gold nanostars as redox probe with catalyst

Sensors & Actuators: B. Chemical 298 (2019) 126866

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Electrochemical aptasensor for detection of acetamiprid in vegetables with graphene aerogel-glutamic acid functionalized graphene quantum dot/gold nanostars as redox probe with catalyst ⁎

⁎

T

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Chu Hongxiaa, Hu Jib, Li Zaijuna, , Li Ruiyia, , Yang Yongqiangc, , Sun Xiulana a

School of Chemical and Material Engineering, School of Pharmaceutical Science, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China School of Materials Science and Engineering, Luoyang Institute of Science and Technology, Luoyang 471023, China c National Graphene Product Quality Supervision and Inspection Center, Jiangsu Province Special Equipment Safety Supervision and Inspection Institute Branch, Wuxi 214071 b

A R T I C LE I N FO

A B S T R A C T

Keywords: Voltammetry Graphene quantum dots Gold nanoparticles Detection of acetamiprid

Glutamic acid-functionalized graphene quantum dots (Glu-GQD) was prepared by pyrolysis of citric acid and glutamic acid and used as reducing agent and stabilizer to produce Glu-GQD/Au in the presence of tannic acid. The resulting Glu-GQD/Au was covalently connected with aptamer (Apt) of acetamiprid to obtain one redox probe with catalyst. The study shows that Glu-GQD/Au offers nanostar-like structure with average particle size of 102.5 nm, composing of several nanometer-sized gold nanocrystals with the rich of sharp edges and corners. The unique structure makes gold nanostars good electrocatalytic activity, which was further improved by combination with Glu-GQD. In the redox probe, Glu-GQD can carry out reversible redox reaction on the electrode surface due to its high electroactivity. Gold nanostars in-situ catalyzes redox reactions of Glu-GQD and leads to an improved electrochemical behavior. Aptamer can specifically bind with acetamiprid and produce sensitive and selective electrochemical response. The electrochemical aptasensor based on Glu-GQD/Au-Apt/ graphene aerogel exhibits ultrahigh sensitivity and selectivity for detection of acetamiprid. The differential pulse voltammetric signal linearly decreases with increasing acetamiprid concentration in the range from 1.0 fM to 1 × 105 fM with detection limit of 0.37 fM (S/N = 3). The aptasensor has been successfully applied in electrochemical detection of acetamiprid in vegetables.

1. Introduction Neonicotinoid insecticides have been extensively applied in agriculture and household pest control because of their high insecticidal efficiency, low biological toxicity and broad-spectrum of insecticidal activity [1]. The annual sale is close to $3.4 billion that is about 20% of global pesticide consumption [2]. Acetamiprid as one insecticide of neonicotinoid group may result in paralysis and death of pests via activation of nicotinic acetylcholine receptors [3]. Due to relatively low mammalian toxicity and special acting characteristics, acetamiprid has been widely used as replacement of organophosphorus and other insecticides [4]. However, growing applications of acetamiprid in agriculture and household may generate serious food and environment pollution, which increases health risk to animals and humans [5]. Therefore, a highly reliable method for detection of acetamiprid in food is critical to ensure human health. Many technologies have been developed for determination of ⁎

acetamiprid, including fluorescence methods using copper nanoparticle [6], carbon dot [7], G-quadruplex [8], upconversion nanoparticle [9] and fluorescent quantum dot [10] as the optical probe, colorimetric methods with tetramethylbenzidine [11] and gold nanoparticle [12], surface-enhanced Raman scattering (SERS) based on gold nanoparticle [13], silver nanorod [14], Ag-coated cellophane [15] and silver dendrite [16], immunochromatographic strip [17], enzyme-linked immunosorbent assay (ELISA) [18], electrochemical method [19], high performance liquid chromatography (HPLC) [20] and gas chromatography-mass spectrometry (GC-MS) [21]. Fluorescent method is rapid, sensitive and selective. However, the method often suffers from serious matrix interference when it is used for food analysis. Many biological macromolecules in food such as proteins and nucleic acids can produce fluorescence emission under ultraviolet light radiation. This may lead to a serious interference to detection of acetamiprid from the background fluorescence. To obtain a reliable result, a complex sample pretreatment is strongly required to separate the interfering components. SERS

Corresponding authors. E-mail addresses: [email protected] (L. Zaijun), [email protected] (L. Ruiyi), [email protected] (S. Xiulan).

https://doi.org/10.1016/j.snb.2019.126866 Received 31 May 2019; Received in revised form 21 July 2019; Accepted 22 July 2019 Available online 24 July 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.

