CN110672589A - A paper sensor for visual detection of nicotine and its preparation method and application - Google Patents

Abstract

Translated fromChinese

一种可视化检测尼古丁的纸质传感器及其制备方法和应用，所述纸质传感器包括附着有介孔二氧化硅纳米孔道的ITO电极基片和纸质盖片；所述ITO电极基片表面的介孔二氧化硅富集电化学发光剂三联吡啶钌，当尼古丁通过氧化去质子化形成强还原自由基，强还原自由基与三联吡啶钌反应生成激发态的三联吡啶钌，根据激发态回到基态释放光子的发光信号强度检测尼古丁溶液的浓度。本发明还包括所述纸质传感器的制备方法和应用。使用本发明进行尼古丁检测，样品消耗少，节约成本，操作简单，灵敏度高，检测限低，稳定性好且抗干扰性好。

A paper sensor for visual detection of nicotine, a preparation method and application thereof, the paper sensor comprises an ITO electrode substrate and a paper cover sheet attached with mesoporous silica nano-channels; Mesoporous silica enriches the electrochemiluminescent agent ruthenium terpyridine. When nicotine is deprotonated by oxidation to form a strong reduction radical, the strong reduction radical reacts with ruthenium terpyridine to generate an excited state of ruthenium terpyridine, which returns to the excited state according to the excited state. The luminescence signal intensity of the released photons in the ground state detects the concentration of the nicotine solution. The invention also includes the preparation method and application of the paper sensor. Using the invention for nicotine detection has the advantages of less sample consumption, cost saving, simple operation, high sensitivity, low detection limit, good stability and good anti-interference.

Description

Translated fromChinese

一种可视化检测尼古丁的纸质传感器及其制备方法和应用A paper sensor for visual detection of nicotine and its preparation method and application

技术领域technical field

本发明涉及一种检测尼古丁的生物传感器，具体涉及一种可视化检测尼古丁的纸质传感器及其制备方法和应用。The invention relates to a biosensor for detecting nicotine, in particular to a paper-based sensor for visually detecting nicotine and a preparation method and application thereof.

背景技术Background technique

吸烟可导致心脑血管病、中风、癌症和哮喘等多种疾病，每年导致约五分之一的死亡，吸烟引起的危害是当今世界最严重的公共卫生问题之一。因此确认吸烟者已成为健康保险的强制要求，吸烟测试经常用于就业和疾病诊断。科学测量烟草暴露量是研究烟草对健康危害的基础。尼古丁是烟草中的主要成分之一，其具有高度的成瘾性，可作用于吸烟者的大脑，使其对烟草产生依赖性。同时，尼古丁具有较长的残存期，可在人和动物体内蓄积而引发中毒。因此，尼古丁是烟草致癌的主要毒物之一，是评价烟草暴露的生物标志物，用于评估人体烟草暴露的剂量和程度。尼古丁摄入量对确认吸烟者具有十分重要的价值。Smoking can cause cardiovascular and cerebrovascular diseases, stroke, cancer and asthma and other diseases, and cause about one fifth of deaths every year. The harm caused by smoking is one of the most serious public health problems in the world today. As a result, identifying smokers has become mandatory for health insurance, and smoking tests are often used for employment and disease diagnosis. Scientific measurement of tobacco exposure is the basis for studying the health hazards of tobacco. Nicotine, one of the main components of tobacco, is highly addictive and acts on the brain of smokers, making them dependent on tobacco. At the same time, nicotine has a long residual period, which can accumulate in humans and animals and cause poisoning. Therefore, nicotine is one of the main carcinogens of tobacco, and it is a biomarker for evaluating tobacco exposure, which is used to evaluate the dose and extent of human tobacco exposure. Nicotine intake is of great value in identifying smokers.

目前，尼古丁的分析检测方法主要有紫外分光光度法、高效液相色谱法、气相色谱法、气相色谱-质谱联用法、酶联免疫法等。然而，由于这些技术存在仪器昂贵、操作复杂、需要专业的操作人员等问题，因此尼古丁在实现低成本检测及现场分析等方面仍存在限制。因此，发展一种快速简便、成本低廉且灵敏度较高的方法以实现对尼古丁的定量检测具有重要的研究意义和广阔的发展前景。At present, the analysis and detection methods of nicotine mainly include UV spectrophotometry, high performance liquid chromatography, gas chromatography, gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay, etc. However, due to the problems of expensive instruments, complicated operations, and the need for professional operators, these technologies still have limitations in achieving low-cost detection and on-site analysis of nicotine. Therefore, developing a fast, simple, low-cost and high-sensitivity method for the quantitative detection of nicotine has important research significance and broad development prospects.

