Electrochemical-Surface Enhanced Raman Spectroscopic (EC-SERS

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Electrochemical-Surface Enhanced Raman Spectroscopic (EC-SERS) Study of 6-Thiouric Acid: A Metabolite of the Chemotherapy Drug Azathioprine Brad H. C. Greene, Dalal S. Alhatab, Cory Christopher Pye, and Christa L. Brosseau J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b01179 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 22, 2017

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The Journal of Physical Chemistry

Electrochemical-Surface Enhanced Raman Spectroscopic (EC-SERS) Study of 6-Thiouric Acid: A Metabolite of the Chemotherapy Drug Azathioprine B.H.C. Greene, D.S. Alhatab, C.C. Pye, C.L. Brosseau*

Department of Chemistry, Street, Saint Mary’s University, Halifax, Nova Scotia, Canada B3H 3C3

* To whom correspondence should be addressed: Christa L. Brosseau ([email protected]) Phone (902) 496-8175 Fax (902) 496-8104

1 ACS Paragon Plus Environment


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ABSTRACT 6-Thiouric acid (6-TUA) has the potential to be an important biomarker for the action of 6mercaptopurine (6-MP), an immunosuppressive drug used in patients suffering from acute lymphoblastic leukemia (ALL). 6-TUA, a non-active metabolite of 6-MP, is excreted in the urine, and routine monitoring of this metabolite can be useful in assessing the efficacy of 6-MP for immune system suppression in patients who have undergone stem cell replacement. In this work, electrochemical surface-enhanced Raman spectroscopy (EC-SERS) is used for the first time to study the adsorption and electrochemical behavior of 6-TUA at a nanostructured silver electrode surface, in both 0.1 M NaF and synthetic urine as supporting electrolytes. In addition, ab-initio calculations were completed in an effort to understand the adsorption behavior. It was found that EC-SERS provided excellent signal for 6-TUA down to µM concentrations in synthetic urine, and highlights the future potential of EC-SERS for rapid detection of important urine biomarkers at the patient point-of-care.


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INTRODUCTION Leukemia is the most common form of childhood cancer, accounting for 25-30% of all cases.1 The most common type of childhood leukemia is acute lymphoblastic leukemia (ALL), which accounts for approximately 75% of cases.1 There are a number of treatment options for ALL including chemotherapy, radiation, and bone marrow transplant (BMT). If BMT is the selected course of treatment, the patient undergoes chemotherapy treatment in order to destroy existing stem cells in the bone marrow, after which bone marrow from a donor is transplanted.1 One obvious issue with bone marrow transplants (and organ transplants more generally) is the rejection of the transplant by the host immune system, which will render the transplant unsuccessful.2 To combat the activity of the immune system, immunosuppressive drugs are used, and rapid and accurate evaluation of the effectiveness of these drugs is vital to adequate care and monitoring after the transplant is completed. One immunosuppressive drug commonly used for bone marrow transplants is 6mercaptopurine (6-MP), which goes by the tradenames Purinethol® and Azathioprine® (prodrug form).3 Monitoring of 6-MP is typically done through analysis of human serum, using techniques such as fluorescence spectroscopy, liquid chromatography, and electrochemistry.4-10 Although these methods have proven to exhibit high selectively and sensitivity, they are often combined with complex sample treatment, expensive equipment and complicated and / or laborious analysis.4 Therefore, advances in the detection of this particular drug and its metabolites is warranted. 6-MP metabolizes mainly into three compounds, one of which, 6-thioguanine, is responsible for the immunosuppression.11 The other two non-active metabolites, 6-thiouric acid (6-TUA) and 6-methyl-mercaptopurine are produced through the action of xanthine oxidase and thiopurine methyltransferase, respectively.11 6-TUA is excreted in the urine5 and quantitative



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detection at low concentrations would be beneficial for evaluating the effectiveness of immunosuppression therapy in cancer patients. For example, elevated levels of 6-TUA in patient urine may indicate that relatively little 6-thioguanine is being produced, rendering the drug less effective for immune suppression. Recently, electrochemical-surface enhanced Raman spectroscopy (EC-SERS), combining both electrochemistry and surface enhanced Raman spectroscopy (SERS) has been developed for routine spectroelectrochemical analyses.12-25 A simple, portable EC-SERS system, consisting of a small benchtop Raman spectrometer, a laptop computer, and a portable USB potentiostat, has been reported previously by our group for real-time on-site monitoring of patient biomarkers, and is used in the present study.26-28 In this paper, the extent to which EC-SERS may be a useful tool for the rapid and selective detection of 6-TUA in urine is investigated. Combined with ab initio calculations, the results indicate that 6-TUA can be readily detected by EC-SERS down to µM levels in synthetic urine, and may indeed be a useful future tool for rapid and routine analysis of patient biomarkers at the point-of-care. To our knowledge, this work represents the first EC-SERS analysis of 6thiouric acid.