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Tris-HCl, 1 mM MgCl2, 1 mM CaCl2 and 5 mM KCl at pH 7.4, and stored at −20 °C. Graphene aerogel (GA) was synthesized by using the reported method [32]. The Glu-GQD was prepared by using a similar procedure reported in literature [38] with the ratio of citric acid and glutamic acid of 1:1. The ultra pure water (18.2 MΩ cm) purified from Milli-Q purification system was used in all experiments.

is highly sensitive and selective, but the instability of detection signal greatly limits the use of SERS method in the routine analysis. ELISA mostly exhibits a relatively poor detection limit and it was mainly used for high throughput screening of pesticide residues. HPLC and GC-MS are mostly used technologies in the routine insecticide analysis. However, these methods requires the use of expensive instruments, high pure organic solvents and high-skilled personnel [22]. In addition, HPLC and GC-MS methods also need the use of sample pretreatment step to eliminate the matrix interference. The pretreatment is not only time-consuming, but also often requires the use of a large number of organic solvents [21]. Electrochemical aptasensor received a more attention because of its low cost, rapid response, specificity recognition and high sensitivity. The development in nanotechnology has exhibited great potential of nanomaterials as basic building blocks and signaling elements for fabrication of electrochemical sensors with excellent analytical performance [23–26]. Due to unique structure and properties, graphene aerogel (GA), graphene quantum dots (GQDs) and gold nanoparticles (Au NPs) are the most important sensing materials and widely applied in the design of electrochemical sensors [27,28]. GA can offer one three-dimensional structure composing of graphene networks. Compared with common graphene, GA has a much higher electrocatalytic activity because of its excellent electron/ion conductivity [29]. In our previous work, several new strategies have been reported for making GA to improve the electrical and mechanical properties and ease of use [30–33]. Different from classical graphene, GQD is composed of small graphene sheets with the rich of functional groups. These groups may give GQDs special functions in catalysis, molecular recognition and linking with other component [34]. As one of noble metal nanomaterials, Au NPs have good electrical conductivity and special catalysis to many molecules [35]. Au NPs are also used for immobilization of biological molecules such as DNA, enzyme and protein on the electrode surface via the Au-S bond. Recently, Au-GQD, GQD-GA and Au-GQDGA hybrid were fabricated and used for fabrication of electrochemical sensors [36–38]. In the comparison to single Au NPs, GA and GQD, the hybrid exhibits a better catalytic activity. More importantly, their hybrid provides an opportunity to meet the multifaceted requirements of biosensor design. However, the present hybrid-based aptasensor still needs the use of additional redox probe to produce electrochemical signal for detection of non-electroactive analyte because the hybrid itself hasn’t the function of redox probe. This not only brings inconvenience to the practical use of aptasensor, but also may bring the interference for detection of acetamiprid because of the interaction between the redox probe and the analyte. Herein, the study reported synthesis of glutamic acid functionalized graphene quantum dot/gold hybrid (Glu-GQD/Au). The as-synthesized hybrid offers nanostar-like structure, composing of several nanometersized gold nanoparticles with the rich of sharp edges and corners and excellent redox behavior. The aptasensor based on Glu-GQD/Au/graphene aerogel provides the advantage of sensitivity, stability and ease of use compared to other electrochemical aptasensors for acetamiprid reported in literatures. The analytical method has been successfully applied in electrochemical detection of acetamiprid in vegetable.

