microspheres were used as the signal probes and were recorded by a smartphone microscopic imaging system after immunoreaction and click chemistry reaction. The number of PS probes is adjusted by aflatoxin B 1. The customized computer vision procedure was used to efficiently identify and count the obtained PS probes. This SMID immunosensor enables sensitive detection of aflatoxin B 1 with a linear range from 0.001 ng/mL to 500 ng/mL, providing a simple, sensitive, and portable tool for food safety supervision. [Display omitted] • Click chemistry-based microsphere counting technology for detection of aflatoxin B 1. • The modified PS microsphere was used as the visual probe for digital signal readout. • Computer vision-based smartphone microscope imaging for automatic counting of PS probes. • The linear range of SMID immunosensor for aflatoxin B 1 is from 0.001 ng/mL to 500 ng/mL. [ABSTRACT FROM AUTHOR]

Abstract Early screening of foodborne pathogenic bacteria is a key to prevent and control foodborne diseases. This study intended to develop a capillary biosensor for rapid and sensitive detection of Salmonella using the multi-column capillary for easy operation, the Fe-nanoclusters (FNCs) for signal amplification and the smart phone APP for image analysis. The multi-column capillary was successively preloaded the magnetic nanoparticle (MNP) column, the FNC column, two phosphate buffer solution with 0.05% Tween 20 (PBST) columns and the HCl column and the columns were separated by air gap. The iron spiral mixer was fabricated to accelerate the mixing, and the multi-ring magnets was developed to transfer the MNPs and their conjugates from column to column. The target bacteria were captured by the MNPs to form the magnetic bacteria and then conjugated with the FNCs to form the nanocluster bacteria. After washing with PBST, the nanocluster bacteria were transferred into the HCl column, and the iron ions were released and reacted with potassium hexacyanoferrate to form the Prussian Blue, which was finally measured and analyzed using the Hue-Saturation-Lightness color space based smartphone APP for the determination of the target bacteria. This proposed biosensor exhibited a wide linear range for detection of Salmonella typhimurium with the lower detection limit of 14 CFU/mL. The mean recovery of the target bacteria in spiked chicken samples was ~105.0%, indicating the applicability of this biosensor. The proposed biosensor had the potential for in-field detection of foodborne pathogens. Highlights • The multi-column capillary was demonstrated to simplify the operation. • Fe-nanocluster was used for effective amplification of biological signals. • Prussian Blue was used with smartphone imaging APP for signal indication. • The detection limit of this biosensor was 14 CFU/mL for Salmonella. • The mean recovery of Salmonella in the spiked chicken samples was ~105.0%. [ABSTRACT FROM AUTHOR]

Abstract We intended to develop a novel biosensor using gold nanoparticles (AuNPs) for indicating different concentrations of E. coli O157:H7 and smart phone imaging APP for monitoring color change of the AuNPs. The magnetic nanoparticles (MNPs) modified with the capture antibodies and the polystyrene microspheres (PSs) modified with the detection antibodies and the catalases were simultaneously used to react with the target bacteria in the first mixing channel of the microfluidic chip, and hydrogen peroxide was injected and catalyzed by the catalases on the MNP-bacteria-PS complexes. After the mixture of the AuNPs and the crosslinking agents were injected to react with the catalysate in the second mixing channel and incubated in the detection chamber, the aggregation of the AuNPs was triggered through the crosslinking agents, resulting in the color of the AuNPs changing from blue to red. Finally, the color was measured using the smart phone imaging APP to determine the amount of the bacteria. This biosensor exhibited a good specificity and sensitivity for detection of E. coli O157:H7 in chicken samples with a lower detection limit of 50 CFU/mL. Highlights • AuNP aggregation were successfully used for signal indication. • HSL-based smart phone imaging was effective to detect color change. • Microfluidic chip was successfully developed for on-chip bioreaction. • The detection limit of this biosensor was 50 CFU/mL for E coli O157:H7. • The mean recovery of E coli O157:H7 in the spiked chicken samples was ~96.8%. [ABSTRACT FROM AUTHOR]

