Recent advances in electrochemical biosensors for antioxidant analysis in foodstuff

Journal Pre-proof Recent advances in electrochemical biosensors for antioxidant analysis in foodstuff Yongli Ye, Jian Ji, Zhanyi Sun, Peili Shen, Xiulan Sun PII:

S0165-9936(19)30482-0

DOI:

https://doi.org/10.1016/j.trac.2019.115718

Reference:

TRAC 115718

To appear in:

Trends in Analytical Chemistry

Received Date: 26 August 2019 Revised Date:

29 October 2019

Accepted Date: 29 October 2019

Please cite this article as: Y. Ye, J. Ji, Z. Sun, P. Shen, X. Sun, Recent advances in electrochemical biosensors for antioxidant analysis in foodstuff, Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2019.115718. 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 B.V.

Recent advances in electrochemical biosensors for antioxidant analysis in foodstuff Yongli Yea, Jian Jia, Zhanyi Sunb, Peili Shenb, Xiulan Suna* a State Key Laboratory of Food Science and Technology, School of Food Science, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, PR China b State Key Laboratory of Bioactive Seaweed Substances, Qingdao Brightmoon Seaweed Group Co Ltd, Qingdao, Shandong 266400, PR China

Corresponding author*: Xiulan Sun (E-mail: [email protected])

1

ABSTRACT

2

Antioxidants play an important role in human health and provide a defense against many diseases.

3

Electrochemical biosensors are considered promising tools for antioxidant research due to their

4

high sensitivity, fast response time, and ease of miniaturization and have penetrated a variety of

5

markets, including food analysis, drug screening, and toxicity research. In this review, recent

6

advances in current state-of-the-art electrochemical biosensors and antioxidant assessment

7

strategies are discussed with a focus on the use of several biosensors, and their advantages and

8

limitations for the rapid and precise analysis of antioxidants in foods. It is concluded that there is

9

widespread applications of electrochaemical biosensors in food quality analysis, the functional

10

evaluation of active factors, and effective components screening. The challenges associated with

11

electrochemical biosensor technology and future directions in this field are also presented.

12

Keywords: Antioxidant, Electrochemical biosensor, DNA, Enzyme, Cell-based biosensor

13 14

1. Introduction

15

In the past few decades, the use of antioxidants has greatly increased in food- and health-related

16

fields, such as food processing, biomedicine, nutrition, physiology, and chemistry. Antioxidants

17

prevent the oxidation of products during storage, processing, distribution, and consumption, which

18

is an important aspect of nutrition and ensuring food quality [1]. It is necessary to identify and

19

characterize antioxidants in food- and health-related products. Antioxidants perform various

20

beneficial functions as a defense mechanism against oxidative damage in living organisms. In this

21

sense, it is necessary to detect and evaluate antioxidant compounds using in vivo and in vitro

22

methods to assess their health benefits. To date, a number of methods with high sensitivity and

23

automation and low detection limits have been used for the detection of antioxidants and the

24

evaluation of their activities through specific mechanisms [2, 3]. Some components are known to

25

have antioxidant properties and the antioxidant effect or quality of food substances can be

26

evaluated by determining the antioxidant content of a single antioxidant but the type of antioxidant

27

in food samples has to be specified in this method.

28

In the case of food products containing various antioxidants, the total antioxidant activity is

29

generally proposed to evaluate the efficacy of the active compounds. The working principles of

30

chemical assays, instrumental assays, and animal assays are well known and have been widely

31

used to quantify antioxidants in food and other products [3]. However, in terms of the connotation

32

of antioxidant, chemical and instrumental methods based on the scavenging of specific reactive

33

oxygen radicals cannot provide a comprehensive evaluation of the antioxidant efficacy of the

34

tested samples. Although animal model/human assays do reflect the antioxidant effect in vivo, the

35

high cost and time requirements render these assays are unsuitable for the antioxidant evaluation

36

in the preliminary research stage in food science. Therefore, simple, reliable, convenient, and

37

low-cost methods that include visualization, miniaturization, and high specificity and can be

38

conducted in real time are required for the monitoring of antioxidants in complex food substrates

39

and for the search and development of new antioxidants.

40

As interdisciplinary studies have become more commonplace, the use of biosensor devices for

41

the evaluation of the total antioxidant capacity (TAC) of foodstuffs or other samples has become a

42

viable alternative method. In this review, we provide an overview of the recent advances in

43

electrochemical biosensor technologies for the detection and evaluation of antioxidants in food

44

samples. Several types of electrochemical biosensors are described, including DNA, enzymatic,

45

and cell-based electrochemical biosensors. The prevailing challenges and future outlook of the use

46

of electrochemical biosensors for antioxidant assessment are also highlighted.

47

2. Electrochemical biosensors

48

Since the first biosensor was developed by using glucose oxidase, as the result of recent

49

developments in many fields (such as nanoscience and biological science), numerous innovations

50

have been integrated into the development of biosensors to endow them with higher performance

51

[4, 5]. The sensitivity of biosensors is closely related to the type of transducer and the

52

immobilization technology, whereas the selectivity and specificity depend on the sensitivity of the

53

materials because the biosensor does not have separation capabilities [6]. The biological

54

recognition elements such as enzymes, aptamers, DNA/RNA, and cells (bacteria, mammal, and

55

plant cells) are the key objective of electrochemical biosensors [7] (as shown in Fig. 1).

56

Electrochemical biosensor monitor of antioxidant activity based on the redox principle has many

57

advantages over conventional chemical methods and is commonly used for the initial screening of

58

antioxidants. This technology does not require sophisticated chemical reagents or solvents nor

59

special sample preparation and provides extensive and reproducible information on

60

electrodynamic processes and rapid execution [8, 9]. The biosensors described below are all

61

electrochemical biosensors unless otherwise specified.

62

3. Application of electrochemical biosensors in food antioxidant assessment

63

3.1. DNA-based biosensors

64

DNA is an excellent biorecognition element and has been used in biosensor technology based

65

on the principle of oxidative damage for the detection and evaluation of antioxidants in food,

66

medicine, environment, and other fields [10]. Compared to other proteins and antibodies, DNA

67

probes have a smaller size, a high degree of stability, and cost-effectiveness [11]. A DNA-based

68

electrochemical biosensor is generally fabricated by attaching DNA molecules to the surface of a

69

working electrode to detect the interaction between DNA and analyte, and induce changes in the

70

DNA structure and electrochemical properties; the signal output is then converted into an

71

electrochemical signal [12]. DNA biosensors are promising devices that can be used to routinely

72

and easily evaluate the antioxidant capacity of samples. The changes in the oxidation peak of the

73

DNA bases before and after the interaction with the analyte can be monitored either without

74

labeling or different amplification strategies can be used to reduce the analysis time, complexity,

75

and to realize miniaturization [13]. DNA-based biosensors for TAC assessment in analytes are

76

designed to simulate an in vivo assay and since DNA oxidative damage is irreversible, the

77

biosensor can only be used once. Disposable DNA biosensors have the unique characteristics of

78

good reproducibility, constant sensitivity, no sample contamination, ability to avoid interferences

79

of colored samples, and having small size and portability, which makes it possible to analyze the

80

antioxidant capacity of samples on-site [14, 15].

