Repetitive Immunoassay with a Surface Acoustic Wave Device and a

Sep 17, 2015 - This work describes a sensor to be incorporated into the on-site monitoring system of airborne house dust mite (HDM) allergens. A surfa...
0 downloads 8 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

Repetitive Immunoassay with a Surface Acoustic Wave Device and a Highly Stable Protein Monolayer for On-Site Monitoring of Airborne Dust Mite Allergens Koji Toma,† Daisuke Miki,‡ Chisato Kishikawa,‡ Naoyuki Yoshimura,§ Kumiko Miyajima,†,∥ Takahiro Arakawa,† Hiromi Yatsuda,§,⊥ and Kohji Mitsubayashi*,†,‡ †

Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan ‡ Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan § Japan Radio Co. Ltd., Saitama 356-8510, Japan ∥ Japan Society for the Promotion of Science, Tokyo 102-0083, Japan ⊥ OJ-Bio Ltd., NE1 4EP Newcastle upon Tyne, United Kingdom S Supporting Information *

ABSTRACT: This work describes a sensor to be incorporated into the on-site monitoring system of airborne house dust mite (HDM) allergens. A surface acoustic wave (SAW) device was combined with self-assembled monolayers of a highly stable antibody capture protein on the SAW surface that have high resistance to pH change. A sandwich assay was used to measure a HDM allergen, Der f 1 derived from Dermatophagoides farinae. Capture antibodies were cross-linked to a protein G based capture layer (ORLA85) on the sensor surface, thereby only Der f 1 and detection antibodies were regenerated by changing pH, resulting in fast repetition of the measurement. The sensor was characterized through 10 repetitive measurements of Der f 1, which demonstrated high reproducibility of the sensor with the coefficient of variation of 5.6%. The limit of detection (LOD) of the sensor was 6.1 ng· mL−1, encompassing the standard (20 ng·mL−1) set by the World Health Organization. Negligible sensor outputs were observed for five different major allergens including other HDM allergens which tend to have cross-reactivity to Der f 1 and their mixtures with Der f 1. Finally, the sensor lifetime was evaluated by conducting three measurements per day, and the sensor output did not substantially change for 4 days. These characteristics make the SAW immunosensor a promising candidate for incorporation into on-site allergen monitoring systems. μL of buffer solution, as a standard for sensitization and development of asthma.13,14 To prevent development of allergy diseases, it is desirable to know the exposure level of airborne HDM allergens and to avoid contact with them because inhalation and skin contact are the main routes of uptake.11,15,16 The concentration distribution of the airborne allergens in a living environment, however, changes over time, influenced by human activity, temperature, or humidity change.17,18 Therefore, there is a need to develop a system combining two functions: collecting airborne allergens and continuously measuring the collected allergens.19−21 Along with development of an airborne allergen collecting device, it is

A

llergies are disorders of the immune system and are becoming the subject of great concern worldwide. They are accompanied by symptoms such as rhinitis or asthmatic attack.1−5 It has been reported that more than 40% of children by the age of 6 years could be diagnosed with allergic rhinitis, and it is estimated that 300 million people suffer from asthma in the world.3,6 One of the major causes of allergy is mite allergen which is found widely in our living environments.7,8 Of particular concern are the house dust mite (HDM) allergens including Dermatophagoides farinae group 1 (Der f 1) and 2 (Der f 2) and Dermatophagoides pteronyssinus group 1 (Der p 1) and 2 (Der p 2) that are known to exhibit strong allergenicities. Therefore, many studies to quantify them in dust collected from floors have been carried out.7,9−12 The World Health Organization (WHO) has set 2 μg HDM allergens/g of dust, equivalent to 20 ng·mL−1 provided 1 g of dust is diluted in 100 © 2015 American Chemical Society

Received: July 10, 2015 Accepted: September 17, 2015 Published: September 17, 2015 10470

DOI: 10.1021/acs.analchem.5b02594 Anal. Chem. 2015, 87, 10470−10474

Article

Analytical Chemistry

Figure 1. (a) Schematic diagram of the SAW immunosensor chip. Shear horizontal (SH)-SAW propagates along a delay line and reflects back to the transducer. (b) Steps used for the ORLA85 protein and PEG-thiol monolayer formation and repetitive sandwich assay. The steps for sandwich assay and regeneration (indicated with yellow arrows) were repeated. cAb, capture antibody; dAb, detection antibody.

