无机离子化学传感器及过氧化氢生物传感器研究
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摘要
离子电极能够直接、灵敏、快速测定无机离子，是电分析化学研究领域中的一个重要方向。过氧化氢传感器在临床、环境、食品等领域具有广阔的应用前景，也是生物传感器研究领域中一个十分活跃的课题。本论文分为两个部分。第一部分着重于设计、合成新型有机物，及其作为离子载体用于PVC溶剂聚合膜离子选择性电极的基础研究，并将电极初步应用于实际样品分析。第二部分着重于生物传感器固定化方法的研究，并将其用于多种高灵敏、高稳定的过氧化氢生物传感器的设计和研制。
第一部分无机离子化学传感器的研究
设计合成了四种有机物或有机金属配合物，并将它们分别作为新型载体，制备和研究了4种高选择性的PVC膜离子电极
1．合成了牛磺酸双核铜配合物为中性载体研制阴离子选择电极。该电极对硫氰酸根有良好的电位响应并呈现出anti-Hofmeister行为，其选择性顺序SCN~-＞I~-＞ClO_4~-＞Sal~-＞NO_3~-＞NO_2~-＞Br~-＞Cl~-＞SO_3~-＞SO_4~(2-)。在20℃pH 5.0的磷酸缓冲溶液中，其线性范围为1.0×10~(-1)～1.0×10~(-6) mol／L，检测下限为8.0×10~(-7) mol／L，斜率为-56.5 mV／pc_(SCN~-)。紫外、红外和交流阻抗研究表明电极的高选择性与载体的立体结构和分析物与中心金属离子的作用相关。将该电极用于废水和人体尿液中硫氰酸根的测定，获得了较满意的结果。
2．合成了Schiff碱金属配合物水杨醛肟铜(Ⅱ)、锌(Ⅱ)、镍(Ⅱ)为中性载体制备阴离子选择性电极。实验结果表明，水杨醛肟铜(Ⅱ)对水杨酸根(Sal~-)具有高选择性及优良的电位响应性能，电极呈现出反Hofmeister选择性行为，其选择性次序为Sal~-＞ClO_4~-＞SCN~-＞I~-＞NO_2~＞NO_3~-＞Br~-＞Cl~-＞Ac~-＞SO_4~(2-)。该电极在20℃pH 4.0的0.01 mol／L磷酸缓冲溶液中对水杨酸根(Sal~-)在1.0×10~(-1)～7.0×10~(-6) mol／L范围内呈近能斯特响应，检出下限为6.0×10~(-6) mol／L，斜率为-58.5mV／pc_(Sal~-)。采用交流阻抗技术和紫外可见光谱技术研究了电极的响应机理。该电极具有响应快、重现性好、检出限低、制备简单等优点。将电极用于药品及人体尿液分析，其结果令人满意。
3．合成了四碘络镉-罗丹明B(TICRhB)离子对化合物，研究了基于该离子缔合物为载体的金属络阴离子选择性电极的电位响应特性。以TICRhB为荷电载体的PVC膜电极在pH 3-6的HNO_3缓冲介质中对Cd~(2+)呈现出良好的电位响应特性。电极的电位响应线性范围为1.0×10~(-2)～2.0×10~(-6)mol／L，检测下限1.0×10~(-6)mol／L,斜率29.0mV／dec.；大多数金属阳离子和阴离子不干扰镉的测定。载体电极的膜中加入阳离子添加剂TOMACl后电极性能无明显改变。但阴离子添加剂NaTPB的加入却使电极的性能明显提高，表现在检测下限降低和斜率增加。由阴离子和阳离子添加剂对电极性能的不同影响的实验结果可得出结论，载体TICRhB遵循荷电载体作用机理。
4．合成了一种新含硫Schiff碱——印三酮-1，3-双缩肼基二硫代甲酸苄酯(NBSB)Ninhydrin-1，3-bis(S-benzyldithiocarbazate)，该分子结构中含有N，S，O三种杂原子，可以提高载体对过渡金属和重金属离子的结合能力。采用摩尔电导法研究了NBSB与重金属离子配合物的稳定性。实验结果表明，这是一种良好的Hg(Ⅱ)电极载体，相对大多数金属离子具有高的选择性，常见离子Cu(Ⅱ)，Pb(Ⅱ)，Cd(Ⅱ)和Ag(Ⅰ)不干扰Hg(Ⅱ)的测定。线性范围为1.0×10~(-2)—2.0×10~(-6)mol／L，检测下限为1.0×10~(-6)mol／L，在pH 3-5的硝酸缓冲介质中斜率为29.0 mV／dec。研究了增塑剂和离子添加剂等因素对电位响应性能的影响。用增塑剂邻硝基苯辛基醚(O-NPOE)制成的电极有最佳的电位响应值：加入2.8wt％KTpClPB能得到最佳的电位响应，斜率接近于能斯特响应理论值(29mV／dec)。由此得出结论，基于载体NBSB的PVC膜Hg~(2+)电极的最佳膜组成为：PVC：NPOE：NBSB：KTpClPB=30.9：61.8：4.5：2.8(wt％)。电极可以成功用作Hg(Ⅱ)浓度的电位滴定的指示电极。
第二部分过氧化氢生物传感器的研究
主要通过相反电荷吸附技术以及聚合物分子的多向效应将比表面积大、吸附能力强的纳米材料固定在电极表面，并以此作为亲和支持体为固载辣根过氧化物酶，研制成多种高灵敏、高稳定的过氧化氢生物传感器。
1．通过在酸性条件下，电聚合硫瑾(thionine)单体分子，形成多孔(由在高电位的作用下氢气的产生形成)高聚复合膜，然后利用NH_2-Au之间的相互作用将纳米Au固定于电极表面，再通过金纳米粒子比表面积大、吸附能力强等特性，将辣根过氧化物酶固定在电极表面，从而制得高灵敏度、高选择的过氧化氢生物传感器。传感器的检测下限为7.5×10~(-7)mol／L H_2O_2，线性范围是9.6×10~(-6)～1.2×10~(-3)mol／L H_2O_2。将传感器用于测定牛奶样品中的过氧化氢含量，获得满意的结果。
