Chem Soc Rev Dynamic Article Links - Semantic Scholar€¦ · This critical review brieﬂy reviews...

2922 Chem. Soc. Rev., 2011, 40, 2922–2942 This journal is c The Royal Society of Chemistry 2011

Cite this: Chem. Soc. Rev., 2011, 40, 2922–2942

Recent advances in molecular imprinting technology: current status,

challenges and highlighted applications

Lingxin Chen,*aShoufang Xu

aband Jinhua Li

a

Received 18th August 2010

DOI: 10.1039/c0cs00084a

Molecular imprinting technology (MIT) concerns formation of selective sites in a polymer

matrix with the memory of a template. Recently, molecularly imprinted polymers (MIPs)

have aroused extensive attention and been widely applied in many fields, such as solid-phase

extraction, chemical sensors and artificial antibodies owing to their desired selectivity,

physical robustness, thermal stability, as well as low cost and easy preparation. With the

rapid development of MIT as a research hotspot, it faces a number of challenges, involving

biological macromolecule imprinting, heterogeneous binding sites, template leakage,

incompatibility with aqueous media, low binding capacity and slow mass transfer, which

restricts its applications in various aspects. This critical review briefly reviews the current

status of MIT, particular emphasis on significant progresses of novel imprinting methods,

some challenges and effective strategies for MIT, and highlighted applications of

MIPs. Finally, some significant attempts in further developing MIT are also proposed

(236 references).

1. Introduction

Molecular imprinting is known as a technique for creation of

tailor-made binding sites with memory of the shape, size and

functional groups of the template molecules. The concept of

molecular imprinting was first proposed by Polyakov in 19311

a Key Laboratory of Coastal Environmental Processes,Yantai Institute of Coastal Zone Research,Chinese Academy of Sciences, Yantai 264003, China.E-mail: [email protected]; Fax: +86-535-2109130;Tel: +86-535-2109130

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

Lingxin Chen

Lingxin Chen received hisPhD in analytical chemistryfrom Dalian Institute ofChemical Physics, ChineseAcademy of Sciences, China,in 2003. After two years post-doctoral experience in theDepartment of Chemistry,Tsinghua University, China,he joined first as a BK21researcher and then as aresearch professor in theDepartment of AppliedChemistry, Hanyang Univer-sity, Ansan, Korea, in 2006.Now he is a professor in

Yantai Institute of Coastal Zone Research, Chinese Academyof Sciences. His current research interests include chromato-graphic separation techniques and optical sensor technologies forenvironmental analysis using novel properties of materials suchas functionalization nanoparticles and molecularly imprintedpolymers.

Shoufang Xu

Shoufang Xu received hermaster degree in materialsscience from East ChinaUniversity of Science andTechnology, China, in 2005.She joined the Laboratory ofEnvironmental Microanalysisand Monitoring in YantaiInstitute of Coastal ZoneResearch, Chinese Academyof Sciences, as doctoralcandidate, in 2009. Hercurrent research interestsfocus on the preparationof molecularly imprintedpolymers (MIPs) and

application of MIPs in chromatographic separation and analysisof typical organic pollutants.

Chem Soc Rev Dynamic Article Links

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This journal is c The Royal Society of Chemistry 2011 Chem. Soc. Rev., 2011, 40, 2922–2942 2923

as ‘‘unusual adsorption properties of silica particles prepared

using a novel synthesis procedure’’. These ‘‘unusual adsorption

properties’’ have been displayed by numerous polymers, which

are defined as molecularly imprinted polymers (MIPs).2 MIPs

can be synthesized by copolymerization of the functional

monomers and cross-linkers in the presence of template

molecules. After removal of the template molecules, recognition

cavities complementary to the template molecule in shape,

size and chemical functionality were formed in the highly

cross-linked polymer matrix, which can selectively rebind

the template molecules from a mixture of closely related

compounds, as shown in Fig. 1.3,4 Compared to other recognition

systems, MIPs possess many promising characteristics,

such as low cost and easy synthesis, high stability to harsh

chemical and physical conditions, and excellent reusability.

Consequently, MIPs have become increasingly attractive in

many fields, particularly as selective adsorbents for solid-phase

extraction (SPE),5–8 chromatographic separation,9 and chemical

sensors.10–20

There are two main methods to form molecular imprinting,

one relying on reversible covalent bonds introduced by

Wulff,21 and the other involving non-covalent interactions

proposed byMosbach,22 between imprinted molecules (templates)

and functional monomers. In covalent imprinting, typically

the templates are bound to appropriate monomers, such as

4-vinylphenylboronic acid and 4-vinylbenzylamine, by covalent

bonds. After polymerization, the covalent linkage is cleaved

and the template is removed from the polymer. Upon rebinding

of the guest molecule by MIPs, the same covalent linkage is

formed. Owing to the greater stability of covalent bonds,

covalent imprinting protocols yield a more homogeneous

binding sites distribution. However, covalent imprinting is

also considered as a less flexible method since the formation

of identical rebinding linkages requires rapidly reversible

covalent interactions between templates and functional

monomers. Therefore, templates suitable for covalent imprinting

are limited. Moreover, it is very difficult to reach thermo-

dynamic equilibrium due to the strong nature of the covalent

interactions and consequent slow binding and dissociation. In

contrast, non-covalent imprinting has no such restrictions. In

an appropriate solvent, template–monomer complexes are

formed relying on various interactions, such as hydrogen

bonding, ionic interactions, van der Waals forces, p–pinteractions, etc. After polymerization and removal of the

template, the functionalized polymeric matrix can then rebind

the target (template) via the same non-covalent interactions, so

the range of applicative compounds which can be imprinted is

greatly expanded. Besides the above-mentioned advantages, a

further factor is simplicity in operation since only mixing of

templates and monomers in a suitable solvent is required.23

Currently, non-covalent imprinting has become the most

popular and general synthesis strategy for MIT.

Interestingly, after a covalently bonded template is

removed, non-covalent rebinding can also be achieved,24

which is defined as a semi-covalent approach, attributed to

Whitcombe et al.25 This method offers an intermediate

alternative in which the template is bound covalently to

functional monomer as in the covalent approach, but the

template rebinding is based on non-covalent interactions. It

is characterized by both the high affinity of covalent binding

and mild operation conditions of non-covalent rebinding,

which will be discussed in detail in section 4.2.

Although MIT enjoys significant benefits and diverse

applications, it is also associated with many problems such

as template leakage, incompatibility in aqueous media, low

binding capacity and slow mass transfer. In the present review,

we focus on the current status of MIPs (including reagents,

traditional synthesis methods), existing problems and several

new appealing strategies to those challenges (involving some

significant advances in preparing well-designed MIPs, e.g.,

surface imprinting, semi-covalent imprinting and dummy

molecular imprinting), as well as highlighted applications of

MIPs. Some novel techniques related to the preparation of

MIPs, such as controlled/living radical polymerization

(CLRP), block copolymer self-assembly, ionic liquid as

solvent, and microwave-assisted heating are also summarized.

Finally, we also make some attempts to explore the future

development direction of MIT.

Fig. 1 Schematic diagram of the molecular imprinting process by

non-covalent imprinting method.

Jinhua Li

Jinhua Li received herdoctorate in analyticalchemistry from the Departmentof Chemistry, Hong KongBaptist University, Hongkong,in 2009. In the same year,she joined the Laboratory ofEnvironmental Microanalysisand Monitoring in YantaiInstitute of Coastal ZoneResearch, Chinese Academyof Sciences, as an assistantprofessor. Her current researchinterests focus on chromato-graphic micro-separation andanalysis of typical waterpollutants.

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2. Reagents for molecular imprinting

Polymerization reaction is known as a very complex process,

which could be affected by many factors, such as type and

concentration of the monomer, cross-linker and initiator,

temperature and time of polymerization, the presence or

absence of magnetic field, and volume of the polymerization

mixture. In order to obtain the ideal imprinted polymer, a

variety of factors should be optimized. Thus synthesis of MIPs

is a time-consuming process. In order to prepare MIPs with

perfect properties, numerous attempts have been made to

investigate such effects on the recognition properties of the

polymeric materials.26 The selection of appropriate reagents is

a crucial step in the molecular imprinting process. The synthesis,

characteristics, effect of molecular recognition and different

preparation methods of MIPs before 2006 have been briefly

discussed,27 so more emphasis is placed here on the development

of MIPs in recent years.

Generally, template molecules are target compounds in

analytical processes. An ideal template molecule should satisfy

the following three requirements. First, it should not contain

groups involved in or preventing polymerization. Second,

it should exhibit excellent chemical stability during the

polymerization reaction. Finally, it should contain functional

groups well adapted to assemble with functional monomers.

Most templates applied in molecular imprinting are derived

from widespread environmental pollutants, which can be

grouped into three broad categories, including endocrine

disrupting chemicals (EDCs), pharmaceuticals and toxic metal

ions. EDCs have attracted extensive attention when referring

to global environmental problems because they can lead to the

disturbance of central regulatory functions in humans and

wildlife. Worse still, some EDCs are even suspected to cause

human infertility or stunt growth of children. Among those

EDCs, hormone drugs,28–31 triazine pesticides,32–35 and bisphenol

A (BPA)36 are the most widely concerning pollutants used in

preparation of MIPs.

Pharmaceuticals, including veterinary and human anti-

biotics, anti-inflammatory agents and b-blockers, have gainedincreasing attention and interest in the last few years due to

their continuous release in the environment, mainly through

excretion, disposal of unused or expired drugs, or directly

from pharmaceutical discharge.15 In addition, they can cause

selective survival of drug-resistant pathogens which may result

in various drug-resistant micro-organisms that would become

dominant species in the environment, causing great harm to

ecosystems as well as mankind. Because of strong polarity and

low volatility, aquatic environments are their primary

reservoirs. Therefore, preparing water compatible MIPs is a

significant development direction for MIT. At present, water

compatible MIPs for determination of quinolones in urine,4,37

milk,38 river water,39 and serum40 have been synthesized.

