Orientationally Fabricated Zwitterionic Molecularly Imprinted

Graduate School of Engineering, Kobe University, 1-1, Rokkodai-cho, Nada-ku, ... 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 6...
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Orientationally Fabricated Zwitterionic Molecularly Imprinted Nanocavities for Highly Sensitive Glycoprotein Recognition Tetsuro Saeki, Hirobumi Sunayama, Yukiya Kitayama, and Toshifumi Takeuchi Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01215 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Orientationally Fabricated Zwitterionic Molecularly Imprinted Nanocavities for Highly Sensitive Glycoprotein Recognition

Tetsuro Saeki, Hirobumi Sunayama†, Yukiya Kitayama and Toshifumi Takeuchi*

Graduate School of Engineering, Kobe University, 1-1, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan

*corresponding author: Toshifumi Takeuchi E-mail: [email protected]

Key words: Molecular imprinting; Glycoproteins; Oriented template immobilization; allergens Controlled/living radical polymerization.

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ABSTRACT Glycoprotein recognition has recently gained a lot of attention, since glycoproteins play important roles in a diverse range of biological processes. Robustly synthesized glycoprotein receptors, such as molecularly imprinted polymers (MIPs), which can be easily and sustainably handled, are highly attractive as antibody-substitutes because of the difficulty in obtaining high-affinity antibodies specific for carbohydrate-containing antigens. Herein, molecularly imprinted-nanocavities for glycoproteins have been fabricated via a bottom-up molecular imprinting approach using surface-initiated atom transfer radical polymerization (SI-ATRP). As a model glycoprotein, ovalbumin was immobilized in a specific orientation onto a surface plasmon resonance sensor chip by forming a conventional cyclic diester between boronic acid and cis-diol. Biocompatible polymer matrices were formed around the template molecule, ovalbumin, using SI-ATRP via a hydrophilic co-monomer, 2-methacryloyloxyethyl phosphorylcholine, in the presence of pyrrolidyl acrylate (PyA), a functional monomer capable of electrostatically interacting with ovalbumin. The removal of ovalbumin left MIPs with binding cavities containing boronic acid and PyA residues located at suitable positions for specifically binding ovalbumin. Careful analysis revealed that strict control over the polymer significantly improved sensitivity and selectivity for ovalbumin recognition, with a limit of detection of 6.41 ng/mL. Successful detection of ovalbumin in an egg white matrix was demonstrated to confirm the practical utility of this approach. Thus, this strategy of using a polymer-based recognition of a 2 ACS Paragon Plus Environment

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glycoprotein through molecularly imprinted nanocavities precisely prepared using a bottom-up approach, provides a potentially powerful approach for detection of other glycoproteins.

Introduction Glycoproteins, which are glycan-conjugated proteins, account for more than 50% of the proteins produced in a human body. They play important roles in biological processes, including the immune response. 1-2 Detection of specific glycoproteins is of great importance in the fields of food production, proteomics, glycomics, and diagnostics. One of the widely used methods for the detection of glycoproteins is mass spectrometry. 3-5 However, this method requires sample pretreatment to remove impurities, thus involving a complex procedure for successful analyte detection. 6-7 Antibodies are also used for glycoprotein detection.

8

Glycoprotein-directed antibodies are generally prepared by

immunization of animals with appropriate glycoprotein antigens. However, it is difficult to obtain specific antibodies for glycoproteins with distinct glycans using this approach because of their non-antigenic property.

9

Thus, synthetic receptors that are capable of selective and sensitive

recognition of specific glycoproteins have gained attention as potential substitutes for antibodies. Boronic acid-based synthetic receptors have been studied as potential recognition elements for glycoproteins because boronic acid can form a cyclic ester linkage with the carbohydrate cis-diol group under basic conditions. 10-13 However, the stability of the cyclic diester formed from boronic acid and 3 ACS Paragon Plus Environment

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cis-diol is low under physiological conditions. Therefore, highly selective recognition of specific glycoproteins using the conventional boronic chemistry may not be easy due to the presence of other competing molecules containing cis-diols. Molecularly imprinted polymers (MIPs) have been recognized as one of the most attractive approaches for preparing antibody-free synthetic molecular recognition materials.

