Design and Fabrication of Biosensing Interface for Waveguide-Mode

Sep 24, 2013 - using triethoxysilane derivatives with anti-leptin antibody to examine ... that the detection limits for human leptin were 50 ng/mL in ...
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Design and Fabrication of Biosensing Interface for Waveguide-Mode Sensor Mutsuo Tanaka,*,† Kyoko Yoshioka,† Yoshiki Hirata,† Makoto Fujimaki,‡ Masashi Kuwahara,‡ and Osamu Niwa† †

Biomedical Research Institute, Advanced Industrial Science and Technology (AIST), Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan ‡ Electronics and Photonics Research Institute, Advanced Industrial Science and Technology (AIST), Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan S Supporting Information *

ABSTRACT: In order to develop a biosensing system with waveguidemode sensor, fabrication of a biosensing interface on the silica surface of the sensing chip was carried out using triethoxysilane derivatives with anti-leptin antibody. Triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized to immobilize the antibody and to suppress nonspecific adsorption of proteins, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moiety suppressed nonspecific adsorption of proteins derived from human serum effectively by rinse with PBS containing surfactant (0.05% Tween 20). On the other hand, it was confirmed that antibody was immobilized on the chip by immersion into antibody solution to show response of antigen−antibody reaction, where the chip was modified with triethoxysilane derivatives bearing succinimide ester moiety. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties, the response of antigen−antibody reaction depended on composition of the mixture and enhanced with the increase of ratio for triethoxysilane derivatives bearing succinimide ester moiety reflecting the antibody concentration immobilized on the chip. While introduction of excess triethoxysilane derivatives bearing succinimide ester moiety induced nonspecific adsorption of proteins derived from human serum, the immobilized antibody on the chip kept its activity after 1-month storage in a refrigerator. Taking into consideration those factors, the biosensing interface was fabricated using triethoxysilane derivatives with anti-leptin antibody to examine performance of the waveguide-mode sensor. It was found that the detection limits for human leptin were 50 ng/mL in PBS and 100 ng/mL in human serum. The results demonstrate that the waveguide-mode sensor powered by the biosensing interface fabricated with those triethoxysilane derivatives and antibody has potential to detect several tens of nanograms per milliliter of biomarkers in human serum with an unlabeled detection method.

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INTRODUCTION Biosensors are of great interest because measurement of biomarkers such as proteins, hormones, and carbohydrates is recognized as a useful tool for diagnosis.1−3 Various methods have been developed to detect biomarkers, and application of affinity and activity derived from protein nature is known as a common method.4−6 In order to fabricate a biosensing interface for biosensor using proteins, immobilization of proteins on the chip is essential. In addition, a countermeasure against nonspecific adsorption of nontargeted proteins to the biosensing interface should be considered, as the nonspecific adsorption can induce undesirable response of the biosensing interface, high background noise resulting in deterioration of sensor performance. Namely, for fabrication of a desirable biosensing interface, not only immobilization of proteins but also suppression of nonspecific adsorption of proteins should be considered.7−11 © 2013 American Chemical Society

For immobilization of proteins, a fundamental point is to achieve enough expression of protein’s functions on biosensing interface. The immobilization technique of proteins can be classified into two groups, physical12−15 and chemical16,17 immobilization methods on a chip surface. The physical immobilization method is based on protein adsorption on a chip surface induced by van der Waals, hydrophobic, and electrostatic interactions between the chip surface and proteins. The chip surface−protein interactions are complicated and still beyond understanding, but the physical immobilization method must be versatile for protein immobilization. On the other hand, formation of a covalent bond between the chip surface and proteins is essential in the chemical modification method. Received: July 22, 2013 Revised: September 19, 2013 Published: September 24, 2013 13111

