Electrochemically assisted fabrication of size-exclusion films of organically modified silica and application to the voltammetry of phospholipids

Abstract

Modification of electrodes with nm-scale organically modified silica films with pores diameters controlled at 10- and 50-nm is described. An oxidation catalyst, mixed-valence ruthenium oxide with cyano crosslinks or gold nanoparticles protected by dirhodium-substituted phosophomolybdate (AuNP-Rh₂PMo₁₁), was immobilized in the pores. These systems comprise size-exclusion films at which the biological compounds, phosphatidylcholine and cardiolipin, were electrocatalytically oxidized without interference from surface-active concomitants such as bovine serum albumin. 10-nm pores were obtained by adding generation-4 poly(amidoamine) dendrimer, G4-PAMAM, to a (CH₃)₃SiOCH₃ sol. 50-nm pores were obtained by modifying a glassy carbon electrode (GC) with a sub-monolayer film of aminopropyltriethoxylsilane, attaching 50-nm diameter poly(styrene sulfonate), PSS, spheres to the protonated amine, transferring this electrode to a (CH₃)₃SiOCH₃ sol, and electrochemically generating hydronium at uncoated GC sites, which catalyzed ormosil growth around the PSS. Voltammetry of Fe(CN)₆³⁻ and Ru(NH₃)₆³⁺ demonstrated the absence of residual charge after removal of the templating agents. With the 50-nm system, the pore structure was sufficiently defined to use layer-by-layer electrostatic assembly of AuNP-Rh₂PMo₁₁ therein. Flow injection amperometry of phosphatidylcholine and cardiolipin demonstrated analytical utility of these electrodes.

Introduction

Deposition of films of sol-gels has been established as a means of modifying the properties of electrodes, as described in recent reviews [1–5]. The general structure of the sol-gel phase of the film is determined by whether acid or base catalysis of the processing is employed. Acid catalysis yields a microporous sol-gel, whereas base catalysis results in a mesoporous structure [6]. Spin- and dip-coating are common methods of modifying electrodes with sol-gel films. Mandler and coworkers described an electrochemically assisted deposition method for the formation of thin films of sol-gels, including organically modified silica (ormosils), on electrodes that provides greater control of film thickness than previously employed procedures [7–9]. This approach, upon which the present study is based, uses a weakly buffered R_nSi(OR)_4-n precursor. Film formation is promoted by either reduction or oxidation of the supporting electrolyte that generated hydroxide or hydronium ion, respectively, thereby catalyzing sol-gel processing at the electrode surface [7–10]. Uniform, nanometer-scale films are obtained by slowly withdrawing the electrode from the solution while continuing to apply potential.

A second level of control of the structure of sol-gel materials is obtained by the use of a templating agent. An early example was the inclusion of dopamine in a spin-coated film derived from tetramethoxysilane (TMOS) and phenyltrimethoxysilane [11,12]. Dopamine was leached from the dried film on the electrode. Voltammetric measurements at the resulting modified electrode showed greater current for dopamine in solution than for molecules of similar or larger structure and for anionic species [11]. Mesoporous sol-gels are formed when the processing is performed in the presence of surfactants under conditions where micelles are present [13–16]. By combining electrochemical generation of hydroxide to catalyze sol-gel processing at an electrode surface, formation of micelles with surfactant in the precursor solution, and orientation of the micelles in the electric field at the electrode surface, mesopores normal to the electrode surface in a silica sol-gel film were obtained [17–19]. Voltammetric measurements showed that facile mass transport of redox probes in a liquid electrolyte across such mesoporous films was achieved. This approach to sol-gel film fabrication, termed electro-assisted self-assembly, yielded an organized pattern of closely spaced mesopores [19].

Mesoporosity also was achieved in sol-gel processing by inclusion of agents other than surfactants in the precursor sol for film deposition by the electrochemically assisted process. One approach was to include β-cyclodextrin in the sol [20,21] and deposit the film by the electrochemically assisted processing method introduced by Mandler and coworkers [7–9]. The selection of β-cyclodextrin as an additive to the tetraethoxysilane (TEOS) precursor was because it stacks into nanotubes that orient perpendicular to the surface of an electrode in the presence of an electric field [22]. The mesopores (diameter, ca. 0.6 nm) that were hypothesized to be normal to the electrode surface were dispersed sufficiently in the silica film to yield voltammetric currents that were limited by hemispherical diffusion [20]. The total pore area was ca. 50% of the geometric area of the base electrode. When the pores are densely packed as in the case of electro-assisted self-assembly, diffusion fields at the film-bulk solution interface overlap so that voltammetric currents will approach linear diffusion control. The pore size in an analogous sol-gel film was increased from 0.6 nm to 10 nm by using generation-4 poly(amidoamine) dendrimer, G4-PAMAM, rather than β-cyclodextrin as the additive [23,24]. The hypothesized model was that adsorption of G4-PAMAM on the electrode, which is known to occur in the form of highly dispersed islands with a density that is a function of concentration [25], will block the electrochemically assisted sol-gel formation on the electrode at those sites, thereby leading to pore formation with the approximate diameter of the G4-PAMAM. That the prediction about the size is valid was supported by electron microscopy data [23]; however, the geometry of the pore network was not determined.

