Graphdiyne: A new promising member of 2D all-carbon nanomaterial as robust electrochemical enzyme biosensor platform

Journal Pre-proof Graphdiyne: A new promising member of 2D all-carbon nanomaterial as robust electrochemical enzyme biosensor platform Lingxia Wu, Xianbo Lu, Juan Gao, Changshui Huang, Dhanjai, Jiping Chen PII:

S0008-6223(19)31000-0

DOI:

https://doi.org/10.1016/j.carbon.2019.09.086

Reference:

CARBON 14656

To appear in:

Carbon

Received Date: 16 July 2019 Revised Date:

21 September 2019

Accepted Date: 29 September 2019

Please cite this article as: L. Wu, X. Lu, J. Gao, C. Huang, Dhanjai, J. Chen, Graphdiyne: A new promising member of 2D all-carbon nanomaterial as robust electrochemical enzyme biosensor platform, Carbon (2019), doi: https://doi.org/10.1016/j.carbon.2019.09.086. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

1

Graphdiyne: a new promising member of 2D all-carbon

2

nanomaterial as robust electrochemical enzyme biosensor platform

3

Lingxia Wua,c, Xianbo Lua,*, Juan Gaob, Changshui Huangb, Dhanjaia, Jiping Chena

4

a

5

Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, PR

6

China

7

b

8

Sciences, Qingdao 266101, PR China

9

c

CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian

Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of

University of Chinese Academy of Sciences, Beijing 100049, PR China

10 11

Abstract: Graphdiyne (GDY), a new two-dimensional all-carbon allotrope composed

12

of benzene rings and alkyne unites, was successfully synthesized via a

13

crossing-coupling reaction with much intriguing properties especially highly

14

π-conjugated structure, attractive electronic and chemical properties, good

15

biocompatibility and dispersion in aqueous solution. The as-prepared graphdiyne was

16

explored for the first time as an extremely attractive matrix for tyrosinase (a model

17

enzyme) immobilization to construct a mediator-free GDY-based biosensor for rapid

18

detection of bisphenol A (BPA). The response of GDY-based tyrosinase biosensor is

19

linear over the range of 1.0 × 10-7 to 3.5 × 10-6 mol L-1 with a high sensitivity of

20

2990.8 mA cm-2 M-1 and a low detection limit of 24 nmol L-1. The proposed

21

GDY-based tyrosinase biosensor exhibited better analytical performances for BPA

*

Corresponding author. Tel: +86-411-84379562. E-mail: [email protected] 1

22

detection than CNTs and graphene based biosensors. The excellent performance of the

23

biosensor should partially be ascribed to the strong π-π interactions between

24

graphdiyne and BPA, which could enrich available BPA concentration on the

25

electrode to react with tyrosinase. The robust GDY-based tyrosinase biosensor was

26

used for BPA detection in drinking bottles and tap water with satisfactory results. As a

27

new 2D all-carbon nanomaterial, graphdiyne is proved to be a powerful

28

electrochemical enzyme biosensor platform for biomolecules (as recognition elements)

29

immobilization and biosensor fabrication, and provides great application prospect for

30

biomedical detection and environmental analyses.

31 32

1. Introduction

33

Chemicals with estrogenic activity have been reported to cause many adverse

34

health effects. Bisphenol A (2,2-bis (4-hydroxyphenyl) propane, BPA), as one of the

35

most important endocrine disrupting chemicals with two hydroxyphenyl groups, can

36

cause cardiovascular diseases, carcinogenicity, neurotoxicity, developmental and

37

behavioral problems [1-3]. In our daily life, BPA is extensively used for its

38

cross-linking properties in the production of epoxy resins (EP) and polycarbonate (PC)

39

plastics. The EP are used as food-contact surface lacquer coatings for cans and metal

40

jar lids, and the PC plastics are widely used in food-packaging and plastic bottles [2].

41

The leaching rate of BPA increases by heating, repeated washing and contacting with

42

either acidic or basic compounds which can destroy the ester bond linking BPA

43

molecules in PC plastics and resins [4]. With the increasing use of plastic productions 2

44

based on EP and PC plastics, more and more people are inevitably exposed to BPA in

45

daily life. BPA has been widely found in human biological fluids, placental and fetal

46

liver tissues, and the risks of BPA in pregnant women and their matching fetuses has

47

been reported [5-8]. Recently, the Regulation (EU) No 2018/213 has updated the

48

specific migration limit of BPA in food contact materials from 0.6 to 0.05 mg/kg.

49

Regarding the high exposure frequency to BPA and its adverse impact on human

50

health, reliable and efficient analytical methods for BPA detection are urgently

51

needed.