In this study, a rapid and sensitive method for detection of Escherichia coli O157:H7 using the coaxial channel-based DNA extraction and the microfluidic PCR was proposed and verified. The magnetic silica beads were first pumped into the coaxial channel, which was captured in the coaxial channel more uniformly by applying the multiring high-gradient magnetic field. After the E. coli O157:H7 cells were lysed with the lysis buffer to release the DNA, the improved coaxial channel was used to efficiently extract the DNA, followed by washing with ethanol to remove the residual proteins and eluting with a small volume of deionized water to obtain the purified and concentrated DNA. Finally, the obtained DNA was amplified and determined using the microfluidic PCR. This proposed bacteria detection method was able to detect E. coli O157:H7 as low as 12 cfu/mL when the large volume (10 mL) of bacterial sample was used, and the recovery of E. coli O157:H7 in the spiked milk samples ranged from 97.4 to 100.6%. This proposed bacteria detection method has shown great potential to detect lower concentration of E. coli O157:H7 from larger volumes of sample. [ABSTRACT FROM AUTHOR]

Magnetic separation was often applied to isolate and concentrate foodborne bacteria using immunomagnetic nanobeads before downstream bacterial detection. However, nanobead-bacteria conjugates (magnetic bacteria) were coexisting with excessive unbound nanobeads, limiting these nanobeads on magnetic bacteria to further act as signal probes for bacterial detection. Here, a new microfluidic magnetophoretic biosensor was elaboratively developed using a rotated high gradient magnetic field and platinum modified immunomagnetic nanobeads for continuous-flow isolation of magnetic bacteria from free nanobeads, and combined with nanozyme signal amplification for colorimetric biosensing of Salmonella. First, the platinum modified immunomagnetic nanobeads were mixed with the bacterial sample to form the magnetic bacteria, and magnetically separated to eliminate non-magnetic background. Then, the mixture of free immunomagnetic nanobeads and magnetic bacteria was injected with sheath flow (PBS) at higher flowrate into the semi-circle magnetophoretic separation channel under rotated magnetic field, which was generated by two repulsive cylindric magnets and their in-between ring iron gear, leading to continuous-flow isolation of magnetic bacteria from free immunomagnetic nanobeads because they suffered from different magnetic forces and thus had different deviating positions at the outlet. Finally, the separated magnetic bacteria and unbound magnetic nanobeads were respectively collected and used to catalyze coreless substrate into blue product, which was further analyzed using the microplate reader to obtain bacterial amount. This biosensor could determinate Salmonella as low as 41 CFU/mL in 40 min. [ABSTRACT FROM AUTHOR]

• A new nanozyme was synthesized by decorating MIL-88 with Pd and Pt NPs. • The nanozyme was verified to greatly enhance peroxidase-mimic activity. • This biosensor enabled detection of 32 CFU/mL Salmonella in 1 h. Pathogen screening is an important step in preventing foodborne diseases. In this study, a hybrid nanozyme, metal organic framework decorated with palladium (Pd) and platinum (Pt) (MIL-88@Pd/Pt), was innovatively synthesized and used with immune magnetic nanobeads (MNBs) for sensitive biosensing of Salmonella. First, immune MIL-88@Pd/Pt nanozymes and immune MNBs were mixed with target pathogens in a large-volume sample, resulting in effective isolation and specific label of target pathogens to form nanobead- Salmonella -nanozyme conjugates. Then, these conjugates were used to catalyze H 2 O 2 -TMB, and its color was changed from colorless to blue. Finally, catalysate absorption was measured to determine pathogen concentration. This colorimetric immunoassay could determine Salmonella typhimurium from 4 × 101 to 4 × 105 CFU/mL in 60 min with a detection limit of 32 CFU/mL. [ABSTRACT FROM AUTHOR]

Rapid screening of contaminated foods is the key to prevent and control the outbreaks of foodborne diseases. In this study, a novel fluorescent biosensor was developed for ultrasensitive and rapid detection of E. coli O157:H7. The proposed fluorescent biosensor used the double-layer channel with the immune magnetic nanoparticles (MNPs) for specific separation and efficient concentration of the target bacteria, and the immune quantum dots (QDs) with the portable optical system for quantitative detection of the bacteria. First, the bacteria were captured by the immune MNPs in the channel at the presence of the high gradient magnetic fields (HGMFs) to form the MNP-bacteria complexes. Then, the immune QDs were used to react with the target bacteria to form the MNP-bacteria-QDs complexes in the channel. Finally, the enriched complexes were collected and detected using the portable optical system to obtain the fluorescence intensity for final determination of the E. coli O157:H7 cells in the sample. This proposed biosensor was demonstrated to be able to detect E. coli O157:H7 as low as 14 CFU/mL within 2 h. The recovery of E. coli in the spiked milk samples ranged from 95.92% to 108.15%, indicating that it was capable of detecting E. coli in real samples. Besides, this proposed biosensor was able to detect the bacteria in the volume of 10 mL, and has the potential to detect the bacteria in a larger volume to increase the sensitivity. [ABSTRACT FROM AUTHOR]

