Ellman's and Aldrithiol Assay as Versatile and Complementary Tools

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Ellman’s and Aldrithiol Assay as Versatile and Complementary Tools for the Quantification of Thiol Groups and Ligands on Nanomaterials Marko Moser, Ralf Schneider, Thomas Behnke, Thomas Schneider, Jana Falkenhagen, and Ute Resch-Genger Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01798 • Publication Date (Web): 02 Jul 2016 Downloaded from http://pubs.acs.org on July 5, 2016

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Ellman’s and Aldrithiol Assay as Versatile and Complementary Tools for the Quantification of Thiol Groups and Ligands on Nanomaterials Marko Moser, Ralf Schneider, Thomas Behnke, Thomas Schneider, Jana Falkenhagen, Ute Resch-Genger* Federal Institute for Materials Research and Testing (BAM), Richard-Willstaetter-Str. 11, D-12489 Berlin, Germany Corresponding Author* U.R.-G.: e-mail, [email protected]; phone, ++49(0)30-8104-1134; fax, ++49(0)30-8104-71134. KEYWORDS quantitative spectroscopy; particle; metal; semiconductor nanocrystal; polyethylene glycol; PEGylation; Ellman’s reagent, aldrithiol, thiol, gold, silver, nanoparticle, quantum dots. ABSTRACT: Simple, fast, and versatile methods for the quantification of thiol groups are of considerable interest not only for protein analysis, yet also for the characterization of the surface chemistry of nanomaterials stabilized with thiol ligands or bearing thiol groups for the subsequent (bio)functionalization via maleimide-thiol chemistry. Here, we compare two simple colorimetric assays, the widely used Ellman’s assay performed at alkaline pH and the aldrithiol assay executed at acidic and neutral pH, regarding their potential for the quantification of thiol groups and thiol ligands on different types of nanoparticles like polystyrene nanoparticles, semiconductor nanocrystals (SC NC) and noble metal particles and derive criteria for their use. In order to assess the underlying reaction mechanisms and to obtain stoichiometry factors mandatory for reliable thiol quantification, both methods were studied photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), thereby demonstrating the influence of different thiols on the reaction mechanism. Our results underline the suitability of both methods for the quantification of directly accessible thiol groups or ligands on the surface of 2D- and 3D-supports, here exemplarily polystyrene nanoparticles. Moreover, we could derive strategies for the use of these simple assays for the determination of masked, i.e., not directly accessible thiol groups like disulfides such as lipoic acid and thiol stabilizing ligands coordinatively bound to Cd and/or Hg surface atoms of II/VI and ternary SC NC and to gold and silver nanoparticles.

Thiols play a critical role in a variety of physiological and biological processes1,2 and have a high binding affinity to noble metals and semiconductors.3 Thus, thiol ligands are among the most common surface ligands of gold and silver nanoparticles (Au NP, Ag NP) and II/VI and IV/VI semiconductor nanocrystals (SC NCs),4-6 enabling size and shape control and the tuning of the surface during particle synthesis.5-8 Moreover, the strength of the thiol bond to certain metals or metal ions is often exploited for surface functionalization with thiolated biomolecules like thiol-bearing DNA or the phase transfer of nanomaterials with hydrophobic surface ligands to the aqueous phase with the aid of thiolated PEG ligands in ligand exchange reactions.9,10 Also, one of the most common bioconjugation strategies, the maleimide-thiol chemistry,11 relies on this functionality, using thiol groups to attach biomolecules to molecular and nanoscale reporters and different types of carriers and supports.12-14 Thus, methods for the quantification of thiol groups and the monitoring of reactions involving thiol ligands are of considerable im-

portance for protein chemistry and the synthesis and characterization of thiol-stabilized nanomaterials as well as nanobioconjugates from polymer and silica particles, dendrimers, micelles, liposomes, oxidic and metallic particles, and inherently fluorescent SC NC and upconversion nanocrystals.3,6,9,15 Particularly interesting for thiol determination are simple, inexpensive, robust, and fast methods which can be used for many different molecular and nanoscale systems. Methods used so far include elemental analysis,16,17 inductively coupled plasma optical emission spectroscopy (ICPOES),8 and mass spectrometry (ICP-MS),8 thermogravimetric analysis (TA),18,19 liquid chromatography with tandem mass spectrometry (LC-MS/MS, HPLC-MS/ MS),20,21 electrospray ionization mass spectrometry (ESI-MS),22 electrochemical methods,23,24 surface enhanced Raman spectroscopy (SERS),25 Fourier transform infrared (FTIR) spectroscopy26,27 as well as photometric28 and fluorometric assays.29,30 The sensitivity of TA and ICP-OES (for light elements like sulfur) is rather low, i.e., in the ppm range, 1


