Gold Bead Electrode Research Articles

FRET Imaging of Nonuniformly Distributed DNA SAMs on Gold Reveals the Role Played by the Donor/Acceptor Ratio and the Local Environment in Measuring the Rate of Hybridization.

Mixed DNA SAMs labeled with a fluorophore (either AlexaFluor488 or AlexaFluor647) were prepared on a single crystal gold bead electrode using potential-assisted thiol exchange and studied using Förster resonance energy transfer (FRET). A measure of the local environment of the DNA SAM (e.g., crowding) was possible using FRET imaging on these surfaces since electrodes prepared this way have a range of surface densities (ΓDNA). The FRET signal was strongly dependent on ΓDNA and on the ratio of AlexaFluor488 to AlexaFluor647 used to make the DNA SAM, which were consistent with a model of FRET in 2D systems. FRET was shown to provide a direct measure of the local DNA SAM arrangement on each crystallographic region of interest providing a direct assessment of the probe environment and its influence on the rate of hybridization. The kinetics of duplex formation for these DNA SAMs was also studied using FRET imaging over a range of coverages and DNA SAM compositions. Hybridization of the surface-bound DNA increased the average distance between the fluorophore label and the gold electrode surface and decreased the distance between the donor (D) and acceptor (A), both of which result in an increase in FRET intensity. This increase in FRET was modeled using a second order Langmuir adsorption rate equation, reflecting the fact that both D and A labeled DNA are required to become hybridized to observe a FRET signal. The self-consistent analysis of the hybridization rates on low and high coverage regions on the same electrode showed that the low coverage regions achieved full hybridization 5× faster than the higher coverage regions, approaching rates typically found in solution. The relative increase in FRET intensity from each region of interest was controlled by manipulating the donor to acceptor composition of the DNA SAM without changing the rate of hybridization. The FRET response can be optimized by controlling the coverage and the composition of the DNA SAM sensor surface and could be further improved with the use of a FRET pair with a larger (e.g., > 5 nm) Förster radius.

Redox-Controlled Energy Transfer Quenching of Fluorophore-Labeled DNA SAMs Enables In Situ Study of These Complex Electrochemical Interfaces.

Interfaces modified by a molecular monolayer can be challenging to study, particularly in situ, requiring novel approaches. Coupling electrochemical and optical approaches can be useful when signals are correlated. Here we detail a methodology that uses redox electrochemistry to control surface-based fluorescence intensity for detecting DNA hybridization and studying the uniformity of the surface response. A mixed composition single-strand DNA SAM was prepared using potential-assisted thiol exchange with two alkylthiol-modified ssDNAs that were either labeled with a fluorophore (AlexaFluor488) or a methylene blue (MB) redox tag. A significant change in fluorescence was observed when reducing MB to colorless leuco-MB. In situ fluorescence microscopy on a single-crystal gold bead electrode showed that fluorescence intensity depended on (1) the potential controlling the oxidation state of MB, (2) the surface density of DNA, (3) the MB:AlexFluor488 ratio in the DNA SAM, and (4) the local environment around the DNA SAM. MB efficiently quenched AlexaFluor488 fluorescence. Reduction of MB showed a significant increase in fluorescence resulting from a decrease in quenching or energy transfer efficiency. Hybridization of DNA SAMs with its unlabeled complement showed a large increase in fluorescence due to MB reduction for surfaces with sufficient DNA coverage. Comparing electrochemical-fluorescence measurements to electrochemical (SWV) measurements showed an improvement in detection of a small fraction of hybridized DNA SAM for surfaces with optimal DNA SAM composition and coverage. Additionally, this coupled electrochemical redox-fluorescence microscopy method can measure the spatial heterogeneity of electron-transfer kinetics and the influence of the local interfacial environment.