微流控芯片是将生物、化学等实验室基本功能和操作微型集成到微纳米级通道网格结构芯片上，其适用于便携式仪器和现场分析。采用纸质材料做为芯片盖片，对电极基片进行简单刻划加工可以避免传统微流控芯片加工的缺点：如步骤繁琐(刻蚀、曝光和显影)，操作复杂耗时长，需要洁净操作室和专业昂贵的仪器设备(紫外光刻机、等离子清洗器和甩胶机等)等。因此，纸质基片和电极简单加工制备的芯片可用于实现快速简单、低成本和高灵敏的尼古丁检测。A microfluidic chip is a micro-integration of basic functions and operations in laboratories such as biology and chemistry into a micro-nano-scale channel grid structure chip, which is suitable for portable instruments and on-site analysis. Using paper material as the chip cover, simply scribing the electrode substrate can avoid the shortcomings of traditional microfluidic chip processing: such as cumbersome steps (etching, exposure and development), complicated and time-consuming operations, and requires cleaning operations Room and professional expensive equipment (ultraviolet lithography machine, plasma cleaner and glue spinner, etc.). Therefore, the chip prepared by simple processing of paper substrate and electrodes can be used to realize fast, simple, low-cost and highly sensitive nicotine detection.

发明内容SUMMARY OF THE INVENTION

本发明所要解决的技术问题是，克服现有技术存在的上述缺陷，提供一种快速简便，成本低廉，灵敏度较高的基于介孔二氧化硅的可视化纸质传感器。The technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art and provide a visual paper sensor based on mesoporous silica, which is fast, simple, low in cost and high in sensitivity.

本发明进一步要解决的技术问题是，克服现有技术存在的上述缺陷，提供一种操作简单，适合工业化生产的可视化检测尼古丁的纸质传感器的制备方法。The further technical problem to be solved by the present invention is to overcome the above-mentioned defects in the prior art, and to provide a preparation method of a paper sensor for visual detection of nicotine, which is simple to operate and suitable for industrial production.

本发明解决其技术问题所采用的技术方案如下：一种可视化检测尼古丁的纸质传感器，包括附着有介孔二氧化硅纳米孔道的ITO电极基片和纸质盖片；所述ITO电极基片表面的介孔二氧化硅富集电化学发光剂三联吡啶钌，当尼古丁通过氧化去质子化形成强还原自由基，强还原自由基与三联吡啶钌反应生成激发态的三联吡啶钌，根据激发态回到基态释放光子的发光信号强度检测尼古丁溶液的浓度。The technical solution adopted by the present invention to solve the technical problem is as follows: a paper sensor for visual detection of nicotine, comprising an ITO electrode substrate and a paper cover sheet attached with mesoporous silica nano-channels; the ITO electrode substrate The mesoporous silica on the surface enriches the electrochemiluminescent agent ruthenium terpyridine. When nicotine is deprotonated by oxidation to form a strong reduction radical, the strong reduction radical reacts with ruthenium terpyridine to generate an excited state of ruthenium terpyridine. According to the excited state The luminescence signal intensity of the released photons back to the ground state detects the concentration of the nicotine solution.

本发明进一步解决其技术问题所采用的技术方案是：一种可视化检测尼古丁的纸质传感器的制备方法，包括以下步骤：The technical solution adopted by the present invention to further solve the technical problem is: a preparation method of a paper sensor for visual detection of nicotine, comprising the following steps:

(1)制备纸质盖片：在印有图案的疏水纸片进行剪裁，形成两个以上的光滑储液池作为独立检测单元，再将其打磨细化，得纸质盖片；(1) Preparation of paper cover sheets: cut out a patterned hydrophobic paper sheet to form two or more smooth liquid storage tanks as independent detection units, and then polish and refine them to obtain paper cover sheets;

(2)制备ITO电极基片：将ITO导电玻璃切割成ITO电极基片，在ITO电极基片中间刻划一条凹槽分割为两部分，然后将ITO电极基片进行清洗，得刻画有电极阵列的ITO电极基片；将介孔二氧化硅纳米孔道附着到刻画有电极阵列的ITO电极基片上，进行酸处理，然后采用电化学表征实验，对酸处理后的 ITO电极基片进行筛选，得合格的ITO电极基片。(2) Preparation of ITO electrode substrate: cut the ITO conductive glass into an ITO electrode substrate, carve a groove in the middle of the ITO electrode substrate and divide it into two parts, and then clean the ITO electrode substrate to obtain an electrode array. The ITO electrode substrate was obtained by attaching the mesoporous silica nanochannels to the ITO electrode substrate engraved with the electrode array, performing acid treatment, and then using the electrochemical characterization experiment to screen the acid-treated ITO electrode substrate. Qualified ITO electrode substrate.

(3)贴合：将步骤(1)所得的纸质盖片与步骤(2)所得的ITO电极基片按顺序排列，使ITO凹槽位于储液池中间，使两层材料通过双面胶带的进行封合，紧密贴合在一起，即成。(3) Lamination: Arrange the paper cover sheet obtained in step (1) and the ITO electrode substrate obtained in step (2) in order, so that the ITO groove is located in the middle of the liquid storage tank, and the two layers of material are passed through the double-sided tape. to seal, fit tightly together, and serve.

进一步，步骤(2)中，所述介孔二氧化硅通过以下方法附着到ITO电极基片上：将ITO电极竖直排列，浸入十六烷基三甲基溴化铵、无水乙醇、超纯水、浓氨水和四乙氧基硅烷混合溶液的密封盒中，恒温水浴锅中静置，将ITO电极取出冷却至室温，用水清洗，洗净后用氮气吹干，置于烘箱中固化过夜，即成。Further, in step (2), the mesoporous silica is attached to the ITO electrode substrate by the following method: the ITO electrodes are arranged vertically, immersed in cetyltrimethylammonium bromide, absolute ethanol, ultrapure Water, concentrated ammonia water and tetraethoxysilane mixed solution in a sealed box, let stand in a constant temperature water bath, take out the ITO electrode and cool it to room temperature, wash it with water, dry it with nitrogen after washing, and put it in an oven to cure overnight. Serve.

