Succinimidyl ester surface chemistry: implications of the competition between aminolysis and hydrolysis on covalent protein immobilization.

N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.

Unusual catalytic effect of the two-dimensional molecular space with regular triphenylphosphine groups.

A novel organic-inorganic hybrid 2D molecular space with regular triphenylphosphine groups (triphenylphosphineamidephenylsilica, PPh(3)APhS) was successfully synthesized through grafting triphenylphosphine groups in the 2D structure of layered aminophenylsilica dodecyl sulfate (APhTMS-DS), which was developed in our previous research, with regular ammonium groups. The 2D structures were kept after the grafting reaction of triphenylphosphine groups in PPh(3)APhS. The catalytic potentials of 2D molecular space with regular triphenylphosphine groups were investigated. An unusual catalytic effect was found in a carbon-phosphorus ylide reaction. The PPh(3)-catalyzed reaction of modified allylic compounds, including bromides and chlorides with tropone yielded a [3 + 6] annulation product. However, an unusual [8 + 3] cycloadduct was obtained in the reaction of modified allylic compounds, including bromides and chlorides with tropone catalyzed by PPh(3)APhS. Otherwise, the stable catalytic intermediate was successfully separated, and the reaction activity of the catalytic intermediate was confirmed in the reaction of modified allylic compounds with tropone catalyzed by PPh(3)APhS. This research is the first successful example of directly influencing catalytic reaction processes and product structures by utilizing the chemical and geometrical limits of 2D molecular spaces with regular catalyst molecules and affords a novel method for controlling catalytic reaction processes and catalyst design.

Strong inclusion of inorganic anions into ß-cyclodextrin immobilized to gold electrode.

The inclusion of inorganic anions such as SO(4)(2-), NO(3)(-), and HPO(4)(2-) into the cavity of ß-cyclodextrin monolayers on Au was examined by X-ray photoelectron spectroscopy (XPS), a quartz crystal microbalance (QCM), and chronocoulometric measurements of the competitive inclusion with ferrocene. The inclusion amounts of ferrocence in 0.2 M Na(2)SO(4), NaNO(3), and Na(2)HPO(4) solutions were less than 6% of the adsorption amount of ß-cyclodextrin on Au, resulting in the apparent inhibition of the ferrocene redox reaction. The surface association constants of these anions reached about 10 on a logarithmic scale and were much higher than those for the inclusion of common organic guest compounds. A stronger anion inclusion was also demonstrated by the QCM response corresponding to the replacement of a preincluded organic guest with sulfate upon the injection of the sulfate solution. Quantitative analysis of the XPS data suggested a 1:1 association for each of these anions per surface ß-cyclodextrin. There was no detectable inclusion for ClO(4)(-), Cl(-), and Br(-).

Characterization and optimization of mixed thiol-derivatized beta-cyclodextrin/pentanethiol monolayers with high-density guest-accessible cavities.

Mixed per-6-thio-beta-cyclodextrin (CD-SH)/pentanethiol (C(5)SH) monolayers were constructed by the sequential immersion of a Au(111) electrode into solutions of CD-SH, ferrocene, and a mixed solution of ferrocene and C(5)SH. Highly compact CD-SH self-assembled monolayers (SAMs) with the surface CD-SH density of 74.0 +/- 6.3 pmol cm(-2) on a true surface area basis were formed in 1 mM CD-SH with the immersion time of more than 48 h as confirmed by reductive desorption voltammetry. Based on the concentration dependence of the adsorption amount, a Langmuir adsorption coefficient was determined to be 1.9 x 10(7) M(-1). Chronocoulometry in a ferrocene solution at the CD-SH SAM and the mixed CD-SH/C(5)SH monolayers revealed the following inclusion properties. (1) All the CD-SH cavities can be used for the inclusion of a guest compound before and after the adsorption of C(5)SH, as shown by the fact that the maximum inclusion amounts of ferrocene, 68.0 +/- 3.4 and 73.0 +/- 2.0 pmol cm(-2) before and after the adsorption of C(5)SH, respectively, were very close to the surface CD-SH density. (2) The association constant between the surface-confined CD-SH and ferrocene (7.6 x 10(4) M(-1)) is greater than the corresponding association constant in solution. (3) The intermolecular vacancies between the adsorbed CD-SH molecules are completely filled with C(5)SH. This ensures that the CD cavities are the only accessible sites for guest compounds and any other reactants.

Surface-enhanced infrared absorption spectroscopic studies of adsorbed nitrate, nitric oxide, and related compounds 1: Reduction of adsorbed NO on a platinum electrode.

