Rapid Prototyping of Chemical Microsensors Based on ...

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Rapid Prototyping of Chemical Microsensors Based on Molecularly Imprinted Polymers Synthesized by Two-Photon Stereolithography

Laura Piedad Chia Gomez, Arnaud Spangenberg,* Xuan-Anh Ton, Yannick Fuchs, Frank Bokeloh, Jean-Pierre Malval, Bernadette Tse Sum Bui, Damien Thuau, Cédric Ayela, Karsten Haupt,* and Olivier Soppera*

L. P. Chia Gomez, Dr. A. Spangenberg, Dr. J.-P. Malval, Dr. O. SopperaInstitut de Science des Matériaux de Mulhouse (IS2M)CNRS - UMR 7361Université de Haute Alsace15 rue Jean Starcky, Mulhouse 68057, FranceE-mail: [email protected]; [email protected]. X.-A. Ton, Dr. Y. Fuchs, F. Bokeloh, Dr. B. Tse Sum Bui, Prof. K. HauptSorbonne UniversitésUniversité de Technologie de CompiègneCNRS Laboratory for Enzyme and Cell EngineeringRue Roger Couttolenc, CS 60319, 60203 Compiègne, FranceE-mail: [email protected]. D. Thuau, Dr. C. AyelaLaboratoire de L’Intégration du Matériau au SystèmeUniversité de Bordeaux351 Cours de la Libération, 33405 Talence cedex, France

DOI: 10.1002/adma.201600218

sensing. We demonstrate the molecular imprinting of polymers prepared by TPS from specifically optimized precursors. Then, we show examples of 3D MIP microsensors to illustrate the potential of TPS. Finally, an all-organic MIP microcantilever is built by TPS and we show the possibility to use it as a label-free microgravimetric chemical sensor.

Unlike in standard photolithography, TPS is based on two photon absorption, a strongly nonlinear absorption process, which amplifies the already nonlinear chemical response of photoresists during the fabrication. Taking advantage of these combined effects, photopolymerization is confined at the focal point of the laser irradiation, in a 3D volume, typically of less than ≈1 μm3.[6] The 3D structure is fabricated by displacing the focal point in a space filled with the photopolymerization for-mulation. The most striking achievements in the field of TPS can be attributed to the development of efficient photoinitia-tors,[7–10] as well as optomechanical strategies to increase writing speed[11–13] and resolution.[14,15] In parallel, research efforts have been devoted to the development of advanced functional mate-rials synthesized through TPS. However, after photostructuring by TPS, most of them involve multistep processes such as phase inversion or thermal annealing to achieve the desired proper-ties.[16–18] In this regard, only few works have been focused on the development of directly crosslinkable functional materials suitable for TPS.[19–22] Indeed, this field of research is still very challenging because it is usually rather difficult to combine antagonist parameters to ensure both integrity and specific properties of the functional material during the processing route. In particular, to the best of our knowledge, there is no report describing the 3D structuring of MIPs by TPS, despite the great potential of these synthetic sensing materials.

Molecularly imprinted polymers are tailor-made antibody mimics obtained by a templating process at the molecular level.[23,24] They are obtained by copolymerizing functional and cross-linking monomers around a template molecule. This leads to a 3D polymer network containing stable cavities that are complementary to the template in terms of size, shape, and position of functional groups. Thus a “molecular memory” is introduced into the polymer, allowing the molecular recognition and binding of target analytes with a high affinity and specificity. MIPs are physically and chemically robust,[25] which facilitates their application in harsh environments and integration into standard industrial fabrication processes and devices. For these reasons, MIPs have been widely used to respond to the growing demands in environmental analysis, clinical diagnostics,

In the last decades, nano- and microtechnologies have shown an accelerated progress toward the development of compact and elaborated systems at nano- and micrometer scale in research fields like microelectromechanical systems (MEMS),[1,2] nano-biotechnology,[3] nanophotonics,[4] and bioengineering.[5] Whereas different strategies have been successfully imple-mented for mass production of 2D/2.5D micro- and nanostruc-tures, fabrication of 3D micro- and nanostructures is usually not trivial and requires time-consuming multistep processes. In this context, rapid prototyping techniques such as two-photon stereolithography (TPS) are of particular interest. TPS is a microfabrication technique that attracts growing interest as it allows the design and direct fabrication of 3D structures based on a photopolymerizable material. TPS can be consid-ered as an extension of 3D printers at the micro- and nanoscale levels, enabling the perfect replication of miniaturized 3D objects. One of the current challenges is to apply the technique to the direct writing of advanced functional materials, with a complex composition. In the context of chemical microsensors or microbiosensors, molecularly imprinted polymers (MIPs) are particularly well adapted as synthetic biomimetic recogni-tion materials, because of their greater chemical and physical stability compared to their biological counterparts like enzymes or antibodies, and easier processing and engineering. Here, we describe the application of TPS to the synthesis of novel com-plex architectural MIP microstructures (Figure 1) for chemical

