Quantification of Amino Groups on Solid Surfaces Using Cleavable

Jul 29, 2015 - Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Langmuir , 2015, 31...
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Quantification of Amino Groups on Solid Surfaces Using Cleavable Fluorescent Compounds Saori Shiota,† Shunsuke Yamamoto,† Ayane Shimomura,† Akio Ojida,‡ Takashi Nishino,† and Tatsuo Maruyama*,† †

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan ‡ Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan S Supporting Information *

ABSTRACT: We quantified amino groups displayed on inorganic and organic surfaces in aqueous solution using different types of cleavable fluorescent compounds and an aldehyde dye. The cleavable fluorescent compounds were designed to bind covalently to amino groups and then liberated under specific conditions. Among the investigated materials, cleavable coumarin was most appropriate for the quantification of amino groups on silica and resin surfaces. The developed method can measure small amounts (∼pmol/cm2) of amino groups on a flat polymeric surface, detecting only amino groups that are exposed to aqueous solution and available for surface immobilization of ligands and biomolecules.

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INTRODUCTION Introduction of reactive functional groups on a solid surface is important for the development of functionalized material surfaces. In particular, amino and carboxy groups are widely used to immobilize biomolecules and ligands on solid surfaces.1 The surface density of functional groups affects the properties and applications of surfaces in, for instance, protein adsorption,2 DNA immobilization,3 cell adhesion,4 and ligand immobilization.5 In some cases, excess surface density of functional groups, like amino and carboxy moieties, can markedly alter the electrostatic charge and microenvironmental pH near a surface,6,7 which indicates the significance of the controlled surface density of functional groups. Many methods have been used to introduce reactive functional groups onto surfaces, including plasma treatment,8 self-assembled monolayer formation,9 chemical vapor deposition,10,11 layer-by-layer assembly,12 dip- and spin-coating,13−16 and physical adsorption of a functional polymer on material surfaces.17 However, it is still challenging to control the density of amino and carboxy groups on surfaces. This is partly because it is difficult to precisely identify and quantify these functional groups on solid surfaces.18 Although there are numerous studies on the quantification of surface amino groups by methods such as colorimetry,19−25 fluorometry,8,26,27 and X-ray photoelectron spectroscopy,1,28,29 these approaches have limitations, such as difficulty in detecting amino groups with a concentration of ∼pmol/cm2. Many of them are only semiquantitative, lacking robustness, and detecting groups buried too deep from the surface to be accessible to reactants in bulk solution. In our previous studies, we developed a cleavable fluorescent compound that can bind to an amino group on a solid surface © XXXX American Chemical Society

and then be liberated from the surface under reducing conditions.15,16 This compound can therefore transform information about the amino groups on a surface to solution, allowing use of a conventional fluorescence spectrophotometer, which can detect even a small amount of amino groups. However, the fluorophore used was sterically bulky fluorescein isothiocyanate (FITC), which might prevent the precise quantification of high-density amino groups on a surface. In the present study, we synthesize three different types of cleavable fluorescent compounds that are then used to quantify small amounts of amino groups displayed on organic and inorganic substrates.

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EXPERIMENTAL SECTION

Descriptions of the materials and syntheses of cleavable fluorescent compounds (cleavable FITC,15 cleavable coumarin, and cleavable anthranilate; Figure 1) and random copolymers of methyl methacrylate (MMA) and 2-aminoethyl methacrylate (AEMA) [poly(MMA-r-(Boc)AEMA)]15 are described in the Supporting Information. Quantification of Amino Groups on Aminated Silica Particles and NH2−Polyethylene Glycol (PEG) Resin Using Cleavable Fluorescent Compounds (Cleavable FITC, Cleavable Coumarin, and Cleavable Anthranilate). In a 2 mL polypropylene tube, silica particles (0.5, 1.25, 2.5, or 5.0 mg) were washed with water (1 mL), dispersed in phosphate buffer solution (PBS; 67 mM, pH 8.0, 1 mL) containing 5 vol % dimethyl sulfoxide (DMSO), 2 mM cleavable fluorescent compound, and 10 mM 4-(4,6-dimethoxy-1,3,5triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM, coupling Received: March 2, 2015 Revised: July 28, 2015