更进一步，所述混合溶液按配方配制：十六烷基三甲基溴化铵0.16g/L，无水乙醇30mL/L，水70mL/L，25wt％浓氨水10μL/L和四乙氧基硅烷80μL/L。Further, the mixed solution is prepared according to the formula: cetyltrimethylammonium bromide 0.16g/L, absolute ethanol 30mL/L, water 70mL/L, 25wt% concentrated ammonia water 10μL/L and tetraethoxy Silane 80 μL/L.

更进一步，所述烘箱的温度为100～130℃，所述固化的时间为6～12h。Further, the temperature of the oven is 100-130° C., and the curing time is 6-12 hours.

更进一步，所述酸化具体步骤如下：是将ITO电极放置在0.1～0.5M盐酸乙醇溶液中，用磁力搅拌器搅拌15～30min后取出，氮气吹干。Further, the acidification specific steps are as follows: the ITO electrode is placed in a 0.1-0.5M hydrochloric acid ethanol solution, stirred with a magnetic stirrer for 15-30 min, taken out, and dried with nitrogen.

进一步，步骤(1)中，所述储液池为矩形、正方形或圆形。Further, in step (1), the liquid storage tank is rectangular, square or circular.

进一步，步骤(2)中，所述凹槽的长为50～60mm，宽为0.2～0.5mm。Further, in step (2), the length of the groove is 50-60 mm, and the width is 0.2-0.5 mm.

进一步，步骤(2)中，所述清洗的具体步骤如下：先置于0.7～1.4M NaOH 溶液中浸泡12～24h，按顺序分别置于丙酮、乙醇、等离子水溶液中超声清洗15～25min。Further, in step (2), the specific steps of the cleaning are as follows: first soak in 0.7-1.4M NaOH solution for 12-24 hours, and then place in acetone, ethanol, and plasma aqueous solution for ultrasonic cleaning for 15-25 minutes in sequence.

进一步，步骤(2)中，所述电化学表征实验具体操作如下：将ITO电极基片分别浸入四种30～60μM探针分子六氨合钌、三联吡啶钌、铁氰化钾和二茂铁甲醇溶液中，依次进行循环伏安测试；所述循环伏安实验测试参数如下，扫速为50mVs^-1：铁氰化钾扫描电位范围为0.5V～0.1V；六氨合钌为0.2V～0.45 V；二茂铁甲醇为0.1V～0.65V；三联吡啶钌为1.5V～2.5V。Further, in step (2), the specific operation of the electrochemical characterization experiment is as follows: the ITO electrode substrate is immersed in four 30-60 μM probe molecules, ruthenium hexaammine, ruthenium terpyridine, potassium ferricyanide and ferrocene, respectively. In the methanol solution, the cyclic voltammetry test was carried out in sequence; the test parameters of the cyclic voltammetry experiment were as follows, the scanning speed was 50mVs⁻¹ : the scanning potential range of potassium ferricyanide was 0.5V～0.1V; the hexaammine ruthenium was 0.2V～ 0.45 V; ferrocene methanol is 0.1V~0.65V; ruthenium terpyridine is 1.5V~2.5V.

本发明更进一步解决其技术问题所采用的技术方案是：可视化检测尼古丁的纸质传感器的应用，向储液池内注入5～15L含有不同浓度的尼古丁溶液和三联吡啶钌的缓冲溶液；所述缓冲溶液为PBS缓冲溶液；含有待测乳酸溶液和鲁米诺的缓冲溶液中PBS的浓度为0.1～0.15mol/L，pH值为6.6～7.5；尼古丁的浓度为0.1～35μM；三联吡啶钌的浓度为0.9～1.5mmol/L。The technical solution adopted by the present invention to further solve the technical problem is as follows: the application of a paper sensor for visual detection of nicotine, injecting 5-15 L of buffer solutions containing nicotine solutions of different concentrations and ruthenium terpyridine into the liquid storage tank; the buffer solution The solution is a PBS buffer solution; the concentration of PBS in the buffer solution containing the lactic acid solution and luminol to be tested is 0.1-0.15mol/L, the pH is 6.6-7.5; the concentration of nicotine is 0.1-35μM; the concentration of ruthenium terpyridine It is 0.9～1.5mmol/L.

