Electrochemical Behavior and Detection of Sulfated Sucrose at a

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Electrochemical Behavior and Detection of Sulfated Sucrose at a Liquid#Organogel Micro-interface Array Bren Mark B. Felisilda, Alan David Payne, and Damien W.M. Arrigan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01710 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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Analytical Chemistry

Electrochemical Behavior and Detection of Sulfated Sucrose at a LiquidǀOrganogel Micro-interface Array Bren Mark B. Felisilda,1,2 Alan Payne,2 Damien W.M. Arrigan1,2,* 1

Curtin Institute for Functional Molecules and Interfaces, 2 School of Molecular and Life Sciences, Curtin University, GPO Box U1987, Perth, Western Australia, Australia 6845.

* Email [email protected] ; Tel. no: +61 08 9266 9735

ABSTRACT: Electrochemical behavior and detection of sulfated carbohydrates was investigated at an array of micro-interfaces 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 phases 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 detection of SOS and related sugars by ion-transfer adsorptive stripping voltammetry.

Polysulfated carbohydrates are considered to be pharmaceutically important substances because of their known biological activity.1,2 A good example of these substances are glycosaminoglycans (GAGs) which manage several biological processes by interacting with their protein binding counterparts via the latter’s basic amino acid residues.1,3 Moreover, these highly sulfated and mostly negatively charged polysaccharides are major constituents in extracellular matrices of several tissues but they are also located inside as well as on the surfaces of cells.2 A number of these polysulfated carbohydrates are known for their biological activities. Specifically, heparan sulfate is known for signal transduction4 while heparin is used to prevent blood coagulation.5 Synthetic counterparts are also known to be useful in the pharmaceutical industry as excipients or drug products.6,7 One of these synthetic sulfated carbohydrates is sucrose octasulfate (SOS). A form of SOS, specifically its aluminium salt, is famously called Sucralfate and is commonly utilized as treatment for duodenal ulcer.8 The interest in SOS can be traced from the proposition that it can promote wound healing9 by its role in the stabilization of fibroblast growth factor (FGF). Additional studies report the application of SOS and its analogues in cancer treatment and wound healing due to the crystallization of its sodium salt within the signal transduction pathway10 as well as its moderate oral bioavailability.6 Conformational changes to proteins were also found to be induced by SOS and sulfated GAGs.11 Given the growing application of SOS and related sulfated carbohydrates in the field of pharmaceuticals as well as their biological importance, a label-free detection method for their measurement would be useful. Current methods used to analyse these sulfated carbohydrates include mass spectrometry,12,13 enzymatic digestion or depolymerization

followed by capillary electrophoresis,14,15 liquid chromatography16 with UV-Vis/fluorescence detection,17,18 and nuclear magnetic resonance spectrometry.19,20 Electro-spray ionization mass spectrometry (ESI-MS) was utilized to survey different counterions and see their effect on the fragmentation of SOS.21 Gunay and co-workers21 reported that quaternary ammonium ions gave good ESI-MS spectra. More recently, Ke and colleagues22 reported a novel liquid chromatography tandem mass spectrometry method to detect SOS in dog plasma and urine samples, by utilising diethylammonium (DEA) to form a stable adduct with SOS that aided its detection to low levels. Electrochemical methods offer prospects for fast, low cost and sensitive detection. For example, potentiometric ion selective electrodes (ISEs) based on polymer membranes containing ion exchangers such as tridodecylmethylammonium (TDMA+) were used to probe substances such as pentosan polysulfate,23 heparin,24,25 and carrageenan.26 In a recent work by Kim and co-workers,27 an ISE was employed to study species of fucoidan, a sulfated polysaccharide derived from brown seaweeds. With the increasing interest in electrochemical methods, the growing field of electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) has also become the basis for novel analytical strategies.28,29 Advantages such as amenability to miniaturisation30 and possibility for label-free detection can be harnessed, leading to the investigation of substances of biological importance including proteins,31,32 neurotransmitters33,34 and carbohydrates.35,36 Several polysaccharides have been the subject of studies utilizing electrochemistry at the ITIES, as exemplified by the detection of heparin.36-39 Reported findings suggest that binding with an ionophore,36 which can also be the organic electrolyte cation used,37 affects the adsorption of heparin at the interface. Guo

