Molecularly Imprinted Polymer Arrays as Synthetic Protein Chips

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Molecularly Imprinted Polymer Arrays as Synthetic Protein Chips Prepared by Transcription-type Molecular Imprinting by Use of Protein-Immobilized Dots as Stamps Takahiro Kuwata, Akane Uchida, Eri Takano, Yukiya Kitayama, and Toshifumi Takeuchi* Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan S Supporting Information *

ABSTRACT: Molecularly imprinted polymer (MIP) arrays were demonstrated for the recognition of proteins. They were prepared via transcription-type molecular imprinting where patterned dots composed of biotinylated nanoparticles were first immobilized on a glass substrate followed by the immobilization of versatile biotinylated proteins via avidin−biotin interactions, yielding a multiple protein-immobilized stamp as a mold that could be transcribed. MIPs were prepared between the stamp and a methacrylated glass substrate, and after the stamp was peeled off, MIP dots were able to be prepared on the methacrylated glass substrate according to the positions of the immobilized proteins on the stamp. We confirmed that the prepared MIP array showed the expected selective binding toward the corresponding template proteins by conducting competitive binding assays using the fluorescently labeled proteins as corresponding competitors. The binding behaviors were consistent with those obtained by a surface plasmon resonance sensing system. We believe that the proposed platform involving the easily handled nanoparticlebased protein stamps for the preparation of MIP arrays can provide a new type of pattern recognition-based protein chip, which can be adopted as a substitute for the use of conventional protein arrays in various research and industrial fields in the life sciences.

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removed, yielding molecular recognition cavities complementary in shape and size to the template molecules; functional monomer residues are assembled within the cavities as binding sites suitable for specific binding. Recently, MIPs have been applied as synthetic molecular recognition elements for the recognition of specific proteins.26−43 Herein, we demonstrated MIP-based novel synthetic protein arrays prepared by transcription-type molecular imprinting using multiple protein-immobilized stamps as molds to obtain transcribed MIP arrays, where a biotinylated nanoparticle-based scaffold was used for immobilizing versatile biotinylated proteins via a unified avidin−biotin interaction protocol. Construction of the transcribed MIP array was carried out by copolymerizing a prepolymerization mixture containing a functional monomer, a comonomer, and a cross-linker between the multiple protein-immobilized stamp and a methacrylated glass substrate that was a transcribed MIP array. To demonstrate the feasibility of the proposed protocol for the preparation of transcribed MIP arrays using the universal nanoparticle-based avidin-immobilized scaffold for multiple protein-immobilized stamp preparation of a synthetic protein array, we prepared different MIP dot arrays via the proposed

he development of highly specific and sensitive protein recognition materials for use in proteomics, disease diagnosis, protein purification, and drug delivery is an important challenge.1−4 In particular, analytical chips with various immobilized antibodies, proteins, and other proteinbinding biomolecules have attracted much attention due to the desire for high-throughput screening of proteins, that is, a wide range of proteins before/after post-translational modifications can be analyzed rapidly and simultaneously, and has great potential for further development of proteomics, metabolomics, and other life science research.4−11 However, various types of antibodies have to be obtained, and highly accurate immobilization technology is necessary for construction of a diverse range of protein arrays.12 Synthetic molecular recognition elements prepared in a tailor-made fashion are desirable as substitutes of natural antibodies in protein arrays due to properties such as an easy and low cost of production, high stability, and capability for any chemical modification in order to introduce further functionalities. Molecularly imprinted polymers (MIPs) are synthetic polymer receptors with highly specific recognition ability for target molecules that are prepared by copolymerization of template molecules with cross-linking agents and functional monomers, which can be conjugated with template molecules covalently or noncovalently during polymerization.13−25 After polymerization is carried out, the template molecules are © XXXX American Chemical Society

Received: August 15, 2015 Accepted: October 22, 2015

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active biotin concentration B (moles per liter) was calculated by use of eq 1:

protocol on a glass substrate targeting various proteins, where cytochrome c, ribonuclease A, myoglobin, and lysozyme were used as model proteins, and we investigated their protein recognition abilities.

