中国科学院海岸带环境过程与生态修复重点实验室知识库

聚合物膜离子选择性电极检测技术具有仪器简单、分析速度快、检测成本低、易于实现微型化等优点，已在生物分析和环境检测等领域得到应用广泛。传统的离子选择性电极检测基于热力学平衡原理，电位响应与待测离子的活度符合能斯特方程。虽然聚合物膜离子选择性电极已被报导用于环境分析，但是受热力学能斯特响应特征的局限以及环境基体的影响，电极测定灵敏度及抗干扰能力有待提高。此外，基于能斯特响应的离子选择性电极仅能用于单一目标物检测，难以实现多种目标物检测。动力学电位分析法具有灵敏度高、选择性好、可逆性强、可实现多组份分析等优势，因此本论文发展一系列基于动力学响应的聚合物敏感膜电极，通过离子通量的调控，实现对待测离子的形态分析和高灵敏检测；采用新的输出方式，降低环境中背景电解质的干扰；开发基于逻辑门和核酸适配体电位传感器，用于不同靶分子的检测，实现在单一聚合物敏感膜上的多组分分析；将生物放大等技术引入电位传感器的设计，实现信号放大和高灵敏电位检测。研究工作概况如下：

1. 聚合物膜钙离子选择性电极检测游离钙和总钙浓度

钙是自然界广泛存的一种元素，许多重要的生理作用与钙的存在形态密切关系。因此，钙的形态分析对于了解自然环境中的生物利用度和反应活性具有重要意义。本文发展了2种聚合物膜钙离子选择性电极，通过改变内充液的组成，分别实现了溶液中游离的钙离子活度以及在络合剂乙二胺四乙酸钠（EDTA）和腐殖酸存在下总钙浓度的检测。研究结果表明：当聚合物膜钙离子选择性电极的内充液为10^-3 M CaCl₂时，可实现低至10^-6 M 游离的钙离子活度的检测；而当聚合物膜钙离子选择性电极的内充液为10^-3 M CaCl₂与5 × 10^-2 M Na₂EDTA （pH 9.0）的混合溶液时，电极在10^-6-10^-5 M Ca²⁺范围内产生从样品溶液相到敏感膜相较强的钙离子通量，并伴随超能斯特现象发生，电位差值约180 mV，电极的电位响应与总钙浓度成线性相关，而与游离的钙离子浓度无关。据此，可实现以聚合物膜钙离子选择性电极对游离钙离子活度和总钙浓度的分析。本方法为进一步开展环境水体形态分析提供了一种简单、有效的技术手段。

2. 基于脉冲恒电流控制的聚合物膜钙离子选择性电极检测海水中钙离子

传统的钙离子选择性电极基于热力学平衡，电极的响应符合能斯特方程，响应斜率仅为30 mV/dec。在这种情况下，1mV电位变化会引起8 %的误差。海水中钙离子浓度变化很小，采用传统的钙离子选择性电极检测时会引起较大的误差。因此，需要发展一种高灵敏度的钙离子选择性电极。本文发展了一种基于脉冲恒电流控制的聚合物膜钙离子选择性电极，实现了海水中钙离子的高灵敏检测。该电极敏感膜以惰性亲脂盐ETH 500代替传统的离子交换剂，在这种情况下，通过膜相的离子通量完全由所施加脉冲恒电流控制。在0.5 M NaCl背景条件下，通过向电极敏感膜施加阴极脉冲恒电流，使得待测溶液中的钙离子被有效萃取到膜相，产生计时电位响应，用于钙离子检测。通过对所施加阴极脉冲电流的时间和大小优化，电极在10^-3—10^-2 M Ca²⁺ 浓度范围内呈超能斯特响应，响应斜率为80 mV/ dec。该电极对海水中高含量的Na⁺、Mg²⁺、K⁺ 具有较高的选择性，且表现出良好的重复性。将该电极应用于实际海水钙离子浓度的检测，测定结果与ICP-AES法相吻合；与传统电位法相比，本方法降低了测定的标准偏差。