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and colleagues38 investigated a number of hydrophobic quaternary ammonium cations that served as selective ionophores for heparin. The results revealed that adsorption of heparin was assisted by complexation with the aforementioned cations. In this work, the electrochemistry of simpler sulfated carbohydrates at an array of microscale ITIES (µITIES array) is reported, and behavior that can be a viable alternative method for their detection is investigated. The analytes studied here SOS, sucrose heptasulfate (SHpS) and sucrose hexasulfate (SHxS), employing an experimental platform of a liquidorganogel microinterface array via voltammetric techniques. The findings indicate that counterion interactions between the analytes and organic phase cations enable the detection of these sulfated sugars. In this way, a detection limit of 0.064 µM SOS following 60 s pre-concentration and 0.036 µM following 180 s, in 10 mM LiCl, was achieved, while detection of 0.16 µM SOS was achieved in a synthetic urine electrolyte mixture.

phase was placed into the silicon micropore array membrane using a pre-warmed glass Pasteur pipette. The assembly was then set aside for at least an hour before use. Once ready, the organic reference solution (saturated BTPPACl or TDDACl or TDMACl in 10 mM LiCl) was introduced into the glass cylinder on top of the organogel. The set-up was then inserted into the aqueous phase and voltammetric measurements were made via a pair of Ag/AgCl electrodes with the cells described in Scheme 1.

EXPERIMENTAL SECTION Reagents. All reagents were purchased from Sigma-Aldrich Australia and used as received unless otherwise stated. The organic electrolyte salts bis(triphenylphosphoranylidene)ammonium tetrakis(4chlorophenyl)borate (BTPPATPBCl) and tridodecylmethylammonium tetrakis(4-chlorophenyl)borate (TDMATPBCl) were prepared by metathesis of equimolar amounts of bis(triphenylphosphoranylidene)ammonium chloride (BTPPACl) and potassium tetrakis(4-chloropheynl)borate (KTPBCl) and tridodecylmethylammonium chloride (TDMACl) and KTPBCl, respectively. Tetradodecylammonium tetrakis(4-chlorophenyl)borate TDDATPBCl was commercially available and used as received. The organic phase comprised the desired organic electrolyte salt (10 mM) in 1,6dichlorohexane (DCH) and was gelled by the addition of low molecular weight poly(vinyl chloride) (PVC) at 10%, to provide mechanical stability.40 Sucrose octasulfate (SOS) was purchased from Toronto Research Chemicals Inc. while sucrose heptasulfate (SHpS) and sucrose hexasulfate (SHxS) were obtained from Santa Cruz Biotechnology, Inc. Stock solutions of these sulfated carbohydrates were prepared weekly in aqueous 10 mM LiCl and stored at 4 °C. Similarly, tetrapropylammonium (TPrA+) chloride was prepared in 10 mM LiCl. Sample matrix effects were tested using a synthetic urine mixture41 that contained creatinine (1.10 g L-1), urea (25 g L-1), sodium sulfate (2.25 g L-1), sodium chloride (2.295 g L1 ), potassium dihydrogen phosphate (1.40 g L-1), potassium chloride (1.60 g L-1), calcium chloride dihydrate (1.103 g L-1) and ammonium chloride (1.00 g L-1). All aqueous solutions were prepared using purified water (resistivity: 18.2 MΩcm) from a USF Purelab with UV system. Apparatus. Electrochemical measurements were conducted using an AUTOLAB PGSTAT302N electrochemical workstation (Metrohm, The Netherlands) operated with the supplied NOVA software. The µITIES array employed was created with a thirty micropore array silicon membrane42 in a hexagonal arrangement. Each pore had a diameter of 22.4 μm, a pore centre-to-pore centre distance of 200 μm. The membrane thickness was 100 μm. These parameters create a total geometric area of 1.18 x 10-4 cm2. The silicon microporous membrane was sealed, using a silicone rubber (Selley’s glass silicone) onto the mouth of a glass cylinder. The gelled organic

Scheme 1. Schematic representation of the electrochemical cells employed, where x represents the sulfated carbohydrate (e.g. SOS) concentrations utilized in the study.

