| As an efficient bioelectronic platform,biosensors can effectively output microscopic biochemical reaction signals in a visual and quantifiable form and provide reliable data for human’s decision-making.Benefiting from the advantages of simple preparation,strong specificity,high sensitivity,fast response,portability and easy integration,etc.,biosensors have shown broad application prospects in biomedical,environmental monitoring,food safety and other fields.Generally,biosensors take bioactive substances（such as enzymes,antibodies,microorganisms,cells,tissues,nucleic acids,etc.）as molecular recognition elements（MRE）,and combine with appropriate signal transducers and amplifiers to realize the monitoring and quantification of target molecules.The key process for consctructing a biosensor is to effectively immobilize these molecular recognition elements on sensing electrode.As a part of the biosensor interface,the structure and properties of the carrier material have great influence on the molecular recognition reaction and signal transmission across the sensor interface.Among all the carrier materials,polymers can not only improve the surface properties for effective immobilization of molecular recognition elements,but also can provide favorable microenvironment for maintaining the biological activity of these recognition elements.As a result,polymers are regarded as ideal carriers for immobilization of molecular recognition elements on the sensor interface.However,there are still some defects with polymers as the carrier material:（1）the non-covalent bonding force is weak,leading to unsatifactory leakage and poor long-term stability;（2）although covalent way can enhance the binding force,the immobilization efficiency is not high due to the complex operation process;（3）the assembly of polymer and molecular recognition elements on the surface of sensor electrode is mainly based on the drop-coating method,and the structure and performance stability of the sensor coating is poor;（4）in addition,the complex of polymers and the molecular recognition elements is usually in the form of membrane or block with a large mass transfer resistance,which limits the sensitivity and response rate of the sensor.Therefore,how to effectively immobilize enzymes from the design of polymer structure and assembled aggregate form for highly effective biosensing platforms is of great significance which would be explored and investigated in this work.Based on above background,this work starts from the design of the structure and aggregation morphology of functionalized macromolecules colloid,and realizes the effective immobilization of biomolecular recognition elements by using functionalized self-assembled colloids as the basic building blocks,and realizes the secondary assembly of these colloid particles through electrical induction.In this process,some inorganic nanomaterials were introduced through co-assembly and co-deposition to prepare the biosensing coatings and finally applied them to the construction of biosensors.The research following below routes:First of all,using pure self-assembled polymer nanoparticles as the basic building unit to prepare particles film on the electrode surface,then using gold nanoparticles（Au NPs）as a bridge to realize antibody immobilization,obtaining the immune-biosensing coating which could specifically recognize target antigen molecule;the advantages of macromolecular colloids in biosensing coatings was demonstrated in this process.Secondly,enzyme-loading polymeric nanoparticles（NPs）were prepared by complex self-assembly between enzymes and photo-cross-linkable polymers,and then the complex NPs was electrically deposited on electrode surface together with silver nanoparticles（Ag NPs）,forming the 0D/0D composite biosensing coating.The coating formation process and mechanism was investigated as well as the structure-activity relationship between the coating structure and sensing performance;Thirdly,a hierarchical 0D/2D composite biosensing coating was fabricated using one step electrophoretic deposition（EPD）of complex enzyme-polymer NPs and graphene oxide（GO）;Finally,a hybrid nanocomposite with molecular recognition function was prepared by co-assembly of enzymes,synthetic polymer and carbon nanotubes（CNTs）,and the hybrid nanocomposites were deposited as composite coatings to fabricate a highly effective biosensing platform with good sensing performance.The specific research contents are as follows:1.Immuno-biosensing coating based on self-assembled composite nanoparticlesIn this part,dopamine（DA）modified poly（γ-glutamic acid）（γ-PGA）was co-assembled with chitosan（CS）through electrostatic interaction,obtaining complex self-assembled nanoparticles（PGA-DA/CS NPs）.Further,PGA-DA/CS NPs were deposited on electrode surface to form NPs film.By controlling the external electric field conditions（deposition time and deposition voltage）,the coating formation process and mechanism were explored.The secondary assembly of colloidal particles on the electrode surface is mainly based on the electrochemical particle aggregation mechanism.Similarly,DLVO theory can well explain the migration and deposition of colloidal particles from the perspective of potential energy.The NPs coating was then applied to anchor Au NPs and further to immobilize carcinoembryonic antigen antibody（CEA-Ab）,obtaining an immuno-biosensing coating which could recognize carcinoembryonic antigen（CEA）.The influence of several factors（γ-PGA-DA contents,pH,deposition potential and times）on the structures and sensing performance of the immuno-biosensing coating was investigated systematically.Finally,the sensing coating was used to construct an immunosensor for CEA sensing,which demonstrates excellent sensing performance than any other previously reported impedimetric immunosensors.The results showed that the colloidal NPs based coating has of significant advantages in biosensors,and it also provided a new idea for the development of new generation of immunosensors.2.Composite biosensing coating based on enzyme-loaded polymeric NPs/silver NPsIn this chapter,complex enzyme-loaded HRP@γ-PGA-AMC NPs was firstly prepared from the co-assembly of a photo-cross-linkableγ-PGA-AMC