81

The assessment of antioxidants mainly relies on DNA damage detection [16] because the use of

82

DNA-based electrochemical biosensors for the assessment of antioxidants is similar to the

83

response of antioxidant activity in biological systems (usually simulating the damage caused by

84

reactive oxygen species (ROS) in vivo) [17, 18]. Single-strand DNA (ssDNA) [19, 20],

85

double-strand DNA (dsDNA) [21, 22], as well as purine and pyrimidine bases [23-25] can serve as

86

the recognition element of a DNA electrochemical biosensor. The detection is based on the

87

disruption of the structure integrity of these probes by the analytes, which causes changes in

88

electrochemical signals [16]. When antioxidants are added to the reaction system, they will

89

compete with DNA for •OH, which enhances the oxidation signals of DNA and reflects the

90

antioxidant capacity of the analytes indirectly [26]. The DNA signal response (the relationship

91

between the signal and the changes in the guanine base) is largely dependent on the DNA structure;

92

therefore, the DNA signal remains almost unchanged during antioxidant detection due to its ability

93

to neutralize the factors causing DNA structural damage [1]. Fig. 2 shows the working principle of

94

DNA-based electrochemical biosensors for antioxidant assessment in vitro.

95

3.1.1. Applications for total antioxidant capacity assessment

96

The antioxidant evaluation of analytes by detecting DNA damage for different treatments using

97

a biosensor purely modified with DNA is based on their ability to scavenge •OH [25]. For instance,

98

adenine or guanine was immobilized on a glassy carbon electrode (GCE) or screen-printed

99

electrode

surface

(SPE)

to

establish

a

purine-based

electrochemical

biosensor

by

100

electro-deposition for the TAC evaluation of antioxidants in beverages [23], coffee [27], flavors

101

and flavored waters [24]. Moreover, Labuda’s group used a dsDNA biosensor to evaluate the TAC

102

of yeast polysaccharides [28], phenolic compounds [29], and flavonols and flavanols [30] in plant

103

extracts. A biosensor consisting of a dsDNA immobilized on a SPE by adsorptive accumulation

104

was also constructed to detect DNA damage for the evaluation of the antioxidant properties of

105

plant extracts [14]. The radicals that formed during the oxidation process after quercetin was

106

bound to dsDNA caused hydrogen bond breaks in the dsDNA, resulting in the generation of

107

8-oxoguanosine (8-oxoG). Two types of DNA-based electrochemical biosensors were developed

108

to investigate the protective effect of quercetin against DNA damage based on this principle [31].

109

In another study, the antioxidant and prooxidant properties of a semi-synthetic flavonolignan

110

7-O-galloylsilybin (7-GSB) were described and the oxidation mechanism of 7-GSB during the use

111

of a DNA-based electrochemical biosensor was proposed [32]. However, because of the different

112

species of free radicals, the results obtained from electrochemical and traditional

113

spectrophotometric methods are different and the specific responsible compounds are difficult to

114

identify.

115

Because •OH is the most potent ROS produced in living organisms and it damages

116

biomolecules, the scavenging activity of antioxidants has to be stronger to protect biomolecules.

117

Researchers determined that it is easier to scavenge H2O2 than •OH radicals for some antioxidants

118

such as ascorbic acid (AA) [17]. Therefore, the type of free radical system affects the precision

119

and suitability of DNA-based biosensors. DNA-based electrochemical biosensor methods based

120

on the scavenging ability of antioxidants for NO• free radicals have been reported [33].

121

Adenine-rich oligonucleotide (dA20) was immobilized onto a carbon paste electrode (CPE)

122

through physical adsorption and square wave voltammetry (SWV) was used for the assessment;

123

the limit of detection (LOD) and limit of quantification (LOQ) were 0.23 mg/L and 0.78 mg/L,

124

respectively. The construction of a dA20-CPE modified biosensor is simple and probably suitable

125

for assessing the TAC in commercial juice samples because the major compound of oranges is AA.

126

DNA-based biosensors using nanomaterials with a large surface area and good electrochemical

127

properties have been proposed (Table 1). Guanine was selected as the biomolecule and was

128

integrated with TiO2 nanoparticles and multiwalled carbon nanotubes (MWCNTs), a GCE was

129

used as the working electrode to construct a guanine/TiO2NPs/MWCNTs/GCE electrochemical

130

biosensor for the antioxidant evaluation of sodium pyrosulfite (Na2S2O5) (commonly used as an

131

antioxidant excipient for drugs and a food preservative) [26].

132

A GCE modified with silver nanoparticles (AgNPs) which was used to amplify the response has

133

also been used for the evaluation of the antioxidant capacity of green tea infusions based on DNA

134

damage in the Fenton system and showed a high sensitivity toward ROS [34]. Notably, some

135

two-dimensional nanomaterials also significantly improve the sensitivity and LOD of DNA-based

136

biosensors [2]. Based on •OH generated by the Fenton system, a GCE was modified with a

137

graphene nanoribbon (GNR) and guanine was used as the biorecognition element to fabricate a

138

new electrochemical biosensor for the evaluation of TAC in fruit juices [35]. Compared with the

139

TAC evaluation results of previously used electrochemical biosensors, the LOD (0.05 mg/L) was

140

improved about 5 times to one order of magnitude and the sensitivity was 4.16 µA/mg/L.

141

Moreover, the current only decreased by about 3.7% after 30 d storage at room temperature,

142

providing a potentially effective tool for the assessment of the TAC of real samples. MWCNTs

143

and chitosan were used to modify a pencil graphite electrode (PGE) before dsDNA was

144

immobilized on the electrode surface. The TAC of several antioxidants (glutathione, AA) and the

145

DNA oxidative damage caused by dopamine in the presence of metal ions were investigated using

146

electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) [36].

147

Since functional polymers exhibit various excellent properties, DNA-based biosensors modified

148

with polymers significantly decrease the detection limit and linear range of analytes in antioxidant

149

assessment when polymers are used as electrode preparation materials or are modified on the

150

electrode surface (Table 1). Among them, chitosan is widely used to fabricate modified electrodes

151

[37, 38]. For instance, a dsDNA-coated chitosan-modified CPE was developed to detect

152

oleuropein. The peak current of the chitosan/CPE was almost twice as high as that of the bare CPE.

