Electronic Supplementary Material s1

Total Page:16

File Type:pdf, Size:1020Kb

Electronic Supplementary Material s1

Electronic Supplementary Material

Cyclovoltammetric acetylcholinesterase activity assay after inhibition and subsequent reactivation by using a glassy carbon electrode modified with palladium nanorods

composited with functionalized C60 fullerene Cui Yea, Xia Zhong*a, Min-Qiang Wangb, Yaqin Chaia, Ruo Yuan*a aKey Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China bChongqing Key Laboratory for Advanced Materials and Technologies of Clean, Electrical Power Sources, Institute for Clean Energy & Advanced Materials, Southwest University, Chongqing 400715, P.R. China

S1 Study of inhibition and reactivation behavior for AChE

The electrochemical behaviour of the AChE inhibited by different inhibitors (ethyl paraoxon and Hup A) and reactivated with 2-PAM was discussed in detail. As shown in Fig.

S1, stable redox peaks were observed at AChE/PdNR/C60-TOAB/GCE in 0.1 M pH 7.5

- phosphate buffer, which were ascribed to the conversion of a redox pair C60/C60 (curve a). When 0.12 mM ATCl was added, the peak currents increased (curve b). When the prepared biosensors were separately exposed to 5 nM ethyl paraoxon (curve c) and 8.7 nM Hup A (curve d), a greatly decreased current was observed compared to the case without the exposure (curve b), which clearly illustrated that the activity of AChE was inhibited after exposure to ethyl paraoxon or Hup A. When the resulting electrode was then immersed in 0.2 mM 2-PAM for 12 min, the activity of ethyl paraoxon-inhibited AChE was well recovered

* Corresponding authors, Tel: +86 23 68252277; fax: +86 23 68253172, E-mail address: [email protected] (X. Zhong); [email protected] (R. Yuan)

1 and the peak current almost recovered to the normal level before exposure in the case of the inhibition treatment by ethyl paraoxon (curve e). However, the recovery of enzyme activity was not good and the biosensor still retained an obviously decreased peak current in the case of inhibition treatment with Hup A (curve f). The decrease of peak currents in the case of the inhibition treatment by ethyl paraoxon is due to the ethyoxyl groups of ethyl parathion blocking serine hydroxyl groups of AChE through covalent interaction. After incubation in 2- PAM, its high nucleophilicity allowed the displacement of the phosphoryl group from the catalytic serine hydroxyl groups of AChE, thus inducing the recovery of AChE catalytic activity. Reactivation of phosphorylated AChE using 2-PAM can be demonstrated by the following equation:

(1)

Whereas, the Hup A did not contain phosphoryl group so that 2-PAM can not recover the catalytic active of AChE after exposure to Hup A.

40

20 A

 0 / i f -20 a d c e -40 b -0.75 -0.50 -0.25 0.00 0.25 0.50 E/VvsSCE Fig. S1. CV responses of the biosensor in 0.1 M pH 7.5 phosphate buffer (a) without, (b) with 0.12 mM ATCl, (c) with 0.12 mM ATCl after exposure to 5 nM ethyl paraoxon, (d) with 0.12 mM ATCl after exposure to 8.7 nM Hup A, (e) with 0.12 mM ATCl and reactivation treatment

2 by 0.2 mM 2-PAM after exposure to 5 nM ethyl paraoxon, (f) with 0.12 mM ATCl and reactivation treatment by 0.2 mM 2-PAM after exposure to 8.7 nM Hup A.

