Electrochemical synthesis of nanostructured gold film for the study of carbohydrate–lectin interactions using localized surface plasmon resonance spectroscopy

Journal of Chromatography A

et al. 2015

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The surface of nanoporous gold (np-Au) monoliths was modified via a flow method with the lectin Concanavalin A (Con A) to develop a substrate for separation and extraction of glycoproteins. Self-assembled monolayers (SAMs) of lipoic acid (LA) on the np-Au monoliths were prepared followed by activation of the terminal carboxyl groups to create amine reactive esters that were utilized in the immobilization of Con A. Thermogravimetric analysis (TGA) was used to determine the surface coverages of LA and Con A on np-Au monoliths which were found to be 1.31 × 1018 molecules m−2 and 1.85 × 1015 molecules m−2, respectively. An in situ solution depletion method was developed that enabled surface coverage characterization without damaging the substrate and suggesting the possibility of regeneration. Using this method, the surface coverages of LA and Con A were found to be 0.989 ×1018 molecules m−2 and 1.32 × 1015 molecules m−2, respectively. The selectivity of the Con A-modified np-Au monolith for the high mannose-containing glycoprotein ovalbumin (OVA) versus negative control non-glycosylated bovine serum albumin (BSA) was demonstrated by the difference in the ratio of the captured molecules to the immobilized Con A molecules, with OVA:Con A = 2.3 and BSA:Con A = 0.33. Extraction of OVA from a 1:3 mole ratio mixture with BSA was demonstrated by the greater amount of depletion of OVA concentration during the circulation with the developed substrate. A significant amount of captured OVA was eluted using α-methyl mannopyranoside as a competitive ligand. This work is motivated by the need to develop new materials for chromatographic separation and extraction substrates for use in preparative and analytical procedures in glycomics.

Section: Resultsmentioning

confidence: 99%

Selective capture of glycoproteins using lectin-modified nanoporous gold monolith

Alla

Andrea

Journal of Chromatography A

et al. 2015

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“…The flat gold surface was prepared by template stripping of sputtered gold from a silicon wafer. The details of this method were previously reported by our group [20]. The water contact angle was determined using a simple home-made setup (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The surface curvature may affect the orientation of alkanethiols and especially the spacing between the terminal functional groups, such as noted in a study showing that the p K a of carboxylic acid-terminated SAMs increases with nanoparticle diameter [16, 17]. The use of SAMs with specific terminal functional groups is important, such as when these groups are activated and coupled to biomolecules for use in development of biosensors [18–20]. In a previous study using thermogravimetric analysis (TGA), we found that the coverage of an alkanethiol SAM within an np-Au monolith was close to that expected on the basis of packing geometry on flat Au [21, 22].…”

Section: Introductionmentioning

confidence: 99%

Electrochemical impedance spectroscopy study of carbohydrate-terminated alkanethiol monolayers on nanoporous gold: Implications for pore wetting

Sharma

Journal of Electroanalytical Chemistry

Nigudkar

et al. 2016

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Electrochemical impedance spectroscopy (EIS) is used to compare the apparent electron transfer rate constant (kapp) for a series of alkanethiol and of carbohydrate-terminated alkanethiol self-assembled monolayers (SAMs) on both flat gold and on nanoporous gold (np-Au). Using the surface area for np-Au determined by oxide stripping, the values of kapp for the alkanethiol modified np-Au are initially over two orders of magnitude smaller than the values found on flat Au. This result provides evidence that the diffusing redox probe Fe(CN)63−/4− only accesses a fraction of the np-Au surface after alkanethiol modification suggesting very limited wetting of the internal pores due to the hydrophobic nature of these surfaces. In contrast, for np-Au modified by carbohydrate-terminated (mannose or galactose) alkanethiols the values of kapp are about 10–40 fold smaller than on flat gold, suggesting more extensive access of the diffusing redox probe within the pores and better but still incomplete wetting, a result also found for modification of np-Au with mercaptododecanoic acid. A short chain PEG thiol derivative is found to result in a comparison of kapp values that suggests nearly complete wetting of the internal pores for this highly hydrophilic derivative. These results are of significance for the potential applications of SAM modified np-Au in electrochemical sensors, especially for those based on carbohydrate–protein recognition, or those of np-Au modified by SAMs with polar terminal groups.

“…and diluted further to the desired concentrations The SAM-modified gold wires were incubated in the desired concentration of lectins solution for 1 h at room temperature and thoroughly rinsed with Tris buffer and water before making the measurement. Our prior studies of Con A binding to SAMs containing Man-C 8 -SH [18] and of binding of the lectin soybean agglutinin to SAMs of a globotriose derivative[14] indicate that one hour is sufficient for binding equilibrium to be reached. Lectin PNA, which binds to galactosyl (β-1,3) N -acetylgalactosamine structures, was used as a negative control in this study.…”

Section: Methodsmentioning

confidence: 99%

“…The K a value for methyl α-D-mannopyranoside reported in the study was 0.82 × 10 4 ( K d = 122 μM). More recently localized surface plasmon resonance [18], surface plasmon resonance (SPR) [19], quartz crystal microbalance (QCM) [20], and electrochemical techniques [21] have been used to investigate biomolecular interactions and have proven to play an important role in investigating carbohydrate–lectin interactions [22, 23]. …”

Section: Introductionmentioning

confidence: 99%

Electrochemical impedance spectroscopy study of Concanavalin A binding to self-assembled monolayers of mannosides on gold wire electrodes

Journal of Electroanalytical Chemistry

Tan

Pandey

et al. 2016

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The interactions of the lectin Concanavalin A (Con A) with self-assembled monolayers (SAMs) of thiolated mono-, di-, and tri-mannosides were studied on the surface of gold wires using electrochemical impedance spectroscopy (EIS). The SAMs of mannosides were prepared either pure or along with thiolated triethylene glycol (TEG) at different molar ratios (1:1, 1:2, 1:4, 1:9, and 1:19) to better understand and optimize the interaction conditions. The charge-transfer resistance of the [Fe(CN)6]3−/4− redox probe was compared before and after the interaction at different concentrations of Con A to determine the equilibrium dissociation constant (Kd) and limit of detection (LOD). Values of Kd were found in the nanomolar range showing multivalent interactions between mannosides and Con A, and LOD was found ranging from 4–13 nM depending on the type of mannoside SAM used. Analysis using the Hill equation suggests negative cooperativity in the binding behavior. Peanut agglutinin was used as a negative control, and cyclic voltammetry was used to further support the experiments. We have found that neither the pure nor the widely dispersed monolayers of mannosides provide the conditions for optimal binding of Con A. The binding of Con A to these SAMs is sensitive to the molar ratio of the mannoside used to prepare the SAM and to the structure of the mannoside. A simple cleaning method has also been shown to regenerate the used gold wire electrodes. The results from these experiments contribute to the development of simple, cheap, selective, and sensitive EIS-based bioassays, especially for lectin–carbohydrate interactions.