Integrated silica membrane-based nucleic acid purification, amplification, and visualization platform for low-cost, rapid detection of foodborne pathogens.

Real-time fluorescence detection of nucleic acid exhibit excellent performance in analytical and diagnostic applications. However, the requirement of laboratory-based instrument and complex nucleic acid extraction greatly limits their application in point-of-care testing (POCT). Herein, a novel integrated silica membrane-based platform incorporating nucleic acid purification, amplification, and detection steps was developed. A universal and portable visualization platform was fabricated by incorporating denaturation bubble-mediated strand exchange amplification (SEA) reaction with silica membrane. The fluorescence signal of SYBR Green I with amplification products was visualized by the naked eye using a simple ultraviolet light on the silica membrane, and significant discrimination between the positive and negative samples could be easily and visually obtained. Besides, chitooligosaccharide-modified silica membrane allows the purification of nucleic acid in a totally aqueous system and enables in situ SEA. With the proposed integrated platform, 102-108 cfu/mL Vibrio parahaemolyticus could be successfully detected and excellent performance was also revealed for gram-positive pathogens. The detection limit of the method for artificially spiked oysters was 103 cfu/g and reached 100 cfu/g after 12 h enrichment. This proof-of-concept method could also be applied to a variety of nucleic acid amplification methods. We believe that the proposed silica membrane-based platform has great potential for the rapid and low-cost detection of nucleic acids especially in low-resource settings. Graphical abstract.

Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

A pattern-free paper enzyme biosensor for one-step detection of fish freshness indicator hypoxanthine with a microfluidic aggregation effect.

In this study, a paper-based enzyme biosensor for hypoxanthine (Hx) was developed, enabling visual and one-step fish freshness detection. Xanthine oxidase and horseradish peroxidase were immobilized on nitrocellulose membranes with 3,3',5,5'-tetramethylbenzidine to output the colour signal. Chitosan oligosaccharide lactate-modified nitrocellulose membranes entrapped the dual-enzyme system and exhibited excellent microfluidic aggregation effect. The developed enzyme biosensor produced a linear response of 0.01-0.16 mmolL-1 with a detection limit of 8.22 µmolL-1, and was selective for Hx with recoveries of 96.13-103.11 % for fish samples. These biosensors were attached directly to the surface of fish samples and the colour was revealed within 3 min. Colour signals can be judged by the naked-eye to distinguish between fresh and spoiled fish samples and analyzed by a smartphone for quantitative analysis. The biosensor shows great potential as a powerful pattern- and reagent-free device for on-site freshness evaluation of fish.

Triple stimuli-responsive ZnO quantum dots-conjugated hollow mesoporous carbon nanoplatform for NIR-induced dual model antitumor therapy.

Aiming at the inefficiency and toxicity in traditional antitumor therapy, a novel multifunctional nanoplatform was constructed based on hollow mesoporous carbon (HMC) to achieve triple stimuli response and dual model antitumor therapy via chemo-photothermal synergistic effect. HMC was used as an ideal nanovehicle with a high drug loading efficiency as well as a near-infrared (NIR) photothermal conversion agent for photothermal therapy. Acid-dissoluble, luminescent ZnO quantum dots (QDs) were used as the proper sealing agents for the mesopores of HMC, conjugated to HMC via disulfide linkage to prevent drug (doxorubicin, abbreviated as Dox) premature release from Dox/HMC-SS-ZnO. After cellular endocytosis, the Dox was released in a pH, GSH and NIR laser triple stimuli-responsive manner to realize accurate drug delivery. Moreover, the local hyperthermia effect induced by NIR irradiation could promote the drug release, enhance cell sensitivity to chemotherapeutic agents, and also directly kill cancer cells. As expected, Dox/HMC-SS-ZnO exhibited a high drug loading capacity of 43%, well response to triple stimuli and excellent photothermal conversion efficiency η of 29.7%. The therapeutic efficacy in 4T1 cells and multicellular tumor spheroids (MCTSs) demonstrated that Dox/HMC-SS-ZnO + NIR had satisfactory chemo-photothermal synergistic effect with a combination index (CI) of 0.532. The cell apoptosis rate of the combined treatment group was more than 95%. The biodistribution and pharmacodynamics studies showed its biosecurity to normal tissues and synergistic inhibition effect to tumor cells. These distinguished results indicated that the Dox/HMC-SS-ZnO nanoplatform is potential to realize efficient triple stimuli-responsive drug delivery and dual model chemo-photothermal synergistic antitumor therapy.

Multi-stimuli responsive nanosystem modified by tumor-targeted carbon dots for chemophototherapy synergistic therapy.

In this work, a tumor-targeted and multi-stimuli responsive drug delivery system combining infrared thermal imaging of cells with thermo-chemotherapy was developed. Oxidized mesoporous carbon nanoparticles (MCNs-COOH) with high photothermal conversion ability (photothermal transduction efficiency η = 27.4%) in near-infrared (NIR) region were utilized to encapsulate doxorubicin (DOX). The outer surfaces of MCNs-COOH were capped with multifunctional carbon dots (CDHA) as simultaneous smart gatekeepers, a tumor targeting moiety and a fluorescent probe. NIR laser irradiation killed cancer cells through NIR-light induced hyperthermia, facilitated chemotherapeutic drug release and enhanced the sensitivity of tumor cells to drugs. The therapeutic efficacy in two-dimensional (2D) and three-dimensional (3D) cells demonstrated that MC-CDHA loading DOX (MC-CDHA/DOX) had good chemo-photothermal synergistic antitumor effects (combination index of CI = 0.448). The biodistribution and pharmacodynamics experiments of MC-CDHA/DOX in the 4T1 tumor model indicated that MCNs-COOH prolonged the residence time of DOX in tumor tissues and therefore actualized effective synergistic photothermal chemotherapy. By combining these excellent capabilities, the tumor-targeted and multi-stimuli responsive drug delivery system can be utilized as a visible nanoplatform for chemophotothermal synergistic therapy.