Recent Developments in Molecularly Imprinted Nanoparticles by Surface Imprinting Techniques

Review

268

Recent Developments in MolecularlyImprinted Nanoparticles by SurfaceImprinting Techniques

Xiaochu Ding,* Patricia A. Heiden*

Molecularly imprinted polymer nanoparticles (MIPNPs) are an increasingly important area ofresearch with potential in applications such as biosensors, solid phase extractions andbioassays. Advantages over the traditionalmolecularly imprinted polymers typically include ahigher binding capacity, greater selectivity and affinity for target species, and aqueouscompatibility. Recent research efforts have sought to impart MIPNPs with additionalcapabilities by introducing nanoparticle size-control, stimuli-responsiveness, biocompatibili-ty, and optoelectronic properties. This short review describes the molecular imprinting
principle and then discusses recent advances inthe field of MIPNPs with particular focus onsurface polymerization techniques to imprintboth small and macro molecules.
X. Ding, P. A. HeidenDepartment of Chemistry, Michigan Technological University,Houghton, MI 49931, USAE-mail: [email protected]; [email protected]

� 2013 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimMacromol. Mater. Eng. 2014, 299, 268–282

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1. Introduction

Natural receptors associated with enzymes, antibodies,

active proteins, etc. all possess excellent binding selectivity

and affinity and are widely studied so their properties

can be reproduced for use in chemo/biosensors,[1–3] bio-

assays,[4–7] and biomedical diagnostics.[8,9] However,

natural receptors also have significant disadvantages that

limit their commercial use. These disadvantages include

poor physical/mechanical stability and high cost to

produce, and their use is limited to moderate conditions

to preserve their binding specificity.[10] In the past few

decades, chemists and biologists have invested time and

effort to develop artificial receptors that possess similar

specific binding and affinity properties for targetmolecules

but are easy to produce and are cost-effective. Molecular

imprinting is an efficient strategy to achieve these

objectives.

Molecularly imprinted polymers (MIPs) are designed to

possess antibody-like selectivity similar to their natural

analogs, but unlike those materials, can be designed to

possess physical/mechanical stability, resistance to harsh

conditions (e.g., high temperature, pressure, acids, bases,

and some organic solvents), and be produced more easily

and economically.[11–13] The key to an effective MIPs is to

produce a ‘‘device’’ that possesses recognition sites with

appropriate spatial arrangement and specific affinity for

a template. The standard approach to prepare MIPs is

illustrated schematically in Figure 1. [11]

Figure 1 shows how vinylic monomers, mixed together

with a ‘‘template,’’ produce a pre-polymerization complex.

The monomers are then polymerized around the template,

together with a difunctional cross-linker, to form a highly

cross-linked polymer network that holds the template

shape. The template is then removed by extraction or

chemical cleavage to leave the shape-specific binding sites.

MIP materials prepared by traditional methodologies

such as bulk polymerization, precipitation polymerization,

and emulsion polymerization, have been extensively

studied for use in solid-phase extractions,[14] chemo/

biosensors,[15,16] andanalytical chemistry.[17,18] Thedimen-

sionsofMIPmaterialsprepared in thesewaysusually range

from ‘‘bulk’’ to micron sized particles. For bulk MIPs, after

the imprinting process is completed, they are usually

ground into a powder and ‘‘sized’’ by passing them through

DOI: 10.1002/mame.201300160m

Xiaochu Ding joined the Department of Chemistryat Michigan Technological University in Au-gust 2008. He studied in the research group ofProf. Patricia A. Heiden, andwill receive his Ph.D. inAugust 2013. He received his B.Sc. in Chemistry(SouthwestUniversity forNationalities, China) andhisM.Sc. in Chemistry (ZhejiangUniversity, China).His research interests include design and synthesisof functionalized organic and inorganic nano-particles for smart self-assembly and self-assem-bled nanofibers for tissue engineering, targetedand controlled drug delivery and biosensing.

Professor Heiden received her B.Sc. in Chemistry(Wright State University, Dayton, Ohio, USA) andher Ph.D. in Polymer Science at University of Akron(Akron,Ohio,USA)working in the researchgroupofDr. Frank Harris. She joined the Department ofChemistry at Michigan Technological University in1994 where she is now a Professor of Chemistryresearching nanoparticles, nanofibers, and bio-polymer use and modification for sustainablematerials.

Recent Developments in Molecularly Imprinted Nanoparticles . . .

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filters having the pre-determined mesh size to isolate the

particles in thedesired size range.Approximatedimensions

of these particles are typically �10�2–10�6m, but other

sizes arepossible. Theparticles typicallypossessa relatively

small number of binding sites on or near the surface

because of the small ratio of surface area to volume.[13] For

perspective, the surface area to volume ratio (S/V ratio)

increases ten times as particle size decreases from 1mm to

100nm.

Although traditional bulk ormicro-sizedMIPs are easy to

prepare, several factors, arising from their highly cross-

linked structures and irregular shapes, limit their applica-

tion as artificial receptors. The specific disadvantages

of bulk and micro-sized MIPs include: (1) difficulty of

removing target molecules from interior binding sites;

(2) the rebinding capacity is limited by the small number of

binding sites on/near the surface; and (3) target molecules

are easily hindered from accessing binding sites deep in

the interior of the particles.[19] These problems need to

be resolved before a truly successful MIP system can

be introduced for recognition of macromolecules (e.g.,

proteins).

Molecular recognition systems for proteins are more

complex than those for small molecular recognition

systems. This is because several other essential factors

must be taken into consideration when preparing MIPs for

protein recognition. First, the reaction conditions must be

carefully controlled so as to make the structure of the

template close to that of the rebinding protein as it exists

in the biological environment where it is to be used,

because themorphology of the protein can be significantly

affected by temperature, pH, ion concentration, etc.[20,21]

Second, the slow rebinding kinetics of a target protein to

binding sites that are deep in the MIP interior, must be

overcome. Molecular imprinting on the surface of particles

is the preferred method to accomplish this. This is done, by

imprintingatemplateproteinonto thesurfaceofmicro-size

particles or solid substrate surfaces.[22–25] The process is

illustrated in Figure 2. First, a solid substrate or particle, a

protein complex (i.e., a protein that is already complexed to

vinylicmonomers that bear functional groups that are able

to interact with the protein), and a difunctional crosslinker

are combined. Then, the copolymerization and cross-

linking are initiated. The resulting product now contains

Figure 1. A schematic representation of a molecular imprinting proc

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the solid support, or ‘‘substrate module,’’ with the

complexed proteins and the cross-linked shell. The com-

plexed protein is then removed, leaving the surface binding

sites that can rebind to the target proteins.

