## 1 Introduction  
More than 100 biothiols, which play a significant role in many biological processes and metabolic pathways (e.g., protein synthesis, antioxidant defense system, cell metabolism, etc. ), have been identified in the human organism [1, 2]. Of these biothiols, glutathione (GSH), cysteine (Cys) and homocysteine (Hcy) are the most abundant, with concentration levels that vary significantly in biological fluids. Under normal (non-pathological) conditions, GSH is present at concentrations as high as 3 mM in whole blood and less than 5 \(\upmu\)M in blood plasma and urine. Cys, on the other hand, typically ranges from 135 to 300 \(\upmu\)M in blood plasma and 20-80 \(\upmu\)M in urine while Hcy concentrations are lower than 15 \(\upmu\)M in both plasma and urine. Abnormal levels of biothiols have been related to clinical disorders and diseases, such as liver damage, Alzheimer's disease, osteoporosis and cardiovascular diseases, among others [3, 4, 5, 6]. Due to the important biological implications of biothiols, there has been a great interest in their determination in biological fluids. To that end, several analytical methods and techniques have been developed: HPLC, LC-MS, UV-Vis spectrometry, fluorescence, chemiluminescence, electrochemical techniques, etc. [7, 8]. Over the past decade, nanomaterials have also been a popular scaffold for the development of biothiol probes and chemosensors [9]. By exploiting the high affinity of the sulfhydryl group for metal nanoparticles, a large variety of optical methods have been proposed. These methods are based on the generation or quenching of an optical event when biothiols bind on the surface of nanomaterials through a variety of mechanisms, such as host-guest interactions of biothiols with receptor molecules on the nanomaterials surface, the replacement of functional molecules from the nanomaterials surface and the disruption of inter-nanoparticle bonds [9]. The main advantage of nanomaterial probes is their high sensitivity as a result of the strong distance- and size-dependent optical properties of metal-based nanomaterials, especially those made of noble metals. However, the use of nanomaterials also faces several shortcomings: although synthesis may be relatively easy in a research laboratory, the reproducible large-scale synthesis and functionalization of nanomaterials for biomedical applications is a resource-demanding task that requires substantial nanotechnology skills and advanced equipment for their proper characterization. Additionally, due to a high surface area, which imbues nanomaterials with high reactivity, and surface functionalization, non-specific interactions with the matrix components may be observed. Furthermore, many of these methods require laboratory facilities, expensive scientific equipment (e.g., plate spectrophotometers or spectrofluorometers) and trained operators for their application, which are probably unavailable at the point-of-need and remote healthcare units. An alternative approach to overcome the challenges associated with the use of nanomaterials is to use thiols as size and growth regulators of nanomaterials. In these methods, thiols firstly react with the precursor metal ions. Upon addition of a reducing agent, the produced nanomaterials exhibit a different size, morphology or aggregation state compared to those obtained in the absence of thiols. The difference in the colorimetric and spectral response in the presence and absence of biothiols is therefore used to determine their concentration. Based on this effect, several analytical methods using gold and silver nanoparticles have been developed, also from our group [10; 11; 12; 13]. The main challenge in these methods, however, is that the kinetics of the reactions evolve over time and thus requiring relatively strict timing to obtain reproducible results [13; 14].