王炳全
论文题目：高稳定性安培生物传感器的研制
作者简介：王炳全，男，1973年01月出生，1995年09月师从于中国科学院长春应用化学研究所董绍俊教授，于2000年12月获博士学位。
摘 要
生物传感器已发展成为一种新型的分析手段，并被广泛应用于临床、环境、食品等领域。本论文从合成新型生物相容性固定化载体、寻找高稳定性的酶及建立新的有机相体系三方面对酶电极进行了研究。主要结果如下：
制备了一种新型的有机-无机掺杂材料，这种材料一方面克服了两种材料各自的缺点，即溶胶-凝胶材料的开裂和水凝胶的溶胀；另一方面保留了两种材料各自的优点，即溶胶-凝胶材料的物理刚性、高稳定性和水凝胶的生物相容性。通过对其性能的表征，我们知道膜中含有大量的羟基和氢键，且两种组分形成互穿网络，可以预测该材料刚柔相济的特点将使其非常适合于生物传感器的制备。
构建了一种新型的有机-无机掺杂材料为基础的葡萄糖氧化酶电极。该酶电极表现出高灵敏度，快速响应和高稳定性。为了降低干扰物质的影响，又制备了以四硫富瓦烯为媒介体的葡萄糖氧化酶电极，由于Nafion
的静电排斥作用，大大地降低了抗坏血酸的干扰，血糖的测定结果表明该生物传感器可以用于进行实际样品的监测。这些结果都表明制备的有机-无机掺杂材料具有较强的生物相容性。考虑到复合材料制备方法的简单性和所用试剂便宜易得，该种溶胶-凝胶/接枝物掺杂材料可望用于制备商品化的生物传感器。
用溶胶－凝胶／水凝胶复合杂化材料为固定化载体，把辣根过氧化物酶（HRP）包埋固定在这种有机－无机复合膜中，构造安培酶电极，检测过氧化氢。用FTIR和EQCM方法表征溶胶－凝胶-有机复合膜和含酶的膜。以亚铁氰化钾为媒介体，表征了pH、适加电位对电极响应的影响，并对传感器的稳定性进行了评估。由此我们发展了一种新型过氧化氢安培酶电极。膜中大量的羟基和氢键为酶提供了生物相容性的微环境，所以此传感器给出很好的响应性能，包括高的灵敏度、快的响应和好的长期稳定性。流动注射实验证明此生物传感器可以用于流动体系实现在线检测。
我们将亚甲基绿包埋到含负电荷的Nafion膜中，然后外盖溶胶－凝胶复合酶膜制备了过氧化氢生物传感器。并且对酶电极的影响因素、操作和储存稳定性等进行了探讨，结果证明被固定的亚甲基绿（MG+）能够有效地传输电子，此种双层膜式构型的酶电极具有较好的稳定性。用Nafion
膜固定染料分子，用溶胶凝胶掺杂膜固定了HRP而制备了过氧化氢传感器。Nafion 膜与溶胶－凝胶膜之间具有很好的黏附作用，同时由于MG+与Nafion之间具有静电相互作用，我们提出的具有两层膜式构型的酶电极具有较好的稳定性。由于染料分子直接固定在电极表面，因而可以不需加入媒介体而直接进行样品的测定。考虑到众多的荷阳离子的电活性分子，我们提出的双层膜式构型为一种普遍适应的方法，可以用于其它多种媒介体。
固定酪氨酸酶于溶胶—凝胶薄膜中，并且对酪氨酶电极进行了表征，包括：灵敏度、稳定性、pH和施加电位对生物传感器电化学响应的影响。溶胶—凝胶杂化材料是固定化酪氨酸酶的优秀材料，强的氢键和溶胶—凝胶网络不仅有效地包埋酶分子，而且增加了其热和长期稳定性。用亲水的溶胶—凝胶前体制备的酶电极响应比较大。氧浓度的涨落对 mM级底物的测定影响不大。鉴于明显优越的分析性能，本文的溶胶—凝胶方法制备的酶电极可以与分离技术相结合实现分别测定单个酚的含量。
现有的过氧化氢生物传感器是以辣根过氧化物酶为基础构建的，但辣根过氧化物酶的最适pH值为中性偏碱，难以在酸性条件下应用；另外，辣根过氧化物酶的热稳定性也不太好。这些原因促使我们寻找具有更低的pH和更高的热稳定性的过氧化物酶。我们确定了一条经济有效的分离路线，它是由双水相萃取、Mono–Q柱层析和Butyl–Superose疏水层析所组成，可使分离的产品达到电泳纯的标准。采用这一方法，从大豆种皮中分离了一种酸性过氧化物酶──大豆过氧化物酶（SBP），活力高于Enzymol