Besides quinolones, propranolol,41–44 tetracycline,45,46 digoxin,47,48

dopamine,49 and sulfonamides50,51 imprinted polymers have

also been prepared.

Heavy metal ions are always the focus of attention owing to

their difficulty of degradation and ease of bio-enrichment.

Ionic imprinted polymers are an important branch of MIPs.

Fig. 2 Structures of commonly used functional monomers and cross-linkers.

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Up to now, ionic imprinted polymers for the determination of

Cu2+,52–54 Eu2+,55 Pd2+,56 Sm3+,57 Cd2+,58 Ni2+,59 Zr4+,60

and Zn2+ 61 have been synthesized and displayed excellent

recognition ability to template ions.

The role of the monomer is to provide functional groups

which can form a complex with the template by covalent or

non-covalent interactions. The strength of the interactions

between template and monomer affects the affinity of

MIPs52,62 and determines the accuracy and selectivity of

recognition sites.63 The stronger the interaction is, the more

stable the complex is, resulting in the higher binding capacity

of the MIPs, and therefore, correct selection of the functional

monomers is very important. Tedious trial-and-error tests are

often required to select a suitable monomer. In order to

perform rational design and convenient synthesis of MIPs,

several strategies, such as spectroscopic measurement (e.g.,

nuclear magnetic resonance,64–67 UV-vis,68,69 Fourier-transform

infrared spectroscopy),70,71 computer simulation,72 and iso-

thermal titration calorimetry73 have been employed to select

optimal functional monomers capable of forming more stable

complexes with templates. Research on how to choose and

evaluate appropriate functional monomers has been reviewed

recently.74

Commonly used monomers for molecular imprinting

include methacrylic acid (MAA), acrylic acid (AA), 2- or

4-vinylpyridine (2- or 4-VP), acrylamide, trifluoromethacrylic

acid and 2-hydroxyethyl methacrylate (HEMA). Structures of

several typical monomers are listed in Fig. 2. MAA has been

used as a ‘‘universal’’ functional monomer due to its unique

characteristics, being capable to act as a hydrogen-bond

donor and acceptor, and showing good suitability for ionic

interactions.75

Attempts have been made to seek new functional monomers

adapted to the synthesis of MIPs. Due to their special structures,

b-cyclodextrins (b-CDs) have aroused extensive interest

as attractive candidate monomers for molecular imprinting.

b-CDs are a series of cyclic oligosaccharides with a hydrophilic

exterior and a hydrophobic cavity,76 which have some unique

superior features when compared with traditional functional

monomers. For example, b-CDs can form complexes with the

template through various interactions, such as hydrogen bond

interaction, van der Waals forces, hydrophobic interaction, or

electrostatic affinity. The hydroxyl group on b-CDs can act as

a polymerization terminal and then form a stable polymer

matrix by virtue of a suitable cross-linker. b-CDs have been

used as functional monomers or co-monomers to imprint

various compounds, such as cholesterol,77 tryptophan,78 ursolic

acid,79 bilirubin,80 dextromethorphan,81 and cyclobarbital.82

Generally, the molar ratios between template and monomer

in the synthesis process affect the affinity and imprinting

efficiency of MIPs. Lower molar ratios induce less binding

sites in polymers due to fewer template–monomer complexes,

but over-high ones produce higher non-specific binding

capacity, diminishing the binding selectivity. So, in order to

gain high imprinting efficiency, the molar ratio of templates to

monomers should be optimized.

The role of the cross-linker is to fix functional groups

of functional monomers around imprinted molecules, and

thereby form highly cross-linked rigid polymer. After removal

of templates, the formed holes should completely complement

to target molecules in shape and functional groups. Types and

amounts of cross-linkers have profound influences on selectivity

and binding capacity of MIPs. When the dosage of cross-linkers

is too low, MIPs cannot maintain stable cavity configurations

because of the low cross-linking degree. However, over-high

amounts of cross-linkers will reduce the number of recognition

sites per unit mass of MIPs. Commonly used cross-linkers

involve ethylene glycol dimethacrylate (EGDMA), trimethylol-

propane trimethacrylate (TRIM), N,N-methylenebisacrylamide

(MBAA) and divinylbenzene (DVB), etc. Their structures are

displayed in Fig. 2.

Porogenic solvent plays an important role in polymerization.

It acts as not only a porogen but also solvent in preparation

process. Besides, it also influences the bonding strength

between functional monomers and templates, the property

and morphology of polymer, especially in non-covalent

interaction system. Aprotic and low polar organic solvents,

such as toluene, acetonitrile and chloroform are often used in

non-covalent polymerization processes in order to obtain good

imprinting efficiency. It is notable that MIPs prepared in

organic solvent work poorly in aqueous media because of

the ‘‘solvent memory’’. The development of water-compatible

MIPs is a notable area and will be discussed in section 4

‘‘Challenges for MIT’’.

3. Synthesis methods of MIPs

The mechanism of MIP formation includes free-radical

polymerization and sol–gel process. Considering the advances

in sol–gel process have been reviewed recently by Gupta,83

here we put more emphasis on free radical polymerization.

Bulk polymerization is the most popular and general method

to prepare MIPs due to its attractive properties, such as

rapidity and simplicity in preparation, with no requirement

for sophisticated or expensive instrumentation, and purity in

the produced MIPs. However, the monolithic polymer

obtained by bulk polymerization has to be crushed, ground

and sieved to an appropriate size, which is time-consuming

and would reduce polymer yield (only 30–40% of polymer

recovered as usable material). In addition, grinding operation

results in irregular particles in shape and size, and some high

affinity binding sites are destroyed and changed into low

affinity sites. Bulk polymerization yields polymers with a

heterogeneous binding site distribution which thus greatly

confines the use of MIPs as chromatographic adsorbents.84

In order to overcome these drawbacks of bulk polymerization,

a variety of attractive polymerization strategies have been

proposed, such as suspension polymerization,46 emulsion

polymerization,85,86 seed polymerization82 and precipitation

polymerization. Since the post-treatment steps required in

bulk polymerization are not necessary when the alternative

polymerisation strategy is used, a more homogeneous binding

site distribution should be obtained.87

Among the mentioned polymerization methods, typically,

MIPs were prepared by conventional suspension polymerization,

where water is used as a continuous phase to suspend a droplet

of pre-polymerization mixtures in the presence of a stabilizer

or surfactant.88,89 There are some very successful suspension

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polymerization reports, widely applied for covalent and

non-covalent MIT.88–91 However, the MIPs prepared by

suspension polymerization are polydisperse in size (a few to

a few hundred micrometres). Moreover, MIP microspheres

made by suspension polymerization displayed poor recognition.

The reason may be that water can weaken the non-covalent

interactions, such as hydrogen bonding and electrostatic

interactions between template molecules and functional

monomers. Moreover, stabilizer or surfactant could interfere

with interactions between template molecules and functional

monomers.

In view of this, recently, two new suspension polymerization

techniques based on droplets of pre-polymerization mixtures

in liquid perfluorocarbon92 or mineral oil (liquid paraffin)93

have been developed. Liquid perfluorocarbons immiscible

with almost all organic solvents, monomers and cross-linkers,

which are employed for MIPs preparation, could be utilized.92

However, perfluoro polymeric surfactant is required. As

for the droplet formation of mineral oil, no stabilizers or

surfactants were required.93 However, neither chloroform,

nor dichloromethane, nor toluene, which is generally used as

porogen in molecular imprinting, could be used with mineral

oil because these solvents are miscible with mineral oil.

Both methods give polydispersed beads as well as suspension

polymerization in water.

As for emulsion polymerization, it has long been regarded

as an effective method to produce high-yield, monodispersed

polymeric particles.85 It is successfully employed for protein

imprinting.85,86 On the other hand, emulsion polymerization

also suffers from the presence of remnants of surfactant.

Another method termed seed polymerization, a typical

multi-step swelling and polymerization, is readily used

to prepare monodispersed MIPs and to perform in situ

modification.82,94 However, the use of water as a continuous

phase interferes with the interactions between template

molecules and functional monomers. In addition, the multi-

step procedure is time-consuming.

Precipitation polymerization, enjoying some advantages in

synthesizing spherical particles such as stabilizers and free of

surfactant, in one single preparative step and with excellent

control over the particle size, is a most promising approach

to produce high-quality, uniform and spherical imprinted

particles.

The four-above mentioned polymerization methods mostly

used to prepare spherical particles all have a number of

advantages and disadvantages. The former three polymer-

ization methods have been comprehensively reviewed,16,94

whereas reviews on precipitation polymerization are relatively

few. Therefore, here, more emphasis has been placed on

precipitation polymerization. Then, as a newer and competitive

synthesis method, surface imprinting is also discussed

comprehensively. The other novel polymerization method

associated technologies including controlled/living free

radical polymerization (CLRP), block copolymer self-

assembly, microwave-assistant heating and ionic liquid

as porogen, are also summarized. In order to illustrate

different polymerization methods clearly, schematic diagrams

concerning the different polymerization methods are displayed

in Fig. 3.

3.1 Precipitation polymerization

In precipitation polymerization (PP), more porogen

(495%, wt) is used than in bulk polymerization method.

The growing polymer chains do not overlap or coalesce but

continue to grow individually by capturing newly formed

oligomers and monomers in this diluted reaction system and

then were separated from the solution with microspherical

morphologies.46 Table 1 summarizes MIPs synthesized by

PP.8,46,84,95–111

To satisfy different application purposes, MIPs with well

controlled physical conformations in different size ranges are

highly desirable. Many attempts have been made to evaluate

the effect of factors upon sizes and shapes of the obtained

particles during PP.97,98 According to Wang et al.,112 an

adequate match between the solubility parameters of the

developing polymer and the polarity of the porogen enables

preparation of polymer beads with permanent pore structures

and highly uniform particle sizes. As a rule of thumb, the

solubility parameter of solvent should be 3–5 (MPa)0.5 away

from that of polymer in order to obtain uniform particles.