2．利用DNA与纳米银复合物固定过氧化物酶。实验结果表明，固定的过氧化物酶HRP能够有效地促进电子的传递。详细研究了过氧化氢在电极HRP／nanosilver／DNA／Au表面的电催化还原过程。考察了电位对复合电极HRP／nanosilver／DNA／Au检测过氧化氢的影响。更重要的是，DNA聚离子复合膜能够增强传感器的稳定性和提高传感器的抗干扰能力。传感器的检测下限为5.0×10~(-7)mol／L(信噪比为3)，线性范围是1.5×10~(-6)～2.0×10~(-3)mol／L。将传感器用于测定消毒剂中过氧化氢的浓度，结果令人满意。
Carrier-based ion-selective electrodes (ISE) have found wide spread application for their response characteristics including fast response time, wide linear dynamic range, low detection limit, and good selectivity. The wide uses of ISE in routine chemical analysis have been accompanied by a search for ionophores that can chemically recognize specific cations or anions and offer either new or improved selectivity to different ions. This thesis focuses on design and synthesis of several novel compounds and studies on solvent polymeric membrane ion-selective electrodes incorporating these compounds. The electrodes can be successfully applied to practical sample analysis.
Biosensors based on enzyme-modified electrodes are valuable in clinical diagnosis, bromatology, environmental monitoring and biochemical analysis. This thesis focuses on the immobilization of the enzyme and involves several novel amperometric hydrogen peroxide biosensor based on nanoparticles.
Part I of the thesis deals with design of some novel carriers for poly (vinyl chloride) (PVC) membrane ion-electrode with high selectivity to Thiocyanate, Salicylate, Cadmium (II) and Mercury (II) ion respectively.
1. The response characteristics of a new potentiometric membrane electrode with unique selectivity towards thiocyanate ion were reported. The electrode was prepared by incorporating bis-taurine-salicylic binuclear copper (II) complex into a plasticized PVC-membrane. The resulting electrode exhibits anti-Hofmeister selectivity sequence: SCN~-> I~-> ClO_4~-> Sal~-> NO_3~- > NO_2~-> Br~-> Cl~-> SO_3~->SO_4~(2-)and a near-Nernstian potential linear range for thiocyanate from 1.0×1.0~(-1) to 1.0×1.0~(-6) mol·L~(-1) with a detection limit of 8.0×10~(-7) mol·L~(-1) and a slope of -56.5 mV/pcscn~- in phosphate buffer solution of pH 5.0 at 20°C. The UV/Vis spectra, IR spectroscopy and AC impedance studies showed that the excellent selectivity to thiocyanate was related to the unique interaction between the central metal and the analyte and a steric effect associated with the structure of the carrier. The electrode was successfully applied to the determination of thiocyanate in wastewater and human urine samples.