153

A linear concentration range of 0.30-12 µM with a LOD of 0.090 µM for oleuropein was obtained

154

by DPV detection and the method was successfully applied to real samples [39]. A biosensor was

155

constituted with a hybridized membrane of poly l-glutamic acid, Ag, and an outside layer of

156

chitosan/dsDNA on a GCE surface for assessment of the TAC in orange juice [40]. Good stability

157

of this biosensor was exhibited when the modified electrode was stored at room temperature for

158

15 d in the air; the mean deviation was less than 5%, demonstrating the potential use in TAC

159

evaluation in foods.

160

3.1.2. Perspectives and challenges

161 162

The performances of DNA-based biosensors for antioxidant assessment still require improvement in the following areas:

163

(1) DNA immobilization technology. The analytical performance of electrochemical

164

biosensors is closely related to DNA immobilization technology. One of the problems is that DNA

165

immobilization decreases the electron transfer rate on the electrode surface. Three conditions must

166

be considered in the selection of immobilization technologies, namely, the stability of the

167

immobilization chemistry, functional retention after DNA attachment, and the biomolecules must

168

have appropriate orientation and configuration [16, 41].

169

(2) Specificity. Most DNA-based biosensors are unable to provide quantitative information on

170

the antioxidant substances in food samples. Additionally, the complex characteristics of the sample

171

matrix present a great challenge to improve the specificity of the biosensors. Pentose nucleic acid

172

has significant sequence specificity and is considered as a potential candidate for DNA oxidative

173

detection [16].

174

(3) Sensitivity. Functional materials with good electrocatalytic properties such as a large

175

surface area and superior electronic conductivity can significantly improve the response of

176

electrical signals to improve the sensitivity of the DNA-based biosensor and the electrochemical

177

performance of the TAC analysis. In addition, the detected signal intensity of the DNA-based

178

biosensor is strongly related to the accessibility of DNA to the electrode surface. ssDNA is

179

considered to be more suitable for TAC evaluation than dsDNA because the bases in ssDNA are

180

more accessible to the electrode surface; the facilitates oxidation and the generated current signal

181

is higher and more sensitive [27].

182

(4) Multi-system. It is impossible to generate only a single ROS when oxidative stress occurs

183

in organisms. In this case, the evaluation of the TAC should be aimed at the scavenging of a class

184

of free radicals and the results do not represent the true antioxidant capacity of the analytes with

185

low TAC values. Therefore, the use of two or more oxidation conditions or multiple free-radical

186

conditions as a requirement for the evaluation of the antioxidant status is a very interesting trend

187

[14].

188

3.2. Enzyme-based biosensors

189

Enzymatic biosensors use enzymes as the biorecognition element and the analysis of the target

190

is based on the inhibition of the enzyme activity. After the enzyme is exposed to a specific

191

inhibitor for a specific period of time, quantitative and qualitative analyses of the analytes are

192

performed by determining the correlation between the inhibition rate of the enzyme and the

193

concentration of the inhibitor [42]. The typical structure of an enzymatic electrochemical

194

biosensor is shown in Fig. 3. Most enzyme-catalyzed reactions can be determined by using simple

195

principles, which is advantageous and widely available biosensors technology can be used,

196

especially electrochemical biosensor methods [43]. The oxidoreductase group (oxidases,

197

dehydrogenases) and the hydrolase and lyase groups are enzymes commonly used in biosensors.

198

At present, a variety of proteases are used for the determination of antioxidants and evaluation of

199

their activity through biochemical oxidation followed by electrochemical reduction [44, 45], such

200

as tyrosinase [46], laccase [47], peroxidase [48] and other proteases, which exhibit single or

201

complex enzyme binding [9]. The electric coupling of oxidoreductase and the electrochemical

202

transducer exhibit excellent characteristics and monitoring is conducted by controlling the reaction

203

of the enzyme in real-time [2, 43]. Specific enzymes can be effectively used for the selective

204

identification of important target compounds in food quality control. Laccase and tyrosinase are

205

the two most widely used enzymes for monitoring antioxidants, especially in phenolic compounds

206

[49].

207

3.2.1. Applications for antioxidant detection

208

The number of reports on biosensors using enzymes for quantification of antioxidants in food

209

products is far greater than that of DNA-based biosensors. Most of the proposed enzyme-based

210

biosensor designs are complex multicomponent systems that combine specific enzymes with

211

functional nanomaterials and polymer membranes or gels (Table 2). The type of the selected

212

enzymes is determined by the analyte specificity whereas a combination of functional biomaterials

213

is required to improve the conductivity of the immobilized matrix or to create redox pathways that

214

electrically connect the active site of the captured enzyme with the electrode surface [50].

215

The detection of polyphenols in samples of wine, beer, fruit and their products, vegetables, tea

216

and its products, herbals and others using enzyme-based biosensors with a variety of modifications

217

is relatively mature [51, 52]. The prevention of the leakage of enzymes during the application, the

218

maintenance of the enzyme activity, and better linkage between the analytes and enzymes, as well

219

as the diffusion of redox products to initiate the electrochemical signal response, are important

220

aspects when evaluating the properties of the biosensors. A gold (Au) electrode surface was

221

deposited with a layer of AgNPs/carboxylated MWCNT/polyaniline (PANI), which after laccase

222

purification was immobilized covalently onto an electrochemically modified electrode to fabricate

223

a polyphenol biosensor [53]. The microenvironment created by the PANI prevented the leakage of

224

enzymes from the external environment. The results showed 0.1-500 µM of linear range, 6 s of

225

response time, and 0.1 µM of LOD, indicating that this biosensor was effective for the

226

determination of the total phenolic content in tea and alcoholic beverages. Another research

227

reported that electrodes modified with nanomaterials (such as graphene oxide (GO) and MWCNTs)

228

and or polymers which were mixed together or layer by layer, were used applied to analyze

229

polyphenols in foodstuffs and other substrates [54, 55]. The practical application of the biosensor

230

was demonstrated by estimating the total polyphenol concentration in juice samples. Similar

231

studies have reported that the free COOH groups of MWCNT can also be conducive to enzyme

232

immobilization via the NH2 groups on the surface of enzymes [56].

233

In order to improve the activity of immobilized enzymes, a biomimetic Langmuir-Blodgett (LB)

234

film of tyrosinase adsorbed onto an arachidic acid layer via COOH group interaction was used to

235

develop a biomimetic amperometric biosensor in which lutetium bisphthalocyanine was used as an

236

electron mediator [57]. LB films used for phospholipid immobilization seem to maintain enzyme

237

activity better than solutions by preserving the conformation of enzymes [58]. It is possible to

238

analyze phenol derivatives using this enzymatic biosensor, the results showed that the

239

voltammetry signal response was related to the redox properties of the enzymatically formed

240

o-quinone while lutetium bisphthalocyanine significantly improved the signal amplification [57].