Another factor affecting the particle size is the cross-linker

used in polymerization. By changing the ratio of TRIM to

DVB, the particle size of the racemic propranolol imprinted

polymer beads altered in the range of 130 nm to 2.4 mm.107 In

addition, polymerization temperature has a profound impact

on the particle size. For poly(MAA-co-TRIM) particles

prepared by PP, when the polymerization temperature was

increased from 4 to 83 1C, the particle diameter decreased

from several micrometres to several nanometres.113,114 Stirring

speed also can affect the mean diameter size of the sedimented

polymer according to classical fluid mechanics. An increase

in the stirring speed considerably increases the amount of

bigger-sized polymer particles.96 It has been pointed out that

the diameter of the particles decreased with increasing of

porogen volume because under dilute conditions less oligomer

and nuclei were formed during polymerization, and therefore,

less radical monomer and cross-linker diffused to the surface

of nuclei and grew up into the particles.107,115 Interestingly,

even under the same synthesis conditions, the size and

size dispersibility of MIPs are not identical with those of

non-imprinted polymers (NIPs),84,107 which can be explained

by that the template altered the solubility of the growing

polymer chain by forming template–monomer complexes,

and thereby influenced the nucleation and growth process of

the cross-linked particles.

The template used in polymerization has a direct influence

on the final morphology of the polymer obtained, as reported

by Cacho et al.97 Fenuron imprinted polymer displayed

discrete uniformly sized microspheres while propazine

imprinted polymer consisted of agglomerates of different sizes.

The same conclusion was obtained by Tamayo et al.98 when

they prepared MIPs by PP for the extraction of phenylurea

herbicides by using linuron or isoproturon as template.

In addition to template, the functional monomer used in

polymerization also has profound influence on the morphology

of obtained polymer. Sambe et al.100 synthesized MIPs for

nicotine via PP by using MAA or TFMAA as functional

monomer and DVB as cross-linker in a mixture of toluene

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and acetonitrile. The nicotine imprinted MAA-co-DVB

polymers were monodisperse microspheres, while the TFMAA-

co-DVB ones were gel-like.

While PP enjoys some advantages, this process is not

without its problems. One problem is that a large volume of

porogen (495%, wt) is necessary during PP, which will also

increase the cost and lead to environmental pollution. Jin

et al.104 proposed a new method to prepare narrowly dispersed

molecularly imprinted microspheres by a modified PP method

by using a smaller amount of porogen (about 50 wt%). The

modified PP used ultraviolet radiation to initiate the process

and used a mixture of mineral oil and toluene as porogen. The

prepared MIPs possess similar binding selectivity and higher

binding capacity compared to microspheres synthesized by

traditional precipitation polymerization (TPP).

Another problem associated with PP is the long polymer-

ization time. Long polymerization time was used in order to

increase the rigidity of the polymer structure and facilitate the

formation of imprinted cavities with better defined shapes.27

However, propranolol-imprinted nanoparticles were obtained

in less than 3 h by using distillation precipitation polymer-

ization (DPP) proposed by Yang et al.114 Unlike TPP

performed at lower temperature for 24 h, DPP was carried

out in neat acetonitrile under refluxing conditions, with half of

the reaction solvent being distilled off in the latter phase of the

polymerization.

3.2 Surface imprinting

There are several compelling reasons to use surface imprinting

approach. Most importantly, extraction of the original

templates located at interior area of the bulk materials is quite

difficult due to the high cross-linking nature of MIPs, which

will result in incomplete template removal, small binding

capacity, and slow mass transfer, as can be seen from Fig. 3.

Fortunately, this problem can be resolved by surface imprinting,

in which the imprinted templates are situated at the surface or

in the proximity of the material’s surface.116 Compared to

traditional MIPs, surface imprinted polymers possess not only

Fig. 3 Schematic diagrams of the molecular imprinting process for

bulk polymerization (1); suspension polymerization or emulsion

polymerization (2a); surface imprinting (3) and hollow imprinted

polymers (4a). Template removal and rebinding in solid spherical

MIPs, core–shell structured MIPs and hollow MIPs are shown in

2b, 3 and 4b, respectively. (m, black, templates in MIPs; & recognition

sites shaped in MIPs after removal of templates; m, red, rebound

templates in recognition sites).

Table 1 MIPs synthesized via precipitation polymerization

Template molecule Target molecule(s) Monomer/cross-linker/porogenApplicationmethod Sample source Ref.

Tetracycline TC antibiotics MAA/TRIM/methanol–ACN MISPE Foodstuffs 46Carbamazepine CBZ, OCBZ MAA/DVB/ACN–toluene MISPE Human urine 84Malachite green MG/GV MAA/EDMA/ACN MISPE-HPLC Aquatic product 8Thiabendazole Benzimidazole compounds MAA/EDMA or DVB/ACN–toluene MISPE Tap, river, well water 95Thiabendazole Benzimidazole fungicides MAA/DVB/ACN–toluene On-line MISPE Tap, river, well water 96Propazine Triazinic herbicides MAA/EDMA/toluene 97Linuron or isoproturon Phenylurea herbicides MAA or TFMAA/EDMA/toluene MISPE Corn 98BPA BPA 4-VP/TRIM/ACN HPLC Tap, lake water 99(S)-Nicotine Nicotine MAA or TFMAA/DVB/ACN–toluene On-line HPLC Cigarette smoke extract 100Ciprofloxacin Fluoroquinolone antibiotics MAA/EDMA/methanol MISPE Soil 101Nickel(II) Nickel(II) 4-VP/DVB/ACN–toluene MISPE Seawater 594-Nitrophenol 4-Nitrophenol MAA/EDMA/ACN On-line MISPE Water 102Theophylline or caffeine Theophylline or caffeine MAA/DVB/ACN–toluene — — 103BPA, TBZ or estradiol BPA, TBZ or estradiol 4-VP/EDMA/mineral oil–toluene — — 1042,4-D 2,4-D 4-VP/EDMA/methanol–water — — 10517b-Estradiol Sterol compounds TFMAA/TRIM/ACN MISPE Milk powder 106R-Propranolol Propranolol MAA/DVB + TRIM/ACN — — 107Cinchonidine Cinchonidine MAA + HEMA/DVB/ACN–toluene HPLC — 108Hydroquinone Hydroquinone MAA/TRIM/ACN–toluene Electrochemical

sensor— 109

p-Acetaminophenol p-Acetaminophenol MAA + GMA/DVB/ACN–toluene — — 110b-Estradiol b-Estradiol MAA/DVB or TRIM/ACN–toluene MISPE Surface water 111

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higher binding capacity but also faster mass transfer and

binding kinetics. Several strategies for surface imprinting have

been examined, such as use of immobilized template,117

initiator on supporting matrix,118 and combined surface

imprinting with CLRP.119 Many particles have been used as

supports in the surface imprinting process, such as activated

silica gel,51,120 Fe3O4 magnetic nanoparticles,34,121 chitosan,122

activated polystyrene beads,118 quantum dots (QDs),123,124

and alumina membranes.125–127

Among the above-mentioned support particles, activated

silica gel shows promising characteristics, because it is a

non-swelling inorganic material, and is stable under acidic

conditions. Besides, activated silica gel has high thermal

resistance. Surface imprinting using silica gel as support has

been applied to imprint EDCs,120,128 pharmaceuticals,129,130

protein,131 and metal ions.58,132

It has been well established that magnetic separation is an

effective technique for separation of complicated samples,

based on its fast recovery, high efficiency, low cost, and direct

purification of crude product from a mixture without any

pretreatment. In recent years, magnetic composite modified

MIPs have become a hotspot based on the significant

advantages of magnetic separation over conventional

methods.45,85,121,125,133 Generally preparation of MIPs-coated

magnetic nanoparticles (MNPs) such as Fe3O4 MNPs involves

three consecutive steps: (1) preparation of Fe3O4 MNPs; (2)

surface modification of Fe3O4 MNPs for hydrophobicity to

favor surface polymerization, with TEOS, oleic acid, ethylene

glycol or poly(vinyl alcohol) and (3) synthesis of surface

imprinted MNPs using a sol–gel process or free radical

polymerization. For example, Wang et al.121 synthesized

MIP-coated MNPs by a sol–gel method for the separation

of estrone. Fe3O4 MNPs were first prepared by coprecipitation

using FeCl2�4H2O and FeCl3�6H2O as raw materials. Then the

Fe3O4 MNPs obtained their hydrophobic shell by reaction

with TEOS. Estrone-imprinted MNPs were synthesized by

condensation of TEOS and IPTS in the presence of estrone.

After binding the target molecule, the black MNPs can be

easily separated by using an external magnetic field to replace

the centrifugation and filtration step in a convenient and

economical way. Compared to silica surface imprinting, the

MNPs surface imprinting had the remarkable superiority of

magnetic separation besides surface imprinting. The advent of

the magnetic surface imprinting technique opens up a new

approach for surface imprinting, and therefore has promoted

the development of MIT.

In recent years, MIT have experienced an explosion towards

nanotechnology motivated by enormous advantages offered

by nanoscale materials. Considering that nanotubes have also

been employed for a broad range of important applications in

biosensors, biocatalysis, biorecognition and drug delivery,

molecular imprinting in nano-porous alumina membranes

(NP AMs) is an effective way to develop MIPs.117,119,125–127

Synthesis of silica nanotubes or nanowires involves several

successive steps.116 First, inner walls of AM pores with a mean

diameter of 100 nm were commonly modified to immobilize

imprinting templates within the pores of AMs. Then template-

immobilized AMs were immersed into the prepolymer mixture

comprised of functional monomer and cross-linker. After

polymerization and removal of AMs and template, molecularly

imprinted nanotubes were produced. Because the binding sites

were situated at the surface of nanotubes, the mass transfer in

imprinted nanotubes is faster.