Early screening of foodborne pathogens is a key to control the outbreaks of foodborne illnesses. In this study, a microfluidic impedance biosensor combined with the immune magnetic nanoparticles (MNPs) for bacteria separation, the urease for biological signal amplification and the microfluidic chip for impedance measurement was developed for rapid, sensitive and continuous-flow detection of E. coli O157:H7. After the streptavidin modified MNPs were conjugated with the biotinylated polyclonal antibodies (PAbs) to form the immune MNPs, the target bacteria was first separated by the MNPs from the background to form the MNP-bacteria complexes. Then, the gold nanoparticles (GNPs) modified with the urease and the aptamers against E. coli O157:H7 were conjugated with the MNP-bacteria to form the MNP-bacteria-GNP-urease complexes. Finally, the complexes were used to catalyze the hydrolysis of urea into ammonium carbonate, leading to the decrease in the impedance. The impedance was online measured by this proposed biosensor and analyzed using the impedance normalization to determine the concentration of E. coli O157:H7. A good linear relationship between the impedance relative change rate of the catalysate and the concentration of the bacteria was obtained with low detection limit of 12 CFU/mL. [ABSTRACT FROM AUTHOR]

Early screening of pathogenic bacteria in foods plays an important role in foodborne disease prevention and control. In this study, we developed an optical biosensor for rapid and sensitive detection of Listeria monocytogenes combining immunomagnetic separation, urease catalysis and pH indication. The magnetic nanobeads (MNBs) conjugated with monoclonal antibodies (MAbs) by streptavidin-biotin binding were used for specifically and efficiently separating the Listeria cells from background to form magnetically labeled bacteria. Then, the gold nanoparticles (AuNPs) modified with urease and polyclonal antibodies (PAbs) by electrostatic adsorption were used to react with the magnetically labeled bacteria to form the MNB-MAb- Listeria -PAb-AuNP-urease complexes. The urease in the complexes could catalyze the hydrolysis of the urea into ammonium carbonate, which could lead to an increase in the pH value of the urea solution. Bromcresol purple was compared with phenol red and bromothymol blue and selected as the best pH indicator to monitor the color change, which could be measured at the characteristic wavelength of 588 nm for bacteria quantification. This proposed biosensor was able to detect Listeria monocytogenes as low as 1.0 × 10 2 CFU/mL, and had the mean recovery of 95.1% for Listeria in the spiked lettuce samples. This optical biosensor has showed its potential to provide a simple, low-cost and sensitive detection method for prevention and control of foodborne diseases. [ABSTRACT FROM AUTHOR]

Combining double-layer capillary based high gradient immunomagnetic separation, invertase-nanocluster based signal amplification and glucose meter based signal detection, a novel biosensor was developed for sensitive and rapid detection of E. coli O157:H7 in this study. The streptavidin modified magnetic nanobeads (MNBs) were conjugated with the biotinylated polyclonal antibodies against E. coli O157:H7 to form the immune MNBs, which were captured by the high gradient magnetic field in the double-layer capillary to specifically separate and efficiently concentrate the target bacteria. Calcium chloride was used with the monoclonal antibodies against E. coli O157:H7 and the invertase to form the immune invertase-nanoclusters (INCs), which were used to react with the target bacteria to form the MNB-bacteria-INC complexes in the capillary. The sucrose was then injected into the capillary and catalyzed by the invertase on the complexes into the glucose, which was detected using the glucose meter to obtain the concentration of the glucose for final determination of the E. coli O157:H7 cells in the sample. A linear relationship between the readout of the glucose meter and the concentration of the E. coli O157:H7 cells (from 10 2 to 10 7 CFU/mL) was found and the lower detection limit of this biosensor was 79 CFU/mL. This biosensor might be extended for the detection of other foodborne pathogens by changing the antibodies and has shown the potential for the detection of foodborne pathogens in a large volume of sample to further increase the sensitivity. [ABSTRACT FROM AUTHOR]