241

In addition to adsorption and encapsulation, Pavinatto et al. [59] reported that another alternative

242

approach is to employ a nanostructured film to immobilize and support the enzyme on the

243

electrode surface to fabricate an all-printed and flexible tyrosinase biosensor for polyphenol

244

detection in wines and olive oils. The high-resolution interdigitated electrodes were directly

245

inkjet-printed on plastic substrates using a nanoparticle-based ink and the tyrosinase was deposited

246

subsequently by large-area rotogravure printing. Cellulose acetate was used as a water-insoluble

247

coating to encapsulate the printed active layer and enzyme leakage was prevented by using a fast

248

dip-coating deposition. The good sensitivity (5.68 Ω/mm) and the LOD (200 µM), as well as the

249

application results using real samples, illustrated the potential applicability of the biosensor for

250

antioxidant analysis [59]. Cross-linked enzyme crystal (CLEC) technology is another robust

251

enzyme immobilization and stabilization method that maintains enzyme activity [60, 61]. A

252

laccase biosensor based on CLECs was developed to analyze the phenol content [62]. Laccase was

253

crystallized, cross-linked, and lyophilized with β-cyclodextrin and was embedded in a 30 %

254

polyvinylpropylidone (PVP) gel and integrated into an electrode to prepare the biosensor. The

255

biosensor showed good sensitivity to phenols with a detection concentration of 50-1000 µmol and

256

good stability for maintaining the enzyme activity over 3 months.

257

In addition to determining antioxidants based on the redox properties of the antioxidants in

258

response to the enzymes, utilizing the antioxidant principle of antioxidants is also a potential tool,

259

such as the detection of superoxide anion (O2•−) free radicals. However, similar methods seem to

260

be less frequently applied to antioxidant analyses of real food samples [63]. It cannot be ignored

261

that the maintenance of the enzyme activity is a critical factor for the commercialization of

262

enzyme-based biosensors. In recent years, the stability and sensitivity of this type of biosensor

263

have been greatly improved through much hard work [54, 55]. An enzyme-modified electrode for

264

a polyphenol biosensor fabrication was used 200 times over 4 months and 300 times over a period

265

of 7 months at 4 °C storage by a research group [53, 64]. A peroxidase biosensor for the

266

assessment of chlorogenic acid in coffee samples was developed by using two different

267

immobilization strategies; which both biosensors exhibited long-term stability of 4 months [38].

268

The useful lifetime of another laccase-based amperometric biosensor for detecting the phenolic

269

compound content in tea infusions was over 6 months and 60% of the signal was detectable after

270

one year at 4 °C storage [65].

271

3.2.2. Applications for antioxidant evaluation

272

Electrochemical biosensors are not significantly affected by colorimetric influences and are,

273

therefore, reliable for evaluating antioxidant activity. In general, there are fewer studies on the

274

antioxidant evaluation of active compounds than the detection of the antioxidant content using

275

enzymatic biosensors (Table 2). Superoxide dismutase (SOD) and tyrosinase- based biosensors

276

have been applied to analyze the antioxidant properties of functional factors in a variety of

277

foodstuffs, especially the scavenging capacity of O2•− and free radicals. Campanella and his group

278

have conducted a series of studies on the evaluation of antioxidant activity based on enzyme

279

biosensors [66-69]. First, the authors developed a biosensor consisting of an amperometric

280

electrode modified with a mixture of SOD enzyme and κ-carrageenan gel to detect hydrogen

281

peroxide (H2O2) [66]. Subsequently, this superoxide biosensor was used to investigate the

282

scavenging properties of several highly effective radical scavengers, such as melatonin, β-carotene,

283

and cysteine. The proposed method can be used as a simple and fast tool for the determination of

284

the antioxidant properties of commercial products [67]. Later, the research group continued to

285

evaluate the scavenging properties of several fruits and other samples by using a SOD biosensor.

286

The results demonstrated that this biosensor was suitable for antioxidant evaluation in food [68].

287

Previous studies did not carry out quantify the effective antioxidant activity, whereas this group

288

continued to use tyrosinase as the recognition element, the antioxidant capacities of phenols in

289

several types of tea were identified and quantified (LOD about 0.1 and 2 µM for the relative

290

antioxidant capacity and content) [69].

291

SOD-based electrochemical biosensors have also exhibited good sensitivity and selectivity

292

regarding the antioxidant activity of food products based on the determination of the O2•− [63, 70,

293

71]. An O2•− biosensor which was modified with SOD, incorporated MWCNT and polymer

294

poly(3,4-ethylenedioxythiophene) in a chitosan dispersion was successfully applied to determine

295

the antioxidant capacity of beverages such as wines and berry juice; an increase in sensitivity, as

296

well as stability of over 2 months, was demonstrated [70]. A phenoloxidase-based biosensor was

297

developed to determine the antioxidant activity of commercial dried herbal extracts, a multivariate

298

statistical model corroborated the feasibility of this electrochemical method [52]. The working

299

electrode was constructed by adding the Laccase modified carbon paste, electrochemical index

300

was used to evaluated the antioxidant activity of herbal samples. This biosensor method

301

eliminated the effect of sulfur dioxide on the signal response, and showed a good calibration curve

302

for rutin in concentration from 0.2 to 1.5µM.

303

3.2.3. Challenges and perspectives

304

Major advancements in enzyme-based biosensors are related to the immobilization and

305

interface functionalization of the biological materials on the electrode surface. However, despite

306

the good performance and significance of this technology for practical and fundamental science, it

307

should be noted that several important aspects have to be considered prior to the commercial

308

application of enzyme biosensors for monitoring active compounds and their antioxidant capacity:

309

(1) Immobilization and stability. Highly efficient enzyme immobilization and the acceleration

310

of the electron transfer rate are challenging tasks in the development of biosensors. As

311

carriers/hosts of immobilized enzymes, nanomaterials and polymers have broad application

312

prospects in improving the electron transfer rate and stability and prolonging the life of

313

enzyme-based biosensors [65].

314

(2) Sensitivity and usage. The bioconjugation of enzymes with the electrode surface has to be

315

considered to improve the sensitivity of the biosensor. Biocompatible materials that meet the

316

requirements of high sensitivity should be developed to fabricate biosensors with high specificity.

317

The interaction with the matrix is also an important factor because it dilutes the effective

318

concentration of the enzymes. An increase in the number of use times of the biosensor by

319

improving the immobilization technology and modification of the biological enzyme is also a key

320

point of enzymatic biosensor development in the future.

321

(3) Matrix interference. Matrix interference is a major hurdle in many research methods and

322

biosensor methods are no exception due to the complexity of real samples [72]. The minimization

323

of the matrix interference not only requires innovative methods in sample pretreatment but also

324

optimization of the specificity and selectivity of the biosensor.