本发明的原理：本发明采用疏水纸制备盖片，ITO电极进行简单刻划加工制备电极阵列，化学生长法修饰介孔二氧化硅后即得基片。采用胶带进行盖片和基片的贴合，制备可视化纸质传感器。ITO电极表面的介孔二氧化硅可富集电化学发光剂三联吡啶钌，尼古丁通过氧化可以去质子化形成强还原自由基，尼古丁自由基可与三联吡啶钌反应生成激发态的三联吡啶钌，激发态回到基态释放光子。采用CCD图像传感器采集发光信号，在一定范围内，尼古丁溶液的浓度越高，发光信号越强，以此实现尼古丁的快速简单、低成本和高灵敏检测。Principle of the present invention: The present invention adopts hydrophobic paper to prepare cover sheet, ITO electrode is simply scribed to prepare electrode array, and mesoporous silica is modified by chemical growth method to obtain substrate. The cover sheet and the base sheet are attached with adhesive tape to prepare a visual paper sensor. The mesoporous silica on the surface of the ITO electrode can enrich the electrochemiluminescent agent ruthenium terpyridine, the nicotine can be deprotonated by oxidation to form a strong reducing radical, and the nicotine radical can react with the ruthenium terpyridine to generate the excited state of the ruthenium terpyridine, The excited state returns to the ground state to release photons. A CCD image sensor is used to collect the luminescence signal. Within a certain range, the higher the concentration of the nicotine solution, the stronger the luminescence signal, so as to realize the rapid, simple, low-cost and highly sensitive detection of nicotine.

本发明的有益效果：(1)采用疏水纸制备盖片，通过打印和剪裁在疏水纸上加工微型储液池，工艺简单，成本较低，可以大规模生产，具有十分优异的经济效益；(2)采用化学合成法在电极表面修饰介孔二氧化硅材料，步骤简单、灵敏度高且抗干扰性好，适合批量大规模生产；(3)仪器运行维护成本低，操作简便，有利于方法的推广和应用；(4)样品消耗少，节约成本；(5)采用电化学发光成像技术检测尼古丁的含量，具有检测速度快，可视性强、灵敏度高、检测限低和稳定性好等优点。The beneficial effects of the present invention are as follows: (1) the cover sheet is prepared by using hydrophobic paper, and the micro liquid storage tank is processed on the hydrophobic paper by printing and cutting, the process is simple, the cost is low, it can be produced on a large scale, and it has very excellent economic benefits; ( 2) The chemical synthesis method is used to modify the mesoporous silica material on the surface of the electrode, the steps are simple, the sensitivity is high and the anti-interference is good, which is suitable for mass production; (3) The operation and maintenance cost of the instrument is low, and the operation is simple, which is beneficial to the method. Popularization and application; (4) Less sample consumption and cost saving; (5) Electrochemiluminescence imaging technology is used to detect nicotine content, which has the advantages of fast detection speed, strong visibility, high sensitivity, low detection limit and good stability. .

附图说明Description of drawings

图1为本发明可视化纸质传感器的尺寸图；Fig. 1 is the dimension drawing of the visual paper sensor of the present invention;

图2为本发明可视化纸质传感器的实物图；FIG. 2 is a physical diagram of the visualized paper sensor of the present invention;

图3为本发明介孔二氧化硅的循环伏安表征图；Fig. 3 is the cyclic voltammetry characterization diagram of the mesoporous silica of the present invention;

图4为本发明介孔二氧化硅的微分脉冲伏安表征图；4 is a differential pulse voltammetry characterization diagram of the mesoporous silica of the present invention;

图5为本发明尼古丁检测的电化学发光成像图和线性图；Fig. 5 is the electrochemiluminescence imaging diagram and linear diagram of nicotine detection of the present invention;

图6为本发明传感器重复性考察的电化学发光图和折线图；Fig. 6 is the electrochemiluminescence graph and the broken line graph of the repeatability investigation of the sensor of the present invention;

图7为本发明传感器专属性考察的柱状图；Fig. 7 is the bar chart of the specificity investigation of the sensor of the present invention;

图8为本发明传感器抗干扰能力考察的柱状图；FIG. 8 is a bar chart of the investigation of the anti-interference ability of the sensor of the present invention;

图9为本发明烟草提取物检测的线性图以及电化学发光成像图。FIG. 9 is a linear diagram and an electrochemiluminescence imaging diagram of the tobacco extract detection of the present invention.

具体实施方式Detailed ways

下面结合实施例和附图对本发明作进一步说明。The present invention will be further described below with reference to the embodiments and accompanying drawings.

本发明实施例所使用的化学试剂，如无特殊说明，均通过常规商业途径获得。The chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified.

实施例1Example 1

如图1所示，本实施例包括以下步骤：As shown in Figure 1, this embodiment includes the following steps:

1、盖片的制备1. Preparation of coverslips

采用Adobe Illustrator绘图软件绘制如附图1所示的盖片图案。采用激光打印机，将绘制的图案打印在疏水纸片上。将印有图案的商业疏水纸进行剪裁，形成六个4×2mm的方形储液池作为独立检测单元，再将其打磨细化，得到纸质盖片。The cover slip pattern shown in Figure 1 was drawn using Adobe Illustrator drawing software. Using a laser printer, print the drawn pattern on a hydrophobic paper sheet. The commercial hydrophobic paper printed with the pattern was cut to form six 4×2 mm square liquid reservoirs as independent detection units, which were then polished and refined to obtain paper cover sheets.