Surface-enhanced infrared absorption spectroscopy (SEIRA) was used to examine the adsorption state of nitrogen monoxide (nitric oxide, NO) and the reduction of the adsorbed species. The SEIRA spectra gave two distinct bands at 1723-1733 and 1575-1607 cm-1 with an additional weak band at 1656-1676 cm-1 at 0.20 V, the frequencies of which are slightly dependent on the surface coverage. The former two bands are attributed to the on-top and bridged NO, respectively. While the on-top NO stably remained on the surface in the potential range of 0.05 -0.60 V, the bridged NO decreased in its intensity with increasing electrode potential. The reduction of the adsorbed NO obeys first-order kinetics with respect to the adsorbed NO. The rate constants are 2.24 +/- 0.03 and 0.24 +/- 0.09 s-1 at -0.10 V for the on-top and bridged NO, respectively. Tafel slopes obtained from the potential dependence of the rate constant indicate that the rate-determining step is the first electron-transfer process.

Surface-enhanced infrared absorption spectroscopic studies of adsorbed nitrate, nitric oxide, and related compounds 2: Nitrate ion adsorption at a platinum electrode.

The adsorbed species formed from the nitrate ion were examined using surface-enhanced infrared absorption spectroscopy (SEIRAS). The main band was observed at 1547-1568 cm-1 at 0.2 V in 0.01 M NaNO3 + 0.1 M HClO4. Although this band is close to that assigned to the adsorbed NO in the literature, it is assigned to the N=O stretching vibration of the chelating bidentate form of nitrate for the following reasons. (i) Nitrate gives a single band, while NO has three bands independent of the coverage. (ii) The band intensity remained constant in the potential range from 0.1 to 0.6 V, while that of the bridged NO at around 1600 cm-1 decreased in this range. (iii) The rate constants for the reduction and/or desorption at negative potentials are about 3 times higher than those of the bridged NO. (iv) The adsorbed species from nitrate is replaced with CO more easily than the bridged NO.

Facile formation of water-insoluble cyclodextrin/Nafion composite films on solid surfaces.

The water-insoluble poly-beta-cyclodextrin (poly-CD)/Nafion composite film was easily prepared by casting a mixed solution of poly-CD and Nafion onto substrate plates. FT-IR measurements showed that the 50/50 wt% poly-CD/Nafion composite film remained stable on the glass and quartz substrates after immersion in water for more than 3 h, while a pure poly-CD film was almost completely dissolved by immersion within 1 h. The film stability was also evaluated from the amount of p-nitrophenol (p-NP) inclusion in the film, which was determined from the decrease in UV-vis absorbance of the p-NP solution into which the film was immersed. The composition dependence of the inclusion amount showed that the film was stable up to 50 wt% CD, but became less stable with further increase in the CD concentration in the film. From the isotherms for the inclusion of p-NP and 1-naphthoate (1-Naph) into the film, the inclusion (stability) constants were determined to be 3.7x10(3) M(-1) and 1.9x10(2) M(-1), respectively. These results show that the selective inclusion of CD is retained after preparation of the composite film.

Construction of mixed mercaptopropionic acid/alkanethiol monolayers of controlled composition by structural control of a gold substrate with underpotentially deposited lead atoms.

Mixed monolayers of 3-mercaptopropionic acid (MPA) and alkanethiols of various chain lengths have been constructed on Au based on a novel concept, namely, control of the composition of the component thiols in mixed monolayers by controlling the surface structure of the substrate. The Au substrate surface was first modified with underpotentially deposited Pb (UPD Pb) atoms, followed by the formation of a self-assembled monolayer (SAM) of alkanethiol. The UPD Pb atoms were then oxidatively stripped from the surface to create vacant site, on which MPA was adsorbed to finally form the mixed monolayers. The surface coverages of Pb, alkanethiol and MPA, and the total numbers of thiols were determined using an electrochemical quartz crystal microbalance, X-ray photoelectron spectroscopy, and reductive desorption voltammetry. These results demonstrate that the surface coverage of MPA in the mixed monolayers is determined by the initial coverage of UPD Pb. Fourier transform infrared spectra also support this conclusion. The observed single peak in the cyclic voltammogram for the reductive desorption shows that MPA and alkanethiol do not form their single-component domains. Scanning tunneling microscopy revealed the single-row pinstripe structure for all the thiol adlayers formed during each step of the preparation. This shows that the surface structure of the mixed monolayers is determined by the structure of the initially formed SAM on Au partially covered with UPD Pb.