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases. • A novel linear displacement isothermal amplification (LDIA) method was developed by using conventional primers. • Using strand displacement (SD) probes, an LDIA-SD method was established to realize probcleic acid-free extraction step and a pocket-sized device were used to realize its POCT applicate-based specific detection. • A nucleic acid-free extraction step and a pocket-sized device were used to realize its POCT applications. [ABSTRACT FROM AUTHOR]

• A new immunoassay using AI model was developed to rapidly detect candidiasis. • The YOLOv5 model was boosted by coordinate attention mechanism to detect pathogens quantitatively. • Transfer learning and multistage training strategy were used to train the model. • This proposed approach enabled the detection of Candida albicans as low as 3.5×102 CFU/mL in 45 min with an accuracy of 92.7%. Rapid detection of pathogens is of great significance to prevent the outbreak of poultry diseases. In this study, a new immunoassay based on coordinate attention boosted YOLOv5 model and quantum dot labeled fluorescence biosensing was developed to rapidly detect a common fungal infectious poultry disease, candidiasis. The target pathogen, Candida albicans , were specifically labeled by the immune quantum dots to form the fluorescent pathogen. Their fluorescent images were collected using a fluorescence microscope. The YOLOv5 model was boosted by coordinate attention mechanism for identifying the fluorescent pathogen in these images to obtain the quantitative results. With the help of transfer learning and multistage training, the intelligent model was successfully trained and applied. This proposed approach enabled the detection of Candida albicans as low as 3.5 × 102 CFU/mL in 45 min with an accuracy of 92.7 %, which was significantly higher than 87.2 % of YOLOv5 and 81.6 % of Faster RCNN. The new approach was featured with shorter detection time, lower cost and simpler operation, and showed its potential to provide a more efficient solution for pathogen screening. [ABSTRACT FROM AUTHOR]

A new method for online detection of peroxidase (POD) using 3D printing, active magnetic mixing, fluidic control, and optical detection was developed and demonstrated in this study. The proposed POD detection system consisted of a 3D printing and active magnetic mixing based fluidic chip for online catalytic reaction, an optical detector with a fluidic flow cell for quantitative determination of the final catalysate, and a single-chip microcontroller based controller for automatic control of two rotating magnetic fields and four precise peristaltic pumps. Horseradish peroxidase (HRP) was used as research model and a linear relationship between the absorbance at the characteristic wavelength of 450 nm and the concentration of HRP of 1/4–1/128 μg mL−1 was obtained as A  =  0.257ln⁡(C) + 1.425 (R2  = 0.976). For the HRP spiked pork tests, the recoveries of HRP ranged from 93.5% to 110.4%, indicating that this proposed system was capable of detecting HRP in real samples. It has the potential to be extended for online detection of the activity of other enzymes and integration with ELISA method for biological and chemical analysis. [ABSTRACT FROM AUTHOR]

As a result of the low concentration of avian influenza viruses in samples for routine screening, the separation and concentration of these viruses are vital for their sensitive detection. We present a novel three-dimensional printed magnetophoretic system for the continuous flow separation of the viruses using aptamer-modified magnetic nanoparticles, a magnetophoretic chip, a magnetic field, and a fluidic controller. The magnetic field was designed based on finite element magnetic simulation and developed using neodymium magnets with a maximum intensity of 0.65 T and a gradient of 32 T/m for dragging the nanoparticle-virus complexes. The magnetophoretic chip was designed by SOLIDWORKS and fabricated by a three-dimensional printer with a magnetophoretic channel for the continuous flow separation of the viruses using phosphate-buffered saline as carrier flow. The fluidic controller was developed using a microcontroller and peristaltic pumps to inject the carrier flow and the viruses. The trajectory of the virus-nanoparticle complexes was simulated using COMSOL for optimization of the carrier flow and the magnetic field, respectively. The results showed that the H5N1 viruses could be captured, separated, and concentrated using the proposed magnetophoretic system with the separation efficiency up to 88% in a continuous flow separation time of 2 min for a sample volume of 200 μL. [ABSTRACT FROM AUTHOR]