B = Z[10−2(0.9AbsA − AbsB)/34]

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(1)

For the preparation of fluorescein-labeled proteins (F-Cyt, FLys, F-Myo, and F-Rna), SureLINK fluorescein labeling kit was used, where concentrations of the conjugated proteins were estimated spectrophotometrically with wavelengths of 420 nm for Cyt and Myo and 280 nm for Lys and Rna. Preparation of Poly(styrene−methacrylic acid) Shell/ Polystyrene Core Nanoparticles (P(S−MAA)/PS). P(S− MAA)/PS were synthesized by two-step emulsifier-free emulsion polymerization. In the first step, styrene (800 mg, 7.69 mmol) was mixed with water (100 mL) containing HCl (40 μL) and V-50 (41.64 mg, 153 μmol), and then the polymerization was carried out under nitrogen atmosphere with 1500 rpm stirring at 80 °C for 24 h. Styrene (47.6 mg, 0.458 mmol) and MAA (92.4 mg, 1.07 mmol) were then added to the obtained PS emulsion (40 g) at room temperature for 24 h with 1500 rpm stirring. After addition of V-50 (41.64 mg, 153 μmol), the seeded polymerization was carried out under nitrogen atmosphere with 1500 rpm stirring at 80 °C for 24 h. The obtained nanoparticles were repetitively washed with methanol and water five times by centrifugation, and finally, P(S−MAA)/PS were dispersed in water. Immobilization of P(S−MAA)/PS on Aminated Glass Substrates. Glass substrates (18 × 26 mm) were treated with UV/O3 for 15 min and then immersed in a 1% APTES dissolved in ethanol/water (95/5 v/v) solution for 1 h. After being washed with ethanol/water solution, the substrates were baked for 1 h at 80 °C. The poly(dimethylsiloxane) (PDMS) plate (Figure 1) was placed on the aminated glass substrate to make 35 wells, and

EXPERIMENTAL SECTION Materials. Styrene, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), cytochrome c (Cyt), ribonuclease A (Rna), avidin (Avi), hydrochloric acid, sodium chloride, and potassium bromide were purchased from Nacalai Tesque Co. (Kyoto, Japan). Acrylic acid, methacrylic acid (MAA), acrylamide (AAm), N,N′methylenebis(acrylamide) (MBAA), 2,2′-azobis(2methylpropionamidine)dihydrochloride (V-50), 4-hydroxyazobenzene-2-carboxylic acid (HABA), acetic acid, methanol, ethanol, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride n-hydrate (DMT-MM), and sodium dodecyl sulfate (SDS) were purchased from Wako Co. Ltd. (Osaka, Japan). 3-Trimethoxysilylpropyl methacrylate was purchased from Tokyo Chemical Industries (Tokyo, Japan). N,N′Bis(acryloyl)cystamine, lysozyme (Lys), and myoglobin (Myo) were purchased from Aldrich (St. Louis, MO). Fluorescein isothiocyanate (FITC)-conjugated avidin (F-Avi) was purchased from Life Technologies Japan (Tokyo, Japan). SureLINK fluorescein labeling kit was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Biotin labeling kit-NH2 was purchased from Dojindo Molecular Technologies Inc. (Kumamoto, Japan). 3-Aminopropyltriethoxysilane (APTES) was purchased from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). (+)-Biotinyl-3,6,9-trioxaundecanediamine (amine-PEG3-biotin) was purchased from Thermo Fisher Scientific (Grand Island, NY). Characterization. Fourier transform infrared (FT-IR) measurements were carried out by KBr method on a Varian 660 KU-IR instrument (Agilent Inc., Santa Clara, CA). Particle sizes and morphologies were observed by VE-9800 scanning electron microscope (Keyence, Osaka, Japan). Particle size distribution was measured on a dynamic light scattering system, Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, U.K.). UV/vis spectral measurements were performed on a V-560 spectrophotometer (Jasco Ltd., Tokyo, Japan). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a Voyager DE-1000 instrument (AB Sciex, Tokyo, Japan). Circular dichroism (CD) spectra were measured on a J-725 instrument (Jasco Corp., Tokyo, Japan). Surface plasmon resonance (SPR) measurements were performed by Biacore 3000 (GE Healthcare Japan, Tokyo, Japan). A desktop programmable dispensing robot (System Instruments Co. Ltd., Tokyo, Japan) was used for small-volume liquid handling. Preparation of Conjugated Proteins. Biotinylated proteins (biotin-Cyt, biotin-Lys, biotin-Myo, and biotin-Rna) were prepared using biotin labeling kit-NH2. The number of active biotin molecules on the biotinylated proteins was estimated by the HABA method.44 HABA (7.0 mg) was dissolved in DMSO (250 μL), and a 100 μL aliquot was added to 10 mM HEPES buffer (pH 7.4) containing 5 mg of Avi (5 mL), and then the solution was diluted to 10 mL with 10 mM HEPES buffer (pH 7.4). Absorbance at 500 nm of the HABA− Avi mixed solution (900 μL) was measured (AbsA). The biotinylated protein solution (100 μL) was added to the HABA−Avi mixed solution (900 μL), and absorbance at 500 nm was measured after incubation for 10 min (AbsB). The

