Mesophilic Argonaute-Mediated Polydisperse Droplet Biosensor for Amplification-Free, One-Pot, and Multiplexed Nucleic Acid Detection Using Deep Learning.

Detection of nucleic acids from a single multiplexed and amplification-free test is critical for ensuring food safety, clinical diagnostics, and environmental monitoring. In this study, we introduced a mesophilic Argonaute protein from Clostridium butyricum (CbAgo), which exhibits nucleic acid endonuclease activity, to achieve a programmable, amplification-free system (PASS) for rapid nucleic acid quantification at ambient temperatures in one pot. By using CbAgo-mediated binding with specific guide DNA (gDNA) and subsequent targeted cleavage of wild-type target DNAs complementary to gDNA, PASS can detect multiple foodborne pathogen DNA (<102 CFU/mL) simultaneously. The fluorescence signals were then transferred to polydisperse emulsions and analyzed by using deep learning. This simplifies the process and increases the suitability of polydisperse emulsions compared to traditional digital PCR, which requires homogeneous droplets for accurate detection. We believe that PASS has the potential to become a next-generation point-of-care digital nucleic acid detection method.

Polystyrene microsphere-mediated optical sensing strategy for ultrasensitive determination of aflatoxin M1 in milk.

Aflatoxin M1 (AFM1) contamination poses a serious threat to human health globally. Hence, it is necessary to develop reliable and ultrasensitive methods for the determination of AFM1 residue in food products at low levels. In this study, a novel polystyrene microsphere-mediated optical sensing (PSM-OS) strategy was constructed to solve the problems of low sensitivity and susceptibility to interference from the matrix in AFM1 determination. Polystyrene (PS) microspheres have the advantages of low cost, high stability, and controllable particle size. They can be useful optical signal probes for qualitative and quantitative analyses attributed to the fact that they have strong ultraviolet-visible (UV-vis) characteristic absorption peaks. Briefly, magnetic nanoparticles were modified with the complex of bovine serum protein and AFM1 (MNP150-BSA-AFM1), and biotinylated antibodies of AFM1 (AFM1-Ab-Bio). Meanwhile, PS microspheres were also functionalized with streptavidin (SA-PS950). In the presence of AFM1, a competitive immune reaction was triggered leading to the changes in AFM1-Ab-Bio concentrations on the surface of MNP150-BSA-AFM1. The complex of MNP150-BSA-AFM1-Ab-Bio binds with SA-PS950 to form the immune complexes due to the special binding of biotin and streptavidin. The remaining SA-PS950 in the supernatant was determined by UV-Vis spectrophotometer after magnetic separation, which positively correlated with the concentration of AFM1. This strategy allows for ultrasensitive determination of AFM1 with limits of detection as low as 3.2 pg/mL. It was also successfully validated for AFM1 determination in milk samples, and a high consistency was found with the chemiluminescence immunoassay. Overall, the proposed PSM-OS strategy can be used for the rapid, ultrasensitive, and convenient determination of AFM1, as well as other biochemical analytes.

Label-Free Microchannel Immunosensor Based on Antibody-Antigen Biorecognition-Induced Charge Quenching.

Pursuing convenient operations and precise testing have become an urgent requirement in clinical diagnosis, treatment, and prognosis. Label-free detection is desirable for obviating the labeling process while maintaining high sensitivity and efficiency. Here, we used the dual properties of highly selective antibody-antigen recognition and potential signaling of biomolecules to construct a label-free electroosmotic flow-driven microchannel (LF-EMB) biosensor based on an antibody-antigen biorecognition-induced charge quenching theory proposed herein. The LF-EMB consists of a one-step immune-reaction, one-button portable device, and supporting microfluidic chip, providing a high-powered tool for rapid on-site testing. The LF-EMB quantified interleukin-6 and kanamycin levels down to 1 pg/mL and 5 pg/mL, respectively, with the whole analysis completed within 35 min. The outstanding sensitivity and detection speed of the constructed LF-EMB provide a convenient option for the quantitative detection of inflammatory markers and antibiotics.

Single-Atom-Thick Active Layers Realized in Nanolaminated Ti3(AlxCu1-x)C2 and Its Artificial Enzyme Behavior.

A Ti3(AlxCu1-x)C2 phase with Cu atoms with a degree of ordering in the A plane is synthesized through the A site replacement reaction in CuCl2 molten salt. The weakly bonded single-atom-thick Cu layers in a Ti3(AlxCu1-x)C2 MAX phase provide actives sites for catalysis chemistry. As-synthesized Ti3(AlxCu1-x)C2 presents unusual peroxidase-like catalytic activity similar to that of natural enzymes. A fabricated Ti3(AlxCu1-x)C2/chitosan/glassy carbon electrode biosensor prototype also exhibits a low detection limit in the electrochemical sensing of H2O2. These results have broad implications for property tailoring in a nanolaminated MAX phase by replacing the A site with late transition elements.

Direct, selective and ultrasensitive electrochemical biosensing of methyl parathion in vegetables using Burkholderia cepacia lipase@MOF nanofibers-based biosensor.

Methyl parathion is one of the most widely used pesticides in agricultural practices. It caused accumulation of acetylcholine and over-stimulation of receptors in synapses which eventually led to damage of the nervous system. Present study developed a direct, sensitive, rapid and reliable method for methyl parathion residues detection in vegetable samples. MOF nanofibers which demonstrated stable framework structure, good thermal/chemical stability, good electrochemical behavior, high porosity, surface area and pore volume was synthesized and used for fabrication of Burkholderia cepacia lipase (BCL)@MOF nanofibers biosensors. BCL@MOF nanofibers/chitosan/GCE biosensor demonstrated high sensitivity for methyl detection with a wide linear range (0.1-38 µM) and low limit of detection 0.067 µM. During the 3 weeks storage stability test at 4 °C, the fabricated biosensor demonstrated good reusability and excellent stability for methyl parathion detection with retainment of more than 80% of its initial response. When applied for detection of methyl parathion residues in vegetable samples, the BCL@MOF nanofibers/chitosan/GCE biosensors demonstrated good recovery rates.

Lipase-catalyzed selective enrichment of omega-3 polyunsaturated fatty acids in acylglycerols of cod liver and linseed oils: Modeling the binding affinity of lipases and fatty acids.

Present study employed molecular modeling method to elucidate the binding affinity of lipases with fatty acids of different chain lengths; and investigated the effects of lipases positional and fatty acids specificity on omega-3 polyunsaturated fatty acids (ω-3 PUFAs) enrichment in cod liver and linseed oils. Among the lipases studied, molecular modeling showed the active sites of Candida rugosa lipase (CRL) had a low C-Docker interactive energy for saturated (SFA) and monounsaturated (MUFA) fatty acids which predicted CRL to have highest preferences to selectively hydrolyze resulting in efficient enrichment of ω-3 PUFAs. Verification experiments showed the SFA and MUFA in the acylglycerol fraction includes monoacylglcyerols (MAG), diacyglycerols (DAG), and triacylglycerols (TAG) of CRL-hydrolyzed cod liver oil decreased from the initial 25.21 to 16.88% and 45.25 to 32.17%, respectively. In addition, CRL-hydrolyzed cod liver oil demonstrated 88.36% of ω-3 PUFAs enrichment. The regio-distribution of fatty acids in CRL-hydrolyzed cod liver oil were not significantly different than that of cod liver oil indicating the ω-3 PUFAs enrichment was due to fatty acids selectivity and not positional selectivity of CRL.