52

Up to now, the conventional analytical techniques, including high performance

53

liquid

chromatographic-fluorescence

detector

54

spectrometry [10], liquid chromatography-tandem mass spectrometry [11], capillary

55

electrophoresis [12], and enzyme-linked immunosorbent assay [13], have been

56

reported for BPA detection with high sensitivity. They usually suffer from some

57

disadvantages including time-consuming sample pretreatment, large and expensive

58

equipments, skilled personnel for analysis and interpretation, and unable to meet

59

on-site applications. Besides, the emerging optical sensors [14-17], based on

60

fluorescence, chemiluminescence, colorimetric and surface plasmon resonance, are

61

also widely used for BPA detection showing simplicity and fast-response. However, a

62

number of factors hinder development of these sensors such as low sensitivity, poor

63

specificity, vulnerable to interference, requirement of fluorescent labels and low

64

efficient recognition of targets. Based on the above disadvantages, the objective of

65

this study is to establish a detection method for BPA analysis with easy-operation, 3

[9],

gas

chromatography-mass

66

fast-response, low-cost, miniaturization, high sensitivity, stability and specificity.

67

By comparison, the electrochemical enzyme biosensors are ideally alternative

68

analytical tools for on-site application. In order to improve the performance of

69

electrochemical enzyme biosensors, the superior biosensing materials should be

70

chosen as immobilization matrix for effective immobilization of enzyme molecules.

71

Recently, a variety of nanomaterials [18-21], including carbon nanomaterials (e.g.,

72

graphene, carbon nanotubes, fullerene, etc.) [22-24], transition metal dichalcogenides

73

(e.g., MoS2, MoSe2, etc.), graphitic carbon nitride, metal-organic frameworks, 2D

74

transition metal carbides or nitrides, have been explored to immobilize enzyme and

75

improve the enzymatic catalysis activity. And among the above nanomaterials, the

76

carbon based nanomaterials are most often used to build electrochemical biosensors

77

because of their promising physical and chemical properties.

78

Over the past two decades, carbon nanomaterials have been the focus of scientific

79

researches, because the carbon atoms with three hybridization states (sp, sp2 and sp3)

80

could be combined with each other to develop many carbon allotropes. The

81

discovered carbon nanomaterials, e.g., graphite, diamond, fullerene, carbon nanotube

82

and graphene, consist of sp2 or/and sp3-hybridized carbon atoms [25-28]. Recently,

83

the carbon-carbon triple bond with high conjugation and linear structure formed by sp

84

hybridization has been of great interest to researchers. In 2010, Li et al. [29] reported

85

a methodology to synthesize large area graphdiyne (GDY) films on the surface of

86

copper, which was occupied by sp and sp2-hybridized carbon atoms. Chemical vapor

87

deposition (CVD) [30] is also used to synthesize GDY, which is easier to control the 4

88

thickness and structure of GDY. However, the wet chemical synthesis route is more

89

cost-effective and scalable for preparing large-area GDY in practical application [31].

90

And then, GDY composed of benzene rings and alkyne unite have drawn much

91

attention from the scientists. In order to regulate the chemical or physical properties of

92

GDY, some doped GDY materials, e.g. boron-GDY [32] and nitrogen-GDY [33] are

93

also prepared. The introduction of B and N could create numerous heteroatomic

94

defects and active sites, showing excellent conductivity and electrochemical

95

properties. Due to the intriguing properties of rich carbon chemical bonds, highly

96

π-conjugated structure, wide plane spacing, tunable electronic properties, high

97

chemical and electrochemical stability, excellent thermal and mechanical stability [34,

98

35], GDY has been extensively applied for a large variety of applications in energy

99

storage and conversion [36-38], optoelectronics [39], catalyst [40], separation

100

membrane [41], and so on. However, up to date, GDY has not been used in

101

electrochemical biosensor field in spite of its intriguing properties.

102

GDY is a new all-carbon nanostructure material after fullerene, carbon nanotubes

103

and graphene. Compared with conventional carbon nanomaterials, GDY possesses

104

richer carbon chemical bonds especially the highly π-conjugated structure, and better

105

dispersion in aqueous solution, which are of great significance for their practical

106

applications. More importantly, the GDY not only has a typical 2D structure similar to

107

graphene, but also has the characteristics of three-dimensional materials including a

108

rigid carbon network and uniformly distributed pores, which can greatly increase

109

active bonding sites [42, 43]. Herein, for the first time, GDY was explored as robust 5

110

biosensing platform for enzyme immobilization and biosensor fabrication. As

111

proof-of-concept demonstrations, tyrosinase was chosen as a model enzyme, and an

112

electrochemical tyrosinase biosensor based on GDY was established for ultrasensitive