Dermatophagoides farinae group 2 allergen (Der f 1) was from Seikagaku Bio Business. Coarse extraction solutions of Dermatophagoides pteronyssinus allergen (Der p 1), ragweedpollen allergen (Amb a 1), and Alternaria alternata allergen (Alt a 1) were purchased from ITEA. Japanese cedar (Cryptomeria japonica) pollen allergen (Cry j 1) was from Hayashibara Biochemical Laboratories. PEGylated bis(sulfosuccinimidyl)suberate (BS(PEG)5) was from Thermo Fisher Scientific. Orla IgGBinder-G kit containing a mixed solution of ORLA85 protein and 11-mercaptoundec-11-ylhexaethylene glycol (HSC11-EG6, PEG-thiol), tris(2-carboxyethyl)phosphine (TCEP), and Tris-HCl buffer solution (150 mM NaCl, pH 7.5) was from Orla Protein Technologies. Phosphate buffered saline (PBS) with Tween 20 (PBS-T) was prepared by adding Tween 20 (0.05%) in PBS solution (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, pH 7.4). Acetate buffered saline with 0.05% Tween 20 (ABS-T) was prepared by adding acetic acid solution to sodium acetate solution until the desired pH is obtained and mixing it with Tween 20. A chromogenic substrate solution for control experiments with ELISA was prepared by mixing 30% H2O2 and 0.05% (w/v) ABTS in citrate phosphate buffer (70 mM, pH 4.2) at a volume ratio of 1/1000. SAW Immunosensor Preparation. The SAW chip was designed to excite a shear-horizontal SAW (SH-SAW) on a 36Y-X quartz substrate with the center frequency of 250 MHz. On the chip, a SAW is excited at an input interdigital transducer and then propagates back and forth between a reflector and an output transducer over a delay line that was used as a sensing region (see Figure 1a). The transducers and the delay line were fabricated by depositing a 90 nm gold film and lift-off process. The SAW chip has an air-cavity which is composed of epoxy walls that surround the transducer and the glass lid. The walls were built by a photolithography technique using a thick epoxybased photo resist; the wall thickness was about 40 μm. The gold coated delay line surface was modified with a SAM of ORLA85 protein and PEG-thiol by forming a sulfur−gold bond as illustrated in Figure 1b. PEG-thiol fills the spaces between ORLA85 proteins on the gold surface, which reduces nonspecific protein adsorption in subsequent protein additions. Prior to the SAM formation, the surface was washed with 95% ethanol (Kanto Chemical) followed by incubation with 2% (v/v) Hellmanex III solution (Hellma Analytics) in ultrapure water for 20 min to clean the Au surface followed by wash with ultrapure water. After blow drying with N2 gas, the surface was passivated by incubation for 5 min in 1% (v/v) BME in ultrapure water followed by a wash with ultrapure water. The

important to develop a sensor measuring them sensitively and selectively with a short measurement time. In general, allergens are quantified by using immunoassay with, e.g., enzyme-linked immunosorbent assay (ELISA).22−24 Although it provides high throughput, sensitivity, and selectivity, this has several disadvantages: the test requires long preparation times; it can only be carried out in suitably equipped laboratory; and it cannot be incorporated into on-site monitoring systems. For practical airborne allergen monitoring applications, a single sensor chip needs to be reused multiple times for long periods of time to prevent frequent exchange of the chip. Up to now, a great effort has been made to realize techniques which allow mitigating nonspecific binding of proteins25 and regenerating a sensor surface.26 In this study, we have focused on developing a sensor which allows the measurement of HDM allergens repeatedly in a short-time (semicontinuously) on the same sensor chip surface by combining a surface acoustic wave (SAW) device27,28 and a highly stable antibody capture protein monolayer−SAW immunosensor. The sandwich immunoassay was used on the sensor surface which was modified with self-assembled monolayers (SAMs) of the ORLA85 protein.29−32 The ORLA85 protein is a fusion of the Fc-binding domains of Streptococcal Protein G with a self-assembling scaffold protein. This allows the cAb to be immobilized on the sensor surface in the correct orientation. The cAb was covalently cross-linked to this capture layer so that only antigens and detection antibodies (dAbs) were removed through regeneration. The ORLA85 protein exhibits high stability against pH change, which enables regenerating the surface without substantial degradation. In this study, we first developed the SAW immunosensor and characterized the optimal conditions for cAb binding, crosslinking, and surface regeneration. Then, multiple measurement of Der f 1 was demonstrated with the SAW immunosensor to investigate a potential of the sensor for airborne allergen monitoring in a living environment.

