Paper-based In Situ Growth Protein-Mn₃?PO₄?₂ Hybrid Material And Its Application In Biological Analysis

With the improvement of health concept,the sensitivity and timeliness of disease diagnosis become more and more important.Point of care?POCT?is a diagnostic technique that enables rapid detection and analysis of medical samples without restriction of time and place.The microfluidic chip is an important tool for modern medical detection.Using a small chip which integrated by micro-reactor and a micro-detection device to complete relatively fast and accurate experimental analysis with a small amount of liquid.Its simple processing,low-cost and disposability result make paper-based microfluidic chip become popular.Especially when it combined with enzyme-catalyzed color reaction,rapid diagnosis can be realized through visual signals,thereby getting rid of dependence on professional instruments.However,the function of the paper chip is relatively simple and researchers hope to use namomaterials to modify the paper chip to overcome this limitation.The enzyme is an expensive biological protein,whether the enzyme and the probe molecules are effectively immobilized affects the performance of the paper-based biochip.The nanomaterials provide a new direction for enhancing the effective immobilization of biomolecules and constructing high-specificity and high-sensitivity biochips because of their unique morphology,catalytic properties and biocompatibility.This paper directly grows protein-inorganic hybrid materials on paper fibers to immobilize enzymes.Further utilizing the catalytic properties of nanomaterials,the artificial enzymes and natural enzymes are directly assembled onto the fibers.Achieve rapid,sensitive and cost-effective detection of SIgA and glucose.In addition,a device combining a pipette tip and a paper chip was developed for biochemical detection.The main research contents of this paper are as follows:1.Paper-based in situ growth of protein G-inorganic hybrid materials for high-sensitivity detection of secretion immunoglobulin A?SSIgA?Protein G can bind to the Fc region of antibody molecules,protein G-inorganic hybrid materials were designed to achieve targeted immobilization of antibodies on paper-based chips and improve the sensitivity of paper-based immunoassays.In this study,the base material was selected by non-specific adsorption contrast of different types of paper base materials.After optimize the concentration of protein G,the inorganic solution?MnSO₄?and protein solution?protein G?were added dropwise to the paper base material in order to synthesize the organic-inorganic hybrid material.Comparing with the method of liquid-phase synthesis of protein inorganic hybrid materials in test tubes,the direct dropping of the reaction solution to the paper-based fiber network can significantly improve the utilization efficiency of the enzyme and avoid the waste of the enzyme;in addition,the use of pipettes for liquids Precise operation improves stability and reproducibility for subsequent immune responses.Protein G binds to the Fc region of the antibody,so that the Fab region,which is the key site for antibody binding,faces in the direction of the addition of the object to be seen.Using paper as a solid substrate,an enzyme-linked immunosorbent assay?ELISA?was performed to determine the content of SIgA in the sample.Visualization results showed that the lowest visible SIgA concentration was 1 ng/mL,which reached the detection line of the ELISA kit based on the microplate reader.Low-cost paper-based immunoassay benefits from the in situ growth of protein G inorganic hybrids,whose immobilized protein G is targeted by antibodies and provides antigen-recognizable antibody domains for immune responses?Fab segment?.More importantly,based on the sensitive protein analysis of paper base,the dependence on special equipment is eliminated and the foundation for POCT application is laid.2.In situ growth of glucose oxidase-inorganic hybrid materials based on paper base for visual detection of glucoseManganese is a metamorphic metal.It has been found that manganese-based nanomaterials have unique catalytic activity.In protein-inorganic hybrid materials,the function of Mn₃?PO₄?₂ is worth digging.Therefore,in order to further explore the catalytic activity of the inorganic part in the hybrid material,BSA-Mn₃?PO₄?₂ was synthesized with bovine serum albumin?BSA?,and by optimizing the phosphate concentration,BSA-Mn₃?PO₄?₂with peroxidase activity was obtained,shows another unique property of protein-inorganic hybrid materials in addition to the function of protein immobilization.Based on this,glucose oxidase?GOx?-Mn₃?PO₄?₂?GOx-Mn₃?PO₄?₂?was synthesized in situ on the paper base,and 0.1M manganese sulfate was added dropwise on Whatman?filter paper.Potassium dihydrogen phosphate containing GOx generates GOx-Mn₃?PO₄?₂ hybrid material for glucose detection.By optimizing the enzyme concentration and the amount of peroxidase substrate?TMB?,a glucose paper-based chip based on enzyme-Mn₃?PO₄?₂ hybrid material was constructed.The sensitivity,stability,reproducibility,and specificity of the paper-based chip were investigated.The results showed that the minimum detectable limit of in situ growth enzyme-Mn₃?PO₄?₂ is 0.1 mM,which can be specific in interfering substances.The detection of glucose and the recovery of repeatability range from 95%to 105%.Sensitive detection benefits from in situ growth of the enzyme-Mn₃?PO₄?₂heteroconjugate:The enzyme?GOx?generates hydrogen peroxide?H₂O₂?during the oxidation of biomolecules?glucose?.The latter is immediately catalytically reduced by Mn₃?PO₄?₂ in the hybrid and oxidizes the TMB substrate.As the enzyme-Mn₃?PO₄?₂ hybrid compound,the biological enzyme and the pseudo-enzyme bind tightly,reducing the diffusion distance of the reaction intermediate product and improving the reaction efficiency.In order to demonstrate its use in real pathological samples,glucose in real-pathological serum samples was monitored and compared with commercial test strips and glucose meters.The results show the potential for in situ growth of enzyme-inorganic hybrid paper chips for real-world applications.3.Rapid detection device combining paper-based microfluidic chip and gun head designThe diagnosis of pathogens or infective samples will be involved in the immediate diagnosis.In order to avoid potentially harmful biological contamination,the detection analysis should be conducted under closed conditions as far as possible.The ordinary paper base is mostly open,and its use has certain risks.In response to this problem,this paper combines a paper-based microfluidic chip with a standard pipette tip to create a fast,safe and pollution-free detection device.The aspiration process of the pipette is used to complete the liquid intake,and the triangular prismatic paper chip is placed in a proper position on the tip of the pipe to realize the simultaneous and non-interfering detection of the three types of samples.The aspiration process of the pipette tip accurately transfers the sample to be tested to the tape,and the detection result is obtained by visualizing the signal.The ability of this device to detect multiple samples was determined by simultaneous detection of glucose,protein and pH in artificial urine.Therefore,the device is simple to manufacture,easy to use,and can be used for biological and chemical experiments.