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In order to form the covalent bond, various methods for surface modification of chips have been developed to introduce functional groups on the chip surface, where the functional groups react with proteins. With an expansion of biosensor studies, various materials have been synthesized for surface modification and examined to suppress nonspecific adsorption of proteins.18−23 It has been reported that functional groups such as polyethylene glycol (PEG),24−31 oligoethylene glycol (OEG),32−38 phosphorylcholine,39−48 and sulfobetaine49−51 are effective to suppress nonspecific adsorption of proteins, and many derivatives bearing those functional groups have been synthesized as surface modification materials. Especially, thiol derivatives are known to form self-assembled monolayers (SAMs) on metal surfaces,52 and SAMs of thiol derivatives bearing those functional groups are reported to suppress nonspecific adsorption of proteins, effectively. On the other hand, it was found that polymer brushes having those functional groups show excellent suppression effect against nonspecific adsorption of proteins, recently.49−51,53,54 In this work, we studied how to fabricate biosensing interface on silica surface of the sensing chip to examine potential of the waveguide-mode sensor55−57 as a biosensor (see Supporting Information: appendix to show difference from waveguide sensor schematically). Chemical immobilization method was adopted in this work, as the proteins immobilized with covalent bond on the chip might be tough enough to lead to sensing stability even in flow system. For this purpose, we synthesized various triethoxysilane derivatives for surface modification of silica chip because triethoxysilane derivatives are easy to purify with common silica-gel chromatography and can afford monolayer-like surface according to our previous work.58 Furthermore, the monolayer-like surface modification is suitable for the waveguide-mode sensor in view of its sensitivity.55−57 Generally, a silica surface is treated by a series of surface modification processes, which are usually introduction of amino, carboxyl, and then active ester groups to the surface, and finally immobilization of proteins on the chip. However, such multistep procedures are essentially not only time-consuming processes but also poor in reproducibility. The lack of reproducibility in surface modification of the chip could induce difficulties in control of protein concentration immobilized on the chip. Therefore, we designed triethoxysilane derivatives bearing active ester, succinimide ester moiety to immobilize proteins on the chip. With those protein immobilization materials, surface modification process of the chip is shortened to a single-step, and proteins are immobilized on the chip only by immersion into protein solution. On the other hand, to resolve nonspecific adsorption of proteins, several triethoxysilane derivatives bearing oligoethylene glycol moieties were also synthesized and evaluated as nonspecific adsorption-resistant materials. In addition, a thin interface such as a monolayer is preferable for the waveguide-mode sensor theoretically.55−57 Therefore, we surveyed surface modification condition of the chip to form a monolayer-like surface using synthesized triethoxysilane derivatives. The biosensing interface fabricated with triethoxysilane derivatives and anti-leptin antibody was evaluated by immunoassay for human leptin in PBS and human serum. The obtained general insight in this work should be instructive for fabrication of biosensing interface with triethoxysilane derivatives on silica chip.

