Research Article,  J Nanomater Mol Nanotechnol Vol: 9 Issue: 1

Quantitative Determination of Thiol Groups Modified Mesoporous Silica Nanoparticles by Ellman’s Reagent

Abdullah M Alswieleh^*

Chemistry Department, College of Science, King Saud University, Saudi Arabia

*Corresponding Author : Abdullah M Alswieleh
Chemistry Department, College of Science, King Saud University, Riyadh 11451, PO Box 2455, Saudi Arabia
E-mail: aswieleh@ksu.edu.sa

Received: January 18, 2020 Accepted: January 28, 2020 Published: February 04, 2020

Citation: Alswieleh AM (2019) Quantitative Determination of Thiol Groups Modified Mesoporous Silica Nanoparticles by Ellman’s Reagent. J Nanomater Mol Nanotechnol 9:1.

Abstract

Keywords: Ellman's reagent; Nanoparticles; Mesoporous silica; Surface modification

Introduction

Nanostructured materials can be fabricated with different size, shape, and surface functionality, which allow for the adjustment of their unique properties over a broad range. There has been an increase in attention to mesoporous nanomaterials during the last decades [1-6]. Mesoporous Silica Nanoparticles (MSNs) have been used for various applications in technology and pharmaceutics, due to their high surface area, excellent physicochemical stability and facile modification [4-9]. The surfaces of silica nanoparticles can be modified through the attachment of silane reagents or coating with polymers [10-14]. Silane reagents can be covalently attached to the surface of silica nanoparticles through a condensation reaction, such as (3-aminopropyl)triethoxysilane or (3-mercaptopropyl) trimethoxysilane [15,16].

3-Mercaptopropyltrimethoxysilane (MPTMS) is often used to functionalize silica surfaces with thiol groups or to deposit silica shells on metallic surfaces [17,18]. Huang and co-workers reported the fabrication of mesoporous silica materials functionalized with MPTMS, followed by conversion of a thiol group to sulfonic acid to be used as a catalyst [19]. Shen et al. have reported the fabrication of PEGylated organosilica-capped mesoporous silica nanoparticles by employing organosilica coated with MPTMS as a gatekeeper to block the mesopores of MSNs [20]. Mosquera et al. developed an efficient coating strategy to stabilize gold nanoparticles (Au NPs) with silica monolayers after immobilization of MPTMS on a gold surface and hydrolyzed under basic conditions [21]. The polymeric silica monolayer on the outer surface of Au NPs can be readily further functionalized using silane coupling agents.

Ellman’s reagent [5,5’-dithiobis-(2-nitrobenzoiaccid)] (DTNB) has been used for the determination of mercapto groups by nucleophilic substitution of the reagent [22]. This reagent is usually used to quantify thiol groups in enzymes and proteins under a wide variety of experimental conditions with satisfactory results [22,23]. Woeppel et al. reported the immobilization of thiolated nanoparticles onto glass and silicon substrates via silane chemistry [24]. Ellman’s reagent was used to quantify the surface thiol concentration grafted on the nanoparticles, which is estimated to be 12.4 nmol of MPTMS per 0.2 mg of thiolated nanoparticles.

In this work, mesoporous silica nanoparticles (MSNs) were fabricated with a surface area of ~900 m²/g, and an average pore size of ~5.5 nm. MSNs were modified with mercapto groups on both inner and outer surfaces. 5,5'-Dithiobis(2-nitrobenzoic acid) (Ellman’s reagent, DTNB) was used to quantify mercapto groups on the surface. When DTNB reacted with materials in PBS solution, the concentration of 2-nitro-5-thiobenzoate (TNB−) increased as the reaction time passes. When the reaction completed, the concentration of 3-mercaptopropyl groups grafted on MSNs was quantified by measuring TNB− via the colorimetric method. This hypothesis is examined using, BET, FT-IR and UV-vis.

