技术领域technical field
本发明属于微流控分析领域，具体涉及一种手持式POCT双性电极-电化学发光装置及其在检测过氧化氢(H2O2)和葡萄糖中的应用。The invention belongs to the field of microfluidic analysis, and in particular relates to a hand-held POCT bipolar electrode-electrochemiluminescence device and its application in detecting hydrogen peroxide (H2 O2 ) and glucose.
背景技术Background technique
POCT(point of care testing)一般称为即时检验，是体外诊断(IVD)的一个分支行业，具有操作简便、快速、效率高、成本低的优势，具有巨大的市场需求和良好的发展前景，主要应用于环境监测、食品安全检测、临床疾病诊断、个人家庭自检与健康管理等领域。POCT (point of care testing) is generally called point-of-care testing. It is a sub-industry of in vitro diagnostics (IVD). It has the advantages of simple operation, fast speed, high efficiency and low cost. It is used in environmental monitoring, food safety testing, clinical disease diagnosis, personal home self-inspection and health management and other fields.
以智能手机等移动互联网设备为基础的POCT具备高灵敏、高特异的多参数定量的检测能力和更加集成便携、操作简便、信息化等诸多潜在优势，促进了远程医疗以及家庭护理等领域的发展，提高了即时诊断的便携性和普遍性，使现场采集的数据能够通过移动设备传送到分析中心，从而避免了试样运输带来的麻烦，已成为分子生物诊断、微流控、电化学检测等领域的最前沿技术。POCT based on mobile Internet devices such as smart phones has high sensitivity, high specificity, multi-parameter quantitative detection capabilities, and many potential advantages such as more integrated portability, easy operation, and informatization, which promotes the development of telemedicine and home care. , which improves the portability and universality of instant diagnosis, and enables the data collected on site to be transmitted to the analysis center through mobile devices, thus avoiding the trouble caused by sample transportation, and has become a tool for molecular biology diagnosis, microfluidic, electrochemical detection The most cutting-edge technology in other fields.
目前，微流控领域中电化学发光检测装置主要应用于实验室，利用PMT(光电倍增管)检测光强或者CCD成像装置捕捉发光图像，与电压源、微流控芯片等组合装置进行成像检测。但是，这些装置集成化不足、成本高、体积庞大、重量不轻，因而导致检测过程繁琐，稳定性和便携性不足。到目前为止，基于智能手机POCT双性电极-电化学发光及其检测应用未被报道过。At present, electrochemiluminescence detection devices in the field of microfluidics are mainly used in laboratories, using PMTs (photomultiplier tubes) to detect light intensity or CCD imaging devices to capture luminescent images, and combined devices such as voltage sources and microfluidic chips for imaging detection . However, these devices are insufficiently integrated, high in cost, bulky, and not light in weight, resulting in cumbersome detection processes and insufficient stability and portability. So far, smartphone-based POCT bipolar electrodes-electrochemiluminescence and its detection applications have not been reported.
1990年，Manz和Widmer等人首次提出微流控全分析系统(MiniaturizedTotal Analysia Systems,μTAS)概念以来，基于微流控芯片的生化分析技术得到了非常快速的发展，已从理论层次开始真正走向实际应用层面。Since Manz and Widmer first proposed the concept of Microfluidic Total Analysis Systems (μTAS) in 1990, biochemical analysis technology based on microfluidic chips has developed very rapidly, and has really moved from the theoretical level to practice. application level.
2007年，Whitesides研究组提出使用纸作为微流控衬底材料，开启了纸芯片的领域。2009年，Henry课题组更首次提出在纸芯片上进行电化学检测，弥补了比色法灵敏度低的缺陷。随后，本领域获得了持续的发展。In 2007, the Whitesides research group proposed the use of paper as a microfluidic substrate material, opening the field of paper chips. In 2009, Henry's research group proposed for the first time to perform electrochemical detection on a paper chip, which made up for the defect of low sensitivity of colorimetry. Subsequently, the field has been continuously developed.