Electrochemical Measurements. Voltammetric investigations were conducted at a sweep rate of 5 mV s-1 unless otherwise stated. Parameters such as analyte concentration (sulfated carbohydrates), potential applied and length of preconcentration time were varied accordingly. The reported detection limits were calculated based on three times the standard deviation of the blank (n=3) divided by the slope of the best-fit linear calibration line. Since the calibration curves were curvilinear, the linear best-fit slope was taken from the four (4) lower concentrations. Potentials reported were transposed to the Galvani potential scale based on the experimental TPrA+ mid-point transfer potential along with its formal transfer potential -0.08 V in the water │DCH system.43 A positive current indicates transfer of cation from aqueous to organic phase (or anion from organic to aqueous phase44.

RESULTS AND DISCUSSION Cyclic Voltammetry. Cyclic voltammetry (CV) was used to examine the behavior of sulfated sucrose at the µITIES array. Initially, different aqueous pH values were screened, with best results obtained in pH 5.5-6 10 mM LiCl. Figure 1(black solid line) illustrates the CV obtained when 10 μM SOS was present in the aqueous phase (Cell 1, Scheme 1). As observed, there was no distinct peak or response different from the CV recorded in the absence of SOS. This indicates that SOS at this concentration was not detected under these conditions. The currents at the negative and positive potential limits of this scan are due to the transfers of background electrolyte ions: the transfer of the cation (BTPPA+) from the organic to the aqueous phase and the anion (Cl-) from the aqueous to the organic phase at the negative potential limit, and the transfer


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Analytical Chemistry of cation (Li+) from aqueous to organic and anion (TPBCl- ) from organic to aqueous phases at the positive limit.29 Based on previous findings45 that the type of organic electrolyte cation impacts the detection of polysulfated analytes, two other organic electrolyte cations were examined to evaluate whether they enabled the detection of SOS at micromolar concentrations. Figure 1 illustrates the CVs recorded when tetradodecylammonium (grey dashed line, TDDA+) and tridodecylmethylammonium (grey solid line, TDMA+), respectively, were used as the organic phase cations together with 10 µM SOS in the aqueous phase.

Figure 1. CV of 10 mM LiCl in the presence of 10 µM sucrose octasulfate using (black solid line) Cell 1, (grey dashed line) Cell 2, and (grey solid line) Cell 3 (Scheme 1).

Utilizing these different alkylammonium cations in the organic phase extended the working potential window range on the negative side. This is due to the characteristic transfer of TDDA+ at a more negative potential46 than BTPPA+, while TDMA+ is assumed to behave similarly to TDDA+. One prime distinction that can be noted in the voltammograms is the difference in the intensity of the SOS response in the presence of TDDA+ (Figure 1, grey dashed line) and TDMA+ (Figure 1, grey solid line) relative to BTPPA+ (Figure 1, black solid line). When 10 µM SOS was present in the aqueous phase, the distinct peaks present (Figure 1, grey dashed and grey solid lines) on the reverse scans indicate that SOS is electrochemically active at the µITIES array. Other polysulfated biomolecules, specifically the oligomeric/polymeric substances heparin37 and fucoidan,45 have similar responses. The peak response on the reverse scan is representative of a desorption process47 which features a rapid decline in current to background levels and represents the depletion of a finite amount of material at the interface. Conversely, an electroadsorption must take place on the forward scan, as illustrated by the increased currents on the forward scans in Figures 1 (grey dashed and grey solid lines). CVs of increasing concentration of SOS concentration (0.25 – 6.0 µM) using Cell 2 (See Scheme 1) are displayed in Figure 2A. Going towards the negative potential (forward scan), the negative current increases with increasing SOS concentrations, as also seen in Figure 1 (grey dashed line). On the other hand, as the potential is scanned in the reverse direction, a peak response was recorded at ca. -0.47 V for 0.5 μM SOS and this peak height intensified with increasing SOS concentrations. The resulting calibration plot has a sensitivity (slope) of 0.887

