A New Method for Accurate Determination of Polyphenol Oxidase

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New Analytical Methods

A New Method for Accurate Determination of Polyphenol Oxidase Activity Based on Reduction in SERS Intensity of Catechol Tingtiao Pan, Da-Wen Sun, Jitendra Paliwal, Hongbin Pu, and qing-yi wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03985 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 13, 2018

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Journal of Agricultural and Food Chemistry

A New Method for Accurate Determination of Polyphenol Oxidase Activity Based on Reduction in SERS Intensity of Catechol

Ting-tiao Pan1,2,3,5, Da-Wen Sun1,2,3,4∗, Jitendra Paliwal5, Hongbin Pu1,2,3, Qingyi Wei1,2,3

1

School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China 2

Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China

3

Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods, Guangzhou Higher Education Mega Center, Guangzhou 510006, China 4

Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland

5

Department of Biosystems Engineering, University of Manitoba, E2-376, EITC, 75A Chancellor’s Circle, Winnipeg, R3T 2N2, Manitoba, Canada

∗

Corresponding author. Tel: +353-1-7167342; Fax: +353-1-7167493.

E-mail address: [email protected]: www.ucd.ie/refrig; www.ucd.ie/sun. 1

ACS Paragon Plus Environment


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Abstract: Rapid and accurate measurement of polyphenol oxidase (PPO) activity is important in the

2

food industry as PPOs play a vital role in catalyzing enzymatic reactions. The aim of this study was

3

to develop surface-enhanced Raman scattering (SERS) approach for accurate determination of PPO

4

activity in fruit and vegetables using the reduction in SERS intensity of catechol in reaction medium.

5

Within a certain catechol concentration, when a purified PPO solution was analyzed, the reduction in

6

SERS intensity (∆I) was linear to PPO activity (Ec) in a wide range of 500-50000U/L, and a linear

7

regression equation of log∆I/∆t = 0.6223 logEc + 0.8072, with a correlation coefficient of 0.9689 and

8

a limit of detection of 224.65 U/L was obtained. The method was used for detecting PPO activity in

9

apple and potato samples, and the results were compared with those obtained from colorimetric assay,

10

demonstrating that the proposed method could be successfully used for detecting PPO activity in

11

food samples.

12

Keywords: Enzyme activity assay, polyphenol oxidase, catechol, SERS, AgNPs

13

2


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Introduction

15

Polyphenol oxidase (PPO) is an oxidoreductase, which can catalyze two different reactions in the

16

presence of molecular oxygen, the o-hydroxylation of monophenols to o-diphenols, and the oxidation

17

of o-diphenols to o-quinones.1-3 The PPO family includes monophenol oxidase (tyrosinase), diphenol

18

oxidoreductase (catechol oxidase), and laccase,4 which are widely dispersed among almost all plant

19

tissues, bacteria, fungi, insects, and animals. PPOs can catalyze the oxidation of phenols to the

20

corresponding quinones. Quinones are slightly colored products that undergo further reactions,

21

leading to high molecular mass dark pigments called melanins.5 PPOs initiate the synthesis of

22

melanins, which are responsible for browning of fruit and vegetables, wound healing in plants and

23

arthropods, and pigmentation of hair and skin.6 In the food industry, enzymatic browning of fruit,

24

vegetables, and processed products is the main cause of quality deterioration, and it is also a major

25

factor that decreases the commercial value of such products.1,7 Nonetheless, PPO-induced enzymatic

26

browning is not always an undesirable reaction. The brown color of tea, cocoa and coffee is

27

developed by enzymatic browning catalyzed by PPO.

28

As PPOs play a vital role in catalyzing enzymatic reactions in the food industry, many studies

29

dealing with the determination of PPO activity and its changes during storage and processing have

30

been presented.8 Colorimetric assay is the most traditional and common method used for analyzing

31

PPO activity.9-12 In addition, the determination of PPO activity by optical, amperometric, and

32

immune biosensors,6,13,14 and its rapid prediction by hyperspectral imaging and other methods have

33

also been reported.15-17 Among these methods, although colorimetric assay is the simplest one, its test

34

result is labile to interference due to the instability of the reaction product. Biosensor-based methods

35

are accurate and sensitive, but the measurement procedure is generally complex, time-consuming,

36

and tedious,13 making such methods unsuitable for rapid determination of the PPO activity.

