M13 Bacteriophage-Assisted Recognition and Signal Spatiotemporal Separation Enabling Ultrasensitive Light Scattering Immunoassay.

The demand for the ultrasensitive and rapid quantitative analysis of trace target analytes has become increasingly urgent. However, the sensitivity of traditional immunoassay-based detection methods is limited due to the contradiction between molecular recognition and signal amplification caused by the size effect of nanoprobes. To address this dilemma, we describe versatile M13 phage-assisted immunorecognition and signal transduction spatiotemporal separation that enable ultrasensitive light-scattering immunoassay systems for the quantitative detection of low-abundance target analytes. The newly developed immunoassay strategy combines the M13 phage-assisted light scattering signal fluctuations of gold nanoparticles (AuNPs) with gold in situ growth (GISG) technology. Given the synergy of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation, the practical detection capability of this strategy can achieve the ultrasensitive and rapid quantification of ochratoxin A and alpha-fetoprotein in real samples at the subfemtomolar level within 50 min, displaying about 4 orders of magnitude enhancement in sensitivity compared with traditional phage-based ELISA. To further improve the sensitivity of our immunoassay, the biotin-streptavidin amplification scheme is implemented to detect severe acute respiratory syndrome coronavirus 2 spike protein down to the attomolar range. Overall, this study offers a direction for ultrasensitive quantitative detection of target analytes by the synergistic combination of M13 phage-mediated leverage effect and GISG-amplified light scattering signal modulation.

Bifunctional M13 Phage as Enzyme Container for the Reinforced Colorimetric-Photothermal Dual-Modal Sensing of Ochratoxin A.

"Point of care" (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL-1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL-1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.

Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading.

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

Quantum Dot Nanobeads Based Fluorescence Immunoassay for the Quantitative Detection of Sulfamethazine in Chicken and Milk.

Sulfamethazine (SMZ) as a broad antibiotic is widely used in livestock and poultry. However, the abuse of SMZ in livestock feed can lead to SMZ residues in food and the resistance of bacteria to drugs. Thus, a method for the detection of SMZ in food is urgently needed. In this study, quantum dot (QD) nanobeads (QBs) were synthesized by encapsulating CdSe/ZnS QDs using a microemulsion technique. The prepared QBs as signal probes were applied in lateral flow immunoassay (LFIA) for the detection of SMZ in chicken and milk. Our proposed method had limits of detection of 0.1138-0.0955 ng/mL and corresponding linear ranges of 0.2-12.5, 0.1-15 ng/mL in chicken and milk samples, respectively. The recovery of LFIA for the detection of SMZ was 80.9-109.4% and 84-101.6% in chicken and milk samples, respectively. Overall, the developed QBs-LFIA had high reliability and excellent potential for rapid and sensitive screening of SMZ in food.

Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn.

Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.

Recent advances in colorimetry/fluorimetry-based dual-modal sensing technologies.

Tailored to the increasing demands for sensing technologies, the fabrication of dual-modal sensing technologies through combining two signal transduction channels into one method has been proposed and drawn considerable attention. The integration of two sensing signals not only promotes the analytical efficiency with reduced assumption, but also improves the analytical performances with enlarged detection linear range, enhanced accuracy, and boosted application flexibility. The two top-rated output signals for developing dual-modal sensors are colorimetric and fluorescent signals because of their outstanding merits for point of care applications and real-time sensitive sensing. Given the rapid development of material chemistry and nanotechnology, the recent decade has witnessed great advance in colorimetric/fluorimetric signal based dual-modal sensing technologies. The new sensing strategy leads to a broad avenue for various applications in disease diagnosis, environmental monitoring and food safety because of the complementary and synergistic effects of the two output signals. In this state-of-the-art review, we comprehensively summarize different types of colorimetric/fluorimetric dual-modal sensing methods by highlighting representative research in the last 5 years, digging into their sensing methodologies, particularly the working principles of the signal transduction systems. Then, the challenges and future prospects for boosting further development of this research field are discussed.