nA µM-1 and a correlation coefficient of 0.989 (n = 4). Additionally, the shape of the peak indicates an adsorption process rather than a diffusion-controlled phenomenon. Consequently, the response mechanism for the detection of SOS is proposed to entail the adsorption of SOS at the polarized soft interface during the forward (negative-going) scan combined with the interaction of the polysulfated analyte with the organic phase cation (TDDA+), in a manner similar to that discussed for heparin.36 The resulting peak on the reverse (positive-going) scan is then associated with the desorption process including the separation of the formed complex between the anionic SOS and the organic phase cation. At the ITIES, these counterionpolyion interactions have been previously reported48-50 and seem to be a generic electrochemical detection mechanism for polyionic analytes. CVs of increasing SOS concentrations (0.25 – 6.0 μM) in a cell with 10 mM TDMA+ in the organic phase (Cell 3) are shown in Figure 2(B). A peak was observed at ca. -0.28 V for 0.25 μM SOS on the reverse scan of the CV. This is an improvement compared to organic phases containing either TDDA+ (the peak was evident only at higher concentrations) or BTPPA+ (no peak was observed in this range of concentrations). Figure 2(B) inset reveals an improved sensitivity of 2.405 nA µM-1 as well as an improved correlation coefficient (n = 4), 0.987, compared to the case of TDDA+ organic phase cation. This enhancement in the response might be attributed to the nature of the interaction of such alkylammonium organic phase cations with SOS, relative to the weak interaction of BTPPA+ with SOS. Other studies have reported the formation of complexes between sulfated polysaccharides, such as heparin, with organic phase cations that served as ionophores.37,39 One such finding discussed that BTPPA+ as the organic phase cation exhibited only weak interactions with heparin, but hexadecyltrimethylammonium (HDTMA+) provided a stronger interaction with heparin. Comparing both cations structurally, it was proposed that HDTMA+ offers more flexibility for heparin interaction versus BTPPA+ which has steric hindrance and shielding of its positively-charged centre by the phenyl rings.37 This was supported by the findings of Guo and colleagues,38 who reported that at the liquid-liquid interface, heparin adsorption was more favored when there was less steric hindrance within the ionophore because this exposed the positive charge on the nitrogen, leaving it more open for electrostatic binding with the negative charges in heparin.



Figure 2. CV of 10 mM LiCl in the absence (grey dashed line) and presence of increasing concentrations (0.25 – 6.0 µM) of SOS using Cell 2 (A) and Cell 3 (B), while (C) is with 6 µM SHpS (black solid line) and (D) is with 6 µM SHxS (black solid line), both using Cell 3 (see Scheme 1).

An equivalent situation can be ascribed to the improved electrochemical signal for SOS detection here when the organic phase cation was TDDA+ and TDMA+, because of their structural resemblance with HDTMA+. In support of this case,

Meyerhoff and group23 employed polyion-sensitive electrodes that were TDMA+-based in detecting pentosan polysulfate (PPS). They reported that the improved ion-pairing interaction strength with PPS was due to the more accessible charge density in TDMA+. Moreover, quaternary ammonium ions were found to be ideal counterions to achieve enhanced ESI-MS analysis by SOS-counterion complex formation.21 Such findings point out that organic phase cation interactions with SOS are more favored in the order: BTPPA+ < TDDA+ < TDMA+. Therefore, it is proposed that at the liquid-organogel interface, the interaction involves the complexation of SOS with TDDA+ or TDMA+, followed by the adsorption of the complex during the forward (negative-going) CV sweep. Then, this adsorbed complex is gradually desorbed during the reverse (positivegoing) scan, with return of the cation to the organic phase and SOS to the aqueous phase. In order to assess the response to other sucrose derivatives that are less sulfated, CVs were recorded (Figure 2 C, D) in the absence (grey dashed line) and presence (black line) of 6 μM SHpS or SHxS (Cell 3, See Scheme 1). Features in the CVs similar to those observed for SOS were seen for these two species, such as the increase of the negative current during the forward (negative-going) scan, indicating a charge transfer that is elevated in the presence of the sulfated sugar, and peaks during the reverse (positive-going) scan, suggesting that both SHpS and SHxS are also electrochemically active at the ITIES. The effect of the lower degree of sulfation on the electrochemical response was not evident. There was no change in the peak currents with sulfation: 10.5 ± 1.60 nA for SOS, 9.7 ± 2.79 nA for SHpS and 9.5 ± 3.22 nA for SHxS (mean ± one standard deviation, n = 3, analyte concentrations 6 μM). As these data show that changes of one or two sulfate groups do not alter the measurements, further experiments were focused on SOS. Adsorptive Stripping Voltammetry. The utilization of adsorptive stripping voltammetry (AdSV) at the microITIES has been instrumental in the investigation of several polyelectrolytes.38,51 The technique involves two stages: first, a constant potential is applied for a chosen time that promotes the adsorption and pre-concentrates the analyte of interest at the interface; second, a voltammetric scan serves as the detection step during which the analyte is desorbed from the interface and yields a current that is the analytical signal. For this investigation, the pre-concentration of SOS was chosen at an appropriate negative potential to stimulate adsorption, then the potential was scanning towards more positive potentials in order to desorb SOS from the interface. This yields a peak current that is shown to be dependent on analyte concentration as well as the chosen pre-concentration time. The optimization of the parameters for SOS adsorption started with examination of the effect of the applied potential during the adsorption stage. Following the determination of the potential range where SOS adsorption occurs, potential values were chosen and applied for a specific time. These were followed by voltammetric scans to desorb the SOS and produce a stripping voltammogram. Displayed in Figure 3(A) are voltammograms showing the effect of varying the adsorption potential for SOS detection with TDMA+ as the organic phase cation. The voltammograms following application of less negative potentials showed no obvious peaks. However, at adsorption potentials ≤ -0.375 V,