Adv. Mater. 2016, DOI: 10.1002/adma.201600218

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production monitoring, biosensors, or biochips.[26,27] Compared to alternative recognition materials based on biomacromol-ecules such as antibodies, enzymes, or DNA, MIPs can be used in a much wider spectrum of operating conditions due to the stability of these crosslinked synthetic polymers.

The integration of the MIP into complex systems or devices has always been a challenge, thus limiting their practical appli-cations. While standard micro- and nanofabrication techniques have been adapted to the synthesis of 2D and 2.5D MIP struc-tures,[28–32] true free-standing 3D structures were not accessible. Photopolymerization is one of the most versatile approaches to achieve this goal. It has two main advantages that pave the road toward fabrication of complex polymer structures: the many dif-ferent physical forms of MIPs that can be produced (monoliths, nanoparticles, supported thin films, nanostructured materials, etc.), and the broad range of available photoinitiators for UV and visible light[33] photopolymerization allowing an accurate spa-tial control of the polymerization. The polymerization kinetics is determined by the intensity of light and can thus easily be fine-tuned, which gives a control over the molecular structure of the polymer (conversion, crosslinking, chain length, etc.) and materials properties (stiffness, Young modulus, transpar-ency, etc.). Last but not the least, photopolymerization can be performed at relatively low temperatures, thus favoring the pre-polymerization complex between the functional monomers and the template, which significantly improves the quality of the

molecular recognition sites.[34,35] It is therefore not surprising that MIPs have during the past years raised increasing interest for applications in chemical sensors,[36–38] such as holographic sensors[39,40] and fiber optics sensors.[41]

However, so far, photopolymerizable MIPs have been restrained to one-photon UV or visible light lithography, thus limiting the process to conventional 2D or 2.5D microstruc-tures. Consequently, extending the microstructuration to arbi-trary 3D microstructures was a major challenge until the recent emergence of two-photon stereolithography.[6,42] The develop-ment of MIPs compatible with TPS thus constitutes a break-through not only in the design of chemical sensors, but also for their integration into complex microfluidic systems or other devices for sensing or molecular separation.

We first developed a MIP formulation showing molecular recognition properties and enabling the fabrication of 3D struc-tures with submicrometric resolution by TPS. The photoinitiator for triggering photopolymerization of the MIP precursors was carefully chosen to fulfill the requirements of both molecular imprinting and two-photon photopolymerization. In order to avoid interferences between the photoinitiator and the tem-plate or the functional monomers, we excluded initiators that require a coinitiator containing an amino group. Thus, initia-tors that react according to a Norrish I mechanism seemed more appropriate for MIPs. Among commercially available Norrish I photoinitiators, phosphine oxides have already been used in MIP



Figure 1. A) Schematized representation of the direct laser writing of MIPs, by displacing a NIR femtosecond laser focused beam inside the MIP precursor solution. B) Procedure to the photoassisted fabrication of the MIP material: (1) Complexation of the template by functional monomers, (2) Photocrosslinking of the functional monomers by two-photon polymerization leading to (3) template embedded in MIP polymer matrix, (4) MIP after template extraction with free cavities for target binding. C) Examples of MIP microstructures imaged by SEM and fluorescence microscopy after binding of a fluorescent derivative of the template (dansyl-l-Phe).

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formulation.[33] Lucirin TPO was thus tested in this study since it exhibits a modest two-photon cross-section but a high yield of production of radicals (φ ≈ 1) from excited states, leading to a rather significant two-photon action cross-section for radicals generation at 800 nm.[43] Moreover, it is liquid at room tempera-ture, which facilitates its incorporation in the MIP precursor mixture.

