Fabrication of Carboxylated Silicon Nitride Sensor Chips for Detection

Sep 11, 2012 - ... Yuki Oba , Yoshihiro Nishida , Tsuyoshi Iwasa , Ken-ichi Tayama ... Fuminori Okada , Yoshikatsu Akiyama , Jun Kobayashi , Hidetaka ...
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Fabrication of Carboxylated Silicon Nitride Sensor Chips for Detection of Antigen−Antibody Reaction Using Microfluidic Reflectometric Interference Spectroscopy Yoshikazu Kurihara,†,‡ Masaaki Takama,‡ Tadanobu Sekiya,‡ Yuka Yoshihara,‡ Tooru Ooya,† and Toshifumi Takeuchi*,† †

Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan Konica Minolta Technology Center, Inc., 1 Sakura-machi, Hino-shi, Tokyo 191-8511, Japan

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S Supporting Information *

ABSTRACT: In this study, we report label-free detection of alphafetoprotein (AFP), which has been used as a biomarker for hepatocellular carcinoma, by a microfluidic reflectometric interference spectroscopy (RIfS) system adopting a simple halogen light source and an inexpensive siliconbased sensor chip. Introduction of carboxy groups on a silicon nitride sensor chip to immobilize anti-AFP monoclonal antibody (anti-AFP) was carried out simply by immersion in aqueous solution containing triethoxysilylpropylmaleamic acid bearing a carboxy group and a silanol group. The RIfS system with the anti-AFP-immobilized sensor chip was found to give a reversible response through 100 on/off cycles using a regeneration buffer with high reproducibility (coefficient of variation (CV) = 5.7%). The limit of detection (LOD) of AFP was 100 ng mL−1, and the measurement range spanned 3 orders of magnitude. Furthermore, the sensor chip showed no cross-reactivity with human serum albumin, Immunoglobulin G, transferrin, or fibrinogen at 100 μg mL−1 without the use of blocking reagents such as bovine serum albumin. Consequently, the proposed RIfS system is a potentially effective tool for biomarker detection and in vitro diagnostics.

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INTRODUCTION Measurements of biomarkers, such as proteins, carbohydrates, nucleic acids, or others, are meaningful for detection in early stage cancer and reduction of risk at developing cancer by appropriate treatment.1,2 Alpha-fetoprotein (AFP) is one of the most widely used biomarkers and commonly used as a serum biomarker to indicate hepatocellular carcinoma.3 AFP was first identified by Bergstrand and Czar as the X component in human cord blood.4 The discovery by Abelev5 that one of the hepatoma-specific proteins he isolated from murine hepatoma was identical to fetoprotein led directly to development of the first serologic test specific for human carcinoma.6,7 AFP is a serum oncofetal glycoprotein and a major component of the globulin fraction. Normally, AFP levels decline rapidly after birth, reaching 1.5) SiO225 and TiO226,27 as interference layers and an incident light irradiated below the substrate. Recently, Ü nlü and coworkers introduced a simple reflectance-based interferometric label-free detection method termed the interferometric Received: June 1, 2012 Revised: August 16, 2012 Published: September 11, 2012 13609

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reflectance imaging sensor (IRIS).28,29 To detect antigen− antibody reaction, this technique uses common-path interferometry through a Si/SiO2 layered substrate acting as the sensor surface by monitoring local optical path length changes attributed to mass accumulation at the surface. In this work, we developed a new RIfS microfluidic flow system for detection of antigen−antibody reactions as shown in Figure 1, equipped with a silicon nitride (SiN) coated Si wafer

ification of the carboxy group was confirmed by X-ray photoelectron spectroscopy (XPS) and through the wavelength shift of the minimum value of reflectance spectra in RIfS system (defined as Δλ) before and after silane coupling reaction. Then, anti-AFP monoclonal antibody (anti-AFP) was immobilized on the SiN sensor chip via amide bonds. Immobilization of antibody was also confirmed by Δλ. A transparent microfluidic flow cell prepared from polydimethylsiloxane (PDMS) was placed on the anti AFP-immobilized sensor chip to construct a continuous-flow microfluidic system for RIfS (Figure 3).