3. 以过渡时间为输出信号的计时电位分析法检测海水中钙离子浓度

对于动力学电位分析法，以电位为输出信号的聚合物膜钙离子选择性电极检测钙离子浓度时，通常会受到背景电解质的干扰。因此，需要发展一种受背景电解质干扰小的检测方法。本章提出以过渡时间作为分析信号的计时电位分析技术，用于海水高背景电解质条件下钙离子浓度的检测。钙离子选择性敏感膜内不包含离子交换剂，当向敏感膜相施加较长时间的阴极恒电流时，钙离子在敏感膜表面耗尽，此时背景离子钠离子随着主离子钙离子一起被萃取到膜相以保持施加的离子通量。钙离子在敏感膜表面耗尽的时间被称为过渡时间，表现为电位随时间瞬态斜率的变化。根据Sand方程，以施加恒电流时获得过渡时间的平方根为定量信号，用于钙离子浓度检测。通过改变施加恒电流的大小以及敏感膜的厚度，可以实现对过渡时间的优化。实验结果表明，过渡时间几乎不受背景电解质的干扰。因此，本章提出的以过渡时间作为分析信号的计时电位分析技术，可用于海水中钙离子浓度的检测。

4. 基于G-四链体脱氧核酶和逻辑门操作的电位型核酸适配体传感器检测卡那霉素和土霉素

传统电位型传感器的响应主要与聚合物敏感膜相的离子载体有关。因而，聚合物膜离子选择性电极主要应用于单一离子的检测，难以用于多种目标的检测。分子逻辑门通常需要分子能够对两种或以上的外界刺激有所响应，并由二进位布尔逻辑规则在分子体系实现输入信号到输出信号的转换。本文首次在单一敏感膜电极基础上，发展了基于G-四链体脱氧核酶和逻辑门操作的双组份检测的电位型核酸适配体传感器。将双功能核酸适配体和G-四链体核酸序列固定在磁珠上，形成DNA杂交结构；采用“OR”或“INHIBIT”逻辑操作，实现对卡那霉素和土霉素的同时电位检测。在“OR”逻辑操作中，以待测的抗生素作为输入信号，以不同抗生素产生的电位响应为输出信号。当有目标物存在时，目标物能够与磁珠上相应的核酸适配体作用，从而释放出G-四链体脱氧核酶信号输出序列；该序列能够与血红素作用形成G-四链体脱氧核酶，进而在H₂O₂存在条件下催化底物生成阳离子反应中间体TMB⁺，产生计时电位响应。在“INHIBIT”逻辑操作中，以不同抗生素以及其特异性识适体分别作为输入信号，各输入信号产生的电位响应为输出信号。当待测抗生素与其适体同时存在时，其适体将抑制抗生素与磁珠上的核酸适配体作用，则不产生G-四链体脱氧核酶信号输出序列释放。在最优条件下，该传感器对卡那霉素和土霉素的检出限为7.5 nM和9.8 nM，并能成功用于海水中卡那霉素和土霉素的检测。

5. 基于酶催化聚合诱导离子通量阻碍效应的电位型核酸适配体传感器检测土霉素

近年来，基于对离子通量阻碍效应的电位传感技术已受到广泛关注。但是，基于聚合物敏感膜表面阻碍被动离子通量的传感技术往往存在电位响应信号小以及重现性差等问题。计时电位分析可以快速精确地控制离子选择性电极的跨膜离子通量，显著提高电极响应的重现性。在此基础上，我们提出了一种原位酶催化聚合诱导离子通量阻碍效应的电位生物传感器，用于对目标物的高灵敏检测。该传感器的分子识别基于核酸适配体与目标物的高亲和性结合作用，其信号放大基于HRP催化多巴胺在聚合物敏感膜表面沉积形成牢固附着的聚多巴胺层。在脉冲恒电流条件下，该聚多巴胺层可以有效阻碍从水相至到敏感膜相的钙离子通量，从而降低电位响应，实现对目标物的高灵敏检测。本文选取抗生素土霉素为目标物。在优化条件下，该计时电位生物传感器对土霉素的检出限为28 pM，并能成功用于海水中土霉素的检测。

