A Flexible Acetylcholinesterase-Modified Graphene for Chiral

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A Flexible Acetylcholinesterase-Modified Graphene for Chiral Pesticide Sensor Yunpeng Zhang, Xiaotong Liu, Shi Qiu, Qiuqi Zhang, Wei Tang, Hongtao Liu, Yunlong Guo, Yongqiang Ma, Xiaojun Guo, and Yunqi Liu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b05724 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 26, 2019

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Journal of the American Chemical Society

A Flexible Acetylcholinesterase-Modified Graphene for Chiral Pesticide Sensor Yunpeng Zhang,†‡ Xiaotong Liu, †‡ Shi Qiu,§ Qiuqi Zhang,§ Wei Tang,§ Hongtao Liu, † Yunlong Guo, †,* Yongqiang Ma, ‡,* Xiaojun Guo, §,* and Yunqi Liu†,* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡ College of Science, China Agricultural University, Beijing 100193, P. R. China. § Department of Electronic Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai 200240, P. R. China. KEYWORDS: Flexible, Acetylcholinesterase, Graphene, Chiral sensor †

ABSTRACT: Sensors based on graphene are promising devices for chemical and biological detection owing to their high sensitivity, biocompatibility, and low costs. However, for chiral recognition, which is very important in biological systems, graphene sensors remain unable to discriminate enantiomers. Here, using chiral pesticide molecules as an example, we realized a highly sensitive graphene chiral sensor by modification with acetylcholinesterase (AChE). Quantum chemical simulations indicate that the inhibition effect of the enantiomer on AChE was transferred to graphene, which allowed for the electrical detection of chiral molecules. Under an operating voltage of 1 V, the sensitivity of the device reached 0.34 μg/L and 0.32 μg/L for (+)/(-)methamidophos, respectively, which is much higher than by circular dichroism (6.90 mg/L and 5.16 mg/L, respectively). Furthermore, real-time, rapid detection was realized by combining with smartphones and wireless transmission.

1. INTRODUCTION Graphene, a single-atom-thick layer with a two-dimensional hexagonal honeycomb structure, has drawn tremendous attention for electronic devices, energy conversion, and storage devices.1-7 In fact, combining excellent electrical conductivity with its biocompatibility and ultrahigh surface area (calculated value, 2630 m2/g) has made graphene a suitable candidate for high-performance sensors.8-12 For producing graphene, several methods have been developed including chemical reduction, thermal reduction, and electrochemical reduction of GO (ERGO). 13-15Among these methods, the ERGO method is one of the most promising for preparing RGO as it is simple, “green,” and scalable. Furthermore, unreduced oxygen-containing functional groups endow the ERGO surface with a certain hydrophilicity, making it a good candidate for immobilizing biomolecules for sensing.16 -19 For biochemical studies, chirality is one of the significant signatures in life since many chemicals and macromolecular substances in biological systems have intrinsic chiral preference. The only difference in the structures of these chiral compounds is that they are mirror images.20 In fact, enantiomers are known to show dramatically different biological and physiological effects; for example, one enantiomer may be physiologically active while the other is inactive or even toxic.21,22 Such enantiomeric selectivity also occurs in chiral pesticides, resulting in significantly different biological activities.22-27 However, this enantiomeric selectivity of chiral pesticides affects not only their insecticidal activities to targeted organisms but also their toxicity to non-targeted organisms, including mammals and human. Numerous chiral pesticides have been used to prevent insect pests or weeds in fruits, vegetables, and other crops. Since it is important for most chiral pesticides to be marketed in their racemic forms (an equimolar mixture of enantiomers),