Figure 1. Schematic illustration of label-free detection of antigen− antibody reaction by RIfS system.

on which an antibody was immobilized. The incident light from a halogen lamp is perpendicularly irradiated from above the sensor chip. When the incident light is irradiated on a sensor chip, a light beam may generate two reflected partial beams, producing a reflectometric interference phenomenon. When a ligand (molecular recognition element) is immobilized on the sensor chip and an analyte (target molecule) is injected, the optical thickness of the interference layer on the sensor chip surface undergoes a change, resulting in modulation of the reflectance spectrum. A SiN-deposited silicon substrate prepared by plasma-enhanced chemical vapor deposition (thickness 65 ± 2.0 nm, evaluated by ellipsometry) was employed as a sensor chip, where the SiN thickness enabled the appearance of only one minimum bottom of reflectance spectra in visible region (Figure 2).30,31 SiN was chosen due to its

Figure 3. Anti-AFP immobilization process and label-free detection of AFP by RIfS.

Effectiveness of the RIfS-based optical sensing system was demonstrated with the highly sensitive detection of AFP even in the presence of abundant human serum proteins. Crossreactivity and within-run reproducibility of the sensor chip were also investigated.

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RESULTS AND DISCUSSION The silane coupling reaction is one of the most commonly used coupling techniques for surface modification of biosensing chips. Among silane coupling agents, in particular, 3-aminopropyltriethoxysilane32−35 and 3-glycidoxypropyltrimethoxysilane36,37 are often chosen to immobilize biomolecules on an oxide (or oxide-like) surface in conjunction with the use of heterobifunctional linkers such as N-succinimidyl-3-(2-pyridyldithio) propionate, N-(6-maleimidocaproyloxy) succinimide, and biotin N-hydroxysuccinimide ester. In this study, we evaluated various silane coupling conditions to introduce a carboxy group onto the sensor chip because formation of a surface oxide layer was confirmed on the SiN sensor chips by XPS depth profile (see Supporting Information Figure S1). Three carboxy-terminated silane coupling agents were examined to modify the surface of SiN sensor chip, including triethoxysilylpropylmaleamic acid, carboxyethylsilanetriol sodium salt, and N-(trimethoxysilylpropyl) ethylenediamine triacetic acid trisodium salt. The sensor chip surface was most effectively covered with triethoxysilylpropylmaleamic acid, and the optimized condition for detection of the antigen− antibody reaction was the 1 h coupling reaction at room temperature, followed by heating for 1 h at 80 °C (see Supporting Information Table S1). The carboxy-introduced sensor chip was characterized by ellipsometry, atomic force microscopy (AFM), and XPS. The ellipsometric thickness of the silane layer was 3.4 ± 1.7 nm (n = 3), which was greater than summation of bond lengths of all atoms contributing to the molecular size (1.86 nm), suggesting

Figure 2. Schematic illustration of the increase in optical thickness with interaction resulting in modulation of reflectance spectra.

appropriate refractive index (n = 2.0−2.5) to a silicon substrate (n = 3.5−5.5) and its high transmission (k < 0.1) in the visible region (400−800 nm). The thin layer of SiN, having 65 nm thickness, gave a minimum reflectance wavelength at around 500−600 nm by attenuation of the reflected light intensity caused by the interference and was purple by visual observation caused by interference color. The halogen light source employed emits high irradiation energy around the wavelength region, contributing to the sensitive RIfS response. To prepare AFP-responsive sensor chips, SiN sensor chips were carboxylated by triethoxysilylpropylmaleamic acid. Mod13610

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chip. Subsequently, we observed that the bottom of the reflectance spectra was red shifted (Δλ was about 10 nm) after immobilization of anti-AFP was completed (Figure 5).

that the propylmaleamic acid layer formed by the silane coupling reaction could have a multilayer structure.38 The arithmetic average roughness (Ra) of the SiN sensor chip before and after silane coupling reaction was examined by AFM images and estimated to be 0.97 ± 0.16 and 0.84 ± 0.17 nm. Their maximum differences of elevation (P − V) were 9.9 ± 2.1 and 9.3 ± 1.7 nm, respectively (n = 4) (see Supporting Information Figure S2). These figures did not show a significant difference, therefore indicating that the silane coupling reaction may proceed homogeneously on the sensor chip surface. Surface elemental analysis of the sensor chip before and after the silane coupling reaction was carried out by XPS (Figure 4).