Article

EXPERIMENTAL SECTION

Waveguide-Mode Sensor. Specific substance analyzer (Chip Type) EVA-001 (OPTEX Co., Ltd. Japan) equipped 38° prism and spectrophotometer USB 4000 (Grating No. 11, measuring range 460− 640 nm, Ocean Optics, Inc.) was used for all measurements. The sensing chip for the waveguide-mode sensor is a gift of Shin-Etsu Chemical Co. Ltd., which consists of the top silica waveguide layer and Si layer on a silica substrate with thickness of 1.2 mm. The thicknesses of the silica waveguide layer and the Si reflecting layer are adjusted to be about 360 and 45 nm, respectively. Mechanism and theory for the waveguide-mode sensor are described in the literature.55−57 Materials and Synthesis. All chemicals were commercially available and used as received without additional purification. Details of synthesis procedures are described in the Supporting Information. Monoclonal anti-leptin antibody (no. 14C9), recombinant human leptin (no. 730027), fibrinogen from bovine plasma (no. F8630-1G), and control serum based on human serum (no. 717438) were purchased from Mikuri Immunology Laboratory, Inc. Japan, ImmunoBiological Laboratories Co. Ltd., Sigma-Aldrich Co. LLC., and Roche Diagnostics K. K. Japan, respectively. Millipore water was used for immunoassay. Surface Modification of Chip with Nonspecific AdsorptionResistant Materials. Before surface modification, the chip was rinsed acetone under ultrasonication for 10 min, and then, dried under vacuum condition for 1 h. The chip was modified in the prepared drytoluene solution of triethoxysilane derivatives. The chip was took out from the solution and rinsed with acetone under ultrasonication for 1 min. Evaluation of Nonspecific Adsorption-Resistant Effect. The prepared chip was set on the waveguide-mode sensor, and the evaluation of nonspecific adsorption-resistant effect was examined using fibrinogen and human serum with batch measurement method at room temperature. First, the attached cell (ca. 0.5 mL) was filled up with PBS (Dulbecco, Mg2+ and Ca2+ free), and wavelength of the dip was read out. Then, PBS solution of fibrinogen (0.4 mL, 5 mg/mL) was introduced to the cell. The cell was stood for 5 min, and then, rinsed with PBS (0.4 mL) three times. The shift of wavelength for the dip was read out to evaluate the nonspecific adsorption-resistant effect. When nonspecific adsorption of human serum was evaluated, not only PBS but also PBS containing 0.05% Tween 20 was examined with the same procedure for fibrinogen. X-ray Photoelectron Spectroscopy (XPS) Analysis of Modified Chip Surface. XPS measurement was performed with PHI 5000 VersaProbe spectrometer (ULVAC-PHI, Inc.) using a monochromatic Al Kα source (1486.6 eV, 100 V) with a take off angle 45.0°. Atomic Force Microscopy (AFM) Analysis of Modified Chip Surface. AFM images were collected by using a NanoScope IIIa Multimode atomic force microscope (Veeco Instruments Inc., Santa Barbara, CA) in tapping mode under ambient conditions. Silicon cantilevers with a nominal resonance frequency of ∼75 kHz, force constant ∼2.8 N/m, tip radius < 10 nm (FM-50, Nano Sensors, Neuchatel, Switzerland) were used in the AFM experiments. Images were analyzed using the software provided by the manufacturer. Ellipsometry Analysis of Modified Chip Surface. Ellipsometry analysis was conducted with VASE (J. A. Woollam Japan, Co., Inc.), and measurement wavelength range was 350−700 nm. Fabrication of Biosensing Interface on Chip. The surface modification procedures for the chip were the same as described in the subsection titled Surface Modification of Chip with Nonspecific Adsorption-Resistant Materials, and a mixture of the protein immobilization materials with the nonspecific adsorption-resistant materials was used for chip modification solution. For immobilization of anti-leptin antibody, anti-leptin antibody solution (20 μg/mL) was prepared with acetate buffer solution (10 mM, pH 5), and the modified chip was immersed in the prepared antibody solution for 24 h at room temperature. The chip was took out and rinsed with water. The prepared chip was used for immunoassay with the waveguidemode sensor. 13112

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Immunoassay. The prepared chip was set on the waveguide-mode sensor, and the immunoassay for PBS and human serum samples containing human leptin was carried out with batch measurement method at room temperature. In the case of immunoassay using PBS, PBS (0.4 mL) was put to the attached cell, and wavelength of the dip, which was observed by the spectrophotometer, was read out. Then, the measurement sample (0.4 mL, PBS containing leptin) was introduced to the cell, and the cell was left to stand for 10 min. The cell was rinsed with PBS (0.4 mL) three times, and wavelength shift of the dip was read out to evaluate the antigen−antibody reaction. When the immunoassay for human serum sample was conducted, PBS containing 0.05% Tween 20 was used with the same procedure for that for PBS sample.