■

EXPERIMENTAL SECTION Reagents and Chemicals. Ethanolamine and β-mercaptoethanol (BME), horseradish peroxidase-streptavidin conjugate (HRP-SA), and 2,2′-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS) were from SigmaAldrich. Sodium dodecyl sulfate (SDS), sodium acetate, and acetic acid were from Wako. Dermatophagoides farinae group 1 allergen (Der f 1) and capture (cAb) and detection (dAb) antibodies for Der f 1 were from Indoor Biotechnologies. 10471

DOI: 10.1021/acs.analchem.5b02594 Anal. Chem. 2015, 87, 10470−10474

Article

Analytical Chemistry ORLA85 protein and PEG-thiol mixture was activated by addition of TCEP-HCl at a final concentration of 25 mM and then applied to the surface. After 10 min of incubation, the protein solution was removed and the surface was washed with 1% (w/v) SDS in ultrapure water to remove protein and PEGthiol that was not covalently bound. The surface was rinsed with PBS solution, and the cycle of addition of protein solution and SDS wash was repeated twice. Finally, the surface was washed once with 100 mM HCl followed by a thorough wash with ultrapure water. For the binding of cAb to the ORLA85 protein, cAb dissolved in PBS-T at a given final concentration was added and incubated for 10 min. Owing to protein G, the bound cAbs were highly oriented on the surface, leading to high accessibility to antigen binding sites of cAb. After rinsing with PBS-T, a cross-linking reagent, 0.5 mM BS(PEG)5 in PBS-T, was added to cross-link cAb to protein G via surface exposed lysine residues of cAb. The sensor was incubated for 10 min and rinsed, followed by terminating the reaction with 1 M ethanolamine pH 8.4. Finally, non-cross-linked cAb was removed by washing with 100 mM HCl and rinsing with PBS-T. The cross-linked cAb hardly dissociates, enabling repetitive binding and regeneration of Der f 1 and dAb. All the above-described preparation processes were conducted at room temperature. As a control, ELISA was used to measure Der f 1. 2.0 μg· mL−1 cAb dissolved in carbonate−bicarbonate buffer solution (80 mM, pH9.5) was added and incubated for 20 h at 4 °C on a plastic microtiter plate. Afterward, the well was incubated with 0.1% (w/w) bovine serum albumin in PBS-T (blocking buffer) for 1 h at room temperature in order to reduce nonspecific bindings of the proteins. After 1 h of incubation with Der f 1 in PBS-T at a given concentration, 2.0 μg·mL−1 dAb in PBS-T was added and incubated for another 1 h followed by 30 min of incubation with 0.25 μg·mL−1 HRP-SA in PBS-T so that HRP bound to biotin-labeled dAb by streptavidin−biotin binding. Three rinses with PBS-T were performed at the end of each step. Finally, the chromogenic substrate solution was added to produce blue colored products through the HRP-mediated enzymatic reaction, followed by stopping the reaction with 1% (w/v) SDS in ultrapure water.

