Surface-Enhanced Raman Analysis of Uric Acid and Hypoxanthine Analysis in Fractionated Bodily Fluids

Abstract

In recent years, the disease burden of hyperuricemia has been increasing, especially in high-income countries and the economically developing world with a Western lifestyle. Abnormal levels of uric acid and hypoxanthine are associated with many diseases, and therefore, to demonstrate improved methods of uric acid and hypoxanthine detection, three different bodily fluids were analysed using surface-enhanced Raman spectroscopy (SERS) and high-performance liquid chromatography (HPLC). Gold nanostar suspensions were mixed with series dilutions of uric acid and hypoxanthine, 3 kDa centrifugally filtered human blood serum, urine and saliva. The results show that gold nanostars enable the quantitative detection of the concentration of uric acid and hypoxanthine in the range 5-50 μg/mL and 50-250 ng/mL, respectively. The peak areas of HPLC and maximum peak intensity of SERS have strongly correlated, notably with the peaks of uric acid and hypoxanthine at 1000 and 640 cm^-1, respectively. The r² is 0.975 and 0.959 for uric acid and hypoxanthine, respectively. Each of the three body fluids has a number of spectral features in common with uric acid and hypoxanthine. The large overlap of the spectral bands of the SERS of uric acid against three body fluids at spectra peaks were at 442, 712, 802, 1000, 1086, 1206, 1343, 1436 and 1560 cm^-1. The features at 560, 640, 803, 1206, 1290 and 1620 cm^-1 from hypoxanthine were common to serum, saliva and urine. There is no statistical difference between HPLC and SERS for determination of the concentration of uric acid and hypoxanthine (p > 0.05). For clinical applications, 3 kDa centrifugal filtration followed by SERS can be used for uric acid and hypoxanthine screening is, which can be used to reveal the subtle abnormalities enhancing the great potential of vibrational spectroscopy as an analytical tool. Our work supports the hypnosis that it is possible to obtain the specific concentration of uric acid and hypoxanthine by comparing the SER signals of serum, saliva and urine. In the future, the analysis of other biofluids can be employed to detect biomarkers for the diagnosis of systemic pathologies.

Keywords: blood serum; bodily fluids; centrifugal filtration; gold nanostars; hypoxanthine; saliva; surface-enhanced Raman spectroscopy; uric acid; urine; vibrational spectroscopy.

Figure 1

UV-vis absorbance spectrum (a) and particle size distribution (b) of AuNS solution.

Figure 2

Nanostars under (a) Scanning and (b) Transmission Electron microscopy.

Figure 3

(a) The uric acid HPLC peak at concentration of 50 μg/mL, (b) SERS spectra of uric acid for concentrations at 5, 10, 20, 25, 50 μg/mL in black, red, blue, purple and green, respectively.

Figure 4

Linear correlation of HPLC and SERS with the concentration of uric acid conentration. (a) The linear correlation of HPLC peak areas with concentration of uric acid. (b) The maximum peak intensity of SERS vs. the concentration of uric acid. The linear correlation from top to bottom is maximum peak at 1000, 442, 1436, 1085, 1343, 1560, 802, 1206, 712 cm⁻¹ to concentration of uric acid. The peak at −1000 cm⁻¹ (red line) showed a linear concentration with. R² of 0.994 (c) The linear correlation of the maximum peak intensity of SERS and peak areas of HPLC of uric acid. R² was 0.971.

Figure 5

(a) The hypoxanthine HPLC peak of 250 ng/mL, (b) SERS spectra of hypoxanthine for concentrations at 50, 100, 150, 200 and 250 ng/mL in black, red, blue, purple and green, respectively.

Figure 6

(a) The linear correlation of HPLC peak areas versus concentration of hypoxanthine (b) Correlation of SERS peak intensities versus concentration of hypoxanthine, from top to bottom. The maximum peak at 640, 1290, 560, 1620, 1206, 802 cm⁻¹. The peak at ~640 cm⁻¹ (red line) showed an R² of 0.994. (c) The correlation of 640 cm⁻¹ maximum peak intensity vs. peak areas of HPLC for hypoxanthine. R² is 0.959.

Figure 7

Repeatability of SERS spectra of 3 kDa filtrates of human serum, saliva and urine samples with added nanostars (nanostar: sample = 1:19 in volume ratio). (a) From five independent experiments of serum. (b) Spectra from five different saliva samples, that from each donor depicted in a different colour. (c) Spectra from five different urine samples, that from each donor depicted in a different colour. For all spectra, source wavelength was 785 nm, laser power 125 mW, and acquisition time was 10 s per spectrum.

Figure 8

Overlap of spectra of 3 kDa filtrates of human serum, saliva and urine with uric acid and hypoxanthine. The intensity of peaks and highest overlap peak in body fluids and uric acid and hypoxanthine. The overlap peaks with uric acid in solid blue line. The overlap peaks with hypoxanthine in vertical dash blue line. The assignment of peaks is provided in Table 1.