CN111781264A - Preparation of 3D paper-based electrochemical glucose sensor based on PtNPs - Google Patents
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 title claims abstract description 60
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical group [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims abstract 4
- 238000000206 photolithography Methods 0.000 claims abstract 2
- 229910052697 platinum Inorganic materials 0.000 claims abstract 2
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 17
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- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
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- 238000004070 electrodeposition Methods 0.000 abstract 1
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Abstract
本发明公开了基于PtNPs的3D纸基电化学葡萄糖传感器的制备方法,该方法首先通过光刻技术和丝网印刷技术制备了3D纸基微流控丝网印刷电极,包括工作电极层和对/参比电极层;然后采用电沉积铂纳米粒子的方法对3D纸基微流控丝网印刷电极的工作电极进行修饰,接着在对/参比电极的醛基化亲水性区域共价固定葡萄糖氧化酶,制得了一种便携式的3D纸基微流控葡萄糖传感器。与现有技术相比,本发明3D纸基微流控葡萄糖传感器的制备方法简便、快捷,通过乙醛基化共价固定葡萄糖氧化酶,对葡萄糖具有较好的重复性、选择性和稳定性,并可用于体表汗液中葡萄糖的检测。
The invention discloses a preparation method of a 3D paper-based electrochemical glucose sensor based on PtNPs. The method firstly prepares a 3D paper-based microfluidic screen printing electrode by photolithography technology and screen printing technology, including a working electrode layer and counter/ Reference electrode layer; then the working electrode of the 3D paper-based microfluidic screen-printed electrode was modified by electrodeposition of platinum nanoparticles, followed by covalent immobilization of glucose in the aldehyde-based hydrophilic region of the counter/reference electrode oxidase, a portable 3D paper-based microfluidic glucose sensor was fabricated. Compared with the prior art, the preparation method of the 3D paper-based microfluidic glucose sensor of the present invention is simple and fast, and the glucose oxidase is covalently immobilized by acetaldehyde, and has good repeatability, selectivity and stability to glucose. , and can be used for the detection of glucose in surface sweat.
Description
技术领域technical field
本发明属于生物医学传感技术领域,具体涉及一种基于PtNPs的3D纸基微流控电化学葡萄糖传感器的制备方法和用于葡萄糖检测的方法。The invention belongs to the technical field of biomedical sensing, and in particular relates to a preparation method of a PtNPs-based 3D paper-based microfluidic electrochemical glucose sensor and a method for glucose detection.
背景技术Background technique
糖尿病是威胁我们人类身体健康的三大重要疾病之一,控制糖尿病及其并发症尤为重要,临床上对糖尿病的诊断主要是通过测量血液中葡萄糖的浓度来进行判断的。因此,血液当中葡萄糖浓度的准确和快速检测对临床上糖尿病及其并发症的控制有着非常重要的意义。目前基于电化学方法对葡萄糖的检测取得了显著成效,并由此开发了商业化电化学葡萄糖检测仪。但这些产品存在成本较高、批间差异大、有创及需要频繁使用消毒措施避免交叉感染、不能连续监测等局限性。人体汗液中存在几种与健康状况有关的有价值的分析物如乳酸、铵盐和葡萄糖等。目前已有大量研究表明,血液中的葡萄糖浓度和汗液中的葡萄糖浓度存在一定的相关性,且汗液中的葡萄糖在10 μM至0.7 mM的浓度范围内。因此,建立一种快速、简便、准确、无痛且非侵入性的从汗液中检测葡萄糖的方法是研究者们关注的焦点。Diabetes is one of the three major diseases that threaten our human health. It is particularly important to control diabetes and its complications. The clinical diagnosis of diabetes is mainly judged by measuring the concentration of glucose in the blood. Therefore, accurate and rapid detection of glucose concentration in blood is of great significance to the clinical control of diabetes and its complications. At present, the detection of glucose based on electrochemical methods has achieved remarkable results, and thus a commercial electrochemical glucose detector has been developed. However, these products have limitations such as high cost, large batch-to-batch variation, invasiveness, frequent use of disinfection measures to avoid cross-infection, and inability to continuously monitor. Several valuable analytes such as lactic acid, ammonium salts, and glucose, which are related to health conditions, are present in human sweat. A large number of studies have shown that there is a certain correlation between the glucose concentration in blood and the glucose concentration in sweat, and the glucose concentration in sweat is in the range of 10 μM to 0.7 mM. Therefore, establishing a fast, simple, accurate, painless and non-invasive method to detect glucose from sweat is the focus of researchers.
