Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo

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Perspective

Molecularly imprinted polymer as an antibody substitution in pseudoimmunoassays for chemical contaminants in food and environmental samples Chaochao Chen, Jiaxun Luo, Chenglong Li, Mingfang Ma, Wenbo Yu, Jianzhong Shen, and Zhanhui Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05577 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 21, 2018

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Journal of Agricultural and Food Chemistry

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Invited Perspective for the Journal of Agricultural and Food Chemistry

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Molecularly imprinted polymer as an antibody

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substitution in pseudo-immunoassays for chemical

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contaminants in food and environmental samples

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Chaochao Chen, Jiaxun Luo, Chenglong Li, Mingfang Ma, Wenbo Yu, Jianzhong Shen,

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Zhanhui Wang*

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Beijing Advanced Innovation Centre for Food Nutrition and Human Health, College of

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Veterinary Medicine, China Agricultural University, Beijing Key Laboratory of

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Detection Technology for Animal-Derived Food Safety, Beijing Laboratory for Food

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Quality and Safety, Beijing 100193, People’s Republic of China

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* Author to whom correspondence should be addressed

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Tel: +86-10-6273 4565

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Fax: +86-10-6273 1032

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E-mail: [email protected]

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1

ACS Paragon Plus Environment


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ABSTRACT

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The chemical contaminants in food and environment are quite harmful to food safety

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and human health. Rapid, accurate and cheap detection can effectively control the

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potential risk derived from these chemical contaminants. Among all detection methods,

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the immunoassay based on specific interaction of antibodies-analyte is one of the most

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widely used technique in the field. However, biological antibodies employed in

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immunoassay usually cannot tolerate extreme conditions, resulting in unstable state in

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both physical and chemical profiles. Molecularly imprinted polymers (MIPs) are a class

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of polymers with specific molecular recognition abilities, which are highly robust,

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showing excellent operational stability under a wide variety of conditions. Recently,

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MIPs have been used in biomimetic immunoassays for chemical contaminants as

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antibody substitute in food and environment. Here, we reviewed these applications of

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MIPs

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immunosorbent assay, fluorescent immunoassay, chemiluminescent immunoassay,

34

electrochemical

35

homogeneous immunoassay, and discussed current challenges and future trends in the use

36

of MIPs in biomimetic immunoassays.

incorporated

in

different

immunoassay,

analytical

microfluidic

platforms, such

paper-based

as

enzyme-linked

immunoassay,

and

37 38

KEYWORDS: molecularly imprinted polymers, biomimetic immunoassay, chemical

39

contaminants, food, environment

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INTRODUCTION

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Food safety and environmental protection is directly related to human health, which

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is a hot topic of study worldwide. Currently, the detection of chemical contaminants in

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food and environment, such as small organic molecules (e.g., phenolic substances,

44

antimicrobial agents, insecticides, and illegal additives), mycotoxins (e.g., aflatoxins and

45

ochratoxins), and small inorganic molecules (e.g., phosphate), is usually achieved by

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physical and chemical analysis like chromatography and chromatography-mass

47

spectrometry,

48

chromatography and chromatography-mass spectrometry, which needs complicated and

49

expensive equipment, immunoassays that are based on antigen-antibody specific

50

interaction have been widely applied due to the high sensitivity, specificity,

51

reproducibility, and analysis speed.

biological

detection,

and

the

immunoassay1.

Compared

with

52

In the past decades, the immunoassay has been continuously improved and

53

developed, and a variety of immunoassays format with different labels have been

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established in the following order: radioimmunoassay, enzyme-linked immunoassay,

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fluorescent immunoassay, and chemiluminescent immunoassay2. These assays have been

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effectively applied in the detection of chemical contaminants in food control and

57

environment monitoring. The development of immunoassay is mainly reflected in the

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synthesis of new labeled probes and the development of highly efficient labeling

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technologies, such as the appearance of nanoprobes (e.g. quantum dots, graphene, and

60

upconversion luminescent materials). Despite the advances in efficient labeling of probes,

61

the sensitivity and stability of the current immunoassay methods based on traditional

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antibodies still do not meet the requirements of highly sensitive detection due to the 3



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complexity of the matrix in food and environmental samples. This is mainly because

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biological antibodies, including the receptor protein, aptamer, and other protein scaffolds,

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are susceptible to matrix interference when the immunoreaction happens, especially

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under acid, alkali, high-salt, high-temperature, and other harsh conditions, thus affecting

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recognition ability and resulting in low sensitivity and stability. The preparation of

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antibodies and bioconjugates not only takes a long time and costly, but also requires a

69

large number of experimental animals. Because of the low chemical and thermal stability,

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biological antibodies is not suitable for long-term preservation. In addition, some toxic

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compounds and immunosuppressive agents have strong toxic effects on immune system,

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causing low yield of effective antibodies from host animals. All above mentioned

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disadvantages make the preparation of biological antibodies and the development of

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immunoassay faces great challenges. Consequently, materials with high affinity, high

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stability, and low cost which could act as antibody function in assay are in high demand3.

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Molecularly imprinted polymers (MIPs) have multiple binding sites on the surface,

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which can selectively recognize target molecules, appearing in response to this demand.

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At present, MIPs have applied to many fields such as solid phase extraction4, chiral

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separation5, drug controlled release6, mimetic enzyme catalysis7, environmental

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monitoring8. Compared with traditional biological molecules, MIPs can tolerate high

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temperature, acid, alkali, and organic solvents, and they do not degrade easily, which

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makes them reusable. Therefore, MIPs as biomimetic antibodies represent a stable and

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low-cost alternative to existing antibodies in immunoassays. In this perspective, we

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elucidated pseudo-immunoassays based on MIPs from the aspects of biomimetic enzyme-

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linked immunosorbent assay (ELISA), biomimetic fluoroimmunoassay, biomimetic

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chemiluminescent immunoassay, biomimetic electrochemical immunoassay, biomimetic

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microfluidic paper-based immunoassay, and biomimetic homogeneous immunoassay

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with the aim of facilitating the development and exploration in the current field for

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researchers (Figure 1).

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PSEUDO-IMMUNOASSAYS

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Biomimetic ELISA. The ELISA is the most widely used detection technology for

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rapid detection of chemical contaminants in food and environment. It has many

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advantages such as being fast, sensitive, and easy to standardize; also, its results are easy

95

to evaluate and capable of detecting lots of samples. Therefore, a variety of biomimetic

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ELISAs (BELISA) have been established by combining with MIPs and applied to detect

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chemical

98

sulfamethazine residue12, trichlorfon13, metolcarb residues14, and methimazole15. In

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BELISA, MIPs, instead of antibodies, are coated on the microplate, and detect the targets

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generally by competitive methods. Compared with the traditional ELISA, there is no need

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for repeated coating and blocking steps, thus assay time is reduced. In addition, MIPs can

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be reused repeatedly without loss of activity, making up for the defects of biological

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antibodies. However, compared with conventional antibody based assay, the sensitivity of

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BELISA is not significant improved. This may due to the low affinity of MIPs.