the chiral detection of pesticides is also critical. Currently, the detection of enantiomers is mostly carried out using expensive and bulky equipment such as circular dichroism (CD) spectrometers or chromatography with chiral columns, which was unable to meet demands for on-line monitoring and rapid determination.28-30 Fast chiral discrimination has been a great challenge in scientific research as pairs of enantiomers have almost the same physical and chemical properties. Recently, electronic sensors based on field-effect transistor (FET) or chemical resistor configurations with the promise of low costs, fast responses, and high sensitivity have gained significant interest for chemical, biological, and physical monitoring.31-36 In 2008, Torsi et al. reported a high-sensitivity chiral sensor based on FETs using chiral bilayer oligomers as semiconductors for chiral sensing.37 Vapor sensors for the distinction of limonene and pinene enantiomers were reported by Kybert.38 Torsi group also measured the weak interactions associated with neutral enantiomers differentially binding to Odorant Binding Protein by an organic bio-electronic transistor.39 The latest report shows that Zhang et al. could distinguish many different chiral compounds by using an ingenious origami hierarchical sensor array.40 However, for the chiral discrimination of chiral pesticides in solution, chiral compounds as recognition elements for sensing was lack of theoretical investigation, and the synthesis of chiral semiconductors was much difficult. Therefore, if we can find an easy way to achieve real-time, low-cost, and highsensitivity chiral sensors in solution, it will be great contributions to bioelectronics. 2. RESULTS AND DISCUSSIONS 2.1 Fabrication and Electrical Properties of Sensors for Chiral Methamidophos Here, we demonstrate a flexible, low-voltage, and ultrasensitive chiral sensor using ERGO sheets immobilized with

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acetylcholinesterase (AChE) for chiral detection of (+)/(-)methamidophos. Methamidophos [(RS)-O, S-dimethyl phosphoramidothioate], a widely used organophosphorus pesticide, comprises a pair of enantiomers with an asymmetric center at the phosphorus atom (Figure 1).41 In previous reports, the two enantiomers of methamidophos were found to have significantly different inhibitory effects on AChE.23,40,42 Hence, considering the above-mentioned enantiomeric selectivity of chiral pesticides toward biological activities, AChE was chosen as the recognition element in sensors for the chiral discrimination of methamidophos.

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cleaned with PBS buffer until its resistance returned to a previous reference value to ensure the validity of the test. After the current of the AChE-ERGO sensors reached a balance by washing with PBS, we recorded and normalized it as a reference for the next test. Upon the addition of the target enantiomer (-)-methamidophos, the resistance of the devices further decreased due to the interactions between the enantiomer and the AChE attached to the ERGO. The sensor response (S), defined by the relative resistance change, was calculated by the following equation: S=R/R0= [(Rsensor  R0)/R0]100%, where R0 is the initial resistance of the sensor and Rsensor is the measured resistance after exposure to enantiomers. As AChE was intended for a selective recognition element for the chiral discrimination of (+)/(-)-methamidophos, the response of AChE-ERGO sensors after exposure to the two enantiomers of methamidophos would be changed differently. The results of (+)-methamidophos for sensing are displayed in Figure S3b.

Figure 1. Flexible sensor array, schematic of one acetylcholinesterase-modified electrochemically reduced graphene oxide (AChE-ERGO) sensor, and the chemical structure of the methamidophos enantiomers. Figure 1 shows the large area flexible array of RGO chiral sensors, the schematic of the AChE-ERGO sensor, and the chemical structure of the methamidophos enantiomers. The separated enantiomers were distinguished via the positive and negative peaks in Circular Dichroism (CD) spectra (shown in Figure 2a). To fabricate the sensor, an electrochemical method for patterning and reducing GO was applied, and the mechanism of the electrochemical process for GO can be explained by the equation: GO + H+ + e- = RGO + H2O, where H+ and e- are provided by the carboxylic acid groups of the GO solution and cathode, respectively. The Raman spectra of GO and ERGO are shown in Figure 2b. A D band at approximately 1350 cm-1 and G band at approximately 1590 cm-1 can be observed in GO spectrum. Then, after reduction, the Raman intensity ratio of ID/IG was increased due to the defectsintroduced into the ERGO, which confirmed the formation of ERGO.43 The morphologies of ERGO were characterized by atomic force microscopy (AFM). As shown in Figure 2c, the flake-like shape of ERGO in the patterned channel, and Figure 2d shows the AFM image of ERGO after immobilization of AChE. Based on the infrared spectrum (Figure S1), we confirmed the existence of residual groups such as hydroxyl groups. These can bind to the amino group of AChE and stabilize it on the ERGO surface during PBS washing. The small dots with a distribution about 40/μm2 that can be clearly seen on the flake-like ERGO are AChE.44 The height of the dots ranged from 5 nm to 7 nm, which is similar to the size of a single AChE molecule (Figure S2). Furthermore, a device based on ERGO was prepared, and typical sensor response curves with the addition of AChE, PBS, and various concentrations of (-)-methamidophos from 1 μg/L to 10 mg/L are shown in Figure S3a. All the devices were operated at 1 V. Linear I-V curves of ERGO before and after the introduction of AChE were observed, which indicated good Ohmic behavior. The immobilization of AChE on ERGO was evidenced by a decrease in current, which was consistent with the AFM images. Before each test, the device was