Figure 2. Effects of (a) pH of cAb solution and (b) cAb concentration on cAb binding to protein G linked to ORLA85 protein. The insets display the binding kinetics of cAb under different conditions.

most efficient pH corresponds to the optimum pH for binding of antibodies to protein G, which occurs under acidic conditions.33 Figure 2b shows the shift ΔPcAb at different cAb concentrations ranging from 3 to 300 μg·mL−1 in PBS-T at pH 6.0. The shift ΔPcAb increases with increasing cAb concentration and approached a plateau. The concentrations over 100 μg·mL−1 provided little change in the phase because this concentration was sufficient to fill the binding sites of ORLA85 on the sensor surface in 10 min of incubation; therefore, 100 μg·mL−1 cAb was used in the subsequent experiments. Repetitive Measurement of Der f 1 with the SAW Immunosensor. To immobilize cAb on ORLA85 protein, cross-linking with BS(PEG)5 was undertaken. As shown in Figure 3, cross-linking was repeated three times so as to

■

RESULTS AND DISCUSSION Influence of pH and Concentration of the cAb Solution on cAb Binding to ORLA85. First, the influence of the cAb solution pH and its concentration on the binding of cAb to ORLA85 protein was investigated. Figure 2a shows the phase shift ΔPcAb attributed to cAb binding to protein G on the gold sensor surface under different pH values. The velocity of the SAW which propagates underneath the gold film is changed due to the binding events on the surface. The phase response of the output signal of the delay line is related to the SAW velocity; thereby, we can estimate the amount of the binding event from the measured phase shift of the output signal of the delay line. The plotted ΔPcAb was obtained by averaging the phase shift from the initial baseline over the last 30 s as shown in the inset of Figure 2a. 100 μg·mL−1 cAb in buffer solution with varied pH from 4.0 to 8.0 was added and incubated for 10 min. Here, two different buffer solutions were used for pH 4.0− 5.0 (ABS-T) and for pH 6.0−8.0 (PBS-T). The shift ΔPcAb increased with increasing pH and peaked at pH 6.0. The maximum shift at pH 6.0 was about 4-fold larger than the shift at pH 4.0 which was the minimum within pH 4.0−8.0. The

Figure 3. Cross-linking between cAb and protein G with BS(PEG)5. The cross-linking step was repeated three times so as to increase the immobilization rate Δa/Δb. CL, cross-linking with BS(PEG)5; R, rinsing; Re, regeneration with HCl; EA, ethanolamine terminating the reaction.

increase the immobilization rate of cAb. The immobilization rate Δa/Δb is a ratio between the phase shift ΔPcAb before and after regeneration, indicating how much cAb remains on the sensor surface through cross-linking. As a result, the first crosslinking resulted in the rate Δa/Δb = 85.5%; however, the third enabled one to attain Δa/Δb = 99.5%, implying that almost all cAbs on ORLA85 were immobilized on the sensor surface. Note that, in this procedure, ethanolamine was applied only after the third cross-linking to eventually terminate the reaction. Prior to repetitive measurement of Der f 1, influence of HCl solution pH on regeneration was investigated. HCl solutions with five different pH values ranging from 1.0 to 3.0 were used 10472

DOI: 10.1021/acs.analchem.5b02594 Anal. Chem. 2015, 87, 10470−10474

Article

Analytical Chemistry to regenerate the sensor surface on which Der f 1 and dAb were bound. Figure 4a represents a regeneration rate at each pH.

Figure 5. (a) Calibration curves of the SAW immunosensor (●) and ELISA (Δ, control) for Der f 1. (b) Sensor responses to a different allergens and a mixture of allergens. The hatched indicates the sample containing Der f 1.

Figure 4. (a) Effect of pH of HCl solution on regeneration rate ΔPRe/ ΔPAg and (b) the 1st (●), 5th (■), and 10th (▲) measurement of Der f 1. These measurements were undertaken successively.