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Furthermore, MIPs are mostly prepared in organic solutions which leads the denaturation

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of enzymes and other used proteins.

contaminants,

such

as

enrofloxacin9,

carbaryl10,

5


malachite

green11,


107

With the development of hydrophilic MIPs, the sensitivity of BELISA has been

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greatly improved. For example, Chianella et al.16 synthesized vancomycin MIP

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nanoparticles (nanoMIPs) by solid-phase synthesis in a special reactor, using vancomycin

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covalently bound on glass beads as a template. The prepared nanoMIPs were then coated

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on a microplate, and a direct competitive BELISA was established to detect vancomycin

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in porcine plasma (Figure 2). The detection sensitivity of this method is three times

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higher than that of traditional ELISA. It can accurately detect vancomycin with the limit

114

of detection (LOD) as low as 2.5 pM, and it was less affected by the plasma matrix.

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Smolinska-Kempisty et al.17 synthesized MIPs of four different small molecules to

116

establish BELISA, and compared them to ELISA with antibodies as the recognition

117

molecule. They found that the BELISA method could detect the target at picomolar

118

concentrations with no cross-reactivity, and performed similarly to or even better than

119

ELISA which is based on biological molecules. In addition, it has a high stability because

120

the microtiter plate coated with nanoMIPs membrane can be stored at room temperature

121

for one month without loss of activity. These findings have laid a solid foundation for

122

commercial applications of MIPs in the field of biological detection.

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Biomimetic Fluoroimmunoassay. The fluorescent detection has the advantage of

125

rapid, simple, and high sensitivity, thus it shows great potential for rapid detection. The

126

biomimetic fluoroimmunoassay (BF), in which MIPs are integrated with fluorescent

127

molecules, takes advantage of the recognition capabilities of fluorescent MIPs (fMIPs)

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for target molecules and uses a fluorescence analyzer for fluorescence signal. It combines

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the selectivity of MIPs with the high sensitivity of fluorescence detection, realizing fast

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and simple detection and analysis for targets. Based on whether the target molecule has

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the property of fluorescence, BF detection can be classified into two categories: direct

132

fluorescence detection and indirect fluorescence detection18. In direct fluorescence

133

detection, the target molecule should possess a fluorophore or chromophore itself. The

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fluorescence intensity changes after MIPs bind with the target molecule thereby enabling

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the detection. For instance, in the work of Ton et al.19, as enrofloxacin (ENRO) is UV-

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excited, it was used as a template to synthesize hydrophilic MIPs in order to detect

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fluoroquinolones in water and milk. However, very few targets themselves possess

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fluorescence property, the application of direct fluorescence detection is restricted. The

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most commonly used technique is indirect fluorescence detection. During the MIPs

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preparation process, fluorescent functional monomers or fluorescent materials such as

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organic dyes, quantum dots(QDs), and upconversion fluorescent molecules are

142

introduced. When the target molecules bind with the MIPs, electron, charge, or energy

143

transfer will occur in the fluorescent functional monomer or fluorescent material, leading

144

to fluorescence quenching or enhancement by which the analytes are detected. We will

145

discuss as follows: i) BF based on organic fluorescent dyes; ii) BF based on quantum dots;

146

iii) BF based on metallic nanomaterials; iv) BF based on optical fiber.

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Biomimetic Fluoroimmunoassay Based on Organic Fluorescent Dyes. Organic

149

fluorescent dyes are often used in the establishment of fMIPs chemosensors (fMIPcs) to

150

detect contaminants, because of their high fluorescence quantum yield in water or organic

151

phase and no radiation. Wu et al.20 established fMIPcs by doping dansyl methacrylate as

152

a functional monomer in order to detect BPA in environmental water, which also can

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monitor the biodegradation of BPA in real time. The prepared fMIPs have dual functions

154

of binding BPA and generating a fluorescent signal. Since BPA has two phenolic

155

hydroxyl groups, it is weakly acidic and can interact with the amino groups of dansyl

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methacrylate which altered the electron density near the fluorophore center, resulting in

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fluorescence quenching. The fMIPcs could accurately detect BPA with a LOD of 13.14

158

pM in environment. Wang et al.21 prepared fMIPs via fluorescein 5(6)-isothiocyanate and

159

3-aminopropyltriethoxysilane/SiO2 particles for detection of λ-Cyhalothrin in chinese

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spirits. By fluorescence quenching mechanism, the fMIPs could detect λ-Cyhalothrin

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with a LOD of 9.17 nM, which demonstrated fMIPcs a potential tool for recognition and

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detection of analytes in food.

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Some researchers used Fe3O4@SiO2 magnetic nanoparticles in combination with

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organic dyes to prepare fMIPs, which have exhibited excellent superparamagnetism and

165

stable fluorescence properties. For example, Gao’s group22 first coated SiO2 and 3-

166

(methacryloxyl) propyl trimethoxysilane (MPS) on Fe3O4 to form Fe3O4/SiO2-MPS,

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which was used as core, and then synthesized fMIPs shell by copolymerization of

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acrylamide with allyl fluorescein on the surface of Fe3O4/SiO2-MPS (Figure 3A), thereby

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detecting λ-cyhalothrin in honey. Due to the unique magnetic properties of Fe3O4

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nanoparticles, the synthesized fMIPs were much easier to separate from the solution,

171

which simplified the whole process. In addition, the fMIPs can be recycled by an external

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magnetic field, and there was almost no fluorescence intensity decrease after being reused

173

for five times, which makes fMIPs potential substitution of antibodies.

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Biomimetic Fluoroimmunoassay Based on Quantum Dots. Besides organic

176

fluorescent dyes, QDs are also widely used in BF. They have stable photochemical

177

properties and high luminous efficiency; the emission spectrum is narrow and

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symmetrical, and the fluorescence signals can be obtained without background

179

interference, when compared with organic fluorescent dyes. Furthermore, changing the

180

conditions of QD growth in synthesis could obtain QDs with different emission

181

wavelengths. Xu et al.23 used trinitrophenol as a mimicking template, 3-

182

aminopropyltriethoxy silane (APTES) as the functional monomer, and coated MIPs on

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CdTe QDs by the sol-gel method to synthesize fMIPs (Figure 3B). The synthesized

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fMIPs were utilized as recognition elements to detect 2,4,6-trinitrotoluene (TNT) in soil

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on the basis of electron transfer induced fluorescence quenching. With the existence and

186

increase of TNT in samples, a Meisenheimer complex can be formed between the TNT

187

and the amino groups of APTES on QDs. The energy of QDs transferred to the

188

Meisenheimer complex which would lead to fluorescence quenching, thereby detecting

189

TNT. The detection range of this method was 0.8–30 µM with a LOD of 0.28 µM.

