Sensitive and Simple Competitive Biomimetic ... - ACS Publications

Subscriber access provided by BOSTON UNIV

New Analytical Methods

A Sensitive and Simple Competitive Biomimetic Nanozyme-Linked Immunosorbent Assay for Colorimetric and SERS Sensing of Triazophos Mengmeng Yan, Ge Chen, Yongxin She, Jun Ma, Sihui Hong, Yong Shao, A. M. Abd El-Aty, Miao Wang, Shanshan Wang, and Jing Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03401 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Journal of Agricultural and Food Chemistry

1

A Sensitive and Simple Competitive Biomimetic Nanozyme-Linked

2

Immunosorbent Assay for Colorimetric and SERS Sensing of Triazophos

3

Mengmeng Yan1, Ge Chen1, Yongxin She1*, Jun Ma1, Sihui Hong1, Yong Shao1, A. M.

4

Abd EI-Aty2, 3, Miao Wang1, Shanshan Wang1, Jing Wang1**

5

1 Institute

6

Academy of Agricultural Sciences, Beijing 100081, P.R China

7

2 Department

8

12211-Giza, Egypt

9

3Department

10

of Quality Standards & Testing Technology for Agro-Products, Chinese

of Pharmacology, Faculty of Veterinary Medicine, Cairo University,

of Medical Pharmacology, Medical Faculty, Ataturk University, 25240-

Erzurum, Turkey

11 12 13 14 15 16 17

*(Y.

S.) Tel: +86 10 82106513; Fax: +86 10 82106567. E-mail: [email protected]

18

**(J.

W.) Tel: +86 10 82106568; Fax: +86 10 82106568. E-mail: [email protected]

19 20 21 22 23 24 25 1

ACS Paragon Plus Environment


26

Page 2 of 33

ABSTRACT

27

Biomimetic enzyme-linked immunosorbent assay (BELISA) is widely used for

28

detection of small molecule compounds due to low cost and reagent stability of

29

molecularly imprinted polymers (MIPs). However, enzymes labels used in BELISA

30

still suffer some drawbacks such as high production cost and limited stability. To

31

overcome the drawbacks, a biomimetic nanozyme-linked immunosorbent assay

32

(BNLISA) based on MIPs and nanozyme labels was first proposed. For nanozyme

33

labels, Pt NPs (platinum nanoparticles) acted as peroxidase by catalysing the oxidation

34

of colourless 3,3′,5,5′-tetramethylbenzidine (TMB) into an ideal SERS marker—blue

35

TMB2+ and BSA-hapten (bovine serum albumin-hapten) showed superior selectivity

36

when competing with targets for binding sites on MIPs, which named Pt@BSA-hapten

37

probe. The BNLISA method was employed to detect triazophos with a limit of detection

38

(LOD) of 1 ng·mL-1 via colorimetric and SERS methods. Replacing traditional

39

enzymes with nanozymes for combination with MIPs may bring about a new prospect

40

for other compound analyses.

41

KEYWORDS: Molecular imprinted polymer; BNLISA; Triazophos; Nanozyme;

42

Horseradish peroxidase

43

INTRODUCTION

44

Owing to the favourable properties such as mechanical, thermal and chemical

45

stability, ease of preparation, and low cost 1, 2, molecularly imprinted polymers (MIPs)

46

have earned a reputation as ‘‘plastic antibodies’’. Thus, MIPs are used in a wide range

47

of applications encompassing the fields of separation, chromatography, immunoassays, 2


Page 3 of 33


48

antibody mimics, (bio)sensors

49

is considered the gold standard for highly sensitive qualitative and quantitative

50

detection of analytes within various samples 7. With the application of MIPs as bionic

51

antibodies for small molecule detection via the ELISA method, biomimetic ELISA

52

(BELISA) came into being. For instance, Piletsky et al. developed MIP microplate

53

wells specific for epinephrine, and the affinity of the polymers was determined by

54

BELISA using a conjugate of horseradish peroxidase and norepinephrine (HRP-N)8. In

55

2003, Chianella et al. synthesized vancomycin MIPs and achieved the detection of

56

vancomycin by BELISA in competitive binding experiments with an HRP–vancomycin

57

conjugate 9. In the same year, Tang et al. also detected metolcarb by direct competitive

58

BELISA10. Subsequently, many other studies on BELISA were also reported11-15.

3-6.

The enzyme-linked immunosorbent assay (ELISA)

59

MIPs possess favourable properties such as mechanical, thermal and chemical

60

stability, ease of preparation, and low cost. However, HRP labels in BELISA method

61

could not adapt to a wide range of temperature and pH conditions. Therefore, the

62

combination between HRP and MIPs may limit the applicability of MIPs as a

63

biomimetic antibody. Recently, the demand for nanozymes has increased due to their

64

advantages, such as simple preparation, high temperature and pH stability, easy surface

65

modification and low cost16-18.

66

Consequently, we proposed a biomimetic nanozyme-linked immunosorbent assay

67

(BNLISA) integrating MIPs with nanozymes for detecting triazophos to broaden

68

applicability of MIPs as a biomimetic antibody. Pt NPs were opted as the nanozyme in

69

our project owing to their advantages of enzyme-like peroxidase activity, simple 3



Page 4 of 33

70

synthesis, good tunability, easy storage and easy surface modification by using DNA

71

and protein19-23. The structural analogues of triazophos (hapten) must be introduced to

72

form a competitive relationship with triazophos when they distinguished the binding

73

sites on MIPs. BSA was used as a “bridge” linker due to its abundant functional groups

74

such as amino, carboxyl, thiol and disulfide that could be reacted with other molecules

75

and nanoparticles24, 25., Hence, the nanozyme label was fabricated by two steps. Firstly,

76

BSA-hapten conjugates were synthesized by carbodiimide method between amino

77

groups from BSA and carboxyl groups from hapten compounds. And then the BSA-

78

hapten conjugates were mixed with Pt NPs colloid solution for forming Pt@BSA-

79

hapten probes. Therefore, the as-obtained Pt@BSA-hapten was not only employed as

80

competitive probe to recognize the MIPs binding site, but also played a crucial role in

81

acting as signal probe due to the presence of massive Pt NPs.