Figure 2. Characteristics of GO, ERGO and AChE-ERGO. (a) CD spectra of (+)/(-)-methamidophos at a concentration of 200 mg/L. (b) Raman spectra of GO and ERGO. (c) AFM image of ERGO in channel. (d) Morphology of ERGO after immobilization of AChE (about 5-7 nm) in channel. The scales of AFM images are both 5 μm× 5 μm. Figure 3a shows the sensor responses to the increasing concentrations of the two enantiomers, clear discrimination could be observed from the results. Each concentration was tested on 5 different devices. And linear equations of (+)/(-)methamidophos were △R/R = 4.12 lg[c/μg L-1] + 16.36 and 5.94 lg[c/μg L-1] + 23.99 with the correlation coefficients of 0.9934 and 0.9993, respectively. The limits of detection (LOD) of (+)/(-)-methamidophos were 0.34 μg/L and 0.32 μg/L (based on 3 times the signal-to-noise ratio), respectively. Furthermore, in order to determine the effects of AChE on the graphene chiral detectors, ERGO sensor without AChE was prepared. The I-V characteristics of the ERGO sensor with different concentrations of (+)/(-)-methamidophos are shown in Figure S3d and Figure S3e. Although ERGO sensors showed a good response to organophosphorus pesticides,45 the detection and discrimination of (+)/(-)-methamidophos were largely uncertain. The responses of ERGO sensors to both enantiomers are shown in Figure 3b. There was no significant different in the slopes of the calibration curves for the two


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Journal of the American Chemical Society enantiomers. The results show that the ERGO sensor could not distinguish the two enantiomers of chiral methamidophos. Thus, AChE as a selective recognition element plays an important role in the chiral discrimination of (+)/(-)methamidophos. Moreover, to verify the sensitivity of the sensor, we compared the above results of AChE- ERGO sensor against CD spectral detection, which is a commonly accepted method for detecting enantiomers. The CD spectra of (+)/(-)-methamidophos are shown in Figure 3c, and the detection wavelength of CD spectra was set at 216 nm. A good linear response was obtained over the (+)/(-)-methamidophos concentration range from 1 mg/L to 200 mg/L with regression equations of y = 0.2664x - 0.1504 (R = 0.9983) and y = 0.2891x + 0.1974 (R = 0.9999) for (+)/(-)-methamidophos, respectively. However, the LOD of (+)/(-)-methamidophos were estimated to be 6.90 mg/L and 5.16 mg/L, respectively, which are much higher than the LODs of the AChE-ERGO sensor (0.34/0.32 μg/L). After bending 90° of our sensors, the test results have no special changes compared to as prepare one. Figure 3d shows the separated signals of the CD spectra and AChE-ERGO sensor for distinguishing (+)/(-)-methamidophos. Separation signal (average value) is defined as S(-)– S(+), where S(-) is signal for (-)-methamidophos and S(+) is signal of (+)methamidophos. It is clearly to see the AChE-ERGO sensor displays a much higher sensitivity than CD spectra at concentrations below 10 mg/mL. Therefore, AChE-ERGO sensor might be promising for the low-cost and highsensitivity detection of (+)/(-)-methamidophos.

