Characterization of protein immobilization on nanoporous gold using atomic force microscopy and scanning electron microscopy

Abstract

Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize a highly efficient approach for protein immobilization on NPG using N-hydroxysuccinimide (NHS) ester functionalized self-assembled monolayers on NPG with pore sizes in the range of tens of nanometres. Comparison of coupling under static versus flow conditions suggests that BSA (Bovine Serum Albumin) and IgG (Immunoglobulin G) can only be immobilized onto the interior surfaces of free standing NPG monoliths with good coverage under flow conditions. AFM is used to examine protein coverage on both the exterior and interior of protein modified NPG. Access to the interior surface of NPG for AFM imaging is achieved using a special procedure for cleaving NPG. AFM is also used to examine BSA immobilized on rough gold surfaces as a comparative study. In principle, the general approach described should be applicable to many enzymes, proteins and protein complexes since both pore sizes and functional groups present on the NPG surfaces are controllable.

Nanoporous Gold Electrodes and Their Applications in Analytical Chemistry

“…Because AFM is ideal for imaging samples under ambient conditions, this technique is useful for examining biomolecules adsorbed on surfaces. In recent work, a nanoporous gold monolith was cleaved and AFM was used to evaluate the presence and orientation of proteins on the surface [90].…”

Molecular release from patterned nanoporous gold thin films

“…The obtained results were in agreement with BET studies of comparable sample morphologies of bulk np-Au used by different groups. 30,47 The gain in surface area, referred to as enhancement factor, scaled linearly with thickness for lms with uniform morphology (T1, T2, T3) (Fig. 3 inset).…”

An overview of dealloyed nanoporous gold in bioelectrochemistry

“…For example, modification of NPG by MnO 2 particles, thin films of tungsten sulfide, silica and conducting polymer can be clearly distinguished by TEM ( Figure 3). The immobilisation of bovine serum albumin (BSA) and immunoglobulin G (IgG) on NPG has been examined using AFM [36], with individual features of proteins observed on the surface of the support. Electrochemical techniques, such as cyclic voltammetry (CV), are primarily applied to measure the electroactive surface areas (Figure 4).…”