PEG-thiol was performed by using ELISA, and the resultant absorbance is together shown in Figure 5a. ELISA showed LOD = 0.58 ng·mL−1 (23 pM). Although ELISA shows about 1 order of magnitude better LOD, the SAW immunosensor has superiority in capability of repetitive measurement, short measurement time, and size. These characteristics are important when incorporating the sensor into the monitoring system. As airborne allergens in a living environment contain not only the allergen derived from HDMs but also a variety of allergens such as pollens or fungi, the selectivity of the SAW immunosensor was also evaluated with 11 different allergen solutions prepared with 6 different allergens, Der f 1, Der f 1, Der p 1, Amb a 1, Cry j 1, and Alt a 1. The concentration of each allergen in a sample solution was 100 ng·mL−1, and the phase shift ΔPAg was measured after dAb binding. Figure 5b shows relative phase shifts to that of Der f 1 (RΔPAg) and from which the shift of a blank sample, containing no antigens, was subtracted. Although all samples containing Der f 1 exhibited similar shift, those without Der f 1 resulted in almost no shift. Especially, allergens Der f 1 and Der p 1, which are derived from a mite body of Dermatophagoides farinae and from Dermatophagoides pteronyssinus, respectively, have similar protein conformations to Der f 1, which is derived from a digestive organ of Dermatophagoides farinae, and therefore tend to show a cross-reactivity.9,34−36 With the SAW immunosensor, however, the relative shift RΔPAg for them were negligibly small, which demonstrates high selectivity of the sensor. Considering the application of the SAW immunosensor to assessment of allergens in a living environment, it is important to maintain a capability of rapid and repetitive measurement for a long period of time. To evaluate the sensor lifetime, 100 ng· mL−1 Der f 1 was measured three times a day, including regeneration. The result presented in S-Figure 2 shows that the sensor performance did not substantially change for the first 4 days, and then, the output decreased as days went by. At day 8, the phase shift was about 83% of that at the day 1. This degradation of the sensor can be attributed to denaturation of cAb through repeated regeneration with HCl.

The regeneration rate was a ratio ΔPRe/ΔPAg, where ΔPAg was a phase difference between that before regeneration and a baseline and ΔPRe was the difference between those before and after regeneration, indicating how much the phase P recovers after regeneration (see Figure 4b). The rates ΔPRe/ΔPAg were larger than 90% when using pH 1.0, 1.5, and 2.0. Especially when using pH 1.0, the highest reproducibility was observed with the smallest standard deviation; thus, HCl solution with pH 1.0 was chosen for regeneration. After cAb immobilization, 100 ng·mL−1 Der f 1 was repeatedly measured 10 times. In the measurement, the SAW immunosensor was incubated with Der f 1 solution for 10 min, followed by rinsing with PBS-T. Then, dAb was added and the sensor was incubated for another 10 min. After rinsing with PBS-T, the sensor surface was regenerated with 100 mM HCl to remove bound Der f 1 and dAb. Figure 4b displays the overlaid phase changes for the 1st, 5th, and 10th measurements. The phase change showed high reproducibility as displayed with the coefficient of variation (C.V.) of the phase shift ΔPAg of 5.6% for 10 measurements. In addition, owing to the crosslinking of cAb, it takes 24 min for a single measurement, whereas it takes almost a day for ELISA. This indicates that the SAW immunosensor holds a potential to measure the airborne allergens twice in an hour. As seen in S-Figure 1, showing 10 successive measurements, with the progress of repetition of the Der f 1 measurement, the baseline gradually shifted to a negative value. It was because of imperfection of surface regeneration. Enough binding sites of ORLA85, however, remained; the influence on the reproducibility was negligible. Afterward, the phase shifts ΔPAg at different Der f 1 concentrations were observed, and they were plotted in Figure 5a. The plots were fitted with the following equation with a coefficient of correlation of 0.992: fluorescence intensity(cps) = A + (B − A)/(1 + ([Der f 1]/C D))

(1)

where A = 0.46, B = 3.7, C = 25, and D = 1.1 are the coefficients and [Der f 1] is the concentration of added Der f 1 in ng·mL−1. The limit of detection (LOD) of the sensor was determined at the concentration where the mean phase shift for a blank sample ΔPblank plus three times the standard deviation σ equals the phase shift ΔPAg. It resulted in the developed sensor LOD = 6.1 ng·mL−1 that is equivalent to 240 pM. As a comparison, the same sandwich assay experiment without ORLA85 protein and

■

CONCLUSIONS In this paper, we have described an immunosensor employing a SAW device and highly stable antibody capture SAMs on the sensor surface in order to enable semicontinuous measurement of indoor airborne allergens. A HDM allergen, Der f 1, was 10473