14-24

MIPs were

synthesized using a template-based polymerization process, where template molecules were assembled from functional monomer(s) and co-polymerized with a mixture of a co-monomer(s) and a crosslinker to form a crosslinked polymer matrix, thereby fixing the three dimensional positions of the template interaction sites. Removal of the template molecule yields nanocavities that are capable of specific recognition of the target molecule. Molecular imprinting provides a high level of tailored construction capability for a wide variety of target molecules 25-35, and recently, MIPs for proteins have been tested as substitutes for biomacromolecules. In this study, glycoprotein-directed, high affinity MIP thin layers were designed and synthesized from the combination of precise bottom-up molecular imprinting via surface-initiated atom transfer radical polymerization using an activator generated by electron transfer (SI-AGET ATRP) 36-39 and a model glycoprotein immobilized in a specific orientation via boronic ester linkage on a substrate. Ovalbumin (OVA), which is a potentially allergenic glycoprotein representing more than 50% of egg white proteins, was used as a model. Food allergens are important detection targets because of the 4 ACS Paragon Plus Environment

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significantly increased numbers of infants and children with food allergies in developed countries. 39 Therefore, sensitive detection of OVA levels is important for food safety in children. 40-41

Figure 1. Scheme detailing the preparation of MIP thin layers for OVA detection.

MIP thin layers were prepared on surface plasmon resonance (SPR) sensor chips, where fluorinated benzeneboronic acid groups with a lowered pKa value of around pH 7 42 were grafted on the chips via formation of a self-assembled monolayer (SAM), which plays a role both in oriented immobilization of the template and interaction within the imprinted cavities of the MIP thin layer. In addition, a polymerizable cyclic secondary amine, pyrrolidyl acrylate (PyA)

29

, was employed as a functional

monomer, to allow electrostatic interaction with OVA in the imprinted nanocavity (Fig. 1). Such 5 ACS Paragon Plus Environment

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orthogonal multiple interaction sites such as benzeneboronic acid and PyA groups can provide precise recognition of the target molecule with high affinity and selectivity. The strict control of MIP thickness by controlled/living radical polymerization (CLRP) resulted in highly sensitive and selective detection of OVA using a SPR sensor equipped with the MIP thin layers as molecular recognition elements. Furthermore, the system detected OVA in real egg white samples, revealing that this strategy can realize antibody-free sensing systems for glycoproteins with the use of abiotic recognition materials in biological systems.

Experimental section Materials Ethanol

(EtOH),

4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium

chloride

n-hydrate (DMT-MM), sodium dihydrogenphosphate, disodium hydrogenphosphate, sodium dodecyl sulfate (SDS), lysozyme, human serum albumin (HSA) and glycine were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). 1-Amino-(ethylene glycol)6-undecanethiol hydrochloride was purchased

from

Dojindo

Laboratories

N-cyclohexyl-2-aminoethanesulfonic acid,

(Kumamoto,

2,2'-bipyridyl,

Japan).

Sodium

CuBr2, L(+)-ascorbic acid,

chloride, avidin,

conalbumin, guanidine hydrochloride and hydrochloric acid (HCl) were purchased from Nacalai Tesque Co. (Kyoto, Japan). Bis[2-(2-bromoisobutyryloxy) undecyl] disulfide and OVA were purchased from 6 ACS Paragon Plus Environment

