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Wang Hui, Chen Ruipeng, Yu Zhixue, He Yue, Zhang Fan, Xiong Benhai. Detection of Phytophthora strawberry by field-effect gas sensor based on porphyrin and semiconductor single-walled carbon nanotubes[J]. Smart Agriculture, 2022, 4(3): 143-151.
WANG Hui, CHEN Ruipeng, YU Zhixue, HE Yue, ZHANG Fan, XIONG Benhai. Porphyrin and semiconducting single wall carbon nanotubes based semiconductor field effect gas sensor for determination of phytophthora strawberries[J]. Smart Agriculture, 2022, 4(3): 143-151.
Detection of Phytophthora strawberry by field-effect gas sensor based on porphyrin and semiconductor single-walled carbon nanotubes
WANG Hui1, CHEN Rui-peng1, YU Zhi-xue1, HE Yue1,2, ZHANG Fan1,2, XIONG Ben-hai1*
(1.State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2.College of Animal Science and Technology, China Agricultural University, Beijing 100193)
Abstract: Phytophthora strawberry can cause strawberry leather rot and crown rot, which affects the economic benefits of strawberry, but the early plants infected with Phytophthora have no obvious symptoms and cannot be diagnosed in time and accurately, so there is an urgent need for low-cost diagnostic methods to achieve early prevention. Infection of strawberry plants with Phytophthora angustifolia releases a unique organic volatile gas, 4-ethylphenol, which can be used as a hallmark gas for rapid diagnosis of the disease. In this study, semiconductor field-effect gas sensors (SWNTs/FETs) were fabricated by using semiconductor single-wall carbon nanotubes (SWNTs) and field effect transistors (FETs), and then modified metal porphyrin MnOEP with high sensitivity and selectivity to 4-ethylphenol to obtain MnOEP-SWNT/FET. Raman spectroscopy, ultraviolet spectroscopy and voltammetry were used to study MnOEP-SWNT/FET, and the physical and chemical properties were analyzed and the detection conditions were optimized, so as to improve the gas sensitivity of MnOEP-SWNT/FET to 4-ethylphenol. Under the optimal detection conditions, the relative standard error of MnOEP-SWNT/FET against 0.25%~100% 4-ethylphenol saturated vapor (20 °C) and 0.15% 4-ethylphenol saturated vapor (S/N=3) was less than 10%. The actual samples were measured, and the results showed that MnOEP-SWNT/FET had false positives for the detection of healthy strawberry plants, but had a high detection accuracy for strawberry plants infected with Phytophthora mosquito.
Key words: strawberry; Fungal infection testing; gas sensors; nanocomposites; field-effect transistors; 4-Ethylphenol; semiconductor single-walled carbon nanotubes; Field Effect Sensors
full text
1
INTRODUCTION
Phytophthora cactorum (P. cactorum) is a highly virulent plant pathogenic fungus, and infection of strawberry plants with Phytophthora cactorum causes leather rot and crown rot. Statistics show that the fruit yield of strawberry plants infected with Phytophthora is reduced by 20%~30% on average, and some can reach 50%, which seriously affects the yield of strawberry fruits. Therefore, Phytophthora has become a major disease in the development of the strawberry industry.
Due to the lack of low-cost treatment methods for Phytophthora strawberry, rapid and accurate diagnosis of this pathogenic fungus is of great significance to prevent the spread of the disease caused by it, control the disease, and reduce the loss of growers. At present, the conventional diagnostic methods include tissue separation, microscopic analysis, polymerase chain reaction (PCR), amplification (polymerase chain reaction and recombinase polymerase amplification), fluorescence in situ hybridization, enzyme-linked immunoassay, etc. Among them, the tissue separation method has low culture cost, simple process, and can realize the quantitative detection of viable bacteria, but the detection time is long and the accuracy is low. Microscopy analysis requires complex equipment and specialized technicians, which is expensive and time-consuming; Immunological detection mainly studies the specific reaction of antigens and antibodies, which is simple to operate and fast to react. The detection of molecular hybridization and PCR amplification has strong specificity and high sensitivity, which can make an accurate diagnosis when the disease does not show symptoms or the symptoms are not obvious, reflecting the severity of the disease. However, the early infection of strawberry plants with Phytophthora infetophthora was unevenly distributed and the content was low, resulting in a large number of samples and inaccurate diagnosis in a timely and accurate manner.