Figure 1. PDMS plate, length 26 mm × width 18 mm × height 5 mm, with 35 (7 × 5) holes (1.25 mm i.d.) therein.

P(S−MAA)/PS emulsion (3 μL, solid content 0.087 wt %) was dispensed in each well. After the emulsion was dried, 25 mM DMT-MM aqueous solution (3 μL) was dropped on and allowed to react for 3 h at room temperature. The substrate was washed in water with sonication for 10 min to remove the physically adsorbed particles. Formation of Immobilized Protein Dots as Multiple Protein-Immobilized Stamps for Transcription-type Molecular Imprinting. First, biotinylation of immobilized P(S−MAA)/PS in the wells was carried out. Amime-PEG3biotin (1.67 mg, 4 μmol) and DMT-MM (1.66 mg, 6 μmol) were dissolved in water (20 mL). The solution (3 μL) was dispensed in P(S−MAA)/PS-immobilized wells, and the wells were incubated for 1 h at room temperature, followed by washing with water. B

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device (CCD) camera (exposure time 0.03 s; binning 3 × 3; Andor Technology, Belfast, U.K.). Competitive Binding Experiments of Target Proteins in the Presence of Fluorescein-Labeled Proteins. HEPES buffer (10 mM, pH 7.4) containing 0−10 nM target protein, 25 nM fluorescein-labeled target protein, and 0−2% bovine serum albumin (BSA; 5 μL) were dispensed into MIP and NIP wells. After incubation for 2 h at room temperature, the wells were washed with HEPES buffer (5 μL) twice. Fluorescence intensity was measured by the same Olympus fluorescence microscope (exposure time 0.03, 0.1, or 0.5 s). Relative fluorescent change was calculated by eq 2, where F is fluorescent intensity derived from bound fluorescein-labeled target proteins in the presence of target proteins (0−10 nM) on the MIPs, F0 is that without the target proteins, and FNIP is that without the target protein on the NIPs.