2、基片的制备2. Preparation of substrate

将ITO玻璃切割成长50mm，宽12.5mm基片。采用玻璃切割器在基片中间刻划一条长50mm，宽0.5mm的凹槽，分割ITO电极为两部分。然后将ITO先置于1.0M NaOH溶液中浸泡过夜，再分别置于丙酮、乙醇、等离子水溶液中超声清洗15min。The ITO glass was cut into 50mm long and 12.5mm wide substrates. A glass cutter was used to scribe a groove with a length of 50 mm and a width of 0.5 mm in the middle of the substrate, and the ITO electrode was divided into two parts. Then, the ITO was first soaked in 1.0M NaOH solution overnight, and then placed in acetone, ethanol, and plasma aqueous solution for ultrasonic cleaning for 15 min.

3、介孔二氧化硅的合成3. Synthesis of mesoporous silica

使用

溶液生长法将介孔二氧化硅纳米孔道修饰到电极基片上。将ITO 电极竖直排列，浸入装有生长溶液的密封盒中，其中十六烷基三甲基溴化铵0.16 g，无水乙醇30mL，超纯水70mL，25wt％浓氨水10μL和四乙氧基硅烷80μL。 60℃恒温水浴锅中静置生长24h。将ITO电极置于100℃烘箱中，固化过夜。将电极放置在0.1M盐酸乙醇溶液中，用磁力搅拌器搅拌15min后取出，氮气吹干。use

The solution growth method modifies the mesoporous silica nanochannels on the electrode substrate. The ITO electrodes were arranged vertically and immersed in a sealed box containing a growth solution, including 0.16 g of cetyltrimethylammonium bromide, 30 mL of anhydrous ethanol, 70 mL of ultrapure water, 10 μL of 25wt% concentrated ammonia water, and tetraethoxylate. Silane 80 μL. Grow for 24h in a constant temperature water bath at 60°C. The ITO electrodes were placed in a 100°C oven and cured overnight. The electrode was placed in a 0.1M hydrochloric acid ethanol solution, stirred with a magnetic stirrer for 15 min, taken out, and dried with nitrogen.

4、传感器的贴合4. Fitting of the sensor

将纸质盖片与筛选合格的电极基片按顺序排列，使ITO凹槽位于储液池中间，使两层材料通过双面胶带的进行封合，紧密贴合在一起，制得基于介孔二氧化硅的可视化纸质传感器。Arrange the paper cover sheet and the qualified electrode substrate in order, so that the ITO groove is located in the middle of the liquid reservoir, and the two layers of materials are sealed by double-sided tape, and they are closely attached together. Visualization paper sensors for silica.

实施例2Example 2

采用基于介孔二氧化硅的可视化纸质传感器检测尼古丁的方法，移取尼古丁和三联吡啶钌的混合缓冲溶液滴加至传感器片上，每个微液池中加入10μL的混合溶液，其中PBS的浓度为0.1mol/L，pH为6.6，尼古丁的浓度为0.1～35μM，三联吡啶钌的浓度为9μM。Using the method of detecting nicotine based on mesoporous silica-based visual paper sensor, the mixed buffer solution of nicotine and ruthenium terpyridine was pipetted and dropped onto the sensor sheet, and 10 μL of the mixed solution was added to each microfluidic cell, in which the concentration of PBS was It is 0.1 mol/L, the pH is 6.6, the concentration of nicotine is 0.1-35 μM, and the concentration of ruthenium terpyridine is 9 μM.

尼古丁通过氧化可以去质子化(α-氢原子)形成强还原自由基，尼古丁自由基可与三联吡啶钌反应生成激发态的三联吡啶钌，激发态回到基态释放光子。根据CCD图像传感器捕获的发光信号强度确定待测混合溶液中的尼古丁浓度。Nicotine can be deprotonated (α-hydrogen atom) through oxidation to form a strong reducing radical, and the nicotine radical can react with ruthenium terpyridine to form an excited state of ruthenium terpyridine, and the excited state returns to the ground state to release photons. The nicotine concentration in the mixed solution to be tested is determined according to the intensity of the luminescent signal captured by the CCD image sensor.