In order to understand this process, a possible sensing mechanism of AChE-ERGO sensor toward (+)/(-)methamidophos was discussed as following. As chiral sensing has been reported by using other materials like DNA, conjugated oligomers, chiral helicene as chiral recognition.37,38,46 These mechanisms of chiral discrimination were attributed to the stereospecific interaction between the chiral materials and target enantiomers.47 In this research, the chiral sensing mechanism was considered to be the stereospecific interaction between AChE and (+)/(-)methamidophos. Therefore, we docked methamidophos back into the active site with the help of theoretical calculations (Figure 4a). The stereospecific interactions between AChE and different conformations of the methamidophos molecule were calculated using the Discovery studio 2.5. The AChE conformation, was searched from the protein databank, and the active amino acid residues for methamidophos were located by ligand expansion.48-50 Then, pesticide molecule (ligand) and receptor were soft docked by the CHARMm. The flexible ligand could be matched to the active site of AChE (see Figure S4). In the presence of ligands, the amino acid chain of the active site was optimized by Chi Rotor. After docking was complete, CDOCKER was used to evaluate the final simulation. From the result of docking, we can see that (-)methamidophos had interactions with ser200, gly118, and glu199 through hydrogen bonding, electrostatic, or Van der Waals forces. Although the (+)-methamidophos had interactions with try121, ser122, and ser81, these are not the major bonding sites for destroying AChE.51,52 Combination with the score given by the scoring program and experimental results, we can deduce that the (-)-methamidophos is more easily matched to AChE than (+)-methamidophos in vitro and thus might transfer a stronger signal to ERGO.

Figure 3. Relationships between different concentrations of methamidophos and resistance of different sensors. (a) Response of AChE-ERGO sensor to (+)/(-)/(±)methamidophos ranged from 1 μg/L to 10 mg/L. Each data point is the average values with five replicates. (b) Resistance changes of sensors based on only-ERGO active layers exposed to different concentrations of (+)-methamidophos,(-)methamidophos, and (±)-methamidophos. (c) Detection of (+)/(-)-methamidophos ranged from 1 mg/L to 200 mg/L by CD. Each data point is the average values with five replicates. (d) Relationship between concentration and separation signal based on CD spectra and AChE-ERGO sensor. Separation signal is defined as S(-)– S(+), where S(-) is signal of (-)methamidophos (average value) and S(+) is signal of (+)methamidophos (average value). 2.2 Mechanism Analysis

Figure 4. Mechanism for AChE and AChE-ERGO sensor working for chiral methamidophos. (a) The interaction


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between AChE and methamidophos. The arrow represents the hydrogen bonding, and direction of the arrow points to the electron donors, green is Van der Waals forces, violet is electrostatic forces. (b) The inhibition mechanism of AChE toward methamidophos. (c) The detection mechanism of AChE-ERGO sensor. The enantioselective inhibition of methamidophos toward AChE was primarily due to the preferential binding of (-)enantiomer compared with (+)-enantiomer. Further, the inhibition could be chemically explained by methamidophos binding to the serine residue of AChE to form the AChEmethamidophos complex, for which the equation is shown in Figure 4b, where Enz-OH is AChE.36 According to previous studies, CH3S- is believed to be the leaving group during the reaction between AChE and methamidophos.53 For graphene sensor, the mechanism for detecting Hg2+, NH3, or NO2 is usually considered to involve electron transfer between target analyte and RGO, which results the change in RGO conductance due to donor or acceptor effects.54-56 In this research, first, a decreased current was observed for the ERGO sheet after the immobilization of AChE, which means the strong electron donor effects from AChE to RGO.10 From the chemical structure of (+)/(-)-methamidophos, we know that it is a strong donor, which is also demonstrated by the RGO sensor without AChE modification. Second, for distinguishing chiral (+)/(-)-methamidophos, (-)-methamidophos associated more readily with AChE than (+)-methamidophos both physically and chemically. In order to clarify the relationship between the three components, we performed a further analysis of our experiment. As shown in Figure S5, the effect of AChE on the device resistance is visually observable. Obviously, we can see that both enzymes and isomers have a strong electron donor effect to ERGO, causing an increase in device resistance.10 As a result, the resistance of AChE-ERGO sensor after inducing with (+)/(-)-methamidophos show different values (shown in Figure 4c).

