| Biosensors have been widely used in the field of fermentation, environmental monitoring, food engineering, clinical medicine, military and military medicine because of its simple, sensitivity, selectivity, and multiplexing capacity. The key step in the preparation of biosensors is the construction of the bio films. In this paper, polymer films prepared by the method of breath figure are applied toconstruct biofilms. Different amphiphilic diblock copolymers have been used to form porous membranes for the immobilization of biocatalyst. Thehydrophobic block with a glass transition temperature higher than room temperature is beneficial for film formation and the hydrophilic block can soluble in water and ionization. Both of them contributed to the formationof the porous membranes based on the method of breath figureon the interface of water/oil, the amphiphilic block copolymer arrangemen orderly, due to its micro phase separation and self-assembly capability. Meanwhile, the electrostatic interaction between the hydrophilic block and the biological molecules will induce the biomolecules arrange orderly. This method was called as charge and amphiphilicity dually driven (CADD) self-assembly. The formation mechanism of the polymer/bio films was investigated by the electron microscopy, IR spectrum, UV spectrum, contact angle measurement,3D stylus surface profilometer, and the parameter for the construction of the biosensor was optimized. XPS and contact angle measurements suggested that the hydrophobic block enriched on the membrane surface. The hydrophilic block pointed toward the internal membrane, meanwhile ordered the biomolecule arrangement at the interface of the polymer and electrode. Several biosensors were developed based on this mothed:(1) PS-b-PAA porous film was constructed on the surface of hemoglobin (Hb) solution in constant temperature and humility via breath figures method. The organized PS-b-PAA assembled Hb by a charge-charge interaction on the W/O interface. The obtained PS-b-PAA/Hb bioelectrode showed a remarkably enhanced direct electron transfer (ET) in a cyclic voltammogram. The direct ET between the electrode surface and the electrically active group of Hb (i.e., Hbheme [Fe(Ⅲ)]) accompanied with single protonation. The results also suggested that the redox on this electrode was a surface-controlled process rather than a diffusion-controlled one. This method was successfully applied to the construction of H2O2 biosensor. The sensitivity of the biosensor was 4.0 mA·M-1·cm-2 with a linear range from 0.12～30 μM. The detection limit was calculated as 12 μM, and the Michaelis constant of immobilized Hb was 1.60 mM.(2) A hydrophilic block with weaker hydrophobility (PMMA) and amphiphilic block PAA with negative charge were chosen for studying the construction of PMMA-b-PAA/Hb electrode via CADD self-assembly. Direct electron transfer of Hb on the PMMA-b-PAA/Hb electrodeis higher than that of the electrode constructed by adsorption and entrapment. The self-assemble of Hb and PMMA-b-PAA makes the film hydrophobic and porous. The PMMA block enriched on the membrane surface, the PAA block pointed toward the internal membrane, meanwhile ordered Hb arrangement at the interface of the polymer and electrode. Furthermore, the Hb active center was exposed outside by the electrostatic interaction, between PAA block with the negative charged and the positively charged Hb. A surface-controlled electrochemical process was observed for PMMA-b-PAA/Hb. The process was on electron transfer accompanied by a proton transfer. Good biological activity of Hb in the PMMA-b-PAA/Hb electrode was observed with Km of 1.2 mM. The PMMA-b-PAA/Hb electrode have high catalytic ability of H2O2 towards the reduction of H2O2 with a sensitivity of 11 mA·M-1·cm-2, detection limit of 0.34 μM and a linear range of 0.57-300 μM. Good reproducibility and stability were also observed for this PMMA-b-PAA/Hb film. (3) A hydrophilic block with stronger hydrophobility (PS) and amphiphilic block P4VP with positive charge were choosed for studying the construction of PS-b-P4VP/PPO electrode via CADD self-assembly on the suface of PPO solution. The organized PS-b-P4VP induced the orderly arrangement of PPO via electrostatic interaction. The phenol biosensor was developed by the construction of the PS-b-P4VP/PPO biofilm on the surface of glass carbon electrode. The obtained biosensor showed high biological activity and has good catalytic performance for phenols, as well as good reproducibility stability. When the PS-b-P4VP/PPO biosensor was used for catechol detection, the sensitivityl was 314 mA·M-1·cm-2. the linear range was 0.12～30 μM, the detection limit was 0.07 μM and the reaction activation energy is 18 kJ·mol-1. The biosensor showed response to various phenol compounds and the sensitivity decreases in the following sequence:phenol> catechol> p-chlorophenol>p-methylthiophenol.(4) The PMMA-b-PAA/GOD bioflim was constructed via CADD self-assembly, which was assembled on the surface of Pt electrode to form a glucose biosensor. The contact angle measurements and SEM showed that the bioflim have a hydrophobic and porous surface, which is benefit to the rapid diffusion of substrate and production during enzymic catalytic reaction. The prepared PMMA-b-PAA/GOD electrode can determine the concentration of glucose quickly. The construction parameters of the electrochemical biosensor and the effect of the pH, potential, and temperature on the response of biosensor were studied in detail. The sensitivity of PMMA-b-PAA/GOD electrode to glucose was 14.3 mA·M-1·cm-2, the linear range was 0.42～ 6000 μM, the Michaelis constant was 17 mM, and the apparent active energy of enzymic catalytic reaction was 34.5kJ mol-1.
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