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Raksawong et al.24 synthesized hybrid MIP-coated QD nanocomposite to detect

191

salbutamol. When salbutamol molecules bind with the recognition sites in MIP, electron

192

transfer happens from QDs to salbutamol, which leads to a fluorescence quenching in the

193

nanocomposite. The synthesized nanocomposite could sensitive and selective detect

194

salbutamol in animal feeds and meat samples with a detection limit of 0.142 pM.

195

However, QDs encapsulated into highly cross-linked MIPs often affect the fluorescence

196

quenching efficiency, leading to poor detection sensitivity18. To increase the fluorescence

197

quenching efficiency and fluorescent signals, mesoporous structures are used by

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198

researchers to synthesize MIPs with nanosized pore wall. For instance, Kim et al.25

199

prepared fMIPs by using mesoporous silica for the detection of BPA. The fMIPs had a

200

detection sensitivity of 0.438 nM and were highly selective. As mesoporous silica

201

usually had a highly cross-linked rigid structure with a large pore volume, the reaction

202

sites of CdSe QDs with mesopores were located in the mesopores. Therefore, QDs could

203

stably exist in mesoporous silica to make the analyte bind to the functional monomer

204

more easily.

205

However, the self-quenching of QDs is prone to occur, which seriously limit its

206

application in bio-analysis. The ion-doped QDs not only retain the advantages of

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traditional QDs, but also have a large Stokes shift, which can effectively avoid self-

208

quenching. Therefore, ion-doped QDs have attracted more and more attention, and the

209

use of them in preparing fMIPs has become a hot research topic. In a study by Ren et

210

al.26, Mn2+ doped ZnS QDs were used as core, and MIPs were encapsulated on its surface

211

to detect cyphenothrin in water samples. As the UV absorption of cyphenothrin was close

212

to the band gap of absorption spectra of MIPs-QDs, when cyphenothrin was added, the

213

conduction band electrons of QDs transited to the lowest unoccupied orbit of

214

cyphenothrin, a charge transfer then occurred, resulting in fluorescence quenching. The

215

LOD of this method was 9 nM with a good linearity in the concentration range of 0.1-80

216

µM, which provides a new way of preparing fMIPs by encapsulating ion-doped quantum

217

dots.

218

Although ion-doped QDs have been used in the field of food and environmental

219

detection, they are all preparation of heavy metal and are highly toxic to humans and

220

environment. Thus, graphene quantum dots (GQDs), which show low toxicity, high

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fluorescence activity, chemical inertness, and excellent water solubility, have become one

222

of the most popular optical elements in multi-field research. Mehrzad-Samarin et al.27

223

embedded GQDs into silica MIPs, then synthesized an optical nanosensor to detect

224

metronidazole in biological samples. The fluorescence of the GQD-MIPs was

225

proportionally quenched in the presence of different concentrations of metronidazole, and

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a LOD of 0.15 µM was obtained. Similar to GQDs, carbon dots are also used in the field

227

of biological detection as a new material because of their unique optical properties, low

228

toxicity, and high biocompatibility. Yang et al.28 encapsulated carbon dots on MIPs by

229

reverse microemulsion method to detect tetracycline in fish samples with a LOD of 9 nM,

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laying a foundation for the establishment of new MIPs based fluorescence biosensors.

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Biomimetic Fluoroimmunoassay Based on Metallic Nanomaterials. Some of

233

metallic nanomaterials have unique luminescent properties and have become a powerful

234

component in the construction of chemical sensors. For example, noble metallic

235

nanoclusters have strong fluorescence due to the aggregation induced effect and are

236

widely used in the preparation of fluorescent probes because of their small size, mild

237

preparation conditions, and non-toxic properties29. Compared with organic fluorescent

238

dyes, noble metallic nanoclusters exhibit stronger and more persistent fluorescence

239

signals; also, they are less biologically toxic and have better biocompatibility than QDs.

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Therefore, they have showed great potential for practical applications in many fields. By

241

combining noble metallic nanoclusters with MIPs and traditional immunoassays, the

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performances can be significantly improved in several aspects. Wu et al.30 covalently

243

bound gold nanoclusters (AuNCs) with SiO2 nanoparticles to form SiO2@AuNCs and

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244

prepared MIPs membrane by the sol-gel method in SiO2@AuNCs using BPA as a

245

template (Figure 3C). When BPA existed, it would form a Meisenheimer complex with

246

the primary amino group on the surface of AuNCs. For the principle of fluorescence

247

resonance energy transfer, the fluorescence energy of AuNCs was transferred to the

248

complex, leading to fluorescence quenching. BPA was detected with the changes in

249

fluorescence intensity and a LOD of 0.963 nM was achieved. In this paper, MIPs showed

250

high selectivity for BPA which makes it a stable alternative to existing antibodies. Based

251

on the fact that silver nanoclusters (AgNCs) in the aqueous solution showed higher

252

fluorescence intensity than AuNCs, which have more superiority in optical detection31,

253

Deng et al.32 synthesized MIPs of BPA on the surface of AgNCs and detected BPA with a

254

LOD of 87.6 pM, which was an order of magnitude higher than that of Wu’s work.

255

Besides noble metallic nanomaterials, lanthanides, as emerging optical materials, are

256

gradually combined with MIPs for applications in bio-analysis field. They have abundant

257

emission spectra, high quantum yield and chelate stability. Liu et al.33 developed core-

258

shell surface molecularly imprinted fluorescent sensor, YVO4:Eu3+@MIPs, using

259

lanthanide doped YVO4:Eu3+ nanoparticles as the signal output and MIPs as recognition

260

elements, while λ-cyhalothrin was employed as the molecular template. Based on the

261

energy transfer principle, When λ-cyhalothrin was combined with MIPs, the donor

262

YVO4:Eu3

263

fluorescence quenching, thereby qualitatively and quantitatively detecting the λ-

264

cyhalothrin in environment with a LOD of 1.76 µM. Liu’s group34 also synthesized

265

fMIPs, mSiO2-Eu (TTA) 3Bpc@MIPs, by Pickering emulsion polymerization with good

266

homogeneity and monodispersity, and successfully applied it to detect λ-cyhalothrin. The

+

transferred its energy to the receptor λ-cyhalothrin, resulting in static

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Eu3+-modified mesoporous silica microspheres mSiO2-Eu(TTA)3Bpc synthesized can

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replace the surfactant as a stabilizer in Pickering emulsion polymerization, making the

269

dispersed droplets more stable. This is a new type of interfacial nanotechnology, which is

270

suitable for the preparation of MIPs, and the final results showed that these MIPs have

271

good stability, as the fluorescence intensity remained stable for nearly a month.