Figure 5. Spectral reflectance of unmodified, carboxylated, and antiAFP immobilized sensor chip.

We then conducted optimization of the antigen−antibody reaction medium, which was flowed into the microfluidic system as a running buffer. Electrostatic interactions and hydrophobic interactions can affect antigen−antibody interactions. At low NaCl concentrations, electrostatic forces should be predominant, whereas hydrophobic interactions should affect the interaction more strongly under high NaCl concentrations (Figure 6a). When the salt concentration was increased, the binding activity was enhanced, and after 138 mM, binding was depressed. We found that a balance of electrostatic interactions and hydrophobic interactions is important to develop high binding activity on the chip, as is the case in biological systems. Therefore, a conventional physiological salt solution containing 138 mM NaCl and 2.68 mM KCl was employed to the antigen−antibody reaction medium. Various types of buffers were tested for further improvement of the binding activity (Figure 6b). HEPES buffer gave the worst binding activity when NaCl and KCl were not added, while the highest activity was observed by PBS or Tris buffer with NaCl and KCl. Furthermore, as an often employed nonionic surfactant for use in immunological detection techniques,39,40 Tween-20 was examined as a blocking ingredient dissolved in the running buffer. When Tween-20 was used, the response based on the antigen−antibody reaction was not affected. On the other hand, the binding affinity decreased when another commonly used BSA was applied as the blocking agent (see Supporting Information Figure S3). From these results, the running buffer constituents were adopted as follows: 9.58 mM PBS buffer pH 7.4 containing 138 mM NaCl, 2.68 mM KCl, and 0.001% Tween-20 (0.001% PBST), and the following reproducibility and cross-reactivity tests were performed. The reproducibility of the AFP response was examined by continuously alternating injections of 1 μg mL−1 AFP and 10 mM glycine−HCl (pH 1.5). Although the baseline declined gradually, which may have been due to denaturation of immobilized anti-AFP by multiple repeated injections of glycine−HCl, we found that the RIfS system provided a highly

Figure 4. XPS data obtained on the silicon nitride sensor chip. Narrow scan in the C1s (a) and N1s (b) region before (triangles) and after (circles) silane coupling reaction.

A narrow scan of the C1s region (Figure 4a) showed an increase in the whole signal, where the signal at 288.6 eV was assigned to the carboxy (C(O)−O) group. A narrow scan of the N1s region (Figure 4b) showed the appearance of an N−C peak (399.9 eV) derived from an amide group of the silane coupling agent. As both of these should arise only from addition of molecules not present in the SiN layer, we thus confirmed that the sensor chip was successfully functionalized with silane coupling agent. After the carboxy residue on the obtained sensor chip was transformed into the corresponding active ester using EDC and NHS, the chip was incubated with anti-AFP for immobilization via amide bonds (Figure 3). Then, ethanolamine was used to quench the unreacted active esters remaining on the sensor 13611

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Figure 6. Optimization of running buffer in the antigen−antibody reaction: (a) Δλ with 1 (open triangle) or 10 μg mL−1 (solid circle) injection of AFP in phosphate buffer containing various NaCl (0, 75, 138, 250, 500, or 1000 mM) concentrations; (b) Δλ with 10 μg mL−1 injection of AFP in various buffers (9.58 mM phosphate, 10 mM TrisHCl, 10 mM HEPES, and 10 mM MOPS) of pH 7.4 containing 138 mM NaCl and 2.68 mM KCl (black) or without NaCl and KCl (white).

Figure 7. Concentration dependence of AFP response: (a) typical sensor gram; (b) calibration curve of AFP (n = 3). Running buffer: 0.001% PBST. Flow rate: 20 μL min−1. AFP: 0.1, 0.3, 1, 3, 10, 30, and 100 μg mL−1. Regeneration buffer: 10 mM Glycine−HCl (pH 1.5).