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Analytical Chemistry a distinct stripping peak starts to show, demonstrating the in-

fluence of the applied potential on the adsorption process.

Figure 3. AdSV of SOS in 10 mM LiCl: (A) 5 µM SOS at varying adsorption potentials applied for 60 s using Cell 3 (Scheme 1). Inset in (A): Corresponding blank profiles for the experiment. (B) 0.25-6.0 µM SOS using the optimized adsorption potential (-0.475 V) following 60 s adsorption (Cell 3). (C) comparison of peak current versus SOS concentration using optimized adsorption potentials with two different organic phase cations, all following 60 s adsorption (Cells 2 and 3). (D) 0.03-0.15 µM SOS using the optimized adsorption potential (0.475 V) following for 180 s adsorption time (Cell 3). The arrow indicates increasing peak height with increasing concentration. Inset in (D): Current versus concentration plot for these experiments.

Equivalent to what was seen in the CV experiments, the AdSV peaks presented shapes consistent with a surfaceconfined process, which agrees with an adsorption/desorption phenomenon at the interface. Optimization of the applied adsorption potential also enables the minimization of background signal due to background electrolyte transfers, which occurs close to the potential where SOS adsorbs. Consequently, the chosen adsorption potential is critical in both maximizing the analyte signal as well as minimizing the background electrolyte signal. The data shown in Figure 3(A) reveals that the optimized adsorption potential for SOS was ca. -0.425 V with TDMA+ in the organic phase. Moreover, increasing concentrations of SOS were examined using the optimized adsorption parameters. Figure 3(B) displays the CVs for 0.25-6.0 μM SOS in the aqueous phase using Cell 3 (Scheme 1). As observed, the peak currents increased proportionately with the concentrations of SOS. The resulting calibration plot (see Figure 3(C)) is linear from 0.25 to 2.0 µM but shows some curvature in the range 4.0 – 6.0 µM. This curvature at higher concentration is more obvious when plotted against that for TDDA+ (Figure 3C). The difference in the slopes (Figure 3(C)) suggests stronger interaction between SOS and TDMA+ than between SOS and TDDA+ and also demonstrates the improvement in sensitivity by using TDMA+ as the organic phase cation. Similar behavior is observed on comparison of the slopes of the calibration plots in Figure 2(A, B). Also, the curvature at higher concentrations of SOS is attributed to surface saturation behavior typical of an adsorption process. Combination of AdSV with TDMA+ as the organic phase cation resulted in a calculated detection limit of 0.064 μM SOS.