近来，本申请发明人所在的实验室首次提出利用纸基双性电极电化学发光方法检测H2O2和三丙胺，然而文中使用的检测装置为专用CCD摄像和数据处理设备和可调电压源等设备，整体检测设备体积和重量都很大，未对装置进行集成以及提出POCT的概念；同时检测过程操作较为复杂，对操作人员的要求较高；文中未对检测系统的应用领域进行拓展，未利用该系统来间接检测待检测物，例如葡萄糖的检测方法为利用葡萄糖氧化酶和葡萄糖反应产生H2O2，通过检测H2O2从而实现间接检测葡萄糖浓度的方法等。Recently, the laboratory of the inventor of the present application proposed for the first time the detection of H2 O2 and tripropylamine by using paper-based bipolar electrode electrochemiluminescence method. However, the detection device used in this paper is a special CCD camera and data processing equipment and an adjustable voltage source And other equipment, the volume and weight of the overall detection equipment are very large, the device has not been integrated and the concept of POCT has not been proposed; at the same time, the operation of the detection process is relatively complicated, and the requirements for operators are high; the application field of the detection system is not expanded in this paper. The system is not used to indirectly detect the substance to be detected. For example, the detection method of glucose is to use glucose oxidase to react with glucose to generate H2 O2 , and to realize the method of indirect detection of glucose concentration by detecting H2 O2 .
发明内容Contents of the invention
本发明的首要目的在于提供一种手持式POCT双性电极-电化学发光装置，该装置将微流控芯片与智能手机等移动互联网设备相结合，具有体积小、重量轻、操作简便、成本低、稳定性好的优点。The primary purpose of the present invention is to provide a handheld POCT bipolar electrode-electrochemiluminescence device, which combines a microfluidic chip with a mobile Internet device such as a smart phone, and has the advantages of small size, light weight, easy operation, and low cost , Good stability.
本发明的另一目的在于提供上述装置在检测H2O2和葡萄糖中的应用，该装置可以达到高灵敏度和低检测限的效果，可实际应用于环境监测、食品安全检测、临床疾病诊断等领域。Another object of the present invention is to provide the application of the above- mentioned device in the detection of H2O2 and glucose.The device can achieve high sensitivity and low detection limit, and can be practically applied to environmental monitoring, food safety detection, clinical disease diagnosis, etc. field.
本发明的目的通过下述技术方案实现：The object of the present invention is achieved through the following technical solutions:
一种手持式POCT双性电极-电化学发光装置，是由一个避光外壳将可充电蓄电池、恒电压电路转换系统和微流控芯片集成在其内；A hand-held POCT bipolar electrode-electrochemiluminescent device, which integrates a rechargeable battery, a constant voltage circuit conversion system and a microfluidic chip in a light-proof casing;
在避光外壳内部，可充电蓄电池连接恒电压电路转换系统，采用粘性导电胶布将恒电压电路转换系统的正负极输出端固定连接到微流控芯片的驱动电极上；微流控芯片与可充电蓄电池、恒电压电路转换系统之间用隔离板隔开，以保证避光外壳闭合时芯片处于一个暗环境中，同时防止电磁信号干扰，尽量提高采集图像时的信噪比；Inside the light-proof shell, the rechargeable battery is connected to the constant voltage circuit conversion system, and the positive and negative output terminals of the constant voltage circuit conversion system are fixedly connected to the driving electrodes of the microfluidic chip with viscous conductive tape; The rechargeable battery and the constant voltage circuit conversion system are separated by an isolation plate to ensure that the chip is in a dark environment when the light-proof casing is closed, and at the same time prevent electromagnetic signal interference and maximize the signal-to-noise ratio when collecting images;
所述的微流控芯片固定在支撑平台上。The microfluidic chip is fixed on the supporting platform.
在避光外壳上嵌装一部智能手机，该智能手机有背部摄像头，同时还具有无线传输功能；其背部摄像头正对着避光外壳内的微流控芯片，能将芯片上发生的双性电极电化学反应成像，然后发送给远程的PC端进行分析；智能手机的显示屏朝外，方便使用者操作。A smart phone is embedded in the light-proof shell, which has a back camera and wireless transmission function; The electrochemical reaction of the electrode is imaged, and then sent to the remote PC for analysis; the display screen of the smartphone faces outwards, which is convenient for users to operate.
智能手机的背部摄像头与微流控芯片之间的距离优选3cm。当距离小于3cm时不便于手机聚焦，导致采集到的数据图像模糊，影响实验数据的分析；当距离大于3cm时摄像头视场较宽，大于整个纸基微流控芯片的面积，一方面有可能产生外界信号干扰，另一方面会导致采集的光强度偏小。3cm的距离既能便于聚焦，又能保证视场和纸基微流控芯片吻合，从而使检测效果达到最佳。The distance between the back camera of the smart phone and the microfluidic chip is preferably 3cm. When the distance is less than 3cm, it is not convenient for the mobile phone to focus, resulting in blurred images of the collected data, which affects the analysis of experimental data; when the distance is greater than 3cm, the field of view of the camera is wider, which is larger than the area of the entire paper-based microfluidic chip. External signal interference will be generated, and on the other hand, the collected light intensity will be too small. The distance of 3cm can not only facilitate focusing, but also ensure that the field of view matches the paper-based microfluidic chip, so that the detection effect can be optimized.