纸基微流控芯片是一种新型微流控芯片,是用纸材料来代替其他材料(如硅、玻璃、高聚物等)作为反应基底,使其形成具有亲/疏水微细通道以及其他相关分析器件的结构。纸基微流控分析器件具有易携带、操作简单、成本低、样品用量少、可进行多元检测以及无需外援设备等优点,有望成为未来最廉价的分析检测器件。2D 纸基微流控芯片是通过物理或化学的方法在纸上制备封闭的疏水性边界来形成微流体通道。3D纸基微流控芯片是通过将多层带微流体通道图案的纸堆叠在一起,并确保相邻纸层间的微流体通道彼此连通制备而成。二者都可用来作为过滤样品、进行色谱分离及发生生化反应的基底,但3D纸基微流控芯片具有能综合复杂微流体网络通道的能力,为微流体通道的制备提供了更多功能。在纸基微流控分析器件上应用的分析方法有比色法、电致化学发光法、化学发光法以及表面增强拉曼光谱法。将纸基微流体器件和电化学分析技术结合使用是现代分析化学的一个新趋势。纸基微流控电化学检测器件因耗样量少,分析速度快,费用低,易于微型化、集成化、便携化等现场POCT优势,已在医学、食品安全以及环境监测等领域得到迅猛发展。Paper-based microfluidic chip is a new type of microfluidic chip, which uses paper material to replace other materials (such as silicon, glass, polymer, etc.) as the reaction substrate, so that it can form hydrophilic/hydrophobic microchannels and other related Analyze the structure of the device. The paper-based microfluidic analytical device has the advantages of easy portability, simple operation, low cost, low sample consumption, multivariate detection, and no need for external equipment. It is expected to become the cheapest analytical detection device in the future. The 2D paper-based microfluidic chip forms a microfluidic channel by preparing a closed hydrophobic boundary on paper by physical or chemical methods. The 3D paper-based microfluidic chip is fabricated by stacking multiple layers of paper patterned with microfluidic channels and ensuring that the microfluidic channels between adjacent paper layers are connected to each other. Both can be used as substrates for filtration of samples, chromatographic separation and biochemical reactions, but the 3D paper-based microfluidic chip has the ability to integrate complex microfluidic network channels, providing more functions for the preparation of microfluidic channels. The analytical methods applied on paper-based microfluidic analytical devices include colorimetry, electrochemiluminescence, chemiluminescence, and surface-enhanced Raman spectroscopy. Combining paper-based microfluidic devices with electrochemical analysis techniques is a new trend in modern analytical chemistry. Paper-based microfluidic electrochemical detection devices have been rapidly developed in the fields of medicine, food safety and environmental monitoring due to their low sample consumption, fast analysis speed, low cost, easy miniaturization, integration, portability and other field POCT advantages. .
近年来,纳米材料在电化学传感器上应用非常广泛,对提高传感器分析性能具有较好的作用。其中,PtNPs由于对过氧化氢具有良好的电催化还原性能,所以被广泛应用于酶传感器的研究。本发明通过电沉积PtNPs的方法对3D纸基微流控电极的工作电极进行修饰,接着在对/参比电极层的醛基化亲水性区域共价固定葡萄糖氧化酶,制得了一种便携式的3D纸基微流控葡萄糖传感器,制备方法简便、快捷,对葡萄糖具有较好的重复性、选择性和稳定性,并可用于体表汗液中葡萄糖的检测。In recent years, nanomaterials have been widely used in electrochemical sensors, which have a good effect on improving the analytical performance of sensors. Among them, PtNPs have been widely used in the research of enzyme sensors due to their good electrocatalytic reduction performance for hydrogen peroxide. In the present invention, the working electrode of the 3D paper-based microfluidic electrode is modified by the method of electrodepositing PtNPs, and then the glucose oxidase is covalently fixed in the aldehyde-based hydrophilic region of the counter/reference electrode layer to prepare a portable The 3D paper-based microfluidic glucose sensor has a simple and fast preparation method, has good repeatability, selectivity and stability for glucose, and can be used for the detection of glucose in body surface sweat.
