Colorimetric nanodiagnostics of biomarkers in biological fluids

The spreading appeal in Point-Of-Care (POC) colorimetric nanosensors, prompted also by the outbreak of COVID-19 pandemic, relies in their portability, ease of use, and rapidity, along with reduced costs respect to laboratory-based assays. These methodologies find application in various fields, for instance as early diagnosis techniques in healthcare, and as quality control tools in food industry and nutrition. Several configurations of POC assays are available, mainly paper-based devices as Lateral Flow Assays (LFAs), and tests in solution, allowing the detection of biomarkers in biological fluids. Most of POC devices are intended for blood testing, given the abundance of targets in this matrix. However, considered the invasiveness related to this fluid, there is nowadays a growing interest in finding more user-friendly alternatives for the patients. To this aim, saliva is among the most suitable noninvasive fluids for POC testing (POCT), showing a good correlation with serum levels for many targets, and being easy to collect and handle. In this thesis, I presented the research activity carried out during the three years of PhD course concerning the development of POC assays for salivary, serum and food testing, employing noble-metal nanoparticles (NPs) as colorimetric tools, given their stability, handiness, and ability to provide different shades of color in the visible range. First, I reported the design and development of a paper-based immunosensor (namely, Lateral Flow Immunoassay, LFIA) for the naked-eye evaluation of salivary cortisol, known as one of the main stress-related biomarkers. The colorimetric strategy exploited the plasmonic properties of two differently shaped gold NPs (i.e., nanospheres and nanostars), combined to get a dual-color system which facilitated an immediate and easy evaluation of cortisol levels, based on a blue-to-pink color change of the detection zone. Then, the technique was employed for the colorimetric evaluation of serum vitamin D (VD), which dysregulations are associated mainly to osteoporosis, diabetes and cardiovascular diseases. To this purpose, a competitive LFIA based on the plasmonic properties of 35 nm gold nanospheres (AuNPs) was developed. The device allowed the discrimination of three ranges of VD levels, according to the color fading of the test line (TL) at the detection zone. Finally, a last paper-based device was developed for the evaluation of Total Antioxidant Capacity (TAC), namely the complete pattern of antioxidant species in a complex medium. To this aim, the peroxidase-like properties of 5 nm platinum NPs (PtNPs) were exploited to design a multi-line PtNP-based LFA, which relied on three sequential TL with increasing concentrations of Pt nanozymes, to get a non-invasive, accurate, and fast colorimetric evaluation of TAC in saliva samples and food matrices. The last part of my thesis concerned the investigation of a colorimetric assay for the assessment of amyloid β peptide in saliva, as one of the main factors associated with the progression of Alzheimer’s disease. The strategy exploited the phenomenon of the plasmon coupling, occurring in solution between 80 nm AuNPs, to get a qualitatively evaluation of the target based on a red-to-violet color change of the solution at a certain limit of detection. Such strategy showed promising results in controlled conditions, and potential applicability in the real matrix.