ANALYTICAL SCIENCES JULY 2015, VOL. 31
725
2015 © The Japan Society for Analytical Chemistry
Notes
An Organic Field-effect Transistor with an Extended-gate Electrode Capable of Detecting Human Immunoglobulin A Tsukuru MINAMIKI,* Tsuyoshi MINAMI,*† Yui SASAKI,* Ryoji KURITA,** Osamu NIWA,*,** Shin-ichi WAKIDA,*** and Shizuo TOKITO* *Research Center for Organic Electronics (ROEL), Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992–8510, Japan **Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305–8566, Japan ***Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761–0395, Japan
We herein report on the development of an extended-gate type organic field-effect transistor (OFET)-based immunosensor for the detection of human immunoglobulin A (IgA). The titration results of IgA exhibited shifts in the transfer characteristics of the OFET sensor device with increasing IgA concentration. A linear detection range from 0 to 10 μg/mL was realized with a detection limit of 2.1 μg/mL, indicating that the OFET-based immunosensor can be potentially applied to the monitoring of infectious diseases and psychological stress in daily life. Keywords Organic field-effect transistor, immunosensor, label-free, immunoglobulin A, self-assembled monolayer (Received February 26, 2015; Accepted April 16, 2015; Published July 10, 2015)
Introduction To date, academic and industrial research on organic electronic devices such as light-emitting diodes,1 solar cells2 and transistors3 has flourished in the field of electronic engineering. Especially, attempts have been made to use organic field-effect transistors (OFETs) as printed flexible integrated-electronic-circuits, screen displays,4 and radio frequency identification (RFID) tags.5 More importantly for analytical scientists, OFETs could be applied for wearable and disposable biosensors because of their intriguing properties including flexibility, compact integration and low fabrication costs.6 OFET devices would thus be able to endow us with novel sensor platforms for various analytical purposes. In this regard, we have succeeded in developing biosensors based on extended-gate type OFETs, which can detect biomolecules such as proteins,7 saccharides,8 amino acids9 and biogenic amines10 in aqueous media. Especially, we recently reported a label-free immunosensing device for a bovine immunoglobulin G (IgG) using an extended-gate OFET.11 Although the preliminary report revealed that extended-gate OFETs can potentially be employed as a platform for immunosensing, label-free immunosensors based on OFETs are still in their infancy, compared to conventional immunosensor devices.12 Therefore, further explorations of OFET-based immunosensors are necessary. Toward that end, we selected human immunoglobulin A (IgA) as a target analyte. IgA can be found in blood serum (i.e. serum IgA) and mucous secretions including saliva and tears (i.e. To whom correspondence should be addressed. E-mail: [email protected]
†
secretory IgA).13 Secretory IgA constitutes a part of mucosaassociated lymphoid tissue. Moreover, the concentration of secretory IgA in saliva might be related to psychological stress.14,15 To date, it has been revealed that the concentration of secretory IgA was obviously reduced in saliva collected from workers who were exposed to psychological stress.16,17 Hence, the detection of IgA levels is very important for the ongoing management of the risk of infection and psychological stress. In this paper, we demonstrate the first label-free immunosensing of human IgA in a simulated saliva sample using an extended-gate type OFET modified with an anti-human IgA antibody (Fig. 1). The expertise we have obtained herein is expected to open up avenues for the development of healthcare devices based on OFETs in daily life.
Experimental General information about chemicals and apparatus, fabrication of the OFET-device, and immobilization of the anti-IgA antibody are summarized in the Supporting Information. The label-free detection of human IgA was carried out as follows. Label-free detection of human IgA The region of the electrode modified with the anti-human IgA antibody was immersed in a phosphate-buffer saline (PBS) solution of analyte human IgA (0 – 50 μg/mL) with 0.1 wt% human serum albumin (HSA), or interfering proteins ([amylase] = 0.4 μg/mL; [lysozyme] = 0.4 μg/mL; [lactoferrin] = 1.0 μg/mL; [myeloperoxidase] = 3.6 μg/mL) for 2 h at 37° C. After the immunoreaction, the IgA level was electrically detected by the OFET sensing device. The Ag/AgCl electrode
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ANALYTICAL SCIENCES JULY 2015, VOL. 31
Fig. 1 Schematic structure for the extended-gate type organic FET designed for IgA detection. The extended-gate electrode is functionalized with the biotinylated anti-IgA antibody.