113

detection of BPA. Due to the unique nanostructure, attractive electronic property,

114

good biocompatibility, high chemical and electrochemical stability, good dispersion in

115

aqueous solution, and strong π-π interactions between GDY and BPA, GDY played a

116

vital role for immobilizing tyrosinase and improving the electrochemical performance

117

of fabricated biosensor. The as-prepared GDY-based tyrosinase biosensor showed

118

remarkable analytical performances for BPA detection with fast response, high

119

sensitivity, good operation repeatability and low detection limit. The GDY proves to

120

be a promising electrochemical biosensing platform for enzyme-based biosensors

121

construction, and the GDY-based tyrosinase biosensor is evidenced to be a powerful

122

tool for realizing rapid detection of BPA.

123

2. Experimental

124

2.1. Materials

125

Tyrosinase (Tyr, from mushroom, ≥ 1000 units mg-1) and chitosan (Chi, from

126

shrimp shells, ≥ 75% deacetylated) were purchased from Sigma (USA). BPA and

127

Copper (Cu) foil were purchased from Tokyo Chemical Industry Co. (Tokyo, Japan)

128

and

129

Tetrabutylammonium fluoride, tetrahydrofuran and pyridine were purchased from

130

Alfa Aesar (USA), tetrahydrofuran and pyridine were dried by distillation before use.

131

All the other reagents were used as received without further purification. Unless

Sinopharm

Chemical

Reagent

6

Co.,

Ltd

(SCRC),

respectively.

132

otherwise stated, 50 mmol L-1 phosphate solutions (pH 7.0, PBS) was used as

133

electrolyte throughout all electrochemical experiments.

134

2.2. Apparatus

135

Transition electron microscopy (TEM) image was recorded using a JEM-2100

136

(JEOL, Japan) instrument with an accelerating voltage of 200 kV. Scanning electron

137

microscopy (SEM) images were obtained by using a field emission scanning electron

138

microscopy JSM-7800F (JEOL, Japan). Fourier transform infrared (FT-IR) spectra

139

were carried out using a NICOLET iS5 FT-IR spectrometer (Thermo, USA). The

140

X-ray diffraction (XRD) pattern was recorded on a Bruker D8 Advance diffractometer

141

(Germany) using Cu Kα radiation (λ=1.5406 Å). Raman spectroscopy measurement

142

was taken using a NanoWizard Ulra Speed & inVia Raman (RENISHAW & JPK,

143

Germany) with a laser excitation wavelength of 532 nm. Atomic force microscopy

144

(AFM) image was obtained using a MultiMode 3D scanning probe microscope

145

(Veeco).

146

using a Quantachrome Autosorb gas-sorption system and calculated through the

147

Brunauer-Emmett-Teller (BET). Electrochemical impedance spectroscopy (EIS)

148

measurements were obtained by using a Metrohm Autolab PGSTAT 302 N

149

Potentiostat/Galvanostat (Eco Chemie, Netherlands). Cycle voltammogram (CV) and

150

amperometric current-time (i-t) curves were obtained by using a CHI 440B

151

electrochemical

152

comprising a working electrode (the modified glassy carbon electrode, GC), a

153

reference electrode (Ag/AgCl electrode), and an auxiliary electrode (platinum wire),

Nitrogen adsorption-desorption measurements were performed at 77 K

workstation

(USA).

A conventional

7

three-electrode

system

154

were used in all the electrochemical measurements.

155

2.3. Synthesis of GDY

156

The GDY was prepared on the surface of copper via a cross-coupling reaction

157

using hexaethynylbenzene (HEB) as monomer according to the reported method with

158

minor revision [29]. The schematic synthesis route of GDY is shown in Fig. S1.

159

Firstly, the precursor hexakis[(trimethylsilyl)ethynyl]benzene (HEB-TMS) was

160

prepared by using the Negishi cross-coupling reaction following the reported

161

synthetic route [44]. Then the HEB monomer was obtained by addition of

162

tetrabutylammonium fluoride to tetrahydrofuran solution of HEB-TMS with stirring

163

for 10 min at 0 ℃. Finally, in the presence of pyridine, the GDY was successfully

164

grown on the surface of copper foils via a cross-coupling reaction of the HEB

165

monomer for 72 h at 60 ℃ under nitrogen atmosphere. After the reaction was

166

completed, the as grown GDY on copper foils was removed by ultrasonicating and

167

concentrated by rotary evaporation, and then washed with heated acetone and

168

N,N-dimethylformamide in turn to remove HEB monomers and oligomers. After that,

169

the GDY powder was refluxed with diluted hydrochloric acid and sodium hydroxide

170

for 3 h at 80 ℃, respectively. Then, it was washed and centrifuged repeatedly. Finally,

171

the black GDY powder was obtained by centrifugation and drying in vacuum.