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thus requiring relatively large amounts of material. Moreover, these methods as well as MS based techniques need comparatively expensive instrumentation, considerable expertise, and can be time consuming. MS methods can be affected by size and charge distributions of particles even under gentle ionization conditions that lead to a broadening of the resulting mass spectra. Albeit very sensitive, fluorometric assays utilizing fluorophore labeling, require knowledge of stoichiometric factors for quantification to consider the reporter´s coupling efficiency like all methods relying on a covalent or adsorptive binding.31-33 Moreover, the binding of relatively large dye molecules to surface functionalities on nanomaterials and microparticles can be affected by material- and surface morphology-specific steric restrictions. In addition, the sensitivity of the signal-relevant absorption and fluorescence properties of the reporter dye in conjunction with the general challenge of analyte quantification from measured relative fluorescence intensities originating from absorbing, scattering and luminescent samples can introduce a systematic bias.

thiolate anion yielding a mixed disulfide and the yellow colored dianion of 2-nitro-5-thiobenzoic acid (NTB2−). The photometrically detectable concentration of NTB2− (Figure 1a) provides a direct measure for the number of thiol groups.

A simple and established photometric method to determine the concentration of free thiols or the number of thiol groups of proteins presents the Ellman’s assay (Scheme 1, left). 12,33-37 This assay utilizes the stochiometric reaction of the Ellman’s reagent (DTNB2-) with a

Figure 1. Wavelength-dependent molar absorption coeffi22cients of DTNB (1), NTB (3), DTDP (2), and 4-TP (4). Insets: calibration curves of both thiol detection reagents (c = 55.7 mM) with L-cys. (a) The Ellman’s assay at 409 nm 22(NTB ) and 327 nm (DTNB and mixed disulfide) and (b) the aldrithiol assay at 324 nm (4-TP) and at 250 nm (DTDP and mixed disulfide). Thiol concentrations can be determined from measured absorbances via the Beer-Lambert law.

Scheme 1. Possible reactions of the Ellman’s reagent (1) and 4-aldrithiol (2).a

Due to some disadvantages of the Ellman’s assay reported in the literature like its susceptibility to matrix effects such as interferences from certain buffers,35 the low stability of the Ellman’s reagent,12,37 variable molar absorption coefficients,12,33-36 and the sometimes incomplete reaction with proteins,33 new derivatives were developed that contain the same or similar photometrically detectable groups but reveal an improved stability and are applicable within a broader pH range.12,37 As uncharged alternative usable at acidic pH values Grassetty and Murray38 proposed 4-aldrithiol (4,4’-dithiodipyridine, DTDP; Scheme 1, right). For thiol quantification, 4-Aldrithiol has to be transformed first to its hydrochloride, which subsequently reacts with thiols in the same way as the Ellman’s reagent in a thiol–disulfide exchange reaction, followed by stoichiometric release of photometrically detectable 4-thiopyridone (4-TP), showing an absorption maximum at 324 nm in acidic solution (Figure 1b). Our general interest in the development of simple methods for surface group and ligand analysis encouraged us to study the potential of the Ellman’s test, recently used by us and others for the quantification of thiol ligands on SC NC like Cd1-xHgxTe36,39 and CdSe/ZnS,40 and the 4-aldrithiol assay for the determination of thiol groups in

a

Reaction of the Ellman’s reagent (1) and 4-aldrithiol (2) 2with L-cys yielding photometrically detectable NTB (3) or 4-TP (4), respectively. 2