272 273

Biomimetic Fluoroimmunoassay Based on Optical Fiber. Unlike MIPs based on

274

organic materials and SiO2 nanoparticles, optical-fiber based MIPs sensors are light in

275

weight, small in volume, and easy to work into a variety of configurations. They possess

276

anti-electromagnetic interference, electrical insulation, corrosion resistance, and can be

277

measured from a long distance. Ton et al.35 prepared micro-scale MIPs at the end of an

278

optical fiber by photo-polymerization, and it only took a few seconds to prepare the

279

fluorescence sensor. Gold nanoparticles were embedded in the polymers to improve

280

signal response. The sensor was used to detect herbicide 2,4-D with a LOD of 250 pM,

281

which is comparable to that of the LC-MS method. This kind of fluorescence

282

enhancement strategy avoids the occurrence of false positives due to non-specific

283

fluorescence quenching, thus it is more advantageous than fluorescence quenching

284

method. Later, Carrasco et al.36 utilized an optical fiber bundle containing about 50,000

285

individual fibers to create microwells via chemical etching, which was then deposited

286

with red-fluorescent-encoded MIPs for ENRO to detect antibiotic. It worked by red-

287

fluorescently-tagged ENRO competing with untagged ENRO. In the presence of low

288

concentration of ENRO, strong fluorescence would be detected. The sensor demonstrated

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289

excellent selectivity which makes it potential for ENRO determination in complex

290

samples.

291

With the developments in science and technology, the new fiber optic sensor came

292

into being, which combines the evanescent wave, BF, and optical fiber into one single

293

sensor. The evanescent wave generates when light is transmitted in the optical fiber in a

294

total reflection manner and stimulates fluorescence MIPs on the surface of the fiber core,

295

causing fluorescent changes. The target molecules attached to the fiber surface could then

296

be detected by the changes in fluorescence intensity. Ton et al.37 developed a disposable

297

optical fiber sensor by coating MIP particles on 4-cm long polystyrene optical

298

waveguides with N-(2-(6-4-methylpiperazin-1-yl)-1,3-dioxo-1H-benzo[de]isoquinolin-

299

2(3H)-yl-ethyl)acrylamide (FIM) as a fluorescent monomer. FIM produces fluorescence

300

under the evanescent wave excitation, and the target molecule with a carboxyl group

301

bound to FIM would enhance its fluorescence. The herbicides 2,4-D and citrinin were

302

detected based on this mechanism. The LOD can reach nanomolar level with high

303

sensitivity. Though the BF has been widely in the detection, the problem of low

304

sensitivity is common in MIPs-based fluorescent assays. There still exists much work to

305

be done to improve the sensitivity while maintaining the selectivity18.

306 307

Biomimetic

Chemiluminescence

Immunoassay.

Unlike

the

fluorescence

308

immunoassay, the chemiluminescence immunoassay does not require external excitation

309

and has the advantages of simplicity and wide detection range. However, the anti-

310

interference ability of the chemiluminescent assay derived from matrices is not enough

311

robust, which would lead to different luminescence signals with remarkable matrix effect

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312

for the same quantity in different of sample matrices.38 In recent years, researchers found

313

that MIPs could effectively eliminate the matrix effect, and used MIPs to specifically

314

identify and capture target molecules, which significantly improves the selectivity of the

315

chemiluminescence immunoassay.

316 317

Flow Injection Biomimetic Chemiluminescence Immunoassay. The most significant

318

feature of flow injection chemiluminescence (FI-CL) is its sensitivity; however, it has

319

poor specificity. By combining FI-CL with MIPs, the problem of selectivity is

320

fundamentally avoided. For example, Qiu et al.39 developed a FI-CL sensor using MIPs

321

as biomimetic antibodies to detect sulfamethoxazole. With high adsorption binding sites

322

of MIPs, the sulfamethoxazole could be absorbed selectively

323

With the popularity of surface imprinting technology based on Fe3O4 magnetic

324

beads (MBs), the amount of its combined application with FI-CL is on the rise. Lu et al.40

325

obtained core-shell magnetic molecularly imprinted polymers (MMIPs) with chrysoidine

326

acting as molecular templates by surface imprinting on Fe3O4@SiO2 MBs. The MMIPs

327

were then combined with FI-CL to detect chrysoidine with a detection limit of 0.617 µM.

328

Based on Lu’s work, Cao et al.41 introduced an oil-based magnetic fluid to successfully

329

prepare surface MIPs with oleic acid (OA) encapsulating Fe3O4 MBs. The OA@Fe3O4

330

has strong magnetic properties and can be well dispersed in the oil phase. It improved the

331

adsorption capacity and separation performance of MIPs, thus enhancing the selectivity

332

of FI-CL.

333

Core-shell imprinted microspheres and solid-core imprinted microspheres prepared

334

by surface imprinting technique only have effective recognition sites on the surface of

15



335

MIPs. Being different with them, porous hollow imprinted microspheres form a high

336

density of binding sites not only on the surface, but also in the interior, thereby greatly

337

improving the recognition efficiency of MIPs. Zhao et al.42 prepared fenpropathrin-

338

imprinted porous hollow microspheres (FIPHMs) by using mesoporous silica particles

339

and encapsulated FIPHMs in a "Y"-channel flow cell as recognition elements, then a flow

340

injection system was connected with to construct a FI-CL-MIP sensor. The sensor could

341

achieve detection of fenpropathrin with a LOD of 87.8 pM.

342 343

Biomimetic electrogenerated chemiluminescence immunoassay. Electrogenerated

344

chemiluminescence immunoassay (ECL) based on MIPs (MIP-ECL) is a new analytical

345

method developed in recent years. It integrates the advantages of MIPs into ECL, and

346

shows great application prospects in food, environment and other fields. Li’s group43

347

obtained MIP membranes by electro-polymerization on the surface of gold electrodes

348

using isoproturon (IPU) as templates, and prepared a new type of MIP-ECL sensor

349

through competitive adsorption of IPU and glucose oxidase-labeled IPU (GOD-IPU) on

350

MIP membranes and the chemiluminescence response of luminol and H2O2 produced by

351

GOD’s hydrolysis of glucose. The sensor was successfully applied to detect IPU in the

352

water sample with a LOD of 3.78 pM. Later, Li’s group44 also established a MIP-ECL

353

method for determination of gibberellin A3 (GA3) based on direct competition (Figure

354

4A). First, the MIPs membrane was synthesized on gold electrodes, and GA3 directly

355

competed with rhodamine B-labeled GA3 (RhB-GA3) to bind with MIPs. When RhB-

356

GA3 was bound to the MIPs membrane, RhB was electrochemically oxidized to produce

357

intermediate RhBox, thereby amplifying the chemiluminescence signal of luminol in the

16


Page 16 of 44

Page 17 of 44


358

solutions to achieve the determination of GA3. The results showed that the MIP-ECL

359

sensor had a good selectivity and a high sensitivity to GA3 with a LOD of 3.45 pM.