Fig. 2 (a) Transfer characteristics (IDS – VGS) of the OFET upon titration with human IgA in a PBS solution with 0.1 wt% HSA. [Human IgA] = 0, 1, 3, 5, 7, 10, 20, 30, and 50 μg/mL. (b) Changes in the threshold voltage (VTH) of the OFET by proteins at various concentrations in a PBS solution with 0.1 wt% HSA. Human IgA (red circle), lactoferrin (green triangle), myeloperoxidase (orange diamond), amylase (black square). [Protein] = 0 – 50 μg/mL. Five repetitions were measured for each analyte.
was used as the reference electrode when we applied gate voltage to the OFET in an aqueous media.
Results and Discussion Firstly, we characterized the step-by-step modification of the Au detection electrode using photoelectron yield spectroscopy in air, measurements of water contact angle and transfer characteristics according to our previously published protocols. A significant difference of the design between IgA and IgG sensors is the length of alkyl chains on the extended-gate electrodes for immobilization of anti-antibodies, meaning that we employed a shorter alkyl chain (n = 3) for the IgA detection. This is because the molecular size of IgA is larger than that of IgG and a far distance from the electrode could decrease the sensitivity and reproducibility.18 As a result, the immobilization of the anti-IgA antibody on the extended-gate electrode was successfully accomplished. For the details of the characterization,
see the Supporting Information (Fig. S1). Next, we measured the electrical characteristics of the OFET device. The results indicate that the OFET device can be applied to biosensing in water, because the device can be operated at low voltages (under 3 V) (Fig. S2, Supporting Information). To achieve label-free immunoassay of human IgA, we immobilized the biotin-conjugated anti-IgA antibody (30 μg/mL) on the extended-gate electrode through the streptavidin-biotin system19 (Fig. S3, Supporting Information). The gate-electrode modified with the anti-IgA antibody was immersed in the PBS solution with the label-free human IgA, and then we measured the transfer characteristics of the OFET device. Figure 2 shows the titration results of the human IgA, showing an observed negative shift of the transfer curve with an increase of the human IgA concentration (Fig. 2a). Nevertheless, the change in the field-effect mobility of the OFET with increasing human IgA concentration was very small (Fig. S4, Supporting Information). In general, FET-based sensors (such as ion sensitive field-effect transistors, ISFETs) measure the potential difference between
ANALYTICAL SCIENCES JULY 2015, VOL. 31 the reference electrode and the gate electrode of the FET in aqueous media. Changes of the potential difference in aqueous media are attributed to the interfacial potential shift at the gate/solution interface because the potential of the reference electrode is constant. Changes of the channel conductance of FETs are caused by shifting the potential difference, which results in shifts of the electrical properties of FETs.20–22 Therefore, the shifts of the transfer curve and the threshold voltage of the fabricated OFET device with increasing human IgA concentration are probably due to the interfacial potential shift at the extended-gate/solution interface, suggesting that the positively charged IgA was captured on the extended-gate electrode. In addition, the gate currents did not significantly change during the titration, suggesting that electrochemical reactions (such as electrolysis) did not occur at the gate electrode21,23 (Fig. S5, Supporting Information). Also notable is the fact that our fabricated immunosensor based on the OFET device could detect human IgA in the presence of large excess amounts of HSA (~1000 μg/mL). Figure 2b shows the relationship between the concentration of analytes and changes in the OFET threshold voltage. As analytes, we selected IgA, amylase,24,25 lactoferrin,26 and myeloperoxidase27 (which are regularly contained in saliva). As a result, the response to the human IgA was highly selective (Fig. 2b and Figs. S6 – S8, Supporting Information). The employed anti-IgA antibody (clone code: IgA5-3B) has high-specificity for the human IgA (which is reported by previous reports),28,29 indicating that the response to human IgA is based solely on the immune-interaction between the anti-IgA antibody and the human IgA on the extended-gate electrode. The addition of the human IgA over 10 μg/mL induced a saturated response because the anti-IgA antibody is fully bound to the human IgA. The limit of detection (LOD)30 was estimated to be 2.1 μg/mL, the sensitivity was comparable to that of electrochemical31 or optical32 IgA sensor devices. Although enzyme linked-immunosorbent assays (ELISAs)33 and radioimmunoassays33 have higher sensitivity for the antibody detection, the protocol of the label-free immunosensor based on the OFET is much simpler than those of conventional methods due to its label-free detection (see Experimental section). To evaluate feasibility of the fabricated OFET sensor for practical use, we finally carried out the detection of the human IgA using a simulated saliva sample containing many other types of proteins ([amylase] = 0.4 μg/mL; [lysozyme] = 0.4 μg/mL; [lactoferrin] = 1.0 μg/mL; [myeloperoxidase] = 3.6 μg/mL).24–27 Consequently, the threshold voltage of the fabricated OFET device was shifted by increasing the human IgA concentration (Fig. 4 and Fig. S9, Supporting Information). These results suggest that the fabricated OFET could be used for the specific detection of the human IgA contained in biological fluids such as saliva.