325

(4) Diversity of enzymes. Due to the specificity of enzyme reactions, one kind of enzyme

326

cannot detect all antioxidants or evaluate the antioxidant properties of all active substances.

327

Laccase, for example, does not catalyze 3-amino phenol and other monophenols because its amino

328

group is in the meta position. Although this means that better specificity is required for the

329

biosensor, for some substrates such as monophenols, this represents a challenge [62]. Therefore, it

330

would be of interest to develop varied enzymatic biosensors that are suitable for monitoring a

331

certain type of antioxidant based on the action mechanisms of the enzymes.

332

3.3. Cell-based biosensors

333

A cell-based biosensor (CBB) is an analytical device that uses living cells as the recognition

334

elements with a suitable physicochemical transducer to detect physiological changes in the cells;

335

the sensor can convert the physiological signals into digital electrical signals [73, 74]. CBB

336

technology has been rapidly developing in the past decades and is widely used for testing of food

337

hazards, environmental toxicity, and other toxic materials, as well as for pharmaceutical

338

evaluations [75]. Mammalian cells (normal cells and cancer cells), microbial cells, plant cells, and

339

their recombinant types are used for CBB fabrication [76]. Mammalian cells reflect physiological

340

responses at the cellular level associated with humans and animals, therefore, mammalian CBBs

341

are suitable for the assessment of antioxidants in food. Another important cell type for CBB

342

development is microbe cells, especially bacteria and yeast due to their properties of ubiquitous

343

presence, rapid growth, ease of culture, low cost, ease of genetic manipulation, and their ability to

344

metabolize a wide range of chemical compounds [77]. It has also been reported that biosensors

345

modified with microorganisms were used for antioxidant evaluation and provided good results

346

(Some applications are shown in Table 3).

347

Cells with excitable cell membranes possess various highly evolved biochemical pathways and

348

they are commonly utilized as the biorecognition element of biosensors. Compared with DNA and

349

other analytical methods, cells provide more comprehensive and complex functional information

350

(for example, protein synthesis and secretion, cell apoptosis, or necrosis) [74, 76]. CBBs represent

351

the next revolution in analytic science, offering a variety of unique and superior advantages,

352

including high sensitivity and stability, rapid response, excellent selectivity, noninvasiveness, and

353

high biocatalytic activity [76]. These characteristics allow cells to provide physiologically relevant

354

data in response to analytes and to sense their functionality or biological activity. Due to the

355

advantages of this technology, CBBs have attracted attention for the assessment of antioxidants.

356

Essentially, most CBBs provide information on the effects of antioxidants on the entire cell,

357

thereby providing a cell-level assessment of active factors such as quantification and function.

358

Gene recombination and immobilization technology, as well as nanomaterials with good

359

biocompatibility, play a key role in maintaining basic cell morphology, improving the stability and

360

sensitivity of CBBs and reducing the detection limit. Cell immobilization and nanomaterials for

361

surface biofunctionalization of CBBs have been reviewed by Banerjee and Bhunia [74],

362

Wongkaew et al. [7], and Liu et al. [73] and the topics are not detailed here.

363

3.3.1. CBBs for antioxidant assessment

364

The changes in intracellular oxidative stress can be evaluated indirectly based on the

365

determination of the production and release degree of cellular ROS and the antioxidant effect of

366

the analytes can be assessed by the level of protection of the added antioxidants. Cells were

367

stimulated by an inducer to produce H2O2, which catalyzes the active site of the compound

368

modified on the electrode surface to be reduced or electro-oxidized. The oxidation current

369

increased with the increased generation of H2O2. Based on this design principle, Ge et al. [78]

370

developed an electrochemical CBB to evaluate the antioxidant capacity of cell-free extracts from

371

Lactobacillus plantarum strains, which were isolated from Chinese dry-cured ham (Fig. 4A).

372

Acidified manganese dioxide (a-MnO2) nanoparticles were dropped onto the surface of a gold

373

electrode (GE) as a catalyst. Then a suspension of RAW 264.7 macrophage cells equally mixed

374

with alginate/graphene oxide (NaAlg/GO) was dropped onto the a-MnO2 to establish a CBB

375

consisting of a three-dimensional (3D) cell culture system. This research group also used silver

376

nanowires (Ag NWs) and platinum nanoparticles (Pt NPs) to modify the GE and immobilized the

377

Caco-2 cells mixed with NaAlg on the electrode surface to construct a CBB for evaluating the

378

antioxidant activity of Asp-Leu-Glu-Glu (DLEE) isolated from dry-cured Chinese Xuanwei ham.

379

The CBB method had an excellent catalytic effect on the reduction of H2O2 and remarkable

380

stability (the current response only decreased 15 % after 15 d of storage at room temperature) and

381

represented a suitable tool for antioxidant evaluation (Fig. 4B) [79].

382

The antioxidant effect of phloretin (Ph) was researched and a CBB based on an A549

383

cell/NaAlg/AuNP-modified working electrode was developed (Fig. 5A) [80]. Under optimized

384

conditions, the response impedance of the biosensor was linear to the Ph concentrations from

385

20 µM to 100 µM with a LOD of 1.96 µM. A significant correlation was also observed between the

386

ROS levels and impedance values following the Ph concentrations. The antioxidant activity of

387

four plant extracts was investigated using a CBB based on fibroblast cell immobilization [81].

388

When the cells were treated with the plant extracts, the cell membrane potential increased, which

389

was attributed to the reduction in the membrane damage. The assay duration was only 3 min;

390

therefore, this CBB may be suitable as a rapid screening method for antioxidants in plant-derived

391

compounds. The bioelectric recognition assay is a whole-cell-based biosensing system that

392

monitors the electric response of different immobilized cells to ligands, which bind to the cells

393

and/or affect their physiologies [82]. Based on this principle, the “membrane-engineered”

394

technology was successfully utilized for CBB development. A method of electroinsertion of SOD

395

molecules into the membranes of Vero fibroblast cells as catalytic units was developed to

396

construct an ultra-sensitive electrophysiological O2•− sensor for monitoring ultra-low

397

concentrations of free radical species and oxidative agents in biological systems [83]. The

398

“membrane-engineered” cells converted O2•− to H2O2, which triggered changes to the cell

399

membrane potential and the CBB instantly responded with a LOD of 0.1 nM. After 4 months

400

storage at room temperature, about 80% of the cells retained their viability, demonstrating that this

401

engineered CBB offers a new perspective for the selective detection of O2•− at nanomolar or even

402

lower concentrations.