As reported by Xie et al.,126 2,4,6-trinitrotoluene (TNT)-

imprinted silica nanotubes were synthesized using the sol–gel

reaction of 3-aminopropyltrimethoxysilane (APTS) and TEOS

in the pores of AMs. First, AMs were chemically modified by

APTS, leading to the formation of APTS monolayers on

the alumina pore walls. Then APTS-modified AMs were

immersed into sol–gel precursors comprised of template

molecule (TNT), APTS and TEOS. After the gelation process,

followed by dissolving AMs with aqueous phosphoric acid and

removal of template, a high density of recognition sites to

TNT molecules were created on the walls of highly uniform

silica nanotubes. Because the inside and outside surface of

silica nanotubes are open to analytes and the wall thickness of

the nanotubes is only 15 nm, most of the binding sites on the

surface or in the proximity of surface, provide good site

accessibility and low mass-transfer resistance. The binding

capacity of imprinted nanotubes (5.6%) is about 8.0 fold that

of non-imprinted nanotubes (0.7%), while the maximum

uptake capacity of the nanotubes is nearly 3.6 times that of

bulk particles. To further increase the binding capacity

and improve binding kinetics, single-hole hollow polymer

microspheres for specific uptake of TNT were prepared by

the same group.134 In this process, carboxyl-capping polystyrene

beads were used as adsorbed substrates for precipitation

polymerization. Then a consecutive two-step procedure

including pre-polymerization and polymerization was carried

out, which resulted in hollow MIPs with a single hole in the

shell. The target molecule can easily diffuse into the interior

sites of hollow MIPs through the open holes, leading to a

higher binding capacity and faster kinetics for the uptake of

target molecule than that of solid microspheres.

Other strategies for surface imprinting, such as the use of

CLRP method, immobilization of imprinting templates on

matrix, and sacrificial supports, will be discussed in section 3.3

and 4.1, respectively.

3.3 Novel technologies for MIPs

Besides the above-mentioned synthesis strategies, many more

novel technologies have also been introduced into MIT for

preparing attractive and competitive well-designed MIPs over

the latest years.

Controlled/living free radical polymerization (CLRP). CLRP

techniques including reversible addition-fragmentation

chain transfer (RAFT) polymerization, metal-catalyzed atom

transfer radical polymerization (ATRP), nitroxide-mediated

polymerization (NMP) and iniferter, have been extensively

studied and exploited for the production of well-defined polymers

with well defined molecular weight, low polydispersity,

controlled composition and functionality.135 CLRP involves

a thermodynamically controlled process with negligible chain

termination and much slower rate for the polymer chain

growth over conventional free radical polymerization. This

greatly improves the match between the chain growth and

chain relaxation rates, and therefore produces a homogeneous

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polymer network with a narrow distribution of the chain

length. Recently, CLRP technique has been utilized to synthesize

MIPs owing to its intrinsic advantages over conventional free

radical polymerization, which opens a new way to develop

MIT.136–139

As discussed in section 3.2, surface imprinting is a most

appealing way to prepare MIPs with cavities at the material

surface or close to the surface, facilitating the mass transfer of

template. However, this procedure is still confronted with a

severe challenge on issues of gelation due to the persistence of

initiator in solution. To overcome this problem, Sulitzky

et al.140 proposed a new way to graft MIP films on silica

supports containing surface-bound free radical initiators.

However, it was associated with the problem that the solution-

phase radical species led to heterogeneous sites, and peak

tailing was serious when the obtained MIPs were used as

stationary phase. Afterward, an improved method by using

photoiniferter (initiator transfer agent terminator) as initiator

was proposed by the same group.141 Under UV irradiation,

the iniferter decomposes into active radical, which bonded to

the support surface and initiates polymerization, and the

non-active radical remaining in solution mainly reacts with

the growing radical to form a ‘‘dormant species’’. Therefore,

the stable dithiocarbamate radical in solution did not initiate

solution-phase polymerization, and thus gelation could

be avoided effectively, significantly different from those of

traditional initiators. So, the method is highly recommended.

Due to its good control over molecular weight and dispersity

of the product, a good tolerance for functional groups, and a

broad selection of monomers, ATRP has become a popular

means for preparing graft polymer brushes. Wang et al.119

grafted ATRP initiator and 2-bromo-2-methylpropionyl

bromide onto APTS silanized porous anodic alumina oxide

(AAO) membrane, then the AAO membrane was used as

macroinitiator to initiate a prepolymer mixture comprised of

b-estradiol as template, 4-VP as functional monomer, and

EGDMA as cross-linker in the presence of CuBr and 1,4,8,11-

tetraazacyclotetradecane as organometallic catalyst. The

ATRP initiated MIP nanotubes have 11-fold higher binding

capacity compared to traditionally formed bulk MIPs. The

result shows that the ATRP route is an efficient way to prepare

MIPs with uniform pores and adjustable thickness.

Recent years have witnessed that RAFT is an ideal candidate

for CLRP, not only due to its versatility and simplicity, but

also because the polymerization products are free from the

contamination of metal catalysts used in ATRP. RAFT has

been applied for preparation of MIPs by bulk polymerization,139

precipitation polymerization,105 and surface imprinting.136,142

Titirici et al.136 synthesized thin films of MIPs via RAFT

polymerization for determination of L-phenylalanine anilide.

The effect of the RAFT agent is well illustrated in Fig. 4.136

Particles prepared by RAFT mediated grafting appear smooth

and show no agglomeration (Fig. 4A and B). By contrast,

during the polymerization in the absence of chain transfer

agent, solution polymerization might easily occur, which leads

to complete immobilization of the silica particles at higher

conversions (Fig. 4C and D).

Lu et al.143 reported a general protocol for preparing

surface-imprinted core–shell nanoparticles via surface RAFT

polymerization using RAFT agent functionalized silica nano-

particles as the chain-transfer agent by copolymerization of

4-VP and EDMA in the presence of 2,4-D as template.

Pan et al.105 prepared MIPs by RAFT precipitation poly-

merization for determination of 2,4-D. The RAFT route is

established as an efficient procedure for the preparation of

MIPs, which can be ascribed to the intrinsic mechanical

characteristics of RAFT.

Block copolymer self-assembly. Recently, polymer self-

assembly, especially block polymer self-assembly has been

rapidly developed. The self-assembly of block copolymers in

selective solvents has been comprehensively reported.143,144

Block copolymers form micelles in the selective solvent with

insoluble block as core and soluble block as shell. Based on

this principle, it is possible to design molecularly imprinted

core–shell nanoparticles via self-assembly of block polymers in

selective solvents. Fig. 5 shows the schematic diagram of the

preparation process for MIP-nanospheres by diblock copolymer

self-assembly.145 First, the diblock copolymer was synthesized

with one block containing functional groups for both

hydrogen bond formation and cross-linking. After forming a

hydrogen-bonding complex with the template, the block

copolymer was allowed to self-assemble to form spherical

micelles in a selective solvent. Then the desired structure was

locked by cross-linking. After removing template, complement

sites to the template were formed in the core–shell structured

nanospheres.

Li et al.145 prepared uniform spherical core–shell nano-

structured MIPs by combining molecular imprinting

and the block copolymer technique. First, the diblock

copolymer, poly[(tert-butylmethacrylate)-block-(2-hydroxylethyl

methacrylate)], was synthesized by CLRP. Then, 2-acrylamide-

6-carboxylbutyl amidopyridine and the cross-linkable

methacryloyl side group was introduced. The resulting diblock

copolymers formed hydrogen bonded complexes with

1-alkyluracil or 1-alkylthymine derivatives. After self-assembly

in cyclohexane followed by cross-linking, a uracil or thymine

template was embedded within the core. After removal of

template, the resultant MIPs demonstrated higher rebinding

capacity, faster and complete removal of template, and better

dispersibility in solvent than traditional bulk MIPs. It should

be noted that MIPs with different properties such as water

solubility can be synthesized by adjusting the length, polarity

and sequences of the blocks.

To our knowledge, there are few reports on MIPs

synthesized by the block copolymer self-assembly technique

due to the complicated procedure. However, the use of block

copolymer self-assembly to prepare MIPs is considered

advantageous for method development.

Microwave-assisted heating. Microwave heating technique

has experienced extensive development and application. Due

to the property of heating speed, integrity, selectivity and

efficiency, microwave heating has been widely applied in

synthesis, sintering, sterilization and other areas. A kind of

novel magnetic MIP (mag-MIP) beads were synthesized,34 via

suspension polymerization by microwave heating, for separation

of trace triazines in spiked soil, soybean, lettuce and millet

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samples. The produced mag-MIP beads are suitable to prepare

samples for trace analysis in complicated matrices. Interestingly,

the polymerization time was dramatically shortened by using

microwave heating in polymerization, which was less than one

tenth that by conventional heating. Moreover, the mag-MIP

beads prepared by microwave heating showed a remarkably

improved imprinting efficiency compared to that prepared by

conventional heating. The microwave heating technique was

proved a novel, advantageous and simple integrated technique

for molecular imprinting.

Ionic liquid as porogen. Room-temperature ionic liquids

(RTILs) are substances with melting points at room temperature,

composed solely by ions. RTILs have some excellent

characteristics, such as low melting point and saturated vapor

pressure, non-flammability, good thermal stability, tunable

viscosity, miscibility with water and organic solvent, RTILs

have been widely employed in many aspects, such as extraction

and separation,146,147 catalysis,148,149 and electrochemistry.150,151

Now the use of RTILs has been extended as solvents in MIPs

preparation process. Molecularly imprinted silica particles

for selective recognition of testosterone were prepared using

1-butyl-3-methylimidazolium tetrafluoroborate as porogenic

solvent by combining sol–gel process with sacrificial spacer

MIT.120 A homogeneous mixture solution was formed in the

sol–gel procedure, showing excellent solvation quality of ionic

liquid. The imprinted silica particles possessed high specific

recognition property and binding capacity towards testosterone.

Subsequently, RTIL was used by Xu et al.152 when they

prepared dichlorvos imprinted polymers. The drawback of

traditionally prepared MIPs, such as their tendency to shrink

or swell when exposed to different solvents and the morphology

change of the polymer network, could be avoided. Furthermore,

ionic liquids with designable structures are more environmentally

friendly than traditional organic solvents due to their negligible

vapor pressure. Using ionic liquid as porogenic solvent has

provided new promising opportunities for the preparation

of MIPs.