S2 Optimization of the experimental conditions

The influence of pH on the response of biosensor was investigated. The response currents of the prepared biosensor were detected in a series of 0.1 M phosphate buffer (pH from 6.0 to 9.0) containing 0.12 mM ATCl. As shown in Fig. S2A, the response current reached the maximum value in pH 7.5. With the pH exceeding 7.5, the response current decreased. Thus, pH 7.5 was chosen as the optimal pH in subsequent work. The effect of ATCl concentration was studied. As shown in Fig. S2B, the peak current increased with increasing concentration of ATCl, and it achieved a relatively stable value when the concentration reached 0.12 mM. Thus, the optimum concentration of ATCl employed in this work was 0.12 mM. The incubation time of AChE was an important factor to achieve good analytical performance. The PdNR/C60-TOAB/GCE was detected in pH 7.5 phosphate buffer containing 0.12 mM ATCl after incubation in AChE solution for 2, 4, 6, 8 and 10 h, respectively. The response currents increased rapidly with the increase of the incubation time from 0 to 6 h, because more AChE could be immobilized onto the electrode surface with the increase of incubation time. When the incubation time increased to 6 h, the response current reached a relatively stable value (Fig. S2C). Consequently, the optimal incubation time of AChE was 6h.

The effect of inhibition time was investigated with ethyl paraoxon. The AChE/PdNR/C60- TOAB/GCE was measured in pH 7.5 phosphate buffer including 0.12 mM ATCl after exposure to 5 nM ethyl paraoxon. The relationship between inhibition time (0, 2, 4, 6, 8, 10, 12, 14 min) and the response current was shown in Fig. S2D. Apparently, the response current showed a decrease with the increase of inhibition time before 8 min and then leveled off. Therefore, the inhibition time of 8 min was chosen as the optimal inhibition time. The incubation time of 2-PAM was also researched. After exposure to 5 nM ethyl paraoxon, the prepared biosensor was immersed in 0.2 mM 2-PAM for AChE reactivation. As seen in Fig. S2E, the response current increased with the increase of incubation time before

3 12 min and then leveled off. Thus, the incubation time of 12 min was selected as the optimal inhibition time.

The incubation concentration of 2-PAM was studied at the AChE/PdNR /C60- TOAB/GCE after exposure to 5 nM ethyl paraoxon in 0.1 M pH 7.5 phosphate buffer containing 0.12 mM ATCl. As shown in Fig. S2F, the peak current increased with the augment of 2-PAM concentration and then leveled off when the concentration reached 0.2 mM. Thus, the optimum concentration of 2-PAM employed in this work was 0.2 mM.

A B 38.0 36

37.5 32

37.0 A A 28  /  i / i 36.5 24 36.0 20 6 7 8 9 pH 0.00 0.05 0.10 0.15 0.20 cATCl/mM

40 C D 39 23

38 22 A    / /

i 21 37 i

36 20

19 0 3 6 9 12 0 4 8 12 16 tAChE/h tethyl paraoxon/min

20

40 F E 36 16 A 

32 /

A i   /

i 12 28 

24 8 0 5 10 15 20 0.05 0.10 0.15 0.20 0.25 0.30 t2-PAM/min c /mM 2-PAM

4 Fig. S2. The optimization of (A) pH on the AChE/PdNR/C60-TOAB/GCE in 0.1 M phosphate buffer containing 0.12 mM ATCl, (B) the concentration of ATCl on the AChE/PdNR/C60- TOAB/GCE in 0.1 M pH 7.5 phosphate buffer, (C) the incubation time of AChE on the

PdNR/C60-TOAB/GCE in 0.1 M pH 7.5 phosphate buffer containing 0.12 mM ATCl, (D) the inhibition time of 5 nM ethyl paraoxon on the AChE/PdNR/C60-TOAB/GCE in 0.1 M pH 7.5 phosphate buffer containing 0.12 mM ATCl, (E) the incubation time of 0.2 mM 2-PAM and

(F) the incubation concentration of 2-PAM on the AChE/PdNR/C60-TOAB/GCE after exposure to 5 nM ethyl paraoxon in 0.1 M pH 7.5 phosphate buffer containing 0.12 mM ATCl.

Fig. S3. Selectivity evaluation of the prepared biosensor for the detection of AChE (250 KU·L-1) activity against interferents. COx, GOx, ChOx were 250 KU·L-1. L-cys, Na +, Ca2+, K+ were 500 nM. Error bars represent the standard deviation of three replicates.