172

2.4. Fabrication of GDY based tyrosinase biosensor

173

The tyrosinase biosensors based on GDY were prepared by a simple casting

174

method. Before modification, the GC electrodes were polished with 1.0, 0.3 and 0.05

175

µm alumina powder successively, then washed and sonicated in ethanol and Milli-Q 8

176

water to remove residual alumina powder. Then the electrodes were dried with

177

purified nitrogen stream. The preparation process of biosensor was as follows: Firstly,

178

10 µL tyrosinase solution (10 mg mL-1) and 20 µL GDY suspension (1.5 mg mL-1)

179

were mixed and shaken for 30 min so that tyrosinase molecules could be adhered to

180

the surface of GDY by adsorption [45]. Then, 10 µL chitosan solution (2 mg mL-1)

181

was injected into the above mixed solution. Finally, a freshly polished GC electrode

182

was casted with 5 µL of the above mixture and covered with a beaker to obtain a

183

uniform film on electrode (Tyr-GDY-Chi/GC). The final loading amounts of

184

tyrosinase, GDY and chitosan on the Tyr-GDY-Chi/GC biosensor are 12.5 µg, 3.75 µg

185

and 2.5 µg, respectively. The fabricated film electrode was stored at 4 ℃ in a

186

refrigerator when not in use.

187

The similar procedures as described above were used to prepare other film

188

electrodes, such as Tyr-Chi/GC, GDY-Chi/GC and Chi/GC electrodes. Before

189

electrochemical measurements, all the as-prepared modified electrodes were

190

immersed in PBS (pH 7.0, 50 mmol L-1) for 30 min to remove residual components.

191

2.5. Electrochemical measurements of BPA with fabricated biosensors

192

CV measurements were studied in PBS with a scan rate of 100 mV s-1 ranging from

193

+0.4 V to -0.4 V. The i-t curves for BPA were performed to comparatively investigate

194

the performance of different biosensors. The measurements were carried out in 8.0

195

mL stirring PBS with an applied potential value of -0.04 V with successive addition of

196

standard BPA solution.

197

3. Results and discussion 9

198

3.1. Physical characterization of GDY

199

In our work, the GDY was prepared on the surface of copper via a cross-coupling

200

reaction [29], as shown in Fig. S1. Fig. 1A shows the schematic chemical structure of

201

GDY consisting of sp and sp2-hybridized carbon atoms with highly π-conjugated

202

structure. To analyze the morphology and structure of GDY powder, the TEM, SEM

203

and AFM were studied. As shown in Fig. 1B, the TEM image demonstrates the

204

uniform and continuous microstructure with stacked layers. The layer distance of

205

GDY film is around 0.365 nm according to the HRTEM image in Fig. S2A. The

206

thickness of bulk GDY is approximately 44 nm, as shown in AFM image of Fig. S3.

207

The BET surface area of GDY powder is 117.7 m2 g-1 with an average pore size of

208

10.8 nm, and the SEM in Fig. S2B also shows the porous structure of bulk GDY

209

powder. This porous structure is very important for the efficient diffusion of BPA

210

substrate on the fabricated biosensor. The unique porous structure of GDY with high

211

π-conjugated acetylenic bonds (sp-hybridized) could provide more binding sites and

212

facilitate strong adsorption to aromatic rings of BPA by π-π interactions [46], which

213

could enrich the available substrate concentration to react with enzyme electrode [32].

10

214 215

Fig. 1. (A) The schematic chemical structure of GDY. (B) Typical TEM image of

216

GDY. (C) XRD of GDY. (D) Raman spectra of GDY.

217 218

The XRD pattern of the GDY powder in Fig. 1C shows a broad peak around 22.4°,

219

and there is no distinguishable diffraction peaks else, indicating its amorphous nature

220

of GDY, which might be due to random conformational fluctuation of GDY at

221

mesoscopic scales [43]. As shown in Fig. 1D, the Raman spectroscopy of GDY

222

exhibits three prominent peaks at 1369, 1585 and 2123 cm-1. The peak at 2123 cm-1

223

could be attributed to the vibration of conjugated diyne links (-C≡C-C≡C-). A D-band

224

at 1369 cm-1 corresponds to the breathing vibration of sp2 carbon domains in aromatic

225

rings. And a G-band at 1585 cm-1 should be ascribed to the first-order scattering of the 11

226

E2g mode observed for in-phase stretching vibration of sp2 carbon domains in

227

aromatic rings. The ratio of the peak intensity of D and G band is 0.67, which

228

indicates that the two-dimensional GDY has relative high structure regularity [47].