Conclusions In conclusion, our fabricated extended-gate type OFET device functioned as a label-free detector of human IgA. The linear range from 0 to 10 μg/mL was obtained with a detection limit of 2.1 μg/mL in a PBS solution. The concentration of IgA in saliva is typically more than 40 μg/mL,34 meaning that the linear detection range was well below salivary IgA levels. However, low levels of IgA in saliva are known as a factor in infectious diseases such as upper respiratory infection.35 In addition, it has also been reported that IgA levels in saliva could be decreased by psychological stress.14–17 Hence, the fabricated OFET device
727
Fig. 3 Changes in the threshold voltage (VTH) of the OFET by the human IgA at various concentrations in a PBS solution with amylase (0.4 μg/mL), lysozyme (0.4 μg/mL), lactoferrin (1.0 μg/mL), and myeloperoxidase (3.6 μg/mL). [Human IgA] = 0, 3, 5, 10, 20, 30, and 50 μg/mL. Five repetitions were measured for each concentration. Relative standard deviations (n = 5) of the IgA response were 4% (0 μg/mL), 2% (3 μg/mL), 3% (5 μg/mL), 2% (10 μg/mL), 3% (20 μg/mL), 2% (30 μg/mL), and 2% (50 μg/mL), respectively.
could be applied for the monitoring of IgA levels for these health conditions in humans. These preliminary results suggest that further modifications at the periphery of the sensor device could provide for more efficient detection systems used in the management of human healthcare conditions in daily life.
Acknowledgements We gratefully acknowledge the financial support from the Japan Science Technology Agency (JST) and the Japan Society for the Promotion of Science (JSPS, Grant-in-Aid for Research Activiry Start-up, No. 26888002). We also thank Professor C. Shepherd, Dr. K. Fukuda, Dr. D. Kumaki of Yamagata University for their technical support and helpful discussion.
Supporting Information General information about chemicals and apparatus, and fabrication of the OFET-device are summarized in the Supporting Information. The reaction scheme for the immobilization of the anti-IgA antibody on the surface of the extended-gate electrode are shown in Scheme S1. Photo-electron yield spectroscopy in air and wettability measurements of the extended-gate electrode are summarized in Fig. S1. The electric characteristics of the fabricated OFET under 3 V are shown in Fig. S2. The titration result of the biotinylated anti-IgA antibody is summarized in Fig. S3. The field-effect mobility of the OFET upon titration with the human IgA is shown in Fig. S4. The gate-source current (IGS – VGS) of the OFET upon titration with the human IgA is summarized in Fig. S5. The transfer characteristics of the OFET upon titration with interfering proteins are shown in Figs. S6 – S8. The transfer characteristics of the OFET upon titration with the human IgA in the presence of interfering proteins are shown in Fig. S9. These materials are
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ANALYTICAL SCIENCES JULY 2015, VOL. 31
available free of charge on the Web at http://www.jsac.or.jp/ analsci/.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