Jellen et al. and Eikemo studied the changes of organic volatile gases in strawberry plants infected with Phytophthora over time, and found that strawberry plants infected with Phytophthora emitted unique organic volatile gases 4-ethylphenol (20 °C vapor pressure 0.13 mmHg) and 4-ethyl-2-methoxyphenol 4-ethylguaiacol (25°C vapor pressure 0.017 mmHg), and the organic volatile gas concentration was directly proportional to the severity of Phytophthora infection, and the concentration range was 1.12~, respectively22.56 mg/kg and 0.14~1.05 mg/kg. Since the concentration of the organic volatile gas 4-ethylphenol is much higher than that of 4-ethylguaiacol concentration, 4-ethylphenol can be selected as the characteristic gas of Phytophthora chinensis infection.
Semiconductor MOSFEs have the advantages of low cost, low power consumption, small size, high sensitivity, and easy integration, which can effectively overcome the shortcomings of gas chromatography-mass spectrometry, high performance liquid chromatography and other analytical methods, and are very suitable for real-time monitoring of organic volatile gases in agricultural environments. Gas-sensitive materials are the core of semiconductor FEG sensors, which directly affect their performance in detecting 4-ethylphenol. Semiconducting Single-Walled Carbon Nanotube (SWNT) can be regarded as a one-dimensional hollow tubular structure formed by a single layer of graphene coiled in a certain direction, with a diameter of several nanometers and a length of 1~100 μm, which has high surface adsorption capacity, good conductivity and electron transport characteristics, and is an excellent gas-sensitive material. However, the semiconductor FEG sensor prepared by SWNT has poor selectivity and low sensitivity, which cannot achieve specific detection of 4-ethylphenol.
Porphyrin is a macromolecular heterocyclic compound formed by four pyrrole rings linked together by methylene, each pyrrole ring is composed of 4 carbon and 1 nitrogen, and all nitrogen atoms located inside the macroring form a central cavity, which can coordinate with almost all metal ions to form metalloporphyrin complexes (Metalloporphyrin, MPs). Since the coordination metal ions of MPs are in an unsaturated state, gas molecules can interact with the central metal ions through van der Waals forces and hydrogen bonds in the axial position of MPs, changing their own optical or electrical properties. Therefore, MPs can change the type of metal ion in the center of the porphyrin molecule, the cyclic structure, and the type of surrounding substituents to adjust the sensitivity and selectivity of the gas response, and realize the detection of specific gases.
In order to achieve the early and rapid diagnosis of Phytophthora strawberry fruit rot, SWNT was deposited in MOSFETs, and metal porphyrins were modified on the surface of SWNTs to improve the sensitivity and specificity of gas sensors prepared by FETs. According to the sensitivity of different metal porphyrins to 4-ethylphenol, semiconductor field-effect gas sensors with high detection sensitivity were screened out. On this basis, the optimal detection parameters of semiconductor FEG sensor were further studied to improve the practicability of early diagnosis of Phytophthora strawberry fruit rot.