(Invited) Improved Thermal Stability of Low-Probe Density DNA SAMs Prepared with Electrodeposition

DNA self-assembled monolayer (SAM) on gold electrodes has been widely used for biosensing.1, 2 The sensor stability and performance determine the reliability of the test results in early disease detection and point-of-care use.3, 4 We have carefully examined the thermal stability of a mixed SAMs composed of a short alkylthiol and alkylthiol modified DNA prepared with electrodeposition vs. those prepared at OCP (Open Circuit Potential) on a single crystal gold bead electrode. The nature of these two types of SAMs were carefully studied in buffer after exposure to different temperatures using in-situ fluorescence microscopy and electrochemical measurements. The analysis showed that surfaces with hexagonal symmetry were thermally less stable than those with rectangular or square symmetry, suggesting that the crystallography of the gold substrate plays an important role in sensor stability and performance in addition to the DNA density.5 We also found that OCP deposition creates a surface with high DNA coverage (>1012 DNA/cm2) but with higher level of irreproducibility. Even though this kind of surface can be more thermally stable, it is not optimal for effective biosensing due to its high coverage.6 It also seems to be challenging to make DNA SAMs with both low coverage and high thermal stability by using current SAM preparation protocols (e.g., OCP). In this study, we show that electrodeposition can prepare DNA SAMs with both higher thermal stability than those made at OCP and with good control of low surface coverage. This study provides valuable insights on making high quality DNA SAMs with better stability and functionality and improved reproducibility.

Electrochemical sensing of the secretion of indoxyl sulfate in a rat intestinal loop using a self-assembled monolayer-modified gold bead electrode

Indoxyl sulfate is a uremic toxin that accumulates in the plasma after a decline in renal function that might progress to chronic kidney disease (CKD). This accumulation is associated with the onset of dialysis and exacerbation of CKD and cardiovascular diseases. In this study, we aimed to demonstrate intestinal secretion as an excretion pathway of indoxyl sulfate in the severe stage of CKD using electrochemical sensing. A self-assembled monolayer (SAM) of 2-mercaptobenzimidazole on gold beads was successfully used for the separate sensing of indoxyl sulfate, uric acid (UA), and ascorbic acid. This electrode could detect indoxyl sulfate at concentrations as low as 50 nM in the presence of UA. No indoxyl sulfate secretion was observed in the intestinal loop of healthy rats or those intravenously administered saline. However, indoxyl sulfate secretion was detected in the 5/6 nephrectomized rats that showed high serum indoxyl sulfate levels, which also occurs in patients with CKD stage 4 or 5 and the healthy rats intravenously injected with indoxyl sulfate. These results suggest that indoxyl sulfate is excreted through the intestine when serum indoxyl sulfate level is high. The results of the present study showed that the SAM-modified gold bead electrode can be used as an easy and sensitive method for evaluating indoxyl sulfate secreted in the intestine over time.

Improved Thermal Stability and Homogeneity of Low Probe Density DNA SAMs Using Potential-Assisted Thiol-Exchange Assembly Methods.

Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor development. The thermal stability of low-coverage DNA SAMs was studied for surfaces prepared using potential-assisted thiol exchange (Edep) and compared to DNA SAMs prepared without control over the substrate potential (OCPdep). Both surface preparation methods were studied using in situ fluorescence microscopy and electrochemistry with fluorophore or redox-modified DNA SAMs on a single-crystal gold bead electrode. Fluorescence microscopy showed that the influence of the underlying surface crystallography was important in both cases. The highest thermal stability was realized for square or rectangular surface atomic structure (e.g., surfaces from 110 to 100). The 111 and related surfaces were the least thermally stable. The low DNA coverage surfaces prepared by Edep had better thermal stability and higher DNA probe mobility as compared to OCPdep-prepared surfaces with the similar coverage. These results were correlated with methylene blue redox-tagged DNA probes, which directly measured the average DNA coverage. Both methods indicated that Edep DNA SAMs were more uniformly distributed across the electrode surface, while the surfaces prepared via OCPdep assembled into clusters with reduced mobility. The potential-assisted thiol-exchange approach to preparing low-coverage DNA SAMs was shown to quickly create modified surfaces that were consistent, had mobility characteristics which should yield superior DNA hybridization efficiencies, and having greater thermal stability which will translate into a longer shelf-life.