4. Challenges for MIT

As mentioned above, appealing features of MIPs might retain

the affinity and selectivity of antibodies, enzymes or biological

receptors, while adding several benefits including greater

stability, ease of production, and lower cost.87 Meanwhile, at

present, MIT still faces severe challenges, such as imprinting

biological macromolecules, template leakage, low binding

capacity, and incompatibility with aqueous media. To address

the existing problems of MIT and to improve the performances

of MIPs, significant attempts have been made during recent

years.

4.1 Protein imprinting

Imprinting water-soluble biological macromolecules is very

challenging in the molecular imprinting field since they need to

be imprinted under conditions close to their natural environments

to ensure conformational integrity. Apart from this, large

structures of biological macromolecules might also result in

restricted mobility within highly cross-linked polymer

networks, and poor rebinding efficiency. Many strategies have

been proposed to prepare biological macromolecule-imprinted

polymers, especially protein-imprinted polymers, such as

surface imprinting,127,131 epitope-mediated imprinting,153–155

micro-contact imprinting,156 metal coordination procedure,157,158

and protein imprinted hydrogel.159 Progress achieved in

protein imprinting before 2007 have also been summarized

by Bossi,160 Janiak,161 Turner,162 and Hansen.163

Among the techniques used for protein imprinting, surface

imprinting is currently the most popular and general method

to solve the problem of diffusion limitation caused by large

size of protein. Controlling the template molecules to locate at

the surface of imprinted materials is typically carried out by

the covalent immobilization of template molecules at the

surface of solid substrates, which has some advantages. For

example, templates insoluble in the prepolymerization mixture

can be easily imprinted through this method while template

aggregation could be minimized and thereby binding sites

could be more homogeneous. Commonly, the process of

protein immobilized imprinting involves five consecutive steps,

as shown in Fig. 6. First, amino-groups are introduced into the

support surface followed by introduction of aldehyde groups

via the reaction of amino and glutaraldehyde. Polymerization

Fig. 4 SEM microphotographs of MIP particles prepared in the

presence (A and B) or absence (C and D) of chain transfer agent.

Adapted from ref. 136.

Fig. 5 Schematic representation of the preparation process for MIP-

nanospheres by diblock copolymer self-assembly. Adapted from

ref. 145.

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occurs after the template protein is immobilized on the support

surface by a covalent bond. After removal of template, specific

binding sites for protein will be generated at the surface of

imprinted materials. This method has been used for protein

imprinting on various particles such as silica,131,164 MNPs,86

and chitosan.165 A novel surface imprinting approach combining

immobilized template with sacrificial support, has proven to

be an effective technique for protein imprinting by Yang

et al.127 when they used nanoporous alumina membranes as

sacrificial support to synthesize surface imprinted nanowires

for the detection of protein. Because the imprinted sites were

located at the surface, the imprinted nanowires exhibit good

site accessibility toward target protein. Meanwhile, the large

surface area of nanowires resulted in large binding capacity.

The disadvantage of this kind of protein imprinting method

is that it requires a multistep treatment of the support matrix

before attaching the protein. To simplify this process,

Menaker et al.166 presented a new approach; instead of

nanoporous alumina membranes, polycarbonate membrane

filter with cylindrical pores (F=8 mm) was used as a sacrificial

microreactor since polycarbonate membrane is hydrophobic

in nature and can adsorb protein molecules, which offers a

straightforward method for fixing the target protein on the

pore walls by simple physical adsorption.

Relying on antibody–antigen interaction mechanism, using

an exposed protein fragment (a short peptide or amino acid

residue (histidine)) as template is another efficient way for

protein imprinting, denoted as epitope-mediated imprinting.153,167

Those imprinted polymers having recognition ability for

peptide or histidine also display selective recognition for the

corresponding proteins. Fig. 7 shows the differences between

epitope-mediated imprinting and protein-engaged imprinting.154

Epitope-mediated imprinting has several intrinsic advantages,

such as avoidance of embedding of target protein and minimal

nonspecific interactions between MIPs and proteins. Moreover,

all of the proteins bonded to the MIPs are arranged in suitable

orientation. This proposed method allowing capturing target

protein is mainly based on genomic information in comparison

with other mentioned protocols. Epitope imprinting facilitates

the removal of the template after the polymerization and

reduces the fabrication cost because it does not require

high-purity protein for the imprinting. However, the

access of macromolecules is still hindered in the highly

cross-linked MIPs.

Proteins are water-soluble biological macromolecules, so it

is invaluable to prepare protein imprinted polymers with

recognition property in aqueous media. There have been

new developments to imprint proteins in water in recent years.

Considering that water can weaken the hydrogen interaction

between templates and monomers, metal-chelates155,157,158 and

hydrophobic interactions168 were employed when imprinting in

aqueous media. The former is based on the fact that strong

metal chelating interactions exist between amino acids on the

protein surface and metal ions, and in terms of strength,

specificity, and directionality, metal coordination interaction

is stronger than hydrogen bonding or electrostatic interaction

in water. For example, Ozcan et al.155 prepared MIPs for

selective separation of cytochrome c (surface histidine exposed

protein) using N-methacroloyl-(L)-hisdidine-copper as a new

metal-chelating monomer via metal coordination interaction

and histidine template. Subsequently, N-(4-vinyl)benzyl

iminodiacetic acid, forming a coordination complex with the

template protein in the presence of Cu ions, was used as

functional monomer to prepare macroporous thermosensitive

imprinted hydrogels for recognition of lysozyme.158 The

results indicated that the imprinted polymer with metal

coordinate interaction had higher adsorption capacity than

that of both the imprinted polymer without the coordinate

interaction and the nonimprinted polymers. However, this

approach was restricted to protein targets that have exposed

histidine residues on their surface.

Hydrophobic interaction, together with shape recognition

mechanism, can provide satisfactory selectivity toward the

template protein as reported by Tan et al.168

4.2 Heterogeneous binding sites

Heterogeneous binding sites often exist in MIPs formed by

non-covalent interactions because the pre-polymerization step

is a non-well defined process, which results in the formation of

complexes with different ratios of template to monomer, and

then leads to different binding sites. On the other hand, excess

monomers are used in order to form template–monomer

complexes, which also might cause the formation of non-

selective binding sites.102

To overcome this limitation, a semi-covalent approach was

proposed,169 also known as sacrificial spacer method, in which

the template is bound covalently to a functional monomer as

in the covalent approach, but the template rebinding is only

based on non-covalent interactions. Because of the coupled

advantages of covalent binding (strict control of functional

group location and more uniform distribution) and non-covalent

binding (reduced kinetic restriction during rebinding), the

semi-covalent approach shows strong template binding and

efficient rebinding.170

The commonly used semi-covalent binding is the thermally

reversible urethane bond formed between an isocyanate and a

phenol which is stable at room temperature, but reversible

Fig. 6 Schematic diagram for protein immobilized surface imprinting

process.

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cleavage occurs at elevated temperatures.171 Various functional

groups can be introduced into the binding cavities via using

different nucleophiles during the removal of the template. The

isocyanato group is converted to an amino group in the

presence of H2O, while it was possible to introduce hydroxyl

groups by adding ethylene glycol instead of H2O. An elegant

example of this approach is that Ki et al.171 used this thermally

reversible urethane bond to imprint estrone on the surface of

silica spheres. The monomer–template complex was prepared

by the reaction of IPTS and estrone, then the estrone

imprinted polymers were prepared by sol–gel process. In

subsequent years, the same thermally reversible urethane bond

was used for imprinting of testosterone,120 estrone121 and

active diosgenin.172 The template can reach the imprinting

sites easily and quickly during the rebinding step in the form of

non-covalent binding.

In addition to the above mentioned semi-covalent

imprinting methods, some others were also exploited. For

example, semi-covalent imprinted polymers were prepared

by using propazine methacrylate as a template molecule,

EGDMA as cross-linker and toluene as porogen via PP for

clean-up of triazines in soil and vegetable samples.24 Propazine

methacrylate was synthesized by the reaction of propazine and

methacryloyl chloride in the presence of triethylamine at room

temperature under argon atmosphere. After polymerization,

the propazine-template was removed by hydrolysis in basic

conditions, and the functional group –COOH capable of

interacting with propazine during the rebinding process

was introduced after acid hydrolysis of the polymer. It was

indicated that the better definition of the template–monomer

complex during the pre-polymerization step yielded a polymer

with fewer non-specific binding sites.

The sacrificial spacer method has also been used to imprint

poorly-functionalized pyridine. Traditionally, imprinting of

nitrogen-containing species employed MAA as functional

monomer to combine the nitrogen base via a carboxylic acid

hydrogen bond (O–H� � �N). However, the specific binding of

pyridine was exclusively attributed to only one hydrogen bond

being formed. To overcome this limitation, Kirsch et al.170

synthesized MIPs for pyridine via a sacrificial spacer method

by using (4-vinyl)phenylbenzoate or phenyldimethylsilyl-

methacrylate as templates. The polymer prepared using

silyl ester as template displayed higher selectivity while the

carboxylic imprinted polymer lacked selectivity. The results

revealed that the bond length in the template is crucial in

establishing specificity of rebinding, and appropriate choice of

template is vital for imprinting simple N-heterocycles.

In addition to improve homogeneous binding sites, the

semi-covalent imprinting approach also can avoid template

leakage.121 Even if a small amount of template was left in

polymer during removal of the template, it is difficult to elute

this from the polymer by organic solvent in a later eluting

process because of the strong interaction between template

and polymer. Polymers prepared by the semi-covalent

approach can fundamentally solve template leakage. However,

application of the semi-covalent approach is restricted by

some limitations, such as the covalent bond formed between

monomer and template should be strong enough to withstand

the polymerization conditions, at the same time, it also must

be readily cleaved for template removal. The covalent bond

should be well designed so that a functional group can act as a

hydrogen bond donor and therefore can be retained in the

polymer after a cleavage step.