2
Materials and methods
2.1 Reagents and Materials
半导体性单壁碳纳米管（SWNT，0.01mg/mL，95%）购自美国NanoIntegris公司；丙酮、异丙醇、氢氧化钠、氨水购自Fisher Scientific公司（中国）；3-氨丙基三乙氧基硅烷（3-aminopropyltriethoxysilane：APTES，99％）购自美国Acros Organics公司；4-乙基苯酚购自上海麦克林公司（中国）；二甲基甲酰胺（Dimethylformamide，DMF）采购自北京化工有限公司（中国）；金属卟啉（tetraphenyl porphyrin （TPP）， iron porphyrin （FeTPP）， zinc porphyrin （ZnTPP）， copper octamethyl porphyrin （CuOEP）and manganese OEP （MnOEP））均由西格玛奥德里奇贸易有限公司（美国）和百灵威科技有限公司（美国）提供。
2.2 Test Instruments
Raman spectroscopy uses the scattering phenomenon of large changes in the frequency of incident light generated by material molecules to detect and identify the vibration (phonon) state of molecules, which is excited by 532 nm laser and measured by Nicolet Almega XR dispersive microscopy. UV-Vis photometry uses the absorption or reflection intensity of monochromatic light radiation of 180~780 nm to perform quantitative, qualitative or structural analysis of substances, and the experiment is collected by Beckman DU800 UV/Vis spectrophotometer (Beckman Coulter, United States); SEM images were acquired by Zeiss Leo SUPRA 55 with a beam energy of 10 kV; Electrochemical performance determinations, including current-voltage (I-V) and current-time (I-T), were analyzed by a semiconductor parameter analyzer (Keithley 2636); High Precision Balances BSA224S Electronic Balances (Sartorius Scientific Instruments Ltd.); Chlorobenzene enrichment column sampling tube procurement self-spectral standard experimental equipment technology co., LTD. (China); Direct-Q8 ultrapure water machine (Millipore Inc., United States); Laboratory tubular electric furnace YG-1206 (Shanghai Yuzhi Electromechanical Equipment Co., Ltd.).
2.3 Preparation of gas sensors
References: Silicon wafers covered with 100 nm SiO2 were placed in acetone, isopropanol and ethanol for ultra-cleaning for 20 min respectively to remove surface organic pollutants and nitrogen to remove residual reagents. The surface of the silicon wafer is spin-coated with positive photoresist, and then the interstitial electrode pattern is printed on the surface of the photoresist through exposure and development. The electron beam evaporation coating machine was used to evaporate the 20 nm chromium film and 180 nm gold film on the surface of the lithography substrate, and placed in a constant temperature environment of 300 °C for heat treatment to enhance the adhesion between chromium gold layers. Finally, the heat-treated silicon wafer was soaked in acetone solution for 12 h, and the photoresist was dissolved to obtain a gap of 10 μm in length and width for the source and drain.
The modification steps for an interstitial FEG sensor are shown in Figure 1. Clean the electrode with acetone, isopropanol and ammonia to remove organic and inorganic residues on the surface; The source and drain of the FET were immersed in APTES for 30 min, and the surface residue was washed away with ultrapure water. The interstitial MOSFET was immersed in a solution of single-walled carbon nanotubes for 60 min. The remaining single-walled carbon nanotubes on the surface of the MOSFET were cleaned with ultrapure water, and placed in a 250 °C tubular atmosphere furnace to remove the active agent on the surface of the single-walled carbon nanotubes. Different MPs were dissolved into dimethylformamide (1 mg/mL), SWNT-FET was immersed in MPs solution for 4 h, and finally annealed at 90 °C for 60 min under inert gas protection conditions.
Fig.1 Schematic diagram of the preparation of a semiconductor FEG sensor
Fig.1 Schematic diagram of semiconducting field effect gas sensor
2.4 Gas Generators
Figure 2 shows the organic volatile gas dilution device, which uses the solid powder 4-ethylphenol at a specific temperature to produce volatile gas saturated vapor, and adjusts the mixing ratio of air flow and 4-ethylphenol organic volatile gas saturated vapor by controlling the gas quality controller to produce different concentrations of 4-ethylphenol vapor; It is then mixed in the gas pipeline and passed through a sealed glass cover of 1.2 cm3 in the gas sensor. Using Keithley 2636 as a data acquisition device, it is connected to a semiconductor FEG sensor through three electrode lines (gate, source and drain), the source and drain are applied with a voltage range of -0.1~ +0.1 V, and the gate is applied to a voltage of 0 V, and the current signal is recorded with the concentration of organic volatile gases.
