Electrodeposited MoS2 Thin Films for Stable Immobilization of Thiolated Biomolecules

Abstract

We report a robust new combination of substrate materials and immobilization chemistry for biosensing that addresses one of the oldest challenges in this field, the stability and reproducibility of biomolecule immobilization. The substrate materials are electrodeposited MoS2 and Cu-doped MoS2 thin films atop glassy carbon electrodes (GCE) created by a two-step process of cathodic electrodeposition, followed by high temperature annealing. Electrodeposition atop a GCE is preceded by an electrochemical pre-treatment process to increase the surface roughness and introduce oxygen functional groups, dramatically improving film adhesion. MoS2 thin films are electrodeposited from 10 mM (NH4)2MoS4 and 0.2 M KCl at pH 6.8, while Cu-doped MoS2 thin films are electrodeposited from 10.0 mM (NH4)2MoS4, 5.0 mM CuSO4, 0.10 M KCl, and 0.50 M KSCN at pH 6.95. Since MoS2 is an active hydrodesulfurization (HDS) catalyst for sulfur removal from hydrocarbons, both thin film materials immediately react with and desulfurize the electrolyte from which they are deposited. The desired MoS2 stoichiometry, which includes coordinately unsaturated Mo sites, can be recovered by high temperature annealing in a reducing environment. High temperature annealing removes the excess sulfur from the as-deposited film, and also partially recrystallizes and partially orients the MoS2 thin films, as suggested by x-ray diffraction (XRD) studies. This annealing procedure regenerates the coordinately unsaturated and highly sulfiphilic Mo sites atop HDS catalyst materials, which can then form strong bonds to thiolated biomolecules. We demonstrate this by disulfide bond reduction at the hinge region of polyclonal antibodies utilizing tris (2-carboxyethyl) phosphine (TCEP) as the reducing agent. Unlike other reducing agents for disulfide bond cleavage, which contain thiol groups, TCEP probably does not compete with antibody fragments for thiol binding sites atop the MoS2 surface. The attached figure illustrates the biosensing platform described here. This new biosensing platform is demonstrated by disulfide bond reduction within polyclonal antibodies to 3-phenoxybenzoic acid (3-PBA), binding of these antibody fragments to newly created MoS2 and Cu-doped MoS2 thin films, and detection of the 3-PBA antigen using electrochemical impedance spectroscopy (EIS). Control experiments demonstrate that only the annealed MoS2 and Cu-doped MoS2 thin films are active towards binding of antibody fragments. In both cases, the charge transfer resistance (Rct) is the equivalent circuit element from the Randles circuit that is most sensitive to antigen binding. Atop un-doped MoS2, the sensitivity for 3-PBA detection by EIS is 2.7x108 Ω-cm2-M-1, and the detection limit is 2.5x10-6 M. Atop Cu-doped MoS2, the sensitivity for 3-PBA detection is 5.9x108 Ω-cm2-M-1, and the detection limit is 3.8x10-6 M. The variation of Rct with antigen concentration atop MoS2 is consistent with the Langmuir adsorption isotherm, with an initial linear relationship, and Rct eventually saturating at high concentrations when all binding sites are occupied. However, two plateau regions are reproducibly observed atop Cu-doped MoS2. The rms surface roughness obtained by atomic force microscopy (AFM) measurements atop MoS2 and Cu-doped MoS2 substrates ranges from 60–140 nm, so these methods are not limited to ultra-smooth substrates. Figure 1