1.传感器的循环伏安表征1. Cyclic voltammetry characterization of the sensor

将电极分别浸入四种30μM探针分子六氨合钌、三联吡啶钌、铁氰化钾和二茂铁甲醇溶液中，依次进行循环伏安测试。循环伏安实验参数如下，扫速为 50mV s^-1：铁氰化钾扫描电位范围为0.5V～0.1V；六氨合钌为0.2V～0.45V；二茂铁甲醇为0.1V～0.65V；三联吡啶钌为1.5V～2.5V。如图3所示，裸ITO 电极上四种探针分子均有较好的氧化还原峰。CTAB/SNC/ITO电极上铁氰化钾、六氨合钌和三联吡啶钌峰形不可见，而二茂铁甲醇有一对氧化还原峰，这是由于介孔硅孔道中充斥着十六烷基三甲基溴化铵分子，中性二茂铁甲醇探针由于其能溶解于该胶束中到达电极表面，产生电信号，而其他三种带电荷的探针均不能穿透胶束层。反之，去除模板剂十六烷基三甲基溴化铵后，由于介孔硅电极对于阳离子具有显著的富集作用，促进阳离子六氨合钌和三联吡啶钌的传输，抑制阴离子铁氰化钾的传输，因此只有二茂铁甲醇、六氨合钌和三联吡啶钌三种探针有较好的电流响应。The electrodes were immersed in four kinds of 30 μM probe molecules, ruthenium hexaammine, ruthenium terpyridine, potassium ferricyanide and ferrocene methanol solutions, respectively, and cyclic voltammetry tests were performed in sequence. The parameters of the cyclic voltammetry experiment are as follows, the scan rate is 50mV s^-1 : the scanning potential range of potassium ferricyanide is 0.5V-0.1V; ruthenium hexaammine is 0.2V-0.45V; ferrocene methanol is 0.1V-0.65V ; Ruthenium terpyridine is 1.5V ~ 2.5V. As shown in Figure 3, the four probe molecules on the bare ITO electrode have good redox peaks. The peak shapes of potassium ferricyanide, ruthenium hexaammine and ruthenium terpyridine are not visible on CTAB/SNC/ITO electrodes, while ferrocene methanol has a pair of redox peaks, which is due to the hexadecyl-filled mesoporous silica channels. The trimethylammonium bromide molecule, the neutral ferrocene methanol probe, can dissolve in the micelle and reach the electrode surface to generate an electrical signal, while the other three charged probes cannot penetrate the micelle layer. On the contrary, after removing the template agent cetyl trimethyl ammonium bromide, the mesoporous silicon electrode has a significant enrichment effect for cations, which promotes the transport of cationic ruthenium hexaammine and ruthenium terpyridine, and inhibits the anion of potassium ferricyanide. Therefore, only three probes, ferrocene methanol, ruthenium hexaammine and ruthenium terpyridine, have better current responses.

2.可视化纸质传感器的微分脉冲表征2. Visualizing Differential Pulse Characterization of Paper Sensors

将电极分别浸入四种30μM探针分子六氨合钌、三联吡啶钌、铁氰化钾和二茂铁甲醇溶液中，依次进行微分脉冲表征。实验参数如下：扫速为50mV s-1：铁氰化钾扫描电位范围为0.5V～0.1V；六氨合钌为0.2V～0.45V；二茂铁甲醇为0.1V～0.65V；三联吡啶钌为1.5V～2.5V。结果如图4所示，原理同上。The electrodes were immersed in methanol solutions of four 30 μM probe molecules, ruthenium hexaammine, ruthenium terpyridine, potassium ferricyanide, and ferrocene, respectively, and differential pulse characterization was performed sequentially. The experimental parameters are as follows: the scanning speed is 50mV s-1; the scanning potential range of potassium ferricyanide is 0.5V~0.1V; the hexaammine ruthenium is 0.2V~0.45V; the ferrocene methanol is 0.1V~0.65V; Ruthenium is 1.5V to 2.5V. The result is shown in Figure 4, and the principle is the same as above.

3.尼古丁溶液的含量测定3. Determination of nicotine solution

移取尼古丁和三联吡啶钌的混合缓冲溶液滴加至纸质传感器上，每个微液池中加入8μL混合溶液。通过电化学工作站施加2.0V恒电位，采集时间为1min，采用CCD检测发光信号。结果如附图5所示，尼古丁浓度在0.1～35μM浓度范围内，发光灰度值与浓度呈良好的线性关系，线性方程为Y＝1094.8+1891.4X(Y 为电化学发光灰度值；X为尼古丁浓度)，线性相关系数0.9996。The mixed buffer solution of nicotine and ruthenium terpyridine was pipetted and dropped onto the paper sensor, and 8 μL of the mixed solution was added to each microfluidic cell. A constant potential of 2.0 V was applied by an electrochemical workstation, the acquisition time was 1 min, and the luminescence signal was detected by CCD. The results are shown in Figure 5. The nicotine concentration is in the concentration range of 0.1-35 μM, and the luminescence gray value and the concentration have a good linear relationship. The linear equation is Y=1094.8+1891.4X (Y is the electrochemiluminescence gray value; X is the nicotine concentration), and the linear correlation coefficient is 0.9996.

制备空白样品并测定多个空白样品的标准偏差取平均值，按基于响应值标准偏差和标准曲线斜率法计算检测限。本发明方法尼古丁的最低检出限为0.017μM。A blank sample was prepared and the standard deviation of multiple blank samples was measured and averaged, and the detection limit was calculated based on the standard deviation of the response value and the slope of the standard curve. The minimum detection limit of nicotine in the method of the present invention is 0.017 μM.

实施例3Example 3

如图1所示，本实施例包括以下步骤：As shown in Figure 1, this embodiment includes the following steps:

1、盖片的制备1. Preparation of coverslips

采用Adobe Illustrator绘图软件绘制如附图1所示的盖片图案。采用激光打印机，将绘制的图案打印在疏水纸片上。将印有图案的商业疏水纸进行剪裁，形成六个3×3mm的方形储液池作为独立检测单元，再将其打磨细化，得到纸质盖片。The cover slip pattern shown in Figure 1 was drawn using Adobe Illustrator drawing software. Using a laser printer, print the drawn pattern on a hydrophobic paper sheet. The commercial hydrophobic paper printed with the pattern was cut to form six 3×3 mm square liquid reservoirs as independent detection units, which were then polished and refined to obtain paper cover sheets.