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molecular systems and on nanomaterials. In this respect, the advantages and limits of both assays were assessed for simple thiol ligands with freely accessible thiol groups in solution and on nanomaterials, masked thiol groups as found in the cyclic disulfide lipoic acid (LA), frequently used, after reduction to the corresponding dithiol dihydrolipoic acid (DHLA), as bidentate ligand for SC NC40 and more or less tightly bound thiol surface ligands.41 Representatively chosen nanomaterials included polystyrene particles (PS NP) surface modified with thiols and thiolated PEG ligands, Ag NP, and Au NP, which are among the most frequently used nanomaterials in the life sciences,4,15,42,43 and thiol-capped SC NC. This comparison included method validation by ICP-OES and the study of the underlying reaction mechanisms as well as potential influences of typical buffers and surfactants used for bioconjugation reactions. Based upon these results, recommendations for the use of both assays and their limitations were derived.

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SPDP and PS NP mixture) at room temperature (rt) at 700 rpm, followed by centrifugation at 15,000 g for 35 min using a Centrifuge 5415D from Eppendorf (Germany). The supernatants were removed, and the PS NP dispersions were washed twice and redispersed in 350 µL phosphate buffer. Each particle dispersion was merged with 100 µL of a TCEP stock solution (14 mM) in phosphate buffer and shaken for 45 min at rt at 700 rpm. After centrifugation, PS NP (PS-MPA and PS-PEG-MPA) were washed twice and redispersed in 300 µL phosphate buffer. The resulting amount of thiol surface groups used for comparison with the results of the two thiol assays was obtained photometrically from the amount of released 2-TP in the supernatant, which absorbs at 343 nm, and by ICP-OES, measuring the sulfur content of the supernatant and the particle suspension, respectively. All relative standard deviations were derived from measurements of six samples. Preparation of Ag NP. A mixture of 12 mL Milli-Q water and 8 mL glycerol, containing 0.25 mM phosphate buffer (pH 7.15) and 2.12 mg MPA was heated to 70 °C. 34.0 mg AgNO3 were added under stirring. In intervals of 60 s, two portions of 100 µL NaBH4 solution (200 mM) were added. After 120 s, the reaction was stopped by pouring the reaction mixture into 80 mL cold water (8 °C). The resulting Ag NP were purified by filtration (Merck Millipore Amicon Ultra centrifugal filters, MWCO 100 kDa) and redispersed in 20 mL pure water. The broad plasmon band at 417 nm matches the particle size of 60 nm determined by DLS.

Materials and Methods Materials. Ellman’s reagent, 4-aldrithiol, 3-mercapto propionic acid (MPA), thioglycolic acid (TGA), tetrahydrofuran (THF), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, sodium phosphate, tetraethylammonium (TEA) cyanide, silver nitrate, chloroauric acid, glycerol, sodium hydroxide, phosphoric acid, LA, and hydrochloric acid (HCl) were purchased from Sigma Aldrich Co. (Germany), L-cysteine (L-cys) from Merck (Germany), L-cystine and nitric acid from AppliChem GmbH (Germany), sodium borohydride from abcr GmbH (Germany). N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP, NHS-PDP), PEG12-SPDP (long chain SPDP crosslinker), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Thermo Fisher Scientific Germany BV & Co KG (Germany) and aminated 100 nm-sized polystyrene particles (PS NP) from Micromod GmbH (Germany), respectively. All solvents were of spectroscopic grade. All solutions and buffers were prepared with Milli-Q water (Millipore). Prior to use, suspensions of all particles were sonicated for 30 s to ensure particle deaggregation. All particle suspensions contained 0.1 mM phosphate buffer; the pH values were adjusted with H3PO4 to ensure a constant pH and electrolyte concentration.

Preparation of Au NP. A solution of 398 mg chloroauric acid (HAuCl4*3H2O) in 40 mL water was mixed at room temperature with 40 mL of a solution of 100 mg thiolPEG(1000 Da)-amine. Subsequently, 420 mg NaBH4 solved in 40 mL water was added dropwise within 15 min. The Au NP obtained were purified by filtration and redispersed in 20 mL pure water. The particles showed a mean diameter of 17 nm as determined by transmission electron microscopy (TEM) on a FEI Tecnai G² 20 S-TWIN microscope at an acceleration voltage of 200 eV and reveal a plasmon resonance peak located at 530 nm. Photometric thiol determination. The Ellman’s reagent was prepared as a 3.4 mM solution in phosphate buffer (0.1 mM) at pH values between 7.3 and 10.0. In the case of the aldrithiol assay, a 3.4 mM solution of 4-aldrithiol was used (pH 4.8, adjusted with HCl). These stock solutions were always freshly prepared and used within one day; the stability of each solution was confirmed photometrically to be within ± 2% over a period of 8 h.