This technique successfully places binding sites on

substrate surfaces after removing the template, but the

density of surface binding sites is still limited due to the

small surface area to volume ratio of these conventional

MIPs.

Nanostructured materials (e.g., nanoparticles) are now

among the most researched alternatives to overcome the

drawbacks associated with conventional MIPs. Indeed, a

search of molecularly imprinted polymer nanoparticles

(MIPNPs) throughWebof Science shows329publications in

last decade. However, almost half of these (158 publica-

tions) are issued in the last twoyears. BecauseMIPNPshave

such a high surface-to-volume ratio the imprinted binding

sites are necessarily created on/near the nano-material’s

surface. This improves the rebinding capacity and imparts

faster rebindingkinetics for the targetmacromolecules, and

so is an efficient strategy to overcome the limitations of

bulk MIPs.

ess. Adapted with permission.[11] Copyright 2004, Springer.

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Figure 2. A surface molecular imprinting process to giveprotein recognition sites on micro-size particle surfaces orflat substrate surfaces is shown. The process if followed byremoval of the template proteins to leave recognition sites onthe substrate surface.

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X. Ding, P. A. Heiden

270

Several recent review papers have described strategies

for the preparation of molecularly imprinted polymer

micro and nanoparticles.[26–31] The advantages of MIPNPs,

compared with traditional MIP materials, have also been

widely discussed with respect to bioassay, biosensor,

environmental analysis, and biomimetic catalytic applica-

tions.[32–37] This review differs from these prior reviews in

its focus on the recent advances inMIPNPpreparation,with

particular attention paid to surface polymerizations on

various nanoparticle (NP) substrates to enhance small

molecule andmacromolecular imprinting and recognition.

These recently developed techniques effectively create

the recognition sites on MIPNP surfaces so that they

are accessible. Also, by selection of an appropriate NP

substrate additional capabilities can be given to the

MIPNP systems, such as magnetic responsiveness for ease

of separation or optoelectronic properties for detection.

Table 1 lists the most commonly used NP substrates,

their surface functional groups, and the corresponding

MIPNP preparation techniques that we are going to

discuss in this review.

The first half of this review focuses on imprinting

and recognition processes, and applications for species

imprinted with small molecules. The second half of

the review addresses imprinting and recognition, appli-

cations, and the latest advances in macromolecular

imprinting and recognition, including discussing the

specific perceived advantages of MIPNPs compared to

conventional MIPs. To avoid overlap within this review,

some of the new advances of MIPNPs described with

Macromol. Mater. Eng. 2

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the small molecularly imprinted MIPNPs will not be

addressed a second time with examples of macro-

molecular imprinted systems.

2. Recent Advances in MIPNPs for SmallMolecule Recognition and TheirApplications

The traditional polymerization strategies used to obtain

MIPNPs that are rich with surface binding sites include

precipitation, emulsion, mini-emulsion, and surface-

initiation on polymer seeds.[38] In recent years more

versatile techniques have been proposed and tested for

thepolymerization of a surface layer on solidNP substrates.

These newmethods give excellent control over MIPNP size

by selection of suitable NP substrates and controlling the

thickness of the imprinted layer. These methods can also

allow use of stimulus-response and biocompatible MIPNP

shell materials for different applications.

2.1. MIPNPs on Silica NP Substrates

Silica NPs are popular substrates for surface imprinted

MIPNPs in part because the synthesis of silica NPs

themselves is a well-established process. Many simple

synthetic methods are described in the literature that give

the conditions that allow the MIPNP particle diameter to

be controlled anywhere within a range of micrometers

to nanometers. Also, silica possesses numerous hydroxyl

groups on its surface, and these are readily employed as

grafting sites for the surface polymer. Therefore, it is a

relatively straightforward matter to prepare silica

MIPNPs with a controlled size and surface.[39,40] There is

also a wide range of reactions and monomers that can

be used for the surface functionalization of silica NPs.

Finally, the chemical/mechanical stability, non-toxicity

and biocompatibility of silica all make it an attractive

substrate for use in many fields.[41]

Silica NPs are probably most often functionalized with

organic or inorganic vinyl functional groups,[42–46] iso-

cyanates,[47] and amine groups.[48,49] The next step is the

imprinting process, using an existing template followed

by polymerization with functional monomers and then

cross-linkers. These methods yield a high density of

effective binding sites on the silica NP surface with good

accessibility, selectivity, and a high rebinding efficiency for

the target molecules.[50,51]

Several variations on the surface imprinting technique

have been described in the recent literature. For example,

Guan et al.[52] reported imprinting 2,4,6-trinitrotoluene

(TNT) molecules on the surface of amine-functionalized

silica NPs (130nm) via a layer-by-layer technique. In this

method, instead of copolymerization of vinylic monomers,

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Table 1. Summaries of nanoparticle substrates, surface functional groups, and MIPNP preparation techniques.

Substrate

Surface functional

group

MIPNP preparation

technique Refs.

Silica NP Vinyl Surface polymerization [42,43,45,46,104]

OH Sol–gel [44]

Isocyanate Condensation polymerization [47]

Amine Layer-by-layer [48,49,52]

RAFT RAFT polymerization [22]

Template molecule Pickering emulsion polymerization [53]

Au NP Thioaniline Electropolymerization [56–58]

Magnetic NP ATRP ATRP polymerization [64,96,97]

Vinyl Radical polymerization [65–67,94]

Amine Sol–gel [98,99]

None Self-polymerization of DPA [92]

None Phase inversion method [103]

QD Vinyl Radical polymerization [73,107]

Amine Cross-linking reaction, sol–gel [74,76]

OH Sol–gel [77,78,82]

Carboxylic acid Sol–gel [79]

ATRP ATRP polymerization [80]

None Encapsulation [81]

Template molecule Sol–gel [108]

None Phase inversion [109]


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a glutaraldehyde (GA) mediated covalent assembly of

gelatin was used to form the imprinted layers. The final

MIPNPs possessed a diameter of 180nm. The authors

found that the rebinding capacity of TNT changed

nonlinearly with the layer number of the TNT-imprinted

gelatin layers, but observed that three layers of

imprinted gelatin yielded the highest rebinding capacity

in this study. The significanceof this layer-by-layer surface

imprinting technique is that it expands the range of

materials that can be used to form the surface-imprinting

layer from vinylic monomers to biopolymers (e.g.,

chitosan, proteins, enzymes), and many of these can be

used in aqueous media by a simple covalent assembly

between GA and the amine groups of those biopolymers.