International Inc. 的高纯级SBP商品酶(130 pyrogallol U/mg)。然后，用溶胶－凝胶薄膜固定了大豆过氧化物酶而构建了一种能在酸性溶液中检测过氧化氢的生物传感器。在pH 5.0时，这种过氧化氢传感器具有灵敏度高、响应时间短（5 s）、热稳定性好和储存稳定性高等优点。而且，该酶电极比辣根过氧化物酶电极具有更高的选择性，流动注射分析证明这种过氧化氢传感器能用于酸性条件下过氧化氢的在线检测。大豆过氧化物酶具有很高的热稳定性，并且在pH 4到10时稳定储存。当该酶被固定在聚乙烯醇接技聚乙烯吡啶的接枝共聚物中时，酶电极在pH 3.0时显示了灵敏的响应和好的稳定性。另外，基于SBP的传感器要较HRP传感器具有更高的检测上限。HRP在pH 3.0时发生卟啉脱落或脱出活性中心，降低甚至丧失了活性；而SBP却可以紧密地抓牢辅基。
许多有机溶剂被环境保护机构认定为致癌产物或有害物质，所以，发展检测这些有害溶剂的方法有重要的实际意义。我们进一步扩展了安培酶生物传感器的使用范围去定量测定极性有机溶剂。定量测定极性有机溶剂是基于底物探针（苯酚或其它的酚）在酶膜与有机溶剂－水介质之间的分配平衡。这种方法可以简单、方便、快速地进行定量分析极性有机溶剂。这种定量分析极性有机溶剂的方法可以用于监测有机化工厂的排水。实验证实了用酪氨酸酶电极安培定量测定电化学惰性的极性有机溶剂的可行性。酪氨酸酶电极的制备方法简单，且可以稳定使用。电化学还原酪氨酸酶催化氧化酚生成的醌为探测信号。定量测定极性有机溶剂是基于底物探针在固定化材料与有机溶剂－水混合介质中的分配平衡。现场计时电流-石英晶体微天平实验证实了其机理：加入的有机溶剂增加了底物探针酚在溶液中的溶解度，从而降低了酶膜中的酚的浓度。此传感器可以用于定量测定甲醇、乙醇、正丁醇、丙酮、乙腈、四氢呋喃等。对某有机溶剂的检测限，灵敏度，线性范围依赖于所使用的固定化材料和底物的疏水性。在选定固定化材料和底物探针的情况下，有机溶剂的疏水性越强，其线性范围越窄。增加底物探针的浓度可以增加检测的灵敏度，因而降低检测限。对所测试的所有有机溶剂的响应时间小于2分钟。此方法检测极性有机溶剂的特点是简单、容易、迅速。用酪氨酸酶电极安培测定电化学惰性的极性有机溶剂极大地扩展了酶电极的应用范围。这种检测方法的优点是定量测定那些还没有发现专一性的酶来构造生物传感器的有机溶剂如丙酮、乙腈、四氢呋喃等。首次将溶胶－凝胶膜固定的酪氨酸酶用于有机相测定酚类化合物。对溶胶－凝胶膜前体和高分子化合物的选择很重要，这是由于固定化材料的亲水性直接影响传感器的灵敏度。由于聚乙烯醇和3-氨基丙基三甲氧基硅烷均具有比较温和的亲水性，并且制备所得的电极的灵敏度较高，故被用作固定化材料。固定化酶的活性要较低温水凝胶和电聚合聚吡咯包埋法高，并且可以通过对酶载量的调节来获得低的检测限或宽的线性范围。该法制备的传感器具有响应快、稳定性好等优点，同时彻底解决了酶泄漏的问题。
将生物膜与传感器结合起来，发展了一种新的固定化方法，实现了酶在生物膜中的直接电化学，为制备第三代生物传感器奠定了基础。
关键词 生物传感器，酶，固定化，溶胶－凝胶，有机－无机杂化，稳定性
Abstract
Fabrication of Highly Stable Amperometric Biosensor
Biosensors have developed into new
analytical tools, and they have been widely applied in the clinical diagnosis,
environmental monitoring and food control etc. In this thesis, the enzyme
electrodes were studied from three aspects: synthesis of new biocompatible
immobilization matrices, purification of highly stable enzymes and
establishment of new organic phase systems. The main results are as follows:
1.