82

To obtain biomimetic 96-well MIPs array plate by directly synthesizing molecular

83

imprinted films onto the plate requiring massive raw materials. Meanwhile, the films

84

were easy to break off resulting in inhomogeneous films from well to well when

85

templates were removed by ultrasonic method12. To avoid these matters, uniform MIP

86

microspheres were got firstly by precipitation polymerization and then they were

87

immobilized onto 96-well array plate by the “grafting to” method with the assistance

88

of ionic liquid (IL) as a binder26, 27.

89

To demonstrate the universal detection capability of the BNLISA method,

90

triazophos was selected as the model target. Triazophos is a broad-spectrum

91

organophosphate pesticide that has been widely used in agriculture to control insect 4


Page 5 of 33


92

pests16, 28. However, owing to its relatively high stability and long half-life, it presents

93

potential risks to human health and the environment29, 30.

94

In this proposal, colorimetric and surface enhanced surface-enhanced Raman

95

scattering (SERS) were selected to evaluate the practicality of the BNLISA method

96

thanks to the catalysate TMB2+. SERS is an advanced Raman technique, and was

97

observed by Fleischmann and Van Duyne group in the 1970s. SERS is highly specific

98

and sensitive for detection molecules due to characteristic fingerprints of SERS spectra

99

and metallic nanoparticles used in SERS enhancing the intrinsically weak Raman signal

100

by 106⁓1014 orders of magnitude31,32. Gold nanoparticles (Au NPs) possess

101

extraordinary SERS activities and have been successfully employed as SERS-active

102

substrates. Here, we prepared gold nanoparticles (AuNPs) within MIL-101, a highly

103

porous and thermally stable metal−organic framework (MOF) which is expected to

104

provide the AuNPs with much better stability, by an in-situ reduction strategy. The

105

AuNPs within MIL-101 were denoted as AuNPs@MIL-101 as the SERS enhanced

106

substrate and was firstly used to enhance the Raman signal of TMB2+.

107

Naturally, the as-prepared BNLISA platform was low-cost, fast, highly sensitive,

108

and excellently stable, which also acted as a model for other pesticides detection. The

109

fabrication and detection process of the proposed BNLISA are illustrated in Scheme. 1.

110

EXPERIMENTAL SECTION

111

Materials and apparatus

112

Triazophos. Parathion, triadimefon, Methomyl, trichlorfon, methamidophos,

113

Malathion and Omethoate (Dr Ehrenstorfer Gmbh Augsburg, Germany). Methacrylic 5



Page 6 of 33

114

acid (MAA), trimethylolpropane trimethacrylate (TRIM) (Alfa Aesar Massachusetts,

115

USA). Free radical initiator 2,2-azobisiso-butyronitrile (AIBN) (no. 4 reagent and H.

116

V. Chemical Co., Ltd. Shanghai, China). Ionic liquid 1-butylpyridinium

117

hexafluorophosphate (BPPF6, 99% purity) (Lanzhou Greenchem, Lanzhou, China).

118

Chromium (III) nitrate (Cr (NO3)3·9H2O), 1,4-benzene dicarboxylic acid (H2BDC),

119

hydrogen tetrachloroaurate trihydrate (HAuCl4·3H2O, > 99.9%), trisodium citrate

120

sodium and citrate dehydrate (Sigma-Aldrich St. Louis, MO, USA). N-

121

Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinate (NHS), Bovine serum

122

albumin (BSA) and polyethylene glycol (PEG) 20000 (Sigma-Aldrich St. Louis, MO,

123

USA), Triazophos hapten (Institute of Pesticide and Environmental Toxicology,

124

Zhejiang University, China). 3, 3´, 5, 5´-tetramethylbenzidine (TMB) ELISA substrate

125

(TransGen Biotech Co., Ltd. Beijing, China). All the other chemicals and organic

126

solvents used in this study were of the highest available purity and at least of analytical

127

grade. Maxisorp polystyrene 96-well plates were obtained from Corning, Inc. (Corning,

128

NY, USA).

129

Maxisorp polystyrene 96-well plates (Corning, Inc. Corning, NY, USA). Ultrapure

130

water (≥ 18.2 MΩ) produced by a Milli-Q water purification system (Millipore, Bedford,

131

MA, USA) was used in all e experimental works. UV absorbance was recorded using a

132

Lab systems 96-well plate reader (TECAN, Switzerland) in dual-wavelength mode

133

(450–650 nm). Scanning electron microscopy (SEM) images were undertaken with a

134

SEM instrument (SU8020, Hitachi, Japan). Scanning electron microscopy (TEM)

135

images were undertaken with a TEM instrument (JEM 1200EX, JEOL). The type and 6


Page 7 of 33


136

relative content of element and the chemical environment surrounding the element were

137

tested by X-ray photoelectron spectroscopy (XPS, Thermo escalab 250X, USA) with

138

Al Kα radiation as the excitation source. The XPS binding energies were adjusted by

139

interior label carbon (C 1s = 284.8 eV), and the other positions of peaks were calibrated

140

depending on the C 1s peak. Matrix-Assisted Laser Desorption/Ionization Time of

141

Flight Mass Spectrometry (MALDI-TOF-MS, ABI 4800Plus, USA).

142

Preparation of biomimetic 96-well MIPs array plate.

143

The preparation of MIP nanoparticles by precipitation polymerization using

144

triazolone as a template has been studied in detail in our laboratory33. The concise

145

synthesis process of MIPs, non-imprinted polymers (NIPs) and the preparation process

146

of the biomimetic 96-well MIPs array plate (Scheme. 1a and Scheme. 1c) are supplied

147

in Supporting Information.

148

Preparation of SERS-enhanced substrate Au NPs@MIL-101

149

MIL-101 and Au NPs@MIL-101 were synthesized (Scheme 1d) according to a

150

previously reported work with appropriate modifications34, 35, and the synthesis process

151

is supplied in the Supporting Information.