403

In another study, a zeolitic imidazolate framework-9 (ZIF-9) was selected as a cobalt-based

404

metal-organic framework precursor; AgNPs were embedded into the ZIF-9-derived cobalt oxide

405

porous carbon material to fabricate a AgNPs/Co3O4@C/GCE non-enzyme biosensor for

406

monitoring O2•− released from living cells [84]. The method provided a linear range of 8 orders of

407

magnitude and a super low LOD of 0.0564 pM; and it was used to estimate the O2•− scavenging

408

capability of four food antioxidants. Moreover, a fibroblast NIH-3T3 cell-based real-time

409

impedance sensing technique was used for the label-free and dynamic measurement of cell

410

responses to phenolic compounds treated with H2O2-induced oxidative stress in a 16-well E-plate

411

to monitor the electrical impedance changes due to changes in cell adhesion and cell number (Fig.

412

5B ) [85]. The results of 12 representative antioxidant phenolic compounds showed that the

413

impedance response had great potential for screening and evaluating phenolic compounds. This

414

cell-based impedance analytical method has strong physiological correlation, non-invasive, high

415

sensitivity, and has the potential to be used for high-throughput screening of antioxidant phenols.

416

The utilization of living microbial cells for active component or toxicological analysis is

417

attractive and microbial CBBs have been developed to demonstrate that the adsorbed cells on an

418

electrode surface remain active for several weeks or even months. This highly stable performance

419

is an attractive approach for developing bacterial biosensors to assess antioxidants. Zhang et al. [3]

420

utilized recombinant E. coli MB275 cells surface-expressing the fusion protein (InaQN)3/WlacD;

421

this was directly deposited and adsorbed onto the GCE surface to develop a reliable and easily

422

regenerated biosensor for monitoring phenols. When this CBB was used to detect phenolics in red

423

wine, tea, and other samples, it showed high accuracy and stability and had good potential for the

424

analysis of phenolic compounds. Next, this research group also used a similar process to develop

425

an electrochemical microbial CBB by immobilizing a highly active E. coli whole-cell

426

laccase-based catalytic system onto a GCE surface (Fig. 5C) [49]. The biosensor exhibited high

427

stability, accuracy, and reproducibility for detecting catechol in red wine and tea samples and

428

showed potential for simple, accurate, and cost-effective analysis of catechol.

429

3.3.2. Challenges and perspectives

430

Due to their intrinsic characteristics, CBBs are mainly used for toxicological evaluation but are

431

used less for antioxidant assessment than DNA and enzyme biosensors. However, with the

432

development and application of related disciplines, CBBs can serve as a bridge between biology

433

and electronics and have shown increasingly significant potential for antioxidant assessment.

434

Since the cells have more complex physiological activities than DNA and enzymes, the following

435

points required consideration:

436

1) The maintenance of cell health, the integration of traditional CBBs into portable devices, the

437

development of new cell immobilization technologies, and the use of cell conjugation technology

438

to develop efficient and stable CBBs are the main challenges. 3D cell culture can mimic in vivo

439

cellular conditions by supporting cell growth, maintaining morphogenesis and cell metabolism,

440

and promoting cell-to-cell interaction normalization, which is a potentially attractive technology in

441

electrochemical biosensor fabrication.

442

2) Recombinant mammalian cells and microbial cells have to be fully developed to improve the

443

sensitivity of antioxidant assessment by the expression of specific molecules. Especially for the

444

analysis targeted by enzymes in cells, since the number of cells is positively correlated with the

445

electrode impedance signal, high-throughput expression can reduce the number of immobilized

446

cells, which is conducive to improving the detection sensitivity.

447

3) New functional nanomaterials with catalytic properties have to be developed. The assessment

448

of antioxidants is highly dependent on the monitoring of the changes in the redox signals on the

449

electrode. Thus, the modified materials on the electrode surface not only require a large specific

450

surface area to promote the conductivity of electrode transfer but also have good biocompatibility

451

and catalytic redox ability.

452

4) The integration with other devices is required to simplify the pretreatment steps, reduce the

453

matrix interference, provide more effective information, and achieve high-throughput detection

454

and analysis, examples include microfluidic chips and microarrays coupled with chromatography

455

or mass spectrometry and others.

456

3.4. Other electrochemical biosensors

457

Other biomolecules such as hemoglobin [86], polypeptides [87], and proteins [88] have been

458

reported for ROS monitoring and antioxidant evaluation. A series of electrochemical biosensors

459

based on the reduction of cytochrome c (cyt c) by O2•− was developed for the assessment of the

460

free radical scavenging ability of antioxidants (Table 3). Since the electron transfer from the

461

radical by cyt c to the electrode is proportional to the radical concentration, cyt c is often used for

462

H2O2 detection or electrochemical biosensor modification that incorporates xanthine oxidase

463

(XOD) as a radical generator [13, 89]. An electrochemical biosensor for antioxidant detection

464

based on cyt c modified on a GE surface and the measurement of steady-state O2•− levels was

465

developed [90]. The antioxidant activity can be evaluated by quantifying the percentage of current

466

decrease in response to the modified electrode. The scavenging capacities of O2•− free radicals of

467

various flavonoids were compared using this biosensor method and the modified electrode could

468

be used for about 1 week with intermittent storage at 4 °C, demonstrating the applicability of this

469

method for antioxidant assessment [90].

470

The electron transfer rate of cyt c on the surface of a bare electrode (such as GE, Ag, and GCE)

471

is very low and often, a redox peak cannot be detected. An effective approach that relies on the

472

utilization of nanomaterials immobilized on the electrode and facilitates long-range cyt c

473

interfacial electron transfer was developed. On the other hand, the modification of proteins by

474

introducing reactive groups, such as thiols, amino, and other active tags, is also an important

475

research aspect [89]. Since the response signal of protein-modified biosensors is usually very

476

small, the enhancement of the electron transfer between the electrode and cyt c through the

477

modification of the electrode surface is the main research aspect in the development of cyt c-based

478

biosensors. Moreover, cyt c-based biosensors for the determination of O2•− lack selectivity because

479

the hemeprotein has no specificity for O2•−, which greatly hinders the application of antioxidant

480

capacity evaluation in real samples. The modification of mercaptan groups enhances the

481

immobilization of cyt c but it also forms a dense non-conductive polymer film, hindering the

482

redox of electroactive substances on the electrode and the electron transfer. At present, AuNPs are

483

mainly used to restore the electrochemical activity of the electrode and the modified materials are

484

relatively simple. The development of new functional materials that promote the immobilization

485

of cyt c and enhance electron transfer is an important aspect in the preparation of protein-based

486

modified biosensor.

487

4. Conclusions and perspectives

488

Electrochemical biosensor technology is suitable for free radical and antioxidant evaluation due

489

to its advantages, such as low color interference, small size, high sensitivity, ease of use, and rapid

490

detection. The assessment of antioxidants by electrochemical biosensors based on DNA, enzymes,

491

cells, and other biorecognition elements were reviewed in this paper, the notable research studies

492

in recent years were summarized, and the challenges and development of several electrochemical

493

biosensors were discussed. DNA-based biosensors are mainly used for the antioxidant capacity

494

evaluation of analytes. This technology is the first choice for antioxidant evaluation because free

495

radicals readily attack DNA in the detection system, which is similar to what occurs in cells.