360

Recently, exploring nanomaterials as MIP-ECL emitters has become a new trend.

361

For instance, as UCNPs possess tremendous ECL performance and prolonged

362

fluorescence lifetime, Jin et al.45 employed them as luminophor in MIP-ECL for detection

363

of clenbuterol with a LOD of 6.3 nM. They first prepared reduced grapheme oxide (rGO)

364

on electrode and fabricated UCNPs; later MIPs were made upon the electrode. When

365

there was no analyte in the K2S2O8 solution, the coreactant S2O82- would react with

366

UCNPs and generate light. With the increasing concentration of clenbuterol, the

367

combination of clenbuterol with MIPs would block electron transfer channels between

368

UCNPs and S2O82-, thereby decreasing the ECL response gradually. In addition, the

369

clenbuterol mixing with S2O82- would be catalyzed into new intermediates, which could

370

quench the ECL intensity of UCNPs. Based on above mechanisms, the clenbuterol was

371

detected. But generally, chemical luminescence is the result of chemical reaction between

372

marked enzymes and substrates. The luminescence is not stable and intermittent. And it is

373

easy to fission in the reaction process, which results in unstable result. Thus, researching

374

in new luminescent materials and how to prolong the luminescence time are imperative.

375 376

Biomimetic Electrochemical Immunoassay. Electrochemical sensors are currently

377

the most sophisticated type of sensor. They selectively convert response to target

378

molecules into electrical signals, which requires the incorporated material of the sensor

379

be capable of molecular recognition ability. Based on the high selectivity of MIPs,

380

combining them with an electrochemical sensor could obtain a biomimetic

17



381

electrochemical sensor, molecular imprinted electrochemical sensor (MIECS), thereby

382

achieving rapid and efficient detection of target molecules. Based on different response

383

signals, MIECS are divided into five categories: amperometric sensors, potentiometric

384

sensors, conductometric sensors, impedimetric sensors, and piezoelectric sensors.

385 386

Biomimetic Amperometric Sensors. Biomimetic amperometric sensors quantitatively

387

detect the changes of the current before and after target molecules binding to MIPs. The

388

key is that there must be certain pores in the MIPs membrane so that the target molecules

389

can penetrate through the membrane to the surface of the electrode surface, and then a

390

redox reaction would occur to generate voltammetric response. For example,

391

Amatatongchai et al.46 prepared a biomimetic amperometric sensor by electro-

392

polymerization using 4-ter-butylcalix [8] arene-carbofuran as the template, and achieved

393

detection of carbofuran with a LOD of 3.8 nM.

394

Since the sensitivity of the MIPs sensor is directly related to the effective

395

recognition ability of MIPs, in order to raise the efficiency of imprinting thereby

396

improving the sensitivity, Xie et al.47 developed PATP-AuNP-gc sensor by molecular

397

imprinting in electro-polymerized poly aminothiophenol (PATP) membranes on glassy

398

carbon electrode which was modified by AuNPs for pesticide chlorpyrifos (CPF) (Figure

399

4B). As the large surface of AuNPs can greatly increase the recognition sites on the

400

surface of the imprinting sensor and good electron transport rate can improve electron

401

conductivity of the imprinted membrane, the amperometric response was 3.2 times better

402

than that of the imprinted PATP-Au sensor, which was made by electro-polymerization

403

of PATP directly on gold electrode, and the LOD was also two orders of magnitude lower

18


Page 18 of 44

Page 19 of 44


404

than that of the imprinted PATP-Au sensor. The biomimetic amperometric sensor can

405

resist acid, alkali, and heavy metals. Thus it has a good stability and has been widely

406

used. However, it is only suitable for the detection of targets with electrical activity. For

407

the detection of targets without electrical activity, the addition of electroactive molecules

408

such as potassium ferricyanide as an electrochemical signal probe is required, but this

409

kind of electroactive molecule is rarely available, making the use of the sensor limited.

410 411

Biomimetic Potentiometric Sensors. Compared with the amperometric sensor,

412

biomimetic potentiometric sensors achieve the detection by the potential signal produced

413

after target molecules bind with the imprinted membrane. The target molecules do not

414

need to pass through the imprinted membrane, so there is no limit to the size of the

415

imprinted template. Its conjunction with MIPs could greatly improve the selectivity of

416

molecular imprinting potentiometric sensors. For instance, Gao et al.48 created a

417

membrane electrode with atrazine-imprinted membrane for detection of atrazine. The

418

LOD was 47 nM. Other conductive materials are also being used to build the sensor.

419

Özkütük et al.49, knowing that carbon nanotubes are highly conductive, used modified

420

multiwalled carbon nanotubes and clenbuterol as the template to obtain MIPs by

421

polymerization, and then used them to synthesize a PVC membrane electrode and

422

nanocarbon paste electrode. The developed metal-chelate based MIPs selective

423

potentiometric sensors was used for detection of clenbuterol, showing a great sensitivity

424

with LODs of 4.9 nM and 9.1 nM, respectively, within only 1−2 minutes. These MIPs,

425

prepared with metal chelates, are effective for the specific recognition of the target, and

19



426

potentionmetric sensors based on these MIPs are not only simple and inexpensive, but

427

also highly selective and sensitive in detecting target molecules.

428 429

Biomimetic Conductometric Sensors. Biomimetic conductometric sensors achieve

430

quantitative detection of the target molecule by measuring the changes in conductivity

431

before and after the MIPs bind with the target molecules. For example, Warwick et al.50

432

developed a conductometric sensor for measuring soluble phosphate in environmental

433

water with MIPs designed to function as the receptor. In the presence of phosphate, it

434

would bind to MIPs, leading the conductance of MIPs membrane changed reversibly, and

435

thereby measuring the phosphate in wastewater. Although the working mechanism of the

436

conductiometric sensor is simple, the preparation and elution of the MIPs membrane will

437

directly affect the performance of the sensor. In addition, its selectivity is not high, as

438

trace impurities in the solution can have an adverse effect on the determination. To some

439

extent, the popularity of MIP-based conductiometric sensor is limited.

440 441

Biomimetic Impedimetric Sensors. Biomimetic impedimetric sensors achieve

442

detection by measuring the changes in impedance before and after the sensor responds to

443

the target. They are of good specificity, high sensitivity, and labeling is not needed.

