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 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.

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.

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.

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.

A finger-actuated microfluidic biosensor for colorimetric detection of foodborne pathogens.

A microfluidic biosensor was developed for rapid detection of Salmonella using a finger-actuated micropump, a finger-actuated micromixer, gold@platinum nanocatalysts (Au@PtNCs) and a smartphone App. First, immune magnetic nanobeads (MNBs), bacterial sample and immune Au@PtNCs were successively finger-actuated pumped into microfluidic chip. Then, they were fully mixed using finger-actuated micromixer to form MNB-Salmonella-Au@PtNC complexes. After hydrogen peroxide-tetramethylbenzidine was pumped into chip and catalyzed by nanocatalysts on complexes, resulting in color change from colorless to blue, the image of catalysate was collected and finally analyzed by self-developed smartphone App or directly compared with Pantone color card in chip to determinebacterial concentration. Experimental results showed this biosensor could quantitatively detect Salmonella from 3.5 × 102 to 3.5 × 105 CFU/mL in 1 h with lower detection limit of 350 CFU/mL. This biosensor has successfully integrated loading, mixing, incubation, washing, separation and detection onto a chip and might pave a promising way for bacterial detection.

A lab-on-a-tube biosensor for automatic detection of foodborne bacteria using rotated Halbach magnetic separation and Raspberry Pi imaging.

A lab-on-a-tube biosensor was established to rapidly, sensitively and automatically detect foodborne bacteria through a rotatable Halbach magnet to form and rotate magnetic nanobead (MNB) chains for specific isolation of target bacteria, gold@platinum nanocatalysts (Au@PtNCs) to label target bacteria for efficient amplification of detection signal and Raspberry Pi App to collect and analyze the image of catalysate. First, the glass tube was successively preloaded with the mixture of MNBs, sample and Au@PtNCs, the washing buffer (skim milk) and the substrate (hydrogen peroxide-3,30,5,50-tetramethylbenzidine), and they were separated by air gaps. After the tube was placed on the biosensor, the MNB chains were stably formed and continuously rotated using the Halbach magnet and the mixture was moved back and forth using a programmable peristaltic pump, thus making the formation of MNB-bacteria-Au@PtNCs complexes. After the washing buffer was moved to wash the complexes, the substrate was then moved to resuspend the complexes, resulting in the catalytic reaction that changed the color of the substrate. Finally, the catalysate was moved to the designated area, the image of which was analyzed by the Raspberry Pi App to quantitatively determine the concentration of bacteria in the samples. This biosensor was able to detect Salmonella in spiked chicken samples in 1 h with lower detection limit of 8 CFU/50 µL and a recovery from 88.96% to 99.74%. This biosensor based on a single tube is very promising to automatically detect foodborne bacteria due to its low cost, high integration and simple operation.

Automatic and multi-channel detection of bacteria on a slidable centrifugal disc based on FTA card nucleic acid extraction and recombinase aided amplification.

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.

Challenges and Perspectives for Biosensing of Bioaerosol Containing Pathogenic Microorganisms.

As an important route for disease transmission, bioaerosols have received increasing attention. In the past decades, many efforts were made to facilitate the development of bioaerosol monitoring; however, there are still some important challenges in bioaerosol collection and detection. Thus, recent advances in bioaerosol collection (such as sedimentation, filtration, centrifugation, impaction, impingement, and microfluidics) and detection methods (such as culture, molecular biological assay, and immunological assay) were summarized in this review. Besides, the important challenges and perspectives for bioaerosol biosensing were also discussed.

Microfluidic Colorimetric Biosensors Based on MnO2 Nanozymes and Convergence-Divergence Spiral Micromixers for Rapid and Sensitive Detection of Salmonella.

In-field screening of foodborne pathogens plays an important role in ensuring food safety. Thus, a microfluidic biosensor was developed for rapid and sensitive detection of Salmonella using manganese dioxide nanoflowers (MnO2 NFs) for amplifying the biological signal, a microfluidic chip with a convergence-divergence spiral micromixer for performing automatic operations, and a smartphone app with a saturation calculation algorithm for processing the image. First, immune magnetic nanoparticles (MNPs), the sample, and immune MnO2 NFs were fully mixed and sufficiently incubated in the spiral micromixer to form MNP-bacteria-MnO2 sandwich complexes, which were magnetically captured in a separation chamber in the microfluidic chip. Then, a 3,3',5,5'-tetramethylbenzidine (TMB) substrate was injected and catalyzed by a MnO2 NF nanomimetic enzyme on the complexes, resulting in the production of yellow catalysate. Finally, the catalysate was transferred into a detection chamber and its image was measured and processed using the smartphone app to determine the number of bacteria. This biosensor was able to detect Salmonella from 4.4 × 101 to 4.4 × 106 CFU/mL in 45 min with a detection limit of 44 CFU/mL, and has the potential to provide a promising platform for on-site detection of foodborne bacteria.