synthesized, where numbers of the oligoethylene glycol unit were 2−5. We adopted oligoethylene glycol monomethyl ethers as hydroxyl group is known to interact with triethoxysilane moiety to decompose. In this study, as our interest was in monolayer-like surface modification of the chip, purification of those triethoxysilane derivatives is definitely important, and the purification was performed with common silica-gel chromatography according to our previous work.58 The synthesis procedures are described in the Supporting Information in detail. Surface Modification of Chip with Nonspecific Adsorption-Resistant Materials. In order to evaluate nonspecific adsorption-resistant effect of the synthesized triethoxysilane derivatives, surface modification of the chip was carried out using 1 mM toluene solution of M2EG∼M5EG, and the nonspecific adsorption-resistant effect of the modified surface was evaluated using fibrinogen solution (5 mg/mL in PBS). We chose fibrinogen for the test of nonspecific adsorption as fibrinogen showed more serious nonspecific adsorption ability than BSA and lysozyme. The amount of material adsorbed on the chip surface is expressed as wavelength shift of the dip, Δnm in the case of the waveguide-mode sensor.55 As shown in Figure 1, nonspecific adsorption of fibrinogen decreased with the modification time in all cases. This tendency seems to reflect increase in the surface concentration of nonspecific adsorption-resistant materials immobilized on the chip as the surface modification proceeded with the modification time resulted in high concentration. In Figure 1a, nonspecific adsorption-resistant effect of M2EG became constant after more than 12 h modification. As it was reported that not only the immobilized concentration30,31 but also the length of oligoethylene glycol moiety is important to show nonspecific adsorption-resistant effect,33,36 M2EG is too short in length to afford enough nonspecific adsorption-resistant effect. While M3EG in Figure 1b demonstrated a good nonspecific adsorption-resistant effect with more than 6 h modification, M4EG in Figure 1c did not show sufficient nonspecific adsorption-resistant effect. In our previous work,58 triethoxysilane derivatives with longer alkyl chains reacted more slowly. Therefore, the surface modification with M4EG was conducted at 50 °C to enhance surface modification. The result in Figure 1d showed a sufficient nonspecific adsorption-resistant effect similar to M3EG. As shown in Figure S1 in the Supporting Information, the reactivity of M5EG decreased significantly, and nonspecific adsorption-resistant effect was not observed when surface modification of the chip was carried out at room temperature. Figure 1e shows that the surface modification with M5EG was still sluggish at 50 °C, and a sufficient nonspecific adsorptionresistant effect was attained with the surface modification at 50 °C in the presence of 5 mM acetic acid (Figure 1f). This significant difference of triethoxysilane derivatives in the reactivity depending on the chain length of incorporated moiety should be paid attention when a mixture of triethoxysilane derivatives is used for surface modification. Similarly, nonspecific adsorption of proteins was examined using human serum without dilution. As it is reported that PBS containing surfactant Tween 20 (0.05%) is effective to remove nonspecifically adsorbed proteins,11 the rinse effect using PBS was compared with that using PBS containing 0.05% Tween 20. The results are summarized in Figure 2. When the chip was rinsed with PBS, slight nonspecific adsorption of proteins was observed for all chips, especially for the chip modified with

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RESULTS AND DISCUSSION Synthesis of Triethoxysilane Derivatives. Synthesized triethoxysilane derivatives for chip modification are shown in Scheme 1. As described in the Introduction, the surface Scheme 1. Synthesized Triethoxysilane Derivatives for Surface Modification of Chip

modification materials to immobilize proteins were designed to be able to introduce active ester moiety to chip surface with single step. It is possible to preserve the chip modified with the protein immobilization materials under dry condition permanently, and proteins are immobilized on the chip only to dip the chip into protein solution when use. In the synthesis of the protein immobilization materials, dioic acids as starting materials were monoesterified with N-hydroxysuccinimide and then chlorinated. As the acyl chloride group reacted with 3aminopropyltriethoxysilane faster than the succinimide ester group, we optimized the reaction condition as much as possible, where only the acyl chloride group reacted with the amino group. Three kinds of protein immobilization materials, in which the difference was in alkyl chain length, were synthesized to investigate in combination with nonspecific adsorption-resistant materials bearing various oligoethylene glycol moieties. In the case of nonspecific adsorption resistantmaterial synthesis, reaction of acyl chloride with 3-aminopropyltriethoxysilane to form amide bond was adopted, similarly. As the nonspecific adsorption-resistant effect is depending on the length of oligoethylene glycol chains,33,36 four kinds of nonspecific adsorption-resistant materials were 13113

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Figure 1. Modification time dependence of nonspecific adsorption-resistant effect modified (a) with M2EG at rt, (b) with M3EG at rt, (c) with M4EG at rt, (d) with M4EG at 50 °C, (e) with M5EG at 50 °C, and (f) with M5EG at 50 °C in the presence of CH3COOH (5 mM).