For the first time,
inorganic glass material was combined with polymer hydrogel by
organic-inorganic hybrid method to fabricate new immobilization matrix. The
fabrication conditions were optimized, and the results demonstrated that the
optimum ratio of tetraethylorthosilicate to water was 1:4 and the optimum
content of hydrogel in the hybrid material was 38.3%. On one hand, this hybrid
material overcomes the respective shortcomings of silica sol and hydrogel, i.
e. it effectively prevents the cracking of sol-gel ceramic material and
eliminates the swelling of the hydrogel. On the other hand, it reserves the
good biocompatibility of hydrogel and physical rigidity and high stability of
silica glass. FT-IR, SEM and QCM experiments illuminate that the hybrid film
contains a large amount of hydroxyl groups and hydrogen bond, and two
components form interpenetrating network. In addition, enzyme leaching was not
observed when enzyme molecules were encapsulated in the composite film.
2.
Glucose oxidase,
horseradish peroxidase and tyrosinase were immobilized in the sol-gel hybrid
material to develop thin-film biosensors. The organic-inorganic thin-film has
short diffusion pathway, so the biosensors had fast response. Moreover, because
of the good biocompatibility and high stability of the hybrid material, the immobilized
enzymes maintained high bioactivity and the biosensors exhibited high
sensitivity and remarkable stability. The immobilization method we proposed was
proved to be a general method for the construction of highly stable biosensors.
3.
A type of extremely
stable peroxidase was purified from Chinese soybean seed coat by successive
aqueous two-phase partition, Chromatography on Mono-Q fast flow and
butylsuperose fast flow. The activity of soybean peroxidase (SBP) is 200
pyrogallol U/mg, which is much higher than that of Sigma product (130
pyrogallol U/mg). The peroxidase maintained its activity in a broad pH range
from 3-10 and exhibited notable thermostability (T1/2 is 20 hours at
60 ℃). The enzyme, for the first time, was used to
construct acid-stable hydrogen peroxide biosensors. At low pH values, The
enzyme electrodes exhibited a fast response, high sensitivity as well as good
long-term stability. These acid-stable enzyme electrodes extend the biosensor
application to a large degree.
4.
New kinds of
amperometric biosensors for the determination of inert organic solvents were
first proposed. EQCM and electrochemical experiments illustrated its mechanism:
the injected organic solvents concentrate the substrate probe and cause the
decrease of substrate concentration in the enzyme membrane, accordingly the
current response decreases. The current drop is proportional to the amount of
organic solvent added, therefore, the electrochemically inert organic solvents
can be determined by amperometric biosensor. The sensitivity and the detection
limit for a certain organic solvent depend on the enzyme loading and the
hydrophobicity of the immobilization matrix and the substrate probe. The
biosensors can be applied in wastewater analysis and environmental monitoring,
which greatly expand the application scope of the biosensor.
5.
Biomimic membrane was
combined with sensor technology to construct bilayer lipid membrane (BLM)
biosensors. New immobilization methods such as dip-coating and casting were
developed to immobilize horseradish peroxidase in bilayer and mutilayer lipid
membrane, respectively. The negative charge in the lipid can exclude
electroactive anion such as ascorbate and uric acid, so it greatly reduces the
interference. In addition, biomimic membrane supply a biocompatible
microenvironment on the surface of the electrode for the enzyme, therefore, the
direct electron transfer between enzyme and the glassy carbon electrode was
realized in the BLM, which lays a foundation for the development of the
third-generation biosensor.
Keywords: Biosensor, Enzyme, Immobilization method,
Sol-gel, Organic-inorganic hybridization, Stability