所述的微流控芯片是中国专利申请(申请号)201410494915.2中所述的双性电极电致化学发光的纸基微流控芯片。The microfluidic chip described in Chinese patent application (application number) 201410494915.2 is a paper-based microfluidic chip with amphoteric electrode electrochemiluminescence.
所述的可充电蓄电池具有体积小、重量轻、容量大的优点，特别适合作为手持式检测装置的供电电源。The rechargeable storage battery has the advantages of small size, light weight and large capacity, and is especially suitable as a power supply for a hand-held detection device.
所述的恒电压电路转换系统具有电压实时显示功能，同时输出电压可调节。The constant voltage circuit conversion system has the function of real-time voltage display, and the output voltage can be adjusted at the same time.
所述的手持式POCT双性电极-电化学发光装置可以用于检测H2O2和葡萄糖；The handheld POCT bipolar electrode-electrochemiluminescent device can be used to detect H2 O2 and glucose;
所述的手持式POCT双性电极-电化学发光装置用于检测H2O2时，包括以下步骤：将鲁米诺溶液与含有H2O2的待检测溶液按体积比1:1混合，滴加到微流控芯片的通道内，调节输出电压5.5-8V，将避光外壳闭合，在芯片上物质充分反应过程中，利用智能手机摄取发光图像，分析得到结果；When the handheld POCT bipolar electrode-electrochemiluminescence device is used to detect H2 O2 , it includes the following steps: mixing the luminol solution with the solution to be detected containing H2 O2 at a volume ratio of 1:1, Drop it into the channel of the microfluidic chip, adjust the output voltage to 5.5-8V, close the light-proof shell, and use the smart phone to capture the luminescent image during the full reaction process of the material on the chip, and analyze the result;
所述鲁米诺溶液的pH值优选9.0-11.5，浓度优选5-10mM。The pH value of the luminol solution is preferably 9.0-11.5, and the concentration is preferably 5-10 mM.
所述的手持式POCT双性电极-电化学发光装置用于检测葡萄糖时，包括以下步骤：将葡萄糖氧化酶滴加在微流控芯片的双性电极阳极上；将葡萄糖溶解到人工尿中形成待测溶液，然后滴加到微流控芯片的通道内，待反应充分后滴加鲁米诺溶液，调节输出电压5.5-8V，将避光外壳闭合，利用智能手机摄取发光图像，分析得到结果；When the handheld POCT bipolar electrode-electrochemiluminescent device is used to detect glucose, it includes the following steps: adding glucose oxidase dropwise on the bipolar electrode anode of the microfluidic chip; dissolving glucose into artificial urine to form The solution to be tested is then added dropwise into the channel of the microfluidic chip. After the reaction is sufficient, the luminol solution is added dropwise, the output voltage is adjusted to 5.5-8V, the light-proof shell is closed, and the luminescent image is taken by the smartphone, and the result is analyzed ;
所述鲁米诺溶液的pH值优选9.0-11.5，浓度优选5-10mM；The pH value of the luminol solution is preferably 9.0-11.5, and the concentration is preferably 5-10mM;
检测葡萄糖的原理为葡萄糖氧化酶法，利用葡萄糖与葡萄糖氧化酶反应产生H2O2,通过检测H2O2的浓度从而来检测葡萄糖的浓度。The principle of glucose detection is the glucose oxidase method, which uses the reaction of glucose and glucose oxidase to generate H2 O2 , and detects the concentration of glucose by detecting the concentration of H2 O2 .
本发明相对于现有技术具有如下的优点及效果：Compared with the prior art, the present invention has the following advantages and effects:
1、以智能手机等移动互联网设备为基础的POCT具备高灵敏、高特异的多参数定量的检测能力和更加集成便携、操作简便、信息化等诸多潜在优势，促进了远程医疗以及家庭护理等领域的发展，提高了即时诊断的便携性和普遍性，使现场采集的数据能够通过移动设备传送到分析中心，从而避免了试样运输带来的麻烦，已成为分子生物诊断、微流控、电化学检测等领域的最前沿技术。1. POCT based on mobile Internet devices such as smart phones has high sensitivity, high specificity, multi-parameter quantitative detection capabilities, and many potential advantages such as more integrated portability, easy operation, and informatization, which promotes the fields of telemedicine and home care. The development of the technology has improved the portability and universality of instant diagnosis, and enabled the data collected on site to be transmitted to the analysis center through mobile devices, thus avoiding the trouble caused by sample transportation. The most cutting-edge technology in chemical detection and other fields.
