Many proteins are capable of causing immune reactions, and the presence of protein aggregates has been identified as an important factor leading to the lowering of immune tolerance [1–3]. For this reason, the precise detection of proteins in the presence of other formulations with high sensitivity and specifity is essential for disease diagnostics, drug screening, and other applications [4, 5]. The most widely used method for the detection and analysis of proteins and their aggregates in formulations is size exclusion chromatography (SEC) . Although useful for the determination of molecular-weights of proteins, its application to the quantitative determination of protein concentrations is more difficult. Another spectroscopic techniques involving the use of light scattering techniques  and Fourier transform infrared spectroscopy (FTIR) [7, 8] are also frequently used for this purpose. Light scattering methods have been used to calculate the mean hydrodynamic radius of protein aggregates and to characterize molecular distribution of protein aggregates. However, high concentrations of protein solutions are needed to accomplish this. As a result, the technique has limitations involving erroneous interpretations and a lack of sensitivity at low concentrations. Although FTIR method has also been used for the determination of changes in protein secondary structure, it is still difficult to quantitatively analyze and differentiate between protein concentrations in aggregates. In order to overcome these limitations and enhance sensitivity, optical methods such as fluorescence spectroscopy have been used . However, strong background or the quenching of spectroscopic signals resulting from the use of labeling dyes has been reported [10, 11]. In this regard, label-free optical methods have been developed and localized surface plasmon-based metallic nanomaterials represent a promising alternative for achieving high sensitivity, and selectivity at low concentrations.
The optical properties of metallic nanomaterials arise from localized surface plasmon resonance (LSPR), which is caused by the collective oscillation of surface conduction electrons by light [12–15]. Changes in the peak intensity and wavelength of plasmon spectra, which are caused by variations in refractive index as the result of the binding of molecules to the metal nanomaterials, are optically detectable parameters that have found use in chemical and biosensensor devices [16–19]. Currently, due to the potential for impacting screening in medical and environmental applicabilities, LSPR sensing systems would be more attractive. However, further improved sensitivity and accuracy of the devices are required, so that the development of a novel nanostructure design with special optical properties for high sensitivity and selectivity has become a priotiry.
Here, we propose a highly sensitive and molecular size-selective detection method for a protein in the presence of its aggregate by utilizing a heteroliganded gold nanoisland on a transparent glass substrate. As a proof-of-concept test, the superoxide dismutase (SOD1) protein was selected for the sensitive and molecular size-selective detection between this protein and aggregates derived from it. SOD1 is a well-known, highly stable dimeric enzyme that catalyzes the dismutation of super radicals to hydrogen peroxide and molecular oxygen. Its aggregated structure in motor neurons is associated with amyotrophic lateral sclerosis (ALS), a neurodegenerative disease [20, 21].
The method focused on several significant factors. First, heteroliganded nanoholes were fabricated on gold nanoislands for protein separation based on its physical dimensions in the presence of aggregates. Second, the detection method is based on sensitive changes in the local dielectric environment of modified gold nanoislands, which are caused by covalent chemical interactions between the proteins and the active sites of nanoholes. Third, the transduction system was modified for application to a chip-based detection method. This would premit the fabrication of materials using off-the-shelf materials with high stability, which would be applicable to simple, low-cost diagnostics. The combination of these factors would result in the more sensitive, molecular size-selective, and simpler method for the determination of a protein.