be noted that the sensor chip also showed no cross-reactivity with highly concentrated bovine serum albumin (BSA, 10 mg mL−1), although a temporary baseline shift was observed while BSA solution was passed over the detection area (see Supporting Information Figure S6). This result was interesting considering that BSA was commonly used for the blocking agent of immunoassays and evaluation of newly developed functional materials which had resistance against nonspecific adsorption of proteins.42−44 The modified SiN surface might have a property of less protein adsorption in the presence of a trace amount of Tween-20. As a result, the chip did not need further surface treatments such as poly(ethylene glycol) grafting as was the case of a gold surface of SPR and QCM systems, revealing that the SiN sensor chips are suitable for evaluation of complicated samples containing other contaminants. Recovery tests of AFP in the presence of human serum were performed (Figure 8). The response upon injecting serum containing AFP increased depending on the amount of AFP added, and also Δλ for human serum itself increased, suggesting that high concentrations of serum proteins may cause high background. When Δλ corresponding to nonspecific binding of human serum (Δλ for each diluted human serum alone) was subtracted from total Δλ observed by injecting each AFP dissolved in 1000-, 100-, or 10-times diluted human serum with the running buffer, the given figures appeared to be almost the same as that of the standard AFP dissolved in the running buffer. Undiluted serum showed a different behavior, having a high response that might mask AFP binding. These results

reproducible response (Δλ (average) = 0.17 nm, CV = 5.4%, n = 100) for such a long operation (35 h, see Supporting Information Figure S4). A calibration curve was drawn for AFP concentrations, and a limit of detection (LOD) was calculated (Figure 7). Concentration dependence was observed between 100 ng mL−1 and 100 μg mL−1, and the LOD was estimated to be 100 ng mL−1. Since the AFP level of 100 ng mL−1 is an index of cumulative intrahepatic recurrence,41 the proposed RIfS system could be applicable to diagnostics for postoperative prognosis of liver diseases, especially hepatocellular carcinoma. It should be noted that the measurement range spanned 3 orders of magnitude. Taking the measurement range of other label-free AFP detection systems into consideration, these results suggest that the RIfS system might have comparable sensitivity with a wider determination range than that of other label-free systems and suitability toward various biomarkers having a wide concentration range of their thresholds. We evaluated the cross-reactivity against abundant human proteins such as HSA, IgG, transferrin, and fibrinogen (see Supporting Information Figure S5). The sensor chip showed no cross-reactivity with the proteins tested at 100 μg mL−1, demonstrating that not only was nonspecific binding to the modified surface of the chip almost nonexistent but also the cross-reactivity of immobilized anti-AFP was very low. It should 13612

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as in the running buffer. Consequently, the proposed RIfS system has the potential for use as an effective tool for exploring protein−protein interactions, in vitro diagnostics, and other investigations of intermolecular interactions in the fields of medical, biological, chemical, and environmental sciences.

