Functionalized nanoporous gold as a new biosensor platform for ultra-low quantitative detection of human serum albumin

The development of a new generation of ultra-sensitive sensors for analytical and bio-diagnostic devices requires a strong signal in front of very small quantity of analyte, often present in complex and interfering matrix. Taking advantage of peculiar plasmonic properties of nanoporous gold (NPG), a promising sensor for selective detection of Human Serum Albumin (HSA), a proof-of-concept bioanalyte, was prepared and tested in a conventional micro-Raman spectrometer using Surface Enhanced Raman Spectroscopy (SERS). NPG was synthesized by chemical de-alloying of an amorphous precursor, Au20Cu48Ag7Pd5Si20, starting from melt spun ribbons. A fully de-alloyed ribbon with ligaments of about 60 nm was obtained after 4 h of de-alloying at 70 °C and 10M HNO3+0.5M HF. This material is self-standing, mechanically resistant, and suitable for wide range of applications. At this stage, NPG is SERS-active toward several molecules, including pyridine, bi-pyridine, and rhodamine at very low concentration. In order to obtain the selective binding properties required to detect molecules in bio-diagnostic applications, immuno-functionalization of NPG was carried out by using an anti-HSA antibody (Ab-anti-HSA) covalently grafted on gold ligaments via the 4-aminothiophenol (4ATP) Raman probe, yielding the SERS-active nanohybrid NPG-4ATP-Ab. SERS signal recorded at increasing HSA concentrations was used for the acquisition of SERS maps and a chemometric regression model allowed to calibrate the sensor. In the best experimental conditions, we were able to get quantitative analysis of HSA at ultra-low concentrations (0.1 ng/l) with SERS. The methodology proposed in this paper has to be considered a major step toward a calibrated device for ultra-sensitive detection of biomolecules by SERS detection