The range of pore size in sol-gels was increased further by inclusion of poly(styrene sulfonate), PSS, nanospheres in inorganic and organically modified sol-gel films. For example, 500-nm PSS spheres were mixed with tetramethoxysilane (TMOS), and a film was formed on a glassy carbon electrode by spin-coating [26]. The PSS was dissolved with chloroform to yield a film with cavities that were open at both the top (ca. 200–220 nm dia.) and bottom. By diluting the TMOS, the thickness of the film was decreased, which was accompanied by exposing more of the PSS spheres. Variation of the size of the PSS in a TEOS, dimethyldiethoxysilane mixed sol resulted in spin-coated films on glassy carbon electrodes with a range of pore diameter (after dissolving the PSS) that reflected the PSS size [27]. A variation of this procedure resulted in free-standing, nanoporous films [28]. Here, the spin-coated sol-gel with PSS as an additive was on a glass support coated by a polystyrene layer so that upon dissolution in chloroform, the template sol-gel was released as a film. Electrochemically assisted deposition from a TEOS, cetyltrimethylammonium bromide mixture (CTAB) [17] on indium tin oxide (ITO) pretreated by immersion in a suspension of ca. 100-nm dia. PSS beads resulted in a templated sol-gel film [29]. The ITO was coated with a 600–1000 nm layer of the PSS beads in this method. The PSS and CTAB were removed thermally at 550 °C to yield a mesoporous sol-gel film (related to the CTAB template) that contained macropores templated by the PSS multilayer. Zhao and Collinson [30] developed templates comprising chemically coupled PSS spheres to achieve moieties with a difference in core and perimeter dimension, e.g. 1200 nm core and 29 nm satellite structure. They were applied to providing an advanced pore structure in sol-gels as well as a surface structure around which a gold film was electrochemically deposited. After removal of the template, a high density of well-defined voids was produced.

The use of sol-gel films with templated porosity for electroanalytical measurements was extended in three ways in the present study. First, in contrast to procedures that yield a dense packing of pores, a method that results in dispersed 50-nm pores was developed to complement our previously reported method for obtaining a dispersion of 10-nm pores. As in the reports by Collinson and coworkers [26–28,30] and Etienne et al. [29], PSS was employed as a templating agent, but in the present case, it was used at sub-monolayer coverage on the base electrode and not included in the precursor sol. Such coverage was obtained by modifying the base electrode with a sub-monolayer of aminopropyltriethoxysilane and electrostatically attaching the PSS to protonated amine sites. Deposition of the sol-gel between these islands was by electrochemically assisted processing [7–9]. Subsequent removal of PSS results in the open pores. Second, these films, which are intended to provide size-exclusion electrochemical response in voltammetry, were modified by electrosynthesis or by assembly of oxidation catalysts in the pore structure. Of particular interest was the use of layer-by-layer electrostatic assembly [31] to modify the PSS-templated, 50-nm pores. Third, the resulting electrocatalytic, size-exclusion films were tested on the oxidation by cyclic voltammetry and flow injection amperometry of two phospholipids, phosphatidylcholine and cardiolipin. The first of these three points has been discussed above.

The second of these points involved deposition of a catalyst at the base of the pores or immobilization of a catalyst with the layer-by-layer (LbL) electrostatic assembly method. The LbL method, which was first described by Decher [31], has been employed in electrochemistry for such diverse applications as energy-related catalysis and sensor design. Review papers suggest that applications to biological sensing have been of particular interest [32,33]. For electrocatalytic applications, polyoxometalates were among the first to be immobilized on electrodes with the LbL approach [34–36]. With this method, multifunctionality was achieved by appropriate selection of the components [34,36], an outcome that was aided by the intercalation of the layers that characterize such assemblies [31]. In this study, dirhodium-substituted phosphomolybdic acid that was adsorbed to gold nanoparticles, AuNP-Rh₂PMo₁₁, was the test catalyst [37]. The Rh^II,III couple mediates the oxidation of such species as phosphatidylcholine and biological compounds with thiol or disulfide functionalities [20,38]. When Rh₂PMo₁₁ was used as a protecting group on AuNPs, there was evidence of synergism in the mediation of the oxidation of compounds such as methionine [37]. Combining the LbL method with sol-gel processing has been reported [39–41], but in these studies a continuous film on an electrode was deposited. As discussed above, the targeted design in this study is a sufficient dispersion of the pores in an otherwise non-electroactive film to provide non-overlapping diffusion fields at the interface of the film and the contacting liquid sample phase. In addition to LbL assembly, electrosynthesis of a catalyst at the base of the pores was studied. Here, mixed-valence ruthenium oxide that is stabilized by cyano crosslinks, RuOx [42] was investigated in that this catalyst is known to promote the electrochemical oxidation of biological compounds [43–45].