The activity of biotin immobilized on P(S−MAA)/PS was examined by using F-Avi. HEPES buffer (10 mM, pH 7.4) containing 50 μM F-Avi solution (2 μL) was dispensed in biotinylated wells and unmodified wells on the same glass substrate, and the wells were incubated for 30 min at room temperature. The glass substrate was sequentially washed with HEPES buffer, 1% Tween-20 dissolved in HEPES buffer, and 500 mM NaCl. Fluorescent images were obtained on a SMZ 1500 stereomicroscope equipped with a B-2E/C fluorescent unit (Nikon Corp., Tokyo, Japan), and the intensity was measured using the imaging analysis software DIIP Image (Ditect Co., Tokyo, Japan). For immobilization of Avi, HEPES buffer containing 50 μM Avi (2 μL) was dispensed in biotinylated P(S−MAA)/PSimmobilized wells, and the wells were incubated for 30 min at room temperature. After being washed with HEPES buffer, the Avi-immobilized substrate was used as a scaffold for immobilization of the biotinylated template proteins by avidin−biotin interaction. Finally, HEPES buffer containing 10 μM biotinylated proteins (20 μL) was dispensed in Avi-immobilized wells, and the wells were incubated for 30 min at room temperature. After being washed with HEPES buffer, the PDMS plate was removed to yield stamps bearing patterned proteins dots as molds for transcription-type molecular imprinting. It should be noted that the stamps were disposable, due to the fact that proteins are fragile. Preparation of Molecularly Imprinted and Nonimprinted Polymer Arrays on Methacrylated Glass Substrates. For preparation of methacrylated glass substrates, UV/O3-cleaned glass substrates (18 × 26 mm) were immersed in 1% (v/v) 3-trimethoxysilylpropyl methacrylate aqueous solution containing 1% (v/v) acetic acid (5 mL) for 1 h at room temperature. After being washed with water, the substrates were baked for 1 h at 80 °C. As the prepolymerization mixture, 10 mM HEPES buffer (pH 7.4) solution (5 μL) containing acrylic acid (14.5 μM), acrylamide (115 μM), MBAA (150 μM), and V-50 (7.37 μM) was interleaved between the methacrylated glass substrate and the stamps bearing 30 protein dots for MIP arrays and five unmodified P(S−MAA)/PS-immobilized dots (nonimprinted polymers, NIP). After 30 min of incubation, polymerization was initiated by photoirradiation (ZUV-C10, Omron, Kyoto, Japan, wavelength: 365 nm, 15 min). Arrays molecularly imprinted with cytochrome c (Cyt-MIP), lysozyme (Lys-MIP), RNase A (Rna-MIP), and myoglobin (Myo-MIP) and nonimprinted polymer dots (NIP) were obtained at the positions corresponding to immobilized protein dots on the stamp, after the stamp was peeled off and the chip was washed with HEPES buffer. Binding Activity of Cytochrome c Molecularly Imprinted Polymer toward Fluorescein-Labeled Cytochrome c. The same 35-hole PDMS plate used in preparation of the stamp was placed on the MIP array chip, where the holes were aligned on the MIP dots to make wells bearing MIP layers on the bottom. F-Cyt dissolved in 10 mM HEPES buffer (pH 7.4) (0−100 nM, 5 μL) was dispensed into the Cyt-MIP wells. After incubation for 2 h at room temperature, the wells were washed with HEPES buffer (5 μL) twice. Fluorescent intensity of MIP dots was measured with a IX73 fluorescent microscope (×40, extinction wavelength 460−495 nm, fluorescence wavelength > 510 nm) with U-HGLGPS light source (Olympus, Tokyo, Japan) and Zyla SCMOS charge-coupled

(F0 − F )/(F0 − FNIP)

(2)

Apparent affinity constants of proteins toward the MIPs were estimated by using the curve-fitting software DeltaGraph 5.4.5v. The fitting equation (eq 3) is generally used for determination of binding constants of 1:1 complex formation. ⎡ Y = ⎣(1 + KH + KG) − ⎛ D ⎞ ⎜ ⎟ ⎝ 2KH ⎠