152

Preparation of the Pt@BSA hapten probe

153

Synthesis of BSA-hapten

154

BSA-hapten conjugates were synthesized by carbodiimide method between amino

155

groups from BSA and carboxyl groups from hapten compounds and identified by UV

156

spectroscopy. Firstly, hapten of triazophos (27.37 mg, 0.1 mmol) and NHS (34.5 mg,

157

0.3 mmol) were dissolved in 0.5 mL DMF and the mixture was stirred for 15 min by 7



Page 8 of 33

158

magnetic stirring, getting solution (a). Then, DCC (30.92 mg, 0.15 mmol) were

159

dissolved in 0.5 mL DMF, and the solution was added in the solution (a) drop by drop,

160

getting solution (b). The solution (b) was stirred over-night at room temperature with

161

avoiding meeting up. Through above steps, the carboxyl groups of the hapten

162

compounds were activated. Solution (b) was centrifugated (5000 rpm, 10min) to

163

received activated hapten supernatant solution and was added dropwise in BSA solution

164

which was obtained by weighing 60 mg BSA in 5 mL carbonate buffer solution (CBS

165

0.01 M pH=9.6) and then stirred for 4h at room temperature to achieve BSA-hapten

166

conjugates. Finally, the BSA-hapten conjugated solution was dialyzed against

167

phosphate buffered solution (PBS 0.01 M pH=7.4) for 3 days to remove the unbound

168

hapten. After purifying, the BSA-hapten conjugated solution was mixed glycerol with

169

the same volume and stored at -20 ℃ before use, and was identified by UV spectroscopy

170

and MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight

171

Mass Spectrometry).

172

Synthesis of Pt NPs

173

The Pt NPs were synthesized according to a previously described method36, and

174

the synthesis process is supplied in the Supporting Information. The obtained Pt NPs

175

were identified by TEM.

176

Preparation of Pt@BSA-hapten probe

177

The pH value of the Pt NPs solution (1 mL) was adjusted to 5.4 with 20 μL K2CO3

178

(0.1 mol⁓L-1), and then different volume of BSA-hapten solution was introduced into

179

the colloidal solution. After with standing for 12 h, the solution was centrifuged for 60 8


Page 9 of 33


180

min at 10000 rpm and the precipitate was dissolved with 1 mL PBS (0.01 M pH=7.4)

181

including 1% PEG 20000 for three times to remove the unbound BSA-haptens.

182

Thereafter, concentrate as the Pt@BSA-hapten probes were resuspended in 200 μL of

183

PBS (0.01 M pH=7.4) including 1% PEG 20000 and stored at 4 ℃. The achieved

184

Pt@BSA-hapten probes were identified by UV spectroscopy and TEM.

185

Competitive BNLISA for triazophos detection based on colorimetric and SERS

186

methods

187

As shown in Scheme. 1c, the detection procedure was carried out using the

188

molecular imprinted film on the 96-well array plate after washing three times with

189

PBST (PBS with 0.05% (v/v) Tween 20). Firstly, 50 μL of triazophos standard solution

190

diluted in Tris-HCl (0.01 M, pH 3.7) and 50 μL of Pt@BSA-hapten probe solution

191

diluted in Tris-HCl (0.01 M, pH 3.7) were sequentially added to the microplate. During

192

incubation for 1 h at room temperature, hapten coating on the Pt@BSA-hapten probe

193

and triazophos competed for binding with the recognition site on the surface of the

194

MIPs. The unbound pesticide molecules and Pt@BSA-hapten probes were removed by

195

three washes with PBST. Then, 100 μL of TMB ELISA substrate was added to the

196

microplate and incubated for 15 min at room temperature, accompanied by the change

197

from TMB to the radical cation TMB2+. The reaction was terminated by adding 50 μL

198

of H2SO4 (2 mol⁓L-1) to each well. Finally, absorbance (450 nm) signals were recorded

199

by a Lab Systems 96-well plate reader (TECAN, Switzerland). Importantly, the radical

200

cation TMB2+ solution was assessed by SERS without adding H2SO4 (2 mol⁓L-1).

201

Firstly, 20 μL of TMB2+ solution was mixed with 200 μL of the Au NPs@MIL-101 9



Page 10 of 33

202

substrate, incubated for 60 s, and then the SERS “fingerprint” information was

203

measured by a portable Raman spectrometer (RT5000, TSINGHUA TONGFANG, Bei

204

Jing, China) with a 785 nm laser as the excitation source. The SERS measurement was

205

carried out within the wavelength range of 350–2000 cm–1. The acquisition time was

206

set to 40 s.

207

RESULTS AND DISCUSSION

208

Preparation and characterization of MIPs and NIPs

209

To obtain MIP nanoparticles with a uniform particle size and an increased

210

number of effective recognition sites, the type of monomer and cross-linker, the ratio

211

of template to monomer and cross-linker, and the kind and volume of dispersing solvent

212

should be optimized. Based on the results of our laboratory studies, the best outcome

213

was obtained when using a 1:6:10 ratio of template, monomer, and cross-linker and 20

214

mL of acetonitrile as the dispersing solvent. The surface morphology of the synthesized

215

MIPs and NIPs was characterized by SEM, as shown in Figure. 1a-b. Uniform MIPs

216

microspheres was obtained and more folds on surface was observed on MIPs in the

217

SEM images than NIPs.

218

Preparation and characterization of Au NPs@MIL-101.

219

The TEM image shown in Figure. S1a-b clearly indicates that the Au NPs were

220

embedded inside MIL-101. The elemental composition of Au NPs@MIL-101 was

221

characterized by X-ray photoelectron spectroscopy (XPS), as shown in Figure. S1c.