DOI: 10.1021/acs.analchem.5b02594 Anal. Chem. 2015, 87, 10470−10474

Article

Analytical Chemistry

(11) Tovey, E. R.; Almqvist, C.; Li, Q.; Crisafulli, D.; Marks, G. B. J. Allergy Clin. Immunol. 2008, 122 (1), 114−118. (12) Antens, C. J. M.; Oldenwening, M.; Wolse, A.; Gehring, U.; Smit, H. a.; Aalberse, R. C.; Kerkhof, M.; Gerritsen, J.; De Jongste, J. C.; Brunekreef, B. Clin. Exp. Allergy 2006, 36 (12), 1525−1531. (13) World Health Organization. Bull. World Health Organ. 1988, 66 (6), 769−780. (14) Miyajima, K.; Itabashi, G.; Koshida, T.; Tamari, K.; Takahashi, D.; Arakawa, T.; Kudo, H.; Saito, H.; Yano, K.; Shiba, K.; Mitsubayashi, K. Environ. Monit. Assess. 2011, 182 (1−4), 233−241. (15) Paufler, P.; Gebel, T.; Dunkelberg, H. Rev. Environ. Health 2001, 16 (1), 65−80. (16) Sander, I.; Zahradnik, E.; Kraus, G.; Mayer, S.; Neumann, H. D.; Fleischer, C.; Brüning, T.; Raulf-Heimsoth, M. PLoS One 2012, 7 (12), e52981. (17) Knibbs, L. D.; He, C.; Duchaine, C.; Morawska, L. Environ. Sci. Technol. 2012, 46 (1), 534−542. (18) Sakaguchi, M.; Inouye, S.; Sasaki, R.; Hashimoto, M.; Kobayashi, C.; Yasueda, H. J. Allergy Clin. Immunol. 1996, 97 (5), 1040−1044. (19) Luczynska, C. M.; Li, Y.; Chapman, M. D.; Platts-Mills, T. A. Am. Rev. Respir. Dis. 1990, 141 (2), 361−367. (20) Miyajima, K.; Suzuki, Y.; Miki, D.; Arai, M.; Arakawa, T.; Shimomura, H.; Shiba, K.; Mitsubayashi, K. Talanta 2014, 123, 241− 246. (21) Custis, N. J.; Woodfolk, J. a.; Vaughan, J. W.; Platts-Mills, T. a E. Clin. Exp. Allergy 2003, 33 (7), 986−991. (22) Merritt, A. S.; Andersson, N.; Almqvist, C. Environ. Sci. Technol. 2013, 47 (8), 3796−3799. (23) Luczynska, C. M.; Arruda, L. K.; Platts-Mills, T. a; Miller, J. D.; Lopez, M.; Chapman, M. D. J. Immunol. Methods 1989, 118 (2), 227− 235. (24) Douwes, J.; Thorne, P.; Pearce, N.; Heederik, D. Ann. Occup. Hyg. 2003, 47 (3), 187−200. (25) Bourquin, Y.; Reboud, J.; Wilson, R.; Zhang, Y.; Cooper, J. M. Lab Chip 2011, 11 (16), 2725−2730. (26) Pritchard, D. J.; Morgan, H.; Cooper, J. M. Anal. Chem. 1995, 67 (19), 3605−3607. (27) Goto, M.; Yatsuda, H.; Kondoh, J. Jpn. J. Appl. Phys. 2013, 52 (7S), 07HD10. (28) Kogai, T.; Yoshimura, N.; Mori, T.; Yatsuda, H. Jpn. J. Appl. Phys. 2010, 49 (7S), 07HD15. (29) Le Brun, A. P.; Soliakov, A.; Shah, D. S. H.; Holt, S. a.; McGill, A.; Lakey, J. H. Biomed. Microdevices 2015, 17 (3), 1−10. (30) Le Brun, A. P.; Holt, S. a; Shah, D. S. H.; Majkrzak, C. F.; Lakey, J. H. Biomaterials 2011, 32 (12), 3303−3311. (31) Le Brun, A. P.; Shah, D. S. H.; Athey, D.; Holt, S. a.; Lakey, J. H. Int. J. Mol. Sci. 2011, 12 (8), 5157−5167. (32) Terrettaz, S.; Ulrich, W.-P.; Vogel, H.; Hong, Q.; Dover, L. G.; Lakey, J. H. Protein Sci. 2002, 11, 1917−1925. (33) Akerstrom, B.; Bjorck, L. J. Biol. Chem. 1986, 261 (22), 10240− 10247. (34) Chruszcz, M.; Pomés, A.; Glesner, J.; Vailes, L. D.; Osinski, T.; Porebski, P. J.; Majorek, K. a.; Heymann, P. W.; Platts-Mills, T. a E.; Minor, W.; Chapman, M. D. J. Biol. Chem. 2012, 287 (10), 7388− 7398. (35) Lind, P.; Hansen, O. C.; Horn, N. J. Immunol. 1988, 140 (12), 4256−4262. (36) Chruszcz, M.; Chapman, M. D.; Vailes, L. D.; Stura, E. a.; SaintRemy, J. M.; Minor, W.; Pomés, A. J. Mol. Biol. 2009, 386 (2), 520− 530.