Rapid screening of pathogenic bacteria contaminated foods is the key to prevent and control the outbreaks of foodborne illness. In this study, an impedance biosensor was developed using immunomagnetic nanoparticles for efficient separation and concentration of the Listeria monocytogenes cells, urease for amplifying the weak signal, and screen-printed interdigitated electrode for quantitative measurement of the impedance change of the catalysate. The magnetic nanoparticles (MNPs) coated by the monoclonal antibodies (MAbs) were used to separate the Listeria cells from the background and concentrate them in small volume of PBS. Then, the gold nanoparticles (GNPs) modified with the urease and the polyclonal antibodies (PAbs) were used to react with Listeria to form the MNP- Listeria -GNP sandwich complexes. The complexes were re-suspended with the urea to catalyze the hydrolysis of the urea into ammonium ions and carbonate ions, which were measured by the electrode. A new equivalent circuit was designed for simulation of the biosensor with a good fitting result. Under the optimized conditions, a linear relationship between the impedance changes and the concentrations of Listeria from 1.9 × 10 3 to 1.9 × 10 6 CFU/mL was obtained. The limit of detection of this biosensor was 1.6 × 10 3 CFU/mL and the recovery of the spiked lettuce sample ranges from 94.7% to 103.8%. This proposed biosensor was developed at much lower cost than our previous studies and could be more applicable for in-field detection of foodborne pathogens. [ABSTRACT FROM AUTHOR]

Early finding of pathogens is significant to avoid foodborne diseases. Here, a novel lab-in-centrifugal-tube colorimetric biosensor was reported for Salmonella typhimurium detection using immune nickel nanowires (NNWs) to form capture nets for specific bacterial separation, gold@platinum nanozymes (GPNs) to mark target bacteria for effective signal amplification, and a smartphone App to analyze color change for quantitative bacterial determination. A 3D-printed cylindrical magnetic separator with air pressure self-regulating structure and NNW capture nets was elaboratively constructed and assembled inside the disposable centrifuge tube to simply perform the bacterial separation, label, wash, coloration and detection. Under optimal conditions, Salmonella typhimurium could be quantitatively detected in 2 h with a low detection limit of 21 CFU/mL. The recovery of target bacteria in spiked pork samples ranged from 87.0% to 97.6% with the averaged recovery of 93.9%. This biosensor was Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free and Deliverable to end-users (ASSURED), and had shown the potential for point-of-care testing of foodborne pathogens to ensure food safety. [ABSTRACT FROM AUTHOR]

A novel colorimetric biosensor was explored for fast, sensitive, and on-site detection of Salmonella on a microfluidic chip employing immune gold@platinum nanoparticles (Au@Pt NPs) for specific bacterial labeling, a finger-driven mixer with two serial air chambers for efficient immunoreaction and a nuclear track membrane as specific-size microfilter for effective bacterial isolation from excessive immune Au@Pt NPs. First, the bacterial sample and immune Au@Pt NPs were pipetted into the mixing chamber and mixed sufficiently through repeated press-release actions on the small air chamber which could precisely control the air volume, leading to the formation of bacteria-immune Au@Pt NP conjugates. Then, the small and large air chambers were pressed simultaneously to push all the solution in the mixing chamber to flow through the microfilter for trap of the formed larger-size conjugates on the membrane and removal of the unbound smaller-size immune Au@Pt NPs. After the H 2 O 2 -TMB substrate was pipetted into the microfilter and catalyzed by the conjugates, ImageJ was used to analyze the color change for bacterial determination. This simple biosensor enabled Salmonella detection as low as 168 CFU/mL in 25 min. [ABSTRACT FROM AUTHOR]