37

Hyperspectral imaging is a rapid and nondestructive technology, but it requires complicated data

38

processing and analysis and the results are easily affected by the tested samples.16 Therefore, it is 3



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necessary to explore more suitable methods for rapid, sensitive, and accurate determination of PPO

40

activity in fruit, vegetables, and other products.

41

As a combination of Raman spectroscopy and nanotechnology, surface-enhanced Raman

42

scattering (SERS) is a rapid, sensitive, and label-free technology collecting fingerprint-like spectra. It

43

can enhance the weak Raman scattering signals tremendously, and the enhancement can often reach

44

up to 106-108 by adsorbing targeted molecules on the vicinity of nanostructured metallic surfaces.18-21

45

As enhanced substrates, metal nanoparticles can magnify the Raman scattering signals via chemical

46

and/or electromagnetic mechanisms.22 Over the past decade, as a novel and useful analytical method,

47

SERS has been widely used for food safety evaluation,23,24 such as microorganisms,19,25 toxins,18,26

48

harmful chemical residues and contaminants.27-32 The main advantages of SERS are high sensitivity

49

and low limit of detection (LOD), thus it is suitable for detecting very low PPO activity present in

50

some food samples, for which the colorimetric assay is not applicable because of the extremely low

51

absorbance reading obtained. Moreover, SERS circumvents the problems of instability of quinones

52

and the absorbance of secondary reaction products in colorimetric assay. However, its signal

53

reproducibility and substrate stability are not satisfactory. To date, the SERS method is rarely used

54

for the determination of enzyme activities in food samples,33-35 especially for the determination PPO

55

activity.

56

Therefore, the aim of the present study is to investigate the potential of SERS method based on the

57

reduction in SERS intensity of catechol in the reaction medium for accurate determination of PPO

58

activity in fruit and vegetables. For this purpose, an easily prepared substrate, silver nanoparticles

59

(AgNps) colloid, was first synthesized. The SERS signals of the reaction medium in different stages

60

were then collected, and a linear relationship between the reduction in SERS intensity (∆I) and PPO

61

activity (Ec) was established, which was finally used to determine the PPO activity of real samples.

62

To the best of our knowledge, this is the first study using SERS method for determination of PPO

63

activity in fruit and vegetables. 4


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Materials and Methods

65

Chemicals and reagents

66

Silver nitrate (99.9%), trisodium citrate (dihydrate, 98%), sodium nitrate, catechol (AR, 99.0%),

67

disodium phosphate, and sodium dihydrogen phosphate were purchased from Shanghai Aladdin

68

Bio-Chem Technology Co. Ltd. (Shanghai, China). Purified PPO from mushroom was purchased

69

from Shanghai Shifeng Biological Technology Co. Ltd. (Shanghai, China). Ultrapure water was

70

generated from a Milli-Q system (EMD Millipore Co., Billerica, MA, USA). Apples and potatoes

71

were obtained from local retail shops in Guangzhou, China.

72

Trisodium citrate solution (1%, m/v) was prepared by dissolving 1.0 g of trisodium citrate in 100.0

73

mL of ultrapure water. Sodium nitrate solution (1 M) was obtained by dissolving 8.5 g of sodium

74

nitrate in 100.0 mL of ultrapure water. Catechol standard solution (0.1 M) was obtained by

75

dissolving 1.1 g of catechol in 100.0 mL of ultrapure water, and different concentrations catechol

76

solutions were prepared by diluting the standard solution with ultrapure water. Na2HPO4-NaH2PO4

77

buffer solution (PBS, 0.2 M, pH 6.4) was created by mixing the Na2HPO4 solution (0.2 M) and

78

NaH2PO4 solution (0.2 M), and then diluted 4 times. The PPO stock solution (1.0×107 U/L) was

79

prepared by dissolving 1.0 mg of purified PPO (25000 U/mg) in 2.5 mL of PBS and a series of PPO

80

standard solutions were obtained by diluting the stock solution.