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different concentrations of methamidophos, resulting in different test currents. The device cleaned with PBS buffer was used as a benchmark and compared with the device with different concentrations of isomers, which eventually translates into real-time online data, where the change in device resistance is reflected in the form of ratios on the screen of the mobile phone. Note that the current displayed on the phone is the lg of the real value. The formulas for calculations are as follows: S=R/R0= [(Rsensor  R0)/R0]100%, Rsensor=1/10n, where R0 is the initial resistance of the sensor, Rsensor is the measured resistance after exposure to enantiomers, and n is the lg (Isensor). As show in Figure S6, the miniaturized test PCB platform can repeat the previous results and effectively distinguish the two chiral isomers of methamidophos. The linear equations of (+)/(-)-methamidophos are △R/R0 =37.64 lg[c/μg L-1] + 159.13 and 60.15 lg[c/μg L-1] + 240.34 with the correlation coefficients of 0.9879 and 0.9796, respectively. Thus, wireless chiral detection by smart phone was realized that can clearly provide different signals between (+) and (-)methamidophos, which is consistent with the data from Keithley 4200 SCS analysis system. 2.4 Effects of Various Chiral Pesticides on ERGO-AChE Sensors In order to further investigate the ability of ERGO-AChE sensors, we chose two additional types of target pesticides. One is acephate, which is similar in structure to methamidophos. Compared with methamidophos, the interaction between acetaphate and AChE is weaker, and the toxicity to mammals is lower.60 The other one is indoxacarb, which is completely different from the structure of methamidophos, is widely used as a sodium ion channel inhibitor to kill insects, and has much less of an effect on AChE than the other two.61-63

2.3 Wireless System for Chiral Pesticides

Figure 5. Portable sensor response of AChE-ERGO sensor to (+)/(-)-methamidophos ranging from 1 μg/L to 10 mg/L. Detector miniaturization and portability afford many advantages such as real-time online monitoring and low energy consumption.57,58 Based on our above experiments, we built a new, portable methamidophos detector using a printed circuit board (PCB) and smart phone via Bluetooth.59 As shown in Figure 5, with power from lithium batteries, programs on the PCB can provide a scan voltage of 03.3 V and a resistor was added to the circuit to maintain 1 V for the graphene sensor. The resistance of the device varies with

Figure 6. Resistance changes of sensors based on AChEERGO and only-ERGO active layers exposed to different concentrations of analytes. (a), (c) Response of AChE-ERGO sensor to (+)/(-)/(±)-acephate and indoxacarb ranging from 1 μg/L to 10 mg/L. (c), (d) Response of ERGO sensor to (+)/(-)/(±)-acephate and indoxacarb ranging from 1 μg/L to 10 mg/L. Each data point is an average of five replicates. Comparing Figure 6a and Figure 6b, we can see that AChE has a significant effect on the test results. Simulations showed that (-)-acephate has interactions with ser200, gly118, and glu199. For (+)-acephate, the amino acid residues interacting


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Journal of the American Chemical Society with it is not the main enzyme active sites of enzymes, and thus it has less of an impact on the devices (Figure S7 and Figure S8). (-)-Acephate, like (-)-methamidophos, associates more easily with AChE than (+)-acephate both physically and chemically, and so the resistance of the AChE-ERGO sensor differs after exposure to (+)/(-)-acephate. Due to its weaker effect on AChE, acephate has less of an influence on device resistance at the same concentrations as methamidophos, which is consistent with previous reports.64,65 In the absence of AChE, there was little difference in device resistance, and the two isomers could not be identified. The I-V characteristics of the two kinds of sensors with different concentrations of (+)/(-)/(±)-acephate are shown in Figure S9. As shown in Figure 6c and Figure 6d, for indoxacarb, the presence and absence of AChE had little effect on the experimental results, and the two isomers could not be identified by AChE-ERGO. As shown in Figure S10 and Figure S11, molecular docking results showed that there is no strong interaction between indoxacarb and AChE active sites like ser200 or gly118. Although both isomers can bind to enzymes and cause changes in device resistance, they do not have a differential effect on the protein, and thus we cannot distinguish the two isomers of indoxacarb using the device. The sensing results of (+)/(-)/(±)-indoxacarb are displayed in Figure S12.