2、本发明装置与实验室目前所采用的CCD成像装置及稳压电源相比具有体积小、操作简单、集成化程度高、便于携带并且可用于现场实时检测等优点，尤其可应用于POCT检测领域。2. Compared with the CCD imaging device and regulated power supply currently used in the laboratory, the device of the present invention has the advantages of small size, simple operation, high degree of integration, easy to carry, and can be used for on-site real-time detection, especially for POCT detection field.
3、本发明的装置中，样品的加样过程约3s(对于葡萄糖检测而言，需要额外30s来产生H2O2)，电化学发光被触发后整个反应过程持续约25s，完成样品成像传感分析仅需30s左右，将得到的数据无线传输给计算机处理，因此从进样到检测的分析速度极快。3. In the device of the present invention, the sample loading process is about 3s (for glucose detection, it takes an additional 30s to generate H2 O2 ), the whole reaction process lasts about 25s after the electrochemiluminescence is triggered, and the sample imaging transmission is completed. The sensory analysis only takes about 30s, and the obtained data is wirelessly transmitted to the computer for processing, so the analysis speed from sample injection to detection is extremely fast.
4、本发明的检测方法中，对检测条件诸如驱动电压、缓冲液pH值、以及鲁米诺溶液浓度进行了优化，得到了较优的检测条件。4. In the detection method of the present invention, the detection conditions such as the driving voltage, the pH value of the buffer solution, and the concentration of the luminol solution are optimized, and better detection conditions are obtained.
5、本发明对H2O2和葡萄糖的检测，降低了检测限，达到了高灵敏度的检测效果。5.The detection of H2O2 and glucose in the present invention reduces the detection limit and achieves a high- sensitivity detection effect.
附图说明Description of drawings
图1是本发明手持式POCT双性电极-电化学发光装置的组成示意图；其中，1-避光外壳，2-可充电蓄电池，3-恒电压电路转换系统，4-智能手机，5-隔离板，6-微流控芯片，7-支撑平台。Fig. 1 is a schematic diagram of the composition of the handheld POCT bipolar electrode-electrochemiluminescent device of the present invention; wherein, 1-light-proof housing, 2-rechargeable battery, 3-constant voltage circuit conversion system, 4-smart phone, 5-isolation Plate, 6-microfluidic chip, 7-support platform.
图2是驱动电压与发光强度的关系曲线图。Fig. 2 is a graph showing the relationship between driving voltage and luminous intensity.
图3是缓冲液pH值与发光强度的关系曲线图。Fig. 3 is a graph showing the relationship between the pH value of the buffer solution and the luminescence intensity.
图4是鲁米诺浓度与发光强度的关系曲线图。Fig. 4 is a graph showing the relationship between luminol concentration and luminous intensity.
图5是H2O2浓度与发光强度的关系曲线图。Fig. 5 is a graph showing the relationship between H2 O2 concentration and luminous intensity.
图6是葡萄糖浓度与发光强度的标准曲线图。Fig. 6 is a standard curve diagram of glucose concentration and luminescence intensity.
具体实施方式detailed description
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.
实施例1Example 1
一种手持式POCT双性电极-电化学发光装置，如图1所示，是由一个避光外壳1将可充电蓄电池2、恒电压电路转换系统3和微流控芯片6集成在其内；A handheld POCT bipolar electrode-electrochemiluminescent device, as shown in Figure 1, is a light-proof housing 1 that integrates a rechargeable battery 2, a constant voltage circuit conversion system 3 and a microfluidic chip 6;
在避光外壳内部，可充电蓄电池2连接恒电压电路转换系统3，采用粘性导电胶布将恒电压电路转换系统3的正负极输出端固定连接到微流控芯片6的驱动电极上；微流控芯片6与可充电蓄电池2、恒电压电路转换系统3之间用隔离板5隔开，以保证避光外壳闭合时芯片处于一个暗环境中，同时防止电磁信号干扰，尽量提高采集图像时的信噪比；Inside the light-proof housing, the rechargeable battery 2 is connected to the constant voltage circuit conversion system 3, and the positive and negative output ends of the constant voltage circuit conversion system 3 are fixedly connected to the driving electrodes of the microfluidic chip 6 by using viscous conductive adhesive tape; The control chip 6 is separated from the rechargeable battery 2 and the constant voltage circuit conversion system 3 by an isolation plate 5, so as to ensure that the chip is in a dark environment when the light-proof casing is closed, and prevent electromagnetic signal interference at the same time, so as to improve the efficiency when collecting images as much as possible. SNR;
微流控芯片6固定在支撑平台7上。The microfluidic chip 6 is fixed on the supporting platform 7 .