This technique also allowed excellent control of the

thickness of the imprinted layers by changing the number

of applied layers.

Another relatively simple procedure was employed

by Zhu et al.[44] to imprint bisphenol A (BPA) on a silica

NP surface using a double-sol–gel method. In the first

step tetraethylorthosilicate (TEOS) was hydrolyzedwith

ammonium hydroxide to obtain spherical silica NPs

with a size of 400 nm. Then the NPs were washed with

anhydrous ethanol. In the second step, the imprinting



process was performed by dispersing the silica NPs

in methanol with the aid of sonication. Then the

template molecules (BPA), the functional monomers

3-aminopropyltriethoxysilane (APTES) and TEOS, and

a small amount of acetic acid were combined. This

mixture was stirred and polymerized at room tempera-

ture for 18 h. After repeatedly washing the product

with a mixture of methanol and 6 M HCl (1:1 v/v) to

remove the templates from MIPNPs, the researchers

tested the efficacy of these MIPNPS using solid phase

extraction, which showed these BPA-imprinted silica

MIPNPs possessed high absorption capacity, high

selectivity, and fast rebinding kinetics for the target

BPA molecules.

Chang et al.[22] recently demonstrated an attractive

method to imprint 2,4-dichlorophenol (2,4-DCP) on silica

micro beads having a polymer layer thickness of�2.27nm.

The author coupled an alkyne-terminated reversible

addition-fragmentation chain transfer agent (RAFT) to

azide-functionalized silica beads via a click conjugation.

The author then grafted a thin, uniform nanofilm on the

silica NP surface that was imprinted with 2,4-DCP. This

technique allowed effective control of the thickness of

the imprinted layer because it used RAFT, a controlled

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radical polymerization, and demonstrated high selectivity

and fast rebinding kinetics towards the target molecule,

2,4-DCP.

Another approach, called interfacial imprinting, was

recently reported by Shen et al.[53] This process, illustrated

inFigure3, employedaPickering emulsionpolymerization

method. The template molecules are first immobilized on

the surface of silica NPs (Figure 3a), often prepared from

TEOS, and these NPs are used to stabilize monomer

droplets (80–240mm diameter) to form a stable oil-in-

water emulsion (left side of Figure 3b). The monomer

droplets, composed of methacrylic acid (MAA) and

ethylene glycol dimethacrylate (EGDMA), are then poly-

merized and cross-linked with azobis(isobutyronitrile;

AIBN; Step 1 in Figure 3b). The silica NPs on the surface of

the microsphere were then removed by stirring the

particles in a solution of HF (30wt%) for 12 h (Step 2 in

Figure 3b), leaving the imprinted pores with specific

affinity for the target molecules. This new type of MIP

microsphere possesses a well-controlled hierarchical

structure with large pores for easily accessible binding

sites. Use of the silica-bound templatemolecule-I (left side

of Figure 3a) allows for selective recognition of a series

of analogous compounds that have the isopropylamino-

propanediol epitope as a common motif, e.g., atenolol,

metoprolol, pindolol, and propranolol, while use of the

silica bound template molecule-II (on the right side of

Figure 3a) gives non-specific recognition to the same series

of compounds. MIP microspheres prepared in this way

Figure 3. Illustration of an interfacial molecular imprinting on silicaimmobilized on silica NPs, while b) showsmonomer droplets in water,These are imprinted by copolymerizing with cross-linker in Step 1, to fophase is removed by stirring in an HF solution (30wt%). The interior of(b). Figure 3a adapted with permission.[53] Copyright 2011, American



possess hydrophilic surfaces, enabling them to be used in

aqueous media.

2.2. MIPNPs on Au NP Substrates

Gold nanoparticles (Au NPs) are another popular substrate

on which to assemble an imprinted layer, because Au NPs

possess a surface plasmon resonance (SPR) which is

sensitive to interparticle distance, and to the absorption

of some target molecules or biomolecules that change

the thickness of the surface layer.[54,55] The SPR feature

makes Au NPs an attractive substrate for use in the field

of biosensors and biodiagnostics.

The value of the SPR is illustrated in recent publications

by Willner’s group.[56–58] They demonstrated the ultra-

sensitive SPR detection of hexahydro-1,3,5-trinitro-1,3,5-

triazine (RDX). The multi-step process, illustrated in

Figure 4,[56] begins with the electropolymerization of

thioaniline-functionalized Au NPs (3.5 nm) onto an Au

electrode that is already coated with a thioaniline-

monolayer. The electropolymerization is done in the

presence of an imprinting template molecule (not shown

in Figure 4a), to give a cross-linked bisaniline network

bound to Au NP substrates that are themselves now

bound onto the Au electrode surface. Because RDX

possesses low solubility in the aqueous electropolymeriza-

tion solution, the authors did not use RDX itself as the

template, but instead used Kemp’s acid as an analogous

template molecule for RDX. Figure 4b shows the extraction

NP-stabilized emulsion drops; a) shows template molecules (I or II)stabilized by the silica NPs with the immobilized templatemolecules.rm cross-linked polymeric microspheres, and then in Step 2, the silicaone of the imprinted sites is expanded and shown on the right side ofChemical Society.

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Figure 4. A schematic illustration that shows MIPNPs with ultrasensitive SPR detection of RDX. a) Thioaniline and mercaptoethane sulfonicacid functionalized Au NPs (3.5 nm) are electropolymerized onto a thioaniline-monolayer-modified Au electrode to form ‘‘sponge’’composites of bisaniline-cross-linked Au NPs associated with Au surface. b) Kemp’s acid-imprinted MIP Au NP composites forrecognition sites for RDX analysis.[56]


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of Kemp’s acid leaving highly sensitive receptor sites for

RDX.

Theauthors foundthatKemp’sacidwasasuitableanalog

for RDX that gave highly sensitive detection of RDX.

This was because their research showed that RDX bonds to

receptor sites through p-donor–acceptor interactions

between RDX and the bisaniline units.[56] Their Au-MIPNPs

generated measurable reflectance changes in the SPR

spectrum with a detection limit as low as 12 fM, which

is 4� 105-fold lower than a non-imprinted sensing matrix.

