Study Of The Application And The Direct Electrochemistry Of Hemoglobin Immobilized On Nanoparticles Modified Glassy Carbon Electrodes

Objective (1) We presented a simple approach to synthesize a novel Fe₃O₄@Pt core-shell nanocomposite, constructed a bionic functional interface based on hemoglobin (Hb) immobilized on Fe₃O₄@Pt-chitosan (CS) film, denoted as"Hb-Fe₃O₄@Pt-CS", and achieved the detection of H₂O₂.(2) A novel Fe₃O₄/APTES/CdS magnetic nanocomposite was synthesized to study the direct electrochemistry and the electrocatalysis of Hb immobilized on Fe₃O₄/APTES/CdS-CS modified glassy carbon electrode (GCE).(3) The influence of Pt nanoparticles (NPs) on the electrogenerated chemiluminescence (ECL) of CdS nanocrystals (NCs) with different fabrication approaches and different amounts of Pt nanoparticles was studied. And a biosensor based on layer-by-layer assembly of Hb-Pt/CdS films was also constructed.(4) Red blood cells (RBCs) were immobilized on Au NPs-CS modified GCE to study the transmembrane electron transfer of Hb inside RBCs.Methods (1) Fe₃O₄@Pt core-shell nanocomposite was synthesized by reduction of Pt⁴⁺ onto the Fe₃O₄-core nanoparticles surfaces using sodium dihydrogencitrate both as a reductant and a stabilizer. Hb with positive charge and Fe₃O₄@Pt nanocomposite with negtive charge were adsorbed alternately onto CS modified GCE. The process was characterized by transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-vis), cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS).(2) Fe₃O₄-core nanoparticles were prepared by coprecipitation of an Fe(â…¡) and Fe(â…¢) chlorides in aqueous solutions without any surfactants. Then TGA-capped CdS nanoparticles were connected onto Fe₃O₄ nanoparticles by 3-aminopropyltriethoxysilane (APTES) to form Fe₃O₄/APTES/CdS magnetic nanocomposite. The magnetic nanocomposite was characterized by TEM, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS), and used to immobilize Hb to construct a H₂O₂ biosensor.(3) A brown stable Pt NPs were prepared without poisoning stabilizers, and then a series of Pt/CdS nanocomposite solutions containing an equal amount of CdS NCs and different amount of Pt NPs were obtained. The influence of Pt NPs on the ECL of CdS NCs with different fabrication approaches and different amounts of Pt NPs were discussed. Meanwhile, a biosensor based on layer-by-layer assembly of Hb-Pt/CdS films was constructed.(4) RBCs were immobilized on Au NPs-CS modified GCE, and characterized by atomic force microscopy (AFM) and scanning electron microscope (SEM). The transmembrane electron transfer and sensing behavior of Hb inside RBCs were systematically studied.Results (1) TEM indicated that Pt4+ shell was successfully reduced on the surface of Fe₃O₄. Hb immobilized on Fe₃O₄@Pt-CS modified GCE remained its secondary structure and exhibited good catalytic activity towards H₂O₂. In pH 7.0 PBS, Hb-Fe₃O₄@Pt-CS modified GCE exhibited a couple of well-defined and quasi-reversible redox peaks. The anodic peak potential and cathodic peak potential were located at -0.38 V and -0.32 V respectively, the formal potential E^0' was about -0.35 V.(2) A novel Fe₃O₄/APTES/CdS magnetic nanocomposite was successfully synthesized. Then Hb was immobilized on the magnetic nanocomposite to construct a H₂O₂ biosensor. In pH 7.0 PBS, a reversible redox peaks of Hb was observed at -0.30 V and -0.38 V respectively. The formal potential E^0' was about -0.34 V. The linear range was from 2.8×10^-6 to 1.7×10^-2 mol/L, with a detection limit of 4.0×10^-7 mol/L (S/N = 3).(3) The influence of Pt NPs on the ECL of CdS NCs with different fabrication approaches and different amounts of Pt NPs were discussed. We found that an appropriate concentration of Pt NPs can enhance the ECL intensity of CdS NCs, of which the excessive concentration showed a quenching effect. Besides, the {Hb-Pt/CdS}₄ multilayer films showed good electrocatalytic activity to H₂O₂.(4) RBCs were successfully immobilized on Au NPs. Hb inside RBCs retained its original biological activity. In pH 7.0 PBS, a characteristic of the Hb-heme Fe(â…¢)/Fe(â…¡) redox couple was observed and the formal potential E^0' was -0.35 V. The electron transfer rate constant was 0.89/s. The RBCs/Au NPs-CS modified GCE also displayed a sensitive electrochemical response to H₂O₂. The linear range was 5.3×10^-6 to 1.4×10^-2 mol/L with a detection limit of 1.8×10^-7 mol/L (S/N = 3).Conclusion: (1) Fe₃O₄@Pt core-shell nanoparticles were successfully synthesized and Hb was immobilized on the nanocomposite modified GCE. A"Hb-Fe₃O₄@Pt-CS"bionic functional interface was then constructed, while direct electron transfer of Hb was successfully achieved.(2) Hb was successfully immobilized on Fe₃O₄/APTES/CdS magnetic nanocomposites, and the electron transfer of Hb was achieved rapidly. The obtained modified GCE also displayed a good catalytic performance to H₂O₂ .(3) We found that an appropriate concentration of Pt NPs could enhance the ECL intensity of CdS NCs, and the obtained {Hb-Pt_0.004/CdS₁}₄-CS modified GCE showed good electrocatalytic activity to H₂O₂.(4) Transmembrane direct electron transfer of Hb inside RBCs immobilized on Au NPs was successfully achieved, and a H₂O₂ biosensor based on RBCs/Au NPs-CS modified GCE was then constructed.