2.2. Structural characterization Scanning electron microscope (SEM) analysis was carried out on HITACHI S4800 field emission scanning electron microscope. Transmission electron microscope (TEM) image was measured on JEOL 2010 FEG microscope. X-ray diffraction (XRD) was measured on the D8 Advance with a Cu Kα radiation. The Infrared spectrum (IR) was measured on Nicolet FT-IR 6700 spectrometer. 2.3. Glu-GQD/Au synthesis The HAuCl4 solution (25 mL, 0.2 mg mL-1) was mixed with the tannic acid solution (2 mL, 5 mg mL-1). Added the Glu-GQD solution (1.0 mL, 50 mg mL-1) and heated at 95℃ until the solution color changes to deep red. To obtain gold seed solution, the temperature was kept for 6 min and then cooled to ambient temperature. The Glu-GQD solution (60 μL, 50 mg mL-1) and the HAuCl4 solution (1.0 mL, 50 mg mL-1) were dispersed into 10 mL of ultrapure water in another vial. After the mixed solution was incubated for 5 min, the AgNO3 solution (0.12 mL, 10 mM), gold seed solution (40 μL) and H2O2 solution (10 mL, 30%) were orderly added. The solution was continued to stir for 40 s after the color of mixed solution is changed into blue. It was treated by centrifugation at 10000 rpm for 10 min. The collected Glu-GQD/Au was washed with ultra pure water for 3 times and then redispersed in the Tris-HCl buffer (pH 7.4, 1.0 mL). The obtained GluGQD/Au solution was stored at 4 °C before use. 2.4. Aptasensor preparation GA (4 mg) was dispersed in ultra pure water (10 mL) and mixed with 1% chitosan solution with volume ratio of 20:1. To obtain a GA/ GCE sensor, its 5 μL was dropped on the surface of glass carbon electrode (GCE, 1.0 mm in diameter) pretreated by using a similar procedure and then dried in N2 [37]. The Glu-GQD/Au dispersion (1.0 mg mL-1) was mixed with 1% chitosan solution with volume ratio of 20:1. Its 5 μL was dripped on the surface of GA/GCE sensor and then dried in N2. Dropped 5 μL of the aptamer solution (1.0 μM), incubated at 37 °C for 2 h, washed with the PBS (pH 7.4, 0.1 M) and finally dried in N2 in sequence. Dropped 5 μL of the MCH solution (1.0 mM) to close the electroactive sites on the surface of Glu-GQD/Au. To obtain one aptasensor, the electrode was incubated 1 h and then washed with the PBS and then dried in N2. The prepared aptasensor was stored in a refrigerator at 4 °C before use. 2.5. Electrochemical measurements Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) measurements were carried out on CHI660D electrochemical workstation (Shanghai, China). Classical three-electrode system was used for all electrochemical measurements. The Ag/AgCI electrode (saturated KCI), platinum wire, and bare or modified GCE were used as the reference electrode, counter electrode and working electrode, respectively. For the EIS measurement, the potential amplitude of ± 5 mV and frequency range of 0.01 to 105 Hz were adopted. For the DPV measurement, the DPV parameters were set to a scan rate of 4 mV s-1, 50 mV pulse amplitude, 20 ms pulse width and -0.2 V initial potential. For the acetamiprid detection, 5 μL of the acetamiprid standard solution with different concentrations or sample solution was dropped on the surface of

2. Experimental 2.1. Materials and reagents Tannic acid (TA), citric acid, glutamic acid (Glu), chloroauric acid (HAuCl4), silver nitrate (AgNO3), acetamiprid, and 6-mercaptohexan-1ol (MCH) were purchased from Sigma-Aldrich. The phosphate buffered saline (PBS, 0.01 M, pH 7.4) was prepared in the laboratory. The aptamer was synthesized and purified by Sangon Biotechnology Limited Company and its sequences is: 5’-SH-(CH2)6TGT AAT TTG TCT GCA GCG GTT CTT GAT CGC TGA CAC CAT ATT ATG AAG A-3’. The probe stock solution was prepared in the Tris/Mg/K buffer containing 20 mM 2

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Fig. 1. Synthesis procedure (A), SEM (B), TEM (C), HRTEM (D), EDS layered electron images (E) and elemental mappings of Au (F) and N (G) of the as-synthesized Glu-GQD/Au.