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Figure 1. Chemical structures of cleavable fluorescent compounds used to quantify surface amino groups: (a) cleavable FITC, (b) cleavable coumarin, (c) cleavable anthranilate, and (d) 5-formylsalicylic acid. For the quantification of amino groups on NH2−PEG resin using 5formylsalicylic acid, NH2−PEG resin slurry (0.15 mg in 100 μL of water or 0.31 mg in 200 μL of water) instead of aminated silica particles was added to phosphate buffer (67 mM, pH 7.4, 1 mL) containing 4 mM 5-formylsalicylic acid and shaken for 2 h at room temperature, followed by the same procedure described above. Preparation of Poly(methyl methacrylate) (PMMA) Substrates with Surface Amino Groups. Amino groups were introduced on PMMA substrates via dip-coating.15 Poly(MMA-r(Boc)AEMA) was dissolved in chloroform/N,N-dimethylformamide (DMF) (70:30, volume ratio) to give a copolymer concentration of 5 wt %. PMMA substrates were immersed in the copolymer solution for a few seconds and then dried in a vacuum desiccator at room temperature overnight. Deprotection of Boc groups was carried out by immersing the substrates in 4 M HCl aqueous solution for 2 h at room temperature. Quantification of Amino Groups on PMMA Substrates Using Cleavable Fluorescent Compounds (Cleavable FITC and Cleavable Coumarin). The dip-coated substrates were washed with water (5 mL) and immersed in phosphate buffer (67 mM, pH 8.0, 2 mL) containing 5 vol % DMSO, 0.2 mM cleavable fluorescent compound (cleavable FITC or cleavable coumarin), and 1 mM DMTMM for 2 h at room temperature. The substrates were washed with PBS (5 mL) and immersed in NaOH aqueous solution (5 mM, 12 mL) for 1 h at 40 °C. The NaOH aqueous solution was replaced with HCl aqueous solution (5 mM, 12 mL), and the mixture was shaken for 1 h at 40 °C. The HCl aqueous solution was replaced with 5 mM NaOH aqueous solution (12 mL) for 1 h at 40 °C. The repeated washing with NaOH and HCl solutions was to solubilize and remove unreacted but adsorbed fluorescent compounds. The substrates were finally rinsed with phosphate buffer (5 mL) and immersed in phosphate buffer (2 mL) containing 2 mM TCEP for 1 h at 40 °C. The fluorescence intensity of the phosphate buffer was measured using a fluorescence spectrophotometer. The measurement conditions were the same as those described above.

agent), and shaken for 2 h at room temperature. After centrifugation, the supernatant was removed and the silica particles were washed with phosphate buffer (1 mL). Silica particles were then dispersed in methanol (1.5 mL) for 1 h at 40 °C. Methanol was removed and replaced with fresh methanol, and then the mixture was shaken for 1 h at 40 °C (termed “methanol-wash procedure”). This methanol-wash procedure was repeated twice to solubilize and remove the unreacted but adsorbed fluorescent compound. After centrifugation, the supernatant was removed. Silica particles were washed with phosphate buffer (1 mL), dispersed in phosphate buffer (1 mL for silica particles and 1.5 mL for resin) containing 4 mM tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and then shaken for 1 h at 40 °C to cleave disulfide bonds. The cleavage of disulfide bonds under the present conditions was confirmed by high-performance liquid chromatography analysis as previously reported.15 It should be noted that the adsorption of reduced fluorophores to substrates and polypropylene tubes was not detected under the present conditions. The fluorescence intensity of the TCEP aqueous solution was measured using a fluorescence spectrophotometer (FP-8200, Jasco, Tokyo, Japan). Excitation (emission) wavelengths were 495 (515) nm for cleavable FITC, 420 (450) nm for cleavable coumarin, and 350 (430) nm for cleavable anthranilate. Excitation and emission band widths were 5 nm. The sensitivity of the apparatus was set at low for cleavable FITC and cleavable coumarin and medium for cleavable anthranilate. Quantification was carried out in triplicate, unless otherwise stated. Error bars represent standard deviations. For the quantification of amino groups on NH2−PEG resin, dry NH2−PEG resin was dispersed in water to prepare a resin slurry with a concentration of 1.54 mg/mL. In a 2 mL polypropylene tube, resin slurry (100 or 200 μL) was added to phosphate buffer (67 mM, pH 8.0, 1 mL) containing 5 vol % DMSO, 2 mM cleavable fluorescent compound, and 10 mM DMT-MM and then vigorously shaken for 2 h at room temperature, followed by the same procedure described above. Quantification of Amino Groups on Silica Particles on NH2− PEG Resin Using 5-Formylsalicylic Acid. In a 2 mL polypropylene tube, aminated silica particles (0.5, 1.25, 2.5, or 5.0 mg) were washed with water (1 mL), dispersed in phosphate buffer (67 mM, pH 7.4, 1 mL) containing 4 mM 5-formylsalicylic acid, and shaken for 2 h at room temperature. After centrifugation, the supernatant was removed and silica particles were subjected to the methanol-wash procedure 3 times. Silica particles were dispersed in HCl aqueous solution (0.1 M, 1 mL) and then shaken for 1 h at room temperature to liberate 5formylsalicylic acid from the silica particles. After centrifugation, NaOH solution (2 M) was added to the supernatant to neutralize it. The fluorescence intensity of the neutralized solution was measured using a fluorescence spectrophotometer. Excitation and emission wavelengths were 325 and 405 nm, respectively. Excitation and emission band widths were 5 nm. The sensitivity of the apparatus was set at medium. Quantification was carried out in triplicate, unless otherwise stated.