222

These revealed the successful formation of Au NPs@MIL-101 SERS enhanced

223

substrate. 10


Page 11 of 33


224

Moreover, the SERS activity of the Au NPs@MIL-101 nanoparticles was

225

quantified and compared to those of Au NPs prepared under the same synthetic

226

conditions as Au NPs@MIL-101. As shown in Figure. 2a, both the Au NPs and Au

227

NPs@MIL-101 produced strong SERS signals corresponding to TMB2+. In contrast,

228

MIL-101 did not produce any detectable SERS signals, indicating that the SERS

229

enhancement ability originated from the Au NPs. Although the Au NPs themselves

230

could produce detectable SERS signals, they were not stable. As shown in Figure. 2b,

231

the intensity of the SERS signal at 558 cm-1 decreased with time. MIL-101, with high

232

porosity and thermal stability, was chosen as a protecting matrix to prevent aggregation

233

of the Au NPs. The Au NPs@MIL-101 exhibited a stable SERS signal at 558cm-1 as

234

shown in Figure. 2c. The reproducibility of the Au NPs@MIL101 substrate was also

235

evaluated, as shown in Figure. 2d. In this figure, the black line indicates the average

236

intensity of the SERS signal at 558 cm-1 and the values of the intensity deviation (D)

237

was calculated to be less than 12% and the definition of D was as shown in Supporting

238

Information. Therefore, Au NPs@MIL-101 was selected as the SERS enhanced

239

substrate in the followed experiments. Moreover, the SERS spectra of TMB and TMB2+

240

was proposed as shown in Figure S2, and the characteristic absorption peaks of TMB2+

241

were assigned in Table S1 in Supporting Information.

242

Preparation and optimization of the Pt@BSA-hapten probe

243

The Pt@BSA-hapten probe consisted of three key parts: target hapten, BSA and

244

Pt NPs. The TEM image in Figure. 1c showed that the prepared Pt NPs exhibited

245

consistent diameter and did not aggregate. As shown in Figure. 1d, after modification 11



Page 12 of 33

246

with hapten, the maximum absorbance peak of BSA shifted from 278 nm to 268 nm.

247

Furthermore, the conjugation ratio between hapten of triazophos and BSA was 21.3: 1

248

that was measured by MALDI-TOF-MS as shown in Figure. S3. Pt NPs were combined

249

with BSA through thiol groups and abundant groups on BSA. To form a solid

250

combination, the pH of the Pt NP colloid solution was adjusted to 4.8, 5.1, and 5.4 near

251

the pI of BSA37. As shown in Figure. 1d, after modification with BSA-hapten, the Pt

252

NPs solution showed the characteristic peak of BSA-hapten at 271 nm.

253

Optimization of the working buffer solutions

254

The prepared Pt@BSA-hapten solution needed to be diluted before use. The dilute

255

buffer solution, named the working buffer solution, influenced the assay sensitivity by

256

changing the characteristics of the analyte solution or affecting the interactions between

257

the MIP recognition sites and the hapten. PBS (0.01 M, pH=4.22), Tris-HCl (0.05 M,

258

pH=4.30), CBS (0.05 M, pH=4.22) and ABS (0.01 M, pH=4.10) were selected for study.

259

In this work, the inhibition rate was used to evaluate the performance of the assays. The

260

inhibition rate (IC (%)) of targets at various volumes was calculated and the definition

261

of IC (%) was as shown in Supporting Information. The dilute buffer solutions were

262

optimized by comparing IC (%). As shown in Figure. 3d, the IC (%) reached a

263

maximum when the dilute buffer solution was Tris-HCl (0.01 mol/L, pH=4.30).

264

Optimization of concentration of the BSA-hapten

265

The concentration of the BSA-hapten mixed in Pt colloid solution had great

266

influence on the detection sensitivity and the linear range of competition reactions. The

267

Pt@BSA-hapten probes were prepared by adding 5 μL, 10 μL, 15 μL, 20 μL and 25 μL 12


Page 13 of 33


268

of BSA-hapten solution in 1 mL of Pt NPs solution at final concentrations of 33.9

269

μg·mL-1, 67.8 μg·mL-1, 101.7 μg·mL-1, 135.6 μg·mL-1, 169.5 μg·mL-1. As the results

270

shown in Figure. 3e, the IC (%) increased accordingly as the concentration increased

271

from 33.9 μg·mL-1 to 67.8 μg·mL-1, and shown a decrease trend as the concentrate

272

continued to increase to 169.5 μg·mL--1. In addition, the TEM images of the Pt@BSA-

273

hapten were provided (Figure. 3a-c) when the concentration of BSA-hapten were 67.8

274

μg·mL-1, 169.5 μg·mL-1 and 678 μg·mL-1. The particle size increased of the

275

composition with the increase of concentration of BSA-hapten, On the one hand, the

276

phenomenon demonstrated the interaction between BSA-hapten and Pt NPs, and on the

277

other hand that explained that why the IC (%) decreased gradually from 67.8 μg·mL-1

278

to 169.5 μg·mL-1. We inferred that the increasing particle produced large steric

279

hindrance that interfered the competition process. Above all, BSA-hapten of 67.8

280

μg·mL-1 was used in subsequent experiments.

281

Optimization of the pH of Tris-HCl

282

The pH value of Tris-HCl was an important factor for the assay. The pH of Tris-

283

HCl (0.05 M) was adjusted in the range of 2.0 – 6.0. As shown in Figure. 3f, we found

284

that the IC (%) increased with the pH from 2.1 to 3.7 and then decreased with the pH

285

from 4.3 to 6.1. The IC (%) was observed to reach a maximum at pH 3.7. Hence, we

286

selected pH 3.7 as the optimum pH for all subsequent experiments. The pKa of the

287

monomer methacrylic acid (MAA) was 5.5; therefore, when the solution pH was 3.7,

288

the imprinted membrane was protonated. Furthermore, at this pH, the surface charge of

289

BSA was positive because its pI is 4.7. Triazophos is a compound with an electron-rich 13



290

structure and thus should combine readily with the protonated imprinted membrane.

291

Analytical performance of the proposed BNLISA method

Page 14 of 33

292

Under the optimal conditions mentioned above, we tested the potential application

293

of competitive BNLISA to triazophos detection by colorimetric and SERS methods.

294

For the colorimetric method, the intensity of the absorbance at 450 nm gradually

295

decreased with increasing triazophos concentration in the range from 0 to 10000 ng·mL-

296

1

297

inhibition rate and the logarithm of the triazophos concentration in the concentration

298

range of 5-1000 ng·mL-1, with a calibration curve of y=34.993 Log c – 21.9 (R2 =

299

0.9641). The limit of detection (LOD) was 1 ng·mL-1. For the SERS method, Figure.