在避光外壳1上嵌装一部智能手机4，该智能手机4有背部摄像头，同时还具有无线传输功能；其背部摄像头正对着避光外壳内的微流控芯片6，摄像头与微流控芯片6之间的距离为3cm，从而能将芯片上发生的双性电极电化学反应成像，然后发送给远程的PC端进行分析；智能手机4的显示屏朝外，方便使用者操作。A smart phone 4 is embedded on the light-proof casing 1. The smart phone 4 has a back camera and also has a wireless transmission function; The distance between the control chips 6 is 3 cm, so that the electrochemical reaction of the amphoteric electrodes on the chips can be imaged, and then sent to a remote PC for analysis; the display screen of the smart phone 4 faces outward, which is convenient for users to operate.
所述的微流控芯片6是中国专利申请(申请号)201410494915.2中所述的双性电极电致化学发光的纸基微流控芯片。The microfluidic chip 6 described in Chinese Patent Application (Application No.) 201410494915.2 is a paper-based microfluidic chip with amphoteric electrode electrochemiluminescence.
智能手机4采用小米手机(型号MI 2SC，北京小米科技有限责任公司出产)，其背部摄像头的参数为：800万像素、光圈数f/2.0。Smartphone 4 adopts Xiaomi mobile phone (model MI 2SC, produced by Beijing Xiaomi Technology Co., Ltd.), and the parameters of its back camera are: 8 million pixels, aperture number f/2.0.
可充电蓄电池采用的是12.6V供电电源(型号XSL-12.6V，深圳市鑫盛力电源有限公司)，容量为4.4Ah(即1A电流放电4.4h)蓄电池，体积大小为65mm×54mm×37mm，重量约为260g，具有过充、过放、过流、短路等保护功能，具有体积小、重量轻、容量大等优点，装置进行检测时整个电路的放电电流约为30mA，因此充电一次可连续工作146.7h(4.4Ah/0.03A＝146.7h)，非常适合作为手持式检测设备的供电电源。The rechargeable battery uses a 12.6V power supply (model XSL-12.6V, Shenzhen Xinshengli Power Co., Ltd.), with a capacity of 4.4Ah (that is, 1A current discharge for 4.4h). The size of the battery is 65mm×54mm×37mm. The weight is about 260g, and it has protection functions such as overcharge, overdischarge, overcurrent, and short circuit. It has the advantages of small size, light weight, and large capacity. It works for 146.7h (4.4Ah/0.03A=146.7h), which is very suitable as a power supply for handheld detection equipment.
恒电压电路转换系统(型号：YS-05，深圳育松创达电子有限公司)由以xl4015芯片为核心的集成电路组成，是开关降压型DC-DC转换芯片；固定频率为180kHz，可减小外部元器件尺寸，方便EMC设计。芯片具有出色的线性调整率和负载调整率，输出电压支持1.25-32V间任意调节，其内部集成过流保护、过温保护、短路保护等安全可靠性模块，具有集成度高、外围器件少、应用灵活等优点，适用于体积小的电压控制调节电路。该恒电压电路转换系统包括极性电容、非极性电容、xl4015芯片、稳压管、电感、电位器等，蓄电池的输出电压正负极分别接到电压控制系统的输入端，输出电压大小为1.25×(1+R2/R1)V，输出电压1.5-12V可调，通过调节电位器R2，可以得到所需的输出电压以用于双性电极-电化学发光的驱动电压源。数码管可以显示输出电压值，便于选择合适的驱动电压进行检测。The constant voltage circuit conversion system (Model: YS-05, Shenzhen Yusong Chuangda Electronics Co., Ltd.) is composed of an integrated circuit with the xl4015 chip as the core. It is a switching step-down DC-DC conversion chip; the fixed frequency is 180kHz, which can reduce Small external component size, convenient for EMC design. The chip has excellent linear adjustment rate and load adjustment rate, and the output voltage supports arbitrary adjustment between 1.25-32V. It integrates safety and reliability modules such as over-current protection, over-temperature protection, and short-circuit protection. It has high integration, few peripheral devices, It has the advantages of flexible application and is suitable for small-sized voltage control and regulation circuits. The constant voltage circuit conversion system includes polar capacitors, non-polar capacitors, xl4015 chips, regulator tubes, inductors, potentiometers, etc. The positive and negative poles of the output voltage of the battery are respectively connected to the input terminals of the voltage control system, and the output voltage is 1.25×(1+R2/R1)V, the output voltage is adjustable from 1.5-12V. By adjusting the potentiometer R2, the required output voltage can be obtained for the driving voltage source of the amphoteric electrode-electrochemiluminescence. The digital tube can display the output voltage value, which is convenient for selecting the appropriate driving voltage for detection.
