Nanobiocatalytic System Based On Microfluidic Paper-based Analytic Device For Whole Blood Analysis

With the rapid development of social economy,people are increasingly concerned about the living environment and human health.Point-of-care testing(POCT),as a low-cost,highly-accurate and highly-portable analysis equipment,has received extensive attention in medical diagnosis,environmental monitoring,and food safety monitoring.Since its discovery,the microfluidic chip has always been the most popular POCT platform for bioassays.The microfluidic chips are an important technology in a miniature full-analysis system and are known as "a laboratory built on a chip." They have an independent analysis system that can perform all laboratory operations in a miniature range on a microdevice.Among them,microfluidic paper-based analytical device(?PAD)is the latest developments in microfluidic chips.It is based on hydrophilic filter paper,and it is manufactured using technologies such as photolithography,paraffin printing,and inkjet printing.It can also be combined with colorimetric,electrochemical,fluorescence,and chemiluminescence detection technologies to achieve simultaneous detection of multiple analytes.Natural enzymes are biocatalysts which have high catalytic efficiency,high selectivity and strong substrate specificity in chemical reactions.However,under harsh conditions such as strong acids,strong bases,and high temperatures,most natural enzymes have poor stability and are easily deactivated due to the degradation of the their molecular structure.Enzyme immobilization has been proven to be an effective method for maintaining the enzyme's catalytic activity and enhancing its stability under harsh conditions.Zare's research group firstly reported that BSA-Cu3(PO4)2 hybrid nanoflowers could be synthesized by co-precipitation method using Cu3(PO4)2·3H2O as the inorganic component and bovine serum albumin(BSA)as the organic component.Compared with the traditional two-step enzyme immobilization method,his self-assembly developed by his group method simplifies the synthesis of the immobilized carrier and the immobilization of the enzyme in one step,and realizes nano-immobilization of various enzymes.The immobilized enzyme prepared by this method has nanomaterial nanostructure and high specific surface area,and it is beneficial to the contact of enzymes and substrates in the catalytic reaction,reducing the mass transfer limit,maintaining the catalytic activity of the enzyme and improving its stability and durability.The self-assembly method can also be used for the simultaneous immobilization of multiple enzymes.The immobilized natural enzymes have good biocompatibility and it is a good nanobiocatalyst and can be used for develop efficient,accurate,rapid and simple nanobiocatalytic systems for the application of POCT.In this study,we have constructed a nanobiocatalytic system based on ?PAD for the analysis of multiple biomarkers in whole blood.In the first chapter of this paper,the physical adsorption method,covalent bonding method,and self-assembly method for enzyme immobilization are introduced.The synthesis method,advantages,types,formation mechanism and application of enzyme-inorganic hybrid nanoflowers were introduced discussed in detail.Subsequently the advantages,production methods,detection techniques and applications of ?PAD are also introduced.Finally,we briefly discuss the research significance and content of this thesis.In Chapter 2,we report the use of glucose oxidase(GOx)and horseradish peroxidase(HRP)as organic components,and Cu3(PO4)2·3H2O as an inorganic component.GOx&HRP-Cu3(PO4)2 hybrid nanoflowers with high catalytic activity,high selectivity and good monodispersity were prepared by coprecipitation at room temperature.Scanning electron microscopy(SEM),X-ray powder diffraction(XRD)and energy dispersive spectroscopy(EDX)were used to investigate the morphology,composition,structure and formation of GOx&HRP-Cu3(PO4)2 hybrid nanoflowers.Then,a nanocatalytic system based on ?PAD combined with GOx&HRP-Cu3(PO4)2 hybrid nanoflowers was designed and applied to the quantitative analysis of glucose in whole blood.Under the optimal conditions,the biosensor designed by us has a detection limit of 25 ?mol·L-1 for glucose,which takes a short time and is highly sensitive.Because the above-mentioned(chapter 2)paper-based nano-biocatalytic system only achieves the analysis of a single substance,it cannot be widely popularized in real life.In particular,in some developing countries and regions where resources and medical equipment are scarce,more diversified POCT analysis equipment are urgently needed.The ?PAD is easy to fold to form branches or design multi-channel microchannels,which is very conducive to the development of a wide range of POCT analysis equipment.In Chapter 3,we developed a diversified POCT analysis device based on ?PAD combined with dual-enzyme inorganic hybrid nanoflowers.The colorimetric detection technology enables the analysis device to simultaneous detection of glucose and uric acid concentrations in whole blood with high sensitivity.In this thesis,we first synthesized UAO&HRP-Cu3(PO4)2 hybrid nanoflowers with uric acid(UAO)and HRP as organic components and Cu3(PO4)2·3H2O as inorganic components.And its morphology,composition,synthesis time,performance and formation mechanism were discussed.Sequentially,under optimal synthesis conditions,we prepare GOx&HRP-Cu3(PO4)2 hybrid nanoflowers and UAO&HRP-Cu3(PO4)2 hybrid nanoflowers suspensions in advance and place them in the dark at 4°C for further use.And a simple wax printer was used to print the designed ?PAD pattern on the filter paper.After heat treatment,the wax was completely melted and infiltrated into the filter paper to form a hydrophobic barrier,resulting in ?PAD equipped with hydrophilic and hydrophobic channels.Afterwards,the hybrid nanoflowers prepared in advance and their respective color indicator are mixed together,respectively added to the corresponding detection area,and dried and used.Finally,a certain amount of whole blood solution was added to the central area,and the sample spontaneously spread from the central area to the detection area by capillary action.After 10 minutes of reaction,the changes in the color signal were recorded with the camera.The recorded image is correspondingly converted to grayscale values correspondingly,and image J,an image processing software,is used to analyze the color intensity and gradient,and the color signal is digitized,which achieves simultaneous rapid and quantitative analysis of glucose and uric acid in whole blood samples.