of alkanethiols.42 Therefore, the mean roughness for the bare chip was measured by AFM, and that was 0.204 nm (Figure S2). This flat and smooth surface of the chip for the waveguidemode sensor can be of great advantage for measurement of human serum samples in view of nonspecific adsorption of proteins. XPS Analysis of Modified Chip Surface. As the waveguide-mode sensor prefers thin interface such as monolayer theoretically,55−57 we evaluated whether the surface modification of the chip proceeded with monolayer or multilayer by XPS analysis. In our previous work,58 we concluded that the complete monolayer surface modification with triethoxysilane derivatives was impossible, but monolayerlike surface modification is possible by controlling modification condition with molecular design of triethoxysilane derivatives. We focused that the surface modification proceeded slowly to show a plateau area in immobilized surface concentration when the immobilized surface concentration reached to the concentration of the close-packed monolayer. In order to examine whether similar phenomenon was observed or not in the case of XPS measurement, the chip was modified with 3mercaptopropyltriethoxysilane in control experiments. In XPS measurement, the S/(S + Si) atom ratio will reach 50% with a multilayer formation as the S/(S + Si) atom ratio for 3mercaptopropyltriethoxysilane is 50%. The results are summarized in Figure S3. A similar phenomenon, where the surface modification proceeded slowly around 20% of the S/(S + Si) atom ratio, was observed, and then the atom ratio reached at 50% finally. On the basis of those results, we judged that the N/ (N + Si) atom ratio obtained by XPS measurement could be an

Figure 2. Nonspecific adsorption of proteins derived from human serum. The surface modifications with M3EG, M4EG, and M5EG were carried out using 1 mM toluene solution with 48 h modification at rt, 15 h modification at 50 °C, and 15 h modification at 50 °C in the presence of CH3COOH, respectively, reflecting the reactivity of triethoxysilane derivatives. The term “-T” represents rinse with PBS containing Tween 20.

M3EG, reflecting a short oligoethylene glycol moiety. The effect of Tween 20 to remove nonspecifically adsorbed proteins derived from human serum was noteworthy, and all modified chips showed excellent nonspecific adsorption-resistant properties where the nonspecifically adsorbed proteins were under the detection limit. It is suggested that nonspecific adsorption of proteins can be induced by the roughness of the surface, even by etching pit produced during self-assembled monolayer (SAM) formation 13114

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Figure 3. Modification time dependence of N1s/(N1s + Si2p) atom ratio in high-resolution XPS analysis for surface modification with (a) M3EG at rt, (b) M3EG at 50 °C, (c) M4EG at 50 °C, and (d) M5EG at 50 °C in the presence of 5 mM CH3COOH, respectively.

indicator whether the chip surface modified with nonspecific adsorption-resistant materials is monolayer-like or multilayer. The N/(N + Si) atom ratio of nonspecific adsorption-resistant materials is 50%; therefore, the N/(N + Si) atom ratio will become 50% with a multilayer formation. On the other hand, when the N/(N + Si) atom ratio shows a plateau area reflecting the slow surface modification in spite of being far smaller than 50%, the immobilized surface concentration could be close to that for the close-packed monolayer. As shown in Figure 3a, the N/(N + Si) atom ratio for surface modification with M3EG afforded a plateau area although the N/(N + Si) atom ratio was far smaller than 50%. Similar tendency was observed when the surface modification was carried out even at 50 °C in Figure 3b. In cases of M4EG (Figure 3c) and M5EG (Figure 3d), the N/ (N + Si) atom ratio was smaller than that for M3EG although the surface modification conditions for M4EG and M5EG were harsher than those for M3EG. In control experiments using 3aminopropyltriethoxysilane (Figure S4), the N/(N + Si) atom ratio reached at 50% to show a multilayer formation similar to 3-mercaptopropyltriethoxysilane. However, we could not find conditions in cases of the nonspecific adsorption-resistant materials where the N/(N + Si) atom ratio became 50%. Therefore, we concluded that surface modification with the nonspecific adsorption-resistant materials afforded a monolayer-like surface. Immunoassay Using Anti-Leptin Antibody. It is known that antibody sometimes loses activity due to denature of the protein by immobilization on substrate.16,17 Therefore, antileptin antibody, where human leptin is recognized as a premarker of diabetes, was immobilized on the chip using the protein immobilization materials, and activity of the antibody was examined. Taking into account the difference in reactivity among the protein immobilization materials, the surface modification of the chip was carried out at rt for C12Es, at 50 °C for C16Es, and at 50 °C in the presence of 5 mM acetic acid for C20Es. The modification time was 15 h in 1 mM toluene solution. As shown in Figure S5, a response reflecting antigen−antibody reaction was detected for 1 μg/mL human leptin in PBS with all chips modified with the protein immobilization materials. In control experiments, such response was not detected without immobilization of anti-leptin

