A reusable capacitive immunosensor with a novel immobilization procedure based on 1,6-hexanedithiol and nano-Au self-assembled layers
Introduction
Capacitive immunosensors, by determining small changes of the interfacial capacitance because of the interaction between an antibody (Ab) and an antigen (Ag), are mainly applied in areas where unlabeled, high sensitivity and high selectivity are required. As for the construction of an immunosensor based on a capacitive transducer, the first and probably most important step is the immobilization of the recognition elements to provide the sites of antibody–antigen interaction. Some earlier works focused on metal oxide and semiconductor thin layers [1], [2], [3], [4]. In these cases, however, the thickness of the dielectric as well as reproducibility of its manufacture is assumed to be mechanically limited. More recently, developments in the physical chemistry of surfaces have led to an increasing interest in using self-assembled monolayers of thiolor sulfur compounds as insulator [5], [6], [7], [8], [9], [10], [11], [12]. The formed single molecular organized structures have numerous advantages [11], such as insulating, nanostructures, free of defects, much more stable, especially in water and other solvents, and so on. However, the method for immobilization of antibody protein, the regenerability, and the reusability are, among other topics, major challenges in capacitive immunosensors development research.
In our work, a novel immobilization procedure for antibodies based on thiolor sulfur compound and gold nanoparticles was investigated. Due to the strong specific interaction between the sulfur atom and the gold surface, the insulating organic monolayer films were first formed by the spontaneous assembly of 1,6-hexanedithiol (HDT) from solution onto gold [13]. The monolayers thus formed are stable enough to withstand further chemistry on the available thiol groups [14]. When these thiol-rich surfaces are exposed to Au colloid, the sulfurs form strong bonds to gold nanoparticles, anchoring the clusters to the electrode substrate [15], [16]. After the assembly of gold nanoparticles layer, the original formed organic thiols surface was restored, and a new negatively charged nano-Au surface was obtained. Thus, the positively charged antibody protein could be immobilized through electrostatic adsorption by controlling the pH of antibody solution about its isoelectric point. The assembled antibody protein layer can be rinsed out, via a saline solution with extreme pH. Therefore, the immunosensor can be regenerated repeatedly. This regeneration is of great significant for capacitive immunosensors.
To investigate its applicability for capacitive immunosensing, the human IgG was used as a model substrate. With the above immobilization procedure, a heterostructure of Au/insulating SAMs/antibody was formed. When such a heterostructure is immersed in a solution that contains the specific antigen, the interaction of antibody with the antigen leads to the increase of the dielectric layer and induces a capacitance decrease, which can be directly related to the amount of antigen to the quantified.
Section snippets
Chemicals
Bovine serum albumin (BSA), goat-anti-human IgG antibody (IgGAb, affinity purification) and normal human reference serum (NHRS, containing 10.9 mg ml−1 of immunoglobulin G (IgG)) were purchased from Sino-Americal Biotechnology Company (Shanghai, China). 1,6-Hexanedithiol (≥97%, Fluck, Switzerland), glutareldehyde (GA) (Chemical Reagents Co., Changsha, China), gold wire (1.0 mm diameter, 99.99%) (Chemical Reagents Co., Changsha, China) and all other chemicals were of analytical reagent grade and
Theoretical background
Electrochemical impedance spectroscopy (EIS) is a useful tool for studying the interface properties of surface-modified electrodes. The typical equivalent scheme of electrochemical interface (Fig. 2a) can be represented as the electrolyte solution resistance (RΩ) in series with the parallel circuit of Faradic impedance (Zf) and double-layer capacitance (Cd) [19]. Here, Zf is composed of electron-transfer resistance (Rct) and Warburg impedance (Zw). For the high frequency domain, the electrode
Conclusions
A novel immobilization strategy combining SAMs and electrostatic adsorption techniques has been used to immobilized human IgG antibodies on capacitive immunosensor for the determination of human IgG. The immunosensor based on the sulfur–Au adsorption procedure shows improved performance in terms of the regeneration compared with those employing conventional covalent immobilization. Moreover, the proposed immobilization procedure results in immobilized entities with relatively high biological
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (Grant Nos. 20075006, 20205004, 20375012) and the Foundation of Ministry of Education for Doctor (No. 20010532008).
Jishan Li graduated from Hunan University in 2000, majoring in chemistry. He is currently a PhD student of the same university.
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2011, Analytica Chimica ActaCitation Excerpt :One way to attempt to improve the detection limit is by using nanoparticles to increase the effective surface area for the immobilized sensing molecules. Such previously developed methods have mostly used gold nanoparticles (AuNPs) [11–13] with only a few applications of silver nanoparticles (AgNPs) have been studied [14,15]. Since silver is cheaper than gold and the preparation of its nanoparticles is also simple we would like to show that AgNPs can effectively enhance the sensitivity and detection limit of a capacitive immunosensor system, comparable to the system with AuNPs.
Jishan Li graduated from Hunan University in 2000, majoring in chemistry. He is currently a PhD student of the same university.
Zaisheng Wu is currently graduate student. His research interests cover chemical and biosensors.
Hua Wang is currently PhD student of Hunan University majoring in chemistry. His research interests cover chemical and biosensors.
Guoli Shen is professor of chemistry, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China; Vice-Chairman of the Chemical Sensor Subcommittee of Chinese Analytical Instrumentation Society and acting deputy editor-in-chief of ‘Chemical Sensor’. He graduated from Department of Chemistry, Fudan University, Shanghai, in 1961. His research interests cover chemical and biosensors.
Ruqin Yu is professor of chemistry, College of Chemistry and Chemical Engineering, Hunan University, Changsha, China; member of Chinese Academy of Sciences since 1991. Editor-in-chief of ‘Chemical Sensor’, editorial adviser of Analytical Chimica Acta (Elsevier) and Journal of Chemometrics (Wiley). He graduated from Department of Chemistry, St. Petersburg University, Russia, in 1959. His research interests cover chemical sensors and chemometrics.