AFM and Cu Electrodeposition Studies of Reduced Graphene Oxide Modified Au(111) Facets Prepared using Electrodeposition and Post-Deposition Pulse Treatment

The structural and electrochemical characteristics of electrodeposited graphene oxide (GO) onto a single crystal gold bead electrode was studied using AFM and Cu electrodeposition. Optimal deposition uniformity was realized by rotation of the electrode, suggesting an initial electrophoretic deposition occurs followed by some reduction of the GO. Potential pulse treatments to reducing potentials for a short time significantly improve the electrochemical characteristics of the electrochemical reduced graphene oxide (ERGO). Both under-potential and over-potential Cu electrodeposition was used to characterize the quality of the ERGO deposit and showed that the gold surface was completely coated with ERGO only after potential pulsing treatments. AFM analysis of ERGO modified Au(111) facet on the gold bead electrode revealed the deposition was composed of sheets of GO/ERGO as well as much rougher deposits. AFM showed that the potential pulsing treatment resulted in the nucleation and growth of electrodeposited Cu nanoparticles over much of the ERGO surface, in contrast to the limited deposition of nanoparticles on the untreated GO deposit. The ERGO modified gold surface prepared using electrodeposition followed by potential pulse treatments results in conductive and electrochemically active ERGO surfaces.

A Fresh Perspective on the Thermal Stability of Thiol-Based DNA SAMs

The stability of gold surfaces modified with DNA self assembled monolayers has drawn increasing attention as the lifespan of the created sensors is an important consideration for biosensor development and applications.1–4 In this study, we investigated the thermal stability of a mixed self assembled monolayer (SAMs) composed of a short alkythiol and alkythiol modified DNA immobilized on a single crystal gold bead electrode at either open circuit potential (OCP) or by electrodeposition.5,6 Using in-situ fluorescence microscopy and electrochemical measurements, different SAMs with varied surface properties were studied, and the relative stability towards increasing temperatures was evaluated. The results show that the stability strongly depends on the preparation method, the DNA coverage and on the underlying surface crystallography.The single crystal bead electrode enabled study of a number of surface crystallographies in a self-consistent manner. For the surfaces made without potential control, the {111} regions were the least thermally stable surface and completely desorbed from the gold surface after heating to 85 °C in some cases. On the other hand, {100} is the most stable region on which SAMs can survive higher than 85 °C in buffer. The 110, 210 and 311 surfaces were also studied. The surfaces with hexagonal symmetry were less stable than those with square or rectangular symmetry. The different methods used to prepare the DNA SAM also showed significant changes in the stability. In general, SAMs made with potential assisted deposition showed higher stability than those made at OCP. This unique finding can be used to comment on typical sensor surface which are polycrystalline flat gold surfaces composed of a variety of grains and grain boundaries. These results may help to explain the irreproducible results from the DNA SAMs and such as the reported inconsistent results during DNA mismatch discrimination while the solution was heated during experiment.7 Modifying the method used to prepare the DNA SAMs and carefully controlling the composition of the gold substrate may result in significant improvements in the SAM stability. Moreover, increasing the thermal stability will result in longer term storage stability.8,9 References(1) Brittain, W. J.; Brandsetter, T.; Prucker, O.; Rühe, J. ACS Appl. Mater. Interfaces 2019, 11, 39397–39409.(2) Civit, L.; Fragoso, A.; O’Sullivan, C. K. Electrochem. commun. 2010, 12, 1045–1048.(3) Flechsig, G. U.; Peter, J.; Hartwich, G.; Wang, J.; Gründler, P. Langmuir 2005, 21, 7848–7853.(4) Ge, D.; Wang, X.; Williams, K.; Levicky, R. Langmuir 2012, 28, 8446–8455.(5) Leung, K. K.; Gaxiola, A. D.; Yu, H.-Z.; Bizzotto, D. Electrochim. Acta 2018, 261,188–197.(6) Yu, Z. L.; Casanova-Moreno, J.; Guryanov, I.; Maran, F.; Bizzotto, D. J. Am. Chem. Soc. 2015, 137, 276–288.(7) Xu, X.; Makaraviciute, A.; Kumar, S.; Wen, C.; Sjödin, M.; Abdurakhmanov, E.; Danielson, U. H.; Nyholm, L.; Zhang, Z. Anal. Chem. 2019, 91, 14697–14704.(8) McAteer, K.; Simpson, C. E.; Gibson, T. D.; Gueguen, S.; Boujtita, M.; El Murr, N. J. Mol. Catal. - B Enzym. 1999, 7, 47–56.(9) Young Choi, J.; Yang, I. M. Pharm. Anal. Acta 2016, 07.Figure 1 Figure 1

Thermal Stability of Thiolated DNA SAMs in Buffer: Revealing the Influence of Surface Crystallography and DNA Coverage via In Situ Combinatorial Surface Analysis.