measured on the SAW immunosensor surface by using a sandwich assay. The sensor LOD for Der f 1 measurement was determined as 6.1 ng·mL−1, and it provided a broad dynamic range to 1000 ng·mL−1 with high selectivity. Additionally, Der f 1 could be measured in 24 min with high reproducibility for 4 days; these results demonstrate the viability of the sensor for semicontinuous measurement of airborne HDM allergens with the monitoring system into which the SAW immunosensor is to be incorporated. Although the LOD encompasses a standards for sensitization to mites (20 ng·mL−1) by the WHO, we plan to pursue further improvement in sensitivity because sampled airborne allergens with a collecting device in the monitoring system can be lost through transport to the sensor. This could be achieved by tuning the center frequency of the SAW device or employing a detection antibody modified with a substance increasing local viscosity.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02594. Sensor response changes over 10 repetitive measurements and over 8 days of successive measurement (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81-3-5280-8094. Notes

The authors declare the following competing financial interest(s): H.Y. is the technical director of a company that commercializes products related to the technologies presented in this study.

■

ACKNOWLEDGMENTS This work was supported by Japan Radio Co. Ltd., the Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research System, the Japan Science and Technology Agency (JST), and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Special Funds for Education and Research “Research Promotion of Neo-Biology”.

■

REFERENCES

(1) Togias, A. J. Allergy Clin. Immunol. 2003, 111 (6), 1171−1183. (2) Beasley, R.; Keil, U.; Von Mutius, E.; Pearce, N. Lancet 1998, 351 (9111), 1225−1232. (3) Pawankar, R.; Canonica, G. W.; Lockey, R. F.; Holgate, S. T., Eds. WAO White Book on Allergy 2011−2012: Executive Summary; WAO: Milwaukee, WI, 2011. (4) Arbes, S. J., Jr.; Gergen, P. J.; Elliott, L.; Zeldin, D. C. J. Allergy Clin. Immunol. 2005, 116 (2), 377−383. (5) Jackson, A. P.; Foster, A. P.; Hart, B. J.; Helps, C. R.; Shaw, S. E. Vet. Dermatol. 2005, 16 (1), 32−38. (6) Wright, A. L.; Holberg, C. J.; Martinez, F. D.; Halonen, M.; Morgan, W.; Taussig, L. M. Pediatrics 1994, 94 (6 Pt1), 895−901. (7) Platts-Mills, T. a; Vervloet, D.; Thomas, W. R.; Aalberse, R. C.; Chapman, M. D. J. Allergy Clin. Immunol. 1997, 100, S2−S24. (8) Ormstad, H.; Johansen, B. V.; Gaarder, P. I. Clin. Exp. Allergy 1998, 28 (6), 702−708. (9) Takai, T.; Kato, T.; Yasueda, H.; Okumura, K.; Ogawa, H. J. Allergy Clin. Immunol. 2005, 115 (3), 555−563. (10) Nuttall, T. J.; Lamb, J. R.; Hill, P. B. Res. Vet. Sci. 2001, 71 (1), 51−57. 10474

DOI: 10.1021/acs.analchem.5b02594 Anal. Chem. 2015, 87, 10470−10474