Polymeric membrane ion-selective electrodes (ISEs) have been widely used in the fields of bioanalysis and environmental monitoring, due to their attractive features including simple instrumentation, rapid response and low cost. The potential response of a conventional ISE based on thermodynamic equilibrium is related to the activity of the ion of interest, which can be expressed by Nernst equation. However, the traditional polymeric membrane ISEs suffer from problems of the low sensitivity during the response slope due to the Nernst equation and of the presence of various interfering ions for environmental monitoring. Additionally, one traditional polymeric membrane ISE only responses to one specific ion, but cannot be used for detection of multiple ananlytes. ISEs based on dynamic responses show the advantages of high sensitivity, good selectivity, excellent reversibility and capability for detection of multiple ananlytes. In this thesis, a series of polymeric membrane ISEs based on dynamic potentiometry are described. By precisely controlling the ion fluxes of polymeric membrane electrodes, the speciation informations and improved sensitivity can be obtained. Transition time, a new readout is proposed to reduce the interference of high background electrolyte. The dual-or multianalyte detection using a single membrane electrode can be achieved by the integration of logic gate operations with potentiometric measurements. A highly sensitive potentiometric biosensor is also proposed based on biological amplification. The contents of the thesis are as follows:

1. Detection of free and total ionic concentrations by using calcium-selective polymeric membrane electrodes

   Calcium is a widespread and important element, and many physiological effects are related to its speciation. The speciation analysis of calcium is of key importance for understanding bioavailability and reactivity in natural and biomedical environments. In this work, we have developed two kinds of calcium-selective polymeric membrane electrodes (Ca²⁺-ISEs) with different inner filling solutions for the detection of free and total ionic concentrations in the presence of EDTA and humic acid. The results indicate that the free Ca²⁺ activity, which could be as low as 10^-6 M, can be obtained by the Ca²⁺-ISE (I) with the inner filling solution of 10^-3 M CaCl₂, while the Ca²⁺-ISE(II) with the inner filling solution of 10^-3 M CaCl₂ and 5 × 10^-2 M Na₂EDTA adjusted to pH 9.0, exhibits an apparently super-Nernstian response（ΔE = 180 mV）in the concentration range of 10^-6 -10^-5 M Ca²⁺. Within the super-Nernstian potential response range, the potential is found to be linearly related to the total Ca²⁺ concentration, rather than the free Ca²⁺ activity. Based on these properities, both the total and free ionic concentrations can be obtained by the proposed Ca²⁺-ISEs with different inner filling solutions. The proposed ion-selective polymeric membrane provides a simple and effective method for speciation analysis.

   2. Current pulse based ion-selective electrodes for chronopotentiometric determination of calcium in seawater

   The theoretical slope of the traditional Ca²⁺-ISE based on thermodynamic equilibrium is about 30 mV/dec, which means that a potential difference of 1 mV causes a 8% change in the activity of Ca²⁺. This may be problemetic for detection of Ca²⁺ in seawater by using the traditional Ca²⁺-selective electrode, due to the slight changes in the activity of Ca²⁺ in seawater. Therefore, it’s required to develop a highly sensitive and accurate method for determination Ca²⁺ in seawater. In this work, we have developed a current pulse based ion-selective electrode with enhanced sensitivity for chronopotentiometric measurements of calcium in seawater. The Ca²⁺-selective membrane containing lipophilic salt ETH 500 instead of traditional ion-exchanger is galvanostatically controlled. Under the background solution containing 0.5 M NaCl, an applied constant cathodic current pulse can leads to the extraction of the calcium ions into the membrane to produce a chronopotential response, which shows a stable and reproducible super-Nernstian response in a narrow calcium activity range. The super-Nernstian region of the electrode depends on not only the magnitude and duration of the applied current pulse but also the interfering ions. Under optimal conditions, the proposed Ca²⁺-ISE exhibits a super-Nernstian response between the calcium concentrations of 10^-2.5–10^-1.5 M with a slope of ca 80 mV/dec. Additionally, the Ca²⁺-ISE shows high selectivity towards Na⁺, Mg²⁺, and K⁺. The current pulse based Ca²⁺-ISE has been applied to determination of calcium in seawater with satisfactory results. Compared with those of the conventional Ca²⁺-ISE, the standard deviations obtained by proposed Ca²⁺-ISE proposed are significantly reduced.