The thermal stability of thiol based DNA SAMs prepared on gold surfaces is an important parameter that is correlated to sensor lifetime. The thermal stability of DNA SAMs was evaluated in aqueous buffer through the use of fluorophore labeled DNA, a single crystal gold bead electrode, and microscopy. The stability of different crystallographic regions on the electrode was studied for thermal treatments up to 95 °C for 90 min. Using a in situ combinatorial surface analytical measurement showed that the crystallography of the underlying gold surface played a significant role, with the square or rectangular lattices (e.g., 110, 100, 210) having the highest stability. Surfaces with hexagonal lattices (e.g., 111, 311, 211) were less stable toward thermal treatments. These crystallographic trends were observed for both high and low coverage DNA SAMs. High coverage DNA SAMs were the most stable, with stability decreasing with decreasing coverage on average. Increasing DNA SAM coverage appears to slow the kinetics of thermal desorption, but the coordination to the underlying surface determined their relative stability. Preparing the DNA SAMs under nominally similar conditions were found to create surfaces that were similar at room temperature, but had significantly different thermal stability. Optimal DNA sensing with these surfaces most often requires low coverage DNA SAMs which results in poor thermal stability, which is predictive of a poor shelf life, making optimization of both parameters challenging. Furthermore, the crystallographically specific results should be taken into account when studying the typically used polycrystalline substrates since the underlying surface crystallography maybe different for different samples. It appears that preparing DNA SAMs with low coverage and significant thermal stability will be challenging using the current SAM preparation procedures.

A Fresh Perspective on the Thermal Stability of Thiol-Based DNA SAMs

The stability of gold surfaces modified with DNA self assembled monolayers has drawn increasing attention as the lifespan of the created sensors is an important consideration for biosensor development and applications.1–4 In this study, we investigated the thermal stability of a mixed self assembled monolayer (SAMs) composed of a short alkythiol and alkythiol modified DNA immobilized on a single crystal gold bead electrode at either open circuit potential (OCP) or by electrodeposition.5,6 Using in-situ fluorescence microscopy and electrochemical measurements, different SAMs with varied surface properties were studied, and the relative stability towards increasing temperatures was evaluated. The results show that the stability strongly depends on the preparation method, the DNA coverage and on the underlying surface crystallography. The single crystal bead electrode enabled study of a number of surface crystallographies in a self-consistent manner. For the surfaces made without potential control, the {111} regions were the least thermally stable surface and completely desorbed from the gold surface after heating to 85 °C in some cases. On the other hand, {100} is the most stable region on which SAMs can survive higher than 85 °C in buffer. The {110}, {210} and {311} surfaces were also studied. The surfaces with hexagonal symmetry were less stable than those with square or rectangular symmetry. The different methods used to prepare the DNA SAM also showed significant changes in the stability. In general, SAMs made with potential assisted deposition showed higher stability than those made at OCP. This unique finding can be used to comment on typical sensor surface which are polycrystalline flat gold surfaces composed of a variety of grains and grain boundaries. These results may help to explain the irreproducible results from the DNA SAMs and such as the reported inconsistent results during DNA mismatch discrimination while the solution was heated during experiment.7 Modifying the method used to prepare the DNA SAMs and carefully controlling the composition of the gold substrate may result in significant improvements in the SAM stability. Moreover, increasing the thermal stability will result in longer term storage stability.8,9 References(1) Brittain, W. J.; Brandsetter, T.; Prucker, O.; Rühe, J. ACS Appl. Mater. Interfaces2019, 11, 39397–39409.(2) Civit, L.; Fragoso, A.; O’Sullivan, C. K. Electrochem. commun. 2010, 12, 1045–1048.(3) Flechsig, G. U.; Peter, J.; Hartwich, G.; Wang, J.; Gründler, P. Langmuir 2005, 21,7848–7853.(4) Ge, D.; Wang, X.; Williams, K.; Levicky, R. Langmuir 2012, 28, 8446–8455.(5) Leung, K. K.; Gaxiola, A. D.; Yu, H.-Z.; Bizzotto, D. Electrochim. Acta 2018, 261,188–197.(6) Yu, Z. L.; Casanova-Moreno, J.; Guryanov, I.; Maran, F.; Bizzotto, D. J. Am. Chem.Soc. 2015, 137, 276–288.(7) Xu, X.; Makaraviciute, A.; Kumar, S.; Wen, C.; Sjödin, M.; Abdurakhmanov, E.; Danielson,U. H.; Nyholm, L.; Zhang, Z. Anal. Chem. 2019, 91, 14697–14704.(8) McAteer, K.; Simpson, C. E.; Gibson, T. D.; Gueguen, S.; Boujtita, M.; El Murr, N. J.Mol. Catal. - B Enzym. 1999, 7, 47–56.(9) Young Choi, J.; Yang, I. M. Pharm. Anal. Acta 2016, 07 .Figure 1 Figure 1