496

Enzyme-based biosensors are utilized for the quantification of antioxidants in foodstuff and other

497

samples. Enzyme biosensors are reusable, the preparation process is simple, but the maintenance

498

of enzyme activity remains an important challenge. Compared with the two former biorecognition

499

molecules, cells have more complex physiological effects, requiring more stringent biosensor

500

preparation and greater detection specificity and stability. There is still a long way for CBBs to

501

become practical in commercial products. The following points should be considered for future

502

development:

503

(1) Explicit mechanism. To date, most studies only considered a variety of possible responses

504

and occurrences and focused less on the mechanisms. For example, for CBBs, key gaps still exist

505

regarding the mechanisms of extracellular electron transfer and the electrical interaction between

506

the cells and electrode interface. Further intracellular and extracellular characterizations involved

507

in the function of the mediators, as well as the molecular mechanism of transmembrane electron

508

transfer, are needed to improve our understanding.

509

(2) Functional material application and model diversification. To commercialize a product,

510

the stability and reliability of electrochemical biosensors are key to development. Currently, most

511

research on improving biosensor performances has focused on the development of new materials,

512

especially conductive nanomaterials and functionalized polymers but the development and

513

application of recombinant engineered biological components (enzymes and cells) are also a

514

fascinating strategy. In addition, different kinds of antioxidants have inconsistent scavenging

515

capabilities with regard to the same type of free radicals, resulting in no response of the electrical

516

signals, which may be interpreted as low or no antioxidant capacity. Different free radical models

517

need to be developed to collect a complete antioxidant fingerprint and to accurately assess the

518

antioxidant capacity of analytes.

519

(3) Modern intelligent monitoring. The application of high technologies is necessary for the

520

sound development of electrochemical biosensors. Artificial intelligence tools can predict

521

quantitative structure-property relationships or quantitative structure-activity relationships of

522

analytes and can be applied to solve nanotechnology-related problems and complex tasks.

523

Furthermore, the application of 3D printing technology can simplify the complicated operation of

524

current electrode modification and achieve various expected functions and designs. The

525

integration of intelligent devices can maximize the simulation of the effect and mechanism of

526

antioxidants in real physiological environments.

527

Acknowledgements

528

This work has been supported by the National Research Program of China (No.

529

2017YFF0211303, 2018ZG003), Open Foundation of the State Key Laboratory of Bioactive

530

Seaweed Substances (SKL-BASS1709), National First-class Discipline Program of Food Science

531

and Technology (No. JUFSTR20180303).

532

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Recent advances in electrochemical biosensors for antioxidant analysis in foodstuff Yongli Yea, Jian Jia, Zhanyi Sunb, Peili Shenb, Xiulan Suna* a State Key Laboratory of Food Science and Technology, School of Food Science, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, PR China b State Key Laboratory of Bioactive Seaweed Substances, Qingdao Brightmoon Seaweed Group Co Ltd, Qingdao, Shandong 266400, PR China

Corresponding author*: Xiulan Sun (E-mail: [email protected])

1

Table 1 Partial applications of DNA-based biosensors for antioxidant assessment Index

Sample

detection technique

Modification

AA

beverages

CV, DPV

DNA(dA21)/CPE

0.05-1.00 µM

50 nM

ascorbic acid, gallic acid, etc.

beverages

SWV

purine /GCE

0.10-4.00 mg/L

0.02-0.47 mg/L

adenine /GCE

0.10-4.00 mg/L

0.10-0.50 mg/L

ascorbic acid, gallic acid, etc.

beverages

SWV

AA

flavored waters

SWV

beverages

SWV

guanine /GCE Adenine/GCE guanine /GCE adenine /GCE guanine/ GCE

0.10-5.00 mg/L 0.10-4.00 mg/L 0.5-5 mg/L 2-19 mg/L 0.10-0.80 mg/L

0.08 mg/L 0.10 mg/L 0.06-0.29 mg/L

beverages

SWV

adenine /GCE DNA(d20)-CPE

0.10-6.00 mg/L 1.0-20.0 mg/L

0.10-0.99 mg/L 0.23 mg/L

guanine /GNR/GCE ds-DNA/CHIT-MWCNTs/PGE ds-DNA/CPE

0.1-4 mg/L 1.0-100.00 mM 0.30-12 µmol/L

0.05 mg/L 1.0 mM

ascorbic acid, gallic acid, etc. AA ascorbic acid glutathione, AA oleuropein AA, resveratrol, gallic acid 2 3 4

fruit juices olive leaf extract orange juice beverage,

CV, DPV EIS, DPV DPV CV

CHI/ds-DNA/Ag-PGL/GCE

Linear range

1.0-50 µmol/L (AA)

LOD

Ref [17] [21] [22] [23] [24] [32] [34] [35] [38] [39]

SWV: square-wave voltammetry; DPV: differential pulse voltammetry; CPE: carbon paste electrode; GNR: graphene nanoribbon; AdSDPV: adsorptive transfer stripping voltammetry; ds-DNA: double stranded DNA; Mn(II) complex: mononuclear complex [Mn(thiophenyl-2-carboxylate)2(H3tea)]; CHI: chitosan; PGA: poly ʟ-glutamic acid; LSV: linear sweep voltammetry; AA: ascorbic acid; PGE: pencil graphite electrode; EIS: electrochemical impedance spectroscopy;

5

Table 2 Partial applications of enzyme-based biosensors for antioxidant assessment Index TPC

Sample

Transducer

red fruits extract tea, alcoholic beverages, etc.

CV, DPV, SWV

polyphenols

wine

CV

polyphenols

fruit juices

CV, CA

TPC

tea leaves extract

CV, EIS

phenols

tea

CV

O2•−

-

EPC

tea infusions

CV, EIS CV, amperometric CV, EIS

Polyphenol

TAC/O2•− catechin and caffeic acid

beverages red wine

catechol

CV, EIS

Modification

Linear range

Limit detection

Ref

Lac/CPE Lac/AgNPs/cMWCNT /PANI/GE Lac (TvL/ThL)-(MWCNTs/SWC NTs)-SPE

0.01-3.5 µM

0.01 µM

[45]

0.1-500 µM

0.1 µM

[53]

Tyr(Lac)/GO/MWCNTs Lac/Fe3O4NPs/cMWCNT/PA NI/GE CLEC Lac/β-CDEP/PVP gel/Au cathode SOD/PtPd-PDARGO/ SPGE Lac /PVA-AWP/SPE

0.1 mg/L for TvL 0.3 mg/L for ThL

[54]