444

Furthermore, they can achieve real-time monitoring, thus have garnered a lot of attention

445

and have been widely used. Zamora-Gálvez et al.51 synthesized MIPs-Fe3O4

446

nanoparticles with screen printing electrodes, and used it as an impedimetric sensor for

447

determination of pesticide tributyltin. The results obtained from the impedance

448

measurement showed that the sensor can detect tributyltin with a LOD of 5.37 pM, which

20


Page 20 of 44

Page 21 of 44


449

was selective and sensitive. In practical applications, the ability to obtain an ultra-thin

450

MIPs membrane with excellent insulation performance is the key to build the imprinting

451

impedimetric sensor, so it needs to be further studied and explored.

452 453

Biomimetic Piezoelectric Sensors. Biomimetic piezoelectric sensors are like quartz

454

crystal microbalance (QCM) sensors. The MIPs film is fixed on the surface of quartz

455

crystal electrode and the mass change before and after the target molecules binding with

456

MIPs is shown by the frequency changes of electrical signal output, thus achieving the

457

quantitative detection of the targets. They can dynamically monitor mass change at the

458

nanogram level by the quartz crystal electrode, which has led to its popularity. Gao et

459

al.52 prepared a QCM sensor for the detection of profenofos, using MIPs ultra-thin film as

460

the

461

mercaptoundecanoic acid to form a self-assembled monolayer, and then the MIPs film

462

was immobilized on the electrode by radical induced polymerization. After the molecular

463

template was eluted, a sensor with specific binding sites of profenofos was obtained. It

464

could sensitively detect profenofos in spiked water and the QCM response showed a

465

LOD of 0.535 fM. Fang et al.53 established a novel 3D biomimetic QCM sensor for the

466

detection of citrinin in cereal grains. First, mesoporous carbon CMK-3 was mixed with

467

AuNPs to obtain AuNPs@CMK-3, it was immobilized directly on Au electrode, then

468

MIP/AuNPs@CMK-3/AuE was prepared by electro-polymerization. The LOD of the

469

imprinting sensor was 1.8 nM for citrinin, and other results showed high selectivity, anti-

470

interference ability, and long-term stability.

recognition

element.

The

QCM

sensor

was

471

21


first

pretreated

with

11-


472

Biomimetic

Microfluidic

Paper-based

Immunoassay.

Page 22 of 44

In

recent

years,

473

microfluidic technology has become an important analytical and detection technique and

474

it has been applied in food and environmental fields for point-of-care testing. The rise of

475

microfluidic paper-based analytical devices (µPADs), which are mainly achieved through

476

preparation of hydrophilic or hydrophobic micro-channels on papers, greatly simplified

477

microfluidic system. It combines the advantages of traditional microfluidic chip such as

478

miniaturization, automation, integration, and portability with that of paper materials such

479

as being low cost, having strong biocompatibility, and being simple to process and easy

480

to degrade. By integrating µPADs with MIPs, the performance of assays can be positively

481

influenced in several aspects, not only in the shortening of time, but also in consuming

482

less reagents and gaining greater selectivity. Wang et al.54 prepared paper-based MIP-

483

grafted multi-disk micro-disk plate(P-MIP-MMP) by a simple in-situ polymerization of

484

MIP layer on the surface of the paper for sensitive and specific competitive

485

chemiluminescence detection of pesticide 2,4-D in water. Under optimal conditions, the

486

P-MIP-MMP could detect 2,4-D at femtomolar level. Although P-MIP-MMP

487

polymerized in-situ on the paper surface can maintain sensitivity and specificity in

488

complex samples, the thickness of the MIPs layer is difficult to control. On account of

489

this, Liu et al.55 prepared MIPs with circle-shaped advice by physical adsorption on the

490

unmodified or activated paper, and a chemiluminescence method was built to detect

491

dichlorvos in vegetables. With an well-controlled depth of MIPs, the paper-based chip

492

can be specific determination for dichlorvos and LOD was 3.6 pM. However, for some

493

samples with low surface tension, if there is insufficient hydrophobicity in the

22


Page 23 of 44


494

hydrophobic area of the paper, it may cause the leakage of the sample, which would

495

affect the detection, thus more effort need to be paid in this area.

496 497

Biomimetic Homogeneous Immunoassay. The homogeneous immunoassay does

498

not need to be coated, washed, or processed in any other way. It simplifies the operations,

499

shortens the analysis time, and is easy to be automated. The homogeneous immunoassay

500

mainly includes three modes: fluorescence polarization immunoassay (FPIA), resonance

501

energy transfer (RET) immunoassay, and luminescent oxygen channeling immunoassay

502

(LOCI). However, the homogeneous immunoassay always suffers from matrix

503

interference and low sensitivity. Integrating MIPs with homogeneous immunoassay could

504

greatly improve its selectivity. The currently reported biomimetic homogeneous

505

immunoassays are FPIA and RET.

506 507

Biomimetic Fluorescence Polarization Immunoassay. As a rapid, simple and high-

508

throughput homogeneous fluorescence immunoassay, FPIA quantitatively determines the

509

analyte by detecting the change in the FP value before and after a fluorescently labeled

510

antigen (tracer) binds to a specific antibody. Based on the nature of MIPs that they can

511

specifically bind to particular target molecules or tracer, tracers have a larger change in

512

molecular weight before and after binding to the MIPs than antibodies, thereby creating a

513

more significant polarization signal. In 2005, Chen et al.56 firstly used anthracene as a

514

template to prepare MIPs and proved that anthracene could closely bind to MIPs through

515

conventional FPIA and time-resolved FPIA, which demonstrated that MIPs could be used

516

as receptor in biomimetic FPIA. Consequently, Ton et al.57 used the inherent fluorescence

23



517

property of the FQs as the signal output and ENRO as the template to prepare a

518

hydrophilic MIPs and established a noncompetitive direct FPIA method for the detection

519

of ENRO. In milk, ENRO could be selectively measured and distinguished from other

520

antibiotics. The method required no tracer, and no separation was needed here because

521

the analyte that bound to MIPs could be distinguished in the solution. However, in FPIA,

522

the tracer may be combined with the matrix, leading to the increase of the FP value,

523

which limits its application.

524 525

Biomimetic Fluorescence Resonance Energy Transfer Immunoassay. Fluorescence

526

resonance energy transfer (FRET) is a photochemical distance dependent process. When

527

the fluorescence emission spectrum of the donor is overlapped with the absorption

528

spectrum of the acceptor in two different fluorescent molecules, and when the distance

529

between two molecules is less than 10 nm, the donor in the excited state would transfer

530

its energy to the receptor in a nonradiative manner by dipole-dipole interaction. In recent

531

years, the homogeneous assay based on the combination of FRET and MIPs has been

532

used in food inspection and environmental testing. Some researchers introduced the

533

fluorescence functional monomer in the preparation of MIPs for homogeneous

534

fluorescence analysis of target molecules20. Some reported homogeneous analysis of

535

target molecules by embedding fluorescent materials in the preparation of MIPs23.