Preparing DNA SAM Electrochemical Sensors Using Potential Assisted Deposition Methods. Controlling the Coverage and Local Organization of the DNA in the SAM.

Typically, DNA SAMs used for electrochemical based sensing are prepared without controlling the gold electrode potential[1]. Applying a potential during DNA deposition enables control over the coverage and DNA organization[2-4]. These characteristics are determined by the composition of the immobilization buffer, the immersion time, the DNA concentration, the extent of backfilling with a small alkylthiol and whether deposition occurs on a bare or alkythiol coverage gold surface[3-4]. Few studies have explored the influence of the underlying surface crystallography. Here, we show the possibilities of using electrochemical potential control to manipulate the characteristics of the DNA SAM. Moreover, the use of a single crystal gold bead electrode, coupled with the use of a fluorophore tagged DNA and fluorescence microscopy, enables examination of the influence of the underlying surface crystallography on the DNA SAM prepared. Various constant potentials were used as well as using a square-wave potential perturbation during deposition. There are significant differences in the DNA SAM produced under these conditions. In some cases, the DNA coverage was low on the <111> surfaces, higher on more open or atomically rough regions. In other cases, the coverage was higher on the <100> surfaces as compared to the other regions. The use of constant positive or negative potentials (vs SCE) resulted in significant differences in the relative coverages among regions of different atomic arrangements (Fig 1). In addition, a significant difference was observed between immobilization buffers that contain Cl and those that are Cl free. Also, the use of constant or square-wave potential perturbations resulted in significantly different local organizations of DNA in the SAM. Examples of these types of DNA SAM surfaces will be presented with some conclusions on the implications for use on polycrystalline planar gold surfaces.

Electrodepositing DNA Self-Assembled Monolayers on Au: Detailing the Influence of Electrical Potential Perturbation and Surface Crystallography.

The preparation of DNA self-assembled monolayers (SAMs) on single-crystal gold bead electrodes using an applied potential is evaluated with in situ electrochemical fluorescence microscopy. Applying a constant deposition potential or a square-wave potential perturbation during the formation of DNA SAMs is compared for two different modification methods: DNA SAM formation on a clean gold surface followed by alkythiol backfilling (as is typically done in the literature) or via thiol-exchange on an alkylthiol-modified gold surface. DNA SAMs prepared from a chloride-containing deposition buffer were not significantly different when using either square-wave potential perturbation or at a constant applied potential even when considering different surface crystallographies. Greater variations were observed when applying more positive potentials for both DNA thiol-exchange and DNA adsorption on clean Au. Our results suggest that using either a constant potential or a square-wave potential perturbation for 5 min both create defects by weakening the gold-thiol interaction. When the deposition is performed with the adsorption of chloride ions from the electrolyte, the electrodeposition results in a similar increase in DNA coverage when compared to depositions performed at open circuit potentials.

Application of FRET Microscopy to the Study of the Local Environment and Dynamics of DNA SAMs on Au Electrodes.