Thiolation of PS NP. 100 nm-sized aminated PS NP were thiolated by reaction with the NHS ester of SPDP or PEG12-SPDP, respectively, and the accordingly formed disulfide bond was then reductively cleaved with TCEP, thereby releasing 2-thiopyridone (2-TP). Stock solutions containing 2.1 µmol SPDP dissolved in 30 µL DMSO and 2.1 µmol PEG12-SPDP dissolved in 50 µL phosphate buffer (pH 7.5) were prepared. 200 µL of aqueous dispersions of PS NP (2.5 wt %) were washed twice with phosphate buffer (pH 7.5) and redispersed in 200 µL phosphate buffer in an ultrasonic bath. The stock solutions of the reagents and the particle dispersions were mixed and shaken (45 min for a SPDP and PS NP mixture; 4 h for a PEG12-

Assay calibration. For the calibration of the Ellman’s and the aldrithiol assay, 3 mL of phosphate buffer (0.1 M) were mixed with defined amounts of the different thiols, i.e. L-cys, TGA, and MPA stock solutions (3 mM; solvent Milli-Q water) and 50 μL of the Ellman’s or 4-aldrithiol stock solution, respectively. For use as stabilizing ligand for SC NC, DHLA was prepared from LA by reduction with NaBH4 following a published procedure.44 In addition, we performed a photochemical reduction. In this case, we 3


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Analytical Chemistry three measurements varied between 1.5 and 4.5%.

exposed the samples in quartz cuvettes for 30 minutes to UV light from a 200 W LC8 Lightningcure UV-lamp from Hamamatsu (250 nm – 450 nm, set to 20% intensity). Subsequently, the absorption spectra of the solutions were measured with a Cary 5000 UV/Vis/NIR spectrophotometer from Varian Inc. (Australia) at rt in 10 x 10 mm quartz cuvettes from Hellma GmbH (Germany).

Results and discussion For the critical assessment of the applicability of the Ellman’s and aldrithiol assay for the determination of thiol groups on different types of nanomaterials, we first studied the underlying reaction mechanisms with representatively chosen mono- and bidentate thiols and then quantified thiol containing surface ligands and surface groups with both assays.

Nanoparticle samples. Thiol containing SC NC and PS NP samples to be analyzed were mixed with 50 µL of the Ellman’s or 4-aldrithiol stock solution, respectively. The reaction time was varied between 12 min and 4 h. In the case of PS NP, typically 0.5 mg of the particles were removed by centrifugation at 15,000 g for 35 min.; depending on their chemical composition, about 0.02 mg of SC NC were dissolved either by addition of the Ellman’s reagent or 4-aldrithiol or by EDTA as previously described.36,39 The remaining supernatant or resulting solution was diluted to a volume of 3 mL with phosphate buffer in a quartz glass cuvette. The thiol concentration was determined from the absorbance of the respective reaction products, i.e., NTB2- at 409 nm and 4-TP at 324 nm, using the absorption coefficients obtained from the calibration curves. Ag NP and Au NP were dissolved prior to assay performance by stirring, 1 mL of the NP dispersion with either 1 mL concentrated HCl (Ag NP) or aqua regia (Au NP) or with a 0.2 M solution of TEA cyanide (Ag NP; Au NP) for two hours. AgCl, resulting as white precipitate for HCl addition, was removed by centrifugation (Hettich Rotina 380R centrifuge, 24,400 g for 30 min.). The acidic supernatants were neutralized with NaOH and diluted to a volume of 5 mL with phosphate buffer and then used for the thiol assays performed as described for the PS NP and SC NC.