Experimental section

Materials: Elga Pure Nanopore was used to obtain deionized water. N-cetyltrimethylammonium bromide (CTAB, 98%), tetraethylorthosilicate (TEOS, 98%), n-hexane (HPLC grade), (3-mercaptopropyl)trimethoxysilane (MPTMS, 95%), methanol (99.8% HPLC grade), ethanol (99.8%, HPLC grade), toluene (analytical grade) and ammonium hydroxide (28 wt%), phosphate buffered saline (PBS) were purchased from Sigma-Aldrich. 5,5'-Dithiobis(2- nitrobenzoic acid) (Ellman’s reagent, DTNB) was obtained from vwr. Ammonium nitrate (95%) was obtained from Fisher Scientific. All the chemicals were used as received.

Mesoporous silica preparations and functionalization: Synthesis of mesoporous silica nanoparticles: 1 g of CTAB was dissolved in 160 mL of deionized water under stirring, followed by addition of 7 mL of concentrated ammonia water (28 wt%). A mixture solution of n-hexane (20 mL) and TEOS (5 mL) was added dropwise to the reaction flask under stirring at 35°C. After 12 h, the final product was centrifuged and washed with deionized water and ethanol and dried in an oven at 100°C for 2 h.

Synthesis of 3-mercaptopropyl-functionalized MSNs (MP-MSNs): Mesoporous silica nanoparticles coated with a 3-mercaptopropyl group was obtained in two steps. First, CTAB templates were removed from prepared mesoporous silica nanoparticles using a solution of ammonium nitrate in ethanol (10 mg/mL) at 80°C for 12 h under stirring. The sample was centrifuged, washed with ethanol, and dried in oven. Secondly, thiol-modified MSNs surface was obtained by suspending the nanoparticles (1 g) in a solution of MPTMS (2.5 mmol) in toluene (40.0 mL). The mixture was heated under reflux overnight. The final product was collected by centrifugation, washed with toluene and ethanol, and dried under vacuum.

Synthesis of the 3-mercaptopropyl-functionalized outer surface of MSNs (MP(outer)-MSNs): 3-Mercaptopropyl group coated outer surface of MSNs was obtained in two steps. First, the thiol-modified outer surface of MSNs surface was obtained by suspending the asprepared nanoparticles (1 g) in a solution of MPTMS (2.5 mmol) in toluene (40.0 mL). The mixture was refluxed overnight. The final product was collected by centrifugation, washed with toluene and ethanol, and dried under vacuum. Secondly, CTAB templates were removed from prepared mesoporous silica nanoparticles using a solution of ammonium nitrate in ethanol (10 mg/mL) at 80°C for 12 h under stirring. The sample was centrifuged, washed with ethanol, and dried in an oven.

Determination of thiol groups functionalized MSNs: Ellman's reagent was used for the determination of thiol groups grafted MSNs. A 0.1 mM solution of DTNB was freshly prepared by dissolving 10 mg of in 250 cm³ phosphate buffer solution (pH=8). To ensure the accuracy of the method used, 0.1 mM Ellman's reagent was reacted with different concentration of an aqueous solution of mercaptoethanol at 25°C. The possible presence of 2-nitro-5-thiobenzoate (TNB−) in sampled solutions was tested using a colorimetric method of determination. Prior to measurement, the solution was allowed to react for 2 h to ensure complete the reaction. Derivatized solutions were measured using a 1 cm absorption cell at an absorbance wavelength of 412 nm by Shimadzu (UV-2600) UV-vis spectrophotometer. Linear regression analysis of concentration vs. absorption gave regression lines of y=0.0035x+0.0057 with a correlation coefficient of 0.9974, as shown in Figure S1.

To determine the quantity of thiol group functionalized MSNs, each functionalized material was conducted to Ellman's reagent solution at different times. Ellman's solutions were freshly prepared with a concentration of 0.1 mM (5 cm³) and stirred with 10 mg of each material. Each mixture was shaken at 25°C for (5, 10, 20, 30, 40, 60, or 90) min. After each time period, solutions were separated by centrifugation and washed two times with 5 cm³ buffer solution. All fragments of each experiment were combined to determine the concentration of 2-nitro-5-thiobenzoate (TNB⁻) in the final solution by colorimetric method, and the concentration was calculated on the basis of a calibration curve.

