Biomolecules/gold nanowires-doped sol–gel film for label-free electrochemical immunoassay of testosterone

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Abstract

A direct, rapid, and label-free electrochemical immunoassay method for testosterone has been described based on encapsulating testosterone antibody into polyvinyl butyral sol–gel film doped with gold nanowires. Gold nanowires prepared by using nanopore polycarbonate membrane were used to conjugate testosterone antibody onto the probe surface. The presence of gold nanowires provided a biocompatible microenvironment for biomolecules, greatly amplified the immobilized amount of biomolecules on the electrode surface, and improved the sensitivity of the immunosensor. In comparison with gold nanoparticle-conjugating probe, the gold nanowire-functionalized probe could avoid the leakage of biomolecules from the composite film, and enhanced the stability of the sensor. The performance and factors influencing the performance of the resulting immunosensor were investigated in detail. Under optimal conditions, the developed immunosensor exhibited a good linear relationship with testosterone ranging from 1.2 to 83.5 ng mL 1 with a detection limit of 0.1 ng mL 1 (at 3δ). Moreover, the proposed immunosensor exhibited high sensitivity, good reproducibility and long-term stability. The as-prepared immunosensors were used to analyze testosterone in human serum specimens. Analytical results suggest that the developed immunoassay has a promising alternative approach for detecting testosterone in the clinical diagnosis. Compared with the conventional ELISAs, the proposed immunoassay method was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate gold nanowires into the solid matrix for biosensing application.

Introduction

Testosterone, a steroid hormone from the androgen group, is primarily secreted in the testes of males and the ovaries of females although small amounts are secreted by the adrenal glands. It is the principal male sex hormone and an anabolic steroid. In both males and females, it plays key roles in health and well-being. Examples include enhanced libido, energy, immune function, and protection against osteoporosis. Depending on age, insufficient testosterone production can lead to abnormalities in muscle and bone development, underdeveloped genitalia, and diminished virility. Moreover, low testosterone levels in elderly men increases the risk of depression. The reference range of serum total testosterone in male adults was shown to be 2.01–7.50 ng mL 1 [1]. Various methods for the detection of testosterone, such as molecular imprinted method, immunofiltration, and ELISA have been reported [2], [3], [4], [5]. Most procedures, unfortunately, need often expensive instrumentation, specialized personnel and suffer from considerable time delays between sampling and obtaining results. Hence, exploring optimum determination methods for testosterone is required. Electrochemical immunosensors have gained growing attention because of their low detection limit, simplicity of the equipment required and the ability to analyze heterogeneous and colored samples [6].

For the development of electrochemical immunosensors, there are two basal issues to realize the application of electrochemical technique. First, the key process is to retain the biologic activity of the immobilized biomolecules on the probe surface in its native status [7]. Polyvinyl butyral (PVB) is a resin usually used for application that require strong binding, optical clarity, adhesion to many surface, toughness and flexibility [8]. PVB membrane has been successfully utilized as useful molecular recognition interface of electrochemical biosensors [9], [10]. From the viewpoint of fabricating an electrochemical recognition layer, the PVB-based membrane is considered to have following advantages: innocuity, scentless, non-corrosive, apyrous and good transmittance, water-and-oil resistance, anti-aging, good cohesion inorganic and organic glass and transparency.

Another key factor is that the immobilized amount and the immobilized firmness of biomolecules in the composite film are very critical [11]. In general, the sensitivity of an immunosensor is relative to the surface coverage of the biomolecules, and the stability depends on the immobilized firmness of the biomolecules in the sensing film. The detection sensitivity can be increased by controlling the orientation of proteins immobilized on the sensor surface and by decreasing the steric-hindrance effect via controlling the structural configuration of immobilized proteins on the sensor surface [12]. Antibody molecules are usually hydrophilic, and it was easily leaked from the sol–gel composite membrane [13]. Tang et al. [9], [10] reported the fabrication procedure of many immunosensors by using gold nanoparticles and PVB. Gold nanoparticles prepared by the reduction method of citric acid, however, are hydrophilic. When antibodies were adsorbed on the surface of nanogold particles, they might be leaked out from the hydrophobic sol–gel film. To avoid the leakage of biomolecules, the gold nanowires were used in this study. The reason is that antibody-functionalized gold nanowires could form crossing-network with PVB sol–gel, and greatly enhanced the stability of the composite membrane. To our knowledge, there was little report focusing on studying non-manually immobilized biomolecules onto gold nanowires-modified complex membranes to fabricate a self-assembled nano-biosensor for the determination of testosterone.

Herein, a new methodology for the construction of testosterone immunosensor was constructed by combining the merits of PVB complex membranes and the characteristics of gold nanowires. The immobilized testosterone antibody exhibited direct electrochemical behavior toward testosterone. The performance and factors influencing the performance of the resulting immunosensor were studied in detail. Moreover, the studied immunosensor exhibited high sensitivity, good reproducibility and long-term stability. Subsequently, the immunoassay system was evaluated by analyzing clinical samples with results compared with those obtained by the ELISA method.

Section snippets

Reagent and apparatus

Monoclonal mouse anti-testosterone IgG antibody (T-Ab) (Testosterone-3, clone number: M021812) was purchased from Fitzgerald Industries International, Inc. (USA). Testosterone (T-Ag) was achieved from Biocell Bioengin. Co. (China). Track-etched porous polycarbonate, and bovine serum albumin (BSA, 96–99%) were obtained from Sigma (USA). Polyvinyl butyral (PVB, 99.8%) was bought from Shanghai Chem. Re. Co. (China). All chemicals and solvents used were of analytical grade and were used as

Results

Fig. 1 shows the SEM image of the gold nanowires. As seen from Fig. 1, the gold nanowires display a highly regular and uniform pattern with a mean diameter of about 200 nm and the length of the nanowire after 15 min of deposition is about 10 μm. To further confirm the interaction between the formed gold nanowires and proteins, UV–vis spectra were used in this study (data not shown). The gold nanowires exhibit absorption bands with a maximum at 522 nm. The absorption peak shifts to 528 nm when

Discussion

This contribution was mainly focused on the development of new and label-free electrochemical immunosensor for quantitative detection of testosterone based on the encapsulation of antibodies into gold nanowires-functionalized composite architecture. To verify the synthesis of gold nanowires and the bioactivity of biomolecules doped into the complex membrane, SEM and UV–vis absorption spectroscopy were used. As seen from Fig. 1, the gold nanowires grew well within the pores of PC template, and

Conclusion

A disposable electrochemical immunosensor for the determination of testosterone was designed based on the immobilization antibody on gold nanowires-derivated sol–gel membrane. Highlights of the immunoassay system are direct, rapid and simple without multiple labeling and separation steps. Moreover, use of the gold nanowires could enhance the surface coverage of the protein, and improved the sensitivity of the immunoassay. The non-specific adsorption of protein species does not show much

Acknowledgments

The authors gratefully acknowledge the financial support of the Natural Science Foundation of Chongqing City and the Education Committee of Chongqing City, China.

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