B. Geffroy, P. Roy, and C. Prat, Polym. Int., 2006, 55, 572. A. Facchetti, Mater. Today, 2013, 16, 123. H. Sirringhaus, Adv. Mater., 2014, 26, 1319. G. Gelinck, P. Heremans, K. Nomoto, and T. D. Anthopoulos, Adv. Mater., 2010, 22, 3778. V. Subramanian, J. M. J. Fréchet, P. C. Chang, D. C. Huang, J. B. Lee, S. E. Molesa, A. R. Murphy, D. R. Redinger, and S. K. Volkman, Proceedings of the IEEE, 2005, 93, 1330. L. Torsi, M. Magliulo, K. Manoli, and G. Plazzo, Chem. Soc. Rev., 2013, 42, 8612. T. Minamiki, T. Minami, R. Kurita, O. Niwa, S. Wakida, K. Fukuda, D. Kumaki, and S. Tokito, Appl. Phys. Lett., 2014, 104, 243703. T. Minami, T. Minamiki, Y. Hashima, D. Yokoyama, T. Sekine, K. Fukuda, D. Kumaki, and S. Tokito, Chem. Commun., 2014, 50, 15613. T. Minami, T. Minamiki, K. Fukuda, D. Kumaki, and S. Tokito, Jpn. J. Appl. Phys., 2015, 54, 04DK01. T. Minamiki, T. Minami, D. Yokoyama, K. Fukuda, D. Kumaki, and S. Tokito, Jpn. J. Appl. Phys., 2015, 54, 04DK02. T. Minamiki, T. Minami, R. Kurita, O. Niwa, S. Wakida, K. Fukuda, D. Kumaki, and S. Tokito, Materials, 2014, 7, 6843. D. R. Shankaran, K. V. Gobi, and N. Miura, Sens. Actuators, B, 2007, 121, 158. S. Fagarasan and T. Honjo, Nat. Rev. Immunol., 2003, 3, 63. H. Nagai, Y. Narita, M. Ohtaki, K. Saito, and S. Wakida, Anal. Sci., 2007, 23, 975. K. Obayashi, Clin. Chim. Acta, 2013, 425, 196. E. A. Shirtcliff, D. A. Granger, E. Schwartz, and M. J. Curran, Psychoneuroendocrinology, 2001, 26, 165. Y. Yang, D. Koh, V. Ng, C. Y. Lee, G. Chan, F. Dong, S. H. Goh, V. Anantharaman, and S. E. Chia, Occup. Environ.
Med., 2002, 59, 836. 18. D. R. Shankaran and N. Miura, J. Phys. D: Appl. Phys., 2007, 40, 7187. 19. P. S. Stayton, S. Freitag, L. A. Klumb, A. Chilkoti, V. Chu, J. E. Penzotti, R. To, D. Hyre, I. L. Trong, T. P. Lybrand, and R. E. Stenkamp, Biomol. Eng., 1999, 16, 39. 20. P. Bergveld, Sens. Actuators, B, 2003, 88, 1. 21. D.-S. Kim, J.-E. Park, J.-K. Shin, P.-K. Kim, G. Lim, and S. Shoji, Sens. Actuators, B, 2006, 117, 488. 22. C. Bian, J. Tong, J. Sun, H. Zhang, Q. Xue, and S. Xia, Biomed. Microdevices, 2011, 13, 345. 23. E. G. R. Fernandes, N. C. S. Vieira, A. A. A. D. Queiroz, F. E. G. Guimarães, and V. Zucolotto, J. Phys. Chem. C, 2010, 114, 6478. 24. J. J. Zakowski and D. E. Bruns, Crit. Rev. Clin. Lab. Sci., 1985, 21, 283. 25. U. M. Nater, N. Rohleder, J. Gaab, S. Berger, A. Jud, C. Kirschbaum, and U. Ehlert, Int. J. Psychophysiol., 2005, 55, 333. 26. P. Glimvall, C. Wickström, and H. Jansson, J. Periodontal Res., 2012, 47, 655. 27. E. L. Thomas, M. M. Jefferson, R. E. Joyner, G. S. Cook, and C. C. King, J. Dent. Res., 1994, 73, 544. 28. J. G. Fox, S. Perkins, L. Yan, Z. Shen, L. Attardo, and J. Pappo, Immunology, 1996, 88, 400. 29. A. S. Flies, C. K. Grant, L. S. Mansfield, E. J. Smith, M. L. Weldele, and K. E. Holekamp, Vet. Immunol. Immunopathol., 2012, 145, 110. 30. J. N. Miller and J. C. Miller, “Statistics and Chemometrics for Analytical Chemistry”, 6th ed., 2010, Pearson, Harlow, UK. 31. S. S. Ordóñez and E. Fàbregas, Biosens. Bioelectron., 2007, 22, 965. 32. K. Sato, M. Tokeshi, T. Odake, H. Kimura, T. Ooi, M. Nakao, and T. Kitamori, Anal. Chem., 2000, 72, 1144. 33. D. S. Hage, Anal. Chem. 1999, 71, 294R. 34. A. Jafarzadeh, A. Mostafaie, M. Sadeghi, M. Nemati, M. T. Rezayati, and G. Hassanshahi, Dent. Res. J., 2008, 5, 89. 35. D. J. Smith, M. A. Taubman, and J. L. Ebersole, J. Dent. Res., 1987, 66, 451.