⎤ (1 + KH + KG) − 4K 2HG ⎦ (3)

where Y = (F0 − F)/(F0 − FNIP), K is the apparent affinity constant, H is the number of binding cavities on the MIPs, G is Cyt concentration, and D is the maximum (F0 − FNIP). The value of H was found by fitting a theoretical curve to the raw data that would result in the smallest deviation error. Selectivity Tests. Samples containing 10 nM of Cyt, Lys, Avi, Myo, or Rna with 25 nM F-Cyt (10 mM HEPES buffer, pH 7.4, 5 μL) were each dispensed into a Cyt-MIP well. After incubation for 2 h, the wells were washed twice with 10 mM HEPES buffer, pH 7.4 (5 μL). Fluorescent intensity at each well was measured by the same Olympus fluorescence microscope (exposure time 0.5 s, binning 3 × 3). All of the binding experiments were carried out in 2% BSA. For Lys-MIP, Myo-MIP, and MIP-Rna, the corresponding fluorescein-labeled proteins (F-Lys, F-Myo, and F-Rna) were used instead of FCyt. Cytochrome c Molecularly Imprinted and Nonimprinted Polymer Arrays on Gold-Coated Glass Substrates for Surface Plasmon Resonance Measurements. Cyt was homogeneously immobilized on the Aviimmobilized glass substrate without the use of the 35-hole PDMS plate, and this was used as the Cyt stamp for preparing Cyt-MIP on the SPR sensor chips. Gold-coated glass substrates were immersed in ethanol (5 mL) containing N,N′-bis(acryloyl)cystamine (3.6 mg, 23.4 μmol) and incubated for 30 min at room temperature. After being washed with ethanol, methacrylated SPR chips were obtained. For preparation of Cyt-MIP, 10 mM HEPES buffer, pH 7.4 (5 μL), containing acrylic acid (14.5 μM), acrylamide (115 μM), MBAA (150 μM), and V-50 (7.37 μM) was interleaved between the methacrylated SPR chip and the Cyt stamp. After 30 min of incubation, polymerization was carried out by photoirradiation (365 nm) for 15 min. NIP was also prepared by the same protocol with P(S−MAA)/PS-immobilized glass substrate instead of Cyt stamp. C

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Scheme 1. (a) Fabrication of Patterned Multiple Protein-Immobilized Dot Stamps for Transcribed MIPs and (b) Protocol for Preparation of MIP Arraya

a

MIP array was prepared on methacrylated glass substrate by transcription-type molecular imprinting by use of the multiple protein-immobilized stamp.

Figure 2. SEM images of P(S−MAA)/PS immobilized well at (A) ×50 and (B) ×10000.

isotherms were drawn by use of ΔRU values for each protein concentration.

Surface Plasmon Resonance Measurements. Cyt, Lys, Rna, Avi, and Myo (0−0.5 μM) were each dissolved in separate 10 mM HEPES buffer (pH 7.4) sample solutions, and 20 μL aliquots were injected with a flow rate of 20 μL/min. Regeneration was performed at each end of the measurement by injecting 200 mM NaCl aqueous solution and 0.3 wt % SDS aqueous solution. The amounts of bound proteins were estimated from signal intensity (RU) at 150 s after injection, where 1 RU was reported to be 1 pg/mm2 bound protein.45 All binding experiments were repeated three times. Binding

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RESULTS AND DISCUSSION

Preparation of P(S−MAA)/PS as a Scaffold for Protein Immobilization. P(S−MAA)/PS was used to form the scaffold of multiple protein-immobilized stamps, as they are easily dispensed at the desired positions by use of a programmable dispensing robot. Such a nanoparticle-based scaffold has never been used for the stamp to prepare D

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Figure 3. (a) Binding behavior of F-Cyt toward Cyt-MIP array. (b) Microscopic images of F-Cyt adsorbed on MIP array.

