Isothermal Amplification and Hypersensitive Fluorescence Dual-Enhancement Nucleic Acid Lateral Flow Assay for Rapid Detection of Acinetobacter baumannii and Its Drug Resistance.

Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatments. In this study, we reported a nucleic acid fluorescent lateral flow assay (NFLFA) to identify A. baumannii and carbapenem-resistant A. baumannii (CRAB) in a rapid and quantitative manner by integrating loop-mediated isothermal amplification (LAMP) and silica-based multilayered quantum dot nanobead tag (Si@MQB). First, a rapid LAMP system was established and optimised to support the effective amplification of two bacterial genes in 35 min. Then, the antibody-modified Si@MQB was introduced to capture the two kinds of amplified DNA sequences and simultaneously detect them on two test lines of a LFA strip, which greatly improved the detection sensitivity and stability of the commonly used AuNP-based nucleic acid LFA. With these strategies, the established LAMP-NFLFA achieved detection limits of 199 CFU/mL and 287 CFU/mL for the RecA (house-keeping gene) and blaOXA-23 (drug resistance gene) genes, respectively, within 43 min. Furthermore, the assay exhibited good repeatability and specificity for detecting target pathogens in real complex specimens and environments; thus, the proposed assay undoubtedly provides a promising and low-cost tool for the on-site monitoring of nosocomial infections.

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.

Production of a Monoclonal Antibody for the Detection of Forchlorfenuron: Application in an Indirect Enzyme-Linked Immunosorbent Assay and Immunochromatographic Strip.

In this study, a monoclonal antibody (mAb) specific to forchlorfenuron (CPPU) with high sensitivity and specificity was produced and designated (9G9). To detect CPPU in cucumber samples, an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold nanobead immunochromatographic test strip (CGN-ICTS) were established using 9G9. The half-maximal inhibitory concentration (IC50) and the LOD for the developed ic-ELISA were determined to be 0.19 ng/mL and 0.04 ng/mL in the sample dilution buffer, respectively. The results indicate that the sensitivity of the antibodies prepared in this study (9G9 mAb) was higher than those reported in the previous literature. On the other hand, in order to achieve rapid and accurate detection of CPPU, CGN-ICTS is indispensable. The IC50 and the LOD for the CGN-ICTS were determined to be 27 ng/mL and 6.1 ng/mL. The average recoveries of the CGN-ICTS ranged from 68 to 82%. The CGN-ICTS and ic-ELISA quantitative results were all confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with 84-92% recoveries, which indicated the methods developed herein are appropriate for detecting CPPU in cucumber. The CGN-ICTS method is capable of both qualitative and semiquantitative analysis of CPPU, which makes it a suitable alternative complex instrument method for on-site detection of CPPU in cucumber samples since it does not require specialized equipment.

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.

Molecular Parameters Promoting High Relaxivity in Cluster-Nanocarrier Magnetic Resonance Imaging Contrast Agents.

We have investigated the mechanism of relaxivity for two magnetic resonance imaging contrast agents that both employ a cluster-nanocarrier design. The first system termed Mn8Fe4-coPS comprises the cluster Mn8Fe4O12(L)16(H2O)4 or Mn8Fe4 (1) (L = carboxylate) co-polymerized with polystyrene to form ∼75 nm nanobeads. The second system termed Mn3Bpy-PAm used the cluster Mn3(O2CCH3)6(Bpy)2 or Mn3Bpy (2) where Bpy = 2,2'-bipyridine, entrapped in ∼180 nm polyacrylamide nanobeads. Here, we investigate the rate of water exchange of the two clusters, and corresponding cluster-nanocarriers, in order to elucidate the mechanism of relaxivity in the cluster-nanocarrier. Swift-Connick analysis of O-17 NMR was used to determine the water exchange rates of the clusters and cluster-nanocarriers. We found distinct differences in the water exchange rate between Mn8Fe4 and Mn8Fe4-coPS, and we utilized these differences to elucidate the nanobead structure. Using the transverse relaxivity from O-17 NMR line widths, we were able to determine the hydration state of the Mn3Bpy (2) cluster as well as Mn3Bpy-PAm. Using these hydration states in the Swift-Connick analysis of O-17 NMR, we found the water exchange rate to be extremely close in value for the cluster Mn3Bpy and cluster-nanocarrier Mn3Bpy-PAm.

Quantum Dot Nanobead-Based Fluorescence-Linked Immunosorbent Assay for Detection of Glycinin in Soybeans and Soy Products.