The third objective was a proof-of-concept that these electrocatalytic size-exclusion films are potentially applicable to the electrochemical oxidation and determination of phospholipids, particularly cardiolipin, in biological matrices. A requirement to meet this objective was a demonstration that electrochemistry at these films was not perturbed by adsorption of concomitants representative of potential interferents in biological samples. Moreover, it was investigated whether these films contained residual ionic sites, which can arise from incomplete removal of the templating agents and/or from silanol, in that such sites can cause departure from diffusion-control as the current-limiting process, which is the desired mode to achieve linear calibration curves with a wide dynamic range.

Experimental

Materials

The precursors to the formation of organically modified silica (ormosil) films were the following: methoxytrimethylsilane (99%), dimethoxydimethylsilane (95%), and trimethoxymethylsilane, (98%). They were obtained from Sigma-Aldrich (Milwaukee, WI). The 2-propanol (99.5%) was from Alfa Aesar (Ward Hill, MA). Bovine serum albumin (BSA, 98%), L-cysteine (98%), and aminopropyltriethoxysilane (99%) were obtained from Sigma-Aldrich (St. Louis, MO). L-α- phosphatidylcholine (bovine heart) powder and cardiolipin (bovine heart, sodium salt) powder were obtained from Avanti Lipids (Alabaster, AL). An aqueous suspension of 2.5% solids (w/v) poly(styrene sulfonate) spheres with a 50-nm mean diameter was obtained from Polysciences, Inc (Warrington, PA). Other chemicals were Reagent Grade. Water used in this study was house-distilled that was further purified with Barnstead NANO pure II system. The glassy carbon (GC) electrodes were 3.0 mm diameter obtained from Bioanalytical Systems (West Lafayette, IN). The indium tin oxide (ITO) was in the form of sheets from Delta Technologies, Limited (Stillwater, MN).

The dirhodium-substituted phosphomolybdate, Rh₂PMo₁₁, was synthesized as previously reported [37,46]. In summary, a hydrothermal reaction (120 °C for 16 h in a Teflon-lined Parr acid digestion bomb) was performed on a solution prepared as follows: sufficient Li₂CO₃ was added to 10 mL of 11.3 mM phosphomolybdic acid (PMo₁₂) to yield pH 4.8, after which the pH was adjusted to 3.0 with HCl. Next, 50 mg of dirhodium acetate was added. The Rh₂PMo₁₁ precipitated upon addition of a 5:1 mole ratio of CsCl. This product was isolated by filtration, rinsed with 2:1 water : ethanol solution, and air-dried overnight. The Rh₂PMo₁₁ was immobilized on gold nanoparticles, AuNP. Here, hexanethiolate (-SR) protected AuNPs were synthesized by reduction of AuCl₄⁻ with NaBH₄ in aqueous solution in contact with hexanethiol in toluene [47]. After purification and suspension in hexane, ligand place-exchange was performed by contacting the AuNP-SRs in hexane with an aqueous solution containing Rh₂PMo₁₁ to yield AuNPRh₂PMo_11(aq) [37]. Mixed-valence ruthenium oxide crosslinked by cyano groups, RuOx, that served as an electron-transfer catalyst in some experiments was formed by voltammetry over the range, 0.5 to 1.1 V vs. Ag | AgCl in 2 mM RuCl₃, 2 mM K₄RuCl₆ with 0.5 M KCl at pH 2 as the supporting electrolyte [43]. Forty cycles at a scan rate of 50 mVs⁻¹ were applied.

Methods and equipment

Electrochemical experiments were performed with models 400 and 660B systems from CH Instruments (Austin, TX). Scanning electron microscopy (SEM) was with a Zeiss Supra 35VP FEG microscope (Oberkochen, Germany). All the samples were sputter-coated with a 20-nm layer of gold (99.9%) prior to imaging. Determination of pores size was with Image-Pro® Plus software. Infrared spectra were obtained with a Perkin Elmer 100 Series FT-IR (Shelton, CT) spectrometer operated in the ATR mode.

As in our previous studies [23,24], the sol-gel and ormosil films were deposited by a method adapted from reports by Mandler and coworkers [7–9]. A positive potential was used to generate hydronium ion as the catalyst in the electrochemically assisted processing so that the solid phases were microporous, and templating agents were used to control the pore structure [23,24]. Typical conditions used to deposit an ormosil film were as follows. A solution was prepared by combining 5 mL of 0.1 M lithium perchlorate in 2-propanol, 2.5 mL of H₂O, and 175 µL of G4-PAMAM. The pH of the solution was adjusted to 5.0 with 1 M HCl, after which 2.0 mL of methoxytrimethylsilane was added. After 30 min, a GC electrode was immersed, and a potential of 1.5 V was applied for an additional 30 min, during which the solution was stirred. The GC electrode was slowly withdrawn from the solution; any excess sol was removed by capillary action onto a tissue contacting the insulator surrounding the electrode (safety note: as (CH₃)₃SiOCH₃ is a potential eye hazard, these manipulations should be performed in a hood). After air-drying overnight, the G4-PAMAM was dissolved with 99% purity methanol for 1 h. From results under analogous conditions [23], the films were estimated as 70 – 100 nm thickness, which depends on deposition time and the deposition potential.