81

Working principle and computing procedures of the measurement

82

The working principle of the SERS method depends on the enzymatic reaction is given below.

83

PPO

Catechol+1/2 O2 Quinone + H2O

(1)

84

As shown in Eq. (1), PPO can catalyze the oxidation of catechol in the presence of molecular

85

oxygen, which causes the consumption of catechol in the reaction medium and eventually leading to

86

a decrease in SERS intensity of the medium. This is because the SERS intensity is related to the

87

concentration of catechol in the medium. Thus, the principle of the measurement was based on the

88

determination of the reduction in SERS intensity of the reaction medium before and after enzymatic 5



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reaction catalyzed by PPO, and the reduction in SERS intensity was related to the activity of PPO

90

added into the medium. When the reaction medium was only catechol solution, there was no

91

enzymatic reaction in the medium and only a stable SERS signal of catechol was monitored. After

92

the addition of PBS and PPO solutions, the SERS signal (I1) of the reaction medium involving

93

catechol solution, PBS and PPO solution was detected. After a brief enzymatic reaction (5 min), the

94

measurement was conducted again and the corresponding SERS signal was referred to I2. The

95

difference between I1 and I2 stemmed from the enzymatic oxidation of catechol, and the degree of

96

oxidation was affected by the PPO activity and reaction time. Thus, the reduction in SERS intensity

97

of the reaction medium with different PPO activities was detected to obtain a regression curve for

98

determining PPO activity.

99

According to the definition of enzymatic activity unit (E) by Enzyme Commission of International

100

Biochemical Association,36 the concentration unit of enzymatic activity (Ec) can be defined as the

101

number of E per volume (U/L). Based on Eq. (1), the concentration unit of PPO activity can be

102

deduced with the reduction in SERS intensity of catechol as given below:

103

PPO activity U⁄L =

∆I ∆t

V

×K×v×L

(2)

104

where, ∆I is the reduction in SERS intensity of catechol in the linear response area, ∆t is the time

105

interval in the linear response area (min), V is the total volume of reaction medium (mL), K is

106

micromolar SERS coefficient of catechol (L µmol-1 cm-1), v is the volume of PPO-containing sample

107

(mL), and L is the optical path length of the colorimetric cup (cm).

108

Silver nanoparticles synthesis and characterization

109

AgNps colloid was synthesized based on the method available with slight modification.37 Briefly,

110

45.0 mg of silver nitrate was dissolved in 250.0 mL of ultrapure water in a clean flask. The solution

111

was stirred and heated to boiling in a constant temperature heating magnetic whisk (DF-101S, Yuhua

112

Instrument Co. Ltd., Yiwu, China). The solution was continuously boiled and stirred, and 2.5 mL

113

trisodium citrate solution (1%, m/v) was quickly added to the boiling solution. The resultant solution 6


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was boiled and stirred for 1 h, and then cooled to room temperature at 25oC.

115

An UV-1800 spectrophotometer (Shimadzu Co., Kyoto, Japan) was used to record the adsorption

116

spectra from 300 to 700 nm at 1 nm interval. The freshly synthesized AgNps colloid was diluted 5

117

times and analyzed in quartz cuvette. Transmission electron microscope (TEM) images were

118

obtained on a field-emission high-resolution JEM-1400 Plus (JEOL Ltd., Tokyo, Japan) microscope

119

at an acceleration voltage of 120 kV. The samples for TEM analysis were prepared by dropping the

120

diluted solution of AgNps colloid on carbon film (Beijing Xinxing Braim Technology Co. Ltd,

121

Beijing, China), and the film was dried at 60 oC. The distribution of diameter of the nanoparticles

122

was measured on a two angle particle and molecular size analyzer (Zetasizer Nano ZS, Malvern

123

Instruments Ltd., Worcestershire, UK) at 25 °C with a scattering angle of 173° at λ = 633 nm.