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MATERIALS AND METHODS

Materials. Triethoxysilylpropylmaleamic acid was purchased from Gelest, Inc. (PA, USA). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide, hydrochloride (EDC) was purchased from Dojindo Laboratories (Kumamoto, Japan). NHS was purchased from Thermo Fisher Scientific Inc. (MA, USA). 2-Morpholinoethanesulfonic acid (MES) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Anti-AFP antibody (clone 1D5, subclass IgG1/k; clone 6D2, subclass IgG2a/k) were purchased from Mikuri Immunology Laboratory, Inc. (Osaka, Japan). AFP was purchased from Acris Antibodies GmbH (Herford, Germany). SiN sensor chips (18 × 26 mm2) and PDMS flow cells (flow cell size L 16 × W 2.5 × H 0.1 mm3, total volume 4 μL) were purchased from Konica Minolta Technology Center, Inc. (Tokyo, Japan). Pooled human serum was purchased from Kohjin Bio Co., Ltd. (Saitama, Japan). Other reagents were all purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Preparation of Carboxy Group-Introduced Sensor Chips. Triethoxysilylpropylmaleamic acid (100 μL) was added to 10 mL of Milli-Q water containing acetic acid (100 μL), and the solution was stirred for 1 h at room temperature. Then the sensor chip was soaked in solution for 1 h at room temperature, and after being washed with Milli-Q water, sensor chips were heated at 80 °C for 1 h to obtain a carboxy-introduced sensor chip. Other reaction conditions are discussed in Table S1(see Supporting Information). Atomic Force Microscopy (AFM). Roughness analysis of the SiN sensor chips was performed using AFM (SPA-400, Seiko Instruments, Inc., Chiba, Japan). All AFM images were taken in dynamic force mode. SI-DF 20 cantilevers (coated on its back with alminium) with a resonance frequency of 118 kHz and a spring constant of 9 N/m were used for imaging in air. Measurements were performed four times. X-ray Photoelectron Spectroscopy (XPS). Surface elemental analysis of the SiN sensor chips was performed by XPS (Quantera SXM, ULVAC PHI, Inc., Kanagawa, Japan). XPS was performed using a PHI 5700 X-ray photoelectron spectrometer equipped with a monochromatic Al Kα X-ray source (1486.6 eV) at a takeoff angle of 45° from the sensor chip surface. Ellipsometry. Thickness measurements were performed using VVASE (J.A. Woollam Co., Inc., NE, USA) equipped with a xenon or halogen lamp at an incident angle of 65°, 70°, and 75°. Thicknesses of the silane layers were determined with a Cauchy model assumed refractive index of 1.472 at 590 nm for triethoxysilylpropylmaleamic acid. This measurement was performed three times. Preparation of Anti-AFP-Immobilized Sensor Chips. The carboxy group-introduced sensor chip was soaked in 50 mM NHS and 200 mM EDC in 25 mM MES buffer (pH 5.0) for 20 min at room temperature. After the sensor chip was washed with Milli-Q water twice and dried by air blowing, the sensor chip was soaked in 20 μg mL−1 anti-AFP solution in 10 mM acetate buffer (pH 6.0) for 30 min at room temperature. The sensor chip was washed with Milli-Q water twice and dried by air blowing again; then the sensor chip was soaked in 1 M ethanolamine−HCl solution (pH 8.5) for 20 min at room temperature. Finally, the sensor chip was washed with Milli-Q water and dried by air blowing. The obtained sensor chip was immediately used for RIfS measurements. RIfS Measurements. The anti-AFP-immobilized sensor chip and PDMS flow cell were equipped to the RIfS apparatus (MI-Affinity LCR-01, Konica Minolta Technology Center, Inc. Tokyo, Japan) to construct a continuous flow microfluidic RIfS system. All RIfS data were collected at 25 °C at a flow rate of 20 μL min−1 using a syringe pump (Econoflo Syringe Pump 70-2205, Harvard Apparatus, MA, USA) or a reciprocating pump (LC-20AD, Shimadzu, Kyoto, Japan). After the baseline of RIfS was stable with a standard running buffer consisting of 9.58 mM phosphate buffer containing 2.68 mM KCl, 138

Figure 8. AFP recovery test in the presence of human serum. Specific signals for AFP (0.1 (blue), 1 (red), and 10 (green) μg mL−1) were calculated by subtracting Δλ obtained by injection of human serum alone from Δλ obtained by injection of human serum containing AFP.

demonstrate that 10-times diluted serum can be used for labelfree detection of AFP by the RIfS system without any interference by the serum proteins. It has been reported that addition of a secondary antibody during binding of antigen to an antibody-immobilized SPR sensor chip enhances sensitivity due to formation of sandwichtype complexes.45 As is in the case of SPR detection, a secondary antibody effect was also observed in the RIfS system, where the binding of the secondary antibody gave a two-times higher response than that before addition (see Supporting Information Figure S7). Therefore, this technique could contribute to further enhancement of sensitivity in the RIfS system. Fluorescence labeling was carried out for the secondary antibody to examine the effect of fluorophores on RIfS sensing. Among them, Alexa 555 and Alexa 594, which have absorptions and fluorescences in the vicinity of the minimum reflectance, are likely to influence the RIfS response. As can be seen, labeling did not affect the response significantly, suggesting that the small amounts of chromophores or fluorophores did not affect detection, as the incident light was strong enough to give a stable RIfS response.

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CONCLUSIONS In this study, we developed label-free detection of AFP based on the antigen−antibody reaction by the RIfS system. The carboxy-introduced SiN sensor chip was obtained using triethoxysilylpropylmaleamic acid, and immobilization of the antibody was accomplished via amide bond formation. The modified sensor chip was fully characterized by AFM, XPS, and the RIfS response. Using the anti-AFP-immobilized SiN sensor chip, selection of running buffer and optimization of salt concentrations for antigen−antibody interaction were carried out to enhance sensitivity. The RIfS system showed a reversible response through 100 on/off cycles with high reproducibility (AFP 1 μg mL−1, CV = 5.4%) and had a wide measurement range (from 100 ng mL−1 to 100 μg mL−1). The anti-AFPimmobilized SiN sensor chip showed no cross-reactivity with abundant human serum proteins at 100 μg mL−1, and AFP dissolved in diluted human sera was detected sensitively as well 13613

