X-ray experimental set-up for in-vitro nail test

In this investigation, the elements Ca and S were evaluated in a single nail clipping using compact and portable X-ray spectrometer model X-123 SDD with Ag target. Experimental conditions for current, voltage and excitation time were investigated. These analyses can be useful for a variety of applications, including nutritional and medical diagnosis, such as, the evaluation of bone dysfunctions by measurement of Ca (bone decalcification) and S for nutrition evaluations (collagen production). This analysis offers some benefits comparatively to blood and serum analyses, such as, non-invasive collection, fast analyses (minutes) and low cost. In addition, nail clippings are simple to obtain, easy to store, and easy to transport. This pilot study show a positive expectation for clinical application using in-vitro nail test.


INTRODUCTION
In recent years, alternative techniques to investigate specifics ions and metals, of clinical relevance, in the human body (blood, serum and urine) have made significant progress. These clinical investigations are very useful for various diagnoses and for routine analysis. In recent years, X-ray Fluorescence (XRF) technique has been applied to this clinical finality at IPEN/CNEN-SP, in collaboration with research centers from Brazil [1][2][3][4][5][6]. The success of this alternative procedure for ions dosage in body fluids, bones, muscles and other biological tissues motivated us to verify the use of portable X-ray Fluorescence Spectrometry for in vitro nail clipping tests for the diagnosis of some specific dysfunctions.
Nails are basically made up of a fibrous protein keratin, a protein of animal origin, composed by several amino acids. The main amino acid that makes up keratin is cysteine (C3H7NO2S), that is, it has the element sulfur in its structure [7]. Therefore, nails contain small quantities of trace elements such as S (its main component), Ca, Cu, Fe, Zn, and others [8; 9; 10]. Like blood and urine, nail samples can be used as biomarkers of several dysfunction : while Fe, Se, and Zn have been correlated with colorectal cancer risk [11], the evaluation of metals (Pb, Cd, Hg) in nails have also been performed to investigate toxicity effects in the organism [12,13,14,15].
All these studies suggest that application of XRF technique for analysis of majoritary and trace elements in nails are very promising for clinical practice.
In this investigation, a portable and compact equipment for X-ray Fluorescence, using target of Ag, low voltages (tens of KV) and current (few µA), was evaluated for in-vitro nail clippings tests for Ca and S evaluation. There are some motivations and positive expectations for clinical applications such as to provide useful data for sports medicine by the evaluation of S, responsible in the organism for collagen production and maintenance of muscle tissues, as well as to check the viability to monitor bone decalcification by Ca evaluation.

MATERIALS AND METHODS
The Energy Dispersive X-ray Fluorescence (EDXRF) analysis was performed using a compact X-ray spectrometer model X-123 SDD with Ag target (Figura 1). The characteristic X-ray fluorescent intensity of Kα lines were measured with a Si Drift detector (25 mm 2 x 500 μm) with Be window (12.5 μm). For the spectrometer calibration, certified standard solutions containing varying concentrations of Ca and S were prepared. All the spectral analysis was performed using WinQxas software [16]. The precision and accuracy of the results were checked by analyzing NIST 1577b Bovine Liver. Brasil) was selected. At least, two nail clippings taken from each donor was collected. The fingernails were preferred to toenails, as they have a faster growth. Nail clippings (mass ~ 5 mg) were performed using a pair of scissors and placed in polyethylene bags. In the laboratory, each sample was placed in a flask with detergent and shacked (mechanical shaking) for ~20 minutes.
After that, the sample was transferred to a beaker and washed with deionized water (Milli-Q) until the detergent was completely removed. In the next step, the samples were placed on filter paper and dried at room temperature. Finally, they were stored in plastic bag until to be used. The experimental conditions for voltage, current, excitation time as well as the appropriate choice of collimators and filters were investigated to reduce radiation exposure, to enable in-vitro analysis on nails. Table 2 presents the optimized experimental conditions for nail clipping tests.

RESULTS AND DISCUSSION
These evaluations were performed using the emission line Kα (2.1 keV for S and 3.6 keV for Ca).
The results for Ca e S concentrations in nail clipping samples using EDXRF technique are shown in  Figure 4 are shown the results of Ca e S concentrations by EDXRF; the mean value as well as the standard deviation (MV ± 1SD) were also included. To illustrate, in figure 2 is presented a XRF spectrum for a nail clipping using the optimized experimental condition for Ag target (5µA, 30kV, 200s). In this figure, the Argon peak (Ar) are due to its presence in air.   (Table 3) show non-significant differences (p > 0.05) when a comparison is performed with NAA [18] and ICP-MS [19] data. The concentration results obtained in this work for Ca e S (figure 3) indicated the viability of using X-ray experimental setup for in-vitro nail clipping test with precision and accuracy, as well as its ability to determine other elements. This pilot study is a fundamental step towards the elementary evaluation of the nail by EDXRF, for future clinical applications. However, several measurements should be explored in future work to extend these assessments to the other elements simultaneously excited. These data will improve the correlation between Ca measurements in nail clipping for evaluation of bone dysfunctions (bone decalcification) as well providing data involving the correlations between S measurements for collagen production (maintenance and fortification muscle tissues), very useful in sports medicine. Finally, the XRF procedure requires no preparation, only that the nails are clean, also offers a cost-effective and rapid measurement approach to help improve our understanding of elements in nails and their correlation to health. Besides, nail clippings are simple to obtain, easy to store and transport, compared to clinical analysis using body fluids.

CONCLUSION
The development of methodology to quickly assess Ca and S concentration from a single nail clipping could be a useful advance for clinical practices. In addition, there is another advantage, the feasibility of using this experimental X-ray set-up in regions lacking a clinical laboratory, for example, its use in a Basic River Health Unit.
Overall, trace element analysis in nail clippings by XRF presents an opportunity for new applications, as well as significant contributions to a better understanding of the role of trace elements in medical and biological sciences.