Measurement and characterization

Surface area analysis: The surface area of fabricated nanoparticle was measured using nitrogen physisorption isotherms on a Micromeritics Gemini 2375 volumetric analyzer. Prior to analysis, each sample was degassed for 5 h at 120°C. The Brunauer-Emmett- Teller (BET) surface areas were determined by experimental points at a relative pressure (P/P°) of 0.05-0.25. The pore volume of each sample was determined from N₂ adsorbed at the P/P° of 0.99. MSNs average pore size distribution was calculated using the Barrett-Joyner- Halenda (BJH) model. Scanning Electron Microscopy (SEM): JEOL JSM-6380 LA was used to collect SEM images. The dried samples were used for imaging without any treatment. Transmission Electron Microscopy (TEM): A JEOL JEM-1230 was used for TEM imaging. A drop of diluted sample suspension in HPLC ethanol was placed on a copper grid and dried at room temperature the measurement. FTIR Spectroscopy: Thermo Scientific Nicolet iS10 instrument was used to obtain the IR spectra of all samples. The spectra were obtained in KBr pellets in the 4000-400 cm^-1 region with a resolution of 4 cm^-1. Thermogravimetric analysis (TGA): SII TGA 6300 instrument was used to determine the decomposition of materials, with a heating rate of 10°C/min under N₂.

Results and Discussion

MSNs were prepared by mixing TEOS with a template (CTAB) in the presence of pore expander (n-hexane), as shown in Scheme 1. 3-Mercaptopropyltrimethoxysilane (MPTMS) can be conveniently anchored onto MSNs to functionalize the internal and external mesopore surfaces.

Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) was used to study the morphology of the MSNs nanoparticles. SEM image reveals that the cleaned MSNs are almost nanospheres, and the particle size is polydisperse, ranging from 150 nm-250 nm, as shown in Figure 1A. TEM image shows that the obtained MSNs are dispersed without aggregation, with a diameter ranging from 150 nm to 250 nm, which agrees with the SEM results (Figure 1B). From TEM images, it can be clearly seen that highly ordered arrays mesopores were formed and pores size was estimated to be ca. 6 nm, due to the pore expanding effect of n-hexane (Table 1).

Table 1: Physiochemical data obtained for the cleaned MSNs, MSN-SH (outer surface), and MSN-SH (outer and inner surface).

All samples of the cleaned MSNs, MSN-SH (outer surface), and MSN-SH (outer and inner surface) were characterized using Brunauer-Emmett-Teller (BET). Table 1 summarized the physicochemical properties of all nanomaterials. After surface modification, there were reductions in surface area, and pore volumes were observed. All fabricated materials have a pore size of about (4-6) nm, which similar to the pore size estimated by the TEM image. In Figure 2, the N₂ adsorption/desorption isotherms are Type IV for all prepared samples, typically for the ordered mesoporous materials. At higher relative pressures, small different capillary condensation steps were noted for MSN-SH (outer surface) and MSN-SH (outer and inner surface) compared with cleaned surfaces. The hysteresis loop was broader for cleaned MSNs, compared to 3-mercaptopropylfunctionalized MSNs, which confirming the modification process.

FTIR was used to characterize the cleaned MSNs, MSN-SH (outer surface), and MSN-SH (outer and inner surface), as shown in Figure 3. FTIR spectra show that wide bands at 1250 cm^-1- 1050 cm^-1 were attributed to siloxane groups (Si-O-Si) bands. The peak at ∼800 cm^-1 was attributed to Si-O stretching vibration. Peak was noted at ∼1640 cm^-1, which attribute to the bending vibration of water. These features were noted in all materials. The absorption peaks of 3-mercaptopropyl-functionalized MSNs were noted at ∼1490 cm⁻¹, which assigned to C-H as bending vibration in -CH₂-, and peaks around ∼2930 cm^-1 ascribed to C-H as stretch vibration. These characters provide evidence of the successful functionalization of 3-mercaptopropyl groups.