229

Besides, GDY may contain a small amount of residual oxygen content due to the

230

adsorption of O2 when exposing to air [29].

231

3.2. FT-IR characterization of GDY and Tyr-GDY nanocomposite

232

The surface functional groups of GDY were also studied by FT-IR, as shown in Fig.

233

2 (curve c). The peaks at 1463 and 1650 cm-1 are assigned to the skeletal vibrations of

234

aromatic rings. The peaks of 2335 and 2377 cm-1 (Fig. 2, inset) corresponds to the

235

typical C≡C stretching vibration, and the intensities are relatively weak as a result of

236

the molecular perfect symmetry of GDY [48]. The stretching vibration and bending

237

vibration of aromatic C-H could also be observed at 3357 and 601 cm-1, respectively.

238

All of these characterization results suggest the formation of the carbon-rich structure.

239

240 241

Fig. 2. FT-IR spectra of tyrosinase (a), Tyr-GDY (b) and GDY (c). Inset: amplified

242

FT-IR of GDY from 2280 - 2420 cm-1. 12

243 244

FT-IR is also considered as an useful technique for characterizing the structural

245

changes of tyrosinase absorbed on the surface of GDY. The secondary structure

246

information of polypeptide chain is proved with the characteristic amide ℃ and amide

247

℃ bands. The peaks at 1700-1600 cm-1 for amide ℃ is the adsorption of C=O

248

stretching vibration of peptide linkages, and the peak at 1620-1500 cm-1 for amide ℃

249

is related to the adsorption of N-H bending and C-N stretching [49]. Fig. 2 shows the

250

FT-IR spectra of tyrosinase (curve a) and tyrosinase absorbed on GDY surface

251

(Tyr-GDY nanocomposite) (curve b). Clearly, the characteristic absorption peaks

252

(amide ℃: 1650 cm-1 and amide Ⅱ: 1544 cm-1) of Tyr-GDY nanocomposite match

253

well with that of tyrosinase and almost all the characteristic absorption peaks of

254

tyrosinase have been retained, which indicates that the native secondary structure of

255

tyrosinase molecules are retained even after forming new Tyr-GDY nanocomposite.

256

The GDY could be applied as a promising matrix for enzyme immobilization and

257

biosensor construction due to its excellent biocompatibility.

258

3.3. EIS characterization of GDY modified electrodes

259

EIS is a very powerful tool for characterizing the electron transfer reaction and the

260

interface properties of different nanocomposite-modified electrodes. The semicircular

261

portion of Nyquist plot is related to the electron transfer limited process, and the

262

diameter equals to the electron transfer resistance (Rct), which controls the electron

263

transfer kinetics of the redox probe at the electrode interface [50]. As shown in Fig.3,

264

the values of Rct fitted by Randles equivalent circuit for different electrodes were as 13

265

follows: Tyr-Chi/GC (2.85 kΩ) > Tyr-GDY-Chi/GC (1.46 kΩ) > Chi/GC (59.2 Ω) >

266

bare GC. Fig. 3a shows a straight line of the impedance spectrum of bare GC

267

electrode, indicating a diffusion-controlled process and fast electron transfer rate. The

268

value of Rct for chitosan modified GC electrode (curve b, inset of Fig. 3) was larger

269

than that of bare GC electrode, indicating that the chitosan film slightly hindered the

270

electron transfer from the redox probe of [Fe(CN)6]3-/4- to the electrode surface. As

271

chitosan is a linear polymer with good film-forming ability, it has been extensively

272

used to construct biosensors [51]. With tyrosinase immobilized on Chi/GC electrode,

273

the Rct increased from 59.2 Ω to 2.85 kΩ, showing that the tyrosinase molecules had

274

been successfully immobilized on the electrode with chitosan film and caused large

275

inhibition of the electron transfer of the redox couple. When the GDY was introduced

276

to the Tyr-Chi nanocomposite, the Rct of GDY-Tyr-Chi/GC electrode decreased to

277

1.46 kΩ, which was lower than that of Tyr-Chi GC electrode. The decreased Rct could

278

be attributed to the introduction of GDY, which could greatly improve the

279

conductivity and the electron transfer process.

280 281

Fig. 3. Nyquist plots of bare GC (a), Chi/GC (b), Tyr-Chi/GC (c) and 14

282

Tyr-GDY-Chi/GC (d) in 1 mmol L-1 Fe(CN)63-/4- containing 0.5 mol L-1 KNO3

283

solution. Insert: amplified Nyquist plot of bare/GC (a) and Chi/GC (b) at low

284

frequency.