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Sigma-Aldrich (MO, USA). 4-Carboxy-3-fluorophenylboronic acid (CFPBA) was purchased from Alfa Aesar (MA, USA). 2-Methacryloyloxyethyl phosphorylcholine (MPC) was purchased from NOF Corporation (Tokyo, Japan). Chicken egg was purchased from Kinoshita (Hyogo, Japan). All water used was obtained from a Millipore Milli-Q purification system. Au-coated SPR sensor chips (superficial area: 120 mm2) were purchased from GE Healthcare Japan (Tokyo, Japan). The Micro BCA Protein Assay Kit was purchased from Thermo Scientific (MA, USA). Bovine serum albumin (BSA) included in the Micro BCA Protein Assay Kit was used for as a standard. PyA was prepared by a procedure reported previously. 29 Preparation of the mixed self-assembled monolayer (mixed SAM) Au-coated SPR sensor chips were rinsed with EtOH. The washed chips were immersed in an ethanolic solution of 0.5 mM bis[2-(2-bromoisobutyryloxy)-undecyl] disulfide and 0.5 mM amino-EG6-undecane thiol hydrochloride for 24 h at 25°C. Afterwards, the mixed SAM-SPR sensor chip was washed with EtOH and dried with nitrogen gas. Subsequently, the mixed SAM-SPR sensor chip was immersed in EtOH containing 1 mM CFPBA and 10 mM DMT-MM for 24 h at 25°C to allow CFPBA immobilization on the surface of the mixed SAM. Afterwards, the chips were washed with EtOH and dried with nitrogen gas. Preparation of MIPs, NIPs by SI-AGET ATRP The typical procedure used for the preparation of MIPs by SI-AGET ATRP was as follows. 7 ACS Paragon Plus Environment

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PyA (2 mM) as a functional monomer, MPC (48 mM) as a co-monomer, CuBr2 (1 mM) as a catalyst, and 2,2-bipyridyl (2 mM) as a ligand were dissolved in 8.25 mL of 20 mM PBS (pH 7.4) (NaCl = 150 mM) as a pre-polymerization solution. SPR sensor chips were fixed in a Teflon cell to expose only the side with the mixed SAM to the solution. The sensor chip and pre-polymerization solution were placed in a 100 mL Schlenk flask, and the solution was degassed by freeze-pump-thaw cycles. Then, 0.25 mL of degassed 20 mM PBS (pH 7.4) (NaCl = 150 mM) containing OVA (final concentration: 100 µg/mL, 2.22 µM) and 0.5 mL degassed 20 mM PBS (pH 7.4) (NaCl = 150 mM) containing ascorbic acid (final concentration 0.5 mM) were added via a syringe to this solution. The solution was degassed and polymerization was carried out in a water bath at 40°C for 60 min (MIP-60) or 45 min (MIP-45). After polymerization, sensor chips were washed with 20 mM PBS (pH 7.4) (NaCl = 150 mM) and pure water. The template protein was removed using a 0.5 wt% SDS aqueous solution or 3 M guanidine chloride in 10 mM glycine-HCl (pH 2.5) using the SPR detector (Biacore 3000, GE Healthcare Japan, Tokyo, Japan) to monitor loss of surface mass until the RU value became constant. Non-imprinted polymer (NIP-60) were also prepared using the same procedure as MIPs without the addition of OVA as a template molecule during the polymerization step. Polymer thickness measurements of MIPs by XRR Various MIPs were prepared using different polymerization times (0 h, 0.5 h, 1.0 h, and 1.5 h). MIPs were prepared on a gold-coated glass substrate (2000-0050, JASCO Ltd., Tokyo, Japan). 8 ACS Paragon Plus Environment

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Polymeric layer thickness was measured by X-ray reflectivity (XRR) (SmartLab 3 kW, Rigaku, Tokyo, Japan). The XRR conditions were as follows: source: CuKα1 radiation, λ= 0.154 nm; measured area: 1.0 cm2; angle range (2θ): 0.0–6.0°. The obtained oscillation patterns of X-ray reflective profiles were analyzed by X-ray reflectivity analysis software GXRR with Parratt theory and parameters related to the thickness, density, roughness of the MIPs was calculated. Protein binding experiments by SPR RU values were monitored using 20 mM PBS (pH 7.4) (NaCl = 150 mM) as a running buffer until the values were stabilized. In the OVA binding experiments using the boronic acid immobilized SPR sensor chip, OVA (final concentrations: 0, 1, 5, 10, 50, 100 µg/mL) was dissolved in 50 mM PBS (final pH values of 6.0, 7.4, or 8.0) (NaCl = 150 mM) or 0.1 M CHES buffer (final pH of 9.0) (NaCl = 150 mM). In the binding experiments using MIP-60 or NIP-60 modified SPR sensor chips, the injected concentrations of OVA, avidin, lysozyme, conalbumin, or HSA were 0, 1, 5, 10, 50, 100 µg/mL. In the case of MIP-45, the injected concentrations of OVA were 0, 0.01, 0.05, 0.1, 0.25, 0.5, and 1 µg/mL. In binding experiments, all proteins were dissolved in 20 mM PBS (pH 7.4) (NaCl = 150 mM). The flow rate was 20 µL/min, the injection volume was 20 µL, the injection interval was 5.5 min, and the data collection point was 5 min after injection. Glycine-HCl (10 mM, pH 2.5) containing SDS (0.5 wt%) or guanidine chloride were selected as regeneration solutions (100 µL) to regenerate the substrates. The amount of bound protein was calculated from the signal intensity (resonance units, RU; 1 RU 9 ACS Paragon Plus Environment