For the MIP formulation, we chose as model template N-car-bobenzyloxy-L-phenylalanine (Z-L-Phe). A fluorescent analogue of this molecule, dansyl-l-phenylalanine (dansyl-l-Phe) is avail-able, which facilitates the evaluation of the binding properties of the MIP. Methacrylic acid (MAA) and 4-vinylpyridine (4-VPY)[44] were employed as functional monomers and pentaerythritol tri-acrylate (PETIA) was used as crosslinker. PETIA has a relatively high viscosity, which is beneficial for 3D structuring. Moreover, MIPs including PETIA as a crosslinker have shown good recog-nition properties.[40] The schematized TPS laser direct writing and the procedure leading to the MIP formation are shown in Figure 1.

The MIP formulation was first optimized by preparing bulk polymers. The photoinitiator lucirin TPO, can also be excited with one-photon UV irradiation. These preliminary studies allowed us to investigate the impact of the different MIP com-ponents and to validate the choice of monomers and photoini-tiator for molecular imprinting, decorrelated from the specifici-ties of TPS. A crucial point was the choice of the solvent, which is usually added during MIP synthesis to generate porosity in the material and thus guarantee access to binding sites.[45] At the same time, it must not interfere with the template-monomers complex. Acetonitrile (ACN) was previously used as solvent for the synthesis of Z-L-Phe or dansyl-l-Phe MIPs;[40,44] however, a high-viscosity medium would be more favorable for 3D microfabrication by two-photon polymerization. Thus, acetonitrile (0.34 cP at 20 °C) and a more viscous solvent, triglyme (2.5 cP at 20 °C), were tested. A MIP precursor solu-tion without any solvent was also tested in parallel. The binding isotherms of dansyl-l-Phe to the three bulk MIPs and the cor-responding non-imprinted control polymers (NIPs) are pre-sented in Figure S1 (Supporting Information). All three MIPs bind dansyl-l-Phe more than the NIPs. With ACN as solvent, an imprinting factor (binding to MIP/binding to NIP) of 2.3 at a polymer concentration of 2 mg mL−1 was observed. The level of adsorption was the highest when ACN was used as solvent for polymer preparation, although the non-specific binding also increased substantially with polymer concentration. How-ever, we later found that ACN could not be used for 3D micro-structuring because the corresponding solution exhibited too low viscosity and too high volatility. The triglyme-based MIP also showed good binding and the highest imprinting effect (IF = 12.3 at 2 mg of polymer per mL). At higher polymer concentrations the non-specific binding to the NIP is high, though. In fact, the number of functional groups per volume of material is the same for MIP and NIP. Therefore, at higher concentrations of one of the interacting partners (polymer or target analyte), the (weaker) non-specific binding to chemical groups in the NIP can indeed reach the level of binding seen in the MIP. This depends in addition on the inner morphology of the polymer, which in turn depends on other factors like the porogenic solvent used. Even the MIP synthesized without the

use of solvent performed well (IF = 2.8 at 2 mg of polymer per mL), though the overall binding was lower probably due to a low porosity. It is possible that some porosity was nevertheless generated in these polymers after the removal of some unre-acted monomers.

Microstructures were then prepared by two-photon poly-merization with the MIP precursor formulations without sol-vent and with triglyme. TPS allows creating arbitrary structures. Figure 2A shows the scanning electron microscopy (SEM) images of MIP squares (20 × 20 μm). NIP squares fabricated in similar conditions were also characterized by SEM and did not show any significant difference in morphology (Figure S2, Supporting Information). Binding of dansyl-l-Phe to the MIP was quantified by fluorescence microscopy. Unexpectedly, the highest fluores-cence signal was obtained with the structures prepared without solvent, contrarily to what had been obtained from the analysis of the bulk MIPs (Figure 2B). Moreover, the IF of the MIP prepared without solvent was higher (4.7 vs 1.6 for the MIP with triglyme). A possible explanation is that first of all, as mentioned above, porosity may be generated in these polymers due to the elution of non-reacted monomers. The resulting microstructure presents a high surface to volume ratio, thus guaranteeing access to suf-ficient imprinted sites. In addition, the quality of the imprinting may be better without the solvent because the precursor concen-trations are higher and an interference of the solvent with the monomer-template interactions is avoided. Anyhow, in the con-text of TPS, the presence of a solvent is a drawback for 3D micro-structuring due to the decrease in viscosity. As a conclusion, other than for solubility reasons, the use of a solvent should be avoided in the MIP recipe when performing TPS.