transcribed MIPs before. P(S−MAA)/PS particles were synthesized by two-step emulsifier-free emulsion polymerization: that is, emulsifier-free emulsion polymerization of styrene (first step) and emulsifier-free seeded emulsion polymerization of styrene and MAA in the presence of PS seed particles (second step) were performed. The size of the obtained particles (240.3 nm) was larger than that of the PS seed particles (234.5 nm) (Figure S1). In FT-IR spectra, new peaks at 3000−3500 and 1700 cm−1 were observed for P(S− MAA)/PS measurements, confirming that the obtained particles possess carboxy groups (Figure S2). Preparation of Multiple Protein-Immobilized Stamps. A 35-hole PDMS plate was placed on the aminated glass substrate, and the P(S−MAA)/PS emulsion (solid content 0.087 wt %) was dispensed into each well. After the emulsion was dried, the coupling reaction between amino groups on the bottom of the wells and carboxy groups on P(S−MAA)/PS was carried out (step A in Scheme 1a). Immobilization of P(S− MAA)/PS on the glass substrate was confirmed by scanning electron microscopy (SEM) after washing with sonication to remove the physically adsorbed P(S−MAA)/PS (Figure 2). In step B (Scheme 1a), the immobilized P(S−MAA)/PS on the bottom of each well was biotinylated. Introduction of biotin on P(S−MAA)/PS was confirmed by adding F-Avi; fluorescence derived from F-Avi was observed in each well (Figure S3). Then avidin (Avi) was dropped into each well (step C, Scheme 1a) to form Avi-immobilized wells, which are ready for use as scaffolds to immobilize versatile biotinylated proteins (step D, Scheme 1a), yielding multiple protein-immobilized stamps. Preparation of Transcribed Cytochrome c Molecularly Imprinted Polymer Array. For preparation of the Cyt stamp, biotin-Cyt was prepared. When biotin-Cyt was subjected to MALDI-TOF MS, m/z values of 1−6 biotinylated Cyt were observed (Figure S4). The HABA method was also used to estimate the amount of biotin introduced (Figure S5), and approximately 2 active biotin molecules existed per Cyt molecule. Furthermore, we measured CD of biotin-Cyt, and

the secondary structure of Cyt was confirmed to be maintained after the biotinylation (Figure S6). Prepared biotin-Cyt was then dispensed into each Aviimmobilized well, and then the PDMS plate was peeled off to obtain the Cyt stamp. To prepare a transcription-type Cyt-MIP array, radical polymerization of acrylic acid (functional monomer), acrylamide (comonomer), and N,N′-methylenebis(acrylamide) (cross-linker) was carried out with V-50 initiator in 10 mM HEPES buffer (pH 7.4) between the Cyt stamp and methacrylated glass substrate by photoirradiation (Scheme 1b). After removal of the Cyt stamp, the Cyt-MIP array was obtained. In this study, MAA was used as the functional monomer, since many MIPs for various proteins have been successfully prepared with this monomer.30−34 Fluorometric Competitive Binding Assay for Cyt. Binding experiments were performed by use of a fluorescence-based competitive binding assay in the presence of a fixed amount of fluorescein-labeled Cyt (F-Cyt), where the number of fluorescein moieties on F-Cyt was estimated to be 2−4 by MALDI-TOF MS (Figure S7), and a similar secondary structure was maintained after the fluorescein labeling, which was confirmed by CD spectra (Figure S8). The PDMS plate was again placed on the Cyt-MIP array, where the holes of the PDMS plate were adjusted to the dots of the MIPs. In competitive binding experiments, the F-Cyt concentration is an important factor. Therefore, we first determined the appropriate concentration of F-Cyt for competitive binding. Various concentrations (0−100 nM) of F-Cyt were incubated; then, the fluorescent intensity was measured using the fluorescent microscope (Figure 3). When 6.25 nM F-Cyt was added, a slight fluorescent change was observed, and it was shown to be a roughly linear response. Therefore, the following binding experiments were conducted with a minimum detectable concentration of 25 nM F-Cyt. To perform the competitive binding experiments, the binding affinity of Cyt toward the MIP array needed to be higher than that of F-Cyt. To investigate the binding abilities of Cyt and F-Cyt toward Cyt-MIP, binding experiments were E