536

Descalzo et al.58 developed a FRET-based competitive biomimetic fluorescence assay for

537

determination of ENRO. The Ru (phen)32+ coated in SiO2 nanoparticles, where the MIPs

538

are synthesized on, acted as the donor and cyanine dye-labeled ENRO (NIR-ENRO)

539

acted as the receptor. NIR-ENRO and ENRO competed for recognition sites on MIPs.

24


Page 24 of 44

Page 25 of 44


540

The binding of ENRO to MIPs was determined according to the changes in the

541

fluorescence signal and the LOD reached 2 µM. Integrating MIPs into the homogeneous

542

assay greatly improved the specificity of the reaction, and the sample does not require

543

purification, thus enhancing the detection efficiency.

544

545

PROSPECTS

546

The biomimetic immunoassay based on MIPs shows good selectivity, high

547

sensitivity and perfect stability, and more importantly it is resistant to harsh conditions,

548

thus lower the requirements for developing sensor which usually work in physiological

549

conditions when biological antibodies are employed. Therefore, it has been applied in the

550

fields of food and environmental safety, and renders the MIPs-based assay a potential

551

candidate for replacement of traditional antibodies. However, it is hard for current

552

biomimetic MIP-based assay to replace traditional immunoassay. There still be some

553

challenges needing to be solved for its practical applications in real world.

554

(1) There usually are a variety of pollutants coexisting in the sample to be tested,

555

which requires that the biomimetic immunoassay achieve co-extraction of a variety of

556

analytes and their simultaneous detection. The computer-aided template molecular design

557

and computer simulation techniques may increase the possibility to obtain the MIPs with

558

broad-specificity. If multiple MIPs with different specificity are used in combination for

559

multi-analyte detection, a suitable detection signal pattern needs to be proposed to

560

distinguish different signal generated by each analyte.

25



561

(2) The MIPs usually have relatively lower affinity than that of antibodies do,

562

resulting in low detection sensitivity of corresponding assay. New technologies may be

563

employed to achieve better performance, such as controlled free radical polymerization,

564

click chemical cycloaddition technology, and multi-template/multi-functional monomer

565

imprinting. In addition, MIPs could be used in conjunction with highly sensitive methods,

566

and a more sensitive signal output mechanism can be used to achieve highly sensitive

567

detection of the target.

568

(3) There are not enough types of functional monomers and cross-linking agents

569

used in the preparation of MIPs to meet the detection of targets with special groups.

570

Therefore, the design and preparation of new monomers and cross-linkers is urgently

571

needed.

572

(4) Template molecules are prone to leakage if they are not completely eluted in the

573

preparation of MIPs. Since the target in the sample is often present in a trace amount, a

574

very small amount of residual template leakage would cause inaccurate determination.

575

Although the use of mimicking templates and fragmented MIPs can partly solve this

576

problem, it is exactly difficult to select suitable molecules as a mimicking template for all

577

possible targets.

578

(5) So far, MIPs are mostly synthesized and exhibited excellent molecular

579

recognition performance in organic phase. The samples of small molecules used in food

580

safety inspection and environmental monitoring are usually in aqueous solution, however,

581

the MIPs that are suitable for aqueous solution are limited. Thus it is imperative to

582

develop MIPs with excellent recognition properties of small molecules in aqueous

583

solutions. Although researchers have developed and synthesized water phase MIPs for

26


Page 26 of 44

Page 27 of 44


584

some small molecules, it is still far from enough. From the aspects of cost, ecology and

585

environmental protection, water phase synthesis or green synthesis methods of MIPs

586

remain to be developed.

587

(6) In addition to chemical contaminants, biological contaminants such as

588

pathogens, proteins and viruses are important challenges for the preparation of MIPs. As

589

MIPs are mostly prepared in organic solvents and protein are almost insoluble in organic

590

solvents, the preparation of MIPs for protein becomes more difficult. Therefore, MIPs

591

preparation for biological macromolecules requires more attention.

592

593

594

We thank the National Science Foundation of China (31622057) and China Agriculture

595

Research System (Grant No. CARS-36) for the financial support.

ACKNOWLEDGMENTS

27



596

597

(1) Fawell, J. K.; Stanfield, G. Drinking Water Quality and Health. Pollution: Causes,

598

Effects and Control. 2001, vol.4, 59−81.

599

(2) Li, Y.; Sun, Y.; Beier, R.; Lei, H.; Gee, S.; Hammock, B.; Wang, H.; Wang, Z.; Sun,

600

X.; She, Y.; Yang, J.; Xu, Z. Immunochemical techniques for multianalyte analysis of

601

chemical residues in food and the environment: A review. TrAC, Trends Anal. Chem.

602

2017, 88, 25−40.

603

(3) Wang, P.; Sun, X.; Su, X.; Wang, T. Advancements of molecularly imprinted

604

polymers in the food safety field. Analyst. 2016, 141, 3540−3553.

605

(4) Liu, H.; Mu, L.; Chen, X.; Wang, J.; Wang, S.; Sun, B. Core-shell metal-organic

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frameworks/molecularly imprinted nanoparticles as absorbents for the detection of

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pyrraline in milk and milk powder. J. Agric. Food. Chem. 2017, 65, 986−992.

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(5) Wu, L.; Liang, R.; Chen, J.; Qiu, J. Separation of chiral compounds using magnetic

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molecularly imprinted polymer nanoparticles as stationary phase by microchip capillary

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electrochromatography. Electrophoresis. 2018, 39, 356−362.

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(6) Zahedi, P.; Fallah-Darrehchi, M.; Nadoushan, S. A.; Aeinehvand, R.; Bagheri, L.;

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Najafi, M. Morphological, thermal and drug release studies of poly (methacrylic acid)-

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614

caprolactone) nanofibers as dexamethasone delivery system. Korean J. Chem. Eng. 2017,

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bisphenol a using molecularly imprinted silica nanoparticles containing quenchable

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using molecularly imprinted microspheres for antibiotic analysis. Chem. Sci. 2015, 6,

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wave fiber optic sensor coated with a molecularly imprinted polymer as a selective

713

fluorescence probe. Biosens. Bioelectron. 2015, 64, 359−366.

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sensor based on core–shell magnetic molecularly imprinted nanoparticles for

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determination of chrysoidine in food samples. Sens. Actuators, B. 2012, 173, 591−598.

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sensor based on magnetic oil-based surface molecularly imprinted nanoparticles for

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determination of bisphenol A. Sens. Actuators, B. 2014, 204, 704−709.