Ellman’s assay. We determined the molar absorption coefficient of NTB2- at (409 ± 2) nm in phosphate buffer (0.1 M, pH 8.0) at rt (25 °C) after 30 min reaction time for different thiol ligands, yielding values of (14,100 ± 200) M-1cm-1 and (13,300 ± 200) M-1cm-1 for MPA and L-cys, respectively. From these calibration curves, detection limits (LOD = 3 SDy/slope) of 1.2 µM were calculated in both cases. Interestingly, the amount of Ellman’s reagent applied enabled the determination of up to 2 equiv. of L-cys (Figure 1a, inset), while for MPA, only 1 equiv. of thiol was detectable. This implies a two-step reaction mechanism for the reaction of L-cys, as shown in Scheme 1, while the reaction of MPA stops after the first reaction step. This difference in reaction mechanism and hence, stoichiometry is confirmed by the absorption spectra shown in Figure 2, measured after reaction of increasing amounts of Ellman’s reagent with constant amounts of L-cys and MPA, respectively. Figure 2 reveals that L-cys can be quantified with 0.5 equiv. of Ellman’s reagent as follows from the matching absorbance of NTB2- spectra at 409 nm for 0.5 equiv. (Figure 2a, full squares) and 1 equiv. (Figure 2a, full triangles) of Ellman’s reagent. In contrast, in the case of MPA, 1 equiv. is required for quantification as indicated by the increase of the absorbance at 409 nm for 1 equiv. of Ellman’s reagent compared to 0.5 equiv. (Figure 2b). ESI-MS studies of the reaction products (Fig-

Mechanistic studies with ESI-TOF-MS. The reaction products of the thiol assays were identified by ESI-TOFMS and a Micromass QToF Ultima instrument (Waters Corp., Manchester, UK) operated in the positive ionization mode by employing multiple reactions monitoring (MRM). The following working parameters were used: capillary voltage: 1.5 kV, cone voltage: 35 V, source temperature: 120 °C, desolvation temperature: 150 °C, desolvation gas flow, 550 L/h (Nitrogen), and cone gas flow of 50 L/h. Nitrogen (99.9% purity) was applied as cone and collision gas. The inter-channel and the inter-scan delays were both set to 0.1 s. The mass spectra of the compounds were obtained for species concentrations of ca. 0.05 0.2 mg/mL in water at pH 8.0 (adjusted with ammonia). 100 µL of 2 wt % formic acid was added to each sample to increase the ionization probability. Due to the different ionization probabilities of the molecules, these measurements are only semi-quantitative. Quantification by ICP-OES. Quantitative elemental analysis of dispersed PS NP and dissolved Ag NP was performed by ICP-OES, using an Ultima 2CHR instrument from HORIBA (Jobin Yvon GmbH, Germany). Ag was detected at a wavelength of 328.068 nm and S at 180.676 nm. The relative standard deviations derived from

Figure 2. Absorption spectra obtained upon addition of increasing amounts of Ellman’s reagent (in portions of 0.1 equiv. compared to the thiol) to a solution of (a) L-cys 2and (b) MPA. Ratios of DTNB -to-thiol 1:2 (full squares) and 1:1 (full triangles). 4



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Figure 3. ESI-TOF mass spectra of the Ellman’s assay with L-cys (left) and MPA (right). Spectra of Ellman’s reagent (I,VI), thiol (II,VII), L-cystine, and MPA dimer (III,VIII), and of the reaction of Ellman’s reagent with the thiol in a ratio of 1:1 (IV,IX) indicat2ing formation of a mixed disulfide in addition to NTB , and for a ratio of 1:2 (V,X) revealing formation of L-cystine (left), yet not of MPA-MPA (right).

ure 3) demonstrate that the formation of the mixed disulfide and L-cystine depends on the ratio of the Ellman’s reagent and L-cys (Figure 3, panels IV-V), while in the case of the reaction with MPA (Figure 3, panels IX-X), the mixed disulfide formed in the first reaction step is the main product. The reason for the different reaction mechanisms of the Ellman’s reagent with L-cys and MPA is probably the different pKa values of the thiol groups of L-cys and MPA of 8.3-8.5 and 10.8, respectively. This could lead to a higher reactivity of L-cys in nucleophilic substitution reactions like the Ellman’s assay.45

Figure 4. pH dependence of the molar absorption coefficient 2of 4-TP (aldrithiol assay) and of NTB (Ellman’s assay) with L-cys in phosphate buffer (0.1 M).