285 286

3.4. Electrochemical characterization of GDY based biosensor for BPA detection

287

Tyrosinase is a copper-containing protein with two distinct substrate binding sites

288

for binding of BPA and oxygen [52]. Compared with laccase of low activity and

289

horseradish peroxidase of poor selectivity [53, 54], tyrosinase has high activities and

290

better specificity toward BPA, and it is more suitable to construct biosensor for highly

291

sensitive and selective detection of BPA. The possible mechanism has been discussed

292

in previous study [55]. The reaction mechanism is simplified as follows:

293 294

In the presence of tyrosinase, BPA is hydroxylated to o-dihydroxybenzene, and then is

295

further oxidized to o-diquinone. At the electrode surface, the current response of

296

biosensor for BPA is obtained with the reduction of o-diquinone into

297

o-dihydroxybenzene. Based on the mechanism presented above, tyrosinase-based

298

electrochemical biosensor could be used as a reliable tool for BPA detection.

15

299 300

Fig. 4. CVs of the Tyr-GDY-Chi/GC biosensor in the absence of BPA (a) and in the

301

presence of 8 µmol L-1 BPA (b) in air-saturated 50 mM pH 7.0 PBS at a scan rate of

302

100 mV s-1.

303 304

In order to estimate the bioelectrocatalytic activity of Tyr-GDY-Chi/GC electrode,

305

the GDY-Tyr-Chi/GC biosensor was evaluated by cyclic voltammetry in the presence

306

of BPA with a potential range from +0.4 V to -0.4 V. As shown in Fig. 4, a pair of

307

significantly increased oxidation and reduction peak current were observed after

308

injecting 8 µM BPA. This indicated the tyrosinase molecules retained high

309

biocatalytic activity for BPA after immobilizing on GDY. And a large response current

310

for BPA were observed at a relatively low potential (-0.04 V), which could effectively

311

minimize the possible interferences in the detection. For the purpose of lower

312

background current and limit of detection (LOD), the optimized working potential

313

(-0.04 V, versus Ag/AgCl) was chosen as constant working potential for further i-t

314

measurements.

16

315 316 317

Fig. 5. (A) The typical amperometric response curves of Tyr-GDY-Chi (a), Tyr-Chi (b)

318

and GDY-Chi (c) with successive additions of BPA standard solution with different

319

concentrations into a stirring PBS (50 mM, pH 7.0, 8 mL). Applied potential: -0.04 V

320

versus Ag/AgCl. (B) The corresponding calibration curves of steady-state currents

321

versus concentrations of BPA.

322 323

Fig. 5A shows the typical i-t curves of Tyr-GDY-Chi/GC (curve a), Tyr-Chi/GC

324

(curve b) and GDY-Chi/GC (curve c) at -0.04 V with successive addition of BPA

325

standard solution into stirring PBS solution. As shown in Fig. 5A (curve c), the 17

326

GDY-Chi/GC biosensor without tyrosinase was responseless after the addition of BPA.

327

However, well-defined i-t curves and fast response were obtained for the BPA

328

additions on the Tyr-Chi/GC and Tyr-GDY-Chi/GC electrodes, indicating that

329

tyrosianse molecules possessed high biocatalytic activity on the modified electrodes.

330

The response time of Tyr-GDY-Chi/GC electrode (achieving 90% of steady state

331

current) was within 20 s. It can be seen from Fig. 5A, the amperometric response

332

signals of Tyr-GDY-Chi/GC biosensor are more stable than Tyr-Chi/GC biosensor, as

333

the response currents of Tyr-Chi/GC biosensor decay over time more obviously.

334

Graphdiyne is an all-carbon nanomaterial with good electronic conductivity and

335

porous structure, and its introduction in the Tyr-GDY-Chi/GC electrode can improve

336

the conductivity of the biosensor, as confirmed by the Nyquist plots of Tyr-Chi/GC

337

and Tyr-GDY-Chi/GC in Fig. 3. When graphdiyne acts as the immobilization matrix

338

of the enzyme electrode, it could obviously improve the signal trapping ability and

339

substrate diffusion velocity of the electrochemical biosensor due to the better

340

conductivity and nanoporous structure, and the electrocatalytic signal of the enzyme

341

electrode to the substrate can be captured in time. So the Tyr-GDY-Chi/GC biosensor

342

shows better response signal stability than that of Tyr-Chi/GC biosensor. Fig. 5B

343

shows the calibration curves of different modified electrodes for BPA, and the

344

Tyr-GDY-Chi/GC electrode exhibits larger response sensitivity than Tyr-Chi/GC

345

electrode, indicating that the GDY with good conductivity and biocompatibility could

346

prominently enhance the performance of tyrosinase biosensor for BPA detection. The

