Secondary Structural Changes in Proteins as a Result of Electroadsorption at Aqueous-Organogel Interfaces.

The electroadsorption of proteins at aqueous-organic interfaces offers the possibility to examine protein structural rearrangements upon interaction with lipophilic phases, without modifying the bulk protein or relying on a solid support. The aqueous-organic interface has already provided a simple means of electrochemical protein detection, often involving adsorption and ion complexation; however, little is yet known about the protein structure at these electrified interfaces. This work focuses on the interaction between proteins and an electrified aqueous-organic interface via controlled protein electroadsorption. Four proteins known to be electroactive at such interfaces were studied: lysozyme, myoglobin, cytochrome c, and hemoglobin. Following controlled protein electroadsorption onto the interface, ex situ structural characterization of the proteins by FTIR spectroscopy was undertaken, focusing on secondary structural traits within the amide I band. The structural variations observed included unfolding to form aggregated antiparallel ß-sheets, where the rearrangement was specifically dependent on the interaction with the organic phase. This was supported by MALDI ToF MS measurements, which showed the formation of protein-anion complexes for three of these proteins, and molecular dynamic simulations, which modeled the structure of lysozyme at an aqueous-organic interface. On the basis of these findings, the modulation of protein secondary structure by interfacial electrochemistry opens up unique prospects to selectively modify proteins.

Electrochemical Behavior and Detection of Sulfated Sucrose at a Liquid|Organogel Microinterface Array.

The electrochemical behavior and detection of sulfated carbohydrates were investigated at an array of microinterfaces between two immiscible electrolyte solutions where the organic phase was gelled. It was found that the electrochemical signal was dependent on the organic phase electrolyte cation. Cyclic voltammetry (CV) of sucrose octasulfate (SOS) with bis(triphenylphosphoranylidene)ammonium BTPPA+ as the organic phase cation did not provide a response to a 10 µM SOS concentration. However, when the organic phase cation was tetradodecylammonium TDDA+, a distinct peak was present in the CV at ca. -0.47 V, indicative of a desorption process following adsorption during the preceding scan. This detection peak shifted to ca. -0.28 V when tridodecylmethylammonium TDMA+ was the organic phase cation, indicating an increased binding strength between this alkylammonium cation and SOS. By combining electroadsorption with TDMA+ as the organic phase electrolyte cation, detection limits of 0.064 µM SOS in 10 mM LiCl and 0.16 µM in a synthetic urine aqueous phase were achieved. The detection limit was improved to 0.036 µM SOS (10 mM LiCl) when the electroadsorption time was increased to 180 s, indicating the analytical capability for the detection of SOS and related sugars by ion-transfer adsorptive stripping voltammetry.

Electroactivity of Aptamer at Soft Microinterface Arrays.

The electrochemical behavior of a synthetic oligonucleotide, thrombin-binding aptamer (TBA, 15-mer), was explored at a liquid-organogel microinterface array. TBA did not display any response when only background electrolytes were present in both phases. On the basis of literature reports that surfactants can influence nucleic acid detection, the response in the presence of cetyltrimethylammonium (CTA+) was examined. With both TBA and CTA+ in the aqueous phase, the transfer current for CTA+ was diminished, signifying the interaction of CTA+ with TBA. Experiments with CTA+ spiked into the organic phase revealed a sharp current peak, consistent with the interfacial formation of a CTA+-TBA complex. However, use of CTA+ as the organic phase electrolyte cation, as the salt with tetrakis(4-chlorophenyl)borate, greatly improved the response to TBA. In this case, a distinctive peak response (at ca. -0.25 V) was attributed to the transfer of CTA+ across the soft interface to complex with aqueous phase TBA. Employing this process as a detection step enabled a detection limit of 0.11 µM TBA (by cyclic voltammetry). Furthermore, the presence of magnesium cations at physiological concentration resulted in the disappearance of the TBA response because of Mg2+-induced folding of TBA. Also, the current response of TBA was decreased by the addition of thrombin, indicating TBA interacted with this binding partner. Finally, the interfacial surfactant-aptamer interaction was explored in a synthetic urine matrix that afforded a detection limit of 0.29 µM TBA. These results suggest that aptamer-binding interactions can be monitored by electrochemistry at aqueous-organic interfaces and open up a new possibility for detection in aptamer-binding assays.

Electrochemical behaviour at a liquid-organogel microinterface array of fucoidan extracted from algae.