As follows from the absorption spectra in Figures 1a and 2a thiol determination from the consumption of Ellman’s reagent is complicated by the overlap of the absorption spectrum of the educt with that of the mixed disulfide. Moreover, this can affect quantification exploiting the absorption of the reaction product at 409 nm. The size of the spectral overlap with the absorption spectrum of NTB2- depends most likely on the chemical nature of the inserted thiol. This could also account for the variations in molar absorption coefficients of NTB2- reported in the literature. 12,33-36 Subsequently, we investigated the influence of pH (Figure 4) and common bioanalytical buffers and additives like surfactants (Table 1) on thiol determination with the Ellman’s assay. Apparently, the results of the Ellman’s assay are independent of pH between pH 7.3 and pH 10, yet the right choice of the buffer is important. Moreover, interferences can arise from surfactants like CTAB, metal ions such as silver ions or anions like cyanide, underlining the importance of blank measurements.

as determined for the reaction with L-cys and MPA, respectively (Figure 1b), providing a LOD of 0.8 µM. The absorption coefficient of 4-TP is slightly pH-dependent, with a linear decrease of 560 M-1cm-1 for a pH increase of 1 (Figure 4). L-cys

reacts with 4-aldrithiol like the Ellman’s reagent in one or two steps depending on the ratio of 4-aldrithiol to L-cys (Figure 5, panels IV-V). With MPA in excess, the MPA dimer is formed contrary to the Ellman’s assay (Figure 5, panels IX-X). 4-Aldrithiol has to be dissolved in 0.1 M HCl prior to use in buffer yielding a mixture of un/protonated species (DTDP, DTDPH+, and DTDPH2+). The absorption spectra of these species, the mixed disulfides, and 4-TP strongly overlap only below 300 nm. This hampers thiol determination via 4-aldrithiol consumption but does not affect quantification via 4-TP absorption at 324 nm. Interferences can arise from certain buffers or surfactant35,46 (Table 1). Hence, the aldrithiol assay is equally suited for the determination of all thiols assessed here and can overcome some of the limitations of the Ellman’s test and vice versa.

Aldrithiol assay. Complementary to the Ellman’s test, requiring pH values between 7.3 and 10.0, the aldrithiol assay is performed in a pH range between 4.5 and 7.8. 4 TP, released upon reaction of 4-aldrithiol with thiols, has an absorption maximum at (324 ± 2) nm. Its molar absorption coefficient is (20,000 ± 200) M-1cm-1 at pH 7.0

Quantification of ligands with accessible thiol groups on particles. In order to prepare NP with a de fined amount of thiol surface groups, we modified 5


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Table 1. Interferences with the thiol assays arising from common buffers and other typical additives. Additives and buffers +

Ellman’s assay

Quantification of masked thiols. Thiols can be “masked” for the Ellman’s or aldrithiol assay chemically by the formation of disulfide bonds or by strong coordinative bonds as found e.g., in complexes of Hg(II) or for the Au- and Ag-thiol bond. The high potential of our two colorimetric assays for thiol quantification encouraged us to develop strategies for their use also for such samples.

Aldrithiol assay

-

H , OH - (pH 7.3-10) + (pH < 7.8) Ag ++ + Ag + + Cl Au 2+ Cd 2+ Hg +++ +++ CN SDS (2%) CTAB (2%) +++ +++ EDTA + + NO3 MES (10 mM, pH 5.0) ++ + Carbonate (50 mM, pH 9.6) +++ TAE (0.2M, pH 8.0) + + Citrate (10 mM, pH 6.0) +++ + Phosphate (0.1 M, pH 8.0) PBS (80 mM, pH 7.6) ++ + HEPES (10 mM, pH 7.5) ++ + TRIS (0.1 M, pH 7.4) +++ +++ - neglectable; + affect < 5%; ++ affect < 10%; +++ interfering