acid, Glu-GQDs are fixed on the surface of gold nanostars by coordination bonds between functional groups in the Glu-GQD such as –OH, -NH2 and –COOH and gold ions on the gold nanostar surface. Different from Glu-GQD, tannic acid is one compound containing many ortho-phenolic hydroxyl groups. In one tannic acid molecule phenolic hydroxyl groups may react with different Au3+ ions to produce the multicenter metal complex. Thus, tannic acid was selected as the functional reagent for making Au nanocrystals with micro/nanostructure. The experiment shows that color of the mixed HAuCl4 with tannic acid changes from light yellow into deep red after added GluGQD, indicating the formation of Au seeds. The as-prepared Au seeds were introduced into the mixed HAuCl4 solution with Glu-GQD to carry out growth of Au nanocrystals. Our investigation also demonstrates that AgNO3 and H2O2 play important roles in improving the growth of Au

aptasensor. The aptasensor was incubated for 1 h, washed with the PBS and dried by N2. Then, the aptasensor was immersed in 10 mM PBS (pH 7.4) and its DPV curves were recorded between -0.3 V and 0.5 V. 3. Results and discussion 3.1. Material synthesis and characterization Glu-GQD/Au was synthesized by using one seed growth method (shown in Fig. 1A). Firstly, HAuCl4 was reduced into Au0 to form Au seeds in the presence of tannic acid and Glu-GQDs. In the step, the GluGQD was used as the reducing agent to change Au3+ into Au0, finally leading to the formation of Au seeds. Furthermore, the Glu-GQD acts as the stabilizer for the formation of gold nanostars. Similar with citric 3

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and bands should be mainly attributed to the Glu-GQD in the hybrid.

seeds and the formation of gold nanostars [39,40]. To investigate the growth behavior of Au nanocrystals, the changes of the growth solution in UV absorption and fluorescence were monitored after added the Au seeds. In the first 1 min, the UV absorption rapidly increases with the prolongation of reaction time. In the growth solution, only gold nanocrystals can produce a special and strong UV absorption. Thus, the UV absorption of the growth solution depends on the amounts of Au nanocrystals in the growth solution. For the above reason, the change in UV absorption can be used for monitoring the formation of Au nanocrystals during the growth of Au seeds. The investigation shows that the increase slows down and tends to stabilize when the time is more than 1.0 min. This is because the growth of Au seeds is close to complete. Interestingly, the fluorescence intensity of Glu-GQDs reduces with the increase of time. As Au nanocrystal has excellent electrical conductivity and Glu-GQD is a typical semiconductor material, their hybrid leads to an ultrafast energy transfer from Glu-GQD to Au nanocrystals in the interface. This causes a fluorescence quenching process of Glu-GQD. With the increase of time, more Glu-GQDs are combined with Au nanocrystals to form more GluGQD/Au hybrids and brings more fluorescence quenching. When the reaction time is more than 1.0 min, the fluorescence intensity of GluGQD quickly reaches to the minimum, verifying that the growth of Au seeds is nearly complete again. The as-synthesized Glu-GQD/Au was characterized by SEM, TEM, XRD and IR techniques. The results of SEM and TEM analysis reveal that the Glu-GQD/Au gives a star-like nanostructure with mean particle size of 102.5 nm, composing of several nanometer-sized Au nanoparticles with the rich of sharp edges and corners. High resolution TEM (HRTEM) indicates a 0.23 nm of interplanar spacing, corresponding to (111) plane of Au nanocrystals. The elemental mappings show the spatial distributions of gold (Au) and nitrogen elements (N) in the Glu-GQD/ Au. Both Au and N have an uniform distribution, verifying that our synthesis achieves to hybrid of Glu-GQD and gold nanocrystals. Fig. 2 presents the XRD pattern and IR spectrum of Glu-GQD/Au. On the XRD pattern there are five diffraction peaks at 38.2°, 44.4°, 66.1°, 78.4° and 81.6°. These peaks are corresponded to the (111), (200), (220), (311) and (222) planes of Au nanocrystals, respectively. As the Glu-GQD offers poor crystallization, the characteristic XRD peak of graphene at 26° is so weak that it can not be observed from Fig. 2A. On the IR spectrum there are six main IR absorption peaks and bands. The absorption band of 3300-3600 cm-1 is the IR absorption caused by symmetric stretching vibration of the O-H and N-H bonds. The absorption peak at 2909 cm-1 is due to symmetric stretching vibration of the saturated C-H bonds. The peak at 1700 cm-1 is the IR absorption of stretch vibration of -C = O bond. The peak at 1380 cm-1 is the IR absorption of the stretching vibration of C-O bond. Because Au nanocrystals have obvious IR absorption between 600 and 4000 cm-1, the above IR absorption peaks