The binding selectivity of the imprinted microstructures was then demonstrated by incubating the polymers with either dansyl-l-Phe or dansyl-d-Phe. Figure 2B shows that binding of dansyl-l-Phe to the MIP is two times higher than that of the d-enanti-omer, whereas no significant difference is obtained for the NIP, which confirms the selectivity of the MIPs synthesized by TPS.

The interest of TPS microstructuring is that it provides access to high-resolution 3D structures with complex architectures. Our setup includes a computer driven piezo-electric system that can automatically reproduce 3D shapes generated by computer-aided design (CAD). Figure 3 exemplifies 2D and 3D MIP structures made by TPS and illustrates the interest of this technique. Figure 3A shows the resolution of 3D-writing that can be reached with the optimized MIP precursor formulation. Crossed lines with a width as small as 250 nm could be gener-ated with a ×100 objective (Numerical Aperture, N.A. = 1.21) and excitation performed at 800 nm. This high resolution is possible owing to the nonlinear two-photon light absorption. Such nanostructures could, for example, be used as diffraction gratings or waveguides for label-free detection as proposed in a previous work from our groups.[39,40]

Another example of structural integration is given in Figure 3B. By sequentially writing different MIP and NIP micro-structures on the same substrate, it is possible to generate mul-tiplexed arrays (microdots in this case). When put in contact with the analyte molecule dansyl-l-Phe, the differential binding between the MIP and the NIP reveals a latent image. This ele-gant approach demonstrates the possibility to create highly mul-tiplexed biochip-like devices allowing the detection of several




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molecules on the same chip. Other potential applications are, for example, anti-counterfeiting features, as from an invisible microscopic structure, a complex fluorescence pattern can be produced by contact with a specific tracer molecule.

The most interesting possibility afforded by the TPS is the generation of 3D arbitrary microstructures. In the con-text of MIPs, this unique feature has several advantages like building hierarchical porous structures with high accessibility of the imprinted binding sites. Moreover, complex designs can be imagined, for example, to facilitate the transduction of the chemical binding signal into an optical signal. To illustrate this, we generated superposed grid structures (high aspect ratio grids) with heights ranging from 2 to 18 μm (Figure S3, Sup-porting Information). Quantitative fluorescence microscopy images (Figure S3, Supporting Information) after binding of dansyl-l-Phe reveal a linear increase in the fluorescence inten-sity with an increasing height of the structures, which dem-onstrates that 3D-structuring does not affect the accessibility of the molecularly imprinted binding sites. This method can therefore be used to generate extremely well defined micro-porous structures to increase the fluorescence signal of MIP microdots, thus improving the sensitivity of detection.

The previous structures are usually qualified as 2.5D micro-structures. More challenging is the fabrication of real 3D suspended structures. Such structures need optimized mechan-ical properties to resist against polymer mass and development step. However, real 3D structures give access to original prop-erties. For instance, 3D photonic crystals (also called woodpile

structures) are very interesting candidates to generate highly sensitive optical sensors. TPS is a means to easily integrate the 3D structure in a complex device. MIP woodpiles generated by TPS are shown in Figure 3C. They were built with 40× (first line) and 100× (second line) microscope objectives. In both cases, the 3D structures are free-standing and the lines used to generate the woodpile structure are well defined. The 100× objective allows generating very small building blocks. From the SEM image, the size of the voxel can be evaluated as 200 × 200 × 800 nm, which is of the order of magnitude of the diffraction limit of the optical setup. Moreover, we measured the fluorescence intensity of two MIP and NIP woodpiles with two different heights after binding of dansyl-l-Phe and found that the fluorescence signal is proportional to the height (Figure 3C). These results demon-strate that the accessibility of the binding sites is preserved even for high 3D structures, while the 3D structuring allows elabora-tion of sensors of high aspect ratio to improve their sensitivity.

Last but not the least, we set out to directly write a free-standing microgravimetric sensing element in the form of a MIP microcantilever, combining the recognition element (MIP) and part of the transducer (cantilever) in one structure. MEMS are used in many applications, including chemical sensors for which they are of particular interest.[46,47] In this context, alternatives to the conventional silicon-based devices are being explored.[17,48] Organic polymers tend to replace these more traditional materials, just like organic light emitting diodes are about to supplant silicon-based light emitting diodes in smart-phones and other displays.[49] Indeed, the tunable properties



Figure 2. A) SEM images of the MIP microstructures synthesized by TPS without the use of solvent (voxel irradiation time: 100 ms, thickness: 3 μm). B) Left: Fluorescence intensities measured on MIP and NIP microstructures after incubation with 10 μm dansyl-l-Phe in acetonitrile for MIP/NIP prepared without solvent and with triglyme (ratio volume monomers:solvent = 2:1). Right: Selectivity tests on MIP/NIP microstructures synthesized without solvent, comparing the binding of dansyl-l-Phe and dansyl-d-Phe (10 μm). Data are means of triplicate measurements, and error bars represent the standard deviation.