antibody, therefore, it was confirmed that those responses were derived from the antigen−antibody reaction, but not nonspecific adsorption of human leptin on the chip. It was also reported that nonspecific adsorption of antibody on substrate can cause activity loss, and the surface modification of substrate with nonspecific adsorption-resistant materials prevents the activity loss effectively.59 Therefore, the chip was modified with various mixtures of protein immobilization materials and nonspecific adsorption-resistant materials, and the antigen−antibody reaction depending on the molar ratio of C12Es and M3EG was examined. The results are summarized in Figure 4. In Figure 4, the response of antigen−antibody

Figure 4. Detection of antigen−antibody reaction with various mixtures of C12Es and M3EG. The chip modification was conducted at rt for 15 h, and the sum of C12Es and M3EG concentrations was 1 mM in toluene.

reaction increased with the increase of C12Es ratio and reached a plateau when the C12Es/M3EG molar ratio was more than 1/200. The maximum response of antigen−antibody reaction would appear when the surface is covered with immobilized antibody. The size of the antibody is known to be 4 × 7 × 9 nm3,60 and the cross-sectional area for M3EG is considered to be similar to that for alkyl chains, 0.0225π nm2. When the antibody is assumed as a sphere with diameter 8 nm, the crosssectional area for the antibody becomes 16π nm2. With this 13115

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As noted above, it is known that nonspecific adsorption of antibody on substrate occurs with time resulted in recognition activity loss of antibody.59 Therefore, to evaluate the activity loss of antibody with time, the chip was modified with 1 mM toluene solution of M3EG and C12Es (10:1 in molar ratio) at 50 °C for 24 h, immersed in 20 μg/mL antibody solution for 24 h at room temperature to immobilize antibody, and preserved at 4 °C in PBS containing 0.09% sodium azide. The response of the antigen−antibody reaction is summarized in Figure 6,

assumption, when the C12Es/M3EG molar ratio was more than 1/700, the surface would be covered completely with antibody to show the maximum response of antigen−antibody reaction. However, C12Es tends to suffer hydrolysis in antibody solution, and it is also possible that a number of C12Es form covalent bond with one antibody. Therefore, it was plausible that the response of antigen−antibody reaction reached a plateau when the C12Es/M3EG molar ratio was more than 1/ 200 in Figure 4. The results imply that the concentration control of antibody immobilized on the chip is possible by changing the molar ratio of protein immobilization materials and nonspecific adsorption-resistant materials. The overview of response for antigen−antibody reaction depending on the chip modified with various mixtures of protein immobilization materials and nonspecific adsorptionresistant materials is shown in Figure 5. In Figure 5a, the

Figure 6. Stability of anti-leptin antibody immobilized on chip.

where the activity loss of antibody is not observed even after 28 days preservation. This result suggests that the nonspecific adsorption-resistant material, M3EG is effective to suppress adsorption of antibody to the chip, and surface modification of the chip with the mixture of protein immobilization materials and nonspecific adsorption-resistant materials is a versatile method not only to suppress nonspecific adsorption of proteins but also to keep the activity of the antibody immobilized on the chip. Performance of Waveguide-Mode Sensor as Biosensor. In order to evaluate performance of the waveguidemode sensor, the response of antigen−antibody reaction depending on human leptin concentration was measured with unlabeled detection method using the interface fabricated with a protein immobilization material, a nonspecific adsorptionresistant material, and anti-leptin antibody. First, we examined the detection limit of the waveguide-mode sensor for human leptin in PBS using the interface fabricated with M3EG, C12Es, and antileptin-antibody. The response of antigen−antibody reaction depending on the human leptin concentration is summarized in Figure 7. The detection limit of the waveguidemode sensor for human leptin in PBS was 50 ng/mL, which is