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corresponds to about 1 pg/mm2 of bound protein at 150 s after protein injection). 43 Apparent limit of detection for OVA were estimated from the binding isotherm using 3SD/m (m: slope of the linear part of the binding isotherm, SD: standard deviation for a value of 0 µg/mL OVA). In order to investigate the selectivity of prepared polymeric thin layers (MIP-60, MIP-45 and NIP-60), avidin, lysozyme, conalbumin, and HSA (10 µg/mL each) were used for binding experiments as reference proteins. Selectivity factor was calculated from following equation:

  =

  

Calculation of binding constants by Scatchard plot To estimate the binding constants, binding isotherms of OVA toward MIPs were analyzed using Scatchard plots. The Scatchard plot had two assumptions: 1) ligands and analytes are bound in a one-to-one ratio by applying the Langmuir equation; 2) compared to the amount of non-bound (free) analyte, the amount of bound analyte is very small. The formula of the Scatchard plot is as follows, where B is the number of moles of OVA bound to the imprinted cavities per mm2 area, F is the molar concentration of free OVA, Ka is the binding constant. B/F = Ka(Bmax - B) B/F (µL/mm2) = B (pmol/mm2) / F (µM) 10 ACS Paragon Plus Environment

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B (pmol/mm2) = ∆RU (pg/mm2) / Mw (g/mol)

Preparation of egg white matrix The egg white matrix was prepared as follows. The chicken egg white was separated carefully without disturbing the egg yolk, and it was then diluted 50-fold with 20 mM PBS (pH 7.4) (NaCl = 150 mM) in a polypropylene tube. The tube was then placed in ultrasound sonication bath for 30 min at room temperature. The diluted egg white matrix solution was filtered three times using a 0.2 µm membrane filter. Measurements of total protein concentration in the prepared egg white matrix The egg white matrix was further diluted 5000-fold with 20 mM PBS (pH 7.4) (NaCl = 150 mM). The total protein concentration in the egg white matrix was measured with a micro BCA Protein Assay Kit according to the manufacturer’s instructions. The sample’s absorbance at 562 nm was measured by UV-Vis (V-560, JASCO Ltd., Tokyo, Japan). BSA was used for preparation of the standard curve. Addition-recovery test The egg white matrix solution was further diluted 5000-fold with 20 mM PBS (pH 7.4) (NaCl = 150 mM). Various concentrations of purified OVA (final concentrations 0, 0.22, 0.44, 1.1, 2.2 nM) were added to the matrix solution, and the SPR binding experiment was performed using a MIP-45 thin 11 ACS Paragon Plus Environment

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layer. The measurement conditions were as described above in “Protein binding experiments by SPR” section. The recovery rates were estimated using the following equation:

The recovery rate (%* =

ΔRU (egg white sample* × 100 ΔRU (buffer*

∆RU (egg white sample) was calculated by subtracting ∆RU at each OVA concentration from ∆RU at 0 nM to eliminate the influence of residual OVA present in the egg white sample.