Fabrication of an ormosil film that had 50-nm pores was performed in a stepwise manner. First, GC was modified with sub-monolayer coverage of aminopropyltriethoxysilane (APTES) by immersion in a 0.5 mM APTES solution for 15 min. Second, after rinsing with water the GC | APTES was dipped into a 1:1 dilution of the commercial PSS suspension for 30 s. Third, after rinsing, film formation was by the above-described electrochemically assisted method except that G4-PAMAM was not included in the sol. Unless otherwise noted, the potential was applied for 30 min. Finally, the PSS was dissolved with chloroform. The deposited films were characterized by cyclic voltammetry and by scanning electron microscopy.

The cells used for the static-solution electrochemical experiments were of two types. With ITO as the working electrode, the sheet was cut into squares with 1.5 cm edges, cleaned with ethanol, and dried under nitrogen prior to use. Using a rubber O-ring, a 0.32 cm² area was isolated for use as a working electrode. A Teflon-coated steel plate with a ca. 1 cm thickness was used to provide pressure to the O-ring. The 1-cm open cylinder above the O-ring connected to a 10 cm³ Teflon cell that held the sample solution. The reference and auxiliary electrodes were in contact with this 10 cm³ portion of the sample solution. When GC was the working electrode, a conventional cell was used. Flow injection amperometry measurements were made at a thinlayer amperometric detector from Bioanalytical Systems (West Lafayette, IN). The injection size was 100 µL. All potentials were measured and reported versus an Ag | AgCl, 3M KCl reference electrode from Bioanalytical Systems (West Lafayette, IN). Platinum wire was the counter electrode. Prior to experiments all the solutions were deaerated with nitrogen gas.

Results and discussion

Fabrication and evaluation of an ormosil film with 10-nm pores on glassy carbon

Silica sol-gel films of controlled porosity on the electrode surface have a significant limitation, namely the residual negative charge on the solid phase influences the rate of transport of ionic species through the pores to the electrode surface. In our previous study, the negatively charged sites on TEOS-derived films with 10-nm pores formed with G4-PAMAM as the templating agent were eliminated by a two-step silanization that was performed after the G4-PAMAM was removed with an oxygen plasma [23]. A more direct approach is to deposit the film from an organically modified precursor, (CH₃)₃SiOCH₃, rather than TEOS. This procedural change required an alternative method for removal of the G4-PAMAM because the oxygen plasma treatment that was used previously degraded ormosils [23]. As described in the Experimental section, extraction with methanol was tested as a means of removing the dendrimer. The initial method used to evaluate whether the G4-PAMAM was removed by methanol extraction was infrared spectroscopy (Figure 1). Lines at 1653 and 1566 cm⁻¹ that are characteristic of this dendrimer were below the detection limit of the method after the extraction. Scanning electron microscopy of the surface was used to obtain an image of the film surface after this extraction step (Figure 2). The primary characteristics of the surface were that the pores were distributed rather than densely packed and the average pore size was 10 nm (diameter). As expected, the surface was the same as that obtained with a TEOS-derived sol-gel film that was prepared in the same manner [23].

Fig. 1.

Infrared spectroscopy of an ormosil film with pores templated by G4-PAMAM. (A) before removal of the dendrimer with methanol, and (B) after treating the deposit in methanol for 60 min

Fig. 2.

Scanning electron microscopy of the surface of a (CH₃)₃SiOCH₃-derived film with G4-PAMAM templated pores after dissolving the G4-PAMAM. Scale bar, 200 nm

A more sensitive method than infrared spectroscopy to test whether the extraction removed all G4-PAMAM is cyclic voltammetry of an anionic species. This test system was 0.5 mM Fe(CN)₆³⁻ in pH 6.0 phosphate buffer. Cyclic voltammetry was performed over the scan rate, v, range of 10 – 100 mVs⁻¹. Analysis of the voltammetry demonstrated that the relationship between the cathodic peak current, i_pc, and v approached the theoretical value for an electrode process limited by semi-infinite linear diffusion, i_pc ∝ v^1/2, given by the Randles - Sevcik Equation. Specifically, a plot of log i_pc vs. log v yielded a slope of 0.52 (r², 0.995). Residual G4-PAMAM, if present, would have preconcentrated Fe(CN)₆³⁻, thereby causing the cathodic current to include a contribution from surface-bound electroactive species. In such a case, the slope will be greater than 0.5 (and approach a limit of 1.0 for current exclusively from a surface-bound species).

