Organic electrochemical transistor based immunosensor for prostate specific antigen (PSA) detection using gold nanoparticles for signal amplification

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Abstract

We demonstrated a highly sensitive organic electrochemical transistor (OECT) based immunosensor with a low detection limit for prostate specific antigen/α1-antichymotrypsin (PSA–ACT) complex. The poly(styrenesulfonate) doped poly(3,4-ethylenedioxythiophene) (PEDOT:PSS) based OECT with secondary antibody conjugated gold nanoparticles (AuNPs) provided a detection limit of the PSA–ACT complex as low as 1 pg/ml, as well as improved sensitivity and a dynamic range, due to the role of AuNPs in the signal amplification. The sensor performances were particularly improved in the lower concentration range where the detection is clinically important for the preoperative diagnosis and screening of prostate cancer. This result shows that the OECT-based immunosensor can be used as a transducer platform acceptable to the point-of-care (POC) diagnostic systems and demonstrates adaptability of organic electronics to clinical applications.

Introduction

Immunosensors, based on the highly specific molecular recognition of an antigen by antibodies, have become the major tools for clinical examinations (Skottrup et al., 2008), biochemical analyses of environmental pollutants (Jiang et al., 2008) and food quality control (Ricci et al., 2007). In immunoassay, the determination of cancer markers associated with certain tumors in patients plays an important role in diagnosing cancer diseases. The prostate specific antigen (PSA), an androgen-regulated serine protease, is the best serum marker currently available for the preoperative diagnosis and screening of prostate cancer. The quantitative detection of PSA has been carried out using optical methods including fluorescent labeling (Ye et al., 2004, Huhtinen et al., 2004), surface-enhanced Raman scattering (SERS) (Grubisha et al., 2003), surface plasmon resonance (SPR) (Cao et al., 2006), as well as electrical and electrochemical signal transduction methods using silicon nanowire field-effect transistors (Zheng et al., 2005), gold nanowire immunosensor (Pampalakisa and Kelley, 2009) and single-walled carbon nanotubes (SWNTs) based biosensors (Li et al., 2005, Okuno et al., 2007). Despite many efforts that have been made in developing the sensors for PSA detection, certain challenges still need to be addressed such as their miniaturization, simplification of the fabrication steps, and cost reduction. Majority of these issues are related to the bulk component and high cost of optical detection methods. Due to these, recently researchers have been increasingly working on the development of point-of-care (POC) systems using electrochemical methods. The technical and commercial merits come from their low production cost, compatibility with modern microfabrication technologies, real time detection, and minimal power requirements. In order to improve the electrochemical sensor performance further, novel technologies for simple fabrication procedure, high sensitivity, fast response, wide dynamic range and low detection limit must be explored and developed.

Among the newly developed electrochemical sensors, the sensors made of organic electrochemical transistor (OECT), based on the electrochemical modulation of a conductive polymer, have many advantages such as compatibility with solution processing, low-cost fabrication route and mechanical flexibility (Bernards et al., 2008, Roberts et al., 2008, Berggren and Richter-Dahlfors, 2007, Badhwar and Narayan, 2008). Several OECT-based sensors have been developed as chemical and biological sensors for water vapor (Nilsson et al., 2002), H+ (Thackeray and Wrighton, 1986), K+ ions (Dabke et al., 1997), glucose (Bartlett and Wang, 1996), IgG antibody (Kanungo et al., 2002), and DNA (Krishnamoorthy et al., 2004) due to their inherent signal amplification capability, miniaturized form factor and suitability for integrated circuits (Mabeck and Malliaras, 2006). However, there have been only a few reports on the application of OECT-based sensors to the disease-specific immunoassay despite their many advantages (Kanungo et al., 2002).

In sandwich-type immunosensors composed of a primary antibody immobilized on a sensor platform and a specific antigen cancer marker from a sample solution, a secondary antibody reacts with the bound antigen and is able to provide the detectable signal with a low noise via enzymes (Zhang and Heller, 2005, Yu et al., 2006), redox mediators (Viswanathan et al., 2009) or nanomaterials (Kerman et al., 2008, Liu and Lin, 2007). Nanoparticles have been used for labeling of biomolecules, facilitating the immobilization of biomolecules, the catalysis of electrochemical reactions, the enhancement of electron transfer, as well as for a reactant (Luo et al., 2006). Among the various nanoparticles, gold nanoparticles (AuNPs) have been popular, because of their large surface area and good biocompatibility (Chumbimuni-To rres et al., 2006).

In this report, AuNPs conjugated with PSA polyclonal antibody (pAb) (AuNPs–PSA pAb) were used for a disease-specific immunosensor. The electrochemical signal produced in response to ad immune-reaction could be significantly amplified by AuNPs. For the first time, we demonstrated the capability of an OECT-based immunosensor with signal amplification by gold nanoparticles for prostate specific antigen detection.

Section snippets

Chemicals

PSA monoclonal antibody (PSA mAb), PSA–ACT complex, and PSA polyclonal antibody were supplied by Fitzgerald, Inc. (North Acton, MA, USA). Poly(3,4-ethylenedioxythiophene)–poly(styrenesulfonate) (PEDOT/PSS), ethylene glycol (EG), aminopropyldiethoxymethylsilane (APTMS), 10 nm dia. of Au colloid, phosphate buffered saline (PBS), Tween 20, N-hydroxysuccinimide (NHS), (N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) (EDC), bovine serum albumin (BSA) were purchased from Sigma–Aldrich.

Characterization of the AuNPs conjugated with PSA pAb

The prepared solution of AuNPs–PSA pAb was red-colored because of the absorption of light in a specific wavelength range as shown in the inset of Fig. 2a. The transmission electron microscopy image of the AuNPs–PSA pAb confirms a homogeneous distribution of spherical 10-nm AuNPs with no significant agglomeration, due to the uniform conjugation of PSA pAb molecules on their surfaces as shown in Fig. 2a. The measurement of the light absorption by the solution using a UV–vis spectrometer showed a

Conclusion

In conclusion, we demonstrated for the first time an organic electrochemical transistor based immunosensor utilizing gold nanoparticles for the amplification of the signal. The use of gold nanoparticles conjugated by PSA pAb enhanced the electrochemical signal significantly between the PSA–ACT complex/PSA mAb and the PEDOT:PSS and, in turn, enlarged the dynamic range and improved the sensitivity. The low concentrations of PSA–ACT complex could be detected with high sensitivity for the

Acknowledgments

This work was supported by the WCU Program (R32-2008-000-10124-0) and the Basic Science Research Program (2010-0015035) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology. We thank Cuong Cao and Jun Pyo Kim for their discussion on the biological experimental setup.

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