2、基片的制备2. Preparation of substrate

将ITO玻璃切割成长50mm，宽12.5mm基片。采用玻璃切割器在基片中间刻划一条长50mm，宽0.5mm的凹槽，分割ITO电极为两部分。然后将ITO先置于1.0M NaOH溶液中浸泡过夜，再分别置于丙酮、乙醇、等离子水溶液中超声清洗30min。The ITO glass was cut into 50mm long and 12.5mm wide substrates. A glass cutter was used to scribe a groove with a length of 50 mm and a width of 0.5 mm in the middle of the substrate, and the ITO electrode was divided into two parts. Then, the ITO was first soaked in 1.0M NaOH solution overnight, and then placed in acetone, ethanol, and plasma aqueous solution for ultrasonic cleaning for 30 min.

3、介孔二氧化硅的合成3. Synthesis of mesoporous silica

使用

溶液生长法将介孔二氧化硅纳米孔道修饰到电极基片上。将ITO 电极竖直排列，浸入装有生长溶液的密封盒中，其中十六烷基三甲基溴化铵0.32 g，无水乙醇60mL，超纯水140mL，25wt％浓氨水20μL和四乙氧基硅烷160 μL。60℃恒温水浴锅中静置生长12h。将ITO电极置于130℃烘箱中，固化过夜。将电极放置在0.5M盐酸乙醇溶液中，用磁力搅拌器搅拌10min后取出，氮气吹干。use

The solution growth method modifies the mesoporous silica nanochannels on the electrode substrate. The ITO electrodes were arranged vertically and immersed in a sealed box containing a growth solution, including 0.32 g of cetyltrimethylammonium bromide, 60 mL of anhydrous ethanol, 140 mL of ultrapure water, 20 μL of 25wt% concentrated ammonia, and tetraethoxylate. Silane 160 μL. Grow for 12h in a constant temperature water bath at 60°C. The ITO electrodes were placed in a 130°C oven to cure overnight. The electrode was placed in a 0.5M hydrochloric acid ethanol solution, stirred with a magnetic stirrer for 10 min, taken out, and dried with nitrogen.

4、传感器的贴合4. Fitting of the sensor

将纸质盖片与筛选合格的电极基片按顺序排列，使ITO凹槽位于储液池中间，使两层材料通过双面胶带的进行封合，紧密贴合在一起，制得基于介孔二氧化硅的可视化纸质传感器。Arrange the paper cover sheet and the qualified electrode substrate in order, so that the ITO groove is located in the middle of the liquid reservoir, and the two layers of materials are sealed by double-sided tape, and they are closely attached together. Visualization paper sensors for silica.

5.重复性5. Repeatability

用低、中、高三种浓度的尼古丁溶液，考察六个传感器的重复性，其结果如附图6所示。低、中、高三种浓度的尼古丁溶液在六片电极上检测结果的RSD 分别为4.72％、3.00％、0.71％，表明该方法的重复性良好。Using low, medium and high concentrations of nicotine solutions, the repeatability of the six sensors was investigated, and the results are shown in Figure 6. The RSDs of the low, medium and high concentrations of nicotine solutions detected on the six electrodes were 4.72%, 3.00% and 0.71%, respectively, indicating that the method has good repeatability.

6.专属性6. Exclusivity

配制与尼古丁溶液相同浓度的氯化钾、BSA、葡萄糖、维生素C、乳酸五种溶液作为对照，分别测定其发光信号，考察检测方法的专属性。结果如附图7 所示，对照组的五种溶液均不产生电化学发光信号(灰度值<5000)，表明检测方法专属性良好。Five solutions of potassium chloride, BSA, glucose, vitamin C, and lactic acid were prepared with the same concentration as the nicotine solution as controls, and their luminescence signals were measured respectively to investigate the specificity of the detection method. The results are shown in FIG. 7 , none of the five solutions in the control group produced electrochemiluminescence signals (gray value <5000), indicating that the detection method has good specificity.

7.抗干扰能力7. Anti-interference ability

将0～300μM梯度浓度的氯化钠、乳糖、葡萄糖三种干扰物分别加入相同浓度的尼古丁标准溶液中，考察传感器的抗干扰能力，结果如附图8所示，在较高浓度干扰物的存在下，尼古丁溶液的检测结果并无明显影响，表明基于介孔二氧化硅的可视化传感器的抗干扰性能良好。The three interfering substances of 0-300 μM gradient concentration of sodium chloride, lactose, and glucose were added to the nicotine standard solution of the same concentration, and the anti-interference ability of the sensor was investigated. The results are shown in Figure 8. In the presence of nicotine solution, the detection results of nicotine solution have no obvious effect, indicating that the visual sensor based on mesoporous silica has good anti-interference performance.

