A label-free electronic biosensor for detection of bone turnover markers

Abstract

This paper describes the development of a biosensor based on label-free immunosensing for the detection of the C-terminal telopeptide bone turnover marker from type-1 collagen. A self-assembled monolayer (SAM) of dithiodipropionic acid was deposited on a gold electrode. Then streptavidin and biotinylated anti-human C-terminal telopeptide antibody were successively conjugated on the self-assembled monolayer. Electrochemical impedance measurements were made to characterize each step of the SAM/streptavidin/biotinylated antibody binding. Subsequently, electrochemical impedance was measured with different concentrations of C-teminal telopeptide. A detection limit of 50 ng/mL and a dynamic range up to 3 μg/mL were achieved. To our knowledge, this is the first attempt to develop a label-free immunosensor based on electrochemical impedance with DC bias for detection of bone-related degradation and rebuilding products. The electronic biosensor might eventually be used for quantitative point-of-care screening of bone health. It is hoped that analysis of bone turnover markers can indicate the beginning of bone diseases such as osteoarthritis and osteoporosis so that treatment might start early when it is most effective.

Keywords: bone turnover marker; electrochemical impedance; immunosensor; label-free.

Figure 1.

Schematic representation of a label-free immunosensor for bone maker detection. Part (A) shows a self-assembled monolayer of dithiodipropionic acid deposited on a gold surface with streptavidin immobilized next as a self-assembled monolayer. Then the biotinylated antibody was bound to the streptavidin. Part (B) illustrates the antigen-antibody binding event and how it hinders the interfacial electron transfer reaction of [Fe(CN)₆]^3−/4−.

Figure 2.

Optical picture of gold array electrodes which were patterned by a lithography technique.

Figure 3.

Electrochemical impedance spectra for a cleaned gold electrode at 0.2 V over a frequency range between 0.1 Hz and 300 kHz. The sinusoidal potential magnitude is 20 mV in 5.0 mM K₃Fe(CN)₆ and 5.0 mM of K₄Fe(CN)₆ in PBS (pH 7.0). The inset is an equivalent circuit model used to fit the experimental data. C_dl, is the double layer capacitance; R_et, is the electron transfer resistance; W, is Warburg impedance; and R_s, is the solution resistance. The final curve-fit theoretical data in curve b matched well with curve a.

Figure 4.

Three dimensional electrochemical impedance spectra of: (A) a self-assembled monolayer with carboxylic terminals on a gold electrode; and (B) a streptavidin immobilized electrode. EIS was done at DC potentials from 0 V to 0.5 V with frequencies between 0.1 Hz and 300 KHz. The sinusoidal potential magnitude was 20 mV in 5.0 mM K₃Fe(CN)₆ and 5.0 mM of K₄Fe(CN)₆ in PBS (pH 7.0).

Figure 5.

Electrochemical impedance spectra response recorded for the biotinylated anti-human C-terminal telopeptides antibody with the incubation time of one-half (a), one (b), two (c), four (d), and 24 hours (e). EIS was done at a DC potential of 0.2 V with frequencies between 0.1Hz and 300 KHz. The sinusoidal potential magnitude is ±20 mV in 5.0 mM K₃Fe(CN)₆ and 5.0 mM of K₄Fe(CN)₆ with PBS (pH 7.0).

Figure 6.

Electrochemical impedance spectra response recorded at the biotinylated anti-human C-terminal telopeptide antibody modified electrode in the presence of increasing concentration of human C-terminal telopeptide: 0 (a), 0.2 (b), 0.5 (c), 1(d), and 10 (e) μg/mL concentration of antigen. EIS was done at a DC potential of 0.2 V at frequencies between 0.1 Hz and 300 KHz. The sinusoidal potential magnitude was ±20 mV in 5.0 mM K₃Fe(CN)₆ and 5.0 mM of K₄Fe(CN)₆ in PBS (pH 7.0).

Figure 7.

The relationship between the change in electron transfer resistance and the concentration of C-terminal telopeptide antigen.

Figure 8.

Fluorescence image of gold electrodes; (a) a gold electrode is coated with antibody and (b) a gold electrode is coated with antibody/antigen/fluorescence dye.

Figure 9.

Cyclic voltammetry of 5.0 mM K₃Fe(CN)₆ and 5.0 mM of K₄Fe(CN)₆ in PBS (pH 7.0) with a 100 mV/s scan rate for: (a) the bare gold electrode; (b) the biotinylated anti-human C-terminal telopeptide antibody immobilized on the electrode after functionalizing with 3,3-dithiodipropionic acid; and (c) with 10 μg/mL human C-terminal telopeptide antigen in the solution.