2. The response characteristics of a new potentiometric membrane electrode with unique selectivity towards salicylate ion are reported. The electrode is prepared by incorporating bis-salicylaldoxime complex of copper (II) into a plasticized PVC-membrane. The resulting electrode exhibits anti-Hofmeister selectivity sequence: Sal~- > ClO_4~- > SCN~- > I~- > NO_2~-> NO_3~-> Br~-> Cl~-> Ac~-> SO_4~(2-)and a near-Nernstian potential linear range for salicylate from 1.0×1.0~(-1) to 3.0×10~(-6) mol·L~(-1) with a detection limit of 7.0×10~(-7) mol·L~(-1) and a slope of -58.5 mV/pcsal~- in pH 4.0 of phosphate buffer solution at 20°C. The UV/Vis spectra and A.C. impedance studies showed that the excellent selectivity for salicylate was related to the unique interaction between the central metal and the analyte and a steric effect associated with the structure of the carrier. The electrode was successfully applied to the determination of salicylate in human urine and pharmaceutical preparations.
3. A novel tetraiodocadmate (II)-selective membrane electrode consisting of tetraiodocadmate (II)-rhodamin B ion pair (TICRhB) dispersed in a PVC matrix plasticized with 2-nitrophenyl octyl ether (o-NPOE) was prepared. The sensor demonstrated a near-Nernstian response for 1×10~(-2) to 2×10~(-6) mol/L cadmium (II) at 25℃with an anionic slope of 29.0. It revealed very good selectivity for Cd~(2+) with negligible interference from many cations and anions, and could be used in a pH range of 3 to 6.
4. In this work, a novel mercury membrane electrode based on ninhydrin-1, 3-bis (S-benzyldithiocarbazate (NBSB) Schiff's base as a neutral carrier is presented. The electrode exhibits a wide linear dynamic range between 1.0×10~(-1) to 2.0×10~(-6) mol/L with a good Nernstian slope of 29.0 mV/decade and a detection limit of 1.0×10~(-6) mol/L. It has a response time of less than 10 s and can be used for at least 10 weeks without any considerable divergence in the potentials. It can be used in the pH range from 3.0 to 5.0. The electrode is highly selective for Hg~(2+) over a large number of cations, such as Cu( II), Pb( II), Cd( II) and Ag( I ). The electrode was used in the direct determination of Hg~(2+) in aqueous solution with satisfactory results.
Part II of the thesis deals with several novel amperometric hydrogen peroxide biosensors based on gold nanoparticles.
1. A new strategy for immobilization of horseradish peroxidase (HRP) has been developed by self-assembling gold nanoparticles to multiporous polythionine (PTH) film modified carbon paste interface. A thionine film was initially electropolymerized onto carbon paste interface in a mildly acidic thionine solution at a bias voltage of -1.0 ~ 1.5 V. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the PTH film with multiporous structure. The multiporous PTH film provided a biocompatible microenvironment for gold nanoparticles and enzyme molecules, greatly amplified the coverage of HRP molecules on the electrode surface. Voltammetric and time-based amperometric techniques were employed to characterize the properties of the biosensor derived. The performance and factors influencing the performance of the biosensor were also proposed. The immobilized HRP displayed a catalytic property to the reduction of H_2O_2. The H_2O_2 biosensor achieved 95% of the steady-state current within 2 s, and exhibited a linear range of 9.6×10~(-6) - 1.2×10~(-3) mol/L H_2O_2 with a detection limit of 7.5×10~(-7) mol/L (S/N =3). Furthermore, the biosensor remained about 90% of its original sensitivity after two weeks' storage.