124

Spectral acquisition, processing, and analysis

125

Raman and SERS spectra were collected using a Raman spectrometer (LabRAM HR, HORIBA

126

Scientific, Longjumeau, France) with a 532 nm laser. The spectra were collected within the range of

127

400-1700 cm-1. 2 accumulations with a total acquisition time of 10 s were used. Each measurement

128

was repeated 3 times. A quartz cuvette (1 cm) was used for all liquid measurements. The LabSpec 6

129

Software (7.1.0.1, HORIBA Scientific, Longjumeau, France) was used for instrument control,

130

spectral acquisition, processing, and data analysis. Smoothing was employed to reduce spectral

131

noises and baseline correction was performed to subtract the baseline shift for all spectra.38,39

132

Detection of catechol solution by SERS method

133

Prior to SERS measurement, 400.0 µL of AgNps colloid and 400.0 µL of catechol solution were

134

added into a centrifuge tube, the resultant mixture was stirred for 5 s. Then 75.0 µL of sodium nitrate

135

solution (1 M) was added into the tube and also stirred for 5 s. Since sodium nitrate has no Raman

136

signal, it promotes the aggregation of AgNps. The resultant solution was measured directly. In order

137

to choose the appropriate catechol concentration for PPO activity determination, concentration

138

response measurements were performed. A series of catechol standard solutions were prepared, and 7



139

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their SERS intensities were measured in triplicate and averaged to represent the SERS responses.

140

In order to provide a comparison for SERS spectrum of catechol, Raman spectra of catechol

141

powder, catechol solution, and AgNps colloid were collected. In addition, the SERS spectrum of the

142

reaction medium involving catechol solution, PBS, and PPO solution was also given.

143

Analytical procedure for purified PPO solution

144

An amount of 0.5 mL of catechol solution (90 mM), 2.0 mL of PBS (0.05 M), and 2.0 mL of

145

ultrapure water were added into a centrifuge tube, respectively. The resultant mixture in the tube was

146

preheated for 10 min in a water-bath at 35 °C, and 0.5 mL of PPO standard solution was added into

147

the mixture. The mixture (reaction medium) was shaken and placed back into the water-bath at 35 °C

148

for enzymatic reaction, and the reaction was stopped by boiling water. Finally, the resultant solution

149

was used for SERS determination. The analytical procedure was the same as the measurement of

150

catechol solution.

151

Analysis of real samples

152

Recovery experiment by SERS method

153

Apple and potato PPO crude extracts were prepared based on the following process. After washing

154

with tap water and distilled water, the samples were wiped dry and about 1 mm thick skin was peeled.

155

Ten grams of fresh tissue was minced finely and added into a mortar containing 15.0 mL of

156

precooled PBS (0.05 M), which was then crushed rapidly in an ice-bath. The homogenate was added

157

into a centrifuge tube and centrifuged at 8000 rpm and 4 °C for 15min, the supernatant was loaded

158

onto a column, and the precipitate was transferred into a new mortar and extracted once again. Two

159

parts of supernatants were mixed together and diluted to 50.0 mL. The activities of the PPO crude

160

extracts were measured by the SERS method, and the recoveries of the crude extracts were tested to

161

examine the method’s reliability. The recovery experiment was performed according to the following

162

procedure: in each centrifuge tube, 1.0 mL of the apple and potato PPO crude extracts with the

163

activities of 0 (inactivated), 2884.00±151.25 U/L, and 0 (inactivated), 7484.00±191.00 U/L were 8


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added, respectively, and then spiked with 1.0 mL of PPO standard solution with two activity levels

165

(10,000 and 20,000 U/L). Finally, the activities of the resultant mixtures were measured. The

166

analytical procedure was the same as the procedure for the PPO standard solution.

167

SERS determination of PPO activity in real samples

168

The validated SERS method was used for determining the PPO activity in real samples. PPO crude

169

extracts of apple and potato were obtained and diluted 2, 5, 8, and 10 times. The activities of the PPO

170

crude extracts and their diluents were measured by the SERS method and the colorimetric assay, and

171

the results of these two methods were compared.