Table S1. Comparison between our method and other electrochemical methods for ethyl paraoxon determinations Methods Detection limit Dynamic range Ref Amperometric 0.5 nM 1.0 nM – 5.0 µM [1] Amperometric 40 nM 0.15 µM – 0.2 µM [2] square wave 1.82 nM 3.62 nM – 1.82 µM [3] voltammetric Amperometric 8.7 pM 0.1 µM – 2.5 µM [4]

5 Cyclic Voltammetry 3.3 pM 10 pM – 0.3 µM This work

Table S2. Figures of merit of recently reported nanomaterial-based methods for determination of the activity of acetylcholineesterase

Methods Detection limit Dynamic range Ref fluorometric 0.2 U·L-1 10 U·L-1 - 70 U·L-1 [5] Liquid crystals 827 U·L-1 ---- [6] Linear Sweep 0.02 U·L-1 0.05 U·L-1 - 5.0 [7] Voltammetry U·L-1 Spectrophotometry ---- 2×10−2 U·L-1 - 0.38 [8] U·L-1 Cyclic Voltammetry 0.83 U·L-1 2.5 U·L-1 - 250 This work KU·L-1

Table S3. Determination of AChE activity in human blood serum (n = 5) by the prepared biosensor. Samples Added AChE Found AChE Recovery RSD (%) (%) 1 25 U·L-1 25.3 U·L-1 101.2 5.43 2 250 U·L-1 252 U·L-1 100.8 6.15 3 2.5 KU·L-1 2.49 KU·L-1 99.6 5.72 4 25 KU·L-1 25.4 KU·L-1 101.6 3.85

Reference 1. Yang YQ, Asiri AM, Du D, Lin YH, (2014) Acetylcholinesterase biosensor based on a gold nanoparticle–polypyrrole–reduced grapheme oxide nanocomposite modified electrode for the amperometric detection of organophosphorus pesticides. Analyst 139: 3055-3060. 2. Upadhyay S, Rao GR, Sharma MK, Bhattacharya BK, Rao VK, Vijayaraghavan R, (2009) Immobilization of acetylcholineesterase–choline oxidase on a gold–platinum

6 bimetallic nanoparticles modified glassy carbon electrode for the sensitive detection of organophosphate pesticides, carbamates and nerve agents. Biosens Bioelectron 25: 832– 838. 3. Yang YQ, Tu HY, Zhang AD, Du D, Lin YH, (2012) Preparation and characterization of Au–ZrO2–SiO2 nanocomposite spheres and their application in enrichment and detection of organophosphorus agents. J Mater Chem 22: 4977–4981. 4. Marinov I, Ivanov Y, Vassileva N, Godjevargova T, (2011) Amperometric inhibition- based detection of organophosphorus pesticides in unary and binary mixtures employing flow-injection analysis. Sensor Actua B-Chem 160: 1098–1105. 5. Han WT, Liao SZ, Zhang CH, Ding HZ, Wu ZY, Shen GL, Yu RQ, (2013) Highly Sensitive Fluorometric Assay Method for Acetylcholinesterase Inhibitor Based on Nile Red-Adsorbed Gold Nanoparticles. Chin J Chem 31: 1-7. 6. Wang Y, Hu QZ, Guo YX, Yu L, (2015) A cationic surfactant-decorated liquid crystal sensing platform for simple and sensitive detection of acetylcholinesterase and its inhibitor. Biosens Bioelectron 72: 25-30. 7. Du D, Tao Y, Zhang WY, Liu DL, Li HB, (2011) Oxidative desorption of thiocholine assembled on core–shell Fe3O4/AuNPs magnetic nanocomposites for highly sensitive determination of acetylcholinesterase activity: An exposure biomarker of organophosphates. Biosens Bioelectron 26: 4231-4235. 8. Cuartero M, Ortuño JA, García MS, García-Cánovas F, (2012) Assay of acetylcholinesterase activity by potentiometric monitoring of acetylcholine. Anal Biochem 421: 208-212.

7

Recommended publications