That i_pc varied with v^1/2 in the above experiment also suggested that the ormosil fabricated from (CH₃)₃SiOCH₃ did not contain residual negative sites. To illustrate the influence of negative sites on the film, GC was modified by an identical procedure except that the precursor was Si(OCH₃)₄, TMOS, rather than (CH₃)₃SiOCH₃. Repeating the cyclic voltammetry of Fe(CN₆)³⁻ showed that the negative sites that are known to exist on TMOS-derived sol-gels (except at the isoelectric point of silica, ca. pH 2) caused i_pc to vary with v^0.3. The exponent below 0.5 is proposed to result from electrostatic repulsion between the TMOS-derived film and the Fe(CN)₆³⁻. Further evidence was obtained by using (CH₃)₂Si(OCH₃)₂ as the precursor from which the film was formed; it gives a film with a negative charge density between that of films derived from TMOS and (CH₃)₃SiOCH₃. In this case, the scan rate dependence was i_pc ∝ v^0.4. Hence, the trend in the scan rate dependence of peak current mirrors that of the negative charge density of the film. This set of experiments supports the view that there is an analogue between a kinetic (electron transfer or chemical) influence on the perturbation of linear diffusion and electrostatic repulsion.

The above interpretations were confirmed by cyclic voltammetry of a cationic species, Ru(NH₃)₆³⁺, at pH 7.2. The electrode comprised a (CH₃)₃SiOCH₃-derived film on GC with templated 10-nm pores, which is designated as GC | ormosil (10-nm). The results obtained as a function of v are shown in Figure 3. Analysis of the voltammetry in Figure 3 demonstrated that i_pc was directly proportional to v^1/2 and, therefore, limited by semi-infinite linear diffusion. Specifically, a plot of log i_pc vs. log v yielded a slope of 0.50 and r² of 0.997. Moreover, the peak current ratio, i_pc/i_pa, was 1.06, which is indicative of no chemical (including electrostatic) complication in this reversible redox process. The identical experiment, except with a TMOSderived film, yielded i_pc ∝ v^0.65, which demonstrates that some preconcentration of Ru(NH₃)₆³⁺ onto the negative sites of the TMOS-derived film occurred.

Fig. 3.

Cyclic voltammetry of 0.5 mM Ru(NH₃)₆³⁺ in pH 7.2 phosphate buffer at glassy carbon modified with the film in Figure 2. v: a → i, 10 → 100 mVs⁻¹ in 10 mVs⁻¹ increments

Fabrication and evaluation of an ormosil film with 50-nm pores on glassy carbon

Considering the objective of devising catalytic, size-exclusion films for amperometric determination of biological compounds, electrode-modifying films with pore sizes significantly greater than10 nm are needed to accommodate some common electron-transfer catalysts such as functionalized metal nanoparticles. The use of PSS to template pores in spin-coated sol-gel films on various substrates [26–28] and in a film formed by an electrochemically assisted process [30] suggested employing these nanoscale beads in the present study. These previous reports dealt with formation of densely packed pores; here, our objective was to form highly dispersed, 50-nm pores in an ormosil film deposited by an electrochemically assisted procedure [7–9]. Deposition at a positive potential was used so that the ormosil phase was microporous (pore size < 2 nm) rather than mesoporous so that the currents obtained in voltammetric and amperometric measurements were related only to transport of the electroactive species through the templated pore structure.

The PSS was immobilized on a GC electrode by ion-exchange with positive sites comprising protonated aminopropyltriethoxysilane (APTES). The conditions, which are detailed in the Experimental section, used to modify the GC with APTES were selected to yield sub11 monolayer coverage. In turn, sub-monolayer coverage by PSS resulted. The ormosil was deposited the PSS-modified surface as described in the Experimental section. After film formation, the PSS was removed by overnight immersion in chloroform. The PSS coverage was dependent upon the amount of APTES immobilized on the GC. As shown in Figure 4, varying the concentration of APTES used to modify the GC resulted in a commensurate variation of the quantity of PSS subsequently deposited, presumably because of a change in surface coverage of APTES. The mean pore diameter in the film in Figure 4 was 50 nm. This electrode was designated as GC | ormosil (50 nm). Of importance in this model is that any PSS on the GC surface results from linking it with APTES. The nearly complete absence of PSS attached to bare GC is illustrated by the SEM in Figure 5; here, the sample was prepared by dipping unmodified GC into a PSS suspension under conditions in the Experimental section.

Fig. 4.

Scanning electron microscopy of GC surfaces with PSS attached when 0.06 M (upper left), 0.03 M (upper right), 0.01 M (lower left) APTES was the modifier. Lower right: SEM of the ormosil that results from using 0.001 M APTES as the modifier. The scale bars are 200 nm

Fig. 5.

Scanning electron microscopy of a glassy carbon surface after immersion in a PSS suspension and rinsing with water. The surface was not modified with APTES in this blank experiment.

