| 摘要: |
| 【目的】制备植物叶片葡萄糖活体检测传感器，以实时检测植物体叶片的葡萄糖浓度，为了解霜霉病侵染黄瓜的抗病状态提供支持。【方法】采用双通道丝网印刷电极为基底电极，使用1 mg/mL氯金酸溶液在传感器表面生成金纳米颗粒用于提高其导电性及活性面积，将5 mg/mL壳聚糖和180 mg/mL葡萄糖氧化酶混合以提高酶的固载量和活性，滴加10%的Nafion水溶液以增加对阳离子的选择性并提高电极稳定性。在此基础上研制出一种适用于活体检测植物扁平叶片中葡萄糖浓度的双通道电化学传感器。采用扫描电镜、X射线能谱、红外光谱、循环伏安法及电化学阻抗谱，对所制备传感器进行形貌、结构及电化学的表征；基于优化条件，对所制备的葡萄糖传感器进行性能测试；进一步使用黄瓜果汁进行传感器的加标试验，并应用该传感器对正常与带菌斑黄瓜叶片的葡萄糖浓度进行测定和比较。【结果】所制备植物叶片葡萄糖活体检测传感器的形貌、结构及电化学表征结果显示电极制备成功，其对葡萄糖具有良好的催化效果，反应为扩散控制过程；所设计的植物活体叶片葡萄糖检测双通道传感器的线性检测范围为1～150 mmol/L，检出限为0.784 mmol/L。采用3种不同浓度黄瓜果汁进行加标回收试验，回收率为97.17%~103.05%，相对标准误差≤ 4.52%，表明该传感器准确可靠。应用所制备的传感器检测结果表明，在双通道检测条件下，正常黄瓜叶片和霜霉菌侵染黄瓜叶片在通道1的葡萄糖浓度分别为（8.03±0.96）和（28.71±2.03） mmol/L，在通道2的葡萄糖浓度分别为（12.15±1.46）和（25.57±1.81） mmol/L，霜霉菌侵染黄瓜叶片的葡萄糖浓度较相应正常叶片增长了257.53%（通道1之间）和110.45%（通道2之间），表明不同生理条件下同种植物叶片的葡萄糖浓度因为霜霉菌侵染而存在显著差异（P＜0.05）；病斑区域葡萄糖浓度的检测结果还显示，通道1的葡萄糖浓度高于通道2，表明通道1检测的侵染区域抗病响应迅速，侵染区相应代谢物质会发生明显变化导致邻近区域的葡萄糖逐步出现积累。【结论】所制备的传感器能够检测植物在不同生理环境下的葡萄糖浓度，可为活体植物叶片葡萄糖的实时检测提供技术支持。 |
| 关键词: 植物叶片 活体检测 葡萄糖检测 电化学技术 生物传感器 |
| DOI： |
| 分类号: |
| 基金项目:国家自然科学基金面上项目（21974012） |
|
| Preparation and application of a dual-channel sensor for in-situ determination of glucose in plant leaves |
|
AI Geng,LI Aixue,XIE Yingge
|
| Abstract: |
| 【Objective】A sensor for in-situ real-time determination of glucose in plant leaves was prepared to provide support for understanding resistance status of downy mildew infection in cucumber.【Method】A dual-channel screen-printed electrode was used as the substrate electrode,and 1 mg/mL chloroauric acid solution was used to generate gold nanoparticles on surface to improve its electrical conductivity and active area.Chitosan and glucose oxidase at concentrations of 5 and 180 mg/mL,respectively,were mixed to improve enzyme loading and activity.The 10% Nafion aqueous solution was added to increase cation selectivity and improve electrode stability.On this basis,a dual-channel electrochemical sensor was developed for the detection of glucose content in plant flat leaves in vivo.Morphology,structure,and electrochemical characteristics of the sensor were characterized by scanning electron microscopy,X-ray energy spectrum,fourier transform infrared spectroscopy,cyclic voltammetry,and electrochemical impedance spectroscopy.Based on the optimized conditions,the performance of the prepared glucose sensor was tested.Finally,cucumber juice was used for the marking recovery test of the sensor,and the glucose contents in normal and stained cucumber leaves were measured and compared.【Result】The morphology,structure and electrochemical characterization of the sensor for glucose detection in plant leaves showed that the electrode was successfully prepared with good catalytic effects on glucose.The reaction was a diffusion controlled process.The linear detection range of the two-channel sensor for glucose detection in living plant leaves was 1-150 mmol/L and the detection limit was 0.784 mmol/L.The recoveries of cucumber juices with different concentrations were 97.17%-103.05% and the relative standard deviation was less than 4.52%,indicating that the enzyme sensor was accurate and reliable.Using the sensor with two-channel condition,glucose contents of normal and stained leaves were (8.03±0.96) and (12.15±1.46) mmol/L in channel 1 and (28.71±2.03) and (25.57±1.81) mmol/L in channel 2,respectively.Compared with normal leaves,glucose contents of stained leaves increased by 257.53% (between channel 1) and 110.45% (between channel 2),indicating significant effects by pathogen infection (P<0.05).The detection of glucose content in the lesion area also showed that glucose content in channel 1 was higher than that in channel 2,indicating that the infected area detected by channel 1 had a rapid resistance response and the corresponding metabolites in the infected area changed significantly,leading to gradual accumulation of glucose in the adjacent area.【Conclusion】The prepared sensor can detect the changes of glucose content of plants in different physiological environments,which could provide support for real-time detection of glucose in plant leaves in vivo. |
| Key words: plant leaves in vivo detection glucose detection electrochemical techniques biosensor |