Fig.2. Dilution device for organic volatile gases
Fig. 2 Organic volatile gas dilution device
2.5 Signal Analysis
Due to the difference in the initial resistance of different MOEP-SWNT/FETs, which affects the detection accuracy, the relative resistance change was used as the response signal in this study, as shown in equations (1) and (2).
Among them, R0 is the original resistance of MnOEP-SWNT/FET placed in dry air, Ω; R is the resistance value of MnOEP-SWNT/FET exposed to VOCs, Ω; is the voltage between the source and drain, V; is the current between the source and drain, A.
The calculation of the carrier mobility μ of semiconductor materials is shown in equation (3).
where and denotes the length and width between the source and drain of the FET, m; is the transfer conductance (slope of IDS/VG), A/V; VG is the base voltage, V; is the gate capacitance, nF/cm2, here 11.6 nF/cm2.
3
Results & Discussion
3.1 MnOEP-SWNT表征
Raman spectroscopy was used to study the changes of MnOEP before and after SWNT/FET modification, and the results are shown in Figure 3(a). Raman spectra of semiconductor single-walled carbon nanotubes show four peaks at 1351 cm-1 (D Band), 1579 cm-1 (G Band), 1600 cm-1 (G+ Band), and 2680 cm-1 (2D Band). After MnOEP modified the semiconductor single-walled carbon nanotubes, the G-band peak narrowed, the G-band shifted to 1598 cm-1, and the D/G intensity ratio increased, indicating that MnOEP and SWNT were bound by non-covalent bonds, and sp2 was converted to sp3 on the surface of SWNT.
Fig. 3 Spectral characteristics of Bare SWNT (black) and MnOEP-SWNT (red).
Fig.3 Spectral characteristics of Bare SWNT （black） and MnOEP SWNT （red）
In order to overcome the poor light transmittance of Si/SiO2, quartz glass with good light transmittance was selected to replace Si/SiO2. The absorption spectrum of the blank quartz glass was used as the reference value, and the absorbance of the SWNT in the range of 200~800 nm was significantly enhanced and there was an obvious absorption peak between 200~400 nm after the SWNT was fixed to the surface of the quartz glass as shown in Figure 3(b), which was consistent with the characteristic absorption peak of SWNT, indicating that the SWNT was well fixed to the surface of the quartz glass through the covalent bond of APTES. When MnOEP modified SWNT, the absorption spectrum of MnOEP-SWNT was significantly higher than that of Bare SWNT, and there were small absorption peaks at 475 and 560 nm, which was consistent with the literature, indicating that MnOEP was modified to the SWNT surface.
Fig.4 Changes in electrochemical performance of SWNT/FET before and after modification of MnOEP
Fig. 4 Changes of electrochemical properties of MnOEP modified by SWNT/FET
The electrochemical analysis method shown in Figure 4(a) characterizes the changes in conductivity of MnOEP-modified SWNT/FETs. The IDS-VDS curves of SWNT/FET and MnOEP-SWNT/FET showed a good linear relationship, but the conductivity of MnOEP-SWNT/FET was significantly reduced, indicating that the interaction between MnOEP and SWNT underwent electron transfer and formed π-π bonds. Fig. 4(b) shows that the gate threshold voltage (VTH) of SWNT/FET is 0.45 V, and the FET curve shifts to the negative direction with a VTH value of -5.3 V after MnOEP modifies SWNT/FET. According to the mobility calculation equation, the mobility of SWNT/FET and MnOEP-SWNT/FET is 525 and 387 cm2/Vs, respectively. Reasons for the deterioration of the conductivity of MnOEP-SWNT/FET [26]: ELECTRON/CHARGE TRANSFER OCCURS BETWEEN MNOEP AND SWNT, AND THE HOLES OF P-TYPE SEMICONDUCTOR SWNT/FET ARE OCCUPIED BY ELECTRONS PROVIDED BY MNOEP, RESULTING IN LOW CARRIER CONCENTRATION AND LOW MOBILITY OF MNOEP-SWNT/FET.