Quantification of LA. In order to increase the stability of organic ligand shells and minimize e.g., ligand adsorption/desorption equilibria of SC NC, bi- or multidentate thiols are increasingly used as stabilizing agents for SC NC and other nanomaterials.40,47,48 A typical example presents LA, which, after reduction to its dithiol analog DHLA, provides excellent stability for SC NC in aqueous media, due to the simultaneous coordination of two thiol groups to surface atoms while the carboxylic group enforces electrostatic stabilization.49 DHLA was obtained by reduction of LA by UV light or chemically.44 Then, the concentration of thiol groups was determined with the aldrithiol assay, which is not influenced by the chemical nature of the thiol. Correlation of these results with the known concentration of LA gave 2.00 ± 0.10 equiv. of thiol groups per DHLA molecule (pH 5.4, ε = 20,900 M-1cm-1). As follows from Figure 6, reoxidation of DHLA by oxygen in aerated solution, masking its thiol groups by disulfide formation, is slow enough to allow a quantitative reaction of both thiol groups with the assay reagent within one hour with an uncertainty of 5 %. This equals the measurement uncertainty derived for the monodentate thiols L-cys, TGA, and MPA. In the case of the Ellman’s assay, we obtained an absorption coefficient of (13,700 ± 200) M-1cm-1 (pH 8.0) assuming a complete reduction of LA. This value agrees very well with the molar absorption coefficients of the calibration of this assay with L-cys and MPA, considering influences of mixed disulfides. Hence, with proper calibration, which is really crucial in the case

100 nm-sized aminated PS NP with SPDP derivatives. The final goal were NP with MPA or PEG-MPA as end groups without/with PEG spacer (PS-MPA and PS-PEG-MPA). The amount of thiol groups introduced was determined photometrically from the amount of released 2-TP during synthesis. These thiol end groups could be quantified with both assays, yielding a average thiol recovery of at least 95%, compared to the results from the 2-TP measurements.

Figure 5. ESI-TOF mass spectra of the aldrithiol assay with L-cys (left) and MPA (right). Spectra of 4-aldrithiol (I,V), thiol (II,VII), L-cystine, and MPA dimer (III,VIII). For reaction of 4-aldrithiol with thiol in a ratio of 1:1 (IV,IX), this indicates the formation of a mixed disulfide in addition to 4-TP, and for a ratio of 1:2 (V,X) it reveals the formation of L-cystine (left) and partial formation of MPA-MPA (right). 6



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dissolution can be achieved either with aqua regia or with cyanide. The suitability of the former approach was demonstrated for both thiol assays, with the matching results of the Ellman’s and 4-aldrithiol assay and ICP-OES demonstrating the absence of thiol oxidation by aqua regia (Table 2). The latter method was not feasible as we observed interferences from cyanide with our thiol assays (see Tables 1 and 2). Figure 6. Time dependence of the reaction of 4-aldrithiol with the masked thiol groups of LA (squares) and free thiol groups of DHLA after photoinduced (triangles, downward) or chemical (triangles, upward) reduction of LA.

of the Ellman’s assay, this assay is also suited for this application. Quantification of stabilizing thiol ligands bound to particle surfaces. For the reliable determination of coordinatively bound, and hence masked surface thiol ligands, unbound thiols, originating from the synthesis and/or adsorption/desorption equilibria in the stock solution, were removed prior to ligand analysis by filtration or precipitation and redispersion to remove the particles to be further analyzed. Here, also quantification of the ligands in the washing solution can be advantageous. Furthermore, complete ligand removal from the particle surface is mandatory and thus, typically particle dissolution prior to assay performance. In this respect, we studied the suitability of our thiol assays for the quantification of typical thiol surface ligands and different representative nanomaterials of varying chemical composition. An overview of the systems studied follows from Table 2, with the reliability of the assay results being typically assessed by comparison with ICP-OES. For CdTe SC NCs,36,39 stabilizing monodentate thiol ligands can be removed from the particle surface solely by addition of the Ellman’s reagent or 4-aldrithiol, even without the need for addition of EDTA for particle dissolution as previously reported.36 In the case of CdHgTe, SC NC, removal of thiol ligands requires, however, particle dissolution, e.g., by addition of EDTA. Here, the higher Hg(II)-sulfur binding constant, exceeding that of Cd(II), is reflected by slower dissolution and reaction kinetics of CdHgTe colloids compared to CdTe colloids.39 A similar influence of the ligand binding strength to surface atoms has been recently reported for the dithiothreitol (DTT)based displacement widely utilized for ligand removal from Au NP.4 In order to circumvent such influences and to shorten reaction times CdHgTe could be also dissolved with a strong oxidizing acid like aqua regia. Ag NP can be simply dissolved by addition of hydrochloric acid. Prior to assay performance, AgCl formed as white precipitate must be removed e.g. by filtration or centrifugation. Depending on the pH sensitivity of the respective thiol assay, also the pH must be readjusted. This was exemplarily shown for MPA-capped Ag NP. Here, both thiol assays provided a recovery rate of 90% concerning the amount of thiol applied for Ag NP synthesis. Moreover, these assay results match within 5 % with the values determined by ICP-OES. In the case of Au NP, particle