As with the GC | ormosil (10 nm) electrode, removal of the templating agent from GC | ormosil (50 nm) was verified by infrared spectroscopy and by cyclic voltammetry of ionic complexes known to have reversible, diffusion-limited behavior at bare surfaces. For example, cyclic voltammetry of 0.5 mM Ru(NH₃)₆³⁺ in 0.1 M phosphate buffer at pH 6.0 was performed over a v range of 10 – 100 mVs⁻¹. A plot of log i_pc vs. log v yielded a slope of 0.48 (r², 0.996), demonstrating that the current was limited by semi-infinite linear diffusion. The results with Fe(CN)₆³⁻ were also consistent with those in the previous sub-section.

The design criteria of these film-coated electrodes included spatial dispersion of the pores as well as control of their diameters. That the procedure developed herein yielded dispersed pores was suggested by the image of a surface in Figure 4. Additional evidence was obtained by voltammetry. A comparison of the cyclic voltammetry of ferrocene, Fc, at bare GC and GC | ormosil (50 nm) was done. The sample was 0.5 mM Fc in acetonitrile with a 0.5 M Bu₄NPF₆ supporting electrolyte. At 100 mVs⁻¹, i_pa decreased from 10.7 µA (GC) to 6.4 µA (GC | ormosil (50 nm) under conditions where analysis of i_pa vs. v demonstrated that the process was limited by semi-infinite linear diffusion in both cases; e.g., log i_pa vs. log v over the range 20–250 mVs⁻¹ had a slope of 0.47 at GC | ormosil (50 nm). Thus, the cross-section of the pores totaled about 60% of the geometric area of the electrode. It should be noted that the electrode used in this experiment was modified with 1.0 mM APTES rather than 0.5 mM APTES as was the case for the film in Figure 4, so the surface cross-section of pores in the image is not the same as the results from voltammetry.

Modification of GC | ormosil (10 nm) and GC | ormosil (50 nm) with electrochemical catalysts

Because they are known to catalyze the electrochemical oxidation of biological compounds [37,38,43–45], this study employed RuOx and AuNP-Rh₂PMo₁₁ as catalysts. The former is deposited from components with sizes at are well-below 10-nm, so it was anticipated that is can be used with both of the modified electrodes, whereas the size of the latter is on the 10-nm scale, making its incorporation into the pores formed by G4-PAMAM problematic. Initial focus was on immobilization of AuNP-Rh₂PMo₁₁. First, the presumed base of the 10-nm and 50- nm pores, i.e. the exposed GC surface, was modified by APTES by immersion in 0.06 M solution for 30 min. The electrodes were transferred for 30 min to an AuNP-Rh₂PMo₁₁ solution in 0.05 M H₂SO₄, which was prepared as described in the Experimental section. After rinsing, these electrodes were tested by cyclic voltammetry of 1.0 mM cysteine in 0.5 M KCl adjusted to pH 2.0 with HCl. At GC | ormosil (50 nm) modified with APTES and AuNP-Rh₂PMo₁₁, i_pa at 100 mVs⁻¹ was 2.9 µA (Figure 6). As a test for the current-limiting step, the parameter, i_pav^−0.5, was determined in the v – range, 10 – 100 mVs⁻¹. A constant value, indicative of i ∝ v^1/2, is evidence of a diffusion-limited process. At 1.15 V, this parameter was 2.9 µA·s^0.5·mV−^0.5, and the relative standard deviation of the 10 data points was 6%. Given that the measurement is complicated by the onset of oxidation of the supporting electrolyte, the result suggests that the oxidation of cysteine under these conditions is diffusion limited, which is the desired case for an analytical application. At the GC | ormosil (10 nm) electrode, the analogous experiment did not yield evidence of oxidation of cysteine, which is consistent with the premise that the 10-nm pores will not accommodate the AuNP-Rh₂PMo₁₁ catalyst. Perhaps more important, this negative result is evidence that APTES did not react with the ormosil. When APTES is included in the precursor sol of an electrochemically assisted film formation process, it co-condenses during the sol-gel processing [48]. In the present case, unreacted (CH₃)₃SiOCH₃, if present, could have caused incorporation of the APTES on the ormosil and led to immobilization of the AuNPRh₂PMo₁₁ onto the film. Redox of this species would have been observed in cyclic voltammetry because the pores can serve as channels for ionic conductivity, as was the case for a sol-gel film with templated 0.6-nm pores [20].

Fig. 6.

Cyclic voltammetry of 1.0 mM cysteine in 0.5 M KCl at pH 2.0 at a GC | ormosil (50 nm) electrode modified with AuNP-Rh₂PMo₁₁. Inner curve, blank at 10 mVs⁻¹; outer curves, presence of cysteine with v ranging from 10 mVs⁻¹ (lowest current) to 100 mVs⁻¹ in 10 mVs⁻¹ increments