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2015 © The Japan Society for Analytical Chemistry
Notes
An Organic Field-effect Transistor with an Extended-gate Electrode Capable of Detecting Human Immunoglobulin A Tsukuru MINAMIKI,* Tsuyoshi MINAMI,*† Yui SASAKI,* Ryoji KURITA,** Osamu NIWA,*,** Shin-ichi WAKIDA,*** and Shizuo TOKITO* *Research Center for Organic Electronics (ROEL), Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jonan, Yonezawa, Yamagata 992–8510, Japan **Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305–8566, Japan ***Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14 Hayashi, Takamatsu, Kagawa 761–0395, Japan
We herein report on the development of an extended-gate type organic field-effect transistor (OFET)-based immunosensor for the detection of human immunoglobulin A (IgA). The titration results of IgA exhibited shifts in the transfer characteristics of the OFET sensor device with increasing IgA concentration. A linear detection range from 0 to 10 μg/mL was realized with a detection limit of 2.1 μg/mL, indicating that the OFET-based immunosensor can be potentially applied to the monitoring of infectious diseases and psychological stress in daily life. Keywords Organic field-effect transistor, immunosensor, label-free, immunoglobulin A, self-assembled monolayer (Received February 26, 2015; Accepted April 16, 2015; Published July 10, 2015)
Introduction To date, academic and industrial research on organic electronic devices such as light-emitting diodes,1 solar cells2 and transistors3 has flourished in the field of electronic engineering. Especially, attempts have been made to use organic field-effect transistors (OFETs) as printed flexible integrated-electronic-circuits, screen displays,4 and radio frequency identification (RFID) tags.5 More importantly for analytical scientists, OFETs could be applied for wearable and disposable biosensors because of their intriguing properties including flexibility, compact integration and low fabrication costs.6 OFET devices would thus be able to endow us with novel sensor platforms for various analytical purposes. In this regard, we have succeeded in developing biosensors based on extended-gate type OFETs, which can detect biomolecules such as proteins,7 saccharides,8 amino acids9 and biogenic amines10 in aqueous media. Especially, we recently reported a label-free immunosensing device for a bovine immunoglobulin G (IgG) using an extended-gate OFET.11 Although the preliminary report revealed that extended-gate OFETs can potentially be employed as a platform for immunosensing, label-free immunosensors based on OFETs are still in their infancy, compared to conventional immunosensor devices.12 Therefore, further explorations of OFET-based immunosensors are necessary. Toward that end, we selected human immunoglobulin A (IgA) as a target analyte. IgA can be found in blood serum (i.e. serum IgA) and mucous secretions including saliva and tears (i.e. To whom correspondence should be addressed. E-mail: [email protected]
†
secretory IgA).13 Secretory IgA constitutes a part of mucosaassociated lymphoid tissue. Moreover, the concentration of secretory IgA in saliva might be related to psychological stress.14,15 To date, it has been revealed that the concentration of secretory IgA was obviously reduced in saliva collected from workers who were exposed to psychological stress.16,17 Hence, the detection of IgA levels is very important for the ongoing management of the risk of infection and psychological stress. In this paper, we demonstrate the first label-free immunosensing of human IgA in a simulated saliva sample using an extended-gate type OFET modified with an anti-human IgA antibody (Fig. 1). The expertise we have obtained herein is expected to open up avenues for the development of healthcare devices based on OFETs in daily life.
