Flexible microfluidic co-recognition coupled with magnetic enrichment and silent SERS sensing for simultaneous analysis of bacteria in food.

Food safety represents a critical global public health issue, with safety challenges posed by foodborne pathogens garnering extensive attention. Therefore, we introduce a co-recognition, enrichment and sensing (CES) all-in-one strategy for analysis of bacteria with low background and high specificity. This method employs antimicrobial peptide (AMP) functionalized magnetic nanoparticles (MNPs) to enrich bacteria and uses aptamer@Au@PBA (KxMFe(CN)6 (M = Pb and Ni)) NPs as silent SERS tags. When both S. aureus and E. coli O157:H7 are present, the silent SERS probes could specifically label the target bacteria, forming a sandwich-like structure. This binding induces silent Raman shifts (2139 cm-1 and 2197 cm-1), enabling quantification of two bacteria. Coupling with the modular flexible microfluidics and magnetic control slider device, this platform facilitates rapid switching between magnetic loading and elution. The CES SERS method demonstrated linear relationships for both S. aureus and E. coli O157:H7 at 50-1600 cfu mL-1, with detection limits of 14 and 18 cfu mL-1, respectively. The method achieved recovery rates of 85.6-112% and relative standard deviations of 1.5-8.6%. Validation using the ELISA method revealed relative errors between -7.5 and 4.3%. The CES approach has potential applications in food safety, environmental monitoring, and biomedical diagnosis.

Ultrasensitive Automatic Detection of Small Molecules by Membrane Imaging of Single Molecule Assays.

Determination of trace amounts of targets or even a single molecule target has always been a challenge in the detection field. Digital measurement methods established for single molecule counting of proteins, such as single molecule arrays (Simoa) or dropcast single molecule assays (dSimoa), are not suitable for detecting small molecule, because of the limited category of small molecule antibodies and the weak signal that can be captured. To address this issue, we have developed a strategy for single molecule detection of small molecules, called small molecule detection with single molecule assays (smSimoa). In this strategy, an aptamer is used as a recognition element, and an addressable DNA Nanoflower (DNF) attached on the magnetic beads surface, which exhibit fluorescence imaging, is employed as the output signal. Accompanied by digital imaging and automated counting analysis, E2 at the attomolar level can be measured. The smSimoa breaks the barrier of small molecule detection concentration and provides a basis for high throughput detection of multiple substances with fluorescence encoded magnetic beads.

ATP-Responsive Strand Displacement Coupling with DNA Origami/AuNPs Strategy for the Determination of Microcystin-LR Using Surface-Enhanced Raman Spectroscopy.

The DNA origami-mediated self-assembly strategy has emerged as a powerful tool in surface-enhanced Raman spectroscopy (SERS). However, these self-assembly approaches typically do not possess high detection specificity. Herein, a novel strategy based on adenosine triphosphate (ATP)-responsive strand displacement (ARSD) coupling with DNA origami/AuNPs for SERS analysis of microcystin-LR (MC-LR) is presented. In the presence of MC-LR and ATP molecules, nucleic acid sensing structures fabricated with anti-MC-LR aptamer (T1) and ATP aptamer (T2) were triggered to release the remaining ATP. In addition, DNA origami-assisted assembly results in the formation of homogeneous plasmonic nanostructures for Raman enhancement via strong plasmonic coupling. After the binding in the gaps of functionalized DNA origami/AuNPs, the Raman shift of the ATP molecules becomes detectable, leading to increased SERS intensity in 734 cm-1. A linear response to MC-LR was obtained in the concentration range of 1.56-50 µg·L-1, and the limit of detection (LOD) was 0.29 µg·L-1. Combined with the solid-phase extraction sample pretreatment for extraction and 10-fold concentration, this proposed method was successfully used to detect MC-LR type in real lake-water samples with good recoveries of 98.4-116% and relative standard deviations of 1.9-6.7%. Furthermore, for the detection of MC-LR in contaminated lake-water samples, the results of the developed method and ultrahigh-performance liquid chromatography-tandem mass spectrometry were found to be in agreement with relative errors between -12 and 2.4%. The proposed strategy provides a sensitive recognition and signal amplification platform for trace MC-LR analysis as well as innovative nucleic acid sensing structures for toxin analysis more generally.

A novel bionic magnetic SERS aptasensor for the ultrasensitive detection of Deoxynivalenol based on "dual antennae" nano-silver.