Recent Advances on Bioaerosol Collection and Detection in Microfluidic Chips.

Bioaerosols containing pathogenic microorganisms have posed a great threat to human and animal health. Effective monitoring of bioaerosols containing pathogenic viruses and bacteria is of great significance to prevent and control infectious diseases. This Feature summarizes recent advances on bioaerosol collection and detection based on microfluidic chips. Besides, the challenges and trends for bioaerosol collection and detection were also discussed.

Magnetic Bead Chain-Based Continuous-Flow DNA Extraction for Microfluidic PCR Detection of Salmonella.

Nucleic acid extraction is crucial for PCR detection of pathogenic bacteria to ensure food safety. In this study, a new magnetic extraction method was developed using 3D printing and magnetic silica beads (MSBs) to extract the target DNA from a large volume of bacterial sample and combined with microfluidic PCR to determine the bacteria. After proteinase K was added into a bacterial sample to lyse the bacteria and release the DNA, it was continuous-flow injected into the serpentine channel of the extraction chip, where magnetic silica bead chains had been formed in advance using a homogeneous magnetic field generated by two concentric semicircle magnets to capture the MSBs. Then, the flowing DNA was captured by the MSB chains, washed with alcohol, dried with gas, and eluted with deionized water to obtain the purified and concentrated DNA. Finally, the extracted DNA templates were injected into a microfluidic PCR chip with lyophilized amplification reagents and determined using a commercial qPCR device. The experimental results showed that the DNA extraction efficiency was more than 90%, and the lower detection limit of Salmonella was 102 CFU/mL. This new Salmonella detection method is promising to provide the rapid, sensitive, and simultaneous detection of multiple foodborne pathogens.

A microfluidic biosensor for rapid and automatic detection of Salmonella using metal-organic framework and Raspberry Pi.

Rapid screening of pathogenic bacteria contaminated foods is crucial to prevent food poisoning. However, available methods for bacterial detection are still not ready for in-field screening because culture is time-consuming; PCR requires complex DNA extraction and ELISA lacks sensitivity. In this study, a microfluidic biosensor was developed for rapid, sensitive and automatic detection of Salmonella using metal-organic framework (MOF) NH2-MIL-101(Fe) with mimic peroxidase activity to amplify biological signal and Raspberry Pi with self-developed App to analyze color image. First, the target bacteria were separated and concentrated with the immune magnetic nanobeads (MNBs), and labeled with the immune MOFs to form MNB-Salmonella-MOF complexes. Then, the complexes were used to catalyze colorless o-phenylenediamine and H2O2 to produce yellow 2,3-diaminophenazine (DAP). Finally, the image of the catalysate was collected under the narrow-band blue light and analyzed using the Raspberry Pi App to determine the bacterial concentration. The experimental results showed that this biosensor was able to detect Salmonella Typhimurium from 1.5 × 101 to 1.5 × 107 CFU/mL in 1 h with the lower detection limit of 14 CFU/mL. The mean recovery for Salmonella in spiked chicken meats was ~112%. This biosensor integrating mixing, separation, labelling and detection onto a single microfluidic chip has demonstrated the merits of automatic operation, fast reaction, less reagent and small size, and is promising for in-field detection of foodborne bacteria.

An ultrasensitive impedance biosensor for Salmonella detection based on rotating high gradient magnetic separation and cascade reaction signal amplification.

An impedance biosensor using rotary magnetic separation and cascade reaction was developed for rapid and ultrasensitive detection of Salmonella typhimurium. First, magnetic nanoparticles (MNPs) modified with anti-Salmonella monoclonal antibodies were injected into a capillary at the presence of a rotary high gradient magnetic field, which was rotated by a stepper motor. Then, a bacterial sample was injected into the capillary and the target bacteria were continuous-flow captured onto the MNPs. After organic-inorganic hybrid nanoflowers were prepared using manganese dioxide (MnO2), glucose oxidase (GOx) and anti-Salmonella polyclonal antibodies (pAbs), they were injected to label the bacteria, resulting in the formation of MNP-bacteria-nanoflower sandwich complexes. Finally, glucose (low conductivity) was injected and oxidized by GOx on the complexes to produce H2O2 (low conductivity) and gluconic acid (high conductivity), leading to impedance decrease. Besides, the produced H2O2 triggered a cascade reduction of MnO2 into Mn2+, leading to further impedance decrease. The impedance changes were measured using an interdigitated microelectrode and used to determine the concentration of target bacteria. This biosensor was able to detect Salmonella ranging from 101 to 106 CFU/mL in 2 h with a low detection limit of 101 CFU/mL and a mean recovery of 100.1% for the spiked chicken samples.