3.2 Parameter optimization
Figure 5 shows the relative resistance of semiconductor FEG sensors prepared with different metal porphyrins to saturated vapors at different concentrations of 4-ethylphenol. The relative resistance of SWNT/FET to saturated vapors of 1%, 10%, and 100% 4-ethylphenol was 0.1, 0.24, and 0.49, respectively. When SWNT/FET modifies different MPs, the sensitivity of the semiconductor FEG sensor changes to MnOEP-SWNT/FET, > ZnTPP-SWNT/FET, > CuOEP-SWNT/FET, > TPP-SWNT/FET, > FeTPP-SWNT/FET, indicating that the special chemical structure of MnOEP has high selectivity for 4-ethylphenol. Therefore, MnOEP was chosen as the gas-sensitive material for semiconductor FEG sensors.
Fig.5 Comparison of saturated vapor performance of 4-ethylphenol detected by different MPs-SWNT/FETs
Fig.5 Performance comparison of different MPs SWNT/FET for detecting saturated vapor of 4-Ethylphenol
Figure 6 shows the relative resistance change of MnOEP-SWNT/FET versus concentration and time. It can be seen from the figure that the relative resistance of MnOEP-SWNT/FET is positively correlated with the detection time of 4-ethylphenol saturated vapor at 1%, 10% and 100% of 4-ethylphenol saturated vapor at low concentration, and reaches the maximum value at 5 min. At high concentrations, the relative resistance of MnOEP-SWNT/FET reaches a maximum value for 2 minutes and then remains constant. Therefore, 5 min was chosen as the detection time for the semiconductor FEG sensor.
Fig.6 Variation of relative resistance and time of MnOEO-SWNT/FET exposure to saturated vapor with 4-ethylphenol at different concentrations
Fig. 6 Changes of relative resistance and time of MnOEO-SWNT/FET exposed to saturated vapor of 4-Ethylphenol at different concentrations
图7 SWNT/FET和MnOEP-SWNT/FET的电压-电阻变化（VG=0 V）
Fig.7 Voltage resistance change of SWNT/FET and MnOEP-SWNT/FET （VG=0 V）
The voltage between source and drain (VDS) is one of the important parameters of semiconductor FEG sensors. In Figure 4(a), the IDS-VDS uses Ohm's law to calculate the resistance at different voltages, as shown in Figure 7. The discontinuity at 0 V is mainly due to the insufficient accuracy of the signal acquisition equipment, which cannot collect ultra-microcurrent signals with high precision. At other voltages, the resistance values of SWNT/FET and MnOEO-SWNT/FET do not change, indicating that the voltage change has no effect on the resistance. Therefore, any voltage can be used as VDS, and 0.1 V was chosen for this study.
3.3 4-ethylphenol detects linear relationships
In order to study the linear detection range and minimum detection limit of the gas sensor, SWNT/FET and MnOEO-SWNT/FET detected different concentrations of 4-ethylphenol saturated vapor for 5 min under the optimal detection conditions, and washed with dry air for 10 min at different concentration intervals. Fig. 8(a) shows the relative impedance changes of SWNT/FET and MnOEO-SWNT/FET on 0.25%~100% of the saturated vapor produced by the gas generator at VDS=0.1 V and VG=0 V, and it can be seen that the relative impedance of MnOEO-SWNT/FET increases with the increase of 4-ethylphenol saturated vapor concentration. As can be seen from the linear plot of Figure 8(b), the relative resistance of MnOEO-SWNT/FET changes rapidly in the concentration range of 0.25%~20% for 4-ethylphenol saturated vapor, and grows slowly in the high concentration range of 20%~100%, but all show a good linear relationship, and the linear regression equations are as follows:
The linear regression correlation coefficients were 0.9411 and 0.9745, respectively, and the detection limit was 0.15% of the saturated vapor of 4-ethylphenol (S/N=3).