Screening of pathogenic bacteria is an essential measure for ensuring food safety. A fast, sensitive and automatic platform for Salmonella determination was presented employing continuous-flow magnetic isolation, enzymatic impedance amplification and smartphone data analysis. First, magnetic nanoparticles coupling with polyclonal antibody, polystyrene spheres coupling with monoclonal antibody and glucose oxidase, and bacterial sample were simultaneously immitted into reaction chamber, and actively mixed for formation of MNP- Salmonella -PS complexes in the microfluidic chip. After the MNP- Salmonella -PS complexes were continuous-flow caught in separation channel using the enhanced magnetic field and successively washed for removing redundant immune PSs and remanent ions, glucose with high impedance was then pumped and catalyzed into hydrogen peroxide and gluconic acid. Finally, impedance change of catalysate was dynamically measured with the on-chip PCB electrode and analyzed by the self-developed smartphone to quantify target bacteria. The microfluidic platform was able to quantitatively detect Salmonella from 1.3 × 102 to 1.3 × 106 CFU/mL in 1.5 h with a detection limit of 53 CFU/mL. The recovery of Salmonella in spiked chicken meats ranged from 88.2% to 112.0%, verifying its good practical applicability. This platform is featured with fast detection, high sensitivity and simple operation, and promising for in-field Salmonella screening. • An active stirring mixer in microfluidic chip was elaborately developed for efficient mixing. • The on-chip PCB electrode was proved for dynamic and accurate measurement of impedance. • Glucose-glucose oxidase-gluconic acid system was employed for effectively signal amplification. • This biosensor integrated whole detection procedures onto a chip to achieve automatic operations. • Salmonella typhimurium was detected to be as few as 53 CFU/mL within 1.5 h. [ABSTRACT FROM AUTHOR]

Early screening of pathogenic bacteria is a key to prevent and control of foodborne diseases. In this study, we developed a fast and sensitive bacteria detection method integrating electrochemical impedance analysis, urease catalysis with microfluidics and using Listeria as model. The Listeria cells, the anti- Listeria monoclonal antibodies modified magnetic nanoparticles (MNPs), and the anti- Listeria polyclonal antibodies and urease modified gold nanoparticles (AuNPs) were incubated in a fluidic separation chip with active mixing to form the MNP- Listeria -AuNP-urease sandwich complexes. The complexes were captured in the separation chip by applying a high gradient magnetic field, and the urea was injected to resuspend the complexes and hydrolyzed under the catalysis of the urease on the complexes into ammonium ions and carbonate ions, which were transported into a microfluidic detection chip with an interdigitated microelectrode for impedance measurement to determine the amount of the Listeria cells. The capture efficiency of the Listeria cells in the separation chip was ∼93% with a shorter time of 30 min due to the faster immuno-reaction using the active magnetic mixing. The changes on both impedance magnitude and phase angle were demonstrated to be able to detect the Listeria cells as low as 1.6×10 2 CFU/mL. The detection time was reduced from original ∼2 h to current ∼1 h. The recoveries of the spiked lettuce samples ranged from 82.1% to 89.6%, indicating the applicability of this proposed biosensor. This microfluidic impedance biosensor has shown the potential for online, automatic and sensitive bacteria separation and detection. [ABSTRACT FROM AUTHOR]

In-field screening of pathogenic bacteria is important for preventing food poisoning. Here, a portable pipette-adapted biosensor using magnetic grid separation and nanocatalyst signal amplification was elaboratively developed for rapid detection of Salmonella typhimurium. A common pipette was innovatively adapted with multiple functions to complete the whole bacterial detection procedure, including mixing, separation, catalysis, washing, detection, analysis and display. The target bacteria were effectively captured by the immune magnetic nanobeads and labeled with immune gold@platinum nanocatalysts through pipette-blowing mixing to form the nanobeads-bacteria-nanocatalyst complexes, which were separated against the magnetic grid separation tip under the magnetic field. The pressure change resulting from oxygen production due to mimicking catalysis of hydrogen peroxide by these nanocatalysts on the complexes was quantified through measuring the moving duration of the conductive liquid in the pipette for bacteria determination. Under optimal conditions, this biosensor could detect target bacteria in 90 min with low detection limit of 180 CFU/mL. This pipette-adapted biosensor is affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to end-users (ASSURED), and has the potential for in-field testing of foodborne pathogens to ensure food safety, especially in resource-constrained areas. [ABSTRACT FROM AUTHOR]

An hourglass-mimicking biosensor was developed to detect target bacteria in 15 mL centrifugal tube using immune magnetic nanobeads to isolate the target from large-volume sample, gold@platinum nanozymes to label the target for amplification of biological signals, and a microplate reader/colorimetric card for determination of the target. First, a centrifugal tube with an iron ball framework was first coaxially assembled in the center of a Halbach ring magnet. After immune magnetic nanobeads, gold@platinum nanozymes and bacterial sample were mixed by repeated bottom-up of the tube using a stepper motor, nanobead-bacteria-nanozyme complexes were formed. Then, colorless H 2 O 2 -TMB was catalyzed by the nanozymes to produce blue TMBox. The color change was finally analyzed using the microplate reader or colorimetric card to determine bacterial concentration. This hourglass-mimicking biosensor could separate ∼95% targets from 10 mL bacteria sample and detect targets from 1.6 × 101 to 1.6 × 106 CFU/mL in 1.0 h with low detection limit of 16 CFU/mL. [ABSTRACT FROM AUTHOR]