Results and discussion Molecular imprinting using immobilized glycoproteins has been previously reported, and the effectiveness of the immobilized glycoproteins via formation of a cyclic diester between boronic acid and

glycans

on

glycoproteins

using

randomly

copolymerized

MIP

matrices

from

N-isopropylacrylamide and acrylamide with N,N’-methylenebisacrylamide 44, self-copolymerization of aniline

45

, and polycondensation of tetraethyl orthosilicate has been demonstrated.46 Since randomly

co-polymerized MIPs are reported to be problematic, less sensitive, and less selective than precisely prepared MIPs using CLRP

31-47

, herein, one of CLRP techniques, SI-AGET ATRP, was employed

using a bottom-up approach to precisely fabricate an MIP thin layer on the gold-coated SPR sensor chip, co-immobilized with an ATRP initiator, 2-bromoisobutyryl group and CFPBA, on which OVA 12 ACS Paragon Plus Environment

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was captured in a specific orientation via its glycans. CFPBA was selected for capturing glycoproteins, because CFPBA possesses a pKa value of 7.2

42

unlike the values of around 9 for common

benzeneboronic acids, making it suitable for using under physiological conditions. The mixed SAM was formed on the sensor chip using 1-amino-(ethylene glycol)6-undecane thiol and bis[2-(2-bromoisobutyryloxy) undecyl] disulfide, which introduced 2-bromoisobutyryl and amino groups on the chip. This was followed by conjugating CFPBA with the amino groups on the chip, yielding a surface co-immobilized with the ATRP initiator and CFPBA. X-ray photoelectron spectroscopy (XPS) measurements before the coupling reaction of CFPBA revealed the presence of a new N1s peak following formation of mixed SAM (Fig. S1). After the coupling reaction of CFPBA, a new XPS peak derived from the B1s orbital was observed, confirming its immobilization on the sensor chip. To verify that the template glycoprotein, OVA (44.3 kDa, pI: 4.5), which was the specifically oriented immobilized template for the MIP preparation, could be captured on the CFPBA-immobilized SPR sensor chip around a neutral pH, binding tests were conducted using four buffered solutions with different pH values (6.0, 7.4, 8.0, and 9.0) (Fig. 2). The binding behavior was similar at pH 7.4 and 8.0, and the estimated association constant (Ka) was calculated to be 1.1 × 106 M-1 (Fig. S2) in both cases. The binding affinity decreased at pH 9.0, probably due to denaturation of OVA at alkaline conditions46. The capture of OVA at pH 6.0 increased linearly, indicating that OVA was not only captured by 13 ACS Paragon Plus Environment

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formation of the cyclic diester, but also via hydrophobic interactions, as the buffer pH was close to the pI of OVA, reducing the charge density. However, hydrophobic interaction is not preferable because of its non-directional manner. Thus, the data showed that OVA can be selectively captured at neutral pH, and herein, pH 7.4 was selected as the condition for OVA capture and MIP preparation.

1600 pH6.0 pH7.4

1200

pH8.0

∆RU

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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pH9.0

800

400

0 0

25

50

75

100

OVA concentration (µg/mL) Figure 2. Binding isotherms of OVA for the immobilized boronic acid at pH 6.0 (circles), 7.4 (triangles), 8.0 (diamonds), and 9.0 (squares).

After capturing OVA on the SPR sensor chip, SI-AGET ATRP was carried out using PyA as a functional monomer, MPC as a co-monomer, and the immobilized 2-bromoisobutyryl groups as 14 ACS Paragon Plus Environment

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initiators to form the hydrophilic polymer matrix around OVA. PyA interacted with OVA via electrostatic interaction, and polyMPC provided biocompatibility, which has been reported to reduce non-specific adsorption of a wide range of proteins.48-50 The P2p peak derived from MPC appeared in XPS measurements after SI-AGET ATRP (Fig. S1), and the polymer thickness estimated by XRR increased linearly with the polymerization time, as shown in Fig. 3, indicating live SI-AGET ATRP process. The polymer thickness reached approximately 12 nm after polymerizing for 1 h at 40 °C from the SAM layer (approximately 2 nm). The template OVA in the polymer matrix was removed using 3 M guanidine-HCl aqueous solution (pH 2.5), yielding an MIP-60 thin layer on the SPR sensor chip.

