Multiplex nanozymatic biosensing of Salmonella on a finger-actuated microfluidic chip.

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.

Cost-Efficient Micro-Well Array-Based Colorimetric Antibiotic Susceptibility Testing (MacAST) for Bacteria from Culture or Community.

Rapid and cost-efficient antibiotic susceptibility testing (AST) is key to timely prescription-oriented diagnosis and precision treatment. However, current AST methods have limitations in throughput or cost effectiveness, and are impractical for microbial communities. Here, we developed a high-throughput micro-well array-based colorimetric AST (macAST) system equipped with a self-developed smartphone application that could efficiently test sixteen combinations of bacteria strains and antibiotics, achieving comparable AST results based on resazurin metabolism assay. For community samples, we integrated immunomagnetic separation into the macAST (imacAST) system to specifically enrich the target cells before testing, which shortened bacterial isolation time from days to only 45 min and achieved AST of the target bacteria with a low concentration (~103 CFU/mL). This proof-of-concept study developed a high-throughput AST system with an at least ten-fold reduction in cost compared with a system equipped with a microscope or Raman spectrum. Based on colorimetric readout, the antimicrobial susceptibility of the bacteria from microbial communities can be delivered within 6 h, compared to days being required based on standard procedures, bypassing the need for precise instrumentation in therapy to combat bacterial antibiotic resistance in resource-limited settings.

Magnetic nanobead chain-assisted real-time impedance monitoring using PCB interdigitated electrode for Salmonella detection.

Pathogen testing is effective to prevent food poisoning. Here, an electrochemical biosensor was explored for Salmonella detection by combining magnetic grid based bacterial separation with enzymatic catalysis based signal amplification on a PCB interdigitated electrode in a microfluidic chip. First, immune magnetic nanobeads, target bacteria, and immune polystyrene microspheres decorated with glucose oxidase were sufficiently mixed to form nanobead-bacteria-microsphere sandwich conjugates. Then, these conjugates were injected into the chip to form conjugate chains right over the electrode under an iron grid enhanced magnetic field. After non-conductive glucose was injected and catalyzed by glucose oxidase on the conjugate chains, conductive glucose acid and non-conductive hydrogen peroxide were continuously produced and rapidly diffused from the conjugate chains to the electrode. Finally, the impedance change was real-timely monitored and used to determine the bacterial amount. This sensor enabled detection of 50 CFU/mL Salmonella typhimurium in 1 h.

Computer vision-assisted smartphone microscope imaging digital immunosensor based on click chemistry-mediated microsphere counting technology for the detection of aflatoxin B1 in peanuts.

Aflatoxin B1 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 B1 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 B1. The customized computer vision procedure was used to efficiently identify and count the obtained PS probes. This SMID immunosensor enables sensitive detection of aflatoxin B1 with a linear range from 0.001 ng/mL to 500 ng/mL, providing a simple, sensitive, and portable tool for food safety supervision.

Rapid detection of multiple antibiotics in chicken samples via a fluorescence nanobiosensor coupled with a homemade fluorescence analyzer.

Antibiotic residues in foods pose a serious threat to human health. However, routine analysis techniques require bulky laboratory instruments and skilled personnel or give single-channel analysis results, exhibiting low practicality. Here, we explored a rapid and easy-to-use detection system combining a fluorescence nanobiosensor with a homemade fluorescence analyzer for the simultaneous identification and quantification of multiple antibiotics. The nanobiosensor assay worked based on the targeted antibiotics competing with signal labels of antigen-quantum dots (IQDs) to bind with recognition elements of antibody-magnetic beads (IMBs). The fluorescence signals of IMB-unbound IQDs in a magnetically separated supernatant, related to antibiotic concentration, were automatically collected and processed by our self-designed and homemade fluorescence analyzer which integrated mechanical control hardware (consisting of a mechanical arm, a ten-channel rotary bench, and an optical detection unit) and user control software (installed on a built-in laptop). The fluorescence analyzer enabled the analysis of 10 samples within 5 min in one round and permitted the real-time uploading of sample data to the cloud. By employing three QDs with emission wavelengths of 525 nm, 575 nm, and 625 nm, this multiplex fluorescence biosensing system demonstrated great sensitivity and accuracy for simultaneously analyzing enrofloxacin, tilmicosin, and florfenicol in chicken samples with detection limits of 0.34 µg kg-1, 0.7 µg kg-1, and 0.16 µg kg-1, respectively. Moreover, the biosensing platform performed well in a wealth of chicken samples covering various breeds from three Chinese cities. This study identifies a generic and user-friendly multiplex biosensor platform with significant potential for use in food safety and regulation.