In subsequent work they also demonstrated chiroselec-

tivity through imprinting of L-glutamic acids or D-glutamic

acids by electropolymerization of thioaniline and cysteine

functionalized Au NPs, using bisaniline as a bridge to form

the chiroselective recognition sites.[57] The chiroselectivity



results from zwitterionic interactions and hydrogen

bonding between the amino acids and the cysteine units.

The rebinding of target chiral amino acids could be

distinguished by the SPR spectrum with a detection limit

of 2 nM. Their studies also showed Au NPs with bisaniline

bridges in themolecularly imprinted composites displayed

quasi-reversible redox properties. When at E< 0.12V,

versus Ag as a quasi-reference electrode (QRE), the bridges

existed in the reduced bisaniline (p-donor state). However,

at E> 0.12V versus Ag QRE, the bridges changed to

the quinoid (p-acceptor state).[58] This potential-induced

reversible uptake and release of p-acceptor molecules

suggests potential applications in chromatographic sepa-

rations, controlled release systems, and removal of

pollutants.

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274

As previously stated, Au NPs possess an SPR which is

highly sensitive to changes in the environment around

the particles. So this feature, along with the chemical

stability, ease of surface modification, and wide range of

controllable particle size at which Au NPs can be synthe-

sized, make Au-MIPNPs and their nanocomposites attrac-

tive substrates for use as sensors for molecular detection

inpreference toothermetallicNPs.[59] A recentmini-review

described the strategies and advantages of Au-MIPNP

composite films used as sensors for various applications

and detections.[60]

2.3. MIPNPs on Magnetic NP Substrates

Magnetic NPs are also of interest as substrates for the

preparation of small molecularly imprinted MIPNPs that

combine the ability to recognize target molecules with

magnetic responsiveness. In the last decade, 113 publica-

tions describe magnetic MIPNPs, and more than half of

thesedescribe smallmolecularly imprintedmagnetic Fe3O4

NPs for uses like separation, solid phase extraction, and

sensors, as described in several recent reviews.[32,61–63] The

strategies used to prepare magnetic NPs that possess

surface functionalities (e.g., vinyl groups, amine groups, or

groups able to function as RAFT chain transfer agents) are

described in the section on macromolecular imprinted

magneticNPs. This is donebecauseof their value inprotein-

imprinted MIPNPs. In this section the focus is on

representative methods to imprint the NP surfaces and

applications of the imprinted particles.

A ‘‘grafting from’’ technique is frequently used to

fabricate molecularly imprinted layers on magnetic NP

surfaces. Generally, the magnetic NP surface is first

decorated with a silica layer, because this provides an

abundance of functional groups that, as described previ-

ously, can be easily converted to other desirable groups.

Figure 5. A representative surface imprinting process is shown that uses a ‘‘graftingfrom’’ technique on ATRP-functionalized magnetic NPs.

Then the magnetic NPs can be further

functionalized with species able to serve

as a radical chain transfer agent for RAFT,

or atom transfer radical polymerization

(ATRP). This step is followed by a surface

imprinting process to create specific

binding sites. For example, Liu et al.[64]

reported a procedure to make Fe3O4 NP

surfaces suitable for ATRP. The process

began by reaction of Fe3O4 NP surfaces

with TEOS, in thepresence of ammonium

hydroxide, to obtain Fe3O4@SiO2 NPs.

Thiswas followed by additional hydroly-

sis with ammonium hydroxide in

the presence APTES to form Fe3O4@-

SiO2@NH2 NPs. Finally, the ATRP initia-

tor, 2-bromoisobutyryl bromide, was

immobilized on the Fe3O4@SiO2@NH2



NP surfaces via reaction of the acyl bromide with the

surface amine groups. Then suitable monomers were

polymerized from the surface, in thepresence of the desired

template, in this case the antibiotic pefloxacin mesylate

(PEF-M), to yield the imprinted magnetic MIPNPs, having a

particle size of 500nm and an imprinted layer having a

depth of 18nm. Testing of PEF-M-imprinted Fe3O4 NPs

showed significant specific affinity towards the target PEF-

M in aqueous media and high rebinding capacity. The PEF-

M-imprinted Fe3O4 NPs were highly magnetic (41.4 emu

g�1) and responded to external magnetic fields allowing

rapid separation of the MIPNPs after bonding with PEF-M

molecules from an egg sample. The general ‘‘grafting

from’’ technique on ATRP-functionalized magnetic NP

surface is shown in Figure 5.

‘‘Grafting to’’ techniques are also employed with

Fe3O4 NPs coated with a SiO2 shell obtained by sol–gel

methods, using TEOS and 3-methacryloxypropyltri-

methoxysilane (MPS), to form Fe3O4@SiO2 NPs with

vinyl groups on the surface of the NPs. Then the

molecularly imprinted layer is formed on the surface

by copolymerizing the vinyl groups with functional

monomers and cross-linkers in the presence of the

desired templatemolecules. This approachwas employed

in several recent publications.[65–67] For instance,

Kong et al.[67] showed that sulfamethazine molecules

were effectively imprinted on vinyl-functionalized

Fe3O4@SiO2 NP surfaces using MAA as the functional

monomer, EGDMA as the cross-linker, and AIBN as

the initiator. The resulting MIPNPs had high binding

capacity and fast re-binding kinetics.

A schematic representation of this ‘‘grafting to’’ tech-

nique on the vinyl-functionalized NP surface will be

illustrated in the macromolecular imprinting section.

Additional surface functionalization methods and newer

surface imprinting technologies using magnetic NPs will

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also be discussed in the section on macromolecular

imprinted MIPNPs.

2.4. MIPNPs on Quantum Dot Substrates

Quantum dots (QDs), such as CdS, CdTe, CdSe, ZnS, etc., are

often used as biolabels or to fabricate chemo/biosensors

because of their optoelectronic characteristics.[68–71] The

techniques to fabricate QD-based MIP chemo/biosensors

were developed only recently. The recognition cavities

capture templates that quench the photoluminescence

emissions from the QDs due to fluorescence resonance

energy transfer between QDs and template molecules.[72]

The QD-based/MIP devices can usually be classified into

four categories for small molecule detections.