Pathogen screening is an important measure for ensuring food safety. A colorimetric biosensor was presented for Salmonella determination employing staggered magnetic bead chains for continuous-flow bacteria isolation, MnO 2 nanozymes for effective signal amplification, and smartphone for accurate catalysate analysis. First, immune magnetic beads were distributed under the staggered magnetic field to form higher magnetic bead chains to occupy more cross-section of separation channel. Then, target bacteria were injected and captured onto magnetic bead chains, followed by labeling with the immune MnO 2 nanozymes to form bead-bacteria-nanozyme complexes. Finally, H 2 O 2 -TMB substrate was catalyzed by MnO 2 nanozymes on these complexes, and color change was monitored and analyzed through smartphone to quantify target bacteria. The sensor enabled Salmonella detection as few as 60 CFU/mL in 2 h. • The staggered magnetic field could enhance the occupation of MB chains in the channel. • The MnO 2 nanoflowers were used to greatly amplify the biological signal. • The smartphone App was developed for accurate analysis of color saturation. • This biosensor could detect Salmonella as low as 60 CFU/mL within 2 h. [ABSTRACT FROM AUTHOR]

In this study, a power-free biosensor was presented to detect Salmonella typhimurium on a microfluidic chip using a slide multivalve for channel selection and a disposable syringe for fluidic transfer. First, bacterial sample with immunomagnetic nanoparticles (IMNPs) and glucose oxidase (GOx) modified immune polystyrene nanoparticles (IPNPs), washing buffer, glucose, and peroxide test strip (PTS) were preloaded in their respective chambers at the periphery of chip. After the slide multivalve was selected to connect sample chamber with common separation chamber, which was connected with a syringe, the mixture of Salmonella , IMNPs and IPNPs was back and forth moved through 3D Tesla-structure micromixer using the syringe, resulting in the formation of IMNP- Salmonella -IPNP complexes, which were captured in the separation chamber using a magnet. Then, two washing chambers were selectively connected respectively to remove sample background and excessive IPNPs, and glucose chamber was connected, allowing the GOx to catalyze glucose to produce hydrogen peroxide in the separation chamber. Finally, PTS chamber was connected and the catalysate was transferred from the separation chamber to the PTS chamber, leading to the color change of PTS, followed by using smartphone App to collect and analyze the image of PTS for bacterial determination. The simple biosensor enabled simple detection of Salmonella as few as 130 CFU/mL within 60 min and is promising for practical applications in the resource-limited regions due to its low cost, simple operation, and small size. [ABSTRACT FROM AUTHOR]

Rapid detection of antibiotic resistance genes (ARGs) of food microorganisms is critical for preventing the spread of ARGs via the food chain. In this study, slipchip-based immunomagnetic separation (IMS) combined with loop-mediated isothermal amplification (LAMP) was developed for rapid, specific, sensitive and multi-sample detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk. First, the magnetic nanoparticles coated with anti- B. cereus polyclonal antibodies were mixed with samples in the magnetic capture chamber of the slipchip, and the capture efficiency of Bacillus cereus (105 CFU/mL) can reach 80% after incubation for 1h. Then, the captured bacteria were boiled in the lysis chamber using a heating block and the extracted DNA was added into the LAMP reaction as templates. Finally, real-time fluorescent LAMP detection for tetL gene of Bacillus cereus was performed at 65 °C for 50 min. Under optimal conditions, the IMS-LAMP could detect 11.6 CFU/mL of Bacillus cereus with tetL gene in pure culture. Furthermore, the limit of detection of Bacillus cereus with tetL gene in pasteurized milk was 21.5 CFU/mL by the slipchip-based IMS-LAMP within 2 h. Therefore, this method is potentially useful for the rapid detection of Bacillus cereus with tetracycline resistance gene tetL in pasteurized milk and could be extended for other resistance genes. • This method could detect 21.5 CFU/mL of B. cereus with tetL in pasteurized milk. • The IMBs could capture more than 80% B. cereus in 1 h. • Real-time fluorescent LAMP enabled specific detection for tetL of B. cereus. • The slipchip made the method easier, quicker and allowed multi-sample detection. • The detection process could be completed within 2 h. [ABSTRACT FROM AUTHOR]