Soybean glycinin, as a major soybean allergen, is difficult to accurately quantify due to its large molecular weight and complex structure. CdSe/ZnS quantum dot nanobead (QB) is a core/shell fluorescent nanomaterial with strong fluorescent signals and high sensitivity at 630 nm. An immunosorbent assay based on CdSe/ZnS quantum dot nanobeads (QBs-FLISA) was developed for the glycinin quantification in soybean and soybean products. Here, the purified glycinin was coated on the microporous plate to serve as the coating antigen, and CdSe/ZnS nanobead conjugated with anti-glycinin polyclonal antibodies was used as fluorescent detection probe. The target glycinin in the sample and the coated antigen on the plate competitively adsorbed the antibody labeled the CdSe/ZnS QBs probes. The limits of detection and quantitation for glycinin were 0.035 and 0.078 µg mL-1, respectively. The recoveries of the spiked samples ranged from 89.8% to 105.6%, with relative standard deviation less than 8.6%. However, compared with ELISA, the sensitivities of QBs-FLISA for the detection of glycinin were increased by 7 times, and the detection time was shortened by two-thirds. This QBs-FLISA method has been effectively applied to the detection of soybean seeds with different varieties and soy products with different processing techniques, which will provide a rapid screening method for soybean and soybean products with low allergens.

Highly sensitive detection of free testosterone assisted by magnetic nanobeads and gap-enhanced SERS nanotags.

The quantitative determination of trace free testosterone (FT) is of great significance for the diagnosis of androgen-related endocrine diseases. Herein, a fascinating detection protocol was developed for highly sensitive FT analysis through a competitive immunoassay mechanism, which was composed of magnetic nanobeads (MNBs) and gap-enhanced surface enhanced Raman scattering (SERS) nanotags. With the MNBs as detection carriers, trace FT could be enriched by simple magnetic separation. The SERS nanotag constructed with silver-gold core-shell nanoparticle was acted as quantitative label, and Raman indicators were located at the interface between silver core and gold shell. It is demonstrated that the as-proposed protocol achieves high detection sensitivity for FT of 12.11 fg mL-1, and wider linear dynamic detection range (LDR) in the concentration of 100 fg mL-1 to 100 ng mL-1 with R2 value of 0.979, which is due to the enhanced Raman signal of the gap-enhanced SERS nanotag and the high surface-to-volume ratio of the MNB, respectively. Taking advantages of such sensitivity and accuracy approach, the as-developed powerful strategy presents potential applications for rapid disease diagnosis through analyzing trace levels of FT, and can also provide guidance for the exploitation of analysis project of other analytes.

Hybrid Nanobeads for Oral Indomethacin Delivery.

The oral administration of the anti-inflammatory indomethacin (INDO) causes severe gastrointestinal side effects, which are intensified in chronic inflammatory conditions when a continuous treatment is mandatory. The development of hybrid delivery systems associates the benefits of different (nano) carriers in a single system, designed to improve the efficacy and/or minimize the toxicity of drugs. This work describes the preparation of hybrid nanobeads composed of nanostructured lipid carriers (NLC) loading INDO (2%; w/v) and chitosan, coated by xanthan. NLC formulations were monitored in a long-term stability study (25 °C). After one year, they showed suitable physicochemical properties (size < 250 nm, polydispersity < 0.2, zeta potential of −30 mV and spherical morphology) and an INDO encapsulation efficiency of 99%. The hybrid (lipid-biopolymers) nanobeads exhibited excellent compatibility between the biomaterials, as revealed by structural and thermodynamic properties, monodisperse size distribution, desirable in vitro water uptake and prolonged in vitro INDO release (26 h). The in vivo safety of hybrid nanobeads was confirmed by the chicken embryo (CE) toxicity test, considering the embryos viability, weights of CE and annexes and changes in the biochemical markers. The results point out a safe gastro-resistant pharmaceutical form for further efficacy assays.

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.

Rapid Quantum Dot Nanobead-mAb Probe-Based Immunochromatographic Assay for Antibody Monitoring of Trichinella spiralis Infection.