发明内容SUMMARY OF THE INVENTION
针对现有技术的不足,本发明的目的在于提供一种新型的可抛弃型的且对环境无污染的可用于体表汗液中葡萄糖定量检测的基于PtNPs的3D纸基微流控传感器的制备方法。In view of the deficiencies of the prior art, the purpose of the present invention is to provide a new type of disposable and environmentally friendly preparation method of a PtNPs-based 3D paper-based microfluidic sensor that can be used for quantitative detection of glucose in body surface sweat .
实现本发明目的的技术方案是:The technical scheme that realizes the object of the present invention is:
本发明一种基于PtNPs的3D纸基微流控电化学传感器,采用电沉积PtNPs的方法对3D纸基微流控电极的工作电极进行修饰,然后在对/参比电极的醛基化亲水性区域固定葡萄糖氧化酶,制得了一种便携式的3D纸基葡萄糖传感器。The present invention is a 3D paper-based microfluidic electrochemical sensor based on PtNPs. The working electrode of the 3D paper-based microfluidic electrode is modified by the method of electrodepositing PtNPs, and then the aldehyde-based hydrophilicity of the control/reference electrode is applied. A portable 3D paper-based glucose sensor was fabricated by immobilizing glucose oxidase in the sexual region.
所述基于PtNPs的3D纸基电化学葡萄糖传感器,包括两层不同的纸基微流控通道,以及印刷在纸基微流控通道上的丝网印刷电极(SPEs);其中,一层纸基微流控通道用来印刷碳工作电极,另一层纸基微流控通道用来印刷碳对电极和银/氯化银参比电极;在所述工作电极上电沉积有PtNPs,在含对电极和参比电极的微流控通道上固定有葡萄糖氧化酶。The PtNPs-based 3D paper-based electrochemical glucose sensor includes two layers of different paper-based microfluidic channels, and screen-printed electrodes (SPEs) printed on the paper-based microfluidic channels; wherein, one layer of paper-based microfluidic channels The microfluidic channel was used to print the carbon working electrode, and another layer of paper-based microfluidic channel was used to print the carbon counter electrode and the silver/silver chloride reference electrode; Glucose oxidase is immobilized on the microfluidic channel of the electrode and the reference electrode.
所述基于PtNPs的3D纸基电化学葡萄糖传感器的制备方法,包括如下过程:The preparation method of the PtNPs-based 3D paper-based electrochemical glucose sensor includes the following processes:
1. 滤纸的醛基化处理1. Formaldehyde treatment of filter paper
首先将滤纸用KIO4浸泡,于恒温箱中浸泡一段时间,待滤纸与KIO4完全反应后,用去离子水洗涤3次,每次均在去离子水中浸泡1分钟;最后用纸巾将滤纸上多余的水分吸干,放置在干燥器中,使滤纸完全干燥。First, soak the filter paper with KIO 4 and soak it in a constant temperature box for a period of time. After the filter paper and KIO 4 are completely reacted, they are washed 3 times with deionized water, and soaked in deionized water for 1 minute each time; Absorb excess moisture and place in a desiccator to allow the filter paper to dry completely.
所述滤纸为Whatman No.1滤纸;Described filter paper is Whatman No.1 filter paper;
所述KIO4的浓度为0.01 M~0.1 M,恒温箱中温度为50~75℃,浸泡时间为1~4小时。The concentration of the KIO 4 is 0.01 M~0.1 M, the temperature in the incubator is 50~75°C, and the soaking time is 1~4 hours.
2.纸基微流控通道的制备2. Fabrication of Paper-Based Microfluidic Channels
取一张滤纸放在匀胶机的托盘上,在滤纸的中心滴加SU-8 2007光刻胶。在匀胶机的作用下,光刻胶均匀分布于滤纸上。先将滤纸放在95℃下软烤10分钟,然后在滤纸上覆盖准备好的掩膜,在紫外灯下对准曝光30 分钟。曝光结束后在95℃条件下硬烤2~3分钟,先将滤纸放入丙酮中浸泡1分钟,随后再用异丙醇洗涤三次,使未聚合的光刻胶被彻底洗掉。其中没有被遮挡的部位在紫外灯的照射下光刻胶发生聚合而形成疏水屏障,被疏水屏障包围的没有被光刻胶覆盖的区域则形成了亲水性区域,最后便形成了带图案的纸基微流控通道。Take a piece of filter paper and place it on the tray of the dispenser, and drop SU-8 2007 photoresist in the center of the filter paper. Under the action of the glue dispenser, the photoresist is evenly distributed on the filter paper. First put the filter paper in a soft bake at 95°C for 10 minutes, then cover the filter paper with the prepared mask and expose it under UV light for 30 minutes. After exposure, hard bake at 95°C for 2~3 minutes, soak the filter paper in acetone for 1 minute, and then wash it with isopropanol three times to completely wash off the unpolymerized photoresist. The parts that are not blocked are polymerized by the photoresist under the irradiation of the UV lamp to form a hydrophobic barrier, and the area surrounded by the hydrophobic barrier that is not covered by the photoresist forms a hydrophilic area, and finally a patterned area is formed. Paper-based microfluidic channels.