172

Analysis steps of colorimetric assay

173

PPO crude extracts of apple and potato were obtained following the procedure described in the

174

above SERS analysis. The activities of the PPO crude extracts were determined according to the

175

colorimetric assay reported previously,40 with some modifications. Briefly, the reaction medium

176

including 0.5 mL of catechol solution, 2.0 mL of PBS, and 0.5 mL of PPO crude extract, was

177

inoculated at 35 °C for 10 min. The reaction was stopped by boiling water, and the PPO activity was

178

determined by measuring the increase in absorbance of the reaction product at 398 nm. In

179

colorimetric assay, E was defined as ∆A398 of 0.01 per min, Ec was expressed in the number of E per

180

gram fresh tissue, which was then converted into the number of E per volume.

181

Results and Discussion

182

Characterization of the AgNps colloid

183

AgNps colloid is one of the most widely used SERS substrate for its ease of synthesis and

184

uniformity across synthesized batches. In the current study, three batches of AgNps colloid were

185

synthesized, characterized, and used as SERS substrates. Their characterization results are presented

186

in Figure 1. As illustrated in the absorption spectra of AgNps colloid (Figure 1a), the full width at

187

half-maximum was 116.10±0.14 nm, and the wavenumber at maximum absorption value was

188

414.56±0.88 nm, indicating the similarity in the properties of the three batches of AgNps. The 9



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189

absorption spectra have broadest band indicating non-uniform distribution of AgNps, which are good

190

agreement with that of previous literature.41,42 Moreover, the overlap in spectra suggests excellent

191

repeatability of AgNps synthesis. Size distribution and average particle diameter of AgNps colloid

192

were obtained by dynamic light scattering (DLS) analysis, and the results are shown in Figure 1b. It

193

was shown that most AgNps have a spherical shape with a diameter ranging from 30 nm to 100 nm,

194

and the average particle diameter reading of 49.30±0.60 nm. TEM image in Figure 1c demonstrated

195

an average particle diameter of 50.08±9.16 nm, which was consistent with the result of DLS analysis.

196

The results of DLS analysis and TEM image indicate that AgNps are a relatively stable structure,

197

which are consistent with most of the previous literature.41,42

198

SERS activity of catechol on AgNps

199

In order to confirm the SERS activity of catechol on AgNps colloid, Raman spectrum of catechol

200

solution (10 mM) and SERS spectrum of the same catechol solution mixing with AgNps colloid are

201

compared in Figure 2. As shown in Figure 2a, no signals were monitored when the catechol solution

202

was measured directly. The reason may be that the concentration of catechol solution was too low to

203

be detected. In contrast, when the catechol solution was mixed with AgNps colloid, the signal was

204

easily detected because it was magnified by several orders of magnitude. One possible reason was

205

that the localized surface plasmon resonance could occur due to the addition of AgNps colloid, and

206

the phenolic hydroxyl groups in catechol molecule could promote its adhesion to silver surface to

207

generate resonance coupling, eventually leading to the enhancement of Raman signal.43,44 It can be

208

seen from Figure 2b that, many characteristic band occurred in the SERS spectrum of catechol

209

solution, especially the ones at 589, 1258, 1494, and 1612 cm-1, which were likely to be associated

210

with various vibrational modes of catechol molecule by comparison with Raman spectrum of

211

catechol powder (Figure 2c). Although some band seems shifted or broadened, the strongest band in

212

the signature corresponds to the published vibrational modes for catechol or other phenolic

213

substances. For example, the dominated SERS band at 1258 cm-1 could be specifically used to 10


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identify the presence of catechol because the band was assigned to the stretching vibrational mode of

215

C-O.18,45 Besides, no characteristic band occurs around 1258 cm-1 in Raman spectra of quinones.46,47

216

Also, it is well known that the amount of enzyme does not change before and after the enzymatic

217

reaction, therefore, the band at 1258 cm-1 is unlikely to be a Raman band produced by PPO. Thus,