Immobilized DNA probe strands self-assembled on an electrode surface are the bases of many electrochemically based biosensors. Control or measurement of the local environment around each DNA molecule tethered to the electrode surface is needed because the local environment can influence the binding or hybridization efficiency of the target in solution. Measurement of this local environment in buffer or under electrochemical control can be challenging. Here we demonstrate the use of fluorescence microscopy and a Förster resonance energy transfer (FRET) methodology to characterize multicomponent DNA SAMs. The DNA SAMs that were studied were composed of a series of mole fraction ratios of alkylthiol-modified DNA which was labeled with either AlexaFluor488 or AlexaFluor647, a FRET donor and acceptor, respectively. The DNA SAMs were hybridized before assembly onto the electrode surface. Wide-field filter-based FRET microscopy was used to study the assembly of DNA SAMs onto gold bead electrodes. These single-crystal gold bead electrodes contain many surface crystallographic regions which enable the comparison of the adsorbed DNA local environment. These surfaces show that most surface modifications are uniformly prepared, and the FRET efficiency can be explained through simple surface density considerations. The FRET efficiency for different compositions of the donor and acceptor for these regions is also explained through 2D FRET modeling. Not all surfaces were similar to the (111) and (110) regions showing deviations from the expected FRET behavior. Also demonstrated is FRET imaging using a confocal microscope. This approach proves useful in the analysis of a more dynamic system, such as the analysis of reductive desorption of the mixed-component DNA SAM. FRET microscopy is useful for surface analysis of the DNA local environment, enabling a measure of the surface modification, local density, and clustering and eventually a new detection modality.

Optimizing Surface Modifications for Quantum Dot Labelled DNA SAMs Using Electrochemistry Coupled Fluorescence Imaging

Electrochemical biosensors are being developed for detecting specific sequences of nucleic acids using DNA modified gold surfaces[1]. A single crystal gold bead electrode was used to study the arrangement on DNA on different gold surfaces[2]. The gold surface was prepared with a mercaptoundecanol (MUDOL) self-assembled monolayer (SAM). Using a specific desorption potential of -0.850 V/SCE, the MUDOL was reductively desorbed from the Au(111) facet. The defects in the SAM were passivated with a shorter alkylthiol, mercaptohexanol. Thiol modified DNA which is also labeled with a fluorophore was allowed to populate the 111 facet specifically. This was studied using fluorescence microscopy coupled with electrochemical control. A fluorescence image of the gold bead (Figure a) showed that only the 111 facets were modified. The density of the DNA modification was also evaluated using the reorientation of DNA based on the electrode charge density. At positive potentials the DNA was positioned close to the electrode and the fluorescence was quenched. At negative potentials the DNA was repelled and the fluorescence increased. This change in fluorescence is indicative of the density of DNA on the surface. The same approach will be used to assemble DNA modified Quantum Dots (QDs) onto the Au(111) facets. A complementary ssDNA was attached to a glutathione capped quantum dot vis a his-tag peptide. The QD labelled ssDNA was allowed to hybridize with the ssDNA on the gold electrode. Figure (b) is the cartoon depiction of such a layer. Recent research results will be described on the characterization of QD labelled DNA SAMs. The QDs due to their broad absorption and narrow emission spectrum also offer the advantage of building more complicated multiplexed systems in future. (1) Hsieh, K.; Ferguson, B. S.; Eisenstein, M.; Plaxco, K. W.; Soh, H. T. Integrated Electrochemical Microsystems for Genetic Detection of Pathogens at the Point of Care. Acc. Chem. Res. 2015, 48, 911–920. (2) Yu, Z. L.; Casanova-Moreno, J.; Guryanov, I.; Maran, F.; Bizzotto, D. Influence of Surface Structure on Single or Mixed Component Self-Assembled Monolayers via in Situ Spectroelectrochemical Fluorescence Imaging of the Complete Stereographic Triangle on a Single Crystal Au Bead Electrode. J. Am. Chem. Soc 2015, 137, 276–288. Figure 1

A non-linear harmonic analysis of potential induced fluorescence modulation of a DNA self assembled monolayer