所述SU-8光刻胶用量为1~5 mL;The amount of the SU-8 photoresist is 1-5 mL;
所述丙酮和异丙醇的用量各为8~30 mL。The consumption of described acetone and isopropanol is respectively 8~30 mL.
3. 纸基丝网印刷电极的制备3. Preparation of Paper-Based Screen-Printed Electrodes
纸基丝网印刷电极为三电极系统,即工作电极、对电极和参比电极,其中工作电极为一层,对电极和参比电极为一层,简称对/参比电极层。印刷前首先要准备印刷用的油墨,主要为碳墨和银/氯化银(Ag/AgCl)油墨两种。纸基丝网印刷电极的工作部分是印刷在纸基微流控设备的亲水区域,而接触部分是印刷在纸基微流控设备的疏水性区域。丝网印刷工艺是由三层制备而成的:第一层是印刷用作工作电极的碳油墨,第二层是印刷用作对电极的碳油墨,第三层是印刷用作参比电极的Ag/AgCl油墨。每一层在丝网印刷机上印刷完后都要在60℃恒温箱中干燥30分钟,并在室温条件下冷却后再进行下一步。The paper-based screen printing electrode is a three-electrode system, that is, a working electrode, a counter electrode and a reference electrode, in which the working electrode is one layer, and the counter electrode and the reference electrode are one layer, referred to as the counter/reference electrode layer. Before printing, the ink for printing should be prepared first, mainly carbon ink and silver/silver chloride (Ag/AgCl) ink. The working part of the paper-based screen-printed electrode is printed on the hydrophilic region of the paper-based microfluidic device, and the contact part is printed on the hydrophobic region of the paper-based microfluidic device. The screen printing process is prepared by three layers: the first layer is printed carbon ink used as working electrode, the second layer is printed carbon ink used as counter electrode, and the third layer is printed Ag used as reference electrode /AgCl ink. After each layer was printed on the screen printer, it was dried in a 60°C incubator for 30 minutes, and cooled at room temperature before proceeding to the next step.
4. 工作电极的修饰4. Modification of the Working Electrode
在工作电极层的亲水性区域分别滴加10 μL 4.0 mM H2PtCl6和10 μL 0.5 M KCl溶液,室温下稳定5分钟,施加-0.6 V的电压100s,使PtNPs电沉积在工作电极表面。10 μL of 4.0 mM H 2 PtCl 6 and 10 μL of 0.5 M KCl solution were added dropwise to the hydrophilic area of the working electrode layer, stabilized at room temperature for 5 min, and applied a voltage of -0.6 V for 100 s to electrodeposit PtNPs on the surface of the working electrode. .
5. 葡萄糖氧化酶的固定5. Immobilization of Glucose Oxidase
用PBS缓冲液配制葡萄糖氧化酶,在纸基丝网印刷电极对/参比电极层的醛基化亲水性区域滴加葡萄糖氧化酶溶液, 4℃冰箱孵育过夜;Glucose oxidase was prepared with PBS buffer, glucose oxidase solution was added dropwise to the aldehyde-based hydrophilic area of the paper-based screen-printed electrode pair/reference electrode layer, and incubated overnight in a 4°C refrigerator;
所述葡萄糖氧化酶溶液的浓度为1 mg/mL~10 mg/mL。The concentration of the glucose oxidase solution is 1 mg/mL to 10 mg/mL.
