Target-induced DNA machine amplification strategy for high sensitive and selective detection of biotoxin

A ratiometric fluorescent biosensor based on cascaded amplification strategy for ultrasensitive detection of kanamycin

Detection of aflatoxin M1 in milk products from China by ELISA using monoclonal antibodies

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M1 (AFM1) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM1 sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM1 in milk. However, if there was no color change, presence of AFM1 at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM1 detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM1 in milk.

Label-free detection technique based on impedance was investigated for aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2) analysis in milk products. The impedance change resulting from antigen-antibody interaction was studied using a two-electrode setup made up of silver (Ag) wire. Processed milk such as drinking yogurt and flavored milk samples were analyzed in a flow-based setup. Two microflow pumps were used to construct the flow system where analytes (AFM1 and AFM2) were injected and impedance was measured using functionalized Ag wire electrodes. The flow system was optimized by adjusting both inlet and outlet flows to maintain the reaction volume optimum for impedance measurements. Using Bode plot, the matrix effect was investigated for detection of AFM1 and AFM2 in various matrices. Good recoveries were obtained even at low-AFM1 concentrations in the range of 1-100pg/mL. The influence of AFM2 on the detection of AFM1 was also investigated. The proposed method provides good scope for online monitoring of such hazardous toxins in milk products.

Aflatoxin M1 (AFM1) is known to be carcinogenic, mutagenic, and teratogenic and poses a serious threat to food safety and human health, which makes its surveillance critical. In this study, an indirect competitive ELISA (icELISA) based on a nanobody (Nb M4) was developed for the sensitive and rapid detection of AFM1 in dairy products. In our previous work, Nb M4 was screened from a Bactrian-camel-immunized phage-displayed library. It exhibits VH-like features, possesses higher thermal stability than monoclonal antibody (mAb 1E6) and tightly binds to AFM1–BSA with a KD value of 2.5 nM. Under the optimal conditions, its half-maximal inhibitory concentration was 0.338 ng/mL, the limit of detection was 0.051 ng/mL, and linearity was noted in the range of 0.168–0.679 ng/mL. Nb M4 displayed almost no cross-reactivity with other mycotoxins. No matrix effect was observed in milk and milk powder samples, and the matrix effect in yogurt samples could be weakened by 2-fold dilution. Furthermore, validation studies in spiked samples (milk, yogurt, and milk powder) resulted in good recoveries of 95.40%–111.33%, with a low coefficient of variation (2.89%–6.78%). High-performance liquid chromatography was used to evaluate the accuracy and reliability of the developed icELISA, which indicated a satisfactory consistent correlation (R2 = 0.9722). This study has provided a novel and ideal strategy for detecting AFM1 in dairy products.

High throughput cellular biosensor for the ultra-sensitive, ultra-rapid detection of aflatoxin M1

Metal-organic frameworks (MOFs): A review of volatile organic compounds (VOCs) detection.

Triple strategy-enhanced immunochromatographic assay based on APCB and AIEFM for the ultrasensitive detection of AFM1

Aflatoxin B1 (AFB1) is a secondary metabolite produced by some Aspergillus spp. fungi affecting many crops and feed materials. Aflatoxin M1 (AFM1), the 4-hydroxylated metabolite of AFB1, is the main AFB1-related compound present in milk, and it is categorized by the International Agency for Research on Cancer (IARC) as a “group 1 human carcinogen”. The aim of this work was to evaluate and compare the analytical performances of two commercial immunoassays widely applied for the detection of AFM1 in milk, namely strip test immunoassay and enzyme linked immunosorbent assay (ELISA). Assay validation included samples at AFM1 levels of 25, 50, 75 ng/kg and blank samples (AFM1 < 0.5 ng/kg). With respect to a screening target concentration (STC) of 50 ng/kg the two assays showed cut-off values of 37.7 ng/kg and 47.5 ng/kg for strip test and ELISA, respectively, a false suspect rate for blanks <0.1% (for both assays) and a false negative rate for samples containing AFM1 at levels higher than STC, of 0.4% (for both assays). The intermediate precision (RSDip) was <32% for the strip test and <15% for the ELISA. Method verification through long-term intra-laboratory quality control (QC) measurements confirmed the results from the validation study. Furthermore, a satisfactory correlation of the results obtained with both immunoassays and the AOAC Official Method 2000.08 was obtained for the analysis of cow milk samples naturally contaminated with AFM1 at levels within “not detected” (< 0.5 ng/kg) and 50 ng/kg. Finally, the extension of the scope of the strip test method to goat and sheep milk was evaluated by applying the experimental design foreseen in the EU regulation.

Tumor markers are one of the important indicators for early cancer diagnosis. As a new analytical method, electrochemical immunosensing analysis has the advantages of high sensitivity, good selectivity, and rapid detection, which is of great significance for the detection of tumor markers. In this work, an AuNP/reduced graphene oxide (AuNP/rGO) composite was synthesized. We used it for electrochemical sensor fabrication with the assistance of the biotin–streptavidin protein (SA) system to further amplify the signal to achieve sensitive detection of carcinoembryonic antigen (CEA). In addition, AuNPs have been incorporated due to their good electrical conductivity and biocompatibility, which can accelerate electron transfer at the electrode interface and improve the loading capacity to capture antibodies. The fabricated AuNPs/SA/rGO has a large working surface area and high material utilization ratio, which improves the catalytic capacity of H2O2 reduction and effectively amplifies the current signal. The linear range of the response current signal of the sensor toward the CEA concentration is 20 fg/ml to 200 ng/ml, and the limit of detection can achieve 6.2 fg/ml. In addition, the fabricated immunosensor has good reproducibility, selectivity, and stability.

Direct visualization of MicroRNA in vivo via an intelligent MnO2-carried catalytic DNA machine

Performance-enhanced lateral flow immunoassay for rapid and ultrasensitive detection of aflatoxin M1 in dairy products using in-situ gold deposition-mediated signal amplification strategy.

Because human immunodeficiency virus (HIV) has been one of the most terrible viruses in recent decades, early diagnosis of the HIV gene is of great importance for all scientists around the world. In our work, we developed a novel electrochemical biosensor based on one-step ultrasonic synthesized graphene stabilized gold nanocluster (GR/AuNC) modified glassy carbon electrode (GCE) with an exonuclease III (Exo III)-assisted target recycling amplification strategy for the detection of HIV DNA. It is the first time that GR/AuNCs have been used as biosensor platform and aptamer with cytosine-rich base set as capture probe to construct the biosensor. With the combination of cytosine-rich capture probe, good conductivity and high surfaces of GR/AuNCs, and Exo III-assisted target recycling amplification, we realized high sensitivity and good selectivity detection of target HIV DNA with a detection limit of 30 aM (S/N = 3). Furthermore, the proposed biosensor has a promising potential application for target detection in human serum analysis.

A multi-functional gold/iron-oxide nanoparticle-CNT hybrid nanomaterial as virus DNA sensing platform

Novel surface plasmon resonance sensor for the detection of heme at biological levels via highly selective recognition by apo-hemoglobin