The first, and most traditional, approach is to embed

QDs into MIP matrices during the molecular imprinting

process. This is done by copolymerization using vinyl

functionalized QDs along with the desired templates,

functional monomers, and cross-linkers. For example,

QD embedded MIPs were synthesized using MAA as

the functional monomer, together with 4-vinylpyridine

functionalized CdSe–ZnS core-shell QDs, and EGDMA as a

cross-linker, in the presence of caffeine as the template

molecule.[73] A post-treatment method can also be used

to embed surface functionalized QDs on MIP matrices

by cross-linking amine-functionalized QDs with MIP

matrices that contain carboxylic acid groups.[74] This

traditional sort of QD embedded MIP material can be cast

into films or ground into particles for molecular detection,

as desired for the intended application. The general process

Figure 6. The traditional QD-embeddedMIPs. a) Molecular imprintingtemplate and cross-linker; b) post-treatment using an amine-functio



to prepare these two QD-embedded MIPs is illustrated in

Figure 6.

The second approach uses a direct surface imprinting

technique on the QD substrate to form an imprinted outer

layer on the QD-MIPNPs. The binding cavities on the outer

layers can be generated by a surface polymerization

technique if vinyl-functionalized QDs are used,[75] or by

sol–gel methods if the QD surfaces possess amines,[76]

silanols,[77,78] or carboxylic acid groups.[79] However,

because of the small size of the QDs, some of the imprinted

outer layers will actually be coated on QD aggregates

comprised of several QDs.[75,76] The surface imprinting

process on vinyl-functionalized QD is similar with other

vinyl-functionalized NP substrates, except QDs slightly

forming aggregations.

In the third category the MIP layer is formed on QD-

embedded nanoparticle substrates to fabricate multi-

functional biosensors with the following structure:

QDs@NPsubstrate@MIP. For example, ZnO nanorods can

be surface functionalized with an ATRP initiator (2-

bromoisobutyryl bromide) and then surface polymeriza-

tion canbe carried out in presence ofmagneticNPs (g-Fe2O3

NP), functional monomers (MAA), cross-linkers (EGDMA),

and templates to form a ZnO@Fe2O3@MIP biosensor that

can give selective antibiotic detection.[80] This is a new

type of biosensor that possesses functionalities that

are reported to be able to give selective recognition,

separation, and detection. Although these biosensors

have not yet been well-studied, they reportedly meet

market requirements for a device suitable for high

throughput analysis, such as might be used in food supply

polymerization using a vinyl-functionalized QD, a vinylic monomer, analized QD, a polymer with carboxylic acid groups and a template.

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Figure 7. General procedure for the preparation of multi-functional QD-embedded magnetic MIPNPs.

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276

management, bio-diagnostics and environmental protec-

tion. The general procedure of preparing QD-embedded

magnetic MIPNPs is shown in Figure 7.

The final category of QD-based MIPs consists of QD-

embeddedMIP nanocomposites. Either surfacemodified or

non-modified QDs can be used to fabricate this type of QD-

basedMIP sensor. This approachwas used by Zhao et al.,[81]

who used an ultrasonication-assisted encapsulation

method that yielded Mn–ZnS embedded QD-MIP nano-

composites for diazinondetection. Thiswas accomplishedby

dispersing non-functionalized Mn–ZnS QDs, the template

(diazinon), and a pre-synthesized copolymer (poly(styrene-

co-methacrylic acid)) into chloroform to give a transparent

solution that was then injected into deionized water under

ultrasonication andwithmagnetic stirring for 6min to form

a diazinon-imprinted QD-MIP nanocomposite suspension.

After washing and collecting by centrifugation, this easily

prepared QD-MIP nanocomposite was tested against diazi-

non in water and showed a good linear correlation, in a

concentration range of 50–600ngmL�1, with a sensitive

detection limit (down to 50ngmL�1 of diazinon in water),

and with excellent selectivity to the analyte.

Another example of this approach used lambda-cyhalo-

thrin (LC) as the template molecule and employed

functionalized QDs. The QD-MIP nanocomposite was

fabricated using APTES functionalized CdSe QDs that can

copolymerize with TEOS in the presence of the template

molecules.[82] The decision to use functionalized or non-

functionalized QDs depends on the type of template

molecule to be imprinted. Surface functionalized QDs are

preferred as assistant monomers if their interaction with

template molecules can increase imprinting efficiency and

rebinding selectivity.

2.5. MIPNPs Prepared by Modified Precipitation

Polymerization and Mini-Emulsion Polymerization

Techniques

Traditional polymerization techniques, such as precipita-

tion polymerization and emulsion/mini-emulsion poly-

merization, have also been useful for the preparation of



MIPNPs for small molecular imprinting, though they have

beenperformedwithmodifications on the traditional route

so they yield MIPNPs. Yang et al.[83] designed a variation

of precipitation polymerization, called distillation precipi-

tation polymerization, to give propranolol-imprinted

MIPNPs within 3h, in contrast to the 24h reaction time

that is common in traditional precipitationpolymerization.

The authors also synthesized core–shell structuredMIPNPs

by this method, where the core contained imprinted

binding sites while a hydrophilic shell was expected

to prevent non-specific adsorption of biomolecules (i.e.,

protein) and allow the small target molecules to enter the

binding sites in the hydrophobic core. Such MIPNPs are

potentially useful for extraction of small organicmolecules

from complex biological samples.

Pan et al.[84] used surface-initiated RAFT polymerization

to introduce poly (N-isopropyl acrylamide; PNIPAAm)

brushes onto MIPNP surface. This not only improved the

surface hydrophilicity, that helped stabilize the imprinted

NPs, but also imparted stimuli-responsive properties to the

MIPNPs’ surface layers.

Recently, Esfandynari-Manesh et al.[85] showed that

carbamazepine-imprinted MIPNPs, prepared by mini-

emulsion polymerization, could be utilized as a drug

delivery system giving a sustainable release of carbamaze-

pine with a higher binding level and slower release rate

than non-imprinted NPs. The study showed that the ratio

of template to functional monomer was a key factor

to obtain the best drug affinity. The release rate of the

loaded drug lasted for more than eight days in 1wt%

sodium dodecyl sulfate aqueous solution.

3. Recent Advances in MIPNPs for ProteinRecognition and Their Applications

Despite the success of small molecular imprinting techni-

ques and their applications in various fields, macromolec-

ularly imprinted MIPNPs remains challenging because of

the difficulties associated with conformational stability,

mass transfer hindrance, and slow rebinding kinetics.

014, 299, 268–282



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In recent years though, significant progress has been

reported in macromolecular imprinting technologies,

especially for applications in bioseparations, biosensors,

and biodiagnostics.