Figure 5. Detection of antigen−antibody reaction with various mixtures of protein immobilization materials and nonspecific adsorption-resistant materials. The surface modification was carried out (a) at rt or (b) at 50 °C for 15 h modification using 1 mM toluene solution.

response of the chip modified with a mixture of M3EG and protein immobilization materials increased with the increase of molar ratio for the protein immobilization materials. On the other hand, the response decreased when the protein immobilization materials had longer alkyl chains. As the reactivity of triethoxysilane derivatives decreases by incorporation of longer alkyl chains, this tendency seems to reflect the low concentration of protein immobilization materials resulted in low concentration of the immobilized antibody. An interesting feature was observed in Figure 5b, where the response using C12Es was not detected in the presence of M4EG. It was considered that the active ester moiety of C12Es might be buried in M4EG and could not react with antibody as M4EG is longer than C12Es. Those results suggest that both reactivity and molecular structure in length should be taken into account to modify the chip with a mixture of protein immobilization materials and nonspecific adsorption-resistant materials.

Figure 7. Detection of human leptin in PBS. The chip was modified with 1 mM toluene solution of M3EG and C12Es (10:1 in molar ratio) at 50 °C for 24 h. 13116

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ethylene glycol moiety, the interface was fabricated with antileptin antibody and the protein immobilization materials in the presence or absence of the nonspecific adsorption-resistant materials, and the response of antigen−antibody reaction for 50 ng/mL human leptin in human serum was examined. The results are summarized in Figure 9 with the data in PBS

comparable or better than that of a common SPR biosensor to show the high performance of the waveguide-mode sensor. Next, the measurement depending on human leptin concentration was conducted using human serum similarly, and the results are shown in Figure 8. When the interface was fabricated

Figure 9. Nonspecific adsorption of proteins derived from human serum to biosensing interface. The term “/PBS” represents the data in PBS. The human serum contained 50 ng/mL human leptin. The chip modifications were carried out for 24 h with M3EG-C12Es, and C12Es, for 24 h in the presence of 5 mM CH3COOH with C16Es and C20Es, or for 48 h in the presence of 5 mM CH3COOH with M4EGC16Es, and M5EG-C20Es in toluene at 50 °C. The molar ratio was 10:1 for M3EG-C12Es, M4EG-C16Es, and M5EG-C20Es. Figure 8. Detection of human leptin in human serum. The chip was modified with 1 mM toluene solution of (a) M3EG and C12Es at 50 °C for 24 h or (b) M5EG and C20Es at 50 °C for 24 h in the presence of 5 mM CH3COOH. The molar ratios for M3EG-C12Es and M5EGC20Es were 10:1.

(represented with light green) for comparison. As the data in PBS represents the net response of the waveguide-mode sensor for 50 ng/mL human leptin, the excess response observed in other data is considered as the response derived from nonspecific adsorption of proteins. When the interface was fabricated in the absence of the nonspecific adsorption-resistant materials, a serious nonspecific adsorption of proteins was observed for all protein immobilization materials. Therefore, it is obvious that the protein immobilization materials induced nonspecific adsorption of proteins derived from human serum. Interestingly, M3EG-C12Es, the combination of the shortest materials, showed the highest nonspecific adsorption-resistant effect being contrary to common tendency where the longer oligoethylene glycol moiety suppresses nonspecific adsorption of proteins more effectively.33,36 In Figure 9, the protein immobilization material bearing longer alkyl chain induces more serious nonspecific adsorption of proteins; therefore, the results reflect that the influence of nonspecific adsorption of proteins induced by the protein immobilization materials was more significant compared with the nonspecific adsorptionresistant effect derived from longer oligoethylene glycol moiety under those conditions. With immobilization treatment of antibody, the succinimide ester moiety of the protein immobilization materials is partially hydrolyzed to form carboxylic acid moiety. This result implies that concentration control for the protein immobilization materials is also important to suppress nonspecific adsorption of proteins effectively, as the carboxylic acid moiety formed by hydrolysis in the interface induces nonspecific adsorption of proteins.