实施例2Example 2
从驱动电压角度对实施例1得到的手持式POCT双性电极-电化学发光装置的检测条件进行优化，包括以下步骤：The detection conditions of the handheld POCT bipolar electrode-electrochemiluminescent device obtained in Example 1 are optimized from the perspective of driving voltage, including the following steps:
使用pH值9.0的Na2CO3-NaHCO3缓冲液稀释鲁米诺至终浓度为5mM、稀释H2O2至终浓度为1mM；将H2O2溶液和鲁米诺溶液按体积比1:1混合，得到含有H2O2和鲁米诺的混合溶液；用移液枪吸取上述混合溶液25μL，滴加在芯片通道上。接着，输出电压(即驱动电压)调节到一定数值后，将避光外壳闭合，电化学发光反应过程中利用智能手机图像采集系统进行图像拍摄、保存(所需时间大约25s)；将得到数据无线传输给计算机进行分析处理。Use Na2 CO3 -NaHCO3 buffer with a pH value of 9.0 to dilute luminol to a final concentration of5 mM, and dilute H2 O2 to a final concentration of 1 mM; : 1 mixed to obtain a mixed solution containing H2 O2 and luminol; use a pipette gun to draw 25 μL of the above mixed solution, and drop it on the chip channel. Then, after adjusting the output voltage (that is, the driving voltage) to a certain value, close the light-proof shell, and use the smart phone image acquisition system to capture and save the image during the electrochemiluminescence reaction (the required time is about 25s); the obtained data will be obtained wirelessly. transmitted to a computer for analysis and processing.
对于每组驱动电压(3V、4V、4.5V、5V、5.5V、6V、7V、8V、9V)做8次以上重复试验。通过ImageJ软件对无线传输得到的图像进行分析，每张图片主要发光区域进行灰度值测量，将灰度值乘以发光区域的像素点得到发光面积的光子数，并将数据导入到Origin进行分析和拟合，得到在不同驱动电压下的双性电极-电化学发光曲线，见图2。For each group of driving voltages (3V, 4V, 4.5V, 5V, 5.5V, 6V, 7V, 8V, 9V), repeat the test more than 8 times. The image obtained by wireless transmission is analyzed by ImageJ software, the gray value of the main luminous area of each picture is measured, the gray value is multiplied by the pixel of the luminous area to obtain the number of photons in the luminous area, and the data is imported into Origin for analysis and fitting to obtain the amphoteric electrode-electrochemiluminescence curves at different driving voltages, as shown in Figure 2.
从图2可以看出在驱动电压为5.5-8V时，电化学发光的强度较大。驱动电压为7V时，检测到的发光强度值最大；电压小于7V时，随着驱动电压增大，电化学发光强度逐渐增加；当电压大于7V时，电化学发光强度减小。It can be seen from Fig. 2 that when the driving voltage is 5.5-8V, the intensity of electrochemiluminescence is relatively large. When the driving voltage was 7V, the detected luminescence intensity was the largest; when the voltage was less than 7V, the electrochemiluminescence intensity gradually increased with the increase of the driving voltage; when the voltage was greater than 7V, the electrochemiluminescence intensity decreased.
实施例3Example 3
从缓冲液pH值角度对实施例1得到的手持式POCT双性电极-电化学发光装置的检测条件进行优化，包括以下步骤：The detection conditions of the handheld POCT bipolar electrode-electrochemiluminescence device obtained in Example 1 are optimized from the perspective of buffer pH value, including the following steps:
配制pH值分别为7.5、8.0、9.0、10.0、10.5、11.0、11.5的Na2CO3-NaHCO3缓冲液，分别用不同pH值的缓冲液将鲁米诺稀释到5mM、H2O2稀释到1mM。保持实施例2优化得到的驱动电压7V，并采用实施例2中检测步骤和数据分析方法，得到缓冲液在不同pH值下的双性电极-电化学发光曲线，见图3。Prepare Na2 CO3 -NaHCO3 buffer solutions with pH values of 7.5, 8.0, 9.0, 10.0, 10.5, 11.0, and 11.5, and dilute luminol to 5 mM and H2 O2 with different pH buffer solutions to 1 mM. Keep the optimized drive voltage of 7V in Example 2, and adopt the detection steps and data analysis method in Example 2 to obtain the amphoteric electrode-electrochemiluminescence curves of the buffer solution at different pH values, as shown in FIG. 3 .