While surface imprinting is an efficient technique to

create binding cavities for proteins on the outer layer of

MIPNPswith good accessibility and fast rebinding kinetics,

the conditions used to accomplish the imprinting process

must be carefully considered. The conformational stability

of proteins is sensitive to their surrounding conditions,

including temperature, pH, ion concentration, surfactant

and its concentration, the ratio of template to functional

monomers, the types of functional monomers used, and

initiator type.[55,86–88] Identifying those reaction conditions

that ensure the conformational integrity of a template

protein and increase binding sites on or near the MIPNP

surface, and that ensure good accessibility, are crucial for

any useful protein recognition system. Several review

papers have described suitable conditions for synthesizing

protein-imprinted MIPNPs.[86,89,90]

A general procedure for protein imprinting on vinyl-

functionalized NP surfaces is shown in Figure 8. The

substrate canbe silicaNPs,magneticNPs, orQDsdepending

on the intended use. Functional monomers, such as MAA,

acrylamide (AAm), and N-isopropylacrylamide (NIPAAm),

are usually used so they can form electrostatic, hydrogen-

bonding, and hydrophobic interactions with appropriate

domains of the template protein. EGDMA or bisacrylamide

(BIS) are often used as cross-linkers to copolymerize with

the functional monomers to give the cross-linked protein-

imprinted layers on theNP substrate surface. The process is

conducted in aqueous media and under appropriate

conditions of pH, salt concentration, reaction temperature,

and with an appropriate ratio of functional monomers to

protein templates.

The sections below describe recent advances in protein

imprinting according to different types of substrate

Figure 8. A general procedure to produce a protein-imprintedMIPNP on a vinyl functionalized NP substrate surface by a surfaceimprinting technique.



categories, and improvements in newer preparation

methods will also be discussed.

3.1. Protein-Imprinted MIPNPs on Magnetic NP

Substrates

Magnetic NPs such as Fe3O4 are among the most useful

substrates for protein imprinting because the magnetic

responsiveness allows these MIPNPs to be easily separated

from complex samples simply by applying an external

magnetic field. This avoids the tedious separations involv-

ing centrifugation or filtration. These NPs are often

prepared by a co-precipitation[91] or a solvothermal[92]

reaction route, usually yielding NPswith a diameter below

25nm that possess superparamagnetism. The major

applications for these MIPNPs are separation and

detection.[93]

One of the most common methods to prepare protein-

imprinted magnetic MIPNPs employs vinyl-functionalized

magneticNP substrates. For example, Jing et al.[94] obtained

a 20nm thick lysozyme-imprinted layer on vinyl function-

alizedFe3O4@SiO2NPswithatotaldiameterof172� 28nm

and with a rebinding capacity of 0.11mg lysozyme/mg

magnetic MIPNP for the target proteins. These researchers

prepared the Fe3O4NPs using a co-precipitation route. First,

they dissolved FeCl3 � 6H2O and FeCl2 � 4H2O in deionized

water with continuous mechanical stirring under a

nitrogen atmosphere. They obtained the desired NPs after

heating to 80 8C with a dropwise addition of ammonium

hydroxide, and maintaining the reaction at that tempera-

ture for 30min. The as-made Fe3O4 NPs were further

modified with a thin layer of silica having silanol surface

functionality via a sol–gel reaction of TEOS in an ammoni-

um hydroxide solution. Then the Fe3O4@SiO2 NPs were

functionalized with vinyl groups obtained by the hydroly-

sis of MPS in acetic acid solution.[94,95] In the subsequent

imprinting process, the selection of the functional mono-

mers and cross-linker must take into consideration the

specific template protein to be used. Although no specific

monomersaredesignated foragivenprotein, combinations

of hydrophobic and hydrophilic monomers are typically

selected that are capable of forming hydrophobic, ionic, or

hydrogen bonding interactions to suitable domains of

the protein. In this study, the authors opted to use MAA

and AAm as functional monomers to form electrostatic

and hydrogen bonding interactions with lysozyme as

a template protein, and N,N0-methylenebisacrylamide

(MBAAm) was employed as a cross-linker. The copolymeri-

zationwas performed in phosphate buffered solution (PBS)

to give the imprinted layer, and then the template protein

waswashed awayby 1MNaCl and deionizedwater to leave

the specific binding sites for lysozyme.

Magnetic NPs have also been prepared with suitable

functionalities so that the imprinted layers canbe prepared

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278

under aqueous conditions by ATRP polymerization.[96,97]

For example, Gai et al.[96] prepared a lysozyme-imprinted

layer with a thickness of 15nm on ATRP-functionalized

Fe3O4@SiO2NPswitha total diameter of 120nm.Again, the

Fe3O4 NPs were surface-modified with a silica–NH2 layer,

introduced by hydrolysis in an APTES/ethanol/water

solution (12 h at 40 8C) to give Fe3O4@SiO2–NH2 NPs. The

Fe3O4@SiO2–NH2 NPs were then reacted with 2-bromoi-

sobutyryl bromide to anchor the ATRP active agent, using a

mixture of tetrahydrofuran (THF) and triethylamine (TEA)

for 20min in an ice bathunder nitrogenatmosphere, giving

Fe3O4@SiO2–ATRP. Finally, the Fe3O4@SiO2–ATRP was

used as the initiator, and combined with the functional

monomers (NIPAAm and AAm) and cross-linker (MBAAm),

along with the template proteins (lysozyme), in a typical

ATRP polymerization process, to give the surface

imprinted layer. This lysozyme-imprinting process was

performed on the magnetic NPs in PBS solution at room

temperature, followed by removal of the lysozyme

template using acetic acid (10% v/v)–SDS (10%w/v), to

give lysozyme-imprinted MIPNPs having excellent desorp-

tion and extraction capability. This study demonstrated

that ATRP-mediated surface imprinting techniques could

be performed under mild reaction conditions and in

aqueous media, and is important because these methods

allow better control over the thickness of the imprinted

layers than non-controlled radical polymerization meth-

ods. This is advantageous because the recognition sites of

thinner imprinted layers are more easily accessible for

target proteins.

Figure 9. Surface imprinting of BHb on amine-functionalized Fe3O4 NPprocess.