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Analytical Chemistry performed by use of an SPR sensor with Cyt-MIP prepared on the sensor chips, because the SPR response is quite consistent with the binding amount. A higher response was observed for Cyt than F-Cyt at all concentrations tested (Figure S9), confirming that Cyt has stronger binding properties than F-Cyt; that is, F-Cyt can be used as a competitor in the competitive binding assay. Competitive binding experiments of Cyt in the presence of 25 nM F-Cyt were performed at various concentrations of Cyt (0−10 nM). The fluorescent intensity on the MIPs decreased when bound F-Cyt was replaced by free Cyt (Figure 4), and the apparent affinity constant was estimated to be 5.3 × 108 M−1 (Figure S10), revealing that a competitive binding assay was successfully achieved.

Figure 5. Binding behavior of Cyt toward Cyt-MIP (red) and NIP (black) prepared on SPR sensor chips.

fluorescent intensity change among the proteins tested, although nonspecific binding of other proteins was also observed, revealing that specific binding cavities toward Cyt were formed (Figure 6). Additionally, positively charged Lys

Figure 4. Competitive binding behavior of Cyt toward Cyt-MIP array in the presence of 25 nM F-Cyt, with (red) and without (black) 2% BSA. Figure 6. Selectivity test of Cyt-MIP array via a competitive binding assay with F-Cyt.

To improve sensitivity, we investigated the suppression of nonspecific binding of F-Cyt for the PDMS plate and/or MIPs in each well by using BSA as a blocking agent. We first investigated whether the presence of BSA affects the fluorescent intensity of F-Cyt. In the presence of 2% BSA, the fluorescent change of F-Cyt on Cyt-MIP in each well, which was caused by competitive binding of Cyt (0−10 nM), was more clearly observed than in the absence of BSA (Figure 4), and the effect was found to be concentration-dependent (Figure S11). The highest fluorescent intensity change was observed at 2% BSA concentration. Therefore, we attempted to demonstrate the highly sensitive detection of Cyt in the presence of 2% BSA in 10 mM HEPES buffer (pH 7.4). From the binding data (Figure 4), the apparent affinity constant was estimated to be 7.1 × 109 M−1 (Figure S10), which is higher than that without the addition of BSA (5.3 × 108 M−1). To verify the imprinting effect on Cyt-MIP, SPR measurements were carried out for the binding of Cyt and other proteins toward Cyt-MIP and NIP prepared on the gold-coated glass substrate, as the obtained SPR response is directly related to the binding of Cyt on Cyt-MIP. NIP was prepared with a P(S−MAA)/PS-immobilized glass substrate instead of the Cytimmobilized stamp. As shown in Figure 5, Cy-MIP had a higher response than NIP, meaning that enhancement of binding activity was acquired during the imprinting process and that the imprinting effect was observed. The selectivity of the Cyt-MIP array was investigated by using Lys (14 kDa, pI 11.1), Avi (66 kDa, pI 10), Rna (14 kDa, pI 9.5), and Myo (17 kDa, pI 7). Cyt gave the highest

although Avi and Myo also interacted slightly with the MIP under the conditions employed. The former possibly interacted nonspecifically with acrylic acid moieties and the latter is thought to have interacted hydrophobically with the MIP due to the minimum charge at pH 7.4, which is close to the pI of Myo. Rna enhanced the fluorescent intensity of F-Cyt, leading to interference in the competitive binding assay. Although a clear reason was not found, this result may be due to interaction between the fluorescein moiety and the positively charged cleft on Rna. To confirm that the fluorescence change in the selectivity test was caused by competitive binding on Cyt-MIP, SPR measurements were again carried out for the binding of Cyt and other proteins (3.12 nM) toward Cyt-MIP prepared on gold-coated glass substrate (Figure S12). The binding amount of Cyt toward the MIP film was the highest compared to the other proteins, which is consistent with the competitive binding data. Targeting Other Proteins. The key technology of this method is multiple protein-immobilized stamps, enabling easy production of a MIP array on which MIP dots can be prepared for different target proteins. To demonstrate such a MIP array, we prepared four different MIP dotsRna-MIP, Lys-MIP, Myo-MIP, and Cyt-MIPwhere multiple protein-immobilized stamps were prepared by using biotin-Lys, biotin-Rna, biotinMyo, and biotin-Cyt. Biotinylation of these proteins was confirmed by the increase in molar mass measured by MALDIF