6. 电化学检测6. Electrochemical detection
(1)将修饰了PtNPs的工作电极和固定了葡萄糖氧化酶的对/参比电极进行组装,构成了完整的3D纸基电化学葡萄糖传感器;(1) The modified PtNPs working electrode and the glucose oxidase-immobilized counter/reference electrode were assembled to form a complete 3D paper-based electrochemical glucose sensor;
(2)用PBS缓冲液配置不同浓度的葡萄糖标准溶液;(2) Use PBS buffer to configure glucose standard solutions of different concentrations;
(3)通过I-t曲线法记录3D纸基电化学葡萄糖传感器在不同浓度葡萄糖溶液中反应产生的电流,获得相应的回归方程和相关系数;(3) The current generated by the reaction of the 3D paper-based electrochemical glucose sensor in different concentrations of glucose solution was recorded by the I-t curve method, and the corresponding regression equation and correlation coefficient were obtained;
所述I-t曲线法的初始电位为-0.4 V;The initial potential of the I-t curve method is -0.4 V;
(4)将待测葡萄糖溶液根据步骤(3)检测,得到I-t曲线电流,将其代入步骤(3)建立的标准曲线中,得到待测样品中葡萄糖的浓度。(4) Detect the glucose solution to be tested according to step (3) to obtain the I-t curve current, which is substituted into the standard curve established in step (3) to obtain the concentration of glucose in the sample to be tested.
与现有技术相比,本发明的有益效果主要体现在:Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:
1.本发明首次通过对纸基微流控通道的亲水性区域进行醛基化处理,用于共价固定葡萄糖氧化酶,克服了传统电化学传感器上需要借助其它材料修饰电极进行生物分子固定的复杂过程,提高了传感器的重现性。1. For the first time, the present invention is used to covalently immobilize glucose oxidase by performing aldehyde treatment on the hydrophilic region of the paper-based microfluidic channel, which overcomes the complex need of modifying electrodes with other materials to immobilize biomolecules on traditional electrochemical sensors. process, which improves the reproducibility of the sensor.
2.本发明通过电沉积PtNPs的方法对3D纸基微流控基底电极的工作电极进行修饰,制备了比表面积大、导电性好的修饰电极,构建了灵敏的电子转移通道,不仅降低了传感器的检测电位,提高了传感器的抗干扰性能,还大大提高了传感器的检测灵敏度。2. In the invention, the working electrode of the 3D paper-based microfluidic substrate electrode is modified by the method of electrodepositing PtNPs, the modified electrode with large specific surface area and good conductivity is prepared, and a sensitive electron transfer channel is constructed, which not only reduces the detection of the sensor The potential of the sensor improves the anti-interference performance of the sensor, and also greatly improves the detection sensitivity of the sensor.
3.利用本发明制备的3D纸基微流控电化学传感器的可渗透性,提供了一种更简便、安全的无创体表在线汗液葡萄糖定量检测装置。3. By utilizing the permeability of the 3D paper-based microfluidic electrochemical sensor prepared by the invention, a simpler and safer non-invasive body surface online sweat glucose quantitative detection device is provided.
附图说明Description of drawings
图1为 3D纸基电化学葡萄糖传感器的结构示意图;Figure 1 is a schematic structural diagram of a 3D paper-based electrochemical glucose sensor;
图中,(a)工作电极层,(b)对/参比电极层,碳工作电极1,碳对电极2,银/氯化银参比电极3,疏水性区域4,亲水性区域5。In the figure, (a) working electrode layer, (b) counter/reference electrode layer,
图2(A)裸工作电极的扫描电镜图,(B)PtNPs修饰工作电极的扫描电镜图,(C)中(a)裸工作电极和 (b)PtNPs修饰工作电极的XPS图,(D)PtNPs的XPS图。Figure 2 (A) SEM image of bare working electrode, (B) SEM image of PtNPs modified working electrode, (C) XPS image of (a) bare working electrode and (b) PtNPs modified working electrode, (D) XPS plot of PtNPs.
图3展示了裸SPEs和PtNPs修饰SPEs在H2O2溶液中的循环伏安图,具体为裸电极在(a) PBS和(b) 1.0 mM H2O2 中的循环伏安图;PtNPs修饰电极在(c) PBS,(d) 0.25 mMH2O2和(e) 0.4 mM H2O2中的循环伏安图。Figure 3 shows the cyclic voltammograms of bare SPEs and PtNPs-modified SPEs in H 2 O 2 solution, specifically the cyclic voltammograms of bare electrodes in (a) PBS and (b) 1.0 mM H 2 O 2 ; PtNPs Cyclic voltammograms of modified electrodes in (c) PBS, (d) 0.25 mM H2O2 and ( e ) 0.4 mM H2O2.