The GC | ormosil (50 nm) electrodes are candidates for modification by macromolecules and nanoparticles using techniques such as LbL electrostatic assembly. Their pore size and, perhaps, non-tortuous geometry are both factors in this regard. Here, LbL assembly in the 50-nm pores was tested initially with phosphomolybdate, PMo₁₂, and G4-PAMAM as the components of a bilayer, a system that we previously characterized at a gold electrode [49]. With GC, it was necessary to first modify the exposed surface at the base of the pores with APTES. The GC | APTES was then immersed sequentially in 5 mM PMo₁₂ and in 1% G4-PAMAM; each step was 60 min. After immersion in PMo₁₂ the system was rinsed in 0.25 M H₂SO₄, and after G4- PAMAM, 0.1 M HCl. Additional bilayers are added by repeating these steps. Cyclic voltammetry of the immobilized PMo₁₂ is shown in Figure 7A. The current from redox of PMo₁₂ doubled when the second bilayer was attached. When applied to the voltammetry of bromate at pH 2, the peak for the second step in the reduction of PMo₁₂ increased and the reversal of this PMo₁₂ reduction at ca. 0.2 V was attenuated (Figure 7B), which is consistent with the well-known mediated reduction of bromate by this species. The results in Figure 7 illustrate the ability to use LbL assembly in CG | ormosil (50 nm). This means of immobilizing a catalyst is particularly important in applications where both bilayer components have catalytic properties or where a single layer of catalyst is not sufficient to attain a diffusion-limited electrode reaction. At the bromate concentration in Figure 7, a diffusion-limited oxidation was obtained with a single bilayer (n), so the current related to bromate concentration with n=2 was the same as that for n=1.

Fig. 7.

Cyclic voltammetry at 50 mVs⁻¹ of a layer-by-layer electrostatic assembly of PMo₁₂ | PAMAM at the base of the pores of a GC | ormosil (50 nm) electrode. A: contacting solution, 0.25 M K₂SO₄ adjusted to pH 2 with H₂SO₄; a, n = 0; b, n = 1; c, n = 2. B: n = 2; a, contacting solution same as A; b, 1.0 mM bromate in the pH 2 supporting electrolyte

Application to the oxidation of phospholipids

Two requirements for application of GC | ormosil (10 nm) and GC | ormosil (50 nm) electrodes to electroanalytical determinations of phospholipids are amelioration of interference caused by adsorption of concomitants in biological samples and promotion of the oxidation of phospholipids by the catalysts that are immobilized in the pores. The first factor addressed was whether adsorption of macromolecules is alleviated by the combination of the surface properties of the ormosil and size-exclusion by the pore design. The test redox system was Fe(CN)₆³⁻, and the possible surface-active interferents were phosphatidylcholine, PC, and bovine serum albumin, BSA. It should be noted that under the conditions of this test, PC was not electroactive. Cyclic voltammetry of 0.5 mM Fe(CN)₆³⁻ in 0.1 M phosphate buffer at pH 6 was performed in the presence and absence of 100 – 300 µM PC at a GC | ormosil (10 nm) electrode. The scan rate was 100 mVs⁻¹, and the electrode was immersed in each solution for 30 min prior to initiating the scan. In the absence of PC, i_pc was 10.8 ± 0.2 µA, and in the presence of 300 µM PC, i_pc was 10.6 ± 0.2 µA. Five replicates on each sample were used to determine the standard deviation. Analysis by the t-test showed agreement at the 95% confidence level. Moreover, statistically identical results were obtained over the 100 – 300 µM PC range. When the experiment was repeated at a bare GC electrode, adsorption of PC significantly decreased the current; the i_pc values for the reduction of 0.5 mM Fe(CN₆)³⁻ were 17.1 and 7.5 µA in the presence and absence, respectively, of 300 µM PC. The result on bare GC is consistent with the reported influence of surface active agents on voltammetry of redox probes [5, 50 and citations therein]. When 0.4% BSA was used as the test interferent with the GC | ormosil (10 nm) electrode, i_pc decreased by 10% unless the electrode was rinsed in the buffer for 5 min between trials. Also demonstrating the protection of GC by an ormosil (10-nm) film is that at bare GC the difference in peak potentials for the cyclic voltammetry of Fe(CN)₆³⁻ is 65 mV in the absence of PC and 440 mV in the presence of 300 µM PC (v, 100 mVs⁻¹; exposure time, 30 min). In contrast, the peak potential difference is not changed by the presence of PC with the GC | ormosil (10 nm) electrode. The 65 mV difference in peak potential approaches the theoretical value of 57 mV for a reversible, diffusion-limited, one-electron transfer, which indicates that the presence of the ormosil does not increase the effective resistance of the working electrode. These results support the hypothesis that the GC | ormosil (10 nm) electrode provides significant protection from adsorption of macromolecules that can passivate bare GC, but when they are present at very high concentration, e.g. 0.4% BSA, application in a flow system rather than static solution may be superior.

The second factor, incorporation of electrochemical catalysts for the oxidation of phospholipids in GC | ormosil (10 nm) and GC | ormosil (50 nm) electrodes, was tested using AuNP-Rh₂PMo₁₁ [37,38] and RuOx [43–45], as discussed in the previous section. The test compounds were PC and cardiolipin, CL. With GC | ormosil (50-nm pores), both catalysts were shown to mediate the oxidation of these phospholipids at ca. 1.0 V; however, consistent with the above results for the oxidation of cysteine, only RuOx was effective when the electrode was GC | ormosil (10-nm pores). A typical result for the oxidation of CL by cyclic voltammetry at GC | ormosil (10 nm) with RuOx as the catalyst is shown in Figure 8. The voltammetric behavior at GC | ormosil (50 nm) with RuOx as the catalyst also showed the oxidation of CL near 1.0 V.