Combining the concept of molecular micelles with MIT, a

remarkably clever approach to ensure homogeneous binding

sites was described by Feliciano et al.,42 which is based

on substitution of functional monomer with a surfactant

monomer by mini-emulsion polymerization. Imprinted

nanoparticles for rac-propranolol analysis were prepared by

using (S)-propranolol, sodium N-undecenoyl glycinate and

EGDMA as template, surfactant monomer and cross-linker,

respectively. Owing to the formation of micelles, all the

binding sites located homogeneously at the surface of

nanosized MIPs. When the obtained nanosized MIPs were

used in capillary electrochromatography, no peak tailing

was observed, which can be attributed to the homogeneous

interactions between template and imprinting sites.

Furthermore, to reduce heterogeneous binding sites and

improve binding homogeneity, several successful strategies

have been developed, such as selective chemical modification

of low affinity sites, and stoichiometric noncovalent imprinting

by using very strong binding contacts, which are also reviewed

by Zimmerman et al.87

4.3 Incompatibility with aqueous media

MIPs synthesized in organic solvent show poor recognition

ability for the target in aqueous environments because the

presence of polar solvent can disturb the hydrogen bond

formed between template and functional monomer. However,

many applications require MIPs capable of working in polar

Fig. 7 Comparison of epitope-mediated imprinting and protein-

engaged imprinting. Adapted from ref. 154.

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solvents, such as aqueous media. For this purpose, many

methodologies have been developed in recent years. One

strategy was to adopt a two-step extraction method.

Commonly, liquid–liquid extraction (LLE) was applied prior

to MIPs-SPE. However, the procedures are complicated and

time-consuming. Hu et al.133 developed a simple and low

organic solvent-consuming method for enhancing selectivity

of MIPs in aqueous samples. The method integrated MIPs

extraction and micro-liquid–liquid extraction (MLLE) into

one step. Target molecules in aqueous media were extracted

into organic phase by MLLE and then bonded to the solid

phase of MIPs. The specificity was significantly improved in a

dual-phase solvent extraction system, as compared to pure

aqueous media. This approach exhibits great potential to

broaden the range of MIPs applications in biological and

environmental samples.

Another practical strategy focuses on the development of

water-compatible MIPs, in which hydrophilic monomers

are employed, such as HEMA, b-CDs.4,9,80,173 Another

representative approach is to use water as porogenic

solvent because of a solvent memory effect. For example,

water-compatible MIPs have been prepared in water–methanol

systems for selective extraction and separation of ciprofloxacin

from human urine samples, and the resulting MIPs displayed

higher affinity for ciprofloxacin in aqueous environment.37

To further achieve recognition in aqueous media, hydrophilic

modification of MIPs surface was proposed, which involved a

two-step polymerization procedure.114,174 Fig. 8 displays the

synthesis process of restricted access material MIPs (RAM-MIPs).

Glycidyl methacrylate was chosen as the co-monomer because

its oxygen atom bonded to two carbons has lower capacity to

form hydrogen bonds than a free hydrogen group, which can

lessen interferences in the formation of the pre-polymerization

complex. Moreover, the epoxide ring opening induces a

hydrophilic behavior to the imprinted particles. As expected,

the hydrophilic external layer formed by the glycidyl

methacrylate epoxide ring opening on the polymeric surface

reduced the non-specific hydrophobic interactions and increased

water compatibility. Ye et al.114 proposed a dramatically

ingenious approach to synthesize core–shell structured

hydrophilic molecularly imprinted nanoparticles by one-pot

distillation precipitation polymerization, where the hydrophobic

core contained prorpanolol-imprinted binding sites and the

hydrophilic shell was nonimprinted. The hydrophilic shell was

grafted to the core particles via copolymerization of the

residual CQC bond remaining in the core with the hydrophilic

monomers added in the second phase. This new kind of

hydrophilic core–shell nanoparticles is expected to reduce

nonspecific protein adsorption while small organic molecules

are permitted to enter specific binding sites in the core, which

opens up a promising way for biological sample analysis.

Specific solutions to the problem of water compatibility can

be adopted for some special template molecules. One typical

example is the synthesis of MIPs for effective recognition

of hexachlorobenzene in water.175 Both hydrophobic and

electron-poor characteristics of these molecules provided

targeted interactions for recognition in aqueous environments

because the template was lipophilic and inherently aromatic.

1,4-Diacryolyloxybenzene was used as functional monomer to

imprint hexachlorobenzene because it can provide an electron-

rich environment to accommodate the template by p-electronstacking.

In terms of strength, specificity and directionality, the metal

coordination interaction is closer to a strong covalent

interaction rather than hydrogen bonding or electrostatic

interactions in water. Thus metal coordination can be used

to prepare highly specific polymers for the recognition of

templates in aqueous media. An 8-hydroxy-2-deoxyguanosine

imprinted quartz crystal microbalance (QCM) sensor was

prepared for selective determination of 8-hydroxy-2-

deoxyguanosine in serum samples by using methacryloyl

aminoantipyrine-Fe(III) and methacryloyl histidine-Pt(II) as

metal-chelating monomers based on double metal coordination

and chelation interactions.176 The double metal chelate

monomer was found to be effective for molecular imprinting

in the aqueous phase.

4.4 Leakage of template molecules

In general, MIPs are synthesized by using the detected object

as template. However, the potential risk of template leakage

during the usage of MIPs can not be ignored, especially the

deleterious effects produced during determining the trace

compounds by virtue of MIP-SPE. Various strategies have

been proposed to resolve this problem, such as isotope molecular

imprinting,177 parallel extraction on blank samples,178 post-

polymerization treatments (thermal annealing, microwave-

assisted extraction, supercritical fluid extraction),179 and dummy

molecular imprinting. To date, the dummy molecular imprinting

technique, using a structural analog of the targeted compound

as template, has been considered more effective to prepare

MIPs than other methods. Even though leakage of the

‘‘dummy template’’ occurs during recovery of samples, it will

not affect the accuracy of the method. According to the

structures of used dummy molecules, this imprinting technique

can be divided into fragment imprinting180–182 and interval

immobilization.183–185 Fragment imprinting utilizes a part of

the target molecule as a pseudo-template to synthesize MIPs.

Kuto et al.181 prepared MIPs for selective separation of

hydroxyl polychlorinated biphenyl by structural recognition

using fragment imprinting technique; p-tert-butylphenol,

p-tert-butylbenzoic acid, biphenyl-4-ol and biphenyl-4-

carboxylic acid were used as template molecules. The resultsFig. 8 Schematic illustration of RAM-MIPs. Adapted from ref. 174.

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of both the experimental evaluations and molecular modeling

indicated that the molecular volumes and pKa values of the

template molecules were related to the retention factor of

hydroxyl polychlorinated biphenyl, and MIPs prepared with

those pseudo-templates displayed selective separation ability

for hydroxyl polychlorinated biphenyls.181

Fragment imprinting is used not only for imprinting targeting

molecules with inflexible and relatively rigid chemical

structures, but also for compounds having flexible chemical

structures. Nenoto et al.180 developed a simple and effective

MIT for a flexible structure compound of domoic acid

using fragment imprinting polymers. o-Phthalic acid, 2,3- or

2,6-naphthalene dicarboxylic acid, and diphenylether-4,40-

dicarboxylic acid, propane-1,2,3-tricarboxylic acid, etc. were

respectively used as template molecules, because they possess

the same carboxylic acid groups as the target compounds.

Imprinting results suggested that multipoint recognition

ability was closely relevant to the structure and spatial

distance of the carboxylic acid group of the template

molecules. Fragment imprinting can also be used for the

determination of some compounds with relatively large

molecular weight that cannot be easily imprinted, such as in

protein imprinting.186

Interval immobilization imprinting involves synthesizing

MIPs by using compounds with similar distances between

two functional groups to target molecule as a dummy

template. By means of this technique, functional monomers

are immobilized at regular distances and those immobilized

groups specifically recognize target molecules with functional

groups separated by the same distance as the template.

Watabe et al.187 prepared uniformly sized MIPs for BPA with

immobilized intervals of functional monomers afforded by

utilizing 4,40-methylenebisphenol as a dummy template, as

shown in Fig. 9. Because of the closely related structure to

BPA, 4,40-methylenebisphenol was chosen as the pseudo-

template, which formed a complex with the functional

monomer of 4-VP. Appropriate intervals of 4-VP were created

by removing 4,40-methylenebisphenol after polymerization

and effective hydrogen bonding between 4-VP and BPA

occurred; the resulting polymers showed significant selectivity

for BPA. Interval immobilization imprinting has also been

employed to synthesize MIPs for cylindrospermopsin183 and

shellfish paralytic poison saxitoxin,185 by using tributyl(4-

carboxybenzyl)ammonium and 4-(tributylammonium-methyl)-

benzyltributylammonium chloride as the pseudo-template,

respectively.

Dummy templates were also used in the synthesis of

MIPs for dopamine,188 organophosphorus nerve agents,189

cyproheptadine190 and 2,4,6-trinitrotoluene.191 In addition to

solve the problem of template leakage, the use of dummy

template also provides an alternative procedure under the

following conditions: (1) when the original template is very

expensive or involves safety consideration in manipulation and

(2) when the condition used to polymerize could result in

unwanted compound degradation, or the low solubility of the

targeted analytes does not allow its use for synthesis of

MIPs.192 It should be pointed out that although the dummy

template imprinting has many advantages, the search for

suitable molecules is not yet straightforward.

Developing novel imprinting or eluting methods, by which

templates can be eluted from the imprinted polymer completely,

is another way to solve the problem of template leaking

fundamentally. For example, by using a novel surface imprinting

method employing immobilized template and sacrificial

support, all of the imprinting sites are located on the surface,

and templates can be removed easily.127 Semi-covalent

imprinting approach also can avoid template leakage, as

discussed in section 4.2. The enormous advantages offered

by the applications of nanotechnology have motivated the

development of nano-based imprinted materials. The main

advantages recognized up to now in the use of nanomaterials

arise from their characteristics of large surface area to volume

ratio which contributes to achieving favourable mass transfer,

as well as entire removal of template. Imprinted nanospheres,

nanorods, nanowires, nanofibers, nanofilms and nanoarrays

have been prepared in the past several years and displayed

superiority in removal of template. In parallel, in recent years,

porous polymers have experienced an explosion in development,

such as hollow polymers and cage-like polymers. Thanks to the

advances of porous polymers, imprinted porous polymers, such

as imprinted hollow microspheres have been prepared and

displayed superiority in removing template entirely.134

At present, Soxhlet extraction is the most popular method

to remove template from crosslinked polymer matrices. Some

novel elution methods require to be further exploited for more

effective removal of template.