polymer and HRP enzyme.Subsequently,HRP@γ-PGA-AMC NPs and silver nanoparticles（Ag NPs）were co-assembled on the surface of the sensor electrode by electrophoretic deposition,forming a binary-particle（0D/0D）composite coating.After photo-crosslinking,by ultraviolet light,a composite biosensing coating was obtained.The enzymatic biosensor constructed based on the composite biosensor coating has excellent comprehensive performance.The influence of HRP concentration,pH,salt concentration on the HRP@γ-PGA-AMC NPs was studied,and the deposition parameters for coating formation was optimized.Also,the deposition mechanism,the relationship between different composite coating structure and sensing performance were explored.When the deposition voltage was 1.0 V and the deposition time was 120 s,the composite biosensing coating was with prepared a smooth and uniform morphology.The co-deposition process included the interaction of particle accumulation flocculation mechanism and electrochemical particle aggregation mechanism.The developed enzymatic biosensor showed good sensing performance for H2O2 detection.UV cross-linking can effectively enhance the structural stability of the composite sensing coating,so as to improve the sensor stability of the enzymatic biosensor.The enzyme biosensor has been successfully applied to the actual detection of milk samples and human urine samples,showing a strong practical application potential in food safety and medical diagnosis.3.Composite biosensing coating based on enzyme-loaded polymeric NPs and graphene oxideIn this chapter,enzyme-loaded polymeric NPs were prepared first through the co-assembly of photo-cross-linkableγ-PGA-HEMA（PGH）and HRP enzymes,generating complex HRP@PGH NPs.The effects of HRP concentration,pH and salt concentration on HRP@PGH NPs were studied.Subsequently,a hierarchical complex coating（0D/2D）was prepared by one-step electrodeposition of HRP@PGH NPs and graphene oxide nanosheets（GO NSs）.After electrochemical reduction of GO NSs into graphene（GNSs）and subsequent photo-cross-linking,a composite biosensing coating（HRP@PGH/GNSs）was prepared.The relationship between different composite coating structure and sensing properties was studied.The composite coating had a good structural integrity prepared from that the deposition voltage was1.5 V and the deposition time was 120 s or above.The co-deposition of HRP@PGH NPs and GO NSs depends on the mutual restraining effect of the two components in electrophoresis migration and the neutralization effect of the charge on the electrode surface.UV cross-linking can enhance the densification of the coating,which can not only effectively prevent the leakage of enzyme molecules,but also improve the structure and performance stability of the composite coating.The constructed enzymatic biosensor has excellent comprehensive sensing performance for H2O2 and has been successfully used in human serum samples,demonstrating practical application potential in medical diagnosis,food safety and other fields.4.Composite biosensing coating based on enzyme-loaded NPs decorating carbon nanotubesIn this chapter,a“necklace-like”hybrid nanocomposite（GOx@PAVE-CNTs NCs）was prepared through the complex assembly of a random copolymer（PAVE）,glucose oxidase（GOx）and multiwalled carbon nanotubes（MWCNTs）.The co-assembly behavior of PAVE and MWCNTs was firstly studied through optimization of polymer molecular structure and polymer/CNTs mass ratios,and the influence of pH on PAVE-CNTs NCs was further studied.Subsequently,glucose oxidase（GOx）was introduced in the co-assembly process of PAVE and MWCNTs to study the influence of GOx content on the formation of the hybrid enzyme-loaded NCs.The composite biosensing coating based on the GOx@PAVE-CNTs NCs was prepared by electrophoretic deposition,and the influence of deposition voltage and deposition time on the morphology and properties of the coating was systematically investigated,so as to reveal the basic law and deposition mechanism of the GOx@PAVE-CNTs NCs in the deposition process.Uniform and continuous coating can be prepared when the deposition voltage is 3.0 V or above and the deposition time is 5 min or above.The main mechanism of migration and deposition is electrochemical particle aggregation mechanism.However,due to this special linear structure,the interfacial assembly of the GOx@PAVE-CNTs NCs is mutually restrained.The developed enzymatic biosensor has excellent comprehensive sensing performance for glucose detection.On the one hand,nano-sized polymer nanoparticles can improve the fixation amount of enzyme and the specific surface area of the coating.The photo-cross-linking can enhance the immobilization effect of the enzyme and improve its overall catalytic activity.The long-term stability of the cross-linked biosensing coating is greatly improved.On the other hand,the presence of MWCNTs facilitates electron transport in the coating,thus improved the response rate and the sensitivity of the sensor.The enzymatic biosensor was also successfully applied in human urine and serum samples,which shows great practical value in the fields of biological medicine and life health.In summary,this work aimed at the efficient immobilization of biological recognition elements through self-assembly of functionalized macromolecules or co-assembly of polymer-bioactive components,which provides a new strategy for the long-term and stable immobilization of bioactive components in solid interface.In addition,soft colloid particles and inorganic conductive materials were co-assembled on electrode surface based on one-step electrophoretic deposition to prepare composite biosensor coating,which offers a new way for the development of biosensor materials and sensors’construction.Through the systematic study of the co-assembly process of soft colloidal particles and the inorganic conductive materials,the rule and mechanism of multi-component and hierarchical assembly were revealed,which would not only provide valuable inspiration for new generation of biosensing materials,but also extend the application of functionalized macromolecular colloids in the field of sensing coatings. |
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