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RESULTS AND DISCUSSION We used four kinds of cleavable fluorescent compounds to quantify amino groups on solid surfaces in the order of picomoles (Figure 1). The cleavable fluorescent compounds were three intramolecular-cleavable fluorescent compounds and a fluorescent aldehyde dye. The intramolecular-cleavable fluorescent compounds consisted of a fluorophore, cleavable moiety (disulfide bond), and reactive moiety (carboxy group) (panels a−c of Figure 1). The fluorescent aldehyde dye was 5formylsalicylic acid. An aldehyde-based dye has already been used as a reversible reactant dye for amino groups on a silica surface, allowing for a colorimetric assay using ultraviolet− visible (UV−vis) spectroscopy.30 However, the sensitivity of B

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Langmuir this process was limited. Here, we used a fluorescent aldehyde dye to improve the sensitivity of amino group detection. In the sensing process, the intramolecular-cleavable fluorescent compounds first bind covalently to amino groups to form amide bonds or 5-formylsalicylic acid forms a Schiff base with amino groups. The intramolecular-cleavable fluorescent compounds can be liberated into aqueous solution under reduction, and 5-formylsalicylic acid can be liberated under acid conditions, respectively (Scheme 1). The concentration of Scheme 1. Schematic Illustration of the Quantification of Amino Groups on a Solid Surface Using Cleavable Fluorescent Compounds: (a) Intramolecular-Cleavable Fluorescent Compound and (b) 5-Formylsalicylic Acid

Figure 2. (a) Effect of the amount of silica particles with amino groups on their surfaces on the fluorescence of liberated fluorophores. (b) Amino groups on the silica particles measured using different cleavable fluorescent compounds.

anthranilate and 5-formylsalicylic acid exhibited relatively high fluorescence in the absence of silica particles, with calculated concentrations of 209 ± 56 and 279 ± 151 nM, respectively. It should be noted that the minimum detection limits of cleavable anthranilate and 5-formylsalicylic acid in fluorescence measurements were relatively high (∼0.5 μM) because of their low intrinsic fluorescence, as shown in the standard curves (Figure S1). Liberated fluorophore increased with the amount of silica particles (Figure 2a). In particular, cleavable FITC and cleavable coumarin showed linear correlations between solution fluorescence and the amount of silica particles with R2 > 0.99, p < 0.01 and R2 > 0.98, p < 0.01, respectively. Such linear correlations suggest that these compounds are suitable for quantification of amino groups. Figure 2b shows the amino groups on the silica particles estimated from the fluorescence of the liberated fluorescent groups in solution (Figure 2a). The amount of amino groups was constant regardless of the amount of silica particles when using cleavable FITC and cleavable coumarin. In contrast, the density of amino groups depended upon the amount of silica particles for cleavable anthranilate and 5-formylsalicylic acid. The incoherent values obtained for these materials would be caused by their high detection limits, non-negligible adsorption to the silica particles and polypropylene tubes, and the low reaction yield of Schiff base formation for 5-formylsalicylic acid. The average amount of amino groups estimated using cleavable coumarin was 0.67 ± 0.02 nmol/mg. The manufacturer stated that the silica particles contained 2 nmol/mg of amino groups on their surfaces and internally. Our present results suggest that one-third of the amino groups of the silica particles was present on their surfaces. Next, amino groups on the surface of a polymeric organic microparticle, NH2−PEG resin particles with a diameter of 10 μm, were quantified using the cleavable fluorescent compounds (Figure 3). For a resin concentration of 0.14 mg/mL, the density of amino groups estimated using cleavable coumarin