从图3可以看出缓冲液pH值为9.0-11.5时，电化学发光的强度较大。缓冲液pH值为10.5时检测到的发光强度值最大；当pH值小于10.5时，随着pH值增大，电化学发光的强度逐渐增加；当pH值大于10.5时，电化学发光强度减小。It can be seen from Fig. 3 that when the pH value of the buffer solution is 9.0-11.5, the intensity of electrochemiluminescence is greater. When the pH value of the buffer solution is 10.5, the detected luminescence intensity value is the largest; when the pH value is less than 10.5, the intensity of electrochemiluminescence increases gradually with the increase of pH value; when the pH value is greater than 10.5, the intensity of electrochemiluminescence decreases .
实施例4Example 4
从鲁米诺浓度角度对实施例1得到的手持式POCT双性电极-电化学发光装置的检测条件进行优化，包括以下步骤：From the perspective of luminol concentration, the detection conditions of the handheld POCT amphoteric electrode-electrochemiluminescent device obtained in Example 1 are optimized, including the following steps:
采用实施例3优化得到的pH值10.5的缓冲液配制鲁米诺溶液，其浓度分别为0、0.5mM、1mM、2.5mM、5mM、7.5mM、10mM、12.5mM；保持实施例2优化得到的驱动电压7V，并采用实施例2中检测步骤和数据分析方法，得到在不同鲁米诺浓度下的双性电极-电化学发光曲线，见图4。Adopt the buffer solution of pH value 10.5 that embodiment 3 optimizes to prepare luminol solution, its concentration is respectively 0,0.5mM, 1mM, 2.5mM, 5mM, 7.5mM, 10mM, 12.5mM; The driving voltage was 7V, and the detection steps and data analysis method in Example 2 were used to obtain amphoteric electrodes-electrochemiluminescence curves at different luminol concentrations, as shown in FIG. 4 .
从图4可以看出在鲁米诺浓度为5-10mM时，电化学发光强度较大。鲁米诺浓度为7.5mM时发光强度最大；当鲁米诺浓度小于7.5mM时，随着鲁米诺浓度增大，电化学发光的强度逐渐增加；当鲁米诺大于7.5mM时，电化学发光强度减小。It can be seen from Fig. 4 that when the concentration of luminol is 5-10 mM, the intensity of electrochemiluminescence is relatively large. When the luminol concentration is 7.5mM, the luminescence intensity is the highest; when the luminol concentration is less than 7.5mM, as the luminol concentration increases, the intensity of electrochemiluminescence increases gradually; when the luminol concentration is greater than 7.5mM, the electrochemiluminescence Luminous intensity decreases.
实施例5Example 5
根据以上实施例2、3、4得到的最优检测条件，实施例1得到的手持式POCT双性电极-电化学发光装置检测不同浓度的H2O2，包括以下步骤：According to the optimal detection conditions obtained in Examples2 , 3, and 4 above, the handheld POCT amphoteric electrode-electrochemiluminescence device obtained in Example1 detects H2O2 of different concentrations, including the following steps:
采用实施例3优化得到的pH值10.5的缓冲液配制不同浓度的H2O2，其浓度分别为0mM、0.005mM、0.01mM、0.02mM、0.05mM、0.1mM、0.2mM、0.5mM、1mM、2mM、5mM；保持实施例2优化得到的驱动电压7V和实施例4优化得到的鲁米诺浓度7.5mM；并采用实施例2中检测步骤和数据分析方法，得到不同浓度H2O2下的双性电极-电化学发光曲线，见图5。Using the buffer solution with a pH value of 10.5 optimized in Example 3 to prepare different concentrations of H2 O2 , the concentrations were 0mM, 0.005mM, 0.01mM, 0.02mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM, 1mM , 2mM, 5mM; keep the driving voltage 7V optimized in Example 2 and the luminol concentration 7.5mM optimized in Example 4; and use the detection steps and data analysis methods in Example 2 to obtain different concentrations of H2 O2 The amphoteric electrode-electrochemiluminescence curve is shown in Figure 5.