EXPERIMENTAL Spectroelectrochemical System: The Raman spectrometer used for these studies consisted of a DeltaNu (Intevac Photonics) bench top dispersive Raman spectrometer equipped with an aircooled CCD, 785 nm diode laser and an optics extension tube. The power at the sample and acquisition time was typically 22.3 - 55.9 mW and 30-60 seconds, respectively. The spectrometer resolution was 4 cm-1. The potentiostat was a Pine Research Instrumentation


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portable USB Wavenow potentiostat. A voltammetry cell, also available from Pine Research Instrumentation, was used as the electrochemical cell. 0.1 M NaF was used as the supporting electrolyte for all fundamental studies due to the low specific adsorption of F- on Ag. All Raman data was smoothed using adjacent averaging (8 point), and corrected for both laser power and acquisition time.

Preparation of Citrate-reduced Colloids: Preparation of the spherical silver nanoparticles was modified from the literature method.29,30 In a typical procedure, 1.0 mL of silver nitrate solution (0.1 M), 3.4 mL of aqueous sodium citrate (0.17 M), and 0.6 mL of citric acid (0.17 M) were added into a flask with 95.0 mL of water. 0.2 mL of freshly prepared sodium borohydride solution (0.1 mM) was then added into the above mixture at room temperature under magnetic stirring. The mixture was allowed to stand at room temperature for 1 min and then brought to boil under reflux within 20 min under magnetic stirring. After boiling for 1 h, the solution was allowed to cool to room temperature.

Preparation of SERS-Active Screen Printed Electrodes: The silver nanoparticles (AgNP) obtained from the above procedure were centrifuged at 8000 rpm for 20 min. The supernatant was then removed and discarded, and the pellet in each Eppendorf tube was collected together and centrifuged again at 8000 rpm for 20 min to obtain a silver nanoparticle concentrate. AgNPmodified electrodes were prepared by drop coating three layers of silver nanoparticle concentrate in 5 µL aliquots onto the carbon working electrode of commercially available screen printed electrodes

(carbon

SPEs,

rectangular

working

electrode,

4x5

mm,

Pine Research

Instrumentation, Durham, USA). The electrodes were allowed to dry completely after each layer



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deposition and prior to use. The SPE features a built-in counter electrode (carbon) and reference electrode (Ag/AgCl). All electrolyte solutions were purged with Argon gas (99.999%, Praxair Canada Inc., Ontario, Canada) prior to measurement.

Reagents: Sodium citrate, citric acid and sodium fluoride were all purchased from Sigma Aldrich (St. Louis, MO) and used without further purification. Silver nitrate (99.9995%) was purchased from Alfa Aesar (Wardhill, MA). 6-thiouric acid (>98%) was purchased from Toronto Research Chemicals (Toronto, ON). Synthetic urine was purchased from Ricca Chemicals (Arlington, TX) and is reported by the manufacturer to contain the following: 97.17% w/w water, 1.90% w/w urea, 0.77% w/w NaCl, 0.09% w/w MgSO4•7H2O, 0.06% w/w CaCl2•2H2O. All solutions were prepared using Millipore water (≥18.2 MΩ cm). All glassware for experimentation was first soaked in neat sulfuric acid overnight, followed by careful rinsing with Millipore water.

Quantum Chemical Calculations: Calculations were performed using Gaussian 03.31 The geometries were optimized using a stepping stone approach, in which the geometries at the levels HF/6-31G*, HF/6-31+G*, B3LYP/6-31G*, B3LYP/6-31+G*, MP2/6-31G*, and MP2/6-31+G* were sequentially optimized, with the geometry and molecular orbital reused for the subsequent level. The MP2 calculations utilize the frozen core approximation. Default optimization specifications were normally used. After each level, a frequency calculation was performed at the same level and the resulting Hessian was used in the following optimization. Z-matrix coordinates constrained to Cs symmetry were used to speed up the optimizations. Since frequency calculations are done at each level, any problems with the Z-matrix coordinates would


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manifest themselves by giving imaginary A” frequencies. The Hessian was evaluated at the first geometry (opt = CalcFC) for the first level in a series in order to aid geometry convergence. The only problem noted was the presence of an imaginary frequency for the aromatic systems at MP2/6-31+G*, which is believed to be an artifact of the calculation method.32

RESULTS AND DISCUSSION ATR-FTIR spectroscopy was used to characterize the solid 6-TUA powder, the result of which is shown in Figure S-1. Analysis of this spectrum shows bands due to ν(=C-S) and ν(C-S) at 590 cm-1 and 767 cm-1, respectively, suggesting that the powder form of 6-TUA exists as an enethiol tautomer. Strong bands at 1594 cm-1 and 1725 cm-1 are suggestive of ν(C=O) modes. Of particular note, vibrational modes associated with the N-C=S moiety are absent.33 The normal Raman spectrum of the powder 6-TUA is shown in Figure S-2. After band assignment, the spectral profile is also consistent with an enethiol tautomer for the solid powder, however the ν(C=O) vibrational modes are not Raman-active. Quantum-chemical calculations were done in order to predict the infrared and Raman frequencies and intensities for the keto form and sixteen tautomers, obtainable from the keto form by a series of 1,3-hydrogen shifts. In the gas-phase, the keto form is most stable, followed by the enol/enethiol tautomers (See Figure S-3 and Table S-1 in the supporting information) in the following order (based on relative energies): 3 < 4

Electrochemical-Surface Enhanced Raman Spectroscopic (EC-SERS

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