Deoxynivalenol (DON), as a mycotoxin produced by Fusarium, showed great harm to human body, crops and animals, so it is urgent to establish an efficient, sensitive and selective method for the detection of DON. Here, a novel bionic magnetic SERS aptasensor based on "dual antennae" nano-silver was designed. ß-CD@AgNPs was modified 4-MBA and the aptamers respectively with chemical bond and host-guest interaction, which was reflected in the "dual antennae" characteristics of identifying target and SERS signal label. In addition, Fe3O4@Au was conjugated with SH- modified complementary DNA to prepare capture probes, enabling fast magnetic separation of the captured target and further enhanced Raman scattering. With the specific recognition and competitive binding of DON and aptamer, the combination of "dual antenna" signal probe and capture probe is significantly reduced, exerting a lower SERS intensity. There was a good linear relationship in the range of 0.0001-100 ng mL-1 between the SERS intensity and the logarithm of DON concentration, and the limit of detection (LOD) was as low as 0.032 pg mL-1. The SERS aptasensor exhibited good selectivity, satisfactory repeatability and expected practicability, presenting a great application prospect in the detection of mycotoxins and biochemical analysis.

Development of Fe3O4@Au nanoparticles coupled to Au@Ag core-shell nanoparticles for the sensitive detection of zearalenone.

Agricultural products are frequently contaminated by mycotoxins; thus, the accurate detection of mycotoxins is important to food safety. Zearalenone (ZEN), a mycotoxin produced by certain Fusarium and Gibberella species, is a group III carcinogen. We developed a universal surface-enhanced Raman scattering (SERS) aptasensor for the detection of ZEN. The SERS biosensor consists of two functional nanomaterials: sulfhydryl (SH)-ZEN aptamer complementary DNA-modified Fe3O4@Au was used as a capture probe and SH-ZEN aptamer-modified Au@Ag core-shell nanoparticles served as reporter probes. In the absence of ZEN, the highest Raman signal was obtained owing to the SERS effects of Fe3O4@Au and Au@Ag core-shell nanoparticles. Conversely, the addition of ZEN triggered the release of Au@Ag core-shell nanoparticles from Fe3O4@Au, leading to a decrease in SERS intensity after magnetic separation. Hybridization of the ZEN aptamer and its complementary strand generated a strong SERS signal from the reporter probe. Moreover, preferential binding of the ZEN aptamer to ZEN was observed. The signal intensity in SERS decreased linearly when the capture probes released the reporter. For ZEN detection, a linear range from 0.005 to 500 ng mL-1, with an R2 of 0.9981, was obtained. The detection limit was 0.001 ng mL-1. The SERS aptasensor showed excellent performance for analytical applications with real-world samples (beer and wine). This study presents a new model for the detection of mycotoxins based on simple changes in aptamers.

Surface-enhanced Raman spectroscopy aptasensor for simultaneous determination of ochratoxin A and zearalenone using Au@Ag core-shell nanoparticles and gold nanorods.

The design and fabrication of a surface-enhanced Raman scattering (SERS) aptasensor for simultaneous detection of zearalenone (ZEN) and ochratoxin A (OTA) in wheat and corn samples is described. The capture and reporter probes were SH-cDNA-modified gold nanorods and SH-Apt-modified Au@Ag core-shell nanoparticles, respectively. After recognizing OTA and ZEN aptamers and complementary strands (SH-cDNA), the reporter probe generated a strong SERS signal. The preferred binding of OTA and ZEN aptamers to OTA and ZEN, respectively, caused reporter probes to release the capture probes, resulting in a linear decrease in SERS intensity. The detection of OTA showed good linearity with an R2 value of 0.986, which could be maintained across a wide concentration range (0.01 to 100 ng/mL), with the limit of detection of 0.018 ng/mL. For detection of ZEN, good linearity with an R2 value of 0.987 could be maintained across a wide concentration range (0.05 to 500 ng/mL), with 0.054 ng/mL as the limit of detection. Good accuracy (relative standard deviation < 4.2%) during mycotoxin determination as well as excellent quantitative recoveries (96.0-110.7%) during the analysis of spiked real samples was achieved. The proposed SERS aptasensor exhibited excellent performance in the detection of OTA and ZEN in real food samples. Hence, by simply changing the aptamer, this new model can be applied to the detection of multiple mycotoxins in the food industry.

A fluorescence aptasensor based on controlled zirconium-based MOFs for the highly sensitive detection of T-2 toxin.