Experimental General information about chemicals and apparatus, fabrication of the OFET-device, and immobilization of the anti-IgA antibody are summarized in the Supporting Information. The label-free detection of human IgA was carried out as follows. Label-free detection of human IgA The region of the electrode modified with the anti-human IgA antibody was immersed in a phosphate-buffer saline (PBS) solution of analyte human IgA (0 – 50 μg/mL) with 0.1 wt% human serum albumin (HSA), or interfering proteins ([amylase] = 0.4 μg/mL; [lysozyme] = 0.4 μg/mL; [lactoferrin] = 1.0 μg/mL; [myeloperoxidase] = 3.6 μg/mL) for 2 h at 37° C. After the immunoreaction, the IgA level was electrically detected by the OFET sensing device. The Ag/AgCl electrode
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ANALYTICAL SCIENCES JULY 2015, VOL. 31
Fig. 1 Schematic structure for the extended-gate type organic FET designed for IgA detection. The extended-gate electrode is functionalized with the biotinylated anti-IgA antibody.
Fig. 2 (a) Transfer characteristics (IDS – VGS) of the OFET upon titration with human IgA in a PBS solution with 0.1 wt% HSA. [Human IgA] = 0, 1, 3, 5, 7, 10, 20, 30, and 50 μg/mL. (b) Changes in the threshold voltage (VTH) of the OFET by proteins at various concentrations in a PBS solution with 0.1 wt% HSA. Human IgA (red circle), lactoferrin (green triangle), myeloperoxidase (orange diamond), amylase (black square). [Protein] = 0 – 50 μg/mL. Five repetitions were measured for each analyte.
was used as the reference electrode when we applied gate voltage to the OFET in an aqueous media.
Results and Discussion Firstly, we characterized the step-by-step modification of the Au detection electrode using photoelectron yield spectroscopy in air, measurements of water contact angle and transfer characteristics according to our previously published protocols. A significant difference of the design between IgA and IgG sensors is the length of alkyl chains on the extended-gate electrodes for immobilization of anti-antibodies, meaning that we employed a shorter alkyl chain (n = 3) for the IgA detection. This is because the molecular size of IgA is larger than that of IgG and a far distance from the electrode could decrease the sensitivity and reproducibility.18 As a result, the immobilization of the anti-IgA antibody on the extended-gate electrode was successfully accomplished. For the details of the characterization,
see the Supporting Information (Fig. S1). Next, we measured the electrical characteristics of the OFET device. The results indicate that the OFET device can be applied to biosensing in water, because the device can be operated at low voltages (under 3 V) (Fig. S2, Supporting Information). To achieve label-free immunoassay of human IgA, we immobilized the biotin-conjugated anti-IgA antibody (30 μg/mL) on the extended-gate electrode through the streptavidin-biotin system19 (Fig. S3, Supporting Information). The gate-electrode modified with the anti-IgA antibody was immersed in the PBS solution with the label-free human IgA, and then we measured the transfer characteristics of the OFET device. Figure 2 shows the titration results of the human IgA, showing an observed negative shift of the transfer curve with an increase of the human IgA concentration (Fig. 2a). Nevertheless, the change in the field-effect mobility of the OFET with increasing human IgA concentration was very small (Fig. S4, Supporting Information). In general, FET-based sensors (such as ion sensitive field-effect transistors, ISFETs) measure the potential difference between
ANALYTICAL SCIENCES JULY 2015, VOL. 31 the reference electrode and the gate electrode of the FET in aqueous media. Changes of the potential difference in aqueous media are attributed to the interfacial potential shift at the gate/solution interface because the potential of the reference electrode is constant. Changes of the channel conductance of FETs are caused by shifting the potential difference, which results in shifts of the electrical properties of FETs.20–22 Therefore, the shifts of the transfer curve and the threshold voltage of the fabricated OFET device with increasing human IgA concentration are probably due to the interfacial potential shift at the extended-gate/solution interface, suggesting that the positively charged IgA was captured on the extended-gate electrode. In addition, the gate currents did not significantly change during the titration, suggesting that electrochemical reactions (such as electrolysis) did not occur at the gate electrode21,23 (Fig. S5, Supporting Information). Also notable is the fact that our fabricated immunosensor based on the OFET device could detect human IgA in the presence of large excess amounts of HSA (~1000 μg/mL). Figure 2b shows the relationship between the concentration of analytes and