In our studies, to quantify the number of accessible thiol groups on the outer and inner MSNs surface, Ellman's reagent (DTNB) was used. When DTNB reacts with the surfaces, 2-nitro-5-thiobenzoate (TNB^-) is generated this absorbs at 412 nm. Figure 4 illustrates the UV-vis spectra of the solution after the reaction between MSN-SH (outer and inner surface) and DTNB at different time. It can be seen that the amount of produced TNB- increased as the reaction time increased which the thiol concentration was calculated based on the calibration curve.

Figure 5 shows the concentration of TNB- in the solution which is corresponding to the concentration of mercapto groups grafted on MSNs at different times. MSN-SH (outer surface) requires about 10 min to reach the maximum amount of released TNB^-, with ca. 0.02 mmol/g. For the MSN-SH (outer and inner surface), the amount of released TNB- dramatically increased to 0.08 mmol/g in 30 min and then remains to 0.11 mmol/g after 40 min. It indicates that outer surface samples display a fast reaction rate and fewer thiol groups, while outer and inner surface samples exhibiting a lower reaction rate and higher thiol groups, due to their thiol groups in opened mesopores and the diffusion of DTNB/TNB^-.

The successful surface reaction between marcapto groups and Ellman's reagent was evaluated by thermogravimetic analysis (TGA) when heating in an N₂ atmosphere to 600°C (Figure 6). After surface modification with MPTMS in both materials (-SH in outer and outer/inner surfaces), ca. 8 wt% of weight loss was observed. When the MSN-SH (outer surface) was reacted with Ellman's reagent, the weight loss was about 10 wt%. The weight loss in the MSN-SH (outer/ inner surface) after reaction with Ellman's reagent was ca. 17%.

Conclusion

MSNs were prepared with a surface area of ~900 m²/g, the large pore volume of 1.3 cm³/g and an average pore size of ~5.5 nm, using the Stöber method in the presence of expander reagent (hexane). MSNs were fabricated with 3-mercaptopropyl groups in both inner and outer surfaces. FT-IR spectra show peaks at ∼1490 cm⁻¹ and ∼2930 cm^-1, which assigned to C-H. Ellman’s reagent was successfully reacted with fabricated materials. The intensity of the absorption peak of TNB− increased as the reaction time passes. The reaction in the outer surface was noticed to be 4 times faster, comparing to the fully coated surface. Thiol groups in the outer surface were calculated to be ca. 0.02 mmol/g, while the total amount of marcapto groups coated outer/inner surfaces was estimated to be ca. 0.11 mmol/g. The amount of thiol groups in the inner surface is almost five times higher than the outer surface.

Acknowledgment

The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through the research project.