20 y = 10.699x + 1.91 R² = 0.9652

15

Thickness (nm)

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10

5

0 0.0

0.5

1.0

1.5

Polymerization time (h) Figure 3. Time course of the MIP thin layer thickness formed on the boronic acid-immobilized gold substrate using SI-AGET ATRP. 15 ACS Paragon Plus Environment

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Binding of the MIP-60 thin layer to OVA was evaluated using SPR at various OVA concentrations (0–100 µg/mL, pH 7.4). SPR analysis revealed that the ∆RU values increased with increasing the concentrations of OVA (Fig. 4). From the Scatchard analysis, Ka value of OVA for MIP-60 was estimated to be 4.7 × 106 M-1 (Fig. S2). An apparent limit of detection of 1.32 µg/mL (29.8 nM) for OVA was estimated from the binding isotherm using 3SD/m (m: slope of the linear part of the binding isotherm, SD: standard deviation for a value of 0 mg/mL OVA). The estimated Ka value of the NIP-60 thin layer, which was prepared without the template OVA, was found to be 6.9 × 105 M-1 (Fig. S2), which was 6.8 times lower than that of MIP-60 layer, confirming the imprinting effect. This implies that the presence of OVA during polymerization drives the imprinting mechanism, enhancing the affinity towards OVA. Furthermore, an estimated Ka value for the SAM-formed SPR sensor chip (before polymerization) was 1.1×106 M-1, which was 4.3 times lower than that of MIP-60, probably due to the presence of multiple interaction sites including boronic acid and pyrrolidyl groups in the imprinted nanocavity of MIP-60. These results indicate that the affinity toward target OVA was improved by formation of OVA recognition nanocavities via the molecular imprinting process.

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200

150

∆RU

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100

50

0

0

25

50

75

100

OVA concentration (µg/mL) Figure 4. Binding isotherms of OVA for MIP-60 (circles) and NIP-60 (square). The error bars represent the standard deviation of three measurements.

In order to understand the selectivity profile of the MIP-60 thin layer, binding behaviors of reference proteins were examined, including lysozyme (Mw: 14.3 kDa, pI: 11), avidin (Mw: 64 kDa, pI: 10), and conalbumin (Mw: 77 kDa, pI: 6.8), which are also found in egg white (avidin and conalbumin are also glycoproteins). In addition, HSA (Mw: 66 kDa, pI:4.7) was selected as another example of albumin. The amounts of bound reference proteins were lower than those of bound OVA. The selectivity factors, defined by ratios of binding amounts of the reference proteins to that of OVA, 17 ACS Paragon Plus Environment

Langmuir

were 0.30, 0.27, 0.24, and 0.14 for avidin, lysozyme, conalbumin, and HSA, respectively, at 10 µg/mL of the proteins (Fig. 5a). In contrast, reference proteins were non-specifically adsorbed onto the NIP-60 thin layer; the selectivity factors in the NIP-60 thin layer were 0.84, 0.36, 1.47, and 1.82 for avidin, lysozyme, conalbumin, and HSA, respectively (Fig. 5b). These results indicate that OVA recognition cavities were successfully formed in the MIP-60 thin layer through the molecular imprinting process.

2.0

2.0

(a) MIP-60 1.5

1.0

0.5

(b) NIP-60 Selectivity factor

Selectivity factor

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1.5

1.0

0.5

0.0

0.0

Figure 5. Protein binding selectivity of the MIP-60 (a) and NIP-60 (b) thin layers. The protein concentration was 10 µg/mL. The tested proteins are OVA (red), avidin (orange), lysozyme (green), conalbumin (blue), and HSA (purple). The error bars represent standard deviation from three measurements.

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60

1.2

(a)

(b) Selectivity factor

1.0 40

∆RU

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 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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20

0.8 0.6 0.4 0.2

0 0

250

500

750

1000

0.0

OVA concentration (ng/mL)

Figure 6. Binding isotherms of OVA for the MIP-45 thin layer. The error bars represent the standard deviation from three measurements.

For molecular imprinting with the orientated immobilized template OVA, thickness of the MIP thin layer is important for forming imprinted cavities capable of OVA recognition. Since the size of OVA is ~5 nm, the ideal polymer thickness is 7–10 nm, including the SAM thickness of ~2 nm. The thickness of the MIP-60 thin layer prepared at 40 °C for 1 h was estimated to be ~12 nm from XRR, which may be slightly thicker than the desirable thickness. Given the linear relationship between polymerization time and layer thickness (Fig. 3), the suitable polymerization time was estimated to be 30 to 45 min. When the MIP thin layer was prepared with the polymerization time of 45 min (MIP-45), an estimated Ka for OVA from Scatchard analysis based on the binding isotherm (Fig. 6a) was about 5.8

