基于微流控阻抗传感器检测番茄环斑病毒

Detection of tomato ring spot virus based on microfluidic impedance sensor

为实现番茄环斑病毒(Tomato Ringspot Virus,ToRSV)高效、准确的快速检测,采用内嵌金叉指阵列微电极的微流控阻抗传感器对ToRSV进行特异性检测。通过将ToRSV抗体固定于金叉指阵列微电极表面免疫结合ToRSV以引起阻抗变化,得到电化学阻抗谱,并建立等效电路分析阻抗变化机理,建立ToRSV浓度和阻抗之间的定量线性关系。等效电路分析表示,ToRSV能造成溶液电阻Rs、电极表面电子转移电阻Ret的显著增加和双电层电容Cdl的减小,从而引起阻抗增加。结果显示在最佳检测频率10.7 Hz下,ToRSV浓度在0.001~10μg/mL范围内检测到的阻抗值范围为248.8~687.2 kΩ,ToRSV浓度与阻抗值具有良好线性关系,R2为0.98。测试该方法的检出限时,阻抗值显示为307.05 kΩ,得到检出限为0.0034μg/mL。试验结果证明该传感器具有检测灵敏度高、特异性好、快速方便等优点,能够实现对ToRSV的特异性定性检测,对于其他植物病毒检测具有可推广性。

Risk of virus propagation is constantly rising in recent years,as the great development of increasing international trade and cross-border electronic commerce service around the world.Therefore,it is necessary for national customs and other health institutes to find an effective way to detect viruses quickly and accurately.In this study,a microfluidic impedance sensor embedded with gold interdigitated array microelectrodes was designed to rapidly detect tomato ring spot virus(ToRSV).The antibodies of ToRSV were fixed on the surface of gold interdigitated array microelectrodes,in order to immunologically bind to the viruses,and thus to trigger the changes in the impedance value.After that,an electrochemical impedance spectrum was measured,while an equivalent circuit was established according to the measurement.Specific electrochemistry tests were carried out to study the influence of each component on the variation of impedance,and thereby to analyze the mechanism of impedance change.An obtained fitting line was formed to verify the correctness of the equivalent circuit.Furthermore,the equivalent circuit analysis showed that the combination of ToRSV and the antibodies can cause a significant increase in the solution resistance Rs,the electron transfer resistance Ret,whereas,a relatively decrease in the capacitance of electric double layer Cdl.It infers that the impedance has a rising trend during the experiment.As such,a simplified formula was established based on the equivalent circuit analysis,showing the relationship between the value of impedance Z and the values of these circuit components.Specifically,there are critical parameters to the circuit in low-frequency area,including the solution resistance,Rs,the electron transfer resistance,Ret,and the decrease in the electric double layer capacitance Cdl.In contrast,in high-frequency area,the solution dielectric capacitor Cdi played a main role in the circuit.Therefore,a proper detecting frequency is necessary to make the obvious impedance response in the certain circuit system.In order to improve the accuracy of detecting system,the samples of the ToRSV in different concentrations were tested three times for the reproducibility,and then to calculate the average values and standard errors for the accurate evaluation of the optimal detecting frequency,indicating the optimum value of 10.7 Hz.Under this detecting frequency,a quantitative linear relationship between ToRSV concentration and impedance was established via different experimental evaluation.The results showed that the ToRSV concentration in the range of 0.001~10μg/mL can have a good linear relationship with the impedance value,where the corresponding impedance values were in the range of 248.8~687.2 kΩ.The R2 values was 0.98,while the detection limit was 0.0034μg/mL,of which the corresponding impedance value was 307.05 kΩ.The effects of different methods to detect ToRSV,such as nano fluorescent particle test strip,real-time fluorescent PCR,HC-RT-PCR-ELISA,and SN-RT-PCR,were then compared with that of the microfluidic impedance sensor.The comparison demonstrated that the microfluidic impedance sensor can serve as an effective and convenient tool to detect ToRSV.In addition,the southern bean mosaic virus(SBMV),Arabis mosaic virus(ArMV)and tobacco ringspot virus(TRSV)were employed as untargeted viruses,to verify the specificity of this detecting system to ToRSV.The samples were separately tested in three times,particularly from the SBMV,ArMV,TRSV,and the mixed solution of all four viruses with the concentration of 10μg/mL.The impedance values of these single-virus samples barely changed,while,that of the mixed solution changed obviously,indicating that the variation was nearly as the same as that of the ToRSV sample.It infers that the detecting system can accurately detect the ToRSV.The detecting system of microfluidic impedance sensor has demonstrated the serval advantages,such as high detection sensitivity,good specificity,effectiveness,and convenience.Thus,the proposed detecting method can provide a sound specific reference for the identification of various virus from other plants.