3.2. Electrochemical performance The CV behavior of Glu-GQD and Glu-GQD/Au were investigated in the PBS of pH 7.0. Fig. 3A indicates that the CV curve of Glu-GQD offers one pair of redox peaks at -0.02/0.12 V. This is due to the conversions between oxidation states of nitrogen-containing functional groups in Glu-GQD [41]. Compared with Glu-GQD, Glu-GQD/Au gives a much bigger peak current. The result confirms that the Au nanostars provide a significant electrocatalytic activity to the redox of Glu-GQD. To evaluate the catalytic activity of Glu-GQD/Au, another two kinds of Au nanomaterials, including common Au nanoparticles (Au NPs) and Au nanostars (Au NSs), were synthesized by using ascorbic acid as the reducing agent and stabilizer without and with tannic acid. Fig. 3B presents the SERS spectra (B) of Rhodamine 6G-modified with Au NPs, Au NSs and Glu-GQD/Au. It can be seen from Fig. 3B that three goldbased materials display different SERS behaviors. The Au NSs brings a much stronger SERS signal than the Au NPs. This is because the introduction of tannic acid during synthesis of Au NSs achieves to the interconnection of different Au nanocrystals. The interconnection greatly reduces the distance between Au nanocrystals, leading to an increased electrical conductivity. If the Au NSs were replaced by GluGQD/Au, the SERS intensity will greatly increase. The result demonstrates that the introduction of Glu-GQD improves the SERS behavior. Compared with Au NSs, Glu-GQD/Au offers a smaller particle size and spiny surface. The small size makes the Au atoms are exposed outside, resulting in more active sites. The spiny surface create a much stronger localized surface plasmon resonance compared to smooth appearance. These factors lead to a much stronger SERS signal. The results of SERS analysis further demonstrates that the Glu-GQD/Au can provide an excellent electrocatalytic activity. The above characteristics determine that the Glu-GQD/Au can be used as one redox probe with catalyst for fabrication of electrochemical sensors. 3.3. Aptasensor fabrication for detection of acetamiprid The as-synthesized Glu-GQD/Au was used for fabrication of electrochemical aptasensor for detection of acetamiprid (shown in Fig. 4). In the scheme, GA is firstly modified on the GCE surface. Here, GA acts as one base material of the modified electrode for providing one threedimensional structure interwoven by graphene networks. The unique structure is beneficial to accelerate the electron transfer via the interconnected graphene networks and electrolyte transport via the rich of porous structures [32]. Then, Glu-GQD/Au was fixed on the surface of GA/GCE. The Glu-GQD/Au was used as one redox probe with catalyst to produce sensitive electrochemical response towards acetamiprid.

Fig. 2. XRD pattern (A) and IR spectrum (B) of the as-synthesized Glu-GQD/Au. 4

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Fig. 3. A: The CV curves of Glu-GQD/GCE (a) and Glu-GQD/Au/GCE (b) in the PBS of pH 7.4 at scan rate of 50 mV s-1. B: The SERS spectra of Rhodamine 6Gmodified with Au NPs (a), Au NSs (b) and Glu-GQD/Au (C) with the excitation wavelength of 785 nm.

currents in both CV and DPV curves decrease after incubated in the acetamiprid solution. The aptamer in the aptasensor can bind with acetamiprid to form the complex. As the complex offers a poor electrical conductivity, its formation on the electrode surface reduces the electrical conductivity of the modification layer. Furthermore, the complex formation will block the part of channels for electrolyte transport between GCE surface and electrolyte solution. The above two factors leads to obvious reduce in the CV peak current. The electrochemical response can be used for detection of trace acetamiprid. In addition, Fig. 6B also indicates that the DPV offers a more sensitive response to acetamiprid compared with CV. Therefore, DPV technology was employed for electrochemical determination of acetamiprid.