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Figure 7. Selectivity tests of Rna-MIP, Lys-MIP, and Myo-MIP arrays by competitive binding assays with F-Rna, F-Lys, and F-Myo, respectively.

to protein analysis as a highly stable, low cost, and tailor-made diagnostic tool.

TOF MS (Figure S13), where 1−2 biotin(s) were conjugated. Preservation of the secondary structures was checked by CD spectra (Figure S14). Competitive binding experiments were also performed in the same manner as in the case of Cyt-MIP, where F-Lys, F-Rna, and F-Myo were also prepared and confirmed by MALDI-TOF MS, where 2−5 fluoresceins were conjugated (Figure S15). Again Cyt, Lys, Avi, Myo, and Rna were employed as testing samples. As seen, Rna-MIP, Lys-MIP, and Myo-MIP showed the highest fluorescence change for the corresponding template proteins and their binding patterns were different, indicating the successful syntheses of different MIPs bearing specific imprinted cavities toward target proteins on the same substrate (Figure 7). Among the MIPs prepared, Myo-MIP showed more selective binding behavior than the others. This may be due to the pI value of Myo, which is around pH 7. Under the Myo imprinting conditions, in addition to electrostatic interactions, hydrophobic interactions may be more favorable than in the cases of Rna and Lys, where Myo/MAA and/or Myo/MBAA complexes are stable, resulting in the formation of highly Myoselective imprinted cavities. In previously reported proteinimprinted polymer arrays prepared by bulk polymerization,36 both acidic and basic proteins could be imprinted by use of a cationic functional monomer, 2-(dimethylamino)ethyl methacrylate (DMA), as well as MAA, and the binding patterns for various proteins were different between MAA- and DMA-based MIPs. Therefore, when functional monomers other than MAA are used, a diverse range of MIP arrays is expected to be obtained. These results strongly suggest that the preparation strategy of MIP arrays using transcription-type molecular imprinting with multiple protein-immobilized stamps is a readily feasible method for preparation of synthetic protein microarrays versatile toward different proteins.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03134. 15 figures showing FT-IR spectra of nanoparticles, fluorescence image of F-Avi immobilized well, MALDITOF-MS and CD spectra of native and conjugated proteins, absorbance of HABA-Avi, estimation of affinity constants by curve fitting, effect of BSA on fluorescence intensity in Cyt-MIP, and binding experiments toward Cyt-MIP (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Tooru Ooya (Kobe University) for his kind suggestion. This work was partially supported by JSPS Kakenhi Grants 24651261 and 15K14943. We also appreciate System Instruments (Tokyo, Japan) for their technical assistance and financial support.

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REFERENCES

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CONCLUSIONS We successfully demonstrated the preparation of MIP-based protein microarrays for detection of target proteins by transcription-type molecular imprinting, where the template proteins were biotinylated and immobilized on a glass substrate bearing the patterned immobilization of biotinylated nanoparticles by avidin. The strategy involved an easy and common process for obtaining stamps possessing dots of a wide range of target proteins, followed by a molecular imprinting process that was carried out between the stamp and the methacrylated glass substrate, resulting in transcribed protein molds for the corresponding positions of the immobilized proteins. We believe that this synthetic protein microarray could be applied G

DOI: 10.1021/acs.analchem.5b03134 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.5b03134 Anal. Chem. XXXX, XXX, XXX−XXX