The characterization of a mixed mercaptohexanol - fluorophore labeled DNA self-assembled monolayer (SAM) on a single crystal gold bead electrode is demonstrated using an electrochemical perturbation and optical harmonic analysis scheme. This approach relies on the non-linear characteristics of the fluorescence intensity response towards a single frequency sinusoidal potential perturbation (163Hz) which causes the DNA SAM to reorient, changing the efficiency of fluorescence quenching by the gold electrode. This results in a modulated fluorescence intensity that is composed of first, second and third harmonic signals which were found to strongly depend on the hybrization state of the DNA SAM, the DNA packing density on the surface, and the surface crystallography. Comparing the results between the different surface crystallographies on the same electrode and between different electrodes was simplified with a self-consistent normalization method reducing the influence of the raw fluorescence intensity. A significant difference in the second harmonic response was observed for single and double stranded DNA SAMs especially on the lower coverage (100) region and surfaces with related crystallographies. The third harmonic signal was weaker, but also showed a distinction between the hybridization states. Higher coverage (311) surface regions showed no significant fluorescence modulation nor any difference based on state of hybridization. Within a limited potential range, the electrochemical response (current) from the modified electrode did not show significant non-linearity revealing that the source of the harmonic signals was due to the non-linear relationship between the fluorescence quenching and fluorophore separation from the electrode surface. Increasing the amplitude of the potential perturbation increased the harmonic signal magnitudes further differentiating the DNA hybridization state.

Influence of surface structure on single or mixed component self-assembled monolayers via in situ spectroelectrochemical fluorescence imaging of the complete stereographic triangle on a single crystal Au bead electrode.

The use of a single crystal gold bead electrode is demonstrated for characterization of self-assembled monolayers (SAM)s formed on the bead surface expressing a complete set of face centered cubic (fcc) surface structures represented by a stereographic projection. Simultaneous analysis of many crystallographic orientations was accomplished through the use of an in situ fluorescence microscopic imaging technique coupled with electrochemical measurements. SAMs were prepared from different classes of molecules, which were modified with a fluorescent tag enabling characterization of the influence of electrical potential and a direct comparison of the influence of surface structure on SAMs adsorbed onto low index, vicinal and chiral surfaces. The assembly of alkylthiol, Aib peptide and DNA SAMs are studied as a function of the electrical potential of the interface revealing how the organization of these SAMs depend on the surface crystallographic orientation, all in one measurement. This approach allows for a simultaneous determination of SAMs assembled onto an electrode surface onto which the whole fcc stereographic triangle can be mapped, revealing the influence of intermolecular interactions as well as the atomic arrangement of the substrate. Moreover, this method enables study of the influence of the Au surface atom arrangement on SAMs that were created and analyzed, both under identical conditions, something that can be challenging for the typical studies of this kind using individual gold single crystal electrodes. Also demonstrated is the analysis of a SAM containing two components prepared using thiol exchange. The two component SAM shows remarkable differences in the surface coverage, which strongly depends on the surface crystallography enabling estimates of the thiol exchange energetics. In addition, these electrode surfaces enable studies of molecular adsorption onto the symmetry related chiral surfaces since more than one stereographic triangle can be imaged at the same time. The ability to observe a SAM modified surface that contains many complete fcc stereographic triangles will facilitate the study of the single and multicomponent SAMs, identifying interesting surfaces for further analysis.

Electrochemistry of a ferrocene-grafted cell-penetrating peptide

A cationic cell-penetrating peptide (CPP) labeled with both a ferrocenyl (Fc) moiety and a biotin (B) was successfully synthesized and investigated by electrochemistry. This original CPP derivative noted as Fc-CPP-B could be electrochemically detected, at a micromolar concentration, at a naked gold bead electrode. The presence of a biotin tag in the Fc-CPP-B complex allowed its complexation with avidin, which was itself tethered to a thiolated self-assembled monolayer. Such an avidin-modified gold surface, characterized by atomic force microscopy (AFM), allowed the immobilization of Fc-CPP-B onto the electrode surface, which greatly enhanced its electrochemical detection. Nevertheless, under these conditions the electrogenerated ferrocenium cation could not be reduced during the backward scan, indicating its unexpected reactivity when tethered within the avidin environment. In terms of detection and redox probe regeneration the best results were obtained at a glassy carbon electrode modified with a cation-exchange polymer. Ion-exchange voltammetry, performed under these conditions, allowed the pre-concentration of the peptide at the electrode surface thanks to the net positive charge of the CPP derivative. Interestingly, the anionic character of the polymer contributed to retain the electrogenerated cation Fc+ in the film so that it could be reduced back to its original neutral form during the reverse voltammetric scans.