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Notes

mM NaCl, and 0.001% Tween-20, pH 7.4 (0.001% PBST), which indicated that a wavelength at minimum value of reflectance was constant with time, measurement was started. Samples (100 μL) were injected by either a manual injector or an autosampler (SIL-20AC, Shimadzu, Kyoto, Japan). When Δλ was determined on a sensorgram, an average and a standard deviation of Δλ at 600 ± 15 s (data acquisition interval was about 1 s) after injection of AFP were employed. Running Buffer Optimization. An optimal NaCl concentration was screened by injecting 10 μg mL−1 AFP dissolved in 0.001% PBST containing various concentrations of NaCl (0, 75, 138, 300, 500, and 1000 mM). These injections were carried out in duplicate. Then, as running buffers, phosphate (9.58 mM), Tris-HCl (10 mM), MOPS (10 mM), and HEPES (10 mM) buffers containing 138 mM NaCl and 2.68 mM KCl were prepared (pH 7.4) and flowed at a flow rate of 20 μL min−1 (25 °C). Glycine−HCl (10 mM, pH 1.5) was injected to wash the surface of the sensor chip at 300 s after starting the RIfS measurement, followed by injection of AFP (1 and 10 μg mL−1). Glycine−HCl (10 mM, pH 1.5) was injected at appropriate intervals (1200, 1800, and 2400 s) to regenerate the surface. Concentration Sependence of AFP Response. Various concentrations of AFP (0.1, 0.3, 1, 3, 10, 30, and 100 μg mL−1) dissolved in 0.001% PBST were injected. Glycine−HCl (10 mM, pH 1.5) was injected before and after injection of AFP. The injection interval of each sample was 600 s. LOD was determined by a minimum AFP concentration, which was satisfied the following conditions: Δλ at injection of AFP solution was bigger than Δλ at injection of running buffer plus 3 standard deviations. The running buffer was 0.001% PBST (20 μL min−1, 25 °C), and each sample concentration was measured three times. Cross-Reactivity with Human Serum Proteins. Purified human serum proteins (0.1 or 1 mg mL−1) including HSA, IgG, transferrin, and fibrinogen were injected. The running buffer was 0.001% PBST (20 μL min−1, 25 °C). AFP Recovery Test. AFP (0.1, 1, and 10 μg mL−1) dissolved in undiluted human serum and diluted human sera by 10, 100, and 1000 times with 0.001% PBST were injected. The running buffer was 0.001% PBST (20 μL min−1, 25 °C). Specific signals for AFP were calculated by subtracting Δλ obtained by injection of human serum alone from Δλ obtained by injection of human serum containing AFP. Sandwich-Type Antigen−Antibody Reaction Using a Secondary Antibody. Anti-AFP (clone 6D2) was labeled with Alexa Fluor 488, 555, 594, and 647 by APEX Antibody Labeling Kits (Life Technologies, CA, USA) and used as a secondary antibody. Existence of antibody labeled with fluorescent dye was confirmed using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, MA, USA). The RIfS measurement was performed using an anti-AFP (clone 1D5)-immobilized sensor chip. After AFP (10 μg mL−1) was injected, each secondary antibody (10 μg mL−1) was injected. The running buffer was 0.001% PBST (20 μL min−1, 25 °C).

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Part of this study was supported by the innovation promotion program of New Energy and Industrial Technology Development Organization (Kanagawa, Japan).

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ASSOCIATED CONTENT

S Supporting Information *

Modification conditions of SiN sensor chip using silane coupling agents; XPS depth profile of SiN sensor chip; AFM images of SiN sensor chip before and after the silane coupling reaction; RIfS measurement data using anti-AFP-immobilized sensor chip with BSA blocking or Tween-20; reproducibility of anti-AFP-immobilized sensor chip; cross-reactivity of anti-AFPimmobilized sensor chip; RIfS measurement data of the nonspecific adsorption (NSA) of BSA for anti-AFP-immobilized sensor chip and sandwich-type antigen−antibody reaction using a secondary antibody. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 13614

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dx.doi.org/10.1021/la302221y | Langmuir 2012, 28, 13609−13615