fluorophores released into the solution can then be measured using a conventional fluorescence spectrophotometer, allowing for the concentration of amino groups available for reaction on a solid surface to be quantitatively determined. Organic fluorescent dye molecules immobilized on a solid surface can undergo intermolecular fluorescence quenching with neighboring dye molecules or surface-induced quenching (e.g., fluorescence quenching by gold substrates).31 The present strategy avoids these problems to facilitate the quantification of amino groups on a solid surface. The standard curves of the cleaved fluorescent compounds in aqueous solution are shown in Figure S1. These standard curves exhibit good linearity within the concentration ranges tested. First, the concentration of amino groups on inorganic materials was quantified using the cleavable fluorescent compounds. Nonporous silica particles that had a diameter of 1 μm and amino groups on their surfaces were used as model inorganic materials. Various concentrations of silica particles were used (0.5, 1.25, 2.5, and 5.0 mg in 1 mL of water). Figure 2a shows the effect of the silica particle concentration on the fluorescence of the solution after cleavage of fluorophores. The experiments without silica particles reveal the concentration of fluorescent compounds physically adsorbed to the sample tubes, which were made of polypropylene. The fluorescence detected in the absence of silica particles was very low for cleavable FITC and cleavable coumarin, suggesting that background fluorescence was negligible. However, cleavable C

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The concentration of amino groups on a flat polymeric surface was quantified using cleavable FITC and cleavable coumarin. PMMA substrates displaying amino groups were prepared by dip-coating PMMA substrates in poly(MMA-r(Boc)AEMA and then deprotecting Boc groups in HCl solution (Figure 4a). Negligible fluorescence (corresponding

Figure 3. Amino groups displayed on NH2−PEG resin surfaces estimated using cleavable fluorescent compounds.

(65.1 ± 9.1 nmol/mg) was nearly twice the values estimated using cleavable FITC and cleavable anthranilate of 29.7 ± 1.3 and 36.1 ± 5.1 nmol/mg, respectively. These differences might arise from the steric hindrance and electrostatic repulsion between the fluorophores. The long axis of the fluorophore was 1.24 nm for cleavable FITC, 0.732 nm for cleavable coumarin, and 0.845 nm for cleavable anthranilate according to their space-filling models.35 Cleavable FITC has carboxy and phenolic hydroxy groups that can form negative charges by dissociation. These negatively charged groups of cleavable FITC immobilized on a surface would affect the accessibility of another molecule to neighboring amino groups. These considerations explain why the surface densities of amino groups estimated using cleavable FITC and cleavable anthranilate were lower than that determined using cleavable coumarin. A different content of resin (0.26 mg/mL) did not affect the amount of the amino groups estimated using cleavable coumarin. The manufacturer stated that the NH2−PEG resin contained 250 nmol/mg of amino groups on its surface and internally. Our results indicate that about a quarter of the amino groups was present on the resin surface. The surface density of amino groups in the NH2−PEG resin estimated using 5-formylsalicylic acid (in aqueous solution) was less than 10 nmol/mg, which was much lower than those determined using the intramolecular-cleavable fluorescent compounds. The formation of a Schiff base between amino and aldehyde groups is reversible in aqueous solution. Therefore, the conversion of amino groups to Schiff bases was not high enough under the present conditions, resulting in the low density of amino groups estimated using 5formylsalicylic acid. Then, we tried the same estimation using 5-formylsalicylic acid in an anhydrous solvent (methanol). The estimation in methanol exhibited 82 ± 3 nmol/mg of amino groups on the resin, which was comparable to the result of cleavable coumarin. Orange II has been used to quantify amino groups on solid surfaces.32−34 Orange II has sulfate and phenolic hydroxy groups that can electrostatically interact with an amino group and dissociate under alkaline conditions. In the present study, the concentration of amino groups on a resin surface was also quantified using Orange II; experimental details are provided in the Supporting Information. The method using Orange II suggested that the resin surfaces had 49 ± 7 nmol/mg amino groups, which is slightly smaller but comparable to that of cleavable coumarin. The orthogonal method supports the present results using the cleavable fluorophores.