   3. Detection of calcium in seawater based on transition time resolved chronopotentiometry

   In dynamic potentiometry, the potentiometric responses of polymeric membrane ISEs are reported to be significantly influenced by the presence of the background electrolyte. Therefore, it’s necessary to develop a promising approach which is less disturbed by the background electrolyte for determination Ca²⁺ concentration in seawater. In this work, an ion-selective electrode with transition time as a readout signal for chronopotentiometric measurement of calcium concentration in seawater is proposed. The proposed Ca²⁺-selective membrane doesn’t contain a traditional ion exchanger. When a constant cathodic current pulse with a long duration is applied on the Ca²⁺-selective membrane, the localized depletion of calcium ions at the transition time occurs, which subsequently results in the co-extraction of calcium and sodium ions from the sample solution into the membrane phase. The square root of transition time, i.e. a potential change (inflection point) of the chronopotentiometry is proportional to the calcium concentration, which is can be expressed by the Sand equation. Additionally, it’s found that the transition time is related to the magnitude of the applied current and the membrane thickness, rather than the presence of the background electrolyte. The proposed chronopotentiometry with transition time as a readout signal is an attractive methodology to detect the calcium concentration in seawater.

   4. Chronopotentiometric aptasensing platform based on a G-quadruplex/hemin DNAzyme and logic-gate operations for detection of KANA and OTC

   Conventional potentiometric ion sensors that rely on a specific ion carrier in a polymeric membrane can hardly achieve multianalyte detection. Inspired by the remarkable ability of built-in logic gates sensors for multianalyte detection, herein we report a potentiometric aptasensing platform based on a G-quadruplex/hemin DNAzyme and logic gate operations for determination of two analytes using a single membrane electrode for the first time. A bifunctional aptamer and a signal reporter nucleic acid are assembled on the magnetic beads to form a DNA hybrid structure. The “OR” and “INHIBIT” logic functions can be performed by using the two aptamers and their targets as inputs, and using the chronopotentiometric response based on the G-quadruplex/hemin DNAzyme-H₂O₂-mediated oxidation of TMB as output. Kanamycin（KANA） and oxytetracycline（OTC）have been employed as the models. Under optimal conditions, the dual targets can be sensed in the range from 10 to 100 nM, with the detection limits of 7.5 and 9.8 nM (3σ) for KANA and OTC, respectively. The potentiometric aptasensing protocol has been evaluated with spiked seawater samples with satisfactory results.

5. Chronopotentiometric aptasensing with signal amplification based on enzyme-catalyzed surface polymerization for detection of OTC

In recent years, potentiometric biosensing based on the blocking effect of ion fluxes has aroused considerable interest. However, the sensing based on the surface blocking of the passive ion fluxes across the polymeric membrane may suffer from problems of relatively small potentiometric signals and poor reproducibilities. Chronopotentiometry can not only rapidly and precisely control the ion fluxes across the polymeric membrane but also improve the reproducibility of the electrode. In this work, chronopotentiometric biosensor based on in situ enzyme-catalyzed dopamine polymerization is proposed for blocking of the active surface area for a high sensitivity. A signal amplification strategy based on HRP induced biocatalyzed polymerization of dopamine and surface coating has been designed and applied for the chronopotentiometric detection of aptamer-target binding events. Using the pulsed galvanostatic measurement protocol, the polydopamine layer adhering to the ion selective membrane (ISM) would effectively block the current-induced indicator ions and subsequently results in a decrease of the potential response. The target binding induced chronopotential changes of the Ca²⁺-ISM is used for highly sensitive detection of target. Oxytetracycline (OTC), as an extensively used antibiotic, has been employed as a model. Under the optimized condition, the chronopotentiometric aptasensor exhibits high sensitivity for the quantitative detection of OTC with a detection limit of 28 pM (3σ/S). The potentiometric aptasensing protocol has been evaluated with spiked seawater samples with satisfactory results.