摘要
将辣根过氧化物酶(HRP)、纳米金、壳聚糖和戊二醛按照一定比例混合均匀,并吸取微量体积滴于玻碳电极表面,4 ℃下放置12 h,于是在玻碳电极表面形成一层稳定固载HRP的壳聚糖膜.由于纳米金能与HRP形成静电复合物,因而有效地防止HRP从壳聚糖膜中泄漏和提供适应酶所需的微环境,高效地保持HRP的生物活性.用对苯二酚作为电子媒介,用计时安培法优化了生物传感器操作参数.此生物传感器测定H2O2的线性范围为3.7×10-6至 1.22×10-3 mol/L,灵敏度为0.31 A L mol-1·cm-2,检测限为3.7 mmol/L,响应时间小于6 s,酶电极的表观米氏常数( Km app)为0.064 mmol/L.实验证明纳米金具有增加固定HRP生物活性、显著延长生物传感器使用寿命及提高测定H2O2的灵敏度等功能.
Horseradish peroxidase (HRP), nano-size particulate gold (nano-Au), chitosan and glutaraldehyde were mixed thoroughly in optimal proportion. Then a microvolume of this mixture was cast onto the surface of glassy carbon electrode (GC). Thus a strongly adherent and stable chitosan film with embedded HRP were obtained on the surface of GC after dried overnight. The presence of nano-Au in film can effectively prevent HRP leaking from chitosan film, provide suitable microenvironment for it and also efficiently retain its activity since the formation of electrostatic complex of nano-Au-HRP. Hydrogen peroxide was determined in the presence of hydroquinone as a mediator to transfer electrons between the electrode and HRP. It was experimentally proved that HRP embedded in chitosan membrane and activated by nano-Au demonstrated excellently electrocatalytical ability to the reduce of H2O2. The influences of experimental parameters on the response of H2O2 biosensor were optimized by using an amperometric method. The linear range of biosensor for the detection of H2O2 was 3.7×10-6 mol/Lto 1.22×10-3 mol /Lwith a sensitivity of 0.31 A L mol-1 cm-2 and a detective limit of 3.7 μmol/L. The response time was less 6 s. The apparent Michaelis-Menten constant ( Km ) for the sensor was determination to be 0.064 mmol l-1. The characteristics of proposed strategy app for immobilizing HRP include well-retained enzyme activity, long lifetime and high sensitivity for the detection of H2O2.
出处
《湖南城市学院学报（自然科学版）》
CAS
2004年第3期51-55,共5页
Journal of Hunan City University:Natural Science