PURPOSE: Sensitive and selective point-of-care biosensor is an urgent pursuit of serological antibody detection to control parasite pathogen. For specific, quantitative and on-site screening of Trichinella spiralis infection in livestock, a quantum dot nanobead-monoclonal antibody (QB-mAb) probe-based immunochromatographic assay (ICA) was developed by introducing a competitive sandwich strategy (QB-CICA). METHODS: In the QB-CICA, QB-mAb probes competed with serum antibody for a particular epitope, followed by immunocomplexes binding to capture antibody on the test line. With the accumulation of target antibody, captured probes served as signal elements for fluorescent readout in a "turn off" mode, along with the fluorescence gradually weakened. The sensitivity and standard calibration curve of the QB-CICA were quantified using swine sera as negative control (n = 200) and artificial infected swine sera (n = 80) compared with a commercial ELISA kit. Besides, Trichinella spiralis-antibody targeting test ability of the QB-CICA, instead of other parasites or viruses antibodies (n = 10), was evaluated. RESULTS: The QB-CICA exhibited a good linear range, a low detection limit of 189.92 ng mL-1 and 100% selectivity that was higher than commercial ELISA kit (90%), as well as the same serological positive rate (100%) with commercial ELISA kit in different infection dose models. CONCLUSION: Taking advantage of its simplicity, short response time (25 min), sensitivity and specificity, the proposed QB-CICA has potential applications for parasite-related antibody monitoring in food safety and clinical diagnosis fields.

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.

Advantages of Lateral Flow Assays Based on Fluorescent Submicrospheres and Quantum Dots for Clostridium difficile Toxin B Detection.

Clostridium difficile colitis is caused by a cytotoxin produced by the anaerobic bacteria C. difficile in the epithelial cells of the large intestine, particularly C. difficile toxin B (TcdB). However, the sensitivity of currently utilized C. difficile endotoxin determination methods has been called into question, and, therefore, more accurate and convenient detection methods are needed. Our study is the first to systematically compare fluorescent submicrosphere-based and quantum-dot nanobead-based lateral fluidity measurement methods (FMs-LFA and QDNBs-LFA) with toxin B quantification in fecal samples via sandwich analysis. The limits of detection (LOD) of FMs-LFA and QDNBs-LFA in the fecal samples were 0.483 and 0.297 ng/mL, respectively. TcdB analyses of the fecal samples indicated that the results of QDNBs-LFA and FMs-LFA were consistent with those of a commercial enzyme-linked immunosorbent assay (ELISA) test kit. The sensitivity of QDNBs-LFA was highly correlated with clinical diagnoses. Therefore, quantum dot nanobeads (QDNBs) are deemed highly suitable for lateral fluidity analyses, which would facilitate the implementation of portable and rapid on-the-spot applications, such as food hygiene and safety tests and onsite medical testing.

Insights into the Formation of DNA-Magnetic Nanoparticle Hybrid Structures: Correlations between Morphological Characterization and Output from Magnetic Biosensor Measurements.

Understanding the binding mechanism between probe-functionalized magnetic nanoparticles (MNPs) and DNA targets or amplification products thereof is essential in the optimization of magnetic biosensors for the detection of DNA. Herein, the molecular interaction forming hybrid structures upon hybridization between DNA-functionalized magnetic nanoparticles, exhibiting Brownian relaxation, and rolling circle amplification products (DNA-coils) is investigated by the use of atomic force microscopy in a liquid environment and magnetic biosensors measuring the frequency-dependent magnetic response and the frequency-dependent modulation of light transmission. This approach reveals the qualitative and quantitative correlations between the morphological features of the hybrid structures with their magnetic response. The suppression of the high-frequency peak in the magnetic response and the appearance of a new peak at lower frequencies match the formation of larger sized assemblies upon increasing the concentration of DNA-coils. Furthermore, an increase of the DNA-coil concentration induces an increase in the number of MNPs per hybrid structure. This study provides new insights into the DNA-MNP binding mechanism, and its versatility is of considerable importance for the mechanistic characterization of other DNA-nanoparticle biosensor systems.

Rapid Enrichment and Ultrasensitive Detection of Influenza A Virus in Human Specimen using Magnetic Quantum Dot Nanobeads Based Test Strips.

Influenza A virus (IAV) possesses a high infectivity and pathogenicity, and can lead to severe respiratory infection with similar symptoms caused by some other common respiratory viruses. Lateral flow assay (LFA) has been widely deployed in remote settings as a rapid and reliable approach for point-of-care detection of infectious pathogens. However, it still remains challenging to detect IAV virions using LFA from clinical samples such as nasopharyngeal or throat swabs, because their various components and high viscosity can decrease flow velocity and lead to the nonspecific adsorption of nanoparticle labels on the sensing membrane. Herein, we demonstrated a magnetic quantum dot nanobeads (MQBs) based LFA for magnetic enrichment and fluorescent detection of IAV virions in clinical specimens. In this study, MQBs were synthesized and then conjugated with IAV-specific antibody to efficiently enrich IAV virions from complex biological matrix, but also serve as highly bright fluorescent probes in lateral flow strips. This assay can achieve quantitative detection of IAV virions with a low limit of detection down to 22 pfu mL-1 within 35 minutes, and show good specificity between influenza B virus and two adenovirus strains. Furthermore, the presented platform was able to directly detect IAV virions spiked in nasopharyngeal swab dilution, indicating its stability and feasibility in clinical applications. Thus, this point-of-care detection platform holds great promise as a broadly applicable approach for the rapid diagnosis of influenza A.