An impedance biosensor based on magnetic nanobead net and MnO2 nanoflowers for rapid and sensitive detection of foodborne bacteria.

Screening of pathogenic bacteria in foods is an effective way to prevent foodborne diseases. In this study, an impedance biosensor was developed for rapid and sensitive detection of Salmonella typhimurium using multiple magnetic nanobead (MNB) nets in a ring channel for continuous-flow separation of target bacteria from 10 mL of sample, manganese dioxide nanoflowers (MnO2 NFs) for efficient amplification of biological signal, and an interdigitated microelectrode for sensitive measurement of impedance change. First, the MNBs modified with capture antibodies were vortically injected from outer periphery of this ring channel to form multiple ring MNB nets at specific locations with high gradient magnetic fields. Then, the bacterial sample was continuous-flow injected, resulting in specific capture of target bacteria onto the nets, and the MnO2 NFs modified with detection antibodies were injected to form MNB-bacteria-MnO2 NF complexes. After the complexes were washed with deionized water to remove excessive nanoflowers and residual ions, H2O2 with poor conductivity was injected to reduce MnO2 NFs to conductive Mn2+ at neutral medium, leading to impedance decrease. Finally, impedance change was measured using the microelectrode for quantitative determination of Salmonella. This biosensor was able to separate ~60% of Salmonella from 10 mL of bacterial sample and detect Salmonella with a linear range of 3.0 × 101 to 3.0 × 106 CFU/mL in 1.5 h with lower detection limit of 19 CFU/mL. This biosensor might be further improved with higher sensitivity using a larger volume (100 mL or more) for routine screening of foodborne bacteria without bacterial pre-culture.

An ultrasensitive biosensor for fast detection of Salmonella using 3D magnetic grid separation and urease catalysis.

Screening of pathogenic bacteria plays a crucial role in preventing foodborne disease outbreaks. In this study, an ultrasensitive biosensor was developed for fast detection of Salmonella using self-assembled magnetic nanoparticle (MNP) chains for continuous-flow separation of Salmonella from large-volume sample, urease coated gold nanoparticles (GNPs) for specific labelling of Salmonella and efficient amplification of signal, and linear scan voltammetry for sensitive detection of catalysate. First, MNP chains were formed and distributed in a 3D spiral channel using mutually repelling cylindrical magnets and ring iron gears to control anti-Salmonella monoclonal antibody coated MNPs. After bacterial sample was continuous-flow drawn into the channel, bacteria-MNP complexes (magnetic bacteria) were formed on the chains, resulting in specific separation of target bacteria from sample background. Then, anti-Salmonella polyclonal antibodies and urease coated GNPs were drawn to label the magnetic bacteria, resulting in the formation of enzymatic bacteria. After washing to remove residual GNPs, urea was drawn and catalyzed by urease on enzymatic bacteria, resulting in the produce of catalysate (ammonium carbonate). Finally, the catalysate was transferred into a microfluidic chip with a thin-film Ag/AgCl reference electrode array for linear scan voltammetric measurement, and the resistance of catalysate was obtained to determine the amount of target bacteria. This biosensor could quantitatively detect Salmonella from 1.0 × 101 to 1.0 × 106 CFU/mL in 1 h with low detection limit of 101 CFU/mL. The mean recovery for Salmonella in spiked milk was about 104.3%.

A Microfluidic Biosensor Based on Magnetic Nanoparticle Separation, Quantum Dots Labeling and MnO2 Nanoflower Amplification for Rapid and Sensitive Detection of Salmonella Typhimurium.