218

the band at 1258 cm-1 was selected for quantitative analysis. Moreover, Raman spectrum of AgNps

219

colloid is also presented in Figure 2d, and it can be seen that there were lots of weak band around

220

610, 948, 1023, 1362, 1397, and 1648 cm-1 in the spectrum, which may come from citrate as

221

reduction reagent in AgNps synthesis step.48,49 However, none of these band disturb the appearance

222

of the characteristic band of catechol. In addition, SERS spectra of the reaction medium involving

223

catechol solution, PBS, and PPO solution were also collected and are shown in Figure 2e and Figure

224

2f. Comparing Figure 2e with Figure 2b, it can be seen that both spectra displayed similar tendency

225

throughout the entire spectral region, but their SERS intensities were not the same, i.e., the addition

226

of PBS and PPO solutions did not introduce new Raman characteristic peaks. Additionally, the weak

227

difference in SERS intensities of these two spectra may result from the difference in fluorescence

228

backgrounds of the two different solutions. As shown in Figure 2f, a low SERS signal was obtained

229

for the same reaction medium after 5 min of enzymatic reaction. The SERS intensity of the resultant

230

solution was weaker than the original one at the same characteristic peaks probably because of the

231

degradation of catechol during enzymatic oxidation.

232

SERS response of catechol concentration

233

In order to select the appropriate concentration range, 10 serial dilutions of solutions were first

234

performed starting from the catechol concentration of 10.0 µM to 0.1 M, and their SERS spectra

235

were monitored to investigate the detection capability of the SERS method. Finally, the catechol

236

concentration ranging from 0.1 mM to 11.0 mM was selected. The SERS spectra of catechol

237

solutions at this concentration range are presented in Figure 3a, indicating an increase in SERS

238

responses with increasing concentration. Figure 3b shows the SERS peak intensity of the vibrational 11



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239

mode at 1258 cm-1 as a function of concentration, and the change trend followed a typical

240

dose-response curve, with SERS intensity increasing as increase in catechol concentration. When the

241

concentration was lower than 0.1 mM, SERS signal of catechol was too weak to be detected.

242

However, when the concentration was higher than 0.1 mM, SERS intensity increased gradually with

243

the concentration increasing from 0.1 mM to 11.0 mM. More specifically, when the concentration

244

was within 0.1-1.0 mM, SERS intensity was low and the increasing rate of intensity was also low.

245

When the concentration was in the range of 1.0-11.0 mM, SERS intensity increased as the

246

concentration increased, and when the concentration was higher than 9.0 mM, the rate of increase in

247

intensity reduced again. The increase of intensity was nonlinear in the whole concentration range, but

248

it was linear within short spans. Thus, the optimum concentration range for this method was 1.0-9.0

249

mM. The relationship between SERS intensity and the concentration varying from 1.0×103 µM to

250

9.0×103 µM is plotted in Figure 3c, showing that the SERS intensity was linearly related to the

251

concentration. Therefore, the recommended starting concentration of catechol for PPO activity

252

determination based on the SERS method was 9.0 mM.

253

PPO activity determination

254

Micromolar SERS coefficient of catechol

255

According to Eq. (2), the micromolar SERS coefficient of catechol (K) is the slope of linear

256

regression curve between SERS intensity and concentration of catechol. If the SERS method was

257

used to measure the PPO activity, the value of K must be obtained first under the specified conditions.

258

The linear regression curve between SERS intensity and catechol concentrations is shown in Figure

259

3c, and with curve fitting the linear regression equation obtained was y = 2.63x + 1869.93, with a

260

correlation coefficient (R2) of 0.9966. Therefore, K = 2.63 L µmol-1 cm-1 and PPO activity can be

261

calculated by substituting the value of K into Eq. (2).