Due to an excellent redox behavior of Glu-GQD/Au, no additional redox probe such as Fe(CN)64+ was required for electrochemical detection of non-electroactive acetamiprid. This avoids the interference come from the interaction between the redox probe and the acetamiprid. Finally, DNA probe (aptamer) was firmly immobilized on the surface of GA/ Glu-GQD/Au/GCE via the Au-S bonds between the Au nanostars and the DNA prove. The introduction of aptamer makes the sensor can produce a specific recognition towards acetamiprid to improve the selectivity. To avoid the nonspecific recognition, 6-mercaptohexan-1-ol (MCH) was used for blocking electroactive sites on the surface of gold nanostars. To understand the effect of different materials on the electrochemical properties, the CV and EIS behaviors of five modified electrodes were investigated in 1 mM of K4Fe(CN)6 in a PBS of pH 7.4. Fig. 5 shows that the bare GCE gives the smallest CV peak currents and the biggest charge transfer impedance (Rct) among all electrodes. After modified with GA, the peak current obviously increases on the CV curve and the Rct reduce on the EIS curve. This is due to high electron/ion conductivity of GA. The modification with Glu-GQD/Au further increases the CV peak current and reduce in the Rct on the EIS curve. On the one hand, the Au nanostar in the hybrid enhance the electron transfer rate in the modification layer due to its high electrical conductivity. On the other hand, the Au nanostars offer excellent catalytic activity towards Fe(CN)64-. The above two factors greatly accelerate the electrode reaction, leading to an increased peak current and reduced Rct value. However, the immobilization of DNA probe (aptamer) results in an decreased peak current and increased Rct. This is because the aptamer has poor electrical conductivity. In addition, the formation of AuS bonds on the Au surface reduces the electroactive sites on the surface of Glu-GQD/Au. These will decrease the catalytic activity of Au towards Fe(CN)64- and leads to decrease in the CV peak current and increase in the Rct value. For the same reason, the blocking of Glu-GQD/Au surface with MCH largely decreases the CV peak current and increases the Rct value. To test the electrochemical response to acetamiprid, the CV and DPV behaviors of as-proposed aptasensor were studied before and after the incubation in the acetamiprid solution. As shown in Fig. 6, the peak

3.4. Condition optimization To optimize the conditions for detection of acetamiprid, the influence of incubation time and aptamer concentration on the change (ΔIp) in the DPV peak current of the as-proposed aptasensor were studied in the PBS of pH 7.4. Fig. 7A indicates that the ΔIp increases with increasing the incubation time if the time is less than 60 min. This is because more acetamiprid molecules are captured by acetamiprid on the surface of aptasensor with the increase of incubation time, leading to an increased ΔIp value. The value reaches to the maximum at the incubation time of 60 min and then remains almost constant, verifying that the capture process for acetamiprid has completed. To obtain a high sensitivity and save the analysis time, 60 min of the incubation time was selected for electrochemical determination of acetamiprid. Fig. 7B presents the relationship curve of ΔIp value with the DNA probe (aptamer) concentration with the incubation time of 60 min. It can see from Fig. 7B that the ΔIp value rapidly increases with increasing the DNA probe concentration. As the DNA probe increases more acetamiprid molecules can be captured by the DNA probes. This will cause a bigger reduce in the DPV peak current, indicating an increased ΔIp value. The ΔIp value reaches the maximum value when the DNA probe concentration is more than 1.0 μM, verifying that the number of DNA probes is large enough to capture all molecules in the solution. Consequently, continuing to increase the DNA probe concentration will not

Fig. 4. Scheme for fabrication of aptasensor for detection of acetamiprid. 5

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Fig. 5. CV (A) at scan rate of 50 mV s-1 and EIS curves (B) of bare GCE (a), GA/GCE (b), GA/Glu-GQD/Au/GCE (c), aptamer/GA/Glu-GQD/Au/GCE (d) and MCH/ aptamer/Glu-GQD/Au/GCE (e) in 1.0 mM K4Fe(CN)6 in the PBS of pH 7.

Fig. 6. The CV curves (A) at scan rate of 50 mV s-1 and DPV curves (B) of the as-prepared aptasensor in the PBS of pH 7.4 after (a) and before (b) incubated in 1.0 × 10-12 M acetamiprid.

Fig. 7. Effects of the incubation time and DNA probe concentration on the DPV response of the aptasensor for 2.0 × 10-14 M acetamiprid in the PBS of pH 7.4.

6

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Fig. 8. A: The DPV curves of aptasensor incubated in 0, 1.0 × 10-15, 2.0 × 10-15, 4.0 × 10-15, 1.0 × 10-14, 2.0 × 10-14, 4.0 × 1014 , 1.0 × 10-13, 4.0 × 10-13, 1.0 × 10-12, 4.0 × 10-11 and 4.0 × 10-12,1.0 × 10-11, 1.0 × 10-10 M acetamiprid solution (from top to bottom). B: The relationship curve of DPV current with logarithm of acetamiprid concentration.