4.5 Hydrophilic compound imprinting

Because of insolubility in non-polar organic solvents, imprinting

of hydrophilic compounds such as water-soluble alkaloids is

another challenge in molecular imprinting. Conversion of the

hydrophilic template to a hydrophobic one by alkyl chain

attachment is a way to solve the problem of insolubility of

target molecules in organic solvent.193 However, the corres-

ponding chemical synthesis and separation of the target

molecules is time-consuming, moreover some functional

groups are consumed during chemical reactions, which limits

the practical applications of this approach. Interestingly, using

a hydrophobic target analogue, instead of the original

hydrophilic target molecule as template, assisted to realize

the determination of a water-soluble compound in a more

facile way.194 However, to find the appropriate analogue is

not straightforward. To date, the simplest method for

water-soluble molecular imprinting is using ion-pairs as

template. Alizadeh et al.195 coupled sodium dodecyl sulfate

to pyridoxine (target molecule) by ion-pair formation and

extracted the ion-pair complex from water to organic solvent

by LLE, and then the ion-pair complex was used as template

to prepare MIPs for determining water-soluble vitamin B6.

Tominaga et al.185 used the ion-pair complex formed between

p-styrenesulfonic acid sodium salt (SSA) and 4-(tributyl-

ammonium-methyl)benzyltributylammonium) as template to

prepare MIPs for the determination of shellfish paralytic

poison saxitoxin. The synthesis concept and procedure is

shown in Fig. 10. Because of the formation of an ionic

complex, shellfish paralytic poison saxitoxin with inherent

hydrophilic nature can be easily dissolved in organic solvent.

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Probably, searching for new monomers and developing

novel imprinting methods in aqueous media are the easiest

and most direct way for imprinting water-soluble molecules in

the future.

For helpful understanding the possible solutions to the

existing problems of MIPs as mentioned above, we summarized

those strategies in Table 2.

5. Highlighted applications

MIPs were most frequently utilized as affinity-based separation

media for sample preconcentration and separation, via

molecularly imprinted solid-phase extraction (MISPE) and

molecularly imprinted solid-phase micro-extraction (MISPME).

In recent years, articles about MIPs used in sensors, artificial

antibodies and catalysts have been increasing in number.

Considering that the highlighted applications of MIPs as

separation media have been summarized in ref. 196–208, more

emphasis is placed here on the application of MIPs in sensors,

artificial antibodies and catalysts. Table 3 summarizes the

relevant studies reported209–225 about applications of MIPs

as separation sorbents, which are divided into four broad

categories: environmental, bioanalytical, food and pharmaceutical

applications. Various compounds such as estron, nonylphenol,

sulfamerazine, fluoroquinlones and triazines have been

extracted from environmental samples (e.g., tap, river, well,

lake, surface, waste, pond water samples, and coastal sediments),

biological samples (e.g., urine, plasma, serum, blood) and/or

food matrices (e.g., pork, milk, tomato, egg).

MIPs have been widely used as the recognition elements for

some sensor technologies, such as QCM sensors,15 electrode-type

sensors,10,13 and optical sensors,17,18 and some breakthroughs

have also been achieved. For example, polymeric membrane

ion-selective electrodes (ISEs) have been used for the

determination of ionic species, but they have been working

poorly for uncharged molecules. Now this limitation has been

eliminated by Qin et al.13 in our institute. They proposed a

novel strategy for selective and sensitive detection of neutral

species, chlorpyrifos, using polymeric membrane ISEs, which

was based on uniform-sized MIPs as sensing element for

molecular recognition and a charged compound, 3,5,6-trichloro-

2-pyridyloxyacetic acid, with a structure similar to that

Fig. 9 Schematic diagram of interval immobilization imprinting

using MBP as the dummy template for determination of BPA.

1: chemical structures of 4,40-methylenebisphenol and BPA; 2:

polymers prepared using 4,40-methylenebisphenol as dummy template

molecule; 3: recognition sites with fixed intervals were created

by removal of the 4,40-methylenebisphenol template molecules; 4:

the obtained recognition sites can bind BPA based on the fact

that BPA has a similar size as 4,40-methylenebisphenol). Adapted

from ref. 187.

Fig. 10 Schematic concept of polymer preparation using ionic

complex by interval immobilization technique. 1: porous polymer as

polymerization matrix; 2: ionic complex is formed between functional

monomer SSA and dummy template TBTA; 3: ionic complex is

immobilized in MIPs after polymerization; 4: recognition sites

are shaped in MIPs after removal of dummy template and anions

remain in recognition cavities; 5: template can bind onto recognition

sites based on interval immobilization mechanism. Adapted from

ref. 185.

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of the analyte as an indicator ion for the transduction

of potential signal. This work may provide a novel methodo-

logy for detecting non-ionic species at trace levels by

using ISEs.

Arrays of carbon-nanotube tips with an imprinted

non-conducting polymer coating can recognize protein with

subpicogram per litre sensitivity using electrochemical

impedance spectroscopy have been prepared recently,226 which

can overcome the limitation of electronic nanosensors for

protein recognition, such as (1) MIP film may attenuate

signals generated in response to template binding due to the

large thickness; (2) the detection mechanisms do not readily

allow for effective signal conversion; (3) the sensor platforms

do not support highly sensitive detection. Fig. 11 shows a

schematic diagram for fabrication of this nanosensor. Vertically

aligned nanotubes were prepared on titanium-coated glass

substrates, followed by embedding in photoresist by spin-coating,

and mechanically polishing to expose the tips. Polyphenol film

was deposited on the exposed nanotube tips by cyclic

voltammetry. To entrap template protein in the polyphenol

film, the corresponding template protein was added to the

polyphenol deposition buffer. Extraction of template resulted

in the reduction of sensor electrical impedance due to electrical

leakage. Subsequent rebinding of template protein led to

an increase in impedance due to the lower conduction of

the protein. This ultrasensitive, label-free electrochemical

detection of protein offers an alternative to biosensors based

on biomolecule recognition.

In addition to the commonly used electrochemical sensors, a

new kind of sensor, a label free colorimetric sensor has been

developed based on colloidal crystals and imprinted polymer.

Fig. 12 shows the protocol for the preparation of photonic

MIP films. From the color change, detection of target compound

can be easily realized by the naked eye. This method has been

used for chiral recognition,19 and excellent determination of

ephedrine,20 cholic acid227 and atrazine.228

Quantum dots (QDs) have attracted intense research interest

in scientific and technological fields owing to their novel

optical properties, such as sharp emission band with broad

excitation, and strong resistance to photobleaching. A number

of QD-based sensors have been reported. The synergetic

combination of the optical property of QDs with the merits

of surface imprinting polymer can improve the selectivity of

QD sensors. Surface molecularly imprinted polymers embedded

QDs have been used to detect pentachlorophenol,124

pyrethroids229 and 4-nitrophenol230 based on room-

temperature phosphorescence optosensing, chemiluminescence

and fluorescence quenching mechanism of electron transfer,

respectively. The general process used to prepare MIPs capped

QDs sensors include the following steps. First, water-soluble

QDs were prepared by refluxing, then silica capped QDs

(QDs@SiO2) were prepared by reverse microemulsion

methods. MIPs embedded QDs can obtained by polymerizing

on the surface of modified QDs via a sol–gel process or a

free-radical polymerization process. As reported,124 the

combination of the room-temperature phosphorescence

property of Mn-doped ZnS QDs with surface imprinted

polymers not only improves the selectivity of QDs but also

makes the MIP-based optosensor applicable for selective

detection of non-phosphorescent analytes without the need

for any inducers and derivatization.

A latest review article about MIPs based chemosensors,

including optical chemosensors (UV-vis spectroscopic

chemosensors, photoluminescent chemosensors, surface plasmon

resonance chemosensor), piezoelectric chemosensors, and

electrochemical sensors (conductometry, capacitance and

impedance measurements, potentiometry, chronoamperometry

and voltammetry) is very comprehensive,231 and is recommend

for reading here.

Immunoassays are a class of analytical techniques based on

the selective affinity of a biological antibody for its antigen.

MIPs have been shown to possess binding characteristics in

terms of affinity and specificity similar to those of antibodies

and biological receptors. Thus MIPs have shown great

potential as replacements for biological antibodies, and have

been used for drug assays, assembly of biomimetic sensors,

and screening of combinatorial libraries. Two attractive review

articles about MIPs used as artificial antibodies have been

Table 2 Solutions to existing challenges for MIPs

Problem Possible solutions

Protein imprinting (1) Surface imprinting (immobilized template)(2) Epitope-mediated imprinting(3) Metal coordination procedure(4) Polyacrylamide soft gel imprinting

Incompatibility with aqueous media (1) Two-step extraction(2) Polymerization in aqueous phase using hydrophilic monomers ornon-hydrogen interaction (metal chelate or hydrophobic interactions)(3) Surface modification of MIPs (RAM-MIP)

Leakage of template molecules (1) Dummy molecular imprinting(2) Nanostructured imprinted polymer(3) Porous imprinted polymer

Heterogeneous binding sites (1) Semi-covalent imprinting or covalent imprinting(2) Selective chemical modification of low affinity sites

Hydrophilic compound imprinting (1) Dummy molecular imprinting (ion-pair as template)(2) Imprinting in aqueous media

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published by Ansell232 and Ye,233 respectively. No futher

examples are thus discussed here.

Significant research efforts have been made in the field of

enzyme-based biosensors, which have been successfully

applied to the detection and quantification of numerous

biologically-relevant molecules. However, in some cases the

limitations of enzyme-based biosensors are primarily those

imposed by the nature of the enzyme itself, such as lack of

Table 3 Selected application examples of MIPs as separation sorbent

Application field Target molecule Template moleculeMonomer/cross-linker/porogen/preparation method

Applicationmethod Ref.