Foodborne probiotics substantially impact human health. Thus, there is an urgent need for real-time and in situ detection of targeted probiotics. In this paper, a novel nanopopcorn fluorescent probe based on DNA-mediated Au@Ag@silica was designed and shown to display plasma resonance characteristics that generated more hot spots. This led to >18-fold greater fluorescence of indocyanine green dye with strong emission in the near-infrared (NIR-I and NIR-II) regions. Moreover, the new fluorescent probe exhibited high stability and low biotoxicity, having a strong linear relationship (R2 = 0.9615) with Lactobacillus Plantarum concentration over the range of 105−109 cfu/mL; it enabled in vivo tracing of exogenous probiotics. Compared with the traditional inductively coupled mass spectrometry (ICP-MS), the results are consistent (Correlation coefficient = 0.994), but the analysis time is much reduced by 89.6%. It is remarkable that the probe enabled real-time and in situ monitoring of target probiotic behavior in a simple, robust, and effective manner. [ABSTRACT FROM AUTHOR]

Early screening for food pathogens, specifically Escherichia coli O157:H7 in ready-to-eat food remains a central challenge, hyperspectral imaging (HSI) has the potential to acquire both image and spectral information of the target and is simple enough to serve as an effective auxiliary technology in food monitoring. Herein, a visual method was developed based on spectral angle mapping (SAM) and dark-field microscopy for rapid detection and quantification of Escherichia coli. First, immunomagnetic nanoparticle (MNP-probe) and gold nanoparticle (AuNP-probe) probes were prepared to form a sandwich complex for efficient concentration of the target bacteria. The complexes were then dropped directly on a glass slide for colony counting with hyperspectral dark field microscopy. The AuNP-probe bound to the complex was counted and SAM was used to evaluate the spectra and HSI image data to distinguish the target pixel from the background. The linear detection range of the method was estimated to be in the range between 6.1 × 101 and 6.1 × 106 CFU/mL in the milk sample, and the detection limit (LOD) of this method was estimated to be ~61 CFU/mL. The average recovery of organisms in the milk sample was 106.0%, indicating that this method can be utilized to detect Escherichia coli in food samples and serve as an effective method to monitor pathogens in a complex sample. • Colorimetric quantification based on spectral angle mapping (SAM) and hyperspectral dark-field imaging. • Detection of Escherichia coli as low as 61 CFU/mL within ~80 min. • Excellent recovery of organisms. • High specificity of detection was also demonstrated. [ABSTRACT FROM AUTHOR]

Rapid and sensitive screening of pathogens is a key to prevent the outbreak of foodborne illnesses. Herein, a new colorimetric immunoassay was proposed based on the release of Ag+ ions from AgNPs and the inhibition of PtNPs, and its supporting microfluidic platform was developed to automatically perform the whole bacterial detection procedure using Raspberry Pi and smartphone App. First, the immune AgNPs and magnetic nanobeads (MNBs) were used to conjugate with Salmonella typhimurium. Then, H 2 O 2 was used to etch the AgNPs for release Ag+ ions. Finally, the colorimetric signal was greatly diminished because of the specific and efficient inhibition of Ag+ toward the peroxidase-like activity of the PtNPs. This colorimetric immunoassay showed a good specificity and an ultralow detection limit of 16.8 CFU/mL, which was about 3 orders of magnitude improvement compared with conventional ELISA, and the averaged recovery for the spiked chicken samples was 95.6%. The combination of this immunoassay with this microfluidic platform might be promising for rapid and sensitive screening of foodborne pathogens to ensure food safety. [Display omitted] • A colorimetric immunoassay was proposed by releasing Ag + ions from AgNPs to inhibit PtNPs for bacterial detection. • A new microfluidic platform was developed to automatically perform the whole bacterial detection procedure. • This platform could detect Salmonella typhimurium as low as 16.8 CFU/mL in 1 h. [ABSTRACT FROM AUTHOR]