347

sensitivity of Tyr-GDY-Chi/GC biosensor was 2990.8 mA cm-2 M-1, which was about 18

348

2 times that of Tyr-Chi/GC biosensor. The linear range of GDY based biosensor was

349

from 1.0 × 10-7 to 3.5 × 10-6 mol L-1 with a correlation coefficient of 0.996. The LOD

350

was estimated to be 24 nmol L-1 (0.0055 mg L-1) at a signal-to-ratio of 3, which was

351

lower than the value of predicted no-effect-concentrations for drinking water quality

352

of China (GB 5749-2006, BPA 0.01 mg L-1). In order to demonstrate the good

353

performance of the resulting biosensor, a comparison of LOD and sensitivity were

354

made with those reported tyrosinase biosensors for BPA detection (Table 1). As shown

355

in Table 1, the developed biosensor is characterized by higher sensitivity and better

356

LOD. The sensitivity of GDY based biosensor is about two times higher than that of

357

CNTs based biosensor [55], which is comparative to that of graphene-based biosensor

358

[59]. As one most important performance parameter of biosensors, the LOD of

359

Tyr-GDY-Chi/GC biosensor is lower than that of graphene, carbon nanotubes,

360

Au-polythionine nanocomposites, Rh2O3/reduced graphene oxide composites and

361

Lanthanum-doped cobalt nanocube based biosensors [55-65]. The excellent

362

performance of the developed biosensor could be partially attributed to the strong π-π

363

stacking interactions between GDY and BPA [39, 46], which would enrich the BPA on

364

the electrode surface and improve the effective concentration of BPA to react with

365

tyrosinase. And the porous structure of GDY also could greatly increase the active

366

bonding sites for BPA adsorption. The GDY based biosensor exhibits high sensitivity

367

and low detection limit for BPA detection, suggesting that it could be applied as a new

368

powerful tool for rapid detection of BPA .

369 19

370 371

Table 1 Comparison of different nanomaterial modified tyrosinase biosensors for BPA detection. Biosensorsa Tyr-GDY-Chi/GCE Tyr-TiO2-MWCNTs-PDDA-Nafion/GCE Tyr/nano-Au/T-NH2/Au electrode Tyr-CoPC/SPE Tyr-NGP-Chi/GCE Tyr-CNTs-Chi/GCE Tyr-thionine/CPE Tyr-MWCNTs-CoPC-SF/GCE Tyr-pTH/GCE Tyr-Au-Nafion/SPE

LOD (uM) 0.024 0.066 0.133 0.08 0.033 0.1 0.15 0.03 23 0.077

Sensitivity (mA M-1) 211 246 188 219 110 85.4 40 -

Linear range (uM) 0.1-3.5 0.28-45.05 39.9-234 0.08-6 0.1-2 0.15-45 0.05-3 0.5-50

References This work [59] [60] [61] [55] [55] [62] [63] [64] [65]

372

a

373

poly(diallyldimethylammonium

374

phthalocyanine,

375

GOx=glucose oxidase, PLT=polymer from L-tyrosine oxidation, SPE= screen printed electrode,

376

CPE= carbon paste electrode.

MWCNTs=multi-walled

carbon

nanotubes,

chloride),

NGP=hydrophilic

PDDA=polycationic

T-NH2=thioctic

nanographene,

SF=silk

acid

amide,

fibroin,

polymer CoPC=cobalt

pTH=poly(thionine),

377 378

3.5. Reproducibility and stability of the Tyr-GDY-Chi/GC biosensor

379

The reproducibility and repeatability of the Tyr-GDY-Chi/GC biosensors were

380

estimated by amperometry for BPA detection, and the results are shown in Fig. S4.

381

The electrode-to-electrode fabrication reproducibility was estimated by determining

382

the response for BPA at four individual electrodes prepared under the same conditions,

383

and the RSD was within 6.5%, indicating acceptable electrode-to-electrode

384

reproducibility. Additionally, we evaluated the repeatability of Tyr-GDY-Chi/GC

385

biosensors with 6 successive determinations of BPA by the addition of 0.25 µM BPA,

386

and the relative standard deviation (RSD) value of 7.0% was obtained for 6 successive

387

determinations, indicating that the biosensor had acceptable repeatability. The

20

388

Tyr-GDY-Chi/GC biosensor was stored dry at 4 ◦C when it was not in use. To

389

examine the long-term storage stability, the biosensor was evaluated by detecting the

390

amperometric response to 0.25 µM BPA every week. The biosensor could retain 94%

391

of its initial response after three weeks storage, demonstrating good long-term

392

stability.