A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for Salmonella Detection.

BACKGROUND: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. METHODS: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. RESULTS: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 102 CFU/mL Salmonella Typhimurium was over 2000 RFU, while 104 CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). CONCLUSIONS: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.

Semi-circle magnetophoretic separation under rotated magnetic field for colorimetric biosensing of Salmonella.

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.

A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases.

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 three-in-one hybrid nanozyme for sensitive colorimetric biosensing of pathogens.

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 H2O2-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.

Power-free colorimetric biosensing of foodborne bacteria in centrifugal tube.

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.

A microfluidic biosensor based on finger-driven mixing and nuclear track membrane filtration for fast and sensitive detection of Salmonella.

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 H2O2-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.

A pipette-adapted biosensor for Salmonella detection.

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.

Hourglass-mimicking biosensor based on disposable centrifugal tube for bacterial detection in large-volume sample.

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 H2O2-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.

An automatic centrifugal system for rapid detection of bacteria based on immunomagnetic separation and recombinase aided amplification.

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.

A Salmonella Microfluidic Chip Combining Non-Contact Eddy Heater and 3D Fan-Shaped Mixer with Recombinase Aided Amplification.

Foodborne pathogenic bacteria have become a worldwide threat to human health, and rapid and sensitive bacterial detection methods are urgently needed. In this study, a facile microfluidic chip was developed and combined with recombinase-aided amplification (RAA) for rapid and sensitive detection of Salmonella typhimurium using a non-contact eddy heater for dynamic lysis of bacterial cells and a 3D-printed fan-shaped active mixer for continuous-flow mixing. First, the bacterial sample was injected into the chip to flow through the spiral channel coiling around an iron rod under an alternating electromagnetic field, resulting in the dynamic lysis of bacterial cells by this non-contact eddy heater to release their nucleic acids. After cooling to ~75 °C, these nucleic acids were continuous-flow mixed with magnetic silica beads using the fan-shaped mixer and captured in the separation chamber using a magnet. Finally, the captured nucleic acids were eluted by the eluent from the beads to flow into the detection chamber, followed by RAA detection of nucleic acids to determine the bacterial amount. Under the optimal conditions, this microfluidic chip was able to quantitatively detect Salmonella typhimurium from 1.1 × 102 to 1.1 × 105 CFU/mL in 40 min with a detection limit of 89 CFU/mL and might be prospective to offer a simple, low-cost, fast and specific bacterial detection technique for ensuring food safety.

Immuno-PET Imaging of TNF-α in Colitis Using 89Zr-DFO-infliximab.

Tumor necrosis factor-alpha (TNF-α) neutralization has become increasingly important in the treatment of inflammatory bowel diseases (IBD). A series of monoclonal antibodies were approved in the clinic for anti-TNF-α therapy. However, a comprehensive assessment of TNF-α levels throughout the colon, which facilitates the diagnosis of IBD and predicts anti-TNF-α efficacy, remains challenging. Here, we radiolabeled infliximab with long-lived radionuclides 89Zr for immuno-positron emission tomography (PET) imaging of TNF-α in vivo. The increased TNF-α level was detected in the inflammatory colon of the dextran sodium sulfate-induced colitis mice. The immuno-PET imaging of 89Zr-desferrioxamine-infliximab reveals a high uptake (7.1 ± 0.3%ID/g) in the inflammatory colon, which is significantly higher than in the healthy control and blocked groups. The colon-to-muscle ratio reached more than 10 and was maintained at a high level for 10 h after injection. The ex vivo biodistribution study also verified the superior uptake in the inflammatory colon. This study provides an in vivo immune-PET approach to molecular imaging of the pro-inflammatory cytokine TNF-α. It is promising in diagnosing and predicting efficacy in both IBD and other autoimmune diseases.

Sample-in-answer-out colorimetric detection of Salmonella typhimurium using non-enzymatic cascade amplification.

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, H2O2 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.

Power-free microfluidic biosensing of Salmonella with slide multivalve and disposable syringe.

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.

Simple and rapid separation of Haematococcus pluvialis and ciliate based on the dean-coupled inertial microfluidics.

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.

DNA-mediated Au@Ag@silica nanopopcorn fluorescent probe for in vivo near-infrared imaging of probiotic Lactobacillus Plantarum.

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.