Table 1 Sensitivity comparison of different analytical techniques for acetamiprid detection. Sensing materials

Analytical method

Liner range (pM)

LOD (pM)

Ref.

Au/MWCNT-rGONR Au-Cu MOF Si NPs Au NPS ZnS:Mn QD Au NPs CdTe QDs, Au NRs, (MWCNTs/rGONRs) AuNPs Cu NP TiO 2 Glu-GQD/Au/GA

Electrochemical impedance spectroscopy Differential pulse voltammetry Differential pulse voltammetry Fluorescence Fluorescence Electrochemiluminescence Electrochemiluminescence Electrochemical impedance spectroscopy Fluorescence Differential pulse anodic stripping voltammetry(DPASV) Differential pulse voltammetry

0.05 - 10000000 0.01- 10000 500- 650000 0.4- 700000 0-150000 800-630000 0.5- 1000000 5000- 600000 5000-500000 10000-2000000 0.001-1000

17 0.0029 153 127 700 62 0.2 1000 2370 200 0.0003

[42] [43] [44] [45] [46] [47] [48] [49] [50] [51] This work

was tested by varying acetamiprid concentration. Fig. 8 presents DPV curves of the aptasensor incubated in different concentrations of acetamiprid and relationship curve of the DPV peak current (Ip) with the logarithm of acetamiprid concentration. With the increase of acetamiprid the DPV response will rapidly reduce. In Fig. 8B the Ip value shows a good linear relationship with logarithm of acetamiprid concentration in the range of 1.0-1 × 105 fM. The corresponding regression equation is: Ip(nA)=-347.13×Log [Cacetamiprid, fM]- 2004.6, with the correlation coefficient of 0.997. Based on the signal/standard deviation of 3 times, the limit of detection (LOD) was found to be 0.37 fM for acetamiprid detection. The sensitivity is much better than that of other analytical technologies for acetamiprid in Table 1. An ultra high sensitivity is mainly attributed to an excellent catalytic activity of GluGQD/Au. Furthermore, the reproducibility of aptasensor was tested via the detection of 100 fM acetamiprid. Fifty duplicate measurement gives a 1.9% of relative standard deviation, verifying high reproducibility. The stability of aptasensor was studied by storing the aptasensor in 4 °C for two weeks. The DPV response towards 100 fM acetamiprid keeps at least 98.4%, indicating high stability. To study on the selectivity of the as-proposed analytical method, the effects of ten insecticides as their mixture on the DPV response of the aptasensor were investigated, respectively. These insecticides are commonly used in vegetable and fruit productions and may coexist in the same sample, including carbendazim (CAR), methamidophos (MET), beta-cypermethrin (CYP), aldicarb (ALD), clothianidin (CLO), chlorpyrifos (CHL), atrazine (ATR), pyridoxine (PYR), thiamethoxam (THI) and acetamiprid (ACE). Fig. 9 indicates that only acetamiprid caused an obvious increase in the DPV signal among all insecticides tested. This verifies that the as-proposed method has an excellent selectivity. This is due to the specificity recognition of DNA probes in the aptasensor towards acetamiprid. As other insecticides can not be bound with the DNA probe to form the complex, their existence doesn’t change

Fig. 9. The change (ΔIp) in DPV peak current of the aptasensor caused by 20 fM of CAR, MET, CYP, ALD, CLO, CHL, ATR, PYR, THI or ACE as well as their mixture.

bring any change in the ΔIp value. To achieve to a high sensitivity, 1.0 μM of the DNA probe was used for making aptasensor.

3.5. Analytical characteristics DPV behavior of the as-proposed aptasensor towards acetamiprid 7

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Project (B13025).