图4显示了在PtNPs修饰电极上连续滴加H2O2后的电流响应;Figure 4 shows the current response after continuous dropwise addition of H 2 O 2 on the PtNPs modified electrode;
图4 (A) PtNPs修饰电极在间隔50s连续滴加1.0 mM H2O2的I-t曲线图,(B)响应电流与H2O2浓度间的线性关系图。Figure 4 (A) It curve of PtNPs-modified electrode with continuous dropwise addition of 1.0 mM H 2 O 2 at intervals of 50 s, (B) the linear relationship between the response current and the concentration of H 2 O 2 .
图5 (A) PtNPs修饰电极在不同浓度葡萄糖溶液(0,0.001,1,3,5,7,9,11mM)中的I-t曲线图,(B)葡萄糖的标准曲线。Figure 5 (A) I-t curves of PtNPs modified electrodes in different concentrations of glucose solution (0, 0.001, 1, 3, 5, 7, 9, 11 mM), (B) standard curve of glucose.
具体实施方式Detailed ways
下面结合附图及实施例对本发明作进一步阐述,实施例仅用于说明本发明而不用于限制本发明。The present invention will be further described below with reference to the accompanying drawings and examples, and the examples are only used to illustrate the present invention and not to limit the present invention.
实施例1Example 1
一种基于PtNPs的3D纸基电化学葡萄糖传感器,如图1所示,其包括两层纸基微流控通道,即工作电极层(图1a)和对/参比电极层(图1b),纸基丝网印刷电极的工作部分印刷在亲水性区域5,接触部分印刷在疏水性区域4,在工作电极层印刷有碳工作电极1,在对/参比电极层印刷有碳对电极2和银/氯化银参比电极3;在工作电极上电沉积有PtNPs,在对/参比电极的亲水性区域固定有葡萄糖氧化酶。A 3D paper-based electrochemical glucose sensor based on PtNPs, as shown in Fig. 1, includes two layers of paper-based microfluidic channels, namely the working electrode layer (Fig. 1a) and the counter/reference electrode layer (Fig. 1b), The working part of the paper-based screen printed electrode is printed on the
实施例2Example 2
一种基于PtNPs的3D纸基电化学葡萄糖传感器的制备,包括如下步骤 :The preparation of a 3D paper-based electrochemical glucose sensor based on PtNPs includes the following steps:
(1)首先将数张Whatman 1号滤纸浸泡于0.03 M的KIO4溶液中,于恒温箱中65 °C孵育2h,以实现对滤纸表面羟基基团的醛基化。醛基化完成之后,用新鲜的去离子水冲洗滤纸2次,每次1 min。最后用干的吸水纸将洗后的醛基滤纸表面多余的水分吸去,于干燥器中充分干燥。(1) First, soak several sheets of Whatman No. 1 filter paper in 0.03 M KIO 4 solution and incubate at 65 °C for 2 h in an incubator to achieve the aldehyde grouping of hydroxyl groups on the surface of the filter paper. After the aldehydeylation was completed, the filter paper was rinsed twice with fresh deionized water for 1 min each time. Finally, use dry absorbent paper to absorb the excess water on the surface of the washed aldehyde-based filter paper and fully dry it in a desiccator.