262

Activity-dependent reductions in SERS intensity

263

In order to determine the linear range of PPO activity for this method, 10 serial dilutions were 12


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264

carried out for PPO stock solution to prepare the reaction medium with PPO activities of

265

1.0×101-1.0×106 U/L. The reduction in SERS intensity of the reaction medium with increasing PPO

266

activities were measured after enzymatic reaction for 5 min, and it was found that the changes in

267

SERS intensity were little for the reaction medium with PPO activities of 1.0×101 U/L and 1.0×102

268

U/L. The decrease in SERS intensity increased gradually at PPO activities ranging from 1.0×103 U/L

269

to 1.0×105 U/L, and the reduction was almost the same for the other two PPO activities of 1.0×105

270

U/L and 1.0×106 U/L. Taking into account these facts, the reduction in SERS intensity of reaction

271

medium with PPO activities of 500, 1000, 5000, 10,000, and 50,000 U/L were selected, and the

272

results are given in Figure 4a-e. The relationship between the reduction in SERS intensity and PPO

273

activities was plotted in double-logarithmic scales and is given in Figure 4f. The fitted linear

274

equation was y (log∆I/∆t) = 0.6223 x (logEc) + 0.8072 in the PPO activity range of 500-50,000 U/L,

275

with R2 = 0.9689. When the reaction medium without PPO solution was used, ∆I/∆t was

276

114.24±46.60, therefore, the LOD of the present method was determined as 224.65 U/L (LOD =

277

3S/K, K=0.6223, S=46.60). Compared with the linear range of 1000-10,000 U/L and LOD of 500

278

U/L for colorimetric assay reported by Jiang et al.,17 the linear range in the current study was wider,

279

being 500-50,000 U/L, and LOD was lower, being 224.65 U/L.

280

Sample analysis

281

Recovery of model samples

282

In order to confirm the feasibility of the SERS method for real sample testing, apple and potato

283

were used to perform recovery experiments. The sum of the activity of the crude extract and the

284

spiked activity was recorded as the total activity. The activity of the spiked samples was determined

285

using the SERS method developed in the current study, and was recorded as the recovery activity.

286

The recovery of samples was calculated as the recovery activity divided by the total activity. The

287

results of the recovery experiments are summarized in Table 1, showing that the recoveries were in

288

the range of 77.86-96.84%. These results indicate that the SERS method is accurate and reliable. 13



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289

Determination of PPO activity in real samples

290

In addition to the recovery experiment, the activities of apple and potato PPO crude extracts and

291

their diluents were measured directly by the SERS method. The results of the measurements are

292

given in supplementary information. Meanwhile, the activities of the same crude extract solutions

293

and diluents were measured by colorimetric assay, and the results are compared in supplementary

294

information, showing that the results of these two methods were similar.

295

Results of comparative analysis

296

The results of comparative analysis of the two methods are presented in Figure 5, and linear

297

relationships between the results of the SERS method and the colorimetric assay were obtained, with

298

R2 of 0.9986 and 0.9891, slope of 1.04 and 1.05, and intercept of 25.88 U/L and 144.63 U/L for

299

apple and potato samples, respectively, which revealed excellent correlation between the SERS

300

method and the traditional colorimetric assay. Therefore, this newly developed method could also be

301

used to detect PPO activities in other food samples.

302

This is the first report of the successful use of a rapid and accurate SERS method for PPO activity

303

determination. The working principle of the measurement was based on the reduction in SERS

304

intensity of catechol before and after enzymatic reaction. A linear regression equation of log∆I/∆t =

305

0.6223 logEc + 0.8072, with an R2 of 0.9689 and a LOD of 224.65 U/L was obtained. The main

306

advantages of this SERS method seem to lie in simple analytical procedure and high accuracy for

307

PPO activity in aqueous solutions. For food samples, PPO activity in solutions was successfully

308

obtained within a short time using the SERS method. The current method was found to be suitable

309

for detecting very low PPO activity in food samples, which may provide a highly practical tool for

310

rapid detection of PPO activity for the food industry. SERS is based on the reduction of reactants to

311

achieve quantitative detection of PPO activity, which may be used for the detection of other enzyme

312

activity. Therefore, the food industry could benefit from the method for improvement of product

313

quality and optimization of processing technology due to accurate control of browning and oxidation. 14


Page 15 of 29

314


Acknowledgments

315

The authors are grateful to the Collaborative Innovation Major Special Projects of Guangzhou