Table 2 The results for detection of acetamiprid in vegetables (N = 5). Samples

Acetamiprid added (fM)

Acetamiprid found (fM)

Recovery (%)

RSD (%)

cucumber

0.0 50.0 0.0 50.0 0.0 50.0 0.0 50.0

1.75 ± 0.15 52.02 ± 0.77 0.19 ± 0.02 50.11 ± 0.41 2.83 ± 0.22 51.77 ± 0.90 0.86 ± 0.14 50.79 ± 1.06

100.5

2.43 1.82 3.17 2.21 1.65 1.06 1.92 1.22

tomato spinach Green beans

99.8 101.8 103.1

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the DPV signal. Furthermore, Fig. 9 also shows that the DPV response incubated with single acetamiprid is similar with that incubated with the mixture of ten insecticides. The result demonstrates that there are no strong interaction between acetamiprid and other nine insecticides. Thus, the co-existence of other nine insecticides cannot interfere with the electrochemical detection of acetamiprid. 3.6. Sample analysis The as-proposed aptasensor has been used for electrochemical detection of acetamiprid in vegetable. All vegetable samples were purchased from farmers markets in Wuxi city. Firstly, vegetable sample was chopped for 10 min using the conventional food processor. After washed three times with ultra pure water, it was placed in a centrifuge tube. Followed by addition of 10.0 mL acetonitrile, vigorous shaking for 2 min and centrifugation at 4000 rpm for 10 min. The collected supernatant was filtered via 0.45 μm of membrane and transferred to a 10 mL volumetric flask. The sample solution was used for electrochemical detection of acetamiprid. In addition, the spiked recovery experiment was also completed by using the same procedure unless adding a known acetamiprid standard solution into the supernatant. Table 2 shows that the acetamiprid recoveries is between 97.6 and 103.1%. The result confirms that the as-proposed analytical method has an acceptable accuracy. 4. Conclusions The study has demonstrated synthesis of glutamic acid-functionalized graphene quantum dot-gold hybrid and the application in electrochemical detection of trace acetamiprid. The graphene quantum dot acts as both reducing agent and stabilizer for formation of gold nanocrystals. The as-synthesized hybrid offers a nanostar-like structure, composing of several nanometer-sized graphene sheets and gold nanoparticles with the rich of sharp edges and corners. The unique structure makes the gold nanoparticle excellent electrocatalytic activity. Interestingly, the graphene quantum dots becomes an electroactive material due to the introduction of glutamic acid. The gold nanostars in the hybrid in-situ catalyzes the redox reactions of the graphene quantum dots and results in an obviously improved the electrochemical behavior. The hybrid coupled with graphene aerogel and aptamer was used as the redox probe and electro catalyst for constructing the acetamiprid aptasensor. The as-proposed aptasensor gives ultra high sensitivity and selectivity for detection of acetamiprid in vegetables. Acknowledgements The authors acknowledge the financial support from The National Key Research and Development Program of China (No.2018YFC1603001), National Natural Science Foundation of China (No.21576115), Science and Technology Department of Henan Province of China (No.192102210184) and MOE & SAFEA for the 111 8

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Chu Hongxia is currently one master student of Professor Li Zaijun in School of Chemical and Materials Engineering, Jiangnan University, China. Her research interest is synthesis of nanomaterials and their application in electrochemical chemistry. Hu Ji is currently associate professor of School of Materials Science and Engineering, Luoyang Institute of Science and Technology, China. He received his doctor degree from Huazhong University of Science and Technology, China, in 2015. Her research interest is synthesis of nanomaterials and their application in electrochemistry. Li Zaijun is one professor of School of Chemical and Materials Engineering, Jiangnan University, China. He received his doctor degree from Jiangnan University, China, in 2004. He is author of more than 200 papers and reviewer of more than 40 journals. His current research interests are synthesis of graphene-based nanomaterials and their applications in sensing, electrocatalysis and energy storage/conversion devices. Li Ruiyi is currently associate professor of School of Pharmaceutical Sciences, Jiangnan University, China. She received his master degree from The Birmingham University, The United Kingdom, 2014. She received her doctor degree from Jiangnan University, China, in 2019. Her research interest is synthesis of graphene-based nanomaterials and their application in pharmaceutical analysis and drug delivery. Yang Yongqiang is senior engineer of National Graphene Product Quality Supervision and Inspection Center, Jiangsu Province Special Equipment Safety Supervision and Inspection Institute Branch, China. He received his doctor degree from Shanghai Jiaotong University, China, in 2014. His current research interests are synthesis of graphene-based materials and application in analytical chemistry. Sun Xiulan is one professor of School of Food and Technology, Jiangnan Unviersity, China. He received her doctor degree from Jiangnan University, China, in 2006. She is author of more than 100 papers. Her current research direction is food safety detection and active prevention and control, focusing on the detection and control of microbial toxins in food processing.

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