Screening of foodborne pathogens is an effective way to prevent microbial food poisoning. A microfluidic biosensor was developed for rapid and sensitive detection of Salmonella Typhimurium using quantum dots (QDs) as fluorescent probes for sensor readout and manganese dioxide nanoflowers (MnO2 NFs) and as QDs nanocarriers for signal amplification. Prior to testing, amino-modified MnO2 nanoflowers (MnO2-NH2 NFs) were conjugated with carboxyl-modified QDs through EDC/NHSS method to form MnO2-QD NFs, and MnO2-QD NFs were functionalized with polyclonal antibodies (pAbs) to form MnO2-QD-pAb NFs. First, the mixture of target Salmonella Typhimurium cells and magnetic nanoparticles (MNPs) modified with monoclonal antibodies (mAbs) was injected with MnO2-QD-pAb NFs into a microfluidic chip to form MNP-bacteria-QD-MnO2 complexes. Then, glutathione (GSH) was injected to dissolve MnO2 on the complexes into Mn2+, resulting in the release of QDs. Finally, fluorescent intensity of the released QDs was measured using the fluorescent detector to determine the amount of Salmonella. A linear relationship between fluorescent intensity and bacterial concentration from 1.0 × 102 to 1.0 × 107 CFU/mL was found with a low detection limit of 43 CFU/mL and mean recovery of 99.7% for Salmonella in spiked chicken meats, indicating the feasibility of this biosensor for practical applications.

Rapid and sensitive detection of Salmonella Typhimurium using nickel nanowire bridge for electrochemical impedance amplification.

Rapid detection of foodborne pathogens is crucial to prevent the outbreaks of foodborne illnesses. In this study, a sensitive electrochemical aptasensor was developed using aptamer coated gold interdigitated microelectrode for target capture and impedance measurement, and antibody modified nickel nanowires (NiNWs) for target separation and impedance amplification. First, the interdigitated microelectrode was modified with the biotinylated aptamers against Salmonella typhimurium through electrostatic absorption of streptavidin onto the microelectrode and streptavidin-biotin binding. Then, the target Salmonella cells were magnetically separated and concentrated using the NiNWs modified with the anti-Salmonella typhimurium antibodies to form the bacteria-NiNW complexes, and incubated on the microelectrode to form the aptamer-bacteria-NiNW complexes. After an external arc magnetic field was developed and applied to control the NiNWs to form conductive NiNW bridges across the microelectrode, the enhanced impedance change of the microelectrode was measured and used to determine the amount of target bacteria. This electrochemical aptasensor was able to quantitatively detect Salmonella ranging from 102 to 106 CFU/mL in 2 h with the detection limit of 80 CFU/mL. The mean recovery for the spiked chicken samples was 103.2%.

Optical Biosensor for Rapid Detection of Salmonella typhimurium Based on Porous Gold@Platinum Nanocatalysts and a 3D Fluidic Chip.

Screening of pathogenic bacteria is a key to avoid food poisoning. The major drawbacks of existing assays for foodborne bacteria detection include long time for culture, complex DNA extraction for the polymerase chain reaction (PCR), and low sensitivity for enzyme-linked immunosorbent assay (ELISA), greatly limiting their practical applications. Here, we developed a sensitive optical biosensor based on porous gold@platinum nanocatalysts (Au@PtNCs) and a passive three-dimensional (3D) micromixer for fast detection of Salmonella typhimurium. The target Salmonella cells were first separated using immunomagnetic nanoparticles and the passive 3D micromixer. Then, immune Au@PtNCs were labeled onto the target cells as signal output to catalyze hydrogen peroxide-3,3',5,5'-tetramethylbenzidine. Finally, the absorbance was measured at 652 nm to calculate the bacterial amount. This optical biosensor could detect Salmonella at concentrations from 1.8 × 101 to 1.8 × 107 CFU/mL in 1 h. Its detection limit was calculated to be 17 CFU/mL. Besides, this passive 3D micromixer could magnetically separate 99% of target bacteria from the sample in 10 min. This biosensor has the potential to be extended to detect other bacteria by changing the antibodies.

Continuous-Flow Separation and Efficient Concentration of Foodborne Bacteria from Large Volume Using Nickel Nanowire Bridge in Microfluidic Chip.

Separation and concentration of target bacteria has become essential to sensitive and accurate detection of foodborne bacteria to ensure food safety. In this study, we developed a bacterial separation system for continuous-flow separation and efficient concentration of foodborne bacteria from large volume using a nickel nanowire (NiNW) bridge in the microfluidic chip. The synthesized NiNWs were first modified with the antibodies against the target bacteria and injected into the microfluidic channel to form the NiNW bridge in the presence of the external arc magnetic field. Then, the large volume of bacterial sample was continuous-flow injected to the channel, resulting in specific capture of the target bacteria by the antibodies on the NiNW bridge to form the NiNW-bacteria complexes. Finally, these complexes were flushed out of the channel and concentrated in a lower volume of buffer solution, after the magnetic field was removed. This bacterial separation system was able to separate up to 74% of target bacteria from 10 mL of bacterial sample at low concentrations of ≤102 CFU/mL in 3 h, and has the potential to separate other pathogenic bacteria from large volumes of food samples by changing the antibodies.