Fucoidans are sulfated polysaccharides mostly derived from algae and used in a number of applications (e.g. nutrition, cosmetics, pharmaceuticals and biomaterials). In this study, the electrochemical behaviour of fucoidans extracted from two algal species (Undaria pinnatifida and Fucus vesiculosus) was assessed using voltammetry at an array of micro-interfaces formed between two immiscible electrolyte solutions (µITIES) in which the organic electrolyte phase was gelled. Cyclic voltammetry revealed an adsorption process when scanning to negative potentials, followed by a desorption peak at ca. -0.50 V on the reverse scan, indicating the electroactivity of both fucoidans. U. pinnatifida fucoidan showed a more intense voltammetric signal compared to F. vesiculosus fucoidan. In addition, use of tridodecylmethylammonium (TDMA+) or tetradodecylammonium (TDDA+) as the organic phase electrolyte cation provided improved detection of both fucoidans relative to the use of bis(triphenylphosphoranylidene)ammonium (BTPPA+) cation. Application of adsorptive stripping voltammetry provided a linear response of current with fucoidan concentration in the range 2-20 µg mL-1 for U. pinnatifida fucoidan (with TDMA+) and 10-100 µg mL-1 for F. vesiculosus fucoidan (with TDDA+). The combination of TDMA+ in the organic phase and adsorptive pre-concentration for 180 s afforded a detection limit of 1.8 µg mL-1 fucoidan (U. pinnatifida) in aqueous phase of 10 mM NaOH and 2.3 µg mL-1 in synthetic urine (pH adjusted). These investigations demonstrate the electroactivity of fucoidans at the µITIES array and provide scope for their detection at low µg mL-1 concentrations using this approach.

Recent advancements in open-tubular liquid chromatography and capillary electrochromatography during 2014-2018.

This review critically discusses the developments on open-tubular liquid chromatography (OT-LC) and open-tubular capillary electrochromatography (OT-CEC) during 2014-2018. An appropriate Scopus search revealed 5 reviews, 4 theoretical papers on open-tubular format chromatography, 29 OT-LC articles, 68 OT-CEC articles and 4 OT-LC/OT-CEC articles, indicating a sustained interest in these areas. The open-tubular format typically uses a capillary column with inner walls that are coated with an ample layer or coating of solid stationary phase material. The ratio between the capillary internal diameter and coating thickness (CID/CT) is ideally ≤ 100 for appropriate chromatographic retention. We, therefore, approximated the CID/CT ratios and found that 22 OT-LC papers have CID/CT ratios ≤100. The other 7 OT-LC papers have CID/CT ratio >100 but have clearly demonstrated chromatographic retention. These 29 papers utilised reversed phase or ion exchange mechanisms using known or innovative solid stationary phase materials (e.g. metal organic frameworks), stationary pseudophases from ionic surfactants or porous supports. On the other hand, we found that 68 OT-CEC papers, 7 OT-LC papers and 4 OT-LC & OT-CEC papers have CID/CT ratios >100. Notably, 44 papers (42 OT-CEC and 2 OT-LC & OT-CEC) did not report the retention factor and/or effective electrophoretic mobility of analytes. Considering all covered papers, the most popular activity was on the development of new chromatographic materials as coatings. However, we encourage OT-CEC researchers to not only characterise changes in the electroosmotic flow but also verify the interaction of the analytes with the coating. In addition, the articles reported were largely driven by stationary phase or support development and not by practical applications.

Investigation of a solvent-cast organogel to form a liquid-gel microinterface array for electrochemical detection of lysozyme.

Ion transfer at aqueous-organogel interfaces enables the non-redox detection of ions and ionisable species by voltammetry. In this study, a non-thermal method for preparation of an organogel was employed and used for the detection of hen-egg-white-lysozyme (HEWL) via adsorptive stripping voltammetry at an array of aqueous-organogel microinterfaces. Tetrahydrofuran solvent casting was employed to prepare the organogel mixture, hence removing the need for heating of the solution to be gelled, as used in previous studies. Cyclic voltammetry of HEWL at the microinterface array revealed a broad adsorption process on the forward scan, at positive applied potentials, followed by a desorption peak at ca. 0.68 V, indicating the detection of HEWL in this region. Application of an adsorption step, where a constant optimized potential of 0.95 V was applied, followed by voltammetric detection provided for a linear response range of 0.02-0.84 µM and a detection limit of 0.030 µM for 300 s adsorption. The detection limit was further improved by utilizing differential pulse stripping voltammetry, resulting in detection limits of 0.017 µM, 0.014 µM, and 0.010 µM for adsorptive pre-concentration times of 60, 120 and 300 s, respectively, in unstirred solutions. These results are an improvement over other methods for the detection of HEWL at aqueous-organic interfaces and offers a basis for the label-free detection of protein.