Table 2. Overview of particle systems and required pretreatment for thiol assays. a

Particle

Ligand

Treatment prior to thiol assay

PS PS CdTe

MPA PEG-MPA TGA

CdTe

MPA

CdHgTe CdHgTe Ag

TGA MPA MPA

Ag

DHLA

Au

HS-PEG

Centrifugation for NP removal Centrifugation for NP removal Dissolution with assay reagent or EDTA Dissolution with assay reagent or EDTA Dissolution with EDTA Dissolution with EDTA Dissolution with HCl or aqua regia, centrifugation Dissolution with HCl or aqua regia, centrifugation, UV light Dissolution with aqua regia

a

: assay-specific pH requirements, and hence, the need to readjust the pH, are not considered.

Conclusion and Outlook In summary, we could demonstrate the reliability of the Ellman’s and aldrithiol assay for the quantification of thiol groups and thiol ligands on nanomaterials by mass balances and comparison with sulfur quantification via optical emission spectroscopy (ICP-OES) and derive assay-specific requirements and limitations. Particularly attractive are their ease of use and the short times of analysis between 5 min and 3 h together with the required inexpensive instrumentation, available in every laboratory. Areas of applications include freely accessible thiol groups on molecules and nanomaterials and other carriers and supports and after chemical treatment, thiols masked as disulfides or coordinatively bound thiols. Our systematic studies of the Ellman’s and aldrithiol assay, utilizing electrospray ionization mass spectrometry (ESI-MS) for species identification, revealed that in both cases, the underlying reactions yield thiol-dependent intermediates, which can absorb in the same wavelength region as the educts. Hence, only the photometric determination of the stoichiometrically formed reaction products NTB2- and 4-TP is suitable for thiol quantification. The aldrithiol assay is slightly more sensitive due to the higher molar absorption coefficient of its reaction product and applicable in a broader pH range, while the Ellman’s assay provides a visible color change. Both the Ellman’s and aldrithiol assays, can quantify freely accessible thiol groups on nanoparticles, e.g., thiolated PS NPs, with a recovery rate of > 95%. Depending on the nanomaterial studied, its removal prior to assay performance, either by centrifugation, dialysis or filtra-


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tion, can be necessary to circumvent distortion of the photometric measurements by scattering and/or absorption. This removal step should be optimized for each sample in a material- and size-dependent-manner. For thiol ligands coordinatively bound to surface atoms of nanomaterials, the strength of the thiol-surface bond must be considered as this determines the need for particle dissolution prior to assay performance. In this respect, both assays may be also utilized for the assessment of the strength of ligand-particle bonds. For particle dissolution under acidic conditions as used for Ag and Au nanoparticles, the aldrithiol assay is recommended due to its applicability in acidic media, thereby circumventing the need for pH readjustment. Although our results clearly show the versatility of both thiol assays, they also highlight potential limitations like the influence of the chemical nature of the thiol and possible interferences from other compounds present in solution. Hence, it is strongly recommended to carefully control assay performance for new samples and targets to be studied including assay calibration and perform control experiments with e.g., buffer components or other sample ingredients not addressed in this study.

AUTHOR INFORMATION Corresponding Author * U.R.-G.: e-mail, [email protected]; phone, ++49(0)308104-1134; fax, ++49(0)30-8104-71134.

ACKNOWLEDGMENT We gratefully acknowledge financial support from the Federal Ministry for Economic Affairs and Energy (BMWi-10/12). We thank Roy Pawliczek for graphic works, S. Penk (BAM) for help with the ICP-OES analyses and A. Dubavik (AK Eychmüller at TU-Dresden) for assistance in synthesis of SC NCs.

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Ellman's and Aldrithiol Assay as Versatile and Complementary Tools

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