Figure 4. (a) Chemical structure of poly(MMA-r-(Boc)AEMA). Boc groups of the copolymer were deprotected to obtain amino groups on the substrate surface. (b) Surface density of amino groups displayed on polymer surfaces measured using cleavable FITC and cleavable coumarin. The substrates were PMMA dip-coated in 5 wt % poly(MMA-r-(Boc)AEMA).

to a surface density of amino groups of less than 1.5 pmol/cm2) was detected from the bare and poly(MMA-r-(Boc)AEMA)coated substrates before deprotection because of the absence of reactive amino groups on their surfaces (Figure 4b). After deprotection, the amino-group-displaying substrates exhibited strong fluorescence. The estimated density of amino groups on the modified PMMA surface was 9.5 ± 1.4 pmol/cm2 for cleavable FITC and 23.9 ± 3.2 pmol/cm2 for cleavable coumarin. The concentration of amino groups estimated using cleavable coumarin was over double that using cleavable FITC. Overall, these results and those obtained for silica particles and resin suggest that cleavable coumarin is appropriate for quantification of a small amount of amino groups with a high surface density on a solid surface. We finally investigated the control of the density of amino groups on the surface and quantified the amino groups using cleavable coumarin. Poly(MMA-r-(Boc)AEMA) and PMMA (Mw = 1.2 × 105 g/mol) were first mixed at varied mixture ratios, and the mixture was then dip-coated onto PMMA substrates. The dip-coated substrates were immersed in 4 M HCl aqueous solution for 2 h at 25 °C to deprotect Boc groups. The liberated fluorophore (amino groups on a surface) increased with the content of poly(MMA-r-(Boc)AEMA) (Figure 5), suggesting that the small amounts (∼10 pmol/ cm2) of amino groups on the solid surfaces can be quantified using cleavable coumarin. The quantification methods based on the electrostatic adsorption of dyes (e.g., Orange II) to an analyte surface often suffer from the unexpected desorption in the analytical procedures, which would be a major drawback. To overcome this drawback, there are methods using the covalent conjugation of fluorescent dyes to amino groups displayed on solid surfaces,8,26,27 which provide the robustness and reproducibility in quantification. However, these methods involved the conjugation reactions in organic solvents, which may alter the surface characteristics as a result of the swelling and reorganization of surface-segregated polymers.36 The D

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Professor A. Mori (Kobe University) for his technical help and advice.

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Figure 5. Effect of the poly(MMA-r-(Boc)AEMA) content in the polymer mixture on amino group density on the dip-coated surfaces. Cleavable coumarin was used to quantify the amino groups on the dipcoated surfaces. Poly(MMA-r-(Boc)AEMA) was mixed with PMMA, and the mixture was dip-coated onto a PMMA substrate, followed by the deprotection of the Boc group using 4 M HCl solution.

present method allows for the quantification of amino groups that are available for immobilization of ligands and biomolecules under aqueous conditions, owing to the conjugation reaction in an aqueous solution. The covalent conjugation also provides the robustness and reproducibility. The cleavable fluorophores allow for the use of a conventional fluorescent spectrophotometer with high sensitivity.

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CONCLUSION We quantified the amino groups displayed on inorganic and organic surfaces in aqueous solution using cleavable fluorescent compounds. Among the four types of fluorescent compounds investigated, cleavable coumarin gave the most rational results. The developed method can measure small amounts (∼pmol/ cm2) of surface amino groups that are exposed to aqueous solution and would be available for surface immobilization of ligands and biomolecules. This method can provide useful information about the available amino groups on solid surfaces, which aids the design of functionalized surfaces.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02548. Descriptions of the materials and syntheses of cleavable fluorescent compounds and random copolymers of MMA and AEMA, quantification of amino groups using Orange II, and standard curves of cleavable fluorescent compounds (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

This work was partially supported financially by a Grant-in-Aid for Challenging Exploratory Research 25630380, the Ogasawara Foundation for the Promotion of Science & Engineering, and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe), Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. E

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