2. The present paper describes the modification of horseradish peroxidase (HRP)-silver nanoparticles (nanosilver)-DNA polyion complex film on a gold electrode surface to develop a novel electrochemical biosensor for the detection of hydrogen peroxide (H_2O_2). With the help of DNA and nanosilver, the immobilized HRP displays a pair of well-defined redox peaks with an electron transfer rate constant of 3.27±0.91 s~(-1) in pH 7.0 PBS. Moreover, the presence of DNA provided a biocompatible microenvironment for silver nanoparticles and enzyme molecules, greatly amplified the immobilized amount of HRP molecules on the electrode surface, and improved the sensitivity of the biosensor. Under the optimal conditions, the proposed biosensor has an electrocatalytic activity towards the reduction of hydrogen peroxide, which shows a linear dependence on the H_2O_2 concentration ranging from 1.5×10~(-6) to 2.0×10~(-3) mol/L with the detection limit of 5.0×10~(-7) mol/L at the signal-to-noise ratio is 3. The K_m~(app) value of HRP in the polyion complex film has been determined to be 1.62 mmol. The properties of polyion composite film, together with the bioelectrochemical catalytic activity, could make them useful in the development of bioelectronic devices and investigation of protein
第一部分无机离子化学传感器的研究
设计合成了四种有机物或有机金属配合物，并将它们分别作为新型载体，制备和研究了4种高选择性的PVC膜离子电极
1．合成了牛磺酸双核铜配合物为中性载体研制阴离子选择电极。该电极对硫氰酸根有良好的电位响应并呈现出anti-Hofmeister行为，其选择性顺序SCN~-＞I~-＞ClO_4~-＞Sal~-＞NO_3~-＞NO_2~-＞Br~-＞Cl~-＞SO_3~-＞SO_4~(2-)。在20℃pH 5.0的磷酸缓冲溶液中，其线性范围为1.0×10~(-1)～1.0×10~(-6) mol／L，检测下限为8.0×10~(-7) mol／L，斜率为-56.5 mV／pc_(SCN~-)。紫外、红外和交流阻抗研究表明电极的高选择性与载体的立体结构和分析物与中心金属离子的作用相关。将该电极用于废水和人体尿液中硫氰酸根的测定，获得了较满意的结果。
2．合成了Schiff碱金属配合物水杨醛肟铜(Ⅱ)、锌(Ⅱ)、镍(Ⅱ)为中性载体制备阴离子选择性电极。实验结果表明，水杨醛肟铜(Ⅱ)对水杨酸根(Sal~-)具有高选择性及优良的电位响应性能，电极呈现出反Hofmeister选择性行为，其选择性次序为Sal~-＞ClO_4~-＞SCN~-＞I~-＞NO_2~＞NO_3~-＞Br~-＞Cl~-＞Ac~-＞SO_4~(2-)。该电极在20℃pH 4.0的0.01 mol／L磷酸缓冲溶液中对水杨酸根(Sal~-)在1.0×10~(-1)～7.0×10~(-6) mol／L范围内呈近能斯特响应，检出下限为6.0×10~(-6) mol／L，斜率为-58.5mV／pc_(Sal~-)。采用交流阻抗技术和紫外可见光谱技术研究了电极的响应机理。该电极具有响应快、重现性好、检出限低、制备简单等优点。将电极用于药品及人体尿液分析，其结果令人满意。
3．合成了四碘络镉-罗丹明B(TICRhB)离子对化合物，研究了基于该离子缔合物为载体的金属络阴离子选择性电极的电位响应特性。以TICRhB为荷电载体的PVC膜电极在pH 3-6的HNO_3缓冲介质中对Cd~(2+)呈现出良好的电位响应特性。电极的电位响应线性范围为1.0×10~(-2)～2.0×10~(-6)mol／L，检测下限1.0×10~(-6)mol／L,斜率29.0mV／dec.；大多数金属阳离子和阴离子不干扰镉的测定。载体电极的膜中加入阳离子添加剂TOMACl后电极性能无明显改变。但阴离子添加剂NaTPB的加入却使电极的性能明显提高，表现在检测下限降低和斜率增加。由阴离子和阳离子添加剂对电极性能的不同影响的实验结果可得出结论，载体TICRhB遵循荷电载体作用机理。
4．合成了一种新含硫Schiff碱——印三酮-1，3-双缩肼基二硫代甲酸苄酯(NBSB)Ninhydrin-1，3-bis(S-benzyldithiocarbazate)，该分子结构中含有N，S，O三种杂原子，可以提高载体对过渡金属和重金属离子的结合能力。采用摩尔电导法研究了NBSB与重金属离子配合物的稳定性。实验结果表明，这是一种良好的Hg(Ⅱ)电极载体，相对大多数金属离子具有高的选择性，常见离子Cu(Ⅱ)，Pb(Ⅱ)，Cd(Ⅱ)和Ag(Ⅰ)不干扰Hg(Ⅱ)的测定。线性范围为1.0×10~(-2)—2.0×10~(-6)mol／L，检测下限为1.0×10~(-6)mol／L，在pH 3-5的硝酸缓冲介质中斜率为29.0 mV／dec。研究了增塑剂和离子添加剂等因素对电位响应性能的影响。用增塑剂邻硝基苯辛基醚(O-NPOE)制成的电极有最佳的电位响应值：加入2.8wt％KTpClPB能得到最佳的电位响应，斜率接近于能斯特响应理论值(29mV／dec)。由此得出结论，基于载体NBSB的PVC膜Hg~(2+)电极的最佳膜组成为：PVC：NPOE：NBSB：KTpClPB=30.9：61.8：4.5：2.8(wt％)。电极可以成功用作Hg(Ⅱ)浓度的电位滴定的指示电极。