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(42) Zhao, P.; Yu, J.; Liu, S.; Yan, M.; Zang, D.; Gao, L. One novel chemiluminescence

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sensor for determination of fenpropathrin based on molecularly imprinted porous hollow

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for

ultratrace

33


oxide-molecularly

isoproturon

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luminescence sensor based on signal amplification for selective determination of trace

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electrochemiluminescence sensor based on upconversion nanoparticles enhanced by

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electrodeposited rGO for selective and ultrasensitive detection of clenbuterol. Biosens.

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Selective amperometric flow-injection analysis of carbofuran using a molecularly-

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organophosphate pesticide imprinting in electropolymerized poly (p-aminothiophenol)

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membranes on a gold nanoparticle modified glassy carbon electrode. Anal. Chem.

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grafting type molecule-Imprinted membrane and potentiometric sensor for atrazine. Sens.

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wastewater. Biosens. Bioelectron. 2014, 52, 173−179.

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carbon

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specific determination of citrinin. Sens. Actuators, B. 2016, 230, 272−280.

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excited analytes in biological and environmental samples using molecularly imprinted

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polymer nanoparticles and fluorescence polarization. Biosens. Bioelectron. 2012, 36,

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three-dimensional

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778

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781

782

783

Corresponding Author

784

*E-mail: [email protected]

785

Notes

786

The authors declare no competing financial interest.

AUTHOR INFORMATION

787

36


Page 36 of 44

Page 37 of 44


788

FIGURE CAPTIONS

789

Figure 1. Application of molecularly imprinted polymers used in pseudo-assays for the

790

detection of chemical contaminants in food and environment.

791 792

Figure 2. Schematic diagram of molecularly imprinted polymers used in biomimetic

793

ELISA: A novel assay for vancomycin using molecularly imprinted polymers

794

nanoparticles as antibodies substitution (modified from reference 16).

795 796

Figure 3. Schematic programs of molecularly imprinted polymers used in a biomimetic

797

fluoroimmunoassay. (A) Schematic illustration of the preparation of Fe3O4/SiO2-

798

MPS/MIPs (modified from reference 22). (B) Schematic illustration for the preparation of

799

dummy MIPs-capped CdTe QDs and the sensing mechanism for TNT (modified from

800

reference 23). (C) Schematic illustration of the process for the preparation of the

801

SiO2@AuNCs-MIPs (modified from reference 30).

802 803

Figure 4. Schematic diagrams of molecularly imprinted polymers used in the

804

chemiluminescence assay and electrochemical sensors. (A) The procedure to construct a

805

MIP-ECL sensor and determination of GA3 (modified from reference 44). (B) Schematic

806

illustrations for the adsorption of the ATP molecule at the AuNP surface and the further

807

self-assembly of CPF at an ATP-modified AuNP-gc electrode (modified from reference

808

47).

37



Page 38 of 44

Table 1. MIPs working as biomimetic antibodies in analysis techniques Detection method

Category

Biomimetic ELISA Based on Organic Fluorescent Dyes

Biomimetic

Based on Quantum Dots

Fluoroimmunoassay Based on Metallic Nanomaterials Based on Optical Fiber Biomimetic

Flow Injection

Chemiluminescence Immunoassay

electrogenerated chemiluminescence

Synthesis

Analyte

LOD

Reference

solid-phase synthesis

vancomycin

2.5 pM

16

bisphenol A

13.14 pM

20

λ-Cyhalothrin

9.17 nM

21

sol-gel method

2,4,6-trinitrotoluene

0.28 µM

23

copolymerization

salbutamol

0.142 pM

24

surface imprinting

cyphenothrin

9 nM

26

precipitation polymerization

metronidazole

0.15 µM

27

sol-gel method

bisphenol A

0.963 nM

30

surface imprinting

bisphenol A

87.6 pM

32


λ-cyhalothrin

1.76 µM

33

photo-polymerization

2,4-D

250 pM

35

coprecipitation

chrysoidine

0.617 µM

40


fenpropathrin

87.8 pM

42

isoproturon

3.78 pM

43

gibberellin A3

3.45 pM

44

clenbuterol

6.3 nM

45


electro-polymerization

38


Page 39 of 44


Table 1. continued Detection method

Category Amperometric Sensor

Biomimetic Electrochemical Immunoassay

Potentiometric Sensor Impedimetric Sensor

Synthesis

Biomimetic Microfluidic Paper-based Immunoassay Fluorescence Polarization

LOD

Reference


carbofuran

3.8 nM

46

pre-graft polymerization

atrazine

47 nM

48


clenbuterol

4.9 nM

49

surface imprinting

tributyltin

5.37 pM

51

profenofos

0.535 fM

52


citrinin

1.8 nM

53


dichlorvos

3.6 pM

55


enrofloxacin

0.1 nM

57

radical induced Piezoelectric Sensor

Analyte

polymerization

Biomimetic Homogeneous

Immunoassay

Immunoassay

Biomimetic FRET


bisphenol A

13.14 pM

20

Immunoassay

surface-grafting

enrofloxacin

2 µM

58

39



Figure 1. Application of molecularly imprinted polymers used in pseudo-assays for the detection of chemical contaminants in food and environment. 137x151mm (300 x 300 DPI)


Page 40 of 44

Page 41 of 44


Figure 2. Schematic diagram of molecularly imprinted polymers used in biomimetic ELISA: A novel assay for vancomycin using molecularly imprinted polymers nanoparticles as antibodies substitution (modified from reference 22). 84x48mm (300 x 300 DPI)



Schematic programs of molecularly imprinted polymers used in a biomimetic fluoroimmunoassay. (A) Schematic illustration of the preparation of Fe3O4/SiO2-MPS/MIPs (modified from reference 22). (B) Schematic illustration for the preparation of dummy MIPs-capped CdTe QDs and the sensing mechanism for TNT (modified from reference 23). (C) Schematic illustration of the process for the preparation of the SiO2@AuNCs-MIPs (modified from reference 30). 80x146mm (300 x 300 DPI)


Page 42 of 44

Page 43 of 44


Figure 4. Schematic diagrams of molecularly imprinted polymers used in the chemiluminescence assay and electrochemical sensors. (A) The procedure to construct a MIP-ECL sensor and determination of GA3 (modified from reference 49). (B) Schematic illustrations for the adsorption of the ATP molecule at the AuNP surface and the further self-assembly of CPF at an ATP-modified AuNP-gc electrode (modified from reference 52). 80x96mm (300 x 300 DPI)



For Table of Contents only. 80x35mm (300 x 300 DPI)


Page 44 of 44

Molecularly Imprinted Polymer as an Antibody Substitution in Pseudo

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