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of organic materials (electrical, optical, mechanical, chemical) allow to tailor them to any given application. This makes them particularly suitable for the elaboration of advanced organic MEMS sensors. However, their fabrication requires multi-step processes, reducing their potential for integration in industrial processes.[2] In this context, TPS can be seen as a key enabling technology for the development of next generations of organic MEMS sensors. In this work, arrays of 20 × 60 μm cantilever sensors (5 μm thick) including their clamped base were written in a single step on a glass support (Figure 4). They were excited to their resonance with a standard piezoelectric element glued onto the support, and the resonance frequency measured with a laser Doppler vibrometer, after elution of the template (binding sites free), with the analyte bound (binding sites occupied), and again after elution of the analyte. The frequency measure-ments have been performed after drying the MIP cantilever. The analyte concentration for incubation experiment was set to 100 μmol L−1. Binding of the target analyte induced a revers-ible shift of the resonance frequency of the sensors (Figure 4C and Figure S4, Supporting Information), with a relative varia-tion higher than 1%. This variation measured for our MEMS is at least one order of magnitude higher when compared to commercially available microgravimetric devices such as quartz

crystal microbalance.[50,51] This demonstrates that these MIP microcantilevers produced by microscale 3D writing can be used as label-free chemical sensors.

To conclude, a MIP formulation compatible with TPS was successfully developed. The choice of the appropriate photo-initiator and other MIP precursors allowed combining good molecular imprinting properties and high-resolution direct-writing in 3D. Polymerization conditions specific to the femto-second laser irradiation for TPS both preserves the imprinting effect and molecular recognition. Moreover, the possibility to generate high surface-to-volume ratio microstructures improved the global binding capacities with no significant impact on specificity. Using TPS for microstructuring MIPs opens many new possibilities for the integration of these smart functional materials into advanced applications and devices, for example, for chemical sensing in biomedical diagnostics or security areas. Finally, to highlight this great potential, proof of concept of advanced all-organic MIP MEMS has been dem-onstrated, paving the way to fabrication of low cost chemical sensors by the increasingly popular 3D printing for rapid pro-totyping, but at the micro- and even nanoscale. The work pre-sented in this paper is focused on chemical sensors prepared by TPS but could be easily extended to other fields.



Figure 3. A) High-resolution MIP microstructures generated by TPS, showing a width of 200 nm. B) Multiplexed NIP and MIP dots polymerized on the same sample. Optical and fluorescence microscopy images after binding of dansyl-l-Phe. C) Left: SEM images of woodpile type porous 3D microstruc-tures prepared with 40× and 100× microscope objectives. Right: Measurement of fluorescence intensity after incubation of MIP and NIP with dansyl-l-Phe.


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Experimental Section

Materials: All chemicals and solvents were of analytical grade and purchased from Sigma-Aldrich (St-Quentin Fallavier, France), unless otherwise stated. All monomers and crosslinkers were employed as received, except 4-VPY which was distilled before use. The photoinitiator Lucirin-TPO was from BASF (Ludwigshafen, Germany). All solvents used for polymerization were anhydrous. Water was purified using a Milli-Q system (Millipore, Molsheim, France).

Synthesis of Bulk MIPs: The polymer precursors solutions consisting of 0.05 mmol of template Z-L-Phe, 0.2 and 0.2 mmol of functional monomers MAA and 4-VPY, 2 mmol of cross-linker PETIA and 0.0192 mmol of photoinitiator Lucirin-TPO were mixed as such or dissolved in a solvent (729 μL of acetonitrile or 274 μL of triglyme) in a four milliliter glass vial fitted with an airtight septum, and the mixture was purged with nitrogen for 2 minutes. The amount of photoinitiator was set to 0.3 mol% relatively to the polymerizable double bonds. Polymerization was carried out with a daylight lamp equipped with a 18W standard tube. After overnight irradiation, the polymers were recovered by breaking the glass vials. The monoliths were manually crushed in a mortar and then transferred to microcentrifuge tubes, suspended in methanol and finely ground with 2.8 mm-diameter ceramic beads in a Precellys 24 homogenizer (Bertin Technologies, Montigny le Bretonneux, France). The polymers were then transferred to 50 mL centrifuge tubes and washed on a tube rotator (SB2, Stuart Scientific) with 3 rounds of methanol-acetic acid (9:1), and then rinsed three times with methanol. The polymer particles were finally dried overnight under vacuum and stored at 4 °C. A control non-imprinted polymer (NIP) was prepared using the same protocol except for the omission of the template.