with the same as Figure 7, the detection limit for human leptin in human serum was 100 ng/mL (Figure 8a). This value is worse than that in PBS due to nonspecific adsorption of proteins derived from human serum; however, the detection limit, 100 ng/mL in human serum with unlabeled detection method is still attractive to measure various biomarkers. The human leptin concentration for health human is about 10 ng/ mL; therefore, it is difficult to measure the human leptin concentration with unlabeled detection method, unfortunately. In the case of the waveguide-mode sensor, an enhancement method of sensitivity using colored nanomaterials will be effective to detect such low concentration proteins.56 When the interface was fabricated with M5EG, C20Es, and anti-leptin antibody, the detection limit for human leptin deteriorated by serious nonspecific adsorption of proteins as shown in Figure 8b. This result is unexpected as it is known that nonspecific adsorption-resistant materials bearing longer oligoethylene glycol moiety are more effective generally.33,36 The reason for this result is discussed in the next section. The chip surface modified with M3EG and C12Es under the same condition was analyzed with ellipsometry and AFM. The thickness of surface layer was determined by ellipsometry as 1.48 ± 0.16 nm. The value is reasonable for the monolayer-like surface modification with M3EG and C12Es. Furthermore, the AFM measurement afforded the mean roughness as 0.308 nm (Figure S6) to show excellent surface flatness again. We consider that this flatness could contribute to this effective suppression against nonspecific adsorption of proteins derived from human serum as surface roughness is reported to induce nonspecific adsorption of proteins.42 In order to scrutinize why nonspecific adsorption of proteins derived from human serum was serious with longer oligo-

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CONCLUSIONS The silica surface of the sensing chip for the waveguide-mode sensor was modified with various protein immobilization materials and nonspecific adsorption-resistant materials to fabricate a biosensing interface, and performance of the waveguide-mode sensor was examined by unlabeled detection method using anti-leptin antibody. In order to fabricate the 13117

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biosensing interface, various triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties were synthesized as protein immobilization materials and nonspecific adsorption-resistant materials, respectively. The chip modified with triethoxysilane derivatives bearing oligoethylene glycol moieties to form a close-packed monolayer-like surface showed excellent nonspecific adsorption-resistant properties for proteins derived from human serum. When the interface was fabricated with antibody and a mixture of triethoxysilane derivatives bearing succinimide ester and oligoethylene glycol moieties, the interface demonstrated a potential to immobilize antibody without activity loss and to resist nonspecific adsorption of proteins derived from human serum. The modified surface analysis by AFM and ellipsometry suggested that chip flatness of the waveguide-mode sensor could contribute to this effective suppression of nonspecific adsorption of proteins. On the other hand, it is suggested that excess introduction of triethoxysilane derivatives bearing succinimide ester moiety to the interface induces nonspecific adsorption of proteins due to formation of the carboxylic acid moiety by hydrolysis. With the interface fabricated using those triethoxysilane derivatives and antileptin-antibody, the performance of the waveguide-mode sensor was confirmed that the detection limits for human leptin were 50 and 100 ng/mL in PBS and human serum, respectively. In this work, the potential of the waveguide-mode sensor as a biosensor with unlabeled detection method was demonstrated.

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ASSOCIATED CONTENT

S Supporting Information *

Additional evaluation data for nonspecific adsorption of proteins and response of antigen−antibody reaction, XPS data, AFM data, and experimental procedure. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +81-29-861-6233. Fax: +81-29-861-6177. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Kuraoka (Kobe University) for practical advise, and thank Mr. Fujitani (AIST) and Mrs. Nagai (AIST) for experimental assistance. We also thank Dr. Yoshida (AIST) for financial support. The chip for the waveguide-mode sensor is a gift of Shin-Etsu Chemical Ltd. This study was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan (No. 25390022).

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