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× 108 M-1 (Fig. S2), which was 123 times higher than that observed for MIP-60, maintaining the high selectivity (Fig. 6b). The limit of detection was estimated to be 6.41 ng/mL (144 pM) from the binding isotherm. These results indicate that strict thickness control of the MIP thin layer is significantly important for optimizing the thickness to maximize the performance of MIPs. Furthermore, binding rate constants were estimated to be 3.2 x 108 M-1⋅s-1 for the association and 8.1 x 10-1 s-1 for the dissociation from a sensorgram of 10 ng/mL OVA in MIP-45 by curve fitting analysis (BIAevaluation Ver 4.1.1 in BIACORE 3000 software ver 4.1.2) (Fig. S3), indicating that the MIP-45 showed fast binding kinetics due to the 10 nm-ordered thin layer. These values are comparable to the previously reported MIP thin layers for VEGF.51 A Ka value estimated by the kinetic analysis is 4.0 × 108 M-1 , which is consistent with that estimated from the affinity analysis using Scatchard plots (5.8 × 108 M-1). 40

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20

10

buffer (MIP-45) Egg white sample

0 0

50

100

OVA concentration (ng/mL)

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Figure 7. Binding of the OVA in buffer solution (circles) and in egg-white (squares) to the MIP-45 thin layer. The error bars represent the standard deviation from three measurements.

Finally, the MIP thin layer was tested with a real sample, i.e. addition-recovery tests with OVA in the presence of total egg white proteins were performed. The real egg-white proteins were obtained by dilution of a sonicated preparation of whole egg-white. Different concentrations of OVA (0, 0.22, 0.44, 1.1, or 2.2 nM) were added to the egg white preparation, and OVA binding was examined. It was assumed that the ∆RU value of 6.16 for the sample without addition of OVA represents binding of endogenous egg white OVA. The endogenous OVA concentration was calculated to be 0.167 nM from the binding isotherm of the MIP-45 thin layer (Fig. 6). Using this value and the various concentrations of OVA spiked into the egg-white samples, the total OVA concentrations were calculated, and the OVA binding isotherm was drawn (Fig. 7). The response was comparable to that of OVA in buffer solution, confirming the utility of this molecularly imprinted thin layer for detection of the target analyte in complex protein mixtures.

Conclusions The MIP thin layers possessing precise glycoprotein recognition capability were successfully prepared using SI-AGET ATRP with immobilization of template OVA in a specific orientation onto a 21 ACS Paragon Plus Environment

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SPR sensor chip via boronic ester linkage. In this design, the boronic acid residues were left inside the imprinted cavities in addition to the pyrrolidyl residues that are capable of interacting with amino acid residues in OVA, resulting in highly sensitive and selective imprinted cavities for OVA bearing two interaction sites with different binding modes. The MIP thin layers showed higher affinity than the NIP thin layer prepared without the template OVA, verifying the efficiency of the molecular imprinting process. Tight regulation of the polymer thickness, achieved by using SI-AGET ATRP, significantly improved the affinity towards OVA. Since the addition-recovery tests for the egg white samples showed high recovery rates, the present approach could be extended for detection of a diverse range of glycoproteins in complex matrices important in disease diagnostics and food safety.

ASSOCIATED CONTENT XPS results for N1s, B1s and P2p orbital, kinetic analysis data and Scatchard plots can be obtained in supporting information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] ORCID 22 ACS Paragon Plus Environment

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Toshifumi Takeuchi: 0000-0002-5641-2333 Present Addresses †

Faculty of Pharmacy, Yasuda Women’s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima

731-0153, Japan Funding Sources Japan Society for the Promotion of Science, JSPS KAKENHI Grant Number 24651261, 15K14943 and 16K18300 Japan Science and Technology Agency, the Matching Planner Program MP27115663368 and MP28116808085.

ACKNOWLEDGEMENTS This work was partially supported by Japan Society for the Promotion of Science, JSPS KAKENHI Grant Number 24651261, 15K14943 and 16K18300, and Japan Science and Technology Agency, the Matching Planner Program MP27115663368 and MP28116808085.

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