T-2 toxin is one of class A trichothecene mycotoxins produced by Fusarium, presenting genotoxic, cytotoxicity, and immunotoxicity for animals and humans. Therefore, It is urgent to establish a rapid test method with high sensitivity, good selectivity and reliability. In this research, by adjusting the synthesis conditions, a kind of NH2-UiO-66 with high quenching efficiency was screened out. On this basis, we constructed a novel fluorescence sensor via Cy3-labeled aptamer (Cy3-aptamer). With the help of π-π interaction, hydrogen bond and coordination, NH2-UiO-66 could adsorb and quench the fluorescence of Cy3-aptamer based on FRET and PET. In the presence of T-2 toxin, it recognized and bound to Cy3-aptamer, leading to the disintegration of the NH2-UiO-66/Cy3-aptamer compound. As the energy transfer process was blocked, the fluorescence intensity was restored, enabling a highly sensitive response to T-2 toxin. There was a good linear correlation between fluorescence intensity and T-2 toxin concentration in the range of 0.5-100 ng ml -1. The LOD of this fluorescence aptasensor was 0.239 ng ml-1 (S/N = 3). Besides, the recoveries of milk and beer were 89.86-108.99% (RSD = 2.0-2.6%) and 92.31-111.51% (RSD = 2.3-2.9%), respectively. The fluorescence aptasensor exhibited advantages of excellent analytical performance, convenient operation procedure and good selectivity. Predictably, the aptasensor was supposed to detect antibiotics and other pollutants, describing an intriguing blueprint and potential application prospect in food safety, biochemical sensing and environmental conservation.

A fluorescence aptasensor for the sensitive detection of T-2 toxin based on FRET by adjusting the surface electric potentials of UCNPs and MIL-101.

T-2 toxin is a class A trichothecene mycotoxin produced by Fusarium, which exhibits genotoxic, cytotoxic, and immunotoxic effects in animals and humans. In this study, we developed an aptasensor for the sensitive detection of T-2 toxin, which was based on fluorescence resonance energy transfer (FRET), and acted by adjusting the electric potential on the surface of upconversion nanoparticles (UCNPs) and MIL-101(Cr). In addition, it combined the excellent spectral properties of UCNPs with the good adsorption quenching abilities of metal organic frameworks (MOFs). Under the action of π-π stacking interactions, the UCNPs-aptamer was adsorbed onto the surface of MIL-101, leading to fluorescence quenching due to the occurrence of FRET. After the addition of T-2 toxin, owing to its selective binding to the UCNPs-aptamer, the UCNPs-aptamer moved away from MIL-101(Cr), resulting in fluorescence recovery. Moreover, the extent of fluorescence recovery was positively correlated with the concentration of T-2 toxin. The limit of detection (LOD) of this sensor was 0.087 ng mL-1 (S/N = 3), and a good linear correlation was observed between the fluorescence intensity and the T-2 toxin concentration in the range of 0.1-100 ng mL-1. Moreover, the recovery of this method was 97.52-109.53% for corn meal samples (relative standard deviation, RSD = 1.7-2.4%) and 90.81-100.02% for beer samples (RSD = 2.4-2.7%). By adjusting the surface electric potentials, the efficient fluorescence aptasensor combined the advantages of UCNPs and MIL-101(Cr) and allowed the first application of such a system in toxin detection, thereby indicating its potential food sample analysis and biochemical sensing.

Recent advances on functional nucleic acid-based biosensors for detection of food contaminants.

It has seen increasing development of reliable, robust, and flexible biosensors for rapid food-safety analysis in the past few decades. Recently, functional nucleic acid-based biosensors have attracted attention because of their programmability, bottom-up characteristics, and structural switches. However, few systematic reviews devoted to categorizing the potential of DNA nanostructures and devices were found for detecting food contaminants. Hence, the applications of functional nucleic acid-based biosensors were reviewed for analyzing food contaminants, including foodborne pathogen bacteria, biotoxins, heavy metals, and et al. In addition to categorizing the various biosensors, multiple signal readout strategies, such as optical, electrochemical, and mass-based signals were also examined. Finally, the future changes and potential opportunities, as well as practical applications of functional nucleic acid-based biosensors were discussed.

A copper monosulfide-nanoparticle-based fluorescent probe for the sensitive and specific detection of ochratoxin A.