CONCLUSION A flexible graphene sensor platform was demonstrated by using AChE-ERGO sensor for the chiral discrimination of (+)/(-)-methamidophos. The AChE-ERGO sensor, which has a relatively simple fabrication process, allowed for the highselectivity and rapid off-line detection of chiral methamidophos. Based on the experimental results, the AChEERGO sensor exhibits a linear response to (+)/(-)methamidophos in the concentration range of 1 μg/L to 10 mg/L, respectively, and LOD values more than 1000 times lower than those of the commonly used CD spectral method. Due to the difference of stereospecific interaction between molecules and AChE, (+)/(-)-methamidophos have different inhibitory effects on the proteins that lead to a distinction in the number of combinations, which eventually translates into a difference in electrical signal. For weaker inhibitors, new proteins or device configurations require further exploration. In addition, we have developed a portable sensor with a smartphone application that has a suitable detection capability and resolution for on-site detection of (+)/(-)-methamidophos. Therefore, AChE-ERGO sensor might be promising for applications in environmental monitoring, food-monitoring, and water-control. Furthermore, the combination of the stereo selective inhibition of chiral pesticide toward the corresponding Target Enzyme-ERGO sensor may be used for the detection of a variety of enantiomers.

until a brown-colored slurry was formed. The suspension was washed with diluted HCl and deionized water. Finally, graphene oxide was obtained after ultrasonication. AFM images of the graphene oxide are given in Figure S13. Fabrication of Sensor Device: Gold electrodes were evaporated through a metal mask with a channel width of 4500 μm and length of 20 μm. A droplet of GO solution was pipetted onto the electrode region on the PET surface and the thickness of ERGO is 5-10 nm (Figure S14). Next, a voltage from 0 V to 4 V was applied between the electrodes for several cycles, and the surface was then dried by N2. A 0.01 U/μL AChE solution (dissolved from 500 U lyophilized power, Sigma-Aldrich) in a phosphate buffered saline (PBS, pH 7.4, Sigma-Aldrich) solution was dropped on the surface of ERGO and incubated at 4 °C for 2 h, followed by washing with PBS and drying by N2 for use. During the experiment, we placed 10 μL of the enantiomers on the surface of the device and waited 3 min, then dried the surface of the device with N2 and performed the test. The process was the same for different concentrations of enantiomers. The devices could be stored at 04 °C, and the longest use time is approximately one week. Biosensing: Before adding the target enantiomer, the devices were washed with PBS until the electrical current achieved stability. At the same time, we recorded the resistance of the device and used it as a reference. Then, the target enantiomer was introduced on the device for 3 min, after which the device was dried by N2. The change in the current of the sensor was measured at 1 V and room temperature using a semiconductor parameter analyzer (Keithley 4200 SCS). After recording the current, we cleaned the device with PBS buffer until its resistance was restored to the previous reference value, then performed the next test. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXX. Experimental details and additional figures and tables.

AUTHOR INFORMATION Corresponding Author * Y. Guo. E-mail: [email protected] * Y. Ma, E-mail: [email protected] *X. Guo, Email: [email protected] *Y. Liu, E-mail: [email protected].

Author Contributions #

Y. Z., and X. L. contributed equally to this work.

Notes The authors declare no competing financial interest

ACKNOWLEDGMENT

EXPERIMENTAL SECTION Synthesis of GO: Graphene oxide was prepared according to Hummers’ method.66 H2SO4 was added to a mixture of graphite and NaNO3 in a three-necked flask. Then, KMnO4 was slowly added to the mixture solution with stirring. The mixture was heated to 40 °C for 10 h. Next, H2O2 (30 wt.% aqueous solution) was added to the mixture and stirred for 2 h

We thank the financial support from the National Natural Science Foundation of China (Nos. 21633012, 51403177, 51233006, and 61334008), the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDB30000000) and Beijing Nova program (Z181100006218034).


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A Flexible Acetylcholinesterase-Modified Graphene for Chiral

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