1-300 µM for ThL 1-340 µM for Tyr 0.1-10 µM 10-500 µM

0.3 µM for ThL 0.5 µM for Tyr

[55]

0.03 µM

[56]

50-1000 µM

50 µM

[62]

0.16-0.24 mM

2 µM

[63]

0.5-250 µM

0.524-35.432 µM

[65]

0.1-18.0 mg/L

SOD/PEDOT/ MWCNT

1-300 µM

1 µM

[70]

CV

DPEM /Lac/Pt electrode

2.0-14.0×10-6 M

1.0×10-6 M

[72]

water

CV，DPV

Lac /GONs/EDOT

TPC

wine, tea

CV

TPC TPC

Honey, propolis plants extracts

potentiometric CV

PBHR/Fc/MWCNT + MO Tyr/prepared electrode Lac/MWCNTs/ CHI

0.036-0.35 µM, 0.032 µM 0.35-2.5 µM 0.05-52 mg/L for t-resveratrol 0.023 mg/L 0.06-69 mg/L for caffeic acid 0.020 mg/L -7 -2 7.3×10−7 M 9.3×10 -8.3×10 M 9.1×10-7-1.21×10-5 mol/L 2.33×10-7 mol/L

[91] [92] [93] [94]

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Phenol content TPC TAC

rutin simulated sample olive oil orange juice

TAC, AA

blueberry, kiwi and orange juice

SWV, CV DPV CV,

CV

PPO/β-CDEP/ graphite/ Nujol /Ir-BMI·PF6 Tyr/SPE AOx/ BSA/PEI/ PU electrode FC60/AOx/CRE FC70/AOx/CRE SWCN/AOx/CRE MWCN/AOx/CRE

1.3×10-7-2.0×10-6 M 10-500 µM 0-20 µM

0-20 µM

7.9×10-8 M

[95]

4 µM 0.26 µM 0.10 µM 0.13 µM 0.2 µM 0.22 µM

[96] [97]

[98]

AOx: Ascorbate oxidase; β-CDEP: β-cyclodextrin; BMI·PF6: 1-butyl-3-methylimidazolium hexafluorophosphate; BSA: bovine serum albumin; CA: chronoamperometry; CRE: carbon rod electrode; CV: cyclic voltammetry; DPEM: derivatized polyethersulfone membrane; EPC: equivalent polyphenolic content; Fc: ferrocene; FC60: fullerene C60; FC70: fullerene C70; GCE: glassy carbon electrode; GE: gold electrode; Lac: laccase; MO: mineral oil; PBHR: Brassica napus hairy roots which can provide peroxidases; PDA: polydopamine; PEI: polyethylenimine; PPO: polyphenol oxidase; PU: polyurethane; PVA-AWP: azide-unit pendant water-soluble photopolymer; PVP: polyvinylpropylidone; RGO: reduced graphene oxide; SPGE: screen-printed gold film electrodes; SOD: enzyme bovine Cu-Zn superoxide dismutase; SPE: screen-printed carbon electrode; TAC: total antioxidant capacity; ThL: Trametes hirsute; TPC: total polyphenolic content; TvL: Trametes versicolor; Tyr: tyrosinase; XOD: xanthine oxidase.

24

Table 3. CBBs and cyt c-based biosensors for antioxidant assessment Target Molecule

Cell line

Modification

Linear range

LOD

Ref

catechol, caffeic acid, gallic acid, and etc. RAC of peptide Asp-Leu-Glu-Glu (DLEE) RAC of Lactobacillus plantarum strains

red wine, tea

Recombinant E. coli MB275 cells

GC

5.0-500.0 µM

1.0-5.0 µM

[3]

dry-cured Xuanwei ham

Caco-2 cell

NaAlg/GO/Pt NPs/Ag NWs/GE

0.2-2 µM

0.12 µM

[79]

catechol

red wine, tea

antioxidants activities

plant extracts

antioxidant assessment antioxidants activities

food antioxidants: VC, βC, OPC and TP longan seed polyphenols and etc. flavonoids

RAW 264.7 macrophage cells Recombinant E. coli MB275 cells Vero fibroblast cells Glioma cells (U87) Fibroblast NIH-3T3 cell cyt c

antioxidants activities

orange juices

cyt c

antioxidants activities

25 26

Sample

Chinese dry-cured ham

NaAlg/GO/a-MnO2/GE

0.05-0.85 µM

0.02 µM

[78]

GC

0.5-300.0 µM

0.1 µM

[79]

alginate

[81]

AgNPs/Co3O4@C/GCE microelectrode arrays GC XOD/MU/MUA/S PGE

1.69×(10–13 - 10−7 M

0.0564 pM

[84] [85]

IC5078nM-0.5mM IC50 34.0 µM for AA, 209.5 µM for Trolox

[90] [99]

cyt c: cytochrome c; βC: β-carotene; Glu: glutaraldehyde; L. plantarum: Lactobacillus plantarum; MU/MUA: mercaptoundecanol/mercaptoundecanoic acid; NaAlg/GO: alginate/graphene oxide; OPC: antholyanin; Pt NPs: platinum nanoparticles; TP: tea polyphenols; VC: vitamin C.

Recent advances in electrochemical biosensors for antioxidant analysis in foodstuff Yongli Yea, Jian Jia, Zhanyi Sunb, Peili Shenb, Xiulan Suna* a State Key Laboratory of Food Science and Technology, School of Food Science, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, PR China b State Key Laboratory of Bioactive Seaweed Substances, Qingdao Brightmoon Seaweed Group Co Ltd, Qingdao, Shandong 266400, PR China

Corresponding author*: Xiulan Sun (E-mail: [email protected])

1

Fig.1 Illustration of electrochemical biosensor system for antioxidants assessment.

2

Fig.2 Working principle diagram of DNA-based electrochemical biosensor.

3

Fig.3 Schematic illustration of the preparation of modified enzyme-based electrode

4

Fig.4 A. Schematic illustration for the antioxidants assessment of A. RAW264.7 cell-based

5

electrochemical biosensor [70] and B. Caco-2 cell-based Pt NPs/Ag NWs/GE biosensor [71].

6

Fig.5 Schematic diagram of cell-based electrochemical biosensor system for antioxidant

7

evaluation. A. A549 cell-based biosensor [80], B. Fibroblast NIH-3T3 cell-based impedance

8

biosensor [74]. C. Construction of engineered E. coli MB275 cells-based biosensor [49].

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Highlights •Electrochemical biosensor strategies are discussed and they have vast potential applications in antioxidant analysis. •Short review on DNA-based electrochemical biosensor used in antioxidant assessment. •Enzyme-based electrochemical biosensors for antioxidants quantification and evaluation were depicted. •The potential of cell-based electrochemical biosensor in antioxidant analysis were demonstrated.

Competing interests: The authors declare no competing interests.