The mycotoxin ochratoxin A (OTA) is a secondary metabolite derived from multiple Aspergillus and Penicillium strains. The development of a rapid, sensitive, and simple method for OTA detection is important to ensure food biosafety and safeguard public health. In this study, we designed a highly specific and sensitive assay for the detection of OTA using copper monosulfide (CuS) nanoparticles conjugated to an anti-OTA antibody (CuS-Ab NPs) and a fluorescent probe for Cu2+. When OTA is present in the solution, the OTA antigen, bound to the microplate, is competed off by the soluble OTA for binding to CuS-Ab NPs. After washing, the CuS-Ab NPs and bound OTA are removed. Subsequently, HCl is added to dissolve the CuS-Ab NPs bound to the OTA antigen, releasing Cu2+ and activating the Cu2+ fluorescent probe. Thus, the resultant fluorescence emission is inversely proportional to the OTA content in the solution. Under optimal conditions, this method detected 0.1-100 ng mL-1 OTA with a limit of detection of 0.01 ng mL-1. The assay was tested using corn, soybean, and coffee samples, with recoveries ranging from 94% to 110%. This strategy provides a new approach for the detection of mycotoxins and other small-molecule analytes with broad application potential in food safety and quality control.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL-1 with a large dynamic range from 10 pg mL-1 to 1 µg mL-1.

Highly sensitive detection of ochratoxin A based on bio-barcode immunoassay and catalytic hairpin assembly signal amplification.

Herein, a highly sensitive ochratoxin A (OTA) detection strategy was developed based on a bio-barcode immunoassay with catalytic hairpin assembly (CHA). Two nanoprobes were designed for the assay: one was a gold nanoparticle (AuNP) harbouring numerous bio-barcode DNA and antibodies, and the other was an antigen-functionalized magnetic nanoparticle (MNP). In the presence of target OTA, the antigens of the MNPs competed with OTA for binding to the antibodies on the AuNPs. After magnetic separation, the unbound AuNPs and target OTA were washed away. Dithiothreitol (DTT) was then added to the MNP-bound AuNPs to elute the bio-barcode DNAs of AuNPs, which triggered the CHA reaction. Under optimal conditions, the proposed method could sensitively detect target OTA ranging from 0.001 to 10000 ng/mL. The limit of detection (LOD, 3 N/S) and limit of quantification (LOQ, 10 N/S) for OTA were 0.54 pg/mL and 1.80 pg/mL, respectively. The bio-barcode immunoassay was used to analyse food samples (corn, wheat, and peanut), and the recovery and relative standard deviations (RSD) ranged from 93.30% to 108.80% and from 3.2% to 6.9%, respectively. The total assay time was 6 h. Therefore, the proposed strategy will provide a new approach for the detection of mycotoxins and other small molecule analytes and can be applied for quality control to ensure food safety.

Competitive fluorometric assay for the food toxin T-2 by using DNA-modified silver nanoclusters, aptamer-modified magnetic beads, and exponential isothermal amplification.

The authors describe an aptamer based assay for the mycotoxin T-2. The method is making use of exponential isothermal amplification reaction (EXPAR) and fluorescent silver nanoclusters (AgNCs). Free T-2 and cDNA (which is a DNA that is partially complementary to the aptamer) compete for binding to aptamer-modified magnetic beads. The cDNA collected by magnetic separation can be used as a primer to trigger EXPAR to obtain ssDNA. The C-base-rich ssDNA binds and reduces Ag(I) ion to form fluorescent AgNCs. Fluorescence is measured at excitation/emission wavelengths of 480/525 nm. T-2 can be detected by fluorometry with a detection limit as low as 30 fg·mL-1. The method was applied to analyse spiked oat and corn, and the average recoveries ranged from 97.3 to 102.3% and from 95.9 to 107.5%, respectively. The results were in good agreement with data of the commercial ELISA kit. The assay is highly sensitive, has a wide analytical range, good specificity and reliable operation. It provides a promising alternative for the standard method for quantitative detection of T-2. Graphical abstract Schematic presentation of fluorometric assay for T-2 based on aptamer-functionalized magnetic beads exponential, isothermal amplification reaction (EXPAR) and fluorescent silver nanoclusters (AgNCs).

Ultrasensitive detection of staphylococcal enterotoxin B in foodstuff through dual signal amplification by bio-barcode and real-time PCR.