References

1. Beck JS,Vartuli J, Roth WJ, Leonowicz M, Kresge C, et al. (1992) A new family of mesoporous molecular sieves prepared with liquid crystal templates. J American Chem Soc 114: 10834-10843.
2. Wu SH, Mou CY, Lin HP (2013) Synthesis of mesoporous silica nanoparticles. Chem Soc Reviews 42: 3862-3875.
3. Reich SJ, Svidrytski A, Holtzel A, Florek J, Kleitz F, et al. (2018) Hindered diffusion in ordered mesoporous silicas: Insights from pore-scale simulations in physical reconstructions of SBA-15 and KIT-6 silica. J Physic Chem 122: 12350-12361.
4. Mal NK, Fujiwara M, Tanaka Y (2003) Photocontrolled reversible release of guest molecules from coumarin-modified mesoporous silica. Nature 421: 350-353.
5. Burkett SL, Sims SD, Mann S (1996) Synthesis of hybrid inorganic-organic mesoporous silica by co-condensation of siloxane and organosiloxane precursors. Chemical Communications 11: 1367-1368.
6. Singh P, Srivastava S, Singh SK (2019) Nanosilica: Recent progress in synthesis, functionalization, biocompatibility, and biomedical applications. ACS Biomater Sci Eng 5: 4882-4898.
7. Lu J, Liong M, Li Z, Zink JI, Tamanoi F (2010) Biocompatibility, biodistribution, and drug‐delivery efficiency of mesoporous silica nanoparticles for cancer therapy in animals. Small 6: 1794-1805.
8. Chen KJ, Chen HL, Tang CC, Wu HH, Jan JS (2019) Synthesis of silica/polypeptide hybrid nanomaterials and mesoporous silica by molecular replication of sheet-like polypeptide complexes through biomimetic mineralization. J Colloid Int Sci 542: 243-252.
9. Tang F, Li L, Chen D (2012) Mesoporous silica nanoparticles: Synthesis, biocompatibility and drug delivery. Advanced Mater 24: 1504-1534.
10. Slowing II, Vivero-Escoto JL, Trewyn BG, Lin V (2010) Mesoporous silica nanoparticles: Structural design and applications. J Mater Chem 20: 7924-7937.
11. Liu C, Guo J, Yang W, Hu J, Wang C, et al. (2009) Magnetic mesoporous silica microspheres with thermo-sensitive polymer shell for controlled drug release. J Mater Chem 19: 4764-4770.
12. Wang Y, Yin M, Lin X, Li L, Li Z, et al. (2019) Tailored synthesis of polymer-brush-grafted mesoporous silicas with N-halamine and quaternary ammonium groups for antimicrobial applications. J Coll Int Sci 533: 604-611.
13. Liu R, Liao P, Liu J, Feng P (2011) Responsive polymer-coated mesoporous silica as a pH-sensitive nanocarrier for controlled release. Langmuir 27: 3095-3099.
14. Zhang X, Yang P, Dai Y, Ma P, Li X, et al. (2013) Multifunctional up‐converting nanocomposites with smart polymer brushes gated mesopores for cell imaging and thermo/ph dual‐responsive drug controlled release. Adv Funct Mat 23: 4067-4078.
15. Zhang S, Zhang Y, Liu J, Xu Q, Xiao H, et al. (2013) Thiol modified Fe3O4 and SiO2 as a robust, high effective, and recycling magnetic sorbent for mercury removal. Chem Eng J 226: 30-38.
16. Ghasemzadeh MA, Abdollahi-Basir MH, Babaei M (2015) Fe3O4 and SiO2-NH2 core-shell nanocomposite as an efficient and green catalyst for the multi-component synthesis of highly substituted chromeno [2,3-b] pyridines in aqueous ethanol media. Green Chem Letters Rev 8: 40-49.
17. Ayala-Orozco C, Liu JG, Knight MW, Wang Y, Day JK, et al. (2014) Fluorescence enhancement of molecules inside a gold nanomatryoshka. Nano letters 14: 2926-2933.
18. Shah KW, Sreethawong T, Liu SH, Zhang SY, Tan LS, et al. (2014) Aqueous route to facile, efficient and functional silica coating of metal nanoparticles at room temperature. Nanoscale 6: 11273-11281.
19. Huang Y, Xu S, Lin VY (2011) Bifunctionalized mesoporous materials with site‐separated bronsted acids and bases: Catalyst for a two‐step reaction sequence. Angewandte Chem Int Ed 50: 661-664.
20. Shen L, Pan S, Niu D, He J, Jia X, et al. (2019) Facile synthesis of organosilica-capped mesoporous silica nanocarriers with selective redox-triggered drug release properties for safe tumor chemotherapy. Biomat Science 7: 1825-1832.
21. Mosquera J, Garcia I, Henriksen LM, Gonzalez RG, Liz-Marzan LM (2018) Reducing protein corona formation and enhancing colloidal stability of gold nanoparticles by capping with silica monolayers. Chem Mat 31: 57-61.
22. Ellman GL (1963) Tissue sulfhydryl groups. Arch Biochem Biophysics 82: 70-77.
23. Webb JL (1963) Enzyme and metabolic inhibitors.
24. Woeppel K, Zheng X, Cui X (2018) Enhancing surface immobilization of bioactive molecules via a silica nanoparticle based coating. J Mater Chem B 6: 3058-3067.