Electrochemical fabrication of a heterogeneous binary SAM on polycrystalline Au

An electrochemical method for generating a phase-segregated mixed monolayer of 2-aminoethanethiol (AET) and 11-mercaptoundecanoic acid (MUA) on a polycrystalline gold bead electrode is presented. Electric potential was used to disrupt a monolayer of AET on Au through numerous cycles of reductive desorption followed by incomplete readsorption. Cyclic voltammetry and differential capacitance were used to monitor the successive removal of AET from the metal with potential cycling; however, capacitance was determined to be a more sensitive measure of fractional coverage. Using differential capacitance as a probe, we determined the most efficient method to make the Au surface completely void of AET involved a potential step to a significantly negative potential for a holding time of 15 min. Using this optimal condition, the negative potential was restricted to limit desorption of AET from only the (1 1 1) sites of the polycrystalline metal. The exposed Au(1 1 1) regions were backfilled with MUA through a separate incubation resulting in the formation of the heterogeneous mixed monolayer with limited thiol exchange of the residual AET for the incoming MUA.

Supramolecular Incorporation of Fullerenes on Gold Surfaces: Comparison of C60 Incorporation by Self-Assembled Monolayers of Different Calix[n]arene (n = 4, 6, 8) Derivatives

[reaction: see text] Two new calix[6]arene derivatives 3 and 4 in a 1,4-anti conformation and one calix[8]arene derivative 5 were synthesized. SAMs of calix[n]arene (n = 4, 6, 8) derivatives 1-5 were formed on gold bead electrodes. Cyclic voltammetry with Ru(NH3)6(3+/2+) as a redox probe, together with impedance spectroscopy and reductive desorption, indicates that SAMs of 5 have a higher coverage than those of 3 and 4 due to the presence of hydrogen bonding and possibly its conformation. Noncovalent immobilization of C60 on gold surfaces was achieved with SAMs of calix[8]arene derivative 5 but not with those of 1-4.

Preparation, Characterization, and Electrocatalytic Activity of Surface Anchored, Prussian Blue Containing Starburst PAMAM Dendrimers on Gold Electrodes

Gold bead electrodes were modified with submonolayers of 3-mercaptopropionic acid or 2-aminoethanethiol and further reacted with poly(amidoamine) (PAMAM) dendrimers (generation 4.0 and 3.5, respectively) to obtain films on which Prussian Blue (PB) was later absorbed to afford mixed and stable electrocatalytic layers. Experiments carried out with these novel materials not only showed an improved surface coverage of PB on the dendrimer modified electrodes as compared to PB modified gold electrodes prepared under acidic conditions, but also showed an increased stability at neutral pH values for one of the dendrimer containing substrates where the PB film on a bare gold electrode is simply not formed. The dendrimer modified electrodes were also tested as electrocatalytic substrates for the electroxidation of L(+)-ascorbic acid (AA), and it was found that their sensitivity as well as the corresponding detection limits were improved as compared to the voltammetric response of a Au-PB modified electrode. On the basis of UV-visible (UV-vis) spectroscopy and electrochemical experiments, it is suggested that the PB molecules are located within the dendritic structure of the surface attached PAMAM dendrimers.

Atomic Force Microscopic and Electrochemical Investigations of an Electrostatically Fabricated Single‐Wall Carbon Nanotubes Modified Electrode

AbstractSingle‐wall carbon nanotubes (SWNTs) sub‐monolayer film has been prepared by simply electrostatically adsorbing nanotubes onto a 2‐aminoethanethiol self‐assembled monolayer (SAM) on a gold bead electrode. Tapping‐mode atomic force microscopy (TM‐AFM) is used to characterize the SWNT film, which exhibits that the orientation of SWNTs on the SAM is horizontal and the surface coverage is quite low. The SWNTs modified electrode shows nearly ideal electrochemical response to Fe(CN)$\rm{ {_{6}^{3-}}}$/Fe(CN)$\rm{ {_{6}^{4-}}}$ redox probe. The electrode with such a low SWNTs coverage, however, shows good electrocatalytic behavior to cytochrome c.