changes in the OFET threshold voltage. As analytes, we selected IgA, amylase,24,25 lactoferrin,26 and myeloperoxidase27 (which are regularly contained in saliva). As a result, the response to the human IgA was highly selective (Fig. 2b and Figs. S6 – S8, Supporting Information). The employed anti-IgA antibody (clone code: IgA5-3B) has high-specificity for the human IgA (which is reported by previous reports),28,29 indicating that the response to human IgA is based solely on the immune-interaction between the anti-IgA antibody and the human IgA on the extended-gate electrode. The addition of the human IgA over 10 μg/mL induced a saturated response because the anti-IgA antibody is fully bound to the human IgA. The limit of detection (LOD)30 was estimated to be 2.1 μg/mL, the sensitivity was comparable to that of electrochemical31 or optical32 IgA sensor devices. Although enzyme linked-immunosorbent assays (ELISAs)33 and radioimmunoassays33 have higher sensitivity for the antibody detection, the protocol of the label-free immunosensor based on the OFET is much simpler than those of conventional methods due to its label-free detection (see Experimental section). To evaluate feasibility of the fabricated OFET sensor for practical use, we finally carried out the detection of the human IgA using a simulated saliva sample containing many other types of proteins ([amylase] = 0.4 μg/mL; [lysozyme] = 0.4 μg/mL; [lactoferrin] = 1.0 μg/mL; [myeloperoxidase] = 3.6 μg/mL).24–27 Consequently, the threshold voltage of the fabricated OFET device was shifted by increasing the human IgA concentration (Fig. 4 and Fig. S9, Supporting Information). These results suggest that the fabricated OFET could be used for the specific detection of the human IgA contained in biological fluids such as saliva.
Conclusions In conclusion, our fabricated extended-gate type OFET device functioned as a label-free detector of human IgA. The linear range from 0 to 10 μg/mL was obtained with a detection limit of 2.1 μg/mL in a PBS solution. The concentration of IgA in saliva is typically more than 40 μg/mL,34 meaning that the linear detection range was well below salivary IgA levels. However, low levels of IgA in saliva are known as a factor in infectious diseases such as upper respiratory infection.35 In addition, it has also been reported that IgA levels in saliva could be decreased by psychological stress.14–17 Hence, the fabricated OFET device
727
Fig. 3 Changes in the threshold voltage (VTH) of the OFET by the human IgA at various concentrations in a PBS solution with amylase (0.4 μg/mL), lysozyme (0.4 μg/mL), lactoferrin (1.0 μg/mL), and myeloperoxidase (3.6 μg/mL). [Human IgA] = 0, 3, 5, 10, 20, 30, and 50 μg/mL. Five repetitions were measured for each concentration. Relative standard deviations (n = 5) of the IgA response were 4% (0 μg/mL), 2% (3 μg/mL), 3% (5 μg/mL), 2% (10 μg/mL), 3% (20 μg/mL), 2% (30 μg/mL), and 2% (50 μg/mL), respectively.
could be applied for the monitoring of IgA levels for these health conditions in humans. These preliminary results suggest that further modifications at the periphery of the sensor device could provide for more efficient detection systems used in the management of human healthcare conditions in daily life.
Acknowledgements We gratefully acknowledge the financial support from the Japan Science Technology Agency (JST) and the Japan Society for the Promotion of Science (JSPS, Grant-in-Aid for Research Activiry Start-up, No. 26888002). We also thank Professor C. Shepherd, Dr. K. Fukuda, Dr. D. Kumaki of Yamagata University for their technical support and helpful discussion.
Supporting Information General information about chemicals and apparatus, and fabrication of the OFET-device are summarized in the Supporting Information. The reaction scheme for the immobilization of the anti-IgA antibody on the surface of the extended-gate electrode are shown in Scheme S1. Photo-electron yield spectroscopy in air and wettability measurements of the extended-gate electrode are summarized in Fig. S1. The electric characteristics of the fabricated OFET under 3 V are shown in Fig. S2. The titration result of the biotinylated anti-IgA antibody is summarized in Fig. S3. The field-effect mobility of the OFET upon titration with the human IgA is shown in Fig. S4. The gate-source current (IGS – VGS) of the OFET upon titration with the human IgA is summarized in Fig. S5. The transfer characteristics of the OFET upon titration with interfering proteins are shown in Figs. S6 – S8. The transfer characteristics of the OFET upon titration with the human IgA in the presence of interfering proteins are shown in Fig. S9. These materials are
728
ANALYTICAL SCIENCES JULY 2015, VOL. 31
available free of charge on the Web at http://www.jsac.or.jp/ analsci/.
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