Portable Real-Time SERS for Glutathione Analysis in Blood

2024-12-18

Introduction

Glutathione (GSH) is a non-protein thiol tripeptide that plays a critical role in defending against oxidative stress. Its concentration is particularly important during the neonatal period, especially for premature infants who are more susceptible to oxidative stress. Monitoring GSH levels can provide valuable insights into the health of neonates, enabling the development of care strategies tailored to their specific needs.

In this context, Abel Albiach-Delgado and colleagues from the Carlos III Health Institute in Spain developed a surface-enhanced Raman spectroscopy (SERS) sensor specifically designed for neonatology, capable of measuring GSH from just 2 μL of whole blood. This new optical sensor utilizes Optosky’s portable ATR3110 Raman spectrometer, offering a rapid and cost-effective solution to assess GSH levels in neonates. This approach promises to significantly improve neonatal care and is also beneficial for health monitoring research in adult populations. The study was published in the scientific journal Talanta under the title "Portable point-of-care surface-enhanced Raman scattering spectroscopy for the quantification of glutathione in whole blood microsamples."

Figure 1. Schematic for detecting GSH in whole blood from neonates using the portable ATR3110 Raman spectrometer

1. Preparation of Silver Colloid

The synthesis of silver colloid was performed according to the method described by Leopold et al. Briefly, at alkaline pH and room temperature, silver nitrate (1 mM) was rapidly reduced with hydroxylamine (1.5 μM) under continuous stirring. The reaction was completed within seconds, and the formation of the silver colloid was confirmed by UV spectroscopy. A maximum absorbance at 416 nm indicated a monodispersed particle size of approximately 100 nm.

2. Preparation of Standards and Filters Containing L-Cysteine-D2 (IS)

A fresh working solution of GSH at 8 mM was prepared by dissolving the GSH standard in water. Eight calibration standards in the range of 0.55-2.40 mM were obtained by serial dilution of this working solution. A 17.9 mM solution of L-cysteine-D2 (IS) was concentrated by evaporation under vacuum at room temperature and stored at -20℃ until use. Prior to analysis, the IS solution was reconstituted in 1.4 mL of ultrapure water. Filters containing IS were prepared by adding 2 μL of the IS solution to a 0.2 μm PTFE syringe filter, drying under a nitrogen stream for 5 minutes, and storing at -20℃.

3. Sample Collection and Preparation

Blood samples were collected from two distinct groups: (i) 35 healthy full-term neonates via heel prick, and (ii) 63 healthy adults via fingertip blood samples. In the neonate group, in addition to samples for SERS measurement, a second 10 μL dried blood spot (DBS) sample was collected for GSH analysis by high-performance liquid chromatography (HPLC).

The samples were analyzed by SERS using two different preparation methods (Figure 2). In Method A, 42 μL of 13% (v/v) PCA aqueous solution was added to 4 μL of whole blood, 4 μL of IS solution (0.895 mM), and 10 μL of water in a polypropylene microtube to precipitate proteins. After homogenization (vortexing for 15 seconds), the sample was centrifuged and the supernatant collected for further analysis. In Method B, 2 μL of whole blood was introduced into the IS filter and evaporated, followed by 1 mL of water to wash the filter for protein precipitation and GSH extraction.

Figure 2. Experimental workflow for quantifying GSH in whole blood (WB) by protein precipitation and centrifugation (Method A) and direct GSH extraction using a filter containing IS (Method B)

4. SERS Analysis

The extracts obtained by Methods A and B were analyzed by SERS under the following conditions (Figure 2): 40 μL of the WB extract was added to 310 μL of silver colloid in a 96-well polystyrene plate. The SERS analysis was performed using the portable ATR3110-532 Raman spectrometer (Optosky Photonics Inc., Xiamen, China). GSH standard spectra were collected by mixing 5 μL of each calibration standard with 5 μL of IS solution (893 μM) and 40 μL of 13% (v/v) PCA. The spectrometer used a 532 nm laser with a maximum power of 100 mW and a -10 °C cooled charge-coupled device (CCD) detector. The measurement parameters were as follows: laser power of 20 mW, acquisition time of 3000 ms, 10 scans, scan interval of 500 ms, and spectral range of 100-3500 cm-1.

5. Results and Discussion

The Raman spectra of the empty plate showed a peak around 1000 cm-1, corresponding to a breathing mode of polystyrene (Figure 3). The PCA solution (13% v/v) showed a peak at 928 cm-1, characteristic of the ν (ClO4-) ion in perchlorate. The SERS spectrum of GSH showed characteristic peaks at 905 cm-1 (ν(C–COO-)), 879 cm-1 (ν(C–C)), and strong peaks at 791 and 653 cm-1 corresponding to amide V and ν(C–S) in Cys. L-cysteine-D2 showed strong peaks at 1069, 982, 881, 699, and 638 cm-1. The spectra obtained after protein precipitation from WB were highly similar to those from the GSH standard solution, with a correlation coefficient of R = 0.98 in the 600-900 cm-1 spectral range.

Figure 3. Raman spectra of 96-well plate, PCA solution, whole blood, GSH, and L-cysteine-D2

Spectral analysis identified characteristic peaks for GSH/WB and IS (L-cysteine-D2) at 791 cm-1 and 982 cm-1, with no interference signals. These peaks were used to quantify GSH concentration by correcting for variations in the SERS signal from the aqueous solutions, thereby improving the precision and accuracy of the measurement. The calibration curves showed a linear range of 0.55-2.4 mM with correlation coefficients (R2) between 0.992 and 0.993.

This method was applied to different populations, including healthy adults (N=63) and neonates (N=35), with measured GSH concentrations ranging from 0.6-1.8 mM and 0.8-2.1 mM, respectively. This new approach provides a rapid and cost-effective solution for evaluating the GSH status of neonates, with potential applications in adult health monitoring. Future research could include the simultaneous quantification of oxidized GSH (GSSG) to assess the GSH/GSSG ratio and improve the evaluation of redox status.

As the leader in Raman spectrometers in China, Optosky offers a full range of handheld, portable, research-grade, and industrial Raman products, capable of providing solutions for a wide variety of applications. All Raman spectra in this study were collected using Optosky’s portable ATR3110 spectrometer.