300

4c shows that the intensity of a number of Raman peaks corresponding to TMB2+

301

decreased with increasing triazophos concentration (0-10000 ng·mL-1). For the

302

intensity of the SERS signal at 558 cm-1, a good linear relationship with triazophos

303

concentration was also obtained, with a calibration curve of y=-736.43 Log c + 3962.3

304

(R2 = 0.9707), as shown in Figure. 4d. The concentration range of triazophos detected

305

by the SERS method was 1-10000 ng·mL-1, which was wider than the range detected

306

by the colorimetric method. The LOD was also 1 ng·mL-1.

(Figure. 4a). As shown in Figure. 4b, there was a good linear relationship between the

307

The recognizing selectivity of the competitive BNLISA were investigated by

308

cross- reactivity studies between the target and analogues of target as shown in Table

309

1. The cross-reactivity rate (CR%) was calculated by the followed Eq: CR% = (IC50

310

(triazophos) /

311

of the compounds containing phosphoester and phosphorothioate groups ((5), (6), (7)

IC50 (Structural analogues)) × 100%. From the results, we found that the IC50 values

14


Page 15 of 33


312

and (8)) were lower than those of the compounds that did not contain these functional

313

groups ((2), (3) and (4)). As a result, we concluded that the phosphorothioate played a

314

very important role in the competition between the target and the Pt@BSA-hapten

315

probe for the binding sites on the MIPs. Both the hapten structure and the selective

316

adsorption ability of the MIPs contributed to the selectivity of the BNLISA. The cross-

317

reactivity studies also showed the selectivity of MIPs were better than the selectivity of

318

NIPs.

319

The properties of the proposed competitive BNLISA were also compared with

320

those of previously reported methods for detecting triazophos, as shown in Table 2. The

321

table shows that the proposed BNLISA for detecting triazophos is a novel, simple and

322

sensitive method.

323

Real Sample Analysis

324

To evaluate the accuracy of BNLISA in real samples, water samples spiked at 10,

325

100, and 500 µg·kg-1 were investigated and pear samples spiked at 20, 100, and 500

326

µg/kg were investigated (with three replicates for each concentration and the sample

327

treatment method as shown in Supporting Information). The results are shown in Table

328

3. The recovery of the BNLISA ranged from 75.6 to 109.8% for the colorimetric

329

method and from 65.6 to 110.7% for the SERS method, with RSDs ranging from 7.8 to

330

9.4% and 10.1 to 15.2%, respectively. The results were in good agreement with the

331

values recorded by HPLC–MS/MS method (Supporting Information). And, these

332

indicated the BNLISA is a practical and accurate method for the detection of triazophos

333

in water and pear samples. 15



Page 16 of 33

334

In summary, a competitive BNLISA for rapid determination of triazophos was

335

constructed. For the low-cost, sensitive and simple method, three major advantages

336

could be concluded: (1) A good performance of 96-well biomimetic MIPs array plate

337

was designed by immobilized MIPs uniformly onto plate by “grafting to” method; (2)

338

The Pt@BSA-hapten probe was proposed instead of HRP-hapten probe benefiting from

339

the peroxidase-like activity of platinum nanoparticles (Pt NPs) and the “bridge”

340

function of bovine serum albumin (BSA) for linking Pt NPs and target hapten. (3) The

341

Au NPs@MIL-101 of SERS enhanced substrate for sensing TMB2+was synthesized

342

successfully. This new strategy is promising for the rapid detection of pesticide residues

343

in the environment with wide working range and low detection limit, and provided a

344

potential method to monitor the other small molecules detection.

345

ASSOCIATED CONTENT

346

Supporting Information

347

Preparation of biomimetic MIPs 96-well array plate.

348

Preparation of SERS enhancement substrate Au NPs@MIL-101.

349

Synthesis of Pt NPs.

350

Sample treatment and HPLC-MS/MS method.

351

Additional tables and figures.

352

AUTHOR INFORMATION

353

Corresponding Authors

354

*E-mail: [email protected]

355

[email protected] 16


Page 17 of 33


356

Notes

357

The authors declare no competing financial interest.

358

ACKNOWLEDGMENTS

359

This work was supported by National Natural Science Foundation of China (contact

360

No. 31471654, 31772071, 31501571) and the China Agriculture Research System (NO.

361

CARS-05-05A-03).

362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 17



Page 18 of 33

378 379 380

References

381

(1) Chen, L. X.; Wang, X. Y.; Lu, W. H.; Wu, X. Q.; Li, J. H. Molecular imprinting:

382

perspectives and applications. Chem. Soc. Rev. 2016, 45, 2137-2211.

383

(2) Pan, J. M.; Chen, W.; Ma, Y.; Pan, G. Q. Molecularly imprinted polymers as

384

receptor mimics for selective cell recognition. Chem. Soc. Rev. 2018, 47, 5574-5587.

385

(3) Masque, N.; Marce, R. M.; Borrull, F.; Cormack, P. A. G.; Sherrington, D.C.

386

Synthesis and evaluation of a molecularly imprinted polymer for selective on-line solid-

387

phase extraction of 4-nitrophenol from environmental water. Anal. Chem. 2000, 72,

388

4122-4126.

389

(4) Zhao, T.; Guan, X. J.; Tang, W. J.; Ma, Y.; Zhang, H. X. Preparation of temperature

390

sensitive molecularly imprinted polymer for solid-phase microextraction coatings on

391

stainless steel fiber to measure ofloxacin. Anal. Chim. Acta. 2015, 853, 668-675.

392

(5) Udomsap, D.; Branger, C.; Culioli, G.; Dollet, P.; Brisset, H. A versatile

393

electrochemical sensing receptor based on a molecularly imprinted polymer. Chem.

394

Commun. 2014, 50 7488-7491.

395

(6) Li, S. H.; Yin, G. H.; Zhang, Q.; Li, C. L.; Luo, J. H.; Xu, Z.; Qin, A. L. Selective

396

detection of fenaminosulf via a molecularly imprinted fluorescence switch and silver

397

nano-film amplification. Biosens. Bioelectron. 2015, 71, 342-347.

398

(7) Farka, Z.; Cunderlova, V.; Horackova, V.; Pastucha, M.; Mikusova, Z.; Hlavacek,

399

A.; Skladal, P. Prussian Blue Nanoparticles as a Catalytic Label in a Sandwich 18


Page 19 of 33


400

Nanozyme-Linked Immunosorbent Assay. Anal. Chem. 2018, 90, 2348-2354.