Evaluation of Binding Properties of Bulk MIPs: The binding properties of the polymers were evaluated by equilibrium binding experiments in anhydrous acetonitrile (ACN), with fluorescence measurements on a Varian Cary Eclipse fluorescence spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). The excitation/emission wavelengths were set to 340/520 nm. A stock solution of dansyl-L-Phe (1 mM) was prepared in ACN and stored in the dark at −20 °C. For the assays, an aliquot of the stock solution was freshly diluted to 100 μM in ACN. The polymers were suspended by sonication in the incubation solvent. From this stock suspension, polymer concentrations ranging from 0 to 9 mg/ml were pipetted in 1.5 ml polypropylene microcentrifuge tubes

and 100 μL of dansyl-L-Phe solution was added. The final volume was adjusted to 1 ml with solvent. The tubes were incubated overnight in the dark at ambient temperature on a tube rotator. They were then centrifuged at 19,000g for 25 min, and the amount of free dansyl-L-Phe in the supernatant was quantified by fluorescence measurements. The amount of analyte bound to the polymers was calculated by subtracting the amount of free analyte from the initial amount of analyte added to the mixture.

Two-photon Polymerization of MIP/NIP Microstructures: For TPS fabrication, a femtosecond laser source (Chameleon Ultra II, 140 fs @ 800 nm) combined with a TeemPhotonics microfabrication 3D system was used. For 2D and 3D microstructures, the beam was introduced via a 40× objective (NA: 0.65), and exposure time and laser power were set to 10 ms and 15 mW (measured after the objective). For the structures in Figure 3C, a 100× oil-immersion objective (NA: 1.21) was used and exposure time and laser power were set to 10 ms and 10 mW. Typical TPS formulations contained 1% to 3%-w of Lucirin-TPO. After laser exposure, the sample was immersed in ethanol to remove the non-polymerized material, and washed with methanol-acetic acid (9:1) to remove the template. Typical writing times of these 3D microstructures prepared with ×40 and ×100 microscope objectives are less than 3 min and ∼10 min respectively.

Fluorescence Microscopy Imaging of Microstructures: The polymer 3D microstructures were incubated 1 hour with 10 μM dansyl-L-Phe or dansyl-D-Phe in ACN, rinsed with ACN. They were imaged by fluorescence microscopy using a Leica DMI6000 B microscope, equipped with 360 nm excitation/470 nm emission filter cubes. The fluorescence intensities were measured using the software ImageJ (public domain, http://rsb. info.nih.gov/ij/). Fluorescence spectroscopic measurements were performed on a Varian Cary Eclipse (Agilent Technologies, Palo Alto, CA, USA).

Scanning Electron Microscopy: Microstructures were characterized using a SEM from FEI, (Quanta 400) or a FEG ESEM from Philips (XL30).

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

Figure 4. A) Schematic view of the MIP cantilever fabricated by TPS with schematic representation of the resonance frequency variation before and after incubation (see the Supporting Information for resonance spectra). B) SEM images of two MIP cantilevers. C) Relative frequency shift after extraction, incubation in Z-L-Phe and second extraction. Error bars correspond to the average of four measurements performed on four distinct microcantilevers.


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AcknowledgementsThe authors acknowledge financial supports by Agence Nationale de la Recherche (ANR Nanoquenching, contract number 10PDOC-009 01, ANR PODCAST, contract number 12-JS03-0003, and ANR HOLOSENSE, contract number 08-BLAN-0236-1). K.H., Y.F., X.-A.T., F.B., and B.T.S.B. thank the European Regional Development Fund and the Regional Council of Picardie (co-funding of equipment under CPER 2007–2013, and project CBI-PEM), and the European Union (FP7 Marie Curie Actions, ITN SAMOSS, PITN-2013–607590) for financial support. The authors thank Stephan Knopf for running SEM experiments.

Received: January 13, 2016Revised: March 9, 2016

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