从图5可以看出，H2O2浓度从0.005mM变化到5mM时，电化学发光的强度逐渐增加。根据图5所示的低浓度下H2O2校正曲线和未加样品时的空白值加上其相对标准偏差三倍作为发光强度值，算出本发明在优化条件下H2O2的检测限为1.75μM。由此可以得出，该发明装置可以实现H2O2高灵敏、宽范围(达到3个数量级)的定量检测。It can be seen from Figure 5 that when the concentration of H2 O2 changes from 0.005mM to 5mM, the intensity of electrochemiluminescence increases gradually. According to the low concentration shown in Figure 5 H2 O2 Calibration curve and the blank value when no sample is added plus three times its relative standard deviation as the luminous intensity value, calculate the present invention under optimized conditions H2 O2 detection limit is 1.75 μM. It can be concluded that the inventive device can realize the quantitative detection of H2 O2 with high sensitivity and wide range (up to 3 orders of magnitude).
实施例6Example 6
利用实施例1的装置检测不同浓度的葡萄糖，包括以下步骤：Utilize the device of embodiment 1 to detect the glucose of different concentrations, comprise the following steps:
(1)将1U/μL的葡萄糖氧化酶分三次滴加到微流控芯片的双性电极阳极上(每次1μL)，保证酶不会扩散到双性电极的阴极一端以及纸芯片的通道中；(1) Add 1U/μL of glucose oxidase three times to the anode of the bipolar electrode of the microfluidic chip (1 μL each time) to ensure that the enzyme will not diffuse into the cathode end of the bipolar electrode and the channel of the paper chip ;
(2)葡萄糖氧化酶需要在弱酸性条件下才能发挥出酶的最佳活性作用，因此采用pH值6.0的人工尿溶解葡萄糖，分别得到0mM、0.1mM、0.5mM、1mM、2mM、5mM的葡萄糖溶液；(2) Glucose oxidase needs to be under weakly acidic conditions to exert the best activity of the enzyme. Therefore, artificial urine with a pH value of 6.0 is used to dissolve glucose to obtain 0mM, 0.1mM, 0.5mM, 1mM, 2mM, and 5mM glucose respectively. solution;
(3)用移液枪吸取上述葡萄糖溶液12.5μL，添加到微流控芯片通道中，等待葡萄糖与葡萄糖氧化酶反应约30s；(3) Use a pipette gun to draw 12.5 μL of the above glucose solution, add it to the channel of the microfluidic chip, and wait for the reaction of glucose and glucose oxidase for about 30 seconds;
(4)滴加体积为12.5μL、实施例4优化得到的浓度为7.5mM鲁米诺溶液到微流控芯片通道中；保持实施例2优化得到的驱动电压7V，并采用实施例2中检测步骤和数据分析方法，得到不同葡萄糖浓度下的双性电极-电化学发光曲线，见图6。(4) Drop the luminol solution with a volume of 12.5 μL and a concentration of 7.5 mM optimized in Example 4 into the channel of the microfluidic chip; keep the driving voltage 7V optimized in Example 2, and use the detection method in Example 2 Steps and data analysis methods, the amphoteric electrode-electrochemiluminescence curves under different glucose concentrations are obtained, see Figure 6.
从图6可以看出，葡萄糖浓度从0.1mM变化到5mM时，电化学发光的强度逐渐增加。根据图6所示的葡萄糖校正曲线和未加样品时的空白值加上其相对标准偏差三倍作为发光强度值，算出本发明在优化条件下葡萄糖的检测限为43μM。It can be seen from Figure 6 that when the glucose concentration changes from 0.1 mM to 5 mM, the intensity of electrochemiluminescence increases gradually. According to the glucose calibration curve shown in Figure 6 and the blank value when no sample is added plus three times its relative standard deviation as the luminous intensity value, the detection limit of glucose under the optimized conditions of the present invention is calculated to be 43 μM.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.
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| CN201610344999.0ACN106018389A (en) | 2016-05-20 | 2016-05-20 | Handheld POCT (Point of Care Testing) bipolar electrode-electrochemical light emitting device and application thereof |
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| CN201610344999.0ACN106018389A (en) | 2016-05-20 | 2016-05-20 | Handheld POCT (Point of Care Testing) bipolar electrode-electrochemical light emitting device and application thereof |
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| CN201610344999.0APendingCN106018389A (en) | 2016-05-20 | 2016-05-20 | Handheld POCT (Point of Care Testing) bipolar electrode-electrochemical light emitting device and application thereof |
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