(2)图1a和图1b分别展示了3D纸基微流控通道的亲水区和疏水区,纸基微流控通道的制备过程如下:取2.5 mL的SU-8 2007负性光刻胶倾倒在经醛基修饰的whatman 1号滤纸的中心位置,在匀胶机的作用下(500 rpm, 15s; 6500 rpm, 60s),使光刻胶均匀的分布并渗透到滤纸内。首先将覆盖了光刻胶的滤纸于烘胶台上95 °C烘烤10 min,以除去滤纸上的溶剂。然后将事先设计好的紫外光掩膜版覆盖在滤纸上,贴紧,于紫外曝光灯下曝光1min。曝光后的滤纸于烘胶台上95 °C烘烤3 min后,先后用15 mL的丙酮浸泡、异丙醇洗涤三次以彻底除去滤纸表面未聚合的光刻胶,最后滤纸上便形成了带图案的微流控通道。(2) Figure 1a and Figure 1b show the hydrophilic and hydrophobic regions of the 3D paper-based microfluidic channel, respectively. The preparation process of the paper-based microfluidic channel is as follows: take 2.5 mL of SU-8 2007 negative photoresist Pour it on the center of whatman No. 1 filter paper modified with aldehyde groups, and under the action of a glue dispenser (500 rpm, 15s; 6500 rpm, 60s), the photoresist is evenly distributed and penetrated into the filter paper. First, the filter paper covered with photoresist was baked on a glue baking table at 95 °C for 10 min to remove the solvent on the filter paper. Then, cover the pre-designed UV mask on the filter paper, stick it tightly, and expose it under the UV exposure lamp for 1 min. After the exposed filter paper was baked at 95 °C for 3 min on the glue drying table, it was soaked in 15 mL of acetone and washed with isopropanol for three times to completely remove the unpolymerized photoresist on the surface of the filter paper. Finally, a band formed on the filter paper. Patterned microfluidic channels.
(3)3D纸基丝网印刷电极的工作电极、对电极和参比电极的结构如图1所示。纸基丝网印刷电极的工作部分印刷在乙醛基功能化微流控装置的亲水性区域,而接触部分印刷在微流控装置的疏水性区域。3D纸基丝网印刷电极由2层微流控装置构成(图1),一层(图1a)用来印刷工作电极,即工作电极层;另一层(图1b)用来印刷对电极和参比电极,即对/参比电极层。其丝印过程是分三步制备而成:首先在工作电极层(图1a)上印刷碳油墨制备碳工作电极1,其次是在对/参比电极层(图1b)上印刷碳油墨制备碳对电极2,最后再在对/参比电极层上印刷银/氯化银油墨制备参比电极3,每层印刷完后都要在60 °C干燥30分钟,并在室温下冷却后再进行下一步。(3) The structures of the working electrode, counter electrode and reference electrode of the 3D paper-based screen-printed electrode are shown in Figure 1. The working part of the paper-based screen-printed electrode is printed on the hydrophilic region of the acetaldehyde-based functionalized microfluidic device, while the contact part is printed on the hydrophobic region of the microfluidic device. The 3D paper-based screen-printed electrode consists of a 2-layer microfluidic device (Fig. 1), one layer (Fig. 1a) is used to print the working electrode, the working electrode layer; the other layer (Fig. 1b) is used to print the counter electrode and the The reference electrode, the counter/reference electrode layer. The screen printing process was prepared in three steps: firstly, carbon ink was printed on the working electrode layer (Fig. 1a) to prepare
(4)在工作电极层的亲水区滴加10 μL 4.0 mM H2PtCl6和10 μL 0.5 M KCl溶液,室温下稳定5分钟,施加-0.6 V的电压100s,使PtNPs电沉积在工作电极表面。然后在对/参比电极层的醛基化亲水区滴加20 μL 6 mg/mL的葡萄糖氧化酶溶液,4℃冰箱孵育12h。制备好后,将2层叠加起来使用即是一个完整的3D纸基微流控检测基底电极。(4) Add 10 μL of 4.0 mM H 2 PtCl 6 and 10 μL of 0.5 M KCl solution dropwise to the hydrophilic area of the working electrode layer, stabilize at room temperature for 5 minutes, and apply a voltage of -0.6 V for 100 s to electrodeposit PtNPs on the working electrode. surface. Then, 20 μL of 6 mg/mL glucose oxidase solution was added dropwise to the aldehyde-based hydrophilic area of the counter/reference electrode layer, and incubated at 4 °C for 12 h. After preparation, the two layers are stacked together to form a complete 3D paper-based microfluidic detection substrate electrode.
采用扫描电子显微镜和X射线光电子能谱对碳工作电极修饰前后的表面形貌进行表征,从图中可以看出3D纸基微流控基底电极的裸工作电极(图2A)表面呈均匀的片状结构,而PtNPs修饰的工作电极表面明显增加了很多纳米颗粒(图2B)。此外,修饰后的工作电极的X射线光电子能谱上增加了Pt元素,说明PtNPs成功沉积在工作电极表面,如图2C、2D所示。Scanning electron microscopy and X-ray photoelectron spectroscopy were used to characterize the surface morphology of the carbon working electrode before and after modification. It can be seen from the figure that the surface of the bare working electrode (Fig. 2A) of the 3D paper-based microfluidic substrate electrode is a uniform sheet However, the surface of the PtNPs-modified working electrode obviously increased many nanoparticles (Fig. 2B). In addition, Pt element was added to the X-ray photoelectron spectrum of the modified working electrode, indicating that PtNPs were successfully deposited on the surface of the working electrode, as shown in Figures 2C and 2D.