This study developed an ultrasensitive method involving (1) gold nanoparticles encoded with a double-stranded DNA as the first signal amplification and goat anti-staphylococcal enterotoxin B (SEB) polyclonal antibody and (2) magnetic microparticles coated with anti-SEB monoclonal antibody to detect SEB. Both functionalized nanoparticles can capture SEB in a sandwich system. The released DNA barcodes were then characterized through SYBR Green real-time polymerase chain reaction and resulted in the second signal amplification. Under optimized experimental conditions, an ultrasensitive bio-barcode for SEB detection was established, and its detection limit could reach 0.269 pg mL-1, which was 1000-fold lower than that of conventional enzyme-linked/immunosorbent assay. The proposed method shows great potential in food security.

Ultrasensitive detection of T-2 toxin in food based on bio-barcode and rolling circle amplification.

A novel and highly sensitive method based on bio-barcode with rolling circle amplification (RCA) was developed for the detection of T-2 toxin. Gold nanoparticles (AuNPs) were modified with anti-T-2 monoclonal antibody and single-stranded thiol-oligonucleotides (SH-ssDNAs) and magnetic microparticles (MMPs) coated with T-2 antigen. The T-2 toxin competes with the antigen on MMPs for the anti-T-2 antibody on AuNPs. Then, the isolating complex system was separated by a magnetic field, and the DNA of the probes was released after washing in dithiothreitol solution. The barcode DNA via RCA and products were stained by SYBR Green I and then detected by fluorescence spectrophotometry. The optimized method was performed on oats, millet, flour, and other substances. This method exhibits a low limit of detection (0.26 pg mL-1) and linear range of 0.002-200 ng mL-1. Moreover, the approach offers good recovery and relative standard deviations ranging from 88.65% to 10.04% and 0.6%-13.1%, respectively. In conclusion, this method exhibits potential for use as an ultrasensitive assay for the detection of a variety of small molecules in complex matrices.

Upconversion Fluorescent Aptasensor for Polychlorinated Biphenyls Detection Based on Nicking Endonuclease and Hybridization Chain Reaction Dual-Amplification Strategy.

A novel upconversion fluorescent aptasensor based on hybridization chain reaction and nicking endonuclease has been developed for detection of polychlorinated biphenyls (PCBs). It combined the dual advantages of UCNPs and HCR. Two harpins (H1 and H2) were first designed according to the partial complementary sequence (cDNA) of the PCB72/106. Since the aptamer specifically recognized the target, the cDNA was detached from the magnetic microspheres (MMPs). The cDNA could initiate hybridization chain reaction (HCR) and open the stems of H1 and H2. After the addition of nicking endonuclease, UCNPs were further away from the quenchers (BHQ-1). Hence, the fluorescence intensity of upconversion nanoparticals (UCNPs) could be restored via fluorescence resonance energy transfer (FRET). Therefore, the fluorescence of UCNPs was directly proportional to concentration of PCB72/106, which was the basis for the quantification of PCB72/106. PCB72/106 could be analyzed within the ranges of 0.004 to 800 ng/mL with a detection limit of 0.0035 ng/mL ( S/ N = 3). The aptasensor was also used for the detection of water and soil samples, and the average recoveries ranged from 93.4% to 109.7% and 83.2% to 118.5%, respectively. The relative standard deviations (RSDs) were all below 3.2%. The signal was first amplified through HCR and further amplified with the help of nicking endonuclease. This work also provided the opportunity to develop fluorescent aptasensors for other targets using this dual-amplification strategy.

Rapid detection of staphylococcal enterotoxin B in milk samples based on fluorescence hybridization chain reaction amplification.

A rapid, simple, and sensitive method has been developed to detect staphylococcal enterotoxin B (SEB). To establish the hybridization chain reaction-based aptasensor, we described the new probes of two hairpins (H1 and H2), which were first designed based on the partial complementary sequence of the SEB aptamer (cDNA). The H1 labeled with a fluorophore and a quencher can act as a molecular fluorescence "switch". Hence, in the presence of SEB, the aptamer binds SEB, while the unbound cDNA triggers HCR to carry out the cyclic hybridization of H1 and H2 so as to turn "ON" the fluorescence through forming long nicked DNA. By using this new strategy, SEB can be sensitively detected within the range of 3.13 ng mL-1 to 100 ng mL-1 with a detection limit of 0.33 ng mL-1 (S/N = 3). Furthermore, the developed method could facilitate the detection of SEB effectively in milk samples.