Amine functionalized Fe3O4 NPs can also be designed to

covalently bond to a template protein, such as bovine

hemoglobin (BHb).[98,99] This is done using GA that bonds

to the BHb forming an imine bond. Then a simple sol–gel

process, using a mixture of TEOS and APTES, is followed

to complete the imprinting process. By this method, Kan

et al.[98] reported that the thickness of BHb-imprinted layer

was about10nm,whichwas close to the spatial size of BHb,

indicating that the imprinted sites were located near the

surface of the magnetic MIPNPs. The imprinted BHb was

then extracted using a mixture of deionized water and

methanol (1:3.2 v/v)with sodiumbicarbonate (�3.0mmol)

at 25 8C for 20h with mechanical stirring under nitrogen

atmosphere. The protein adsorption tests showed a rapid

rebinding equilibrium, achieved within 1h, with a rebind-

ing capacity of 10.52mg BHb g�1magneticMIPNP. Figure 9

shows the imprinting process of BHb on the surface of

amine-functionalized Fe3O4 NPs, as described above.

Recently, 3-aminophenylboronic acid (APBA), and dop-

amine (DA) have been explored as new functional

monomers for use in magnetic MIPNPs.[92,100,101] DA

is a neurotransmitter that is biocompatible and bio-

degradable. DA contains several functional groups and

can self-polymerize (to PDA) in a weakly basic aqueous

solution, as shown in Figure 10. [102] Zhou et al.[92] used DA

as the functional monomer to imprint human hemoglobin

to the surface of a magnetic NP by mixing the Fe3O4 NPs

(100mg) and hemoglobin (20mg) in 20mL Tris buffer

(10mM, pH 8.0). After mechanically stirring for 2h at room

temperature, DA (40mg) was added and the reaction was

surface based on covalent bond of glutaraldehyde-amine and sol–gel

014, 299, 268–282


Figure 10. Illustration of dopamine self-polymerization in weaklybasic aqueous solutions.


www.mme-journal.de

continued for 3h at room temperature. The product was

collected onamagnet andwashedwith a3%v/v solutionof

acetic acid containing 0.1% w/v SDS. TEM images of the

protein-imprinted MIPNP show a diameter of �100nm

with an outer layer of �10nm. The authors reported that

the thickness of the imprinted layer could be controlled by

the DA self-polymerization time. Binding experiments

showed that the MIPNPs were highly selective to human

hemoglobin. The ease of producing theseMIPNPs using the

self-polymerizing DAmakes this an attractive approach for

imprinting suitable proteins.

Interestingly, Lee et al.[103] reported a simple method to

fabricate active enzyme (amylase) imprintedmagnetic NPs

with an average size of �100nm by a phase inversion

method of poly(ethylene-co-vinyl alcohol) (27–44 mol% of

ethylene) solutions in the presence of amylase templates.

Theamylase remainedactive for50cycles, asestablishedby

measuring glucose production from starch hydrolysis. By

this method, amylase-imprinted MIPNPs were prepared

that possessed high surface area, were easily separated

from the reaction mixture, and afforded rapid enzyme

reloading. MIPNPs that combine the superparamagnetism

of Fe3O4 with high selectivity and affinity to target

molecules are expected to be a valuable therapeutic and

biodiagnostic tool. Theymay also find value as amethod to

clear cytotoxic peptides from the bloodstream, as reported

by Hoshino et al.[112] Also, unlike other MIPNPs, an

applied external magnetic field might be used to guide

these MIPNPs to desired locations for action, and then aid

removal from the body. This alleviates the risk of NP

accumulation in the liver if theyarenoteffectively removed

from the body by the mononuclear phagocytic system.

3.2. Protein-Imprinted MIPNPs on Silica NP

Substrates

Most protein imprinted silica NPs are intended for use as

biosensorsor for solid-phaseextraction.Althoughmagnetic



MIPNPs are more popular for protein separation, several

papers[104–106] reported the use of protein-imprinted silica

NPs for protein separation in solid phase extraction. Such

papers are representative of much of the literature on

designing protein-imprinted MIPNPs.

He et al.[104] imprinted lysozyme onto vinyl-functional-

ized silica NPs via radical polymerization using a low

concentration (0.4wt%) of functional monomers, which is

less than one-tenth of the concentration that is typically

used. The advantage of this low concentration is that

the possible aggregation of MIPNPs is avoided. The well-

dispersed MIPNP system allows a very thin protein-

imprinted film formed on the silica NPs surface, which

wasnotvisiblebyTEM.However, rebindingstudies showed

that the adsorption equilibriumwas achievedwithin 5min

with specific recognition towards the target protein.

This result indicates of importance of vinyl density on

the NP surface, when imprinting with vinyl monomers, on

the thickness of the imprinted layer and the binding site

density. This is corroborated by the study reported by Lu

et al.[45]. They changed the vinyl group spacing on the silica

NP surface, through control of the polymerization con-

ditions, and compared the thickness of the imprinted shell.

Therefore, both the shell thickness and density of binding

sites can be controlled in this way to achieve efficient and

accessible surface binding sites on MIPNPs.

3.3. Protein-Imprinted QD-Based MIPNPs

QD-based MIPNPs are a new type of chemo/biosensor

for protein detection. They combine the merits of the

optoelectronic properties of QDs with the recognition

specificity of MIPNPs. The principle of QD-based MIPNPs

was explained in the small molecule imprinting section. A

surface imprinting technique can also be used to fabricate

protein-imprinted QD-MIPNPs with excellent conforma-

tional integrity of the template protein and rapid rebinding

kinetics to target proteins. Different surface-modified

QDs have been developed to imprint different proteins.

For example, Tang et al.[107] synthesized bovine serum

albumin (BSA)-imprinted QD-MIP nanocomposites using

L-cysteine modified CdS QDs and a radical polymerization.

SEM images showed that the synthesized QD-MIPNPs

were aggregates with the imprinted layer coated on the

QD nanocrystals. After rebinding of the template BSA,

photoluminescence emission of QDs was quenched due

to fluorescence resonance energy transfer between QDs

and template. Zhang et al.[108] used a denatured bovine

serum albumin (dBSA) modified CdTe QDs and sol–gel

process to perform surface imprinting process. They used

APTES as a functional monomer and TEOS as a cross-linker

to form dBSA-imprinted QD-MIPNPs with particle sizes

of 30–50nm. Imprinted layers were formed on slightly

aggregated QDs.