第二部分过氧化氢生物传感器的研究
主要通过相反电荷吸附技术以及聚合物分子的多向效应将比表面积大、吸附能力强的纳米材料固定在电极表面，并以此作为亲和支持体为固载辣根过氧化物酶，研制成多种高灵敏、高稳定的过氧化氢生物传感器。
1．通过在酸性条件下，电聚合硫瑾(thionine)单体分子，形成多孔(由在高电位的作用下氢气的产生形成)高聚复合膜，然后利用NH_2-Au之间的相互作用将纳米Au固定于电极表面，再通过金纳米粒子比表面积大、吸附能力强等特性，将辣根过氧化物酶固定在电极表面，从而制得高灵敏度、高选择的过氧化氢生物传感器。传感器的检测下限为7.5×10~(-7)mol／L H_2O_2，线性范围是9.6×10~(-6)～1.2×10~(-3)mol／L H_2O_2。将传感器用于测定牛奶样品中的过氧化氢含量，获得满意的结果。
2．利用DNA与纳米银复合物固定过氧化物酶。实验结果表明，固定的过氧化物酶HRP能够有效地促进电子的传递。详细研究了过氧化氢在电极HRP／nanosilver／DNA／Au表面的电催化还原过程。考察了电位对复合电极HRP／nanosilver／DNA／Au检测过氧化氢的影响。更重要的是，DNA聚离子复合膜能够增强传感器的稳定性和提高传感器的抗干扰能力。传感器的检测下限为5.0×10~(-7)mol／L(信噪比为3)，线性范围是1.5×10~(-6)～2.0×10~(-3)mol／L。将传感器用于测定消毒剂中过氧化氢的浓度，结果令人满意。
Carrier-based ion-selective electrodes (ISE) have found wide spread application for their response characteristics including fast response time, wide linear dynamic range, low detection limit, and good selectivity. The wide uses of ISE in routine chemical analysis have been accompanied by a search for ionophores that can chemically recognize specific cations or anions and offer either new or improved selectivity to different ions. This thesis focuses on design and synthesis of several novel compounds and studies on solvent polymeric membrane ion-selective electrodes incorporating these compounds. The electrodes can be successfully applied to practical sample analysis.
Biosensors based on enzyme-modified electrodes are valuable in clinical diagnosis, bromatology, environmental monitoring and biochemical analysis. This thesis focuses on the immobilization of the enzyme and involves several novel amperometric hydrogen peroxide biosensor based on nanoparticles.
Part I of the thesis deals with design of some novel carriers for poly (vinyl chloride) (PVC) membrane ion-electrode with high selectivity to Thiocyanate, Salicylate, Cadmium (II) and Mercury (II) ion respectively.
1. The response characteristics of a new potentiometric membrane electrode with unique selectivity towards thiocyanate ion were reported. The electrode was prepared by incorporating bis-taurine-salicylic binuclear copper (II) complex into a plasticized PVC-membrane. The resulting electrode exhibits anti-Hofmeister selectivity sequence: SCN~-> I~-> ClO_4~-> Sal~-> NO_3~- > NO_2~-> Br~-> Cl~-> SO_3~->SO_4~(2-)and a near-Nernstian potential linear range for thiocyanate from 1.0×1.0~(-1) to 1.0×1.0~(-6) mol·L~(-1) with a detection limit of 8.0×10~(-7) mol·L~(-1) and a slope of -56.5 mV/pcscn~- in phosphate buffer solution of pH 5.0 at 20°C. The UV/Vis spectra, IR spectroscopy and AC impedance studies showed that the excellent selectivity to thiocyanate was related to the unique interaction between the central metal and the analyte and a steric effect associated with the structure of the carrier. The electrode was successfully applied to the determination of thiocyanate in wastewater and human urine samples.