A simple magnetic nanoparticle-poly-enzyme nanobead sandwich assay for direct, ultrasensitive DNA detection.

A simple magnetic nanoparticle (MNP)-poly-enzyme nanobead sandwich assay for direct detection of ultralow levels of unlabeled target-DNA is developed. This approach uses a capture-DNA covalently linked to a dense PEGylated polymer encapsulated MNP and a biotinylated signal-DNA to sandwich the target-DNA. A DNA ligation is then followed to offer high discrimination between the perfect-match and single-base mismatch target-DNAs. Only the presence of a perfect-match target can covalently link the biotinylated signal-DNA onto the MNP surface for subsequent binding to a polymer nanobead tagged with thousands of copies of high-activity neutravidin-horseradish peroxidase (NAV-HRP) for great enzymatic signal amplification. Combining the advantages of the dense MNP surface PEGylation to reduce non-specific adsorption (assay background) and the powerful signal amplification of poly-enzyme nanobead, this assay can directly quantify the target-DNA down to single digit attomolar with a large linear dynamic range of 5 orders of magnitude (from 10-18 to 10-13M).

Dual-color magnetic-quantum dot nanobeads as versatile fluorescent probes in test strip for simultaneous point-of-care detection of free and complexed prostate-specific antigen.

Simultaneous detection of free and complexed prostate-specific antigen (f-PSA and c-PSA) is critical to the prostate cancer (PCa) diagnostic accuracy for clinical samples with PSA values in the diagnostic gray zone between 4 and 10 ng mL-1. Herein, red and green magnetic-quantum dot nanobeads (MQBs) with superior magnetic property and high luminescence were fabricated via polyethyleneimine-mediated electrostatic adsorption of numerous quantum dots onto superparamagnetic Fe3O4 magnetic cores, and were conjugated with f-PSA antibody and c-PSA antibody, respectively, as versatile fluorescent probes in test strip for immune recognition, magnetic enrichment, and simultaneous detection of f-PSA and c-PSA analytes in complex biological matrix with t-PSA antibody on the test line. A low-cost and portable smartphone readout device with an application was also developed for the imaging of dual-color test strips and data processing. This assay can simultaneously detect f-PSA and c-PSA with the limits of detection of 0.009 ng mL-1 and 0.087 ng mL-1, respectively. Clinical serum samples of PCa and benign prostatic hyperplasia patients were evaluated to confirm the clinical feasibility. The results suggest that the proposed dual-color MQBs-based fluorescent lateral flow immunoassay is a promising point-of-care diagnostics technique for the accurate diagnosis of PCa even in resource-limited settings.

Magnetic Tracking of Protein Synthesis in Microfluidic Environments-Challenges and Perspectives.

A novel technique to study protein synthesis is proposed that uses magnetic nanoparticles in combination with microfluidic devices to achieve new insights into translational regulation. Cellular protein synthesis is an energy-demanding process which is tightly controlled and is dependent on environmental and developmental requirements. Processivity and regulation of protein synthesis as part of the posttranslational nano-machinery has now moved back into the focus of cell biology, since it became apparent that multiple mechanisms are in place for fine-tuning of translation and conditional selection of transcripts. Recent methodological developments, such as ribosome foot printing, propel current research. Here we propose a strategy to open up a new field of labelling, separation, and analysis of specific polysomes using superparamagnetic particles following pharmacological arrest of translation during cell lysis and subsequent analysis. Translation occurs in polysomes, which are assemblies of specific transcripts, associated ribosomes, nascent polypeptides, and other factors. This supramolecular structure allows for unique approaches to selection of polysomes by targeting the specific transcript, ribosomes, or nascent polypeptides. Once labeled with functionalized superparamagnetic particles, such assemblies can be separated in microfluidic devices or magnetic ratchets and quantified. Insights into the dynamics of translation is obtained through quantifying large numbers of ribosomes along different locations of the polysome. Thus, an entire new concept for in vitro, ex vivo, and eventually single cell analysis will be realized and will allow for magnetic tracking of protein synthesis.

Rapid and Sensitive Detection of Campylobacter jejuni in Poultry Products Using a Nanoparticle-Based Piezoelectric Immunosensor Integrated with Magnetic Immunoseparation.