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280

Lin et al.[109] reported a simple method to prepare

protein-imprinted QD-MIP nanocomposites by a phase

inversion technique. The host polymer solution (poly

(ethylene-co-vinyl alcohol)/DMSO solution) was mixed

together with a suitable amount of commercially available

QDs and template proteins, and the mixture was then

dispersed into non-solvent solution, such as deionized

water/isopropanol (2:3w/w), to form protein-imprinted

nanocomposites. Using this phase inversion technique,

they prepared creatinine-, albumin-, and lysozyme-

imprinted QD-MIP nanocomposites with particle size

ranging from 140 to 255nm. That study demonstrated

that the particle size of protein-imprinted QD-MIP nano-

composites and their morphologies were significantly

affected by reaction temperature when dispersing the

polymer/DMSO host solution in non-solvent. After remov-

ing the template proteins by dialysis, in 1wt% SDS solution

(15min) and deionized water several times, all three

protein-imprinted QD-MIP nanocomposites were used to

test real urine samples and showed good selective

recognition to the target proteins of creatinine, albumin,

and lysozyme, and possessed good accuracy.

Two different types of surface-modified QDs and one

convenient technique have been discussed above on the

fabrication of protein-imprinted QD-MIPNPs. Which type of

surface-modifiedQDsandmethods are suitable to fabricate a

specific protein-imprinted QD-MIPNP is mainly dependent

on theproperty of the template protein. In combinationwith

the techniques shown in small-molecularly imprinted QD-

MIPNPs, diverse techniques have been proposed to modify

QD surface functionalities, which make QD-MIPNPs a

promising platform for protein recognition and detection.

3.4. Protein-Imprinted MIPNPs by Precipitation

Polymerization

Precipitation polymerization is well-established polymeri-

zation method, and some recent advances in protein-

imprinted MIPNPs have been reported using this

method. The advances are achieved by reducing particle

size to enrich binding sites on/near the MIPNP surface

with easy accessibility for targets.[110,111] The examples

described in this section are selected to illustrate the

key factors for the protein imprinting process by precipita-

tion polymerization, including forming particles of a

suitable size and with good imprinting efficiency. To

accomplish this, mild reaction conditions in aqueous

solution are usually required, alongwith suitable function-

al monomers at a ratio that is empirically determined for

the specific template protein. These conditions and

procedures are usually required to avoid denaturization

of the protein, as well as to form effective interactions

between the functionalmonomers andappropriate regions

of the template proteins during the imprinting process.



For example,Wang et al.[110] used precipitation polymer-

ization to prepare a MIPNP for specific adsorption of atrial

natriuretic peptide (ANP). The particle size was measured

at 215.8� 4.6 nm. The NH2–SLRRSS–CONH2 used as the

template peptide, which is a short peptide fromANP,MAA,

and NIPAAm as functional monomers, and MBAAm as

cross-linker. The optimal ratio of these reagents was

reported as MAA:NIPAAm:MBAAm at 1:3:10mol/mol.

The precipitation polymerization was carried out at room

temperature in aqueous media, with the assistance of a

small amount of surfactant (SDS), and initiated by

ammonium persulfate (APS) and N,N,N0, N0-tetramethyl-

ethylenediamine (TMED). The template peptide was

removed using 10wt% of acetic acid solution and washing

several times, leaving the recognition sites for both

template peptide and ANP. The binding kinetics test

revealed that MIPNPs reached protein-adsorption equilib-

rium within 30min, and showed a binding capacity of

106.7mmol g�1 for the template peptide and 36.0mmol g�1

for ANP, but showed little affinity to BSA or scrambledANP.

In 2010, Hoshino et al.[112] reported a type of MIPNP by

precipitation polymerization in mild conditions designed

to neutralize and clear cytotoxic target peptides (melittin)

from the bloodstream, functioning in the same way as

natural antibodies. The precipitation polymerization was

conducted inaqueous solutionwithvery lowconcentration

of surfactant at room temperature using an optimized

composition of functional monomers to yield melittin-

imprinted MIPNPs with a size distribution of 10–100nm.

In thiswork, they studied the effect of functionalmonomer

types and monomer ratio on the MIPNP size and yield.

When the functionalmonomerswere tert-butyl acrylamide

(TBAm) and acrylamide (AAm), used as a mole ratio of

40:5 the MIPNPs were 63nm and were obtained in 81%

yield. Similarly, when acrylic acid (AAc) was used instead

of AAm, still at the same 40:5, MIPNP was a 54nm and

obtained in 79% yield. When the researchers used TBAm,

AAm, and AAc at a mole ratio of 40:5:5, a 56nm MIPNP

was obtained in 88% yield.

Considering that template proteins possess complex

structures with hydrophobic, hydrophilic, and ionic

domains, a combination of the functional monomers

TBAm, AAm, and AAc (40:5:5 molar ratio) was chosen

that could provide hydrophobic interactions, hydrogen

bonding and electrostatic interactions with template

proteins, to yield effective recognition sites.[113] These

MIPNPs were termed ‘‘plastic antibodies’’ and expected

to perform in a manner similar to natural antibodies.

They were tested in the bloodstream of living mice with

effective adsorption of the toxin (melittin) and finally

accumulated in the liver to be expelled from the body.

This is the first report of MIPNPs being used as artificial

antibodies in the bloodstream. This interesting result

opens up new and promising applications for MIPNPs.

014, 299, 268–282



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4. Conclusion

The subject of MIPNPs is advancing rapidly. This short

review summarized recent progress in the area of MIPNPs,

with a focus on various surface imprinting techniques to

design MIPNPs using different NP substrates, such as silica

NPs, magnetic NPs, Au NPs, and QDs, and described some

of the advantages and applications of these MIPNPs.

Both small molecule and macromolecule imprinting were

described to show factors favoring binding sites formation,

accessibility, specific affinity to target molecules and fast

rebinding kinetics. The advantages of different synthetic

methods for the preparation of MIPNPs were illustrated

from literature examples, along with factors affecting

the surface imprinting process. This review also addressed

recent advances in designing MIPNPs to possess multiple

capabilities, such as combining magnetic responsiveness

withQDs or SPR properties into a singleMIPNP. Significant

advances have already been made that improve control

over surface imprinting, increase and allow MIPNPs to be

prepared that possess multi-functionality and can per-

form specific recognition, separation, and detection for

complex samples. It is expected that efforts to improve

specificity and impart multiple capability to MIPNPs

will continue, along with efforts to allow these MIPNPs

to be mass produced using cost-effective methods to

provide high throughput analyses to meet market

requirements.

Acknowledgements: The authors thank the Department ofChemistry at Michigan Technological University for financialsupport during the writing of this review article.

Received: April 14, 2013; Revised: June 16, 2013; Published online:August 19, 2013; DOI: 10.1002/mame.201300160

Keywords: biosensor; molecular imprinting; molecular recogni-tion; nanoparticles; surface imprinting

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