Fig. 8.

Cyclic voltammetry of (B) cardiolipin and (A) the supporting electrolyte at a GC | ormosil (10-nm pores) electrode with RuOx as the catalyst. Supporting electrolyte: 0.25 M Na₂SO₄ at pH 2.0; v, 50 mVs⁻¹

Analytical applications of an electrode process involving mediated electron transfer are better performed by a potentiostatic measurement than by cyclic voltammetry because the baseline current from the mediator is negligible in the former case. Here, flow injection amperometry (FIA) was used as the potentiostatic method with 1.0 V as the applied potential. A comparison of results obtained for PC at GC | ormosil (50 nm) with these catalysts is shown in Figure 9. The FIA current obtained with AuNP-Rh₂PMo₁₁ as the catalyst was somewhat greater than that with RuOx; however, given that the current for the oxidation of PC was diffusion limited with both catalysts, these relative values probably are related to a somewhat greater effective surface area when the nanoparticles were employed. A possible limitation of this electrode system that was observed was a gradual loss of sensitivity. The GC | ormosil (50 nm) electrode with AuNP-Rh₂PMo₁₁ as the catalyst was evaluated on two sets of 8 consecutive injections under the conditions in Figure 9; between these data sets, the supporting electrolyte was flowed briefly. With injections on a freshly prepared electrode, the peak current was 2.34 ± 0.04 µA. The subsequent set of eight injections had a peak current of 2.20 ± 0.06 µA. At the 95% confidence level this decrease in current is statistically significant. The decrease in peak current may be related to gradual loss of the AuNP-Rh₂PMo₁₁ catalyst. In contrast, deposited RuOx films were stable in both 10-nm and 50-nm pores. Data on 12 injections of with GC | ormosil (10 nm) as the working electrode are shown in Figure 10. With the conditions in Figures 9 and 10, peaks for blank injections were not observed. The stability of RuOx was not surprising in that a single preparation has been used previously for more than one month [42].

Fig. 9.

Flow injection amperometry of phosphatidylcholine at a GC | ormosil (50-nm pores) electrode with (A) AuNP-Rh₂PMo₁₁ electrostatically bound to the base of the pore by APTES and (B) RuOx as catalysts. Electrolyte, 0.25 M Na₂SO₄ at pH 2.0; flow rate, 1.0 mL·min⁻¹; injections, 100 µL of 10 µM phosphatidylcholine; applied potential, 1.0 V

Fig. 10.

Flow injection amperometry of cardiolipin at a GC | ormosil (10-nm pores) electrode with RuOx as the catalyst. Electrolyte, 0.25 M Na₂SO₄ at pH 2.0; flow rate, 1.0 mL·min⁻¹; injections, 100 µL of 10 µM cardiolipin; applied potential, 1.0 V

These preliminary data suggest that with the GC | ormosil (50-nm) electrode both electrochemical deposition and LbL electrostatic assembly may be applicable to electroanalytical determination of biological compounds such as phospholipids, but the electrode with a pore size of 10 nm places restrictions on the size of the catalyst. The stability of the LbL assembly of AuNP-Rh₂PMo₁₁ and G4-PAMAM is questionable for extensive duty cycles. Further work with this catalytic system will focus on the use of a conducting polymer with positive sites as the counter layer in the LbL assembly.

Conclusions

Electrochemically assisted deposition of organically modified silica (ormosil) films with pore sizes determined by inclusion of G4-PAMAM (10 nm) or by immobilizing a controlled distribution of poly(styrene sulfonate) nanobeads on the surface (50 nm) was demonstrated. In the latter case, the nanobeads were attached electrostatically to surface-bound aminopropyltriethoxysilane, a procedure that results in a continuous link between the base electrode and the outer surface of the film. Whether this link is a network or follows the proposed model of non-tortuous “nanowells” upon dissolution of the beads could not be determined because of lack of cutting equipment to obtain clear images of a cross-sections. However, the 50-nm pores have characteristics that are amenable to conversion to electrocatalytic reactors using layer-by-layer electrostatic assembly of moieties such as gold nanoparticles modified with a sheath of dirhodium phosphomolybdate. By a combination of the surface properties of the ormosil and the size-exclusion of the pores, these modified electrodes resist passivation by adsorption of bovine serum albumin and phospholipids. This property in conjunction with catalysis by mixed-valence ruthenium oxide with cyano crosslinks and by the modified gold nanoparticles permitted the oxidation of phosphatidylcholine and cardiolipin in a manner useful for electroanalytical measurements. To our knowledge, the latter is the first report of the electrochemical oxidation of cardiolipin, which is a biologically important phospholipid.