Environmentalapplication

Tap water,wastewater

Cibacron reactive red dye Red dye MAA/EDMA/chloroform/bulk MISPE 209

Tap, river, well water Benzimidazole fungicides Thiabendazole MAA/DVB/ACN–toluene/PP MISPE 96River water Ethynylestradiol Ethynylestradiol MAA/EDMA/ACN/bulk MISPE 210Well water, lake water Estrone Estrone APTS/TEOS/surface imprinting MISPE 211Tap, river, lake water Diphenylguanidene Diphenylguanidene MAA/EDMA/chloroform/bulk MISPE 212Water Parachion Parachion MAA/EDMA/chloroform/PP MISPE 213Wastewater Polybrominatediphenyl

ethersBED-209 PTMOS/TEOS/sol–gel process SPME 214

River water Antiepileptics Cyclobarbital 4-VP/EDMA/multi-step swelling HPLCcolumn

82

Tap, river, well water Benzimidazole Thiabendazole MAA/EDMA/ACN–toluene/PP MISPE 95Drinking water Estrone Estrone AAm/MPTS/DMSO–toluene/bulk MISPE 215Surface water, wastewater

Diclofenac Diclofenac 2-VP/EDMA/toluene/bulk MISPE 65

River water Fluoroquinlones Enrofloxacin MAA/EDMA/ACN/bulk MISPE 39Sludge Nonylphenol and ethoxy-

lated derivatesNonylphenol MAA or 4-VP/DVB/toluene/PP MISPE 216

Ponder water, Sulfamerazine Sulfamerazine MAA/EDMA/THF/PP MISPE 37Coastal sediments,Waste water

PAHs 16 PAHcompounds

MAA/EDMA/ACN/bulk MISPE 217

Water Nonylphenol Nonylphenol TFMAA/EDMA/ACN/bulk MISPE 218

Bioanalyticalapplication

Human serum Fluoroquinoloneantibiotics

Enrofloxacin MAA/EDMA/ACN/bulk MISPE 9

Human serum Metronidazole Metronidazole MAA/EDMA/DMF/bulk MISPE 219Urine Nicotine Nicotine MAA/EDMA/ACN/bulk On-line

MISPE220

Tissue TC antibiotics Oxytetracycline MAA/DVB/water/surfaceimprinting

extraction 45

Urine, plasma Beta-blockers Propranolol MAA/TRIM/ACN/coating SPME 44Human urine CBZ, OCBZ Carbamazepine MAA/DVB + EDMA/ACN–

toluene/PPMISPE 84

Fishery 17b-Estradiol 17b-Estradiol MAA/EDMA/ACN/bulk MISPE 7Human serum, blood Isoxicam Isoxicam MAA/EDMA/DMF/bulk MISPE 6Human plasma Ephedrine and analogues Ephedrine MAA/EDMA/chloroform/bulk MISPE 221Serum Fluoroquinolones Ofloxacin MAA/EDMA/methanol–water/

bulkSPD 40

Human urine Ciprofloxacin Ciprofloxacin MAA/EDMA/methanol–water/Bulk

HPLCcolumn

4

Urine Quinolones Ofloxacin MAA/TRIM/methanol–water/bulk MISPE 37Food application Baby food Fluoroquinolones Ciprofloxacin MAA/EDMA/methanol/PP MISPE 222

Tomatoes Fenitrothion Fenitrothion MAA/EDMA/dichloromethane/bulk

MISPE 223

Lobster, duck, egg,honey,

TC antibiotics TC MAA/TRIM/methanol–ACN/PP MISPE 46

Soybean, millet,lettuce

Triazines Atrazine MAA/TRIM + DVB/toluene/suspension

Extraction 34

Rice, maize, onion Triazine herbicides Ametryn MAA/EDMA/ACN/monolith SPME 32Grass carp, shrimp,shellfish

Malachite green, violet Malachite green MAA/EDMA/ACN/PP MISPE 224

Pork Ractopamine Ractopamine ATPES/TEOS/surface sol–gelprocess

On-lineMISPE

129

Milk ENF, CIP Ofloxacin HEMA/EDMA/methanol–water/bulk

MISPE 38

Citrus Thiabendazole Thiabendazole MAA/EDMA/toluene–water/monolith

MIP-CEC 225

Pharmaceutical Herb Diosgenin Diosgenin IPTS/TEOS/Sol–gel process MISPE 14

1 Abbreviations: CIP, ciprofloxacin; DMF, dimethyl formamide; DMSO, dimethyl sulfoxide; ENF, enrofloxacin; OCBZ, oxycarbamazepine; SPE,

solid-phase extraction; PTMOS, phenyltrimethoxysilane; MPTS, 3-methylacryloxyprolyl trimethoxysilane.

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native enzymes corresponding to a target analyte, poor

availability and highly specific conditions—so artificial

enzymes are required. A comprehensive review about artificial

enzyme-based biosensors was given by Vial et al.234 As a

promising material, MIPs have been employed to mimic

natural enzyme systems with catalytically active arrangements

in designed receptors. Enzymes are basically composed of

substrate-binding sites and catalytic sites which are close to

each other and cooperatively accelerate specific reactions.

Molecular imprinting method is an effective way to synthesize

artificial enzymes by orderly placing of these two sites.

For example, Takeuchi et al.235 synthesized an artificial

enzyme for decomposition of atrazien by copolymerization

of MAA, 2-sulfothyl methacrylate and EGDMA. Using a

transition-state analog as a template to prepare catalytic

antibodies is another development direction for mimicking

catalytic antibodies. Increasing the transition-state binding

and correctly incorporating and positioning the functional

groups is essential for the construction of an effective enzyme

model. Liu et al.236 reported new MIPs which exhibited

strong enhancement for carbonate hydrolysis by oriented

amidinium group and Zn2+ in a transition-state analog

imprinted cavity. This is the first example in which molecularly

imprinted catalysts is clearly much more efficient than catalytic

antibodies.

6. Conclusions and outlook

The current status, challenges, and highlighted applications of

MIPs have been described in this critical review. Much work

have been performed to resolve the problems hindering the

development of MIT during past years. Protein imprinting has

been achieved by surface imprinting and epitope-mediated

imprinting techniques, while heterogeneous binding sites can

be overcome by a semi-covalent imprinting approach. The

combining of water-soluble functional monomer with non-

hydrogen interactions is an effective approach to solve the

problem of MIPs working poorly in aqueous media. For the

challenge of template leakage during trace analysis process,

dummy molecule imprinting is the most widely used approach.

Owing to their high selectivity, high sensitivity, low cost, and

ease of preparation, MIPs have been extensively utilized as

chromatographic media, sensors, and artificial antibodies to

detect various compounds in environmental, bioanalytical,

pharmaceutical and food samples.

Although remarkable achievements have been attained in

MIT, there are still substantial development challenges and

opportunities. We attempt to tackle some important exploration

initiatives as follows: (1) to explain the molecular imprinting

and recognition mechanism at the molecular level with the aid

of advanced equipment; (2) to transfer the imprinting process

from organic phase to aqueous phase, reaching the level of

natural molecular recognition; (3) to design and synthesize

new monomers in order to imprint those molecules without

functional groups, broadening the application field of MIT;

(4) to exploit new polymerization methods for molecular

imprinting for higher imprinting efficiency and binding

Fig. 11 Schematic diagram for nanosensor fabrication and template

protein detection. The supporting polymer is spin-coated on a glass

substrate containing nanotube arrays. Template proteins trapped in

the polyphenol (PPn) coating are removed to reveal the surface

imprints. Inset: hypothetical sensor impedance responses at critical

stages of fabrication and detection. Adapted from ref. 226.

Fig. 12 Schematic representation for preparation of imprinted

photonic polymers: (A) SiO2 colloidal crystals on glass substrate; (B)

infiltration of complex solution into colloidal template followed by

photopolymerization; (C) photonic MIP film after the removal of SiO2

nanoparticles and template molecules; (D) complex of monomer and

template molecule; (E) imprinted molecules within the polymer matrix;

and (F) imprinted cavities with complementary shape and binding sites

to the template molecule.

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capacity; and (5) to broaden imprinting targets from small

molecules to proteins, and even to living cells, in order to make

molecular imprinting a truly practical approach for molecular

recognition.

List of abbreviations and acronyms

AA acrylic acid

AAO anodic alumina oxide

APTS 3-aminopropyltrimethoxysilane

ATRP atom transfer radical polymerization

b-CDs b-cyclodextrinsCLRP controlled/living radical polymerization

DPP distillation precipitation polymerization

DVB divinylbenzene

EDCs endocrine disrupting chemicals

EGDMA ethylene glycol dimethacrylate

HEMA 2-hydroxyethyl methacrylate

IPTS 3-isocyanatopropyltriethoxysilane

LLE liquid–liquid extraction

ISEs ion-selective electrodes

MAA methacrylic acid

MBAA N,N-methylenebisacrylamide

MIPs molecularly imprinted polymers

MNPs magnetic nanoparticles

MIT molecular imprinting technology

MISPE molecularly imprinted solid-phase extraction

MISPME molecularly imprinted solid-phase

microextraction

NP Ams nanoporous alumina membranes

NMP nitroxide-mediated polymerization

PP precipitation polymerization

QDs quantum dots

QCM quartz crystal microbalance

RAFT reversible addition-fragmentation chain

transfer

RAM-MIPs restricted access material MIPs

RTILs room-temperature ionic liquids

SPE solid-phase extraction

TEOS tetraethoxysilane

TPP traditional precipitation polymerization

TRIM trimethylolpropane trimethacrylate

2- or 4-VP 2- or 4-vinylpyridine

2,4-D 2,4-dichlorophenoxyacetic acid

Acknowledgements

Financial support from the National Natural Science

Foundation of China (20975089), the Innovation Projects of

the Chinese Academy of Sciences (KZCX2-EW-206), the

Department of Science and Technology of Shandong Province

of China (2008GG20005005) and the 100 Talents Program of

the Chinese Academy of Sciences is gratefully acknowledged.

Notes and references

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