401

(8) Piletsky, S. A.; Piletska, E. V.; Chen, B. N.; Karim, K.; Weston, D.; Barrett, G.;

402

Lowe, P.; Turner, A. P. F. Chemical grafting of molecularly imprinted homopolymers

403

to the surface of microplates. Application of artificial adrenergic receptor in enzyme-

404

linked assay for beta-agonists determination. Anal. Chem. 2000,72, 4381-4385.

405

(9) Chianella, I.; Guerreiro, A.; Moczko, E.; Caygill, J. S.; Piletska, E. V.; Sansalvador,

406

I. M. P. D.; Whitcombe, M. J.; Piletsky, S. A. Direct Replacement of Antibodies with

407

Molecularly Imprinted Polymer Nanoparticles in ELISA-Development of a Novel

408

Assay for Vancomycin. Anal. Chem. 2013, 85, 8462-8468.

409

(10) Tang, Y. W.; Fang, G. Z.; Wang, S.; Sun, J. W.; Qian, K. Rapid Determination of

410

Metolcarb Residues in Foods Using a Biomimetic Enzyme-Linked Immunosorbent

411

Assay Employing a Novel Molecularly Imprinted Polymer Film as Artificial Antibody.

412

J. Aoac. Int. 2013, 96, 453-458.

413

(11) Bi, X. D.; Liu, Z. Facile Preparation of Glycoprotein-Imprinted 96-Well

414

Microplates for Enzyme-Linked Immunosorbent Assay by Boronate Affinity-Based

415

Oriented Surface Imprinting. Anal. Chem. 2014, 86, 959-966.

416

(12) Hong, S. H.; She, Y. X.; Cao, X. L.; Wang, M.; Zhang, C.; Zheng, L. F.; Wang, S.

417

S.; Ma, X. B.; Shao, H.; Jin, M. J.; Jin, F.; Wang, J. Biomimetic enzyme-linked

418

immunoassay based on a molecularly imprinted 96-well plate for the determination of

419

triazophos residues in real samples. Rsc. Adv. 2018, 8, 20549-20556.

420

(13) Li, L.; Peng, A. H.; Lin, Z. Z.; Zhong, H. P.; Chen, X. M.; Huang, Z. Y. Biomimetic

421

ELISA detection of malachite green based on molecularly imprinted polymer film. 19



Page 20 of 33

422

Food Chem. 2017, 229, 403-408.

423

(14) Sun, Q.; Xu, L. H.; Ma, Y.; Qiao, X. G.; Xu, Z. X. Study on a biomimetic enzyme-

424

linked immunosorbent assay method for rapid determination of trace acrylamide in

425

French fries and cracker samples. J. Sci. Food. Agr. 2014, 94, 102-108.

426

(15) Shi, C.; Liu, X. Y.; Song, L. Y.; Qiao, X. G.; Xu, Z. X. Biomimetic Enzyme-linked

427

Immunosorbent Assay Using a Hydrophilic Molecularly Imprinted Membrane for

428

Recognition and Fast Determination of Trichlorfon and Acephate Residues in

429

Vegetables. Food Anal, Method. 2015, 8, 2496-2503.

430

(16) Wang, Q. Q.; Wei, H.; Zhang, Z. Q.; Wang, E. K.; Dong, S. J. Nanozyme: An

431

emerging alternative to natural enzyme for biosensing and immunoassay. Trac-Trend.

432

Anal. Chem. 2018, 105, 218-224.

433

(17) Gao, L. Z.; Zhuang, J.; Nie, L.; Zhang, J. B.; Zhang, Y.; Gu, N.; Wang, T. H.;

434

Feng, J.; Yang, D. L.; Perrett, S.; Yan, X. Intrinsic peroxidase-like activity of

435

ferromagnetic nanoparticles. Nat. Nanotechnol. 2007, 2, 577-583.

436

(18) He, W. W.; Liu, Y.; Yuan, J. S.; Yin, J. J.; Wu, X. C.; Hu, X. N.; Zhang, K.; Liu,

437

J.B.; Chen, C. Y.; Ji, Y. L.; Guo, Y. T. Au@Pt nanostructures as oxidase and peroxidase

438

mimetics for use in immunoassays. Biomaterials 2011, 32, 1139-1147.

439

(19) Borodko, Y.; Thompson, C. M.; Huang, W. Y.; Yildiz, H. B.; Frei, H.; Somorjai,

440

G.A.

441

Compounds: Structure and Stability. J. Phys. Chem. C. 2011, 115, 4757-4767.

442

(20) Knecht, M. R.; Weir, M. G.; Myers, V. S.; Pyrz, W. D.; Ye, H. C.; Petkov, V.;

443

Buttrey, D. J.; Frenkel, A. I.; Crooks, R. M. Synthesis and Characterization of Pt

Spectroscopic Study of Platinum and Rhodium Dendrimer (PAMAM G4OH)

20


Page 21 of 33


444

Dendrimer-Encapsulated Nanoparticles: Effect of the Template on Nanoparticle

445

Formation. Chem. Mater. 2008, 20, 5218-5228.

446

(21) Wang, X.Y.; Zhang, Y.C.; Li, T.F.; Tian, W.D.; Zhang, Q.; Cheng, Y.Y.

447

Generation 9 Polyamidoamine Dendrimer Encapsulated Platinum Nanoparticle Mimics

448

Catalase Size, Shape, and Catalytic Activity. Langmuir 2013, 29, 5262-5270.

449

(22) Li, W.; Bin, C.; Zhang, H. X.;

450

stabilized Pt nanozyme for peroxidase mimetics and its application on colorimetric

451

detection of mercury(II) ions. Biosens. Bioelectron. 2015, 66, 251-258.

452

(23) Ke, H.; Zhang, X.; Huang, C. S.; Jia, N. Q. Electrochemiluminescence evaluation

453

for carbohydrate antigen 15-3 based on the dual-amplification of ferrocene derivative

454

and Pt/BSA core/shell nanospheres. Biosens. Bioelectron. 2018, 103, 62-68.