316

City (201604020007) for its support. This research was also supported by the Guangdong Provincial

317

Science and Technology Plan Projects (2015A020209016, 2016A040403040), the Fundamental

318

Research Funds for the Central Universities (2017MS067, 2017MS075), the International and Hong

319

Kong - Macau - Taiwan Collaborative Innovation Platform of Guangdong Province on Intelligent

320

Food Quality Control and Process Technology & Equipment (2015KGJHZ001), the Guangdong

321

Provincial R & D Centre for the Modern Agricultural Industry on Non-destructive Detection and

322

Intensive Processing of Agricultural Products, the Common Technical Innovation Team of

323

Guangdong Province on Preservation and Logistics of Agricultural Products (2016LM2154) and the

324

Innovation Centre of Guangdong Province for Modern Agricultural Science and Technology on

325

Intelligent Sensing and Precision Control of Agricultural Product Qualities. In addition, Tingtiao Pan

326

acknowledged the support from China Scholarship Council (201806150089) and China Association

327

for Science and Technology for his visiting scholarships in Canada.

328

15



Page 16 of 29

329

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Figure captions

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Figure 1 (a) The absorption spectra of AgNps colloid; (b) size distribution and average particle diameter of AgNps

450 451 452 453 454 455 456 457 458

colloid; (c) TEM image of AgNps. Figure 2 Raman signals of (a) catechol solution, (c) catechol powder, and (d) AgNps colloid; SERS signals of (b) catechol solution, (e) and (f) reaction medium before and after enzymatic reaction. Figure 3 (a) Spectra of the catechol solutions at different concentrations; relationship between the SERS intensity of the peak at 1258.0 cm-1 and catechol concentrations in the range of (b) 0.1-11.0 mM, and (c) 1.0-9.0 mM. Figure 4 Reduction in SERS intensity at the activity of (a) 500, (b) 1000, (c) 5000, (d) 10000, and (e) 50000 U/L, (f) the relationship between the reduction in SERS intensity and PPO activity. Figure 5 Relationship between the results of the SERS method and the colorimetric assay, (a) apple sample and (b) potato sample.

459

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Figure 1

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Figure 2

23



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Figure 3

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Figure 4

25



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Figure 5

26


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Table 1 Results of recovery experiments for apple and potato samples. Samples (n = 5)

Activity (before adding) (U/L)

Added activity (U/L)

Total activity (U/L)

Recovery activity (U/L)

Recovery (%)

10,000

5000

4469.31±421.40

79.32-99.27

20,000

10,000

9490.48±359.54

89.90-99.91

10,000

6447.34

6243.34±340.90

90.35-103.27

20,000

11,447.34

10,407.15±724.46

82.78-98.98

10,000

5000

3892.93±378.83

70.18-85.52

20,000

10,000

8365.51±457.68

77.19-90.12

10,000

8729.32

6960.08±366.34

74.92-84.48

20,000

13,729.32

10,994.22±749.19

73.75-86.71

0

Apple 2894.68±142.59

0

Potato 7458.63±187.75

27



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TOC graphic 460

28


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Supplementary information

Activities of PPO crude extracts and their diluted solutions obtained by SERS method and colorimetric assay Sample (n=5)

Apple

Potato

SERS method (U/L)

Colorimetric assay (U/L)

PPO crude extract

2747.05±122.26

2884.00±151.25

diluted 2 times

1439.40±103.85

1516.00±96.33

diluted 5 times

480.90±71.64

576.80±30.25

diluted 8 times

316.90±41.39

360.50±18.91

diluted 10 times

297.96±32.22

288.40±15.13

PPO crude extract

7120.80±215.47

7484.00±191.00

diluted 2 times

3659.17±186.59

4364.00±143.81

diluted 5 times

1454.71±110.27

1788.00±137.55

diluted 8 times

937.32±88.74

935.50±23.87

diluted 10 times

632.99±64.05

748.40±19.10

461

29


A New Method for Accurate Determination of Polyphenol Oxidase

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