8.烟草提取液检测配制尼古丁、烟草提取物和三联吡啶钌的混合缓冲溶液，其中PBS的浓度为 0.01M，pH为6.6，尼古丁的浓度为0、1、5、10、15、20μM，烟草提取液的体积为5μL，三联吡啶钌的浓度为9μM。往每个储液池中加入8μL混合溶液，施加2.0V恒电位，采集时间为1min，CCD采集发光信号。其结果如附图9所示，计算可得烟草提取液中尼古丁的含量为4.27μM。8. Tobacco extract detects and prepares the mixed buffer solution of nicotine, tobacco extract and ruthenium terpyridine, wherein the concentration of PBS is 0.01M, the pH is 6.6, the concentration of nicotine is 0, 1, 5, 10, 15, 20 μM, tobacco The volume of the extraction solution was 5 μL, and the concentration of ruthenium terpyridine was 9 μM. Add 8 μL of mixed solution to each reservoir, apply 2.0V constant potential, collect time for 1min, and collect luminescence signal by CCD. The results are shown in FIG. 9 , and the content of nicotine in the tobacco extract can be calculated to be 4.27 μM.

Claims (10)

1. A paper sensor for visually detecting nicotine is characterized in that: comprises an ITO electrode substrate and a paper cover plate, wherein the ITO electrode substrate is attached with a mesoporous silica nanometer pore canal; the mesoporous silicon dioxide on the surface of the ITO electrode substrate is rich in electrochemical luminescent agent ruthenium terpyridyl.

2. A method for preparing a paper sensor for visually detecting nicotine according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:

(1) preparing a paper cover sheet: cutting the hydrophobic paper sheet printed with the patterns to form more than two smooth liquid storage tanks as independent detection units, and polishing and refining the liquid storage tanks to obtain a paper cover plate;

(2) preparing an ITO electrode substrate: cutting ITO conductive glass into an ITO electrode substrate, etching a groove in the middle of the ITO electrode substrate, dividing the ITO electrode substrate into two parts, and then cleaning the ITO electrode substrate to obtain the ITO electrode substrate with an electrode array; attaching the mesoporous silica nanopores to an ITO electrode substrate on which an electrode array is engraved, then carrying out acidification treatment, and screening the ITO electrode substrate subjected to acidification treatment by adopting an electrochemical characterization experiment to obtain a qualified ITO electrode substrate;

(3) attaching: and (3) arranging the paper cover plate obtained in the step (1) and the ITO electrode substrate obtained in the step (2) in sequence, so that the ITO groove is positioned in the middle of a liquid storage tank of the paper cover plate, and sealing and closely adhering the two layers of materials through a double-sided adhesive tape.

3. The method for preparing the paper sensor for visually detecting nicotine according to claim 2, comprises the following steps: in the step (2), the mesoporous silica is attached to the ITO electrode substrate by the following method: vertically arranging the ITO electrodes, immersing the ITO electrodes in a sealed box of mixed solution of cetyl trimethyl ammonium bromide, absolute ethyl alcohol, ultrapure water, concentrated ammonia water and tetraethoxysilane, placing the sealed box in a constant-temperature water bath kettle, standing, taking out the ITO electrodes, cooling to room temperature, washing with water, drying with nitrogen after cleaning, and placing the ITO electrodes in an oven for curing to obtain the ITO electrodes.

4. The method for preparing the paper sensor for visually detecting nicotine according to claim 3, wherein the method comprises the following steps: the mixed solution is prepared according to the following formula: 0.16g/L of hexadecyl trimethyl ammonium bromide, 30mL/L of absolute ethyl alcohol, 70mL/L of ultrapure water, 10 mu L/L of 25 wt% concentrated ammonia water and 80 mu L/L of tetraethoxysilane.

5. The method for preparing the paper sensor for visually detecting nicotine according to claim 3 or 4, wherein the method comprises the following steps: the temperature of the oven is 100-130 ℃, and the curing time is 6-12 h.

6. The method for preparing a paper sensor for visually detecting nicotine according to any one of claims 3 to 5, wherein the method comprises the following steps: the specific steps of acidification are as follows: and (3) placing the ITO electrode in 0.1-0.5M ethanol hydrochloride solution, stirring for 15-30 min by using a magnetic stirrer, taking out, and drying by using nitrogen.

7. The method for preparing a paper sensor for visually detecting nicotine according to any one of claims 2 to 6, wherein the method comprises the following steps: in the step (1), the liquid storage tank is rectangular, square or circular.

8. The method for preparing a paper sensor for visually detecting nicotine according to any one of claims 2 to 7, wherein the method comprises the following steps: in the step (2), the length of the groove is 50-60 mm, and the width of the groove is 0.2-0.5 mm.

9. The method for preparing a paper sensor for visually detecting nicotine according to any one of claims 2 to 8, wherein the method comprises the following steps: in the step (2), the specific steps of cleaning are as follows: soaking in 0.7-1.4M NaOH solution for 12-24 h, and then sequentially and respectively placing in acetone, ethanol and plasma aqueous solution for ultrasonic cleaning for 15-25 min.

10. Use of a paper sensor for visual detection of nicotine according to any of claims 2-9, characterized in that: respectively injecting 5-15L of buffer solutions containing nicotine solution and terpyridyl ruthenium with different concentrations into different liquid storage tanks; the buffer solution is PBS buffer solution; the concentration of PBS in the buffer solution containing the lactic acid solution to be detected and the luminol is 0.1-0.15 mol/L, and the pH value is 6.6-7.5; the concentration of nicotine is 0.1-35 mu M; the concentration of the terpyridyl ruthenium is 0.9-1.5 mmol/L.

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