图3展示了裸SPEs和PtNPs修饰SPEs在H2O2溶液中的循环伏安图。由图可知,裸SPEs在H2O2溶液(曲线b)中均没有明显的氧化还原峰。而PtNPs修饰SPEs在H2O2溶液中还原电流明显增大,且随H2O2浓度的增大,还原电流也随之增加(曲线d,e)。这是由于H2O2在电极表面被Pt催化还原所致,说明修饰电极对H2O2具有明显的催化响应。Figure 3 shows the cyclic voltammograms of bare SPEs and PtNPs-modified SPEs in H 2 O 2 solution. It can be seen from the figure that the bare SPEs have no obvious redox peaks in the H2O2 solution ( curve b ) . However, the reduction current of PtNPs-modified SPEs in H 2 O 2 solution increased significantly, and the reduction current also increased with the increase of H 2 O 2 concentration (curves d, e). This is due to the catalytic reduction of H 2 O 2 by Pt on the electrode surface, indicating that the modified electrode has an obvious catalytic response to H 2 O 2 .
图4显示了在PtNPs修饰电极上连续滴加H2O2后的电流响应。图4A展示了随H2O2浓度的增加,还原电流呈阶梯状上升的趋势。并且从图4B中可以看出还原电流与增加的H2O2浓度间呈良好的线性关系,说明PtNPs修饰电极可用于H2O2的连续检测。Figure 4 shows the current response after continuous dropwise addition of H2O2 on the PtNPs - modified electrode. Figure 4A shows a stepwise increase in the reduction current with increasing H2O2 concentration . And it can be seen from Fig. 4B that there is a good linear relationship between the reduction current and the increasing H2O2 concentration, indicating that the PtNPs modified electrode can be used for the continuous detection of H2O2 .
使用I-t曲线法检测不同浓度的葡萄糖溶液的电流响应,结果如图5A所示;当葡萄糖溶液在1 μM~12 mM之间,得到的响应电流与葡萄糖浓度间存在良好的线性关系,如图5B所示。The I-t curve method was used to detect the current response of glucose solutions with different concentrations, and the results are shown in Figure 5A; when the glucose solution was between 1 μM and 12 mM, there was a good linear relationship between the response current and the glucose concentration, as shown in Figure 5B shown.
为评价该3D纸基微流控葡萄糖传感器的实际应用价值,使用该3D纸基微流控传感器对人体汗液中的葡萄糖进行测定。选取3名健康志愿者,经过30 min剧烈运动,待身体稍微出汗后,分别从3名志愿者的皮肤表面收集汗液样品至传感器的检测区,使用计时电流法检测其在-0.4 V时的响应电流。为了验证此方法的可靠性,3个汗液样品还用商品化的试剂盒进行了分光光度测定,对比检测结果如表1所示,从表中数据可知,2种方法检测结果差异较小,表明该3D纸基微流控葡萄糖传感器对汗液中葡萄糖测定的可行性较高。In order to evaluate the practical application value of the 3D paper-based microfluidic glucose sensor, the 3D paper-based microfluidic sensor was used to measure glucose in human sweat. Three healthy volunteers were selected, after 30 minutes of vigorous exercise, and after sweating a little, the sweat samples were collected from the skin surface of the three volunteers to the detection area of the sensor, and the chronoamperometry was used to detect the temperature at -0.4 V. response current. In order to verify the reliability of this method, the three sweat samples were also measured spectrophotometrically with a commercial kit. The comparative test results are shown in Table 1. From the data in the table, it can be seen that the difference between the test results of the two methods is small, indicating that The 3D paper-based microfluidic glucose sensor has high feasibility for the determination of glucose in sweat.
表1 电化学法和分光光度法对汗液中葡萄糖测定的结果比较Table 1 Comparison of the results of electrochemical and spectrophotometric determination of glucose in sweat
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