455

(24) Zhang, A. M.; Huang, C. S.; Shi, H.Y.; Guo, W. W.; Zhang, X.; Xiang, H. K.; Jia,

456

T.; Miao, F.; Jia, N. Q. Electrochemiluminescence immunosensor for sensitive

457

determination of tumor biomarker CEA based on multifunctionalized Flower-like

458

Au@BSA nanoparticles. Sensor. Actuat. B-Chem. 2017, 238, 24-31.

459

(25) Goswami, N.; Giri, A.; Bootharaju, M. S.; Xavier, P. L.; Pradeep, T.; Pal, S. K.;

460

Copper Quantum Clusters in Protein Matrix: Potential Sensor of Pb2+ Ion. Anal. Chem.

461

2011, 83, 9676-9680.

462

(26) Yao, T.; Gu, X.; Li, T. F.; Li, J. G.; Li, J.; Zhao, Z.; Wang, J.; Qin, Y. C.; She, Y.

463

X. Enhancement of surface plasmon resonance signals using a MIP/GNPs/rGO nano-

464

hybrid film for the rapid detection of ractopamine. Biosens. Bioelectron. 2016, 75, 96-

465

100.

Sun, Y. H.; Wang, J.; Zhang, J. L.; Fu, Y. BSA-

21



Page 22 of 33

466

(27) Zhu, X. D.; Zeng, Y. B.; Zhang, Z. L.; Yang, Y. W.; Zhai, Y. Y.; Wang, H. L.;

467

Liu, L. Y.; Hu, J.; Li, L. A new composite of graphene and molecularly imprinted

468

polymer based on ionic liquids as functional monomer and cross-linker for

469

electrochemical sensing 6-benzylaminopurine. Biosens. Bioelectron. 2018, 108, 38-45.

470

(28) Chen, C.; Wang, Y. H.; Zhao, X. P.; Wang, Q.; Qian, Y. Z. The combined toxicity

471

assessment of carp (Cyprinus carpio) acetylcholinesterase activity by binary mixtures

472

of chlorpyrifos and four other insecticides. Ecotoxicology 2014, 23, 221-228.

473

(29) Lv, S.; Zhang, K. Y.; Lin, Z. Z.; Tang, D. P. Novel photoelectrochemical

474

immunosensor for disease-related protein assisted by hemin/G-quadruplex-based

475

DNAzyme on gold nanoparticles to enhance cathodic photocurrent on p-CuBi2O4

476

semiconductor. Biosens. Bioelectron. 2017, 96, 317-323.

477

(30) Meher, H. C.; Gajbhiye, V. T.; Singh, G.; Kamra, A.; Chawla, G. Persistence and

478

Nematicidal Efficacy of Carbosulfan, Cadusafos, Phorate, and Triazophos in Soil and

479

Uptake by Chickpea and Tomato Crops under Tropical Conditions. J. Agr. Food. Chem.

480

2010, 58, 1815-1822.

481

(31) Xu, M. L.; Gao, Y.; Han, X. X.; Zhao, B. Detection of pesticide residues in food

482

using surface-enhanced Raman spectroscopy: A review. J. Agric. Food Chem. 2017, 65,

483

6719−6726.

484

(32) Liu, Y.; Zhou, H. B.; Hu, Z. W.; Yu, G. X; Yang, D. T; Zhao, J. S. Label and label-

485

free based surface-enhanced Raman scattering for pathogen bacteria detection: A

486

review. Biosens. Bioelectron. 2017, 94, 131–140.

487

(33) Zhao, F. N.; She, Y. X.; Zhang, C.; Cao, X. L.; Wang, S. S.; Zheng, L. F.; Jin, M. 22


Page 23 of 33


488

J.; Shao, H.; Jin, F.; Wang, J. Selective solid-phase extraction based on molecularly

489

imprinted technology for the simultaneous determination of 20 triazole pesticides in

490

cucumber samples using high-performance liquid chromatography-tandem mass

491

spectrometry. J. Chromatogr. B. 2017, 1064, 143-150.

492

(34) Zhang, J. J.; Sun, L.; Chen, C.; Liu, M.; Dong, W.; Guo, W. B.;. Ruan, S. P. High

493

performance humidity sensor based on metal organic framework MIL-101(Cr)

494

nanoparticles. J. Alloy. Compd. 2017, 695, 520-525.

495

(35) Hu, Y. L.; Liao, J.; Wang, D. M.; Li, G. K. Fabrication of Gold Nanoparticle-

496

Embedded Metal-Organic Framework for Highly Sensitive Surface-Enhanced Raman

497

Scattering Detection. Anal. Chem. 2014, 86, 3955-3963.

498

(36) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Nucleic acid-functionalized Pt

499

nanoparticles: Catalytic labels for the amplified electrochemical detection of

500

biomolecules. Anal. Chem. 2006, 78, 2268-2271.

501

(37) Zhang, C.; Du, P. F.; Jiang, Z. J.; Jin, M. J.; Chen, G.; Cao, X. L.; Cui, X. Y.;

502

Zhang, Y. D.; Li, R. X.; Abd El-Aty, A. M.; Wang, J. A simple and sensitive

503

competitive bio-barcode immunoassay for triazophos based on multi-modified gold

504

nanoparticles and fluorescent signal amplification. Anal. Chim. Acta. 2018, 999, 123-

505

131.

506

(38) Guo, Y. R.; Liu, R.; Liu, Y.; Xiang, D. D.; Liu, Y. H.; Gui, W. J.; Li, M. Y.; Zhu,

507

G. N. A non-competitive surface plasmon resonance immunosensor for rapid detection

508

of triazophos residue in environmental and agricultural samples. Sci. Total. Environ.

509

2018, 613, 783-791. 23



Page 24 of 33

510

(39) Liu, B. B.; Gong, H.; Wang, Y. L.; Zhang, X. S.; Li, P.; Qiu, Y. L.; Wang, L. M.;

511

Hua, X. D.; Guo, Y. R.; Wang, M. H.; Liu, F. Q.; Liu, X. J.; Zhang, C. Z. A gold

512

immunochromatographic assay for simultaneous detection of parathion and triazophos

513

in agricultural products. Anal. Methods-Uk. 2018, 10, 422-428.