393

3.6 Applications of the Tyr-GDY-Chi/GC biosensors

394

To further estimate the performance of Tyr-GDY-Chi/GC biosensors for actual

395

sample analysis, the biosensors were used to detect BPA leaching from drinking

396

bottles or in tap water. Briefly, commercial drinking bottles made of different

397

materials were purchased from a local supermarket and cut into small pieces of about

398

1 cm×1 cm size and washed thoroughly with Milli-Q water by sonicating within 3

399

minutes. Then 1.0 g of drinking bottle pieces were immersed in 20 mL water and kept

400

48 h at 78 ℃. The liquid phase was filtrated and collected in 50 mL volumetric flasks

401

and the volume was adjusted to 50 mL with Milli-Q water. As for the tap water

402

sample, it was filtered through a 0.45 µm filter membrane before use. A

403

known-amount of the obtained samples and the samples added BPA standard solution

404

(0.5 µM) were studied and used to analyze the recovery of BPA by amperometric

405

method. As shown in Table 2, the content of BPA in water bottle (PC) and beverage

406

bottle (Al) samples were calculated to be 6.4 µg/g and 4.2 µg/g, respectively. In other

407

samples including coffee spoon (PP), mineral water bottle (PET), beverage bottle (Tin)

408

and tap water in our laboratory, BPA was not found. The recoveries for actural simples

409

were in the range of 86.4%~114%, indicating excellent performance of the GDY 21

410

based tyrosinase biosensor.

411 412

Table 2 Determination of BPA leaching from drinking bottles or in tap water. Measured (uM)b

Added (uM)

Found (uM)b

Recovery (%)

Water bottle (PC)

0.564

0.5

1.134

114.0

Beverage bottle (Aluminium)

0.366

0.5

0.866

100.0

c

0.5

0.432

86.4

Samplea

Coffee spoon (PP)

n.d.

Beverage bottle (Tinplate)

n.d.

0.5

0.468

93.6

Mineral water bottle (PET, Brand A)

n.d.

0.5

0.454

90.8

Tap water

n.d.

0.5

0.444

88.8

413

a

PC=polycarbonate; PP=polypropylene; PET=polyethylene terephthalate.

414

b

The average value of three determinations.

415

c

n.d. means "not detectable".

416 417

BPA is primarily used as a material for the production of PC and EP, which can be

418

used in food packaging, plastic bottles and as food-contact surface lacquer coatings

419

for cans and metal jar lids [2]. Due to the unstable ester bond linking BPA molecules

420

in PC plastics and resins, BPA can leach from the bottles into water or foods by

421

heating [4]. With the prohibition of the use of BPA, bisphenol S are widely used

422

instead of BPA. However, the tyrosinase based biosensor shows no response to

423

bisphenol S [66]. And other co-existing components, e.g. dimethyl phthalate and octyl

424

phthalate, also did not interfere with the detection of BPA [55]. The specific

425

biocatalytic activity of tyrosinase molecules enables the good selectivity of the

426

Tyr-GDY-Chi/GC biosensor for BPA detection. It should be noted that the

427

Tyr-GDY-Chi/GC biosensor is highly sensitive, selective, low-cost and portable, 22

428

which is particularly suitable for the rapid detection of BPA in food-contact packaging

429

materials, such as PC or EP products.

430

4. Conclusions

431

In this study, 2D all-carbon nanomaterial graphdiyne had been successfully

432

synthesized and explored as a robust matrix for tyrosinase immobilization to construct

433

the GDY-based tyrosinase biosensor for BPA detection. The prepared GDY based

434

tyrosinase biosensor showed high sensitivity, low detection limit, wide linear range,

435

good repeatability and long-term stability, resulting from the excellent properties of

436

GDY, especially the unique chemical and physical structure, attractive electronic

437

property, acceptable biocompatibility, good aqueous dispersion, and strong π-π

438

interactions between GDY and BPA. With those remarkable advantages, GDY-based

439

tyrosinase biosensor is proved to be a promising and reliable tool for rapid detection

440

of BPA in drinking bottles and food contact packing materials. As a new promising

441

2D all-carbon nanomaterial after graphene, graphdiyne with intriguing properties

442

would inevitably attract the general interest of scientists and promote the development

443

of analytical field, biosensor, biocatalysis, electronic devices, etc.

444

Acknowledgements

445

This work was financially supported by the National Natural Science Foundation of

446

China (Grant No. 21577139), the Natural Science Foundation of Liaoning Province

447

(2019-MS-317) and the Special Fund for Agro-scientific Research in the Public

448

Interest of China (Grant No. 201503108). This work is dedicated to the 70th

449

anniversary of Dalian Institute of Chemical Physics, CAS.

450

23

451

References

452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: