Photopolymerization and photostructuring of molecularly imprinted polymers for sensor applications—A review

Analytica Chimica Acta 717 (2012) 7–20

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Review

Photopolymerization and photostructuring of molecularly imprinted polymers for sensor applications—A review Yannick Fuchs a , Olivier Soppera b , Karsten Haupt a,∗ a b

Compiègne University of Technology, CNRS UMR 6022, BP20529, Compiègne 60205, France Mulhouse Institute for Material Sciences, CNRS LRC 7228, BP2488, Mulhouse 68200, France

a r t i c l e

i n f o

Article history: Received 26 September 2011 Received in revised form 12 December 2011 Accepted 14 December 2011 Available online 22 December 2011 Keywords: Molecularly imprinted polymers Biomimetic sensors Microchips Photopolymerization Photostructuring

a b s t r a c t Biosensors are already well established in modern analytical chemistry, and have become important tools for clinical diagnostics, environmental analysis, production monitoring, drug detection or screening. They are based on the specific molecular recognition of a target molecule by a biological receptor such as an antibody or an enzyme. Synthetic biomimetic receptors like molecularly imprinted polymers (MIPs) have been shown to be a potential alternative to biomolecules as recognition element for biosensing. Produced by a templating process at the molecular level, MIPs are capable of recognizing and binding target molecules with similar specificity and selectivity to their natural analogues. One of the main challenges in MIP sensor development is the miniaturization of MIP structures and their interfacing with the transducer or with a microchip. Photostructuring appears thereby as one of the most suitable methods for patterning MIPs at the micro and nano scale, directly on the transducer surface. In the present review, a general overview on MIPs in biosensing applications is given, and the photopolymerization and photopatterning of MIPs are particularly described. © 2011 Elsevier B.V. All rights reserved.

Contents 1.

2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Molecularly imprinted polymers as sensing materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1. Molecularly imprinted polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. MIP-based biomimetic sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Photopolymerization of molecularly imprinted polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Photoinduced free-radical polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. 2.2. Photopolymerization in the context of molecular imprinting of synthetic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Photostructuring of molecularly imprinted polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mask-lithography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Contact or proximity photolithography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Projection photolithography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Focused laser-beam polymerization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Near-field assisted optical lithography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +33 (0)3 44234455; fax: +33 (0)3 44203910. E-mail addresses: [email protected] (Y. Fuchs), [email protected] (O. Soppera), [email protected] (K. Haupt). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.12.026

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Yannick Fuchs studied polymer chemistry at the European Engineering School of Chemistry, Polymers and Materials Science (ECPM) in Strasbourg, France, where he graduated in 2009 with a degree in chemistry engineering. The same year, he received his M.S. degree in polymer sciences from the Strasbourg University. Yannick is currently a Ph.D. student at Compiègne University of Technology (UTC), in the group of Prof. Karsten Haupt, and his main research interests are molecularly imprinted polymers, material nanostructuring and chemical sensors. In 2011, Yannick received a Young Scientist Award from the European Material Research Society in recognition of his work for Bioinspired and Biointegrated Materials as New Frontiers Nanomaterials.

Dr Olivier Soppera is graduated from Ecole Normale Supérieures Cachan (Aggrégation de chimie in 1998). He completed his PhD in Polymer and Photochemistry (Université de Haute-Alsace) in 2003 and then went to Porto University-Portugal for a post-doctoral position (European Marie Curie Grant). He joined CNRS in 2004 as a Senior Researcher and he is now at Institut de Sciences des Matériaux de Mulhouse (IS2M – CNRS LRC 7228). He is heading a team of 6 permanent people devoted to develop photomaterials for applications in optics and nanotechnologies. His current research activities are focused on photomaterials for micro and nano-fabrication for applications in optics, photonics and biology. In particular, he developed optical setup and suitable materials for photofabrication in the DUV and the NIR wavelengths. Olivier Soppera received the Médaille de Bronze du CNRS in 2009 for his research work.

Karsten Haupt studied Biochemistry at the University of Leipzig, Germany, where he received an MSc Degree in 1991. In 1994 he obtained his PhD in Bioengineering from Compiègne University of Technology, France. After a one-year lectureship at Compiègne University, he spent three years as a research fellow at Lund University, Sweden, where he worked on molecular imprinting with Klaus Mosbach. After returning to France he spent one year as a researcher at INSERM, Paris, before joining the University of Paris 12 as an associate professor in 1999. In 2003 he was appointed full professor of Nanobiotechnology at Compiègne University of Technology, France, where he is currently the Head of the Enzyme and Cell Engineering Institute (GEC – CNRS UMR 6022). Professor Haupt is also one of the founders and scientific advisor of the French company PolyIntell that commercializes molecularly imprinted polymer-based products for biomedical, food and environmental analysis. His present research interests include affinity technology, chemical and biosensors, molecularly imprinted polymers and synthetic receptors, biomimetic polymers and nanostructured materials.

1. Introduction 1.1. Molecularly imprinted polymers as sensing materials 1.1.1. Molecularly imprinted polymers Biological recognition elements such as enzymes or antibodies have been widely used over the years for the development of biosensors. However, although they are perfectly fitting with their natural targets, biological receptors are unstable when not in their native environment, they are not always easy to obtain for a given target, and can be difficult to fine-tune for a specific application. In this context, researchers have developed synthetic tailor-made receptors capable of selectively recognizing and binding target molecules with high affinity, but that are at the same time

more stable, easier to produce, available at low costs, and easy to integrate into standard industrial fabrication processes. One of the most straightforward strategies to create such artificial receptors is molecular imprinting of synthetic polymers [1–5]. This technique is based on the co-polymerization of functional monomers and crosslinking monomers in the presence of an imprint molecule, also called the molecular template (the target molecule or a derivative thereof). Initially, the template and functional monomers form a complex. After co-polymerization the functional groups are “frozen” in a specific position by the cross-linked polymeric network. Subsequent removal of the template molecules leads to empty cavities in the polymer structure, which are complementary in size, shape and positioning of chemical groups to the template (Fig. 1). The molecularly imprinted polymer (MIP) thus has a molecular memory and is now able to specifically recognize and bind the target molecule. Polymethacrylate or polyvinyl type polymers obtained by free radical polymerization are most often used as matrix for molecular imprinting, although other organic polymers, and also sol–gel materials, are becoming increasingly popular. Two approaches were developed for imprinting synthetic polymers at the molecular level, differing by the nature of the bonds formed between template and functional monomers prior to polymerization. The first procedure was introduced by Wulff in 1972 and consists in covalent coupling the monomers to the template prior to polymerization [3]. As an alternative, Mosbach and coworker developed a non-covalent approach, in which the functional monomers form a complex with the target molecules by selfassembly [4]. A combination of both covalent and non-covalent methods can sometimes also be used [6]. Despite the fact that the use of weak non-covalent bonds results in association–dissociation equilibria with the template, yielding a heterogeneous population of binding sites in terms of the positioning of the binding groups, this strategy allows for a much larger choice of functional monomers and can be adapted to a wide range of templates. It is more similar to biological recognition processes since biomolecular interactions are most often of the non-covalent type. Being more straightforward in practice, non-covalent imprinting is by far the most widely used method for MIP fabrication. Molecular imprinting of synthetic polymers can be applied to a wide range of target molecules ranging from small organic molecules (pharmaceuticals, steroids, sugars, amino-acids, etc.) [7–10] to peptides [11–13], and proteins [14–16]. However, imprinting macromolecules, such as proteins, in a synthetic matrix is not easy and still remains one of the major challenges in the area. MIPs are employed in a broad range of domains such as solid-phase extraction (SPE) [17,18], controlled drug delivery [19,20], affinity separation [21], immunoassays [22], chemical sensors [23], directed synthesis and catalysis [24–26] and others. While the use of MIPs as antibody mimics in immunoassays, thus as a diagnostic tool, has been proposed by Mosbach as early as 1993 [27], their application as therapeutics had never been shown. However, the group of Shea recently published outstanding results about the potential use of MIPs as therapeutic antibody mimics [12]. They demonstrated MIP nanoparticles to be capable of efficiently binding and neutralizing the cytotoxic peptide melittin in the bloodstream of mice, which is the first example of biocompatible MIPs for in vivo applications [28]. Similarly, in our group we have recently shown that MIP nanogels can be used as specific enzyme inhibitors [16], and thus are potential drug candidates. Although affinity separation remains the most important application for molecularly imprinted synthetic polymers from a commercial point of view, the development of MIP-based sensors and biochips has raised increasing interest during the past decade [29]. This is due to the growing demand in clinical diagnostics, food analysis, environmental pollution measurements, production monitoring, or drug detection [23,30].

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Fig. 1. Principle of the molecular imprinting process.

1.1.2. MIP-based biomimetic sensors In biosensors, upon binding of the analyte molecule to the recognition element, a chemical or physical signal is generated. The transducer, which is in close contact with the recognition element, will then transform this signal into a measurable output signal that can be correlated with the analyte concentration (Fig. 2). Biosensors are based on biological receptors (e.g. enzymes, DNA, antibodies) that are in most cases immobilized on the surface of the transducer [31,32]. Although they exhibit high molecular affinity for the template, their use is limited because of their high fabrication costs and limited stability (pH, temperature, ionic strength, organic solvents and other additives), resulting in difficult handling and storage. Researchers are therefore trying to develop stable and low cost biomimetic synthetic receptors, such as molecularly imprinted polymers, which can be implemented as recognition elements in biosensors and biochips as substitutes of natural receptors. MIPs were combined to different types of transducers. Electrochemical sensors can translate electrochemical reactions of the analyte, or differences of the electrochemical properties of the system in the presence of the analyte, into an electrical signal [33]. Acoustic sensors can detect changes in the propagation velocity or in the frequency of an acoustic wave upon analyte binding [34], and calorimetric sensors measure the heat released upon a chemical reaction or a recognition phenomenon in which the analyte is involved [35]. Optical detection techniques, for example fluorescence or luminescence, were also used with MIPs for sensing applications [36,37]. A recent trend goes toward the development of label-free optical sensors, in which the detection can be performed without the need of a specific property of the analyte, and without the use of labels. Changes in more general properties of the MIP like absorption, reflectivity, or refractive index can be used for analyte detection and quantification if they can be translated into a suitable output signal. A nice example is the work published by Wu et al. in 2008 who developed a label-free optical sensor based

on molecularly imprinted photonic polymers [38]. They were able to detect traces of the herbicide atrazine at low concentrations in aqueous solution using a 3D-ordered interconnected macroporous inverse polymer opal. Atrazine adsorption into the binding sites resulted in a change in Bragg diffraction of the polymer characterized by a color modification (Fig. 3). Since this color change was visible with the naked eye, this is in fact an example where the recognition element acts at the same time as the transducer. Other examples of label-free optical sensors with MIPs are based on surface plasmon resonance [39], or on reflectometric interferometry [40]. The development of highly sensitive MIP-based sensors normally requires a perfect interfacing between the recognition element and the transducer. For that, either a preformed MIP is coated onto the surface of the transducer [40–43], or the MIP is polymerized in situ on the surface of the transducer [40,44]. One of the advantages of photopolymerization is the versatility of this method for spatially controlled in situ polymerization. For example, molecularly imprinted polymer thin films can be fabricated with good control of both film thickness (down to the nanometer range) and inner morphology (porosity, optical properties), and have found applications in the sensor area [45,46]. Standard microfabrication techniques such as spin-coating have been used for the deposition of pre-polymerization formulations that after photopolymerization yield porous thin molecularly imprinted films [44,47]. Another example is the generation of nanostructured films by nanomolding the photopolymerized MIP on a porous alumina substrate. This yields layers of parallel MIP nanofilaments of 50–200 nm in diameter and 0.5–5 ␮m in length [48]. Thin films can also be the starting point for the patterning of MIPs on surfaces, for example for the development of biochips. In fact, light can be used not only to initiate polymerization, but also to structure MIP films. During the past few years, polymer photostructuring resulted in a number of innovations and advances in the field of MIP-based

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sensor development. A number of more or less standard approaches and techniques have been adapted to MIP fabrication, as outlined in the following part of this review. 2. Photopolymerization of molecularly imprinted polymers 2.1. Photoinduced free-radical polymerization As mentioned earlier, the vast majority of MIPs is synthesized by free radical polymerization (FRP). Highly flexible in terms of reaction conditions, FRP can be initiated either thermally or photochemically, and thus be carried out in a wide range of temperatures. It can be performed in bulk format or in solution, and is not water sensitive. Moreover, a large number of vinyl-based monomers are commercially available, carrying different types of functional groups. This makes FRP particularly suitable for the development of functional materials such as molecularly imprinted polymers. FRP is a three-steps process, consisting of initiation, propagation and termination as outlined below [49]. Polymerization initiation: Free radicals R• are generated from excited states of an initiator species in the system to be polymerized. The addition of a free radical to the first monomer yields a chain initiating radical, as shown in Eq. (1). ki

R• + M1 −→RM1 •

Fig. 2. Schematic representation of a MIP-based chemical sensor.

(1)

Production of the reactive species from the initiator is possible by using heat or light as energy source. Of particular interest for polymer structuring is light-mediated initiation (also called photoinitiation). Free radicals are created by converting the energy of the light into chemical energy. Basically, photons are absorbed by the photoinitiator and promote it into singlet state and then to a triplet state by intersystem conversion, from which chemical reactions then start [50]. Concerning the generation of reactive radicals, two types of photoinitiators can be distinguished. The dissociation of photoinitiators of type I yields to two free radicals through homolytic bond cleavage. Acetophenone, diazo, or benzoine etherbased molecules are examples for type I initiators, which absorb light in the UV range. Acylphosphine oxide derivatives are also used as type I initiators, absorbing in the visible range. In the case of type II photoinitiators, electron/proton or direct hydrogen transfer

Fig. 3. (A) SEM image of a MIP photonic polymer film. (B) Color changes induced in the MIP photonic polymer by the rebinding of atrazine at different concentrations. (C) Optical response of the MIP photonic polymer after incubation with atrazine at different concentrations. Adapted from [38] with permission from Wiley-VCH Verlag.

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Table 1 Non-exhaustive list of the main photoinitiators used for MIP photopolymerization and photostructuring. Name

Dissociation

Decomposition

Use

Refs.

2,2 -Azobis(2-methylpropionitrile) (AIBN)

Type I Homolytic scission

Near UV

Bulk

[64,67]

2,2-Dimethoxy-2phenylacetophenone

Type I Homolytic scission

Near UV

Nanomolding Soft lithography Films Spin-coated films

[48,62] [71] [34] [47]

Mechanical micro-spotting Photolithography Nanomolding

[74,75] [82–84] [87]

Type I Homolytic scission Type I Homolytic scission

Near UV

Photolithography

[80]

Near UV

Bulk

[66]

Surface initiated thin films Microstereolithography

[66] [91]

Near UV

Photolithography

[81]

Near UV

Photolithography

[81]

Films

[46]

Benzoin

Benzoin ethyl ether

2-Isopropylthioxanthone

2-(Dimethylamino)-1-(4morpholin-4-ylphenyl)-2(phenylmethyl)butan-1-one

Molecular structure

Type II Hydrogen transfer Type I Homolytic scission

1-Hydroxy-cyclohexyl-phenylketone

Type I Homolytic scission

Near UV

Photolithography

[84]

Phosphine oxide, phenyl bis (2,4,6-trimethyl benzoyl)

Type I Homolytic scission

VISIBLE

Near-field photopolymerization

[68]

Benzyl N,N-dimethyl dithiocarbamate

CRP Active and dormant radicals

Near UV

Core–shell, nanoparticles

[57]

between the photoinitiator (or photosensitizer) and a co-initiator produces the primary radicals. Benzophenone, thioxanthones, or anthraquinone are typical photosensitizers of type II absorbing in the UV range. Type II photoinitiators absorbing in the visible range are also available, such as camphroquinone (sensitive to blue light), eosin Y (absorbing in the green), or methylene blue (absorbing in the red). Conforming co-initiators, which are associated to these photosensitizers are for example n-phenylglycine, diethanolamine, ethyl p-dimethylaminobenzoate, methyl

p-toluenesulfinate, or 2-mercaptobenzoxazole, all of them bearing a labile hydrogen to be exchanged during initiation. Due to the choice of available wavelengths, photoinitiators have opened new reaction possibilities. Table 1 gives a non-exhaustive list of the most common photoinitiators used for MIP photopolymerization and photostructuring. Propagation: Once the chain initiating radicals RM1 • are formed, the propagation step takes place and the polymer is built by successive additions of a large number of monomers to intermediate

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radicals, leading in a very short time to high-molecular-weight macromolecules (Eq. (2)). kp

RMn • + M−→RMn+1 •

(2)

In the reaction mixture, the monomer concentration decreases throughout the time, while the number and the size of macromolecules increase. Termination: At some point of the reaction, the growth of the macromolecules stops and the polymerization terminates. Bimolecular termination usually occurs at the early time of the reaction, when the viscosity of the precursor solution is low, and is favored by high light intensity or high photoinitiator concentration. It can lead either to radical coupling or to disproportionation (Eqs. (3) and (4)). • Coupling ktb,c

R1 Mn • + R2 Mm • −→R1 Mn+m R2

(3)

• Disproportionation ktb,d

R1 Mn • + R2 Mm • −→R1 Mn + R2 Mm

molecularly imprinted one, the binding sites were still accessible for the target molecule (the drug propranolol).

(4)

Since multifunctional monomers (cross-linkers) are employed for the fabrication of MIPs, the increasing viscosity of the reactive medium during the polymerization reaction restricts the mobility of the reactive species, which can thus be trapped inside of the polymer network. Another important termination pathway to consider is due to the inhibitory role of oxygen in free-radical polymerization. Oxygen is indeed known to be a strong scavenger of free-radicals, producing peroxyde radicals that are unable to induce further polymerization. Inhibitors contained in the monomers are usually removed before polymerization. Interestingly, in the context of spatially controlled photopolymerization, the presence of inhibitors was proven to be an efficient way to confine the polymerized volume, and is thus quite useful to reach microscale or nanoscale resolution [51]. Due to irreversible termination reactions and atom transfers, FRP does not allow a control of the size, architecture and number of macromolecules synthesized. By controlling the growth and the termination steps of the polymerization reaction, macromolecular engineering appears easier and new polymer structures can be designed. In this context, controlled radical polymerization (CRP) is well suited. While FRP allows slow initiation, but fast chain propagation at early stages of the reaction, CRP methods are characterized by fast activation–deactivation cycles, and include atom transfer radical polymerization (ATRP) [52], nitroxide-mediated polymerization (NMP) [53], iniferter-mediated polymerization (iniferter standing for initiator–transfer agent–terminator) [54], and reversible addition-fragmentation chain-transfer (RAFT) [55]. For cross-linked polymers such as MIPs, the use of CRP is somewhat more complex, and the potential benefits are still under discussion [56]. With respect to MIP synthesis, iniferter-initiated and RAFT polymerization seem to be the most versatile CRP methods since they are compatible with most of the functional monomers used in molecular imprinting. They are also the only CRP methods that have been used for the photopolymerization of MIPs [57,58]. For the synthesis of functional materials, and especially for MIPs, CRPs as pseudo-living polymerization methods have the distinct advantage to enable the grafting of multiple successive polymer layers resulting in hierarchical structures. Pérez-Moral and Mayes reported for example the synthesis of core–shell particles using a dithiocarbamate iniferter as photoinitiator [57]. The iniferter species were grafted over a polystyrene core and different complex polymer shells were sequentially built using UV-initiated polymerization. Despite the two additional shells that were grown over the first

2.2. Photopolymerization in the context of molecular imprinting of synthetic polymers Molecularly imprinted polymers are templated materials synthesized from pre-polymerization mixtures combining target molecules, mono- and multi-functional monomers, initiator and solvents. Because of the complexity of MIP formulations, the success of the synthesis depends on the combination of the ingredients, but also on polymerization parameters, that is, the type of polymerization, the rate of growth, the reaction time, and the initiation strategy. These have to be optimized for each molecularly imprinted polymer. The effect of the initiation mechanism on the imprinting effect has already been widely studied in the past. Thermal [59], electrochemical [60], microwave-assisted [61], or photoinduced [62] initiation modes were applied to the fabrication of MIPs, the latter being probably the most versatile approach [63]. Photopolymerization exhibits very fast initiation rates compared to other polymerization modes, and can be carried out at room temperature and below [49]. Several research groups focused their studies on the effect of polymerization temperature on MIP properties and have shown that, as expected, the complexation between template and functional monomers through non-covalent bonds prior and during polymerization is more efficient at lower temperatures [63]. In an early paper, Mosbach and co-workers reported the fabrication of MIPs imprinted with L-phenylalanine anilide for high-performance liquid chromatographic applications, using AIBN as either thermoinitiator at 60 ◦ C, or as photoinitiator at 0 ◦ C [64]. Their results suggested that the polymers prepared by photolytic homolysis of AIBN exhibited higher separation factors for the phenylalanine anilide enantiomers than those obtained by thermopolymerization. It is suggested that the increase of temperature required for thermal polymerization will increase the kinetic energy of the functional monomer–template complex and consequently shift the equilibrium toward uncomplexed species [65]. A distinct advantage of photopolymerization is that almost all physical forms of MIP are accessible (monoliths, nanoparticles, supported thin films, nanostructured materials, etc.) [66]. This together with the broad range of commercially available photoinitiators make it a very versatile method. In addition, photopolymerization allows for an accurate spatial control of the irradiation, as well as a good control over the polymerization kinetics since the intensity of light can easily be fine-tuned, which is beneficial for MIP structuring in the x, y and z dimensions. Photoinitiated polymerization has, of course, also a number of limitations. The main problem is the low penetration depth of light in opaque or diffusing materials. This makes the method less suitable for the synthesis of larger monoliths, which may become cured non-homogeneously. For example, if the light intensity is lower inside the material, the polymerization kinetic is also slower resulting in differences in phase separation between polymer and solvent, and thus in a MIP with gradient porosity [47]. On the other hand, with proper stirring, photoinitiation can still be used for diluted solutions, for precipitation or emulsion polymerization. Another main issue of MIP photopolymerization is linked to the potential sensitivity of the template molecule to light, and in particular UV light. Nevertheless, some examples of light-sensitive templates imprinted under photopolymerization conditions have been published and showed efficient imprinting and molecular recognition [62,67]. A workaround in this context may be the use of visible light for the photopolymerization. Two examples of visiblelight photoinitiators for the synthesis of MIPs and similar vinylic polymers have appeared recently [68,69].

Y. Fuchs et al. / Analytica Chimica Acta 717 (2012) 7–20

Fig. 4. Fluorescent image of polymerized dots and lines drawn on a silicon wafer with a nanofountain pen filled with an imprinting mixture containing fluorescein as template. Reproduced from [74] with permission from American Institute of Physics.

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MIPs, a non-contact approach, seems to be the method of choice. Photostructuring is a technique well established in microelectronics and microsystem fabrication to pattern polymeric photoresists with a micro scale resolution [76]. However, due to the usual complexity of MIP formulations, special efforts are necessary to adapt them to these standard techniques. From the MIP point of view, a high degree of cross-linking is required to maintain the structural integrity of the binding sites in the polymeric network. The creation of porosity in the polymer network is also essential to provide access to the binding sites and to facilitate mass transfer, although fast photopolymerization often prevents the formation of pores through phase separation between solvent and the forming polymer chains. Also, the regular vinyl-based monomers used for MIP synthesis and the organic solvents employed as porogens often lead to low-viscosity pre-polymerization mixtures, whereas for photostructuring low-viscosity solutions are more difficult to deposit onto the chip surfaces and to structure. It is therefore not surprising that only few examples have been reported so far in

3. Photostructuring of molecularly imprinted polymers For the development of sensors and biochips based on MIPs as recognition element, interfacing of the polymer with a substrate is required, often in the form of a pattern, which usually implies a resolution between a few micrometers and tens of nanometers. To achieve soft matter structuring at the micro scale, the different approaches commonly used can be classified in two groups, contact or non-contact techniques. Among the contact approaches, soft lithography, also called nanoimprint lithography [70], has been shown to be suitable to achieve MIP microstructuring. Yan et al. reported the fabrication of MIP microstructures using micromolding in capillaries (MIMIC) and could design 2,4-dichlorophenoxyacetic acid imprinted micro-monolith with a cross-sectional dimension of 20 ␮m × 20 ␮m [71]. More recently, Lalo et al. used soft-lithography to engineer MIP films at the nanoscale. They generated arrays of lines on silicon substrates, capable to specifically bind the fluorescent amino acid derivative dansyl-(L)-Phe [72]. Unfortunately, soft-lithography, which necessitates elastomeric (PDMS) stamps with patterned relief structures on its surface, may be incompatible with some MIP precursor mixtures [71]. The presence of organic solvents can swell or damage the PDMS stamp, and makes the solution to be transferred less viscous and thus more difficult to process by soft lithography. Moreover, it is usually difficult to avoid a thin residual polymer layer between structures, which can be a problem for practical applications. Recently, the group of Moreno-Bondi reported an example of MIP film patterning by micro-transfer molding based on SiO2 /Si molds, avoiding thus the use of conventional elastomeric stamps [73]. Alternatively, mechanical microspotting techniques can be used for the deposition of MIP arrays on a surface. As pictured in Fig. 4, we recently demonstrated in collaboration with Gheber’s group the feasibility of creating MIP microstructures, consisting of dots and lines, using an AFM-operated nanofountain pen [74]. The parallel deposition of several MIP dots using a robotized microfountain pen has also been reported [75]. However, mechanical microspotting approaches are serial patterning techniques and thus tend to be time consuming. One of the challenges for scientists working in the MIP field are therefore to develop low-cost MIP-based biosensors and biochips by (i) polymerizing patterned MIP films in situ by controlling size and shape of the patterns with a good resolution down to the nanometer scale, (ii) depositing a number of different MIPs in parallel on the same chip (parallelization, multiplexing), (iii) integrating the patterned MIP with a sensor for direct label-free detection, and (iv) enabling mass production of microbiochips. To achieve this, photostructuring of

Fig. 5. Schematic representation of (A) contact photolithography, (B) proximity photolithography, and (C) projection photolithography.

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the scientific literature on photopatterning of MIPs with standard techniques. In the following, this review will focus on some recent examples of MIP biosensor engineering using innovative photopatterning approaches. 3.1. Mask-lithography Mask-lithography, also called photolithography, is an optical method for wafer patterning introduced for the first time in the sixties. While in 1970 photolithography was able to provide structures with 10 ␮m linewidth, in 2000 patterns with a period of 180 nm were achievable in volume production [77]. Nowadays, thanks to the evolution of laser technology, deep-UV light (DUV) from excimer lasers with wavelengths of 248 nm and 193 nm allows feature sizes down to 32 nm. 3.1.1. Contact or proximity photolithography Contact photolithography was the first photolithographic technique commercially available, introduced in 1964 (Fig. 5). The photoresist substrate to be patterned is brought into contact with a photomask containing the pattern to be reproduced [78,79]. This photomask is made from both opaque areas stopping the incident light, and transparent areas through which the light can pass. Of course, a close and stable contact between a large photomask and photoresist is always difficult to maintain, and may induce defects in the photoresist and pollution of the mask. An alternative is to keep a small gap between the mask and the substrate. This approach, called proximity printing, exhibits a decreased resolution as the gap increases, but defects are significantly reduced since the mask is not in contact with the photoresist. The pattern resolution obtained by proximity printing is limited by light diffraction at the edges of the opaque area. Therefore, instead fabricating the theoretical crenellations-like patterns, proximity printing yields more sinusoidal-like structures. Eq. (5) gives the theoretical maximum pattern resolution, which can be obtained using proximity printing technology: 2bmin = 3 ·

 

 s+

1 e 2

 (5)

where b is the half-period of the pattern,  is the wavelength of incident light, s is the distance between photomask and photoresist, and e is the thickness of photoresist. Usually, the thickness of the photoresist is negligible compared to the distance between the photomask and the photoresist, and in this case the theoretical maximum pattern resolution can be simplified as shown in Eq. (6): 2bmin ≈ 3 ·



·s

such as clenbuterol and terbutaline. Compared to their previous work, they enhanced the pattern resolution to 20 ␮m, and onchip MIP parallelization seemed to be in close reach. Later, in collaboration with Ayela’s team, our group reported an example of spin-coated MIP films patterned by contact photolithography using mask aligners and other standard equipment for the large-scale fabrication of microbiochips. In contrast to Huang’s group, instead of using a pre-polymerized functional polymer layer as MIPh, we employed conventional monomer-based MIP precursor mixtures [82]. Patterning was very efficient and reproducible and different features could be generated with a 1 ␮m resolution (Fig. 7). Parallel printing of a large number of chips on the same wafer, combined with multiplexing through sequential printing of different MIPs, paved the road to mass production of MIP chips. The group of Byrne demonstrated photopatterning by photolithography being potentially applicable to polymer gel matrices [83]. A novel copolymer containing poly(ethylene glycol)n dimethacrylate and acrylamide was developed and used as a matrix to be imprinted with d-glucose [84]. The polymer gel patterns exhibited thicknesses of about 13 ␮m and a number of geometrical configurations as presented in Fig. 8. Incubation of these patterns with a fluorescent glucose derivative indicated that binding was not only on the surface of the structures, but homogeneously distributed throughout the matrix. The above examples of MIP photostructuring are encouraging for the development of MIP based microbiochips. Moreover, in 2011 Lautner et al. reported the possibility of imprinting not only small molecules, but also proteins in photostructured polymer substrates [85]. Avidin-imprinted poly(3,4-ethylenedioxythiophene)/poly(styrenesulfonate) (PEDOT/PSS) conducting polymer microbands were prepared directly on surface plasmon resonance (SPR) chips by photolithographic patterning (Fig. 9). An elegant aspect of this work was the introduction, between the micropatterned PEDOT/PSS lines, of an additional sacrificial polymer (polycarbonate) exhibiting high protein affinity, and thus generating protein binding sites only on the lateral walls of the PEDOT/PSS bands. The binding tests showed submicromolar dissociation constants for protein binding.

(6)

In 2004, Huang et al. published a paper on the combination of photolithography with molecularly imprinted polymers for the construction of a voltammetric sensor for albuterol [80]. The MIP structuring was performed onto a gold working electrode, and the pattern resolution was about 50 ␮m. However, instead of the conventional methacrylate or vinyl-based monomers, polynorbornene containing various functional groups was synthesized by ring opening metathesis and used as MIP photoresist (MIPh), which limits the applicability of this technique. More recently, the same group published another example of combination between MIPs and photolithographic printing [81], describing the integration of acrylic MIP patterns into a miniaturized three-electrode cell, using a photolithographic approach (Fig. 6). By pre-polymerizing a MIPh made of benzyl methacrylate, methacrylic acid and 2hydroxyethyl methacrylate, they obtained a sensing layer that could discriminate the target albuterol from interfering analogues

3.1.2. Projection photolithography The mechanical problems encountered with contact or proximity photolithography triggered the development of projection photolithography in the early eighties. The photoresist and the mask are kept at some distance from each other and an optical system is inserted between both in order to focus the image from the mask onto the photoresist [86]. It is thereby possible to achieve patterning with good resolution, without damaging the photoresist. As for proximity printing, diffraction of the light at the edges of the opaque zone of the mask is limiting the pattern resolution. As the light is collected in the far-field, the Fraunhofer diffraction theory is applicable. The diffraction pattern is calculated by summing at each point of the image plane all the contributions from the wave fronts coming from the diffracting object, and considering for each light-path the path-length difference. Since lenses are used to focus the image from the mask onto the substrate, their numerical aperture is of main relevance. The bigger the numerical aperture, the better the resolution of the pattern. The minimal separation between two objects imaged by a lens is calculated using the Rayleigh criterion given in Eq. (7), where  is the wavelength of the light, and N.A. the numerical aperture: Lmin =

0.61 N.A.

(7)

The only example so far of MIP patterning using projection printing has been published in 2011 by our group in collaboration with Gheber’s group [87]. We combined projection photolithography

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Fig. 6. Preparation of MIP photoresist-based chip (MIPC) developed by Huang et al. via photolithographic approach. Reproduced from [81] with permission from Elsevier.

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Fig. 7. Example of MIP patterns obtained by photolithography. SEM images of (A) a matrix of spiral and (B) linear features used for the determination of the maximum resolution achievable (numbers indicate the linewidth). (C) Four-inches silicon substrate with multiplexed MIPs. Features defining one chip are repeated on the entire wafer. Inset: A, MIP templated with dansyl-L-Phe; B, MIP templated with Boc-L-Phe; C, non-imprinted control polymer. Adapted from [82] with permission from The Royal Society of Chemistry.

and nanomolding for the microfabrication of MIP arrays. The MIP photoresist was a standard mixture of vinyl-based monomers, and dot arrays were obtained by coating the mixture and projecting the pattern on a flat substrate. Using a 10× microscope objective and a 0.5 mm feature size on the mask, dot sizes of 70 ␮m were obtained (Fig. 10), although much smaller structures are possible. Moreover, if the pattern was projected on a nanoporous alumina oxide substrate coated with the MIP precursors, nanostructured dots were obtained composed of parallel nanofilaments, 150 nm in diameter and 4 ␮m long. In that way, the surface-to-volume aspect ratio of the MIP dots is considerably increased, and the binding site accessibility improved compared to porous dots. Indeed, the technique allowed for the imprinting of a protein (myoglobin) in the filament surface with an imprinting factor of IF = 4.3 (IF = binding to MIP dots/binding to non-imprinted control dots).

3.2. Focused laser-beam polymerization For some sensor formats, the controlled generation of 3D MIP microstructures is required. Focused laser-beam polymerization allows for 3D polymer patterning and includes approaches such as microstereolithography (␮SL) [88], 2D and 3D interferometry [89], and multiphoton lithography [90]. The only example reported so far concerning 3D MIP patterning using focused laser beam polymerization has been published by Shea’s group [91]. Microstereolithography is a photopolymerization technique derived from conventional stereolithography, which was the first prototyping process introduced for the fabrication of polymer molds [92]. Using computer-assisted design software, the desired 3D model image is horizontally sliced into a series of 2D layers, with uniform thickness. The focused UV scanning beam is absorbed

Fig. 8. Three-dimensional projection of micropatterned rectangular array of a biomimetic recognitive polymer network obtained utilizing a confocal microscope. Adapted from [84] with permission from American Chemical Society.

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Fig. 9. (A) Schematic representation of the surface imprinting procedure for fabricating MIP microbands on bare Au SPR chips by photolithographic approach. Cross-sectional SEM images of (B) PC and PEDOT/PSS bands, (C) surface imprinted (SIP) PEDOT/PSS. Adapted from [85] with permission from Wiley-VCH Verlag.

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Fig. 10. (A) Schematic representation of MIP microarrays nanopatterning by combining projection photolithography and nanomolding. (B) Fluorescence microscope image of nanostructured MIP microarrays imprinted with fluorescein by projection photolithography. Inset: SEM image. Adapted from [87] with permission from Wiley-VCH Verlag.

by the curable monomer mixture, and the synchronized motion of the x–y plane enables to photopolymerize a 2D polymer layer [93]. As shown in Fig. 11A, translation along the z-axis allows the next monomer layer to be polymerized on top of the previous one. Finally, a complicated 3D polymer structure can be fabricated in a layer-by-layer fashion. In their work, Shea and coworkers fabricated 3D waffle patterns (Fig. 11C) imprinted with ethyladenine, and showed these patterns to exhibit the same affinity for the target molecule as the corresponding monolayer patterns and bulk MIPs [91]. Piletsky’s group also reported focused laser-beam polymerization by using a low frequency IR beam working at a wavelength of 10.6 ␮m. However, in this example the polymerization itself is initiated thermally. By focusing the IR laser onto the precursor mixture,

they could locally heat the monomers and polymerize microdots about 70 ␮m diameter [94]. 3.3. Near-field assisted optical lithography Basic research on near-field optics has been started in the early 1980s by several groups in Europe, US and Japan. Rapid progress has been made by an increasing number of research groups in the area since the 1990s [95]. Near-field optics concerns phenomena involving non-propagating inhomogeneous fields (evanescent electromagnetic waves) and their interactions with matter, and are particularly interesting since the optical interactions they are entailed with are on sub-wavelength scales [96]. Optical near-fields

Fig. 11. Schematic representation of (A) stereolithography and (B) a ␮SL apparatus developed at Penn State including an Ar+ laser, a beam delivery system, computercontrolled precision x–y–z stages and a CAD design tool, and an in situ process monitoring system. (C) SEM images of a 3D adenine imprinted polymer microstructure fabricated by microstereolithography. (A and B) Adapted from [93] with permission from Elsevier. (C) Adapted from [91] with permission from Wiley-VCH Verlag.

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Fig. 12. (A) Experimental setup used for photopolymerization by evanescent waves. (B) AMF scan of the surface of 1D-patterned polymer films using interferometric evanescent wave irradiation (pitch = 160 nm), and (C) AFM scan of the surface patterned by double exposure irradiation with interferometric evanescent wave configuration (x–y pitch = 0.5 ␮m; z amplitude = 120 nm). (A) Adapted from [98] with permission from Wiley-VCH Verlag. (B and C) Adapted from [101] with permission from Wiley-VCH Verlag.

are located at the source region of optical radiation, or at the surface of materials interacting with free radiation, and in many situations they are explored for their ability to localize optical energy to length scales smaller than the diffraction limit of roughly /2n, with  being the wavelength of light and n the refractive index [97]. In photostructuring, the pattern resolution is determined by the minimum volume in which light may be confined. This volume, also called voxel, is defined by the minimum surface that can be irradiated and by the thickness of the layer. Taking advantages of near-field optics, Lougnot’s group reported the first procedure for the fabrication of very thin polymer parts by the use of evanescent wave photopolymerization (PEW) [98–100]. They built on the concept of total reflection of a laser beam on a substrate in order to create an evanescent field in this substrate (Fig. 12). When a photosensitive monomer mixture was placed on top of the substrate, the thickness of the monomer layer in which actinic light is confined was similar to the penetration depth of the evanescent field, resulting in polymeric parts with a z-resolution of only a few tens of nanometers. In 2007, Soppera et al. illustrated the potential of evanescent fields to fabricate 1D and 2D periodically patterned thin polymer films. Outstanding resolution was reported and lines with sub-100 nm width could be easily patterned, thus opening new doors toward the development of functional materials [101]. Similarly, the evanescent field generated by surface plasmon resonance at a metal surface can be used for localizing photopolymerization of thin films on this surface [69]. Our group in collaboration with Soppera’s group has recently reported the first proof of concept of MIP photopatterning using near-field assisted optical lithography [68]. We showed the feasibility of fabricating ultra-thin MIP microdot arrays in only a few seconds. The MIP precursors mixture had to be specifically

optimized to be compatible with this technique. It uses for the first time photopolymerization of a MIP under visible-light (405 nm). Moreover, compromises had to be found with respect to a high viscosity of the precursor solution being required for near-field polymerization by evanescent wave, and the necessity to use solvents in the mixture to render the MIP porous. The MIP microdots exhibited sub-100 nm thicknesses and a mesoporous morphology. They were imprinted with the amino acid derivative Z-(L)-Phe, and incubation tests with the fluorescent derivative dansyl-(L)-Phe revealed specific target binding and a certain degree of enantioselectivity. Owing to the simplicity of the optical setup enabling to rapidly obtain reproducible polymer nanoparts, and the possibility of using “close-to-conventional” MIP formulations, near-field assisted optical lithography appears promising for the miniaturization of MIP sensors and biomimetic microchips.

4. Conclusions Photopolymerization and optical structuring of molecularly imprinted polymers are well adapted for the development of miniaturized MIP-based microchips and microsensors. Compared to other initiation modes, photoinduced reactions have been shown to be particularly well-suited for non-covalent imprinting of synthetic polymers, since polymerization can be carried out at low temperature, resulting in a better imprinting efficiency. MIP films patterning by the use of optical methods is relatively new compared to other structuring approaches such as soft lithography or mechanical microspotting, but the recent increase in the number of publications in the field reflects the high potential of the concept. Contact and proximity printing, projection photolithography,

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microstereolithography, and near-field assisted optical lithography were all successfully combined with MIPs, resulting in innovative high-resolution patterns exhibiting molecular specificity and selectivity. Photopolymerization and photostructuring facilitate interfacing MIPs with a transducer, and the fabrication of multiplexed MIP chips at the wafer scale. Acknowledgments The authors gratefully acknowledge the French National Research Agency (ANR – Project HOLOSENSE, ANR-08-BLAN-023602), and the Regional Council of Picardy (France) as well as the European Union for funding of equipment under CPER 2007-2013. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46]

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