514

(40) Boroduleva, A. Y.; Wu, J.; Yang, Q. Q.; Li, H.; Zhang, Q.; Li, P. W.; Eremin, S.

515

A. Development of fluorescence polarization immunoassays for parallel detection of

516

pesticides carbaryl and triazophos in wheat grains. Anal. Methods-Uk. 2017, 9, 6814-

517

6822.

518

(41) Xu, Z. L.; Wang, Q.; Lei, H. T.; Eremin, S. A.; Shen, Y. D.; Wang, H.; Beier, R.

519

C.; Yang, J. Y.; Maksimova, K. A.; Sun, Y. M. A simple, rapid and high-throughput

520

fluorescence

521

organophosphorus pesticides in vegetable and environmental water samples. Anal.

522

Chim. Acta. 2011, 708, 123-129.

523

Legends

524

Scheme 1. Representative illustration of the design strategy and fabrication process of

525

competitive BNLISA.

526

Figure 1. Analysis and characterization of MIPs/NIPs and Pt@BSA-hapten probe.

527

Figure 2. Analysis enhanced ability of Au NPs@MIL-101 for SERS signal.

528

Figure 3. Optimization of the competition conditions for BNLISA.

529

Figure 4. Analytical performance of the competitive BNLISA.

530

Tables

531

Table 1. Cross- reactivity studies between triazophos and structural analogues.

polarization

immunoassay

for

simultaneous

detection

of

24


Page 25 of 33


532

Table 2. Comparison of various methods for triazophos detection.

533

Table 3. Results of the detection of triazophos in water samples by the proposed method.

534

25



Figures

Scheme 1. Representative illustration of the design strategy and fabrication process of competitive BNLISA. (a). Synthesis of MIP microspheres; (b). Fabrication of Pt@BSA-hapten signal probe; (c). Design of competitive BNLISA; (d). Synthesis of the SERS-enhanced substrate Au NPs@MIL-101.


Page 26 of 33

Page 27 of 33


Figure 1. Analysis and characterization of MIPs/NIPs and Pt@BSA-hapten probe. (a). SEM of MIPs; (b). SEM of NIPs; (c). TEM of Pt NPs; (d). UV–vis absorption spectra of Pt NPs, BSA, BSA-hapten and Pt@BSA-hapten.



Figure 2. Analysis enhanced ability of Au NPs@MIL-101 for SERS signal. (a) SERS spectra of TMB2+ with Au NPs, Au NPs@MIL-101 and MIL-101; (b) The varieties of Raman signals of TMB2+ from 0 to 9 min for Au NPs substrate; (c) The varieties of Raman signals of TMB2+ from 0 to 9 min for Au NPs@MIL-101 substrate; (d) The relative intensity of SERS signals at 558 cm-1 from 13 different batches Au NPs@MIL101.


Page 28 of 33

Page 29 of 33


Figure 3. Optimization of the competition conditions for BNLISA. (a). TEM of Pt@BSA-hapten with BSA-hapten of 67.8 μg·mL-1 ; (b). TEM of Pt@BSA-hapten with BSA-hapten of 169.5 μg·mL-1; (c). TEM of Pt@BSA-hapten with BSA-hapten of 678 μg·mL-1. Optimization of assay conditions for detection of triazophos; (d). Different work buffer solution; (e) different concentration of the BSA-hapten; (f). Different pH of work buffer solution.



Figure 4. Analytical performance of the competitive BNLISA. (a). UV–vis absorption spectra for triazophos detection in the concentration range of 0 – 10000 ng·mL-1 with the colourimetric method; (b). Calibration curve obtained from the absorbance values at 450 nm; (c). SERS spectra for triazophos detection in the concentration range of 0 – 10000 ng·mL-1 with the SERS method; (d). Calibration curve obtained from the SERS signal intensity at 558 cm-1.


Page 30 of 33

Page 31 of 33


Tables Table 1. Cross- reactivity studies between triazophos and structural analogues

MIPs Analogues

Structure

NIPs

IC50 (ng·mL-1)

CR (%)

IC50 (ng·mL-1)

CR (%)

Triazophos (1)

112

100

1513

100

Methomyl (2)

> 106

< 0.01

> 103.5

< 49

Triazolone (3)

> 106

< 0.01

> 103.5

< 49

Carbaryl (4)

> 106

< 0.01

> 106.0

< 0.01

Trichlorfon (5)

> 103.8

< 1.8

103.2

93

Parathion (6)

> 103.8

< 1.8

> 103.5

< 49

Methamidophos (7)

> 103.8

< 1.8

> 103.5

< 49

Chlorpyrifos (8)

> 103.8

< 1.8

> 103.5

< 49



Page 32 of 33

Table 2. Comparison of various methods for triazophos detection

Linear range Assay format

LOD

Method

Ref -1

-1

(ng⸱mL )

(ng⸱mL )

0.98 - 8.29

0.096

38

Colorimetric

-

50

39

fluorescence

40 - 200

40

40

fluorescent polarization immunoassay

fluorescence

16.09 - 511.84

5.86

41

Competitive-BELISA

Colorimetric

0.001 - 10000

0.001

12

Colorimetric

5 – 1000

1

This

SERS

1 – 10000

1

work

surface plasmon Non-competitive direct SPR immunosensor resonance competitive gold immunochromatographic assay competitive fluorescence polarization immunoassays

Competitive-BNLISA

Table 3. Results of the detection of triazophos in water samples by the proposed method

Sample

Water

Pear

Recoveries/RSD (%)

Spiking concentration (µg·kg-1)

Colourimetric

SERS

HPLCMS/MS

10

92.0/7.8

80.5/10.1

99.7/2.3

100

103.4/8.1

97.9/10.9

103.2/1.4

500

109.8/9.4

110.7/13.7

100.8/1.2

20

78.4/11.2

65.6/15.2

89.8/.1.0

100

75.6/9.8

80.2/13.5

98.2/2.1

500

81.2/9.3

76.3/11.8

101/2.6


Page 33 of 33


Graphical Abstract: Competitive Biomimetic Nanozyme-Linked Immunosorbent Assay for Colorimetric and SERS Sensing of small molecular.


Sensitive and Simple Competitive Biomimetic ... - ACS Publications

Recommend Documents