2. The response characteristics of a new potentiometric membrane electrode with unique selectivity towards salicylate ion are reported. The electrode is prepared by incorporating bis-salicylaldoxime complex of copper (II) into a plasticized PVC-membrane. The resulting electrode exhibits anti-Hofmeister selectivity sequence: Sal~- > ClO_4~- > SCN~- > I~- > NO_2~-> NO_3~-> Br~-> Cl~-> Ac~-> SO_4~(2-)and a near-Nernstian potential linear range for salicylate from 1.0×1.0~(-1) to 3.0×10~(-6) mol·L~(-1) with a detection limit of 7.0×10~(-7) mol·L~(-1) and a slope of -58.5 mV/pcsal~- in pH 4.0 of phosphate buffer solution at 20°C. The UV/Vis spectra and A.C. impedance studies showed that the excellent selectivity for salicylate was related to the unique interaction between the central metal and the analyte and a steric effect associated with the structure of the carrier. The electrode was successfully applied to the determination of salicylate in human urine and pharmaceutical preparations.
3. A novel tetraiodocadmate (II)-selective membrane electrode consisting of tetraiodocadmate (II)-rhodamin B ion pair (TICRhB) dispersed in a PVC matrix plasticized with 2-nitrophenyl octyl ether (o-NPOE) was prepared. The sensor demonstrated a near-Nernstian response for 1×10~(-2) to 2×10~(-6) mol/L cadmium (II) at 25℃with an anionic slope of 29.0. It revealed very good selectivity for Cd~(2+) with negligible interference from many cations and anions, and could be used in a pH range of 3 to 6.
4. In this work, a novel mercury membrane electrode based on ninhydrin-1, 3-bis (S-benzyldithiocarbazate (NBSB) Schiff's base as a neutral carrier is presented. The electrode exhibits a wide linear dynamic range between 1.0×10~(-1) to 2.0×10~(-6) mol/L with a good Nernstian slope of 29.0 mV/decade and a detection limit of 1.0×10~(-6) mol/L. It has a response time of less than 10 s and can be used for at least 10 weeks without any considerable divergence in the potentials. It can be used in the pH range from 3.0 to 5.0. The electrode is highly selective for Hg~(2+) over a large number of cations, such as Cu( II), Pb( II), Cd( II) and Ag( I ). The electrode was used in the direct determination of Hg~(2+) in aqueous solution with satisfactory results.
Part II of the thesis deals with several novel amperometric hydrogen peroxide biosensors based on gold nanoparticles.
1. A new strategy for immobilization of horseradish peroxidase (HRP) has been developed by self-assembling gold nanoparticles to multiporous polythionine (PTH) film modified carbon paste interface. A thionine film was initially electropolymerized onto carbon paste interface in a mildly acidic thionine solution at a bias voltage of -1.0 ~ 1.5 V. This process is accompanied by the hydrogen evolution reaction, and the released hydrogen gas made the PTH film with multiporous structure. The multiporous PTH film provided a biocompatible microenvironment for gold nanoparticles and enzyme molecules, greatly amplified the coverage of HRP molecules on the electrode surface. Voltammetric and time-based amperometric techniques were employed to characterize the properties of the biosensor derived. The performance and factors influencing the performance of the biosensor were also proposed. The immobilized HRP displayed a catalytic property to the reduction of H_2O_2. The H_2O_2 biosensor achieved 95% of the steady-state current within 2 s, and exhibited a linear range of 9.6×10~(-6) - 1.2×10~(-3) mol/L H_2O_2 with a detection limit of 7.5×10~(-7) mol/L (S/N =3). Furthermore, the biosensor remained about 90% of its original sensitivity after two weeks' storage.
2. The present paper describes the modification of horseradish peroxidase (HRP)-silver nanoparticles (nanosilver)-DNA polyion complex film on a gold electrode surface to develop a novel electrochemical biosensor for the detection of hydrogen peroxide (H_2O_2). With the help of DNA and nanosilver, the immobilized HRP displays a pair of well-defined redox peaks with an electron transfer rate constant of 3.27±0.91 s~(-1) in pH 7.0 PBS. Moreover, the presence of DNA provided a biocompatible microenvironment for silver nanoparticles and enzyme molecules, greatly amplified the immobilized amount of HRP molecules on the electrode surface, and improved the sensitivity of the biosensor. Under the optimal conditions, the proposed biosensor has an electrocatalytic activity towards the reduction of hydrogen peroxide, which shows a linear dependence on the H_2O_2 concentration ranging from 1.5×10~(-6) to 2.0×10~(-3) mol/L with the detection limit of 5.0×10~(-7) mol/L at the signal-to-noise ratio is 3. The K_m~(app) value of HRP in the polyion complex film has been determined to be 1.62 mmol. The properties of polyion composite film, together with the bioelectrochemical catalytic activity, could make them useful in the development of bioelectronic devices and investigation of protein
引文
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