Campylobacter jejuni is one of the leading causes of foodborne human gastrointestinal diseases. Poultry and poultry products have been identified as the major transmission routes to humans for this pathogenic bacterium. The objective of this research was to develop a rapid and sensitive immunosensor for detection of C. jejuni in poultry products on the basis of a quartz crystal microbalance (QCM) using magnetic nanobeads (MNBs) for separation of target pathogen and gold nanoparticles for amplification of the measurement. A QCM sensor in a flow cell was prepared by immobilizing the mouse anti- C. jejuni monoclonal antibody (mAb1) on the sensor surface to specifically capture C. jejuni. Rabbit anti- C. jejuni polyclonal antibody (pAb1) was conjugated with MNBs to capture and separate C. jejuni from food matrices. MNB-pAb1- C. jejuni complexes were injected into the flow cell to bind with the mAb1 immobilized on the QCM sensor surface. Goat anti-rabbit immunoglobulin G polyclonal antibody conjugated with gold nanoparticles was injected into the flow cell to bind with pAb1 on MNBs. Finally, resonant frequency was measured with a QCM analyzer, and the change in resonant frequency was correlated to the cell number of C. jejuni. The specificity of this immunosensor was confirmed with different strains of Campylobacter, Salmonella, and other foodborne pathogens commonly colonized in the broiler gastrointestinal tract. Samples of broiler carcass wash and ground turkey were spiked with C. jejuni at different concentrations for use in tests. Results showed that the QCM immunosensor could rapidly detect C. jejuni in poultry products, with a detection limit of 20 to 30 CFU/mL without preenrichment, and a total detection time of less than 30 min. Characteristics of C. jejuni captured by the antibody immobilized on the surface of the QCM sensor were visualized by using atomic force microscopy. This highly adaptive and flexible method could provide the poultry industry a more rapid, sensitive, and effective method for detection of major foodborne pathogens in poultry products.

Quantum dot nanobead-based multiplexed immunochromatographic assay for simultaneous detection of aflatoxin B1 and zearalenone.

Immunochromatographic assay (ICA) is a promising technology for on-site detection. Nonetheless, the wide-scale application of ICA is hindered by several disadvantages, such as poor reproducibility, low sensitivity, and single-target detection. Thus, a novel quantum dot nanobead (QB)-based multiplexed ICA (QB-ICA) with multiple test lines was developed in this study for the simultaneous quantitative detection of aflatoxin B1 (AFB1) and zearalenone (ZEN), where QBs with high luminescence were used as labels to enhance the analytical sensitivity of the ICA. Moreover, a streptavidin (SA)-biotin system, which was undisturbed by the target mycotoxins, was introduced as the signal output for the control line. Consequently, stable and reliable T/C values (ratios of signals on the test line to that of the control line) were obtained as quantitative signals. The proposed QB-ICA demonstrated high sensitivity for the simultaneous detection of AFB1 and ZEN, of which the half-maximal inhibitory concentrations reached as low as 38.98 pg mL-1 and 1.23 ng mL-1, respectively. At 10% competitive inhibition concentration, the limit detections (LOD) were 1.65 and 59.15 pg mL-1 for AFB1 and ZEN, respectively. The average recoveries of the intra- and inter-assays ranged from 81.77% to 119.70% and from 94.18% to 111.4% for AFB1 and ZEN quantification, respectively, and the variation coefficients were less than 12%, thereby indicating that the proposed method is highly accurate and robust. These findings suggest that QB-ICA using SA-biotin system as the signal output of control line is an excellent point-of-care platform for the rapid screening of mycotoxins.

An On-Site, Ultra-Sensitive, Quantitative Sensing Method for the Determination of Total Aflatoxin in Peanut and Rice Based on Quantum Dot Nanobeads Strip.

An on-site, ultra-sensitive, and quantitative sensing method was developed based on quantum dot nanobeads (QDNBs) and a test strip for the determination of total aflatoxins (AFTs) in rice and peanuts. The monoclonal antibody against AFT (mAbAFT) was homemade and labeled with QDNB. After the pre-coating of the AFT antigen on the test line (T line), the competitive immunoreactions were conducted between AFT and AFT antigen on the T line with QDNBs-mAbAFT. Under optimal conditions, this approach allowed a rapid response towards AFT with a considerable sensitivity of 1.4 pg/mL and 2.9 pg/mL in rice and peanut matrices, respectively. The put-in and put-out durations were within 10 min. The recoveries for AFT in rice and peanut sample matrices were recorded from 86.25% to 118.0%, with relative deviations (RSD) below 12%. The assay was further validated via the comparison between this QDNB strip and the conventional HPLC method using spiked samples. Thus, the design provided a potential alternative for on-site, ultra-sensitive, and quantitative sensing of AFT that could also be expanded to other chemical contaminants for food safety.