Fig.8. Response of FEG sensor to saturated vapor with different concentrations of 4-ethylphenol
Fig. 8 Response of field effect gas sensor to saturated vapor of 4-ethylphenol with different concentrations
3.4 Consistency
To study the consistency of the sensors, four MnOEP-SWNT/FETs were prepared by the above method for the detection of saturated vapors of 4-ethylphenol at different concentrations, as shown in Figure 9. Compared with the relative resistance changes of different MnOEP-SWNT/FET after the detection of the same concentration of 4-ethylphenol, the relative resistance was positively correlated with the concentration of 4-ethylphenol and the change trend was consistent, and the relative standard deviation (RSD) at each concentration was less than 10%, indicating that MnOEP-SWNT/FET had good consistency.
Fig.9 Changes in the relative resistance of 4-ethylphenol saturated vapor detected by different MnOEO-SWNT/FETs
Fig. 9 Change of relative resistance after detecting saturated vapor of 4-ethylphenol with different MnOEO-SWNT/FET
3.5 Analysis of actual samples
In order to verify the effect of MnOEO-SWNT/FET on the concentration of 4-ethylphenol in actual samples, chlorobenzene enrichment column sampling tubes were used to enrich the volatile organic volatile gases of healthy strawberry plants (1 and 2) and phytophthora infected strawberry plants (3 and 4), respectively. The collected organic volatile gases were analyzed using MnOEO-SWNT/FET and then mixed with 4-ethylphenol saturated steam at an equal volume of 1:1, and the results are shown in Table 1. After analysis, it can be seen that MnOEO-SWNT/FET has a small signal and false positive in the detection of strawberry health, mainly because the semiconductor gas sensor is susceptible to environmental interference. For strawberry infected plants, the detection signal of MnOEO-SWNT/FET was significantly greater than 10% 4-ethylphenol saturated vapor concentration, and the diagnosis accuracy of MnOEO-SWNT/FET was high.
表1 MnOEO-SWNT/FET检测草莓健康植株和感染植株
Table 1 Detection of healthy and infected strawberry plants by MnOEO-SWNT/FET
4
Conclusions
The organic volatile gas 4-ethylphenol is a gas marker for the diagnosis of Phytophthora chinensis infection, and its concentration is directly related to the severity of Phytophthora infection in strawberry plants. The detection of 4-ethylphenol by semiconductor FEG sensor can effectively overcome the shortcomings of traditional tissue separation, microscopic analysis, PCR amplification technology, fluorescence in situ hybridization, enzyme-linked immunoassay, etc., and has the advantages of simple operation, easy use and low cost of analysis.
In this study, the coordination metal ions of metal porphyrins were in an unsaturated state, so that the gas molecules could interact with the central metal ions at the axial position of MPs through van der Waals force and hydrogen bonding, and the sensitivity and selectivity of semiconductor single-walled carbon nanotubes were screened out, and the semiconductor field effect gas sensor was fabricated by combining with SWNT and FET. The analysis showed that MnOEO-SWNT/FET had high selectivity, sensitivity and response time for the organic volatile gas 4-ethylphenol, which could achieve an accurate diagnosis of Phytophthora infection in strawberry plants, but there was a false positive in the diagnosis of healthy strawberry plants.
In view of the fact that MnOEO-SWNT/FET is susceptible to the interference of temperature and organic volatile gases in the measured environment, it is necessary to explore the interference law of the response signal of variable temperature and multi-gas coupling to MnOEO-SWNT/FET, and propose a decoupling interference method to improve the accuracy of MnOEO-SWNT/FET in the detection of 4-ethylphenol in complex environments.
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