To further lower the detection limit, experiments with a longer adsorption or pre-concentration time at 180 s and a lower concentration range, 0.03-0.15 μM SOS, were undertaken. The resulting voltammograms are displayed in Figure 3(D). With the longer pre-concentration time and lower concentration range, the sensitivity was improved, and the calculated detection limit was 0.036 μM SOS. Matrix Effects. Most investigations involving SOS revolve around its array of biological activities so that detection in physiological matrices like urine, blood serum, or plasma is vital and has been the subject of studies.22,52 For this investigation, a synthetic urine mixture was examined as a working matrix for SOS detection to aid assessment of matrix effects on the analyte response. The synthetic urine was prepared according to a published recipe41 and used as the aqueous phase for the electrochemical measurements (Cell 4, Scheme 1). Figure 4(A) displays a CV of the prepared synthetic urine (grey dashed line) overlaid on the CV in which 10 mM LiCl (black line) was the aqueous phase. These results show that the potential window was shorter when using synthetic urine as the aqueous phase, because of the various ionic components of the mixture. This was similar to observations in a previous study45 when the component ions were studied individually and revealed that the cations NH4+, K+, Ca2+ transfer at lower potentials. These results also support findings of a study of synthetic saliva.41 However, in spite of the shortened potential window, SOS was still detected, as shown in Figure 4(B), when 6 μM SOS was spiked into the synthetic urine. The peak at ca. -0.30 V on the reverse (positive-going) scan reveals the detection of SOS in the synthetic urine. This peak shape is suggestive of an adsorption/desorption process, as previously



discussed. AdSV using optimized parameters was assessed for SOS detection in this biomimetic matrix. Figure 4(C) shows AdSV of increasing SOS concentrations. The calibration plot (Inset, Figure 4(C)) for SOS in synthetic urine is linear within the lower concentration range used. However, in comparison with the calibration plot for SOS in regular aqueous phase

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electrolyte (10 mM LiCl) (inset, Figure 3(B)), the slope of the best fit line is lower. This lowered sensitivity is due the additional ions present in the synthetic urine matrix which increase the background current and lower the available potential window and, therefore, the potential required to drive the adsorption.

Table 1. Summary of analytical characteristics of SOS at the polarized liquid-organogel micro-interface array with the different voltammetric methods employed. Limit of Detection (LOD) / µM

Concentration Range / µM

Correlation Coefficient / R

Detection Method

Organic Cation

Sensitivity (calibration graph)# /nA µM-1

CV

BTPPA+

ND‡

ND

ND

ND

+

0.887

0.320

0.25-2.0

0.989

CV

+

TDMA

2.405

0.207

0.25-2.0

0.987

†

TDMA+

1.922

0.285

0.25-2.0

0.989

AdSV (60 s)

+

TDDA

0.976

0.267

0.25-2.0

0.992

AdSV (60 s)

TDMA+

3.011

0.064

0.25-2.0

0.986

AdSV (180 s)

TDMA+

5.589

0.036

0.03-0.12

0.997

TDMA+

1.465

0.162

0.25-2.0

0.977

CV

CV

†

AdSV (60 s)

TDDA

†

Corresponds to experiments with synthetic urine as the aqueous phase. All other aqueous phases were 10 mM LiCl; ‡ND = not detected; Due to the curvilinear nature of the calibration curves, the slope refers to the linear best-fit line through the lower four (4) concentrations only.

#


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Analytical Chemistry when the organic phase contained TDDA+ and this peak shifted to ca. -0.28 V when the organic phase cation was TDMA+. This observation is attributed to the increased binding strength between SOS and these specific organic electrolyte cations. The shape of the peak suggests a desorption process, in agreement with an adsorption process during the forward (negative-going) scan. Examination of the optimized adsorption potential for SOS showed that maximum adsorption occurred at a potential near the potential for background electrolyte transfer. With AdSV, having TDMA+ in the organic phase and an adsorption time of 60 s resulted in a detection limit of 0.064 μM in 10 mM LiCl and of 0.162 μM in a synthetic urine mixture. When adsorption time was increased to 180 s, the detection limit improved to 0.036 μM (in 10 mM LiCl). The behaviour reported in this study supports the capacity of employing electrochemistry at the liquid-organogel μITIES array as a bioanalytical label-free tool for the detection of sulfated sucrose. Selectivity (such as distinction between less sulfated species) is a parameter for further investigations.

AUTHOR INFORMATION Corresponding Author * Email [email protected] Tel. no: +61 08 9266 9735

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT BMBF extends his thanks to Curtin University for the award of a research scholarship.

REFERENCES

Figure 4. (A) Cyclic voltammograms of synthetic urine (Cell 4, grey dashed line) in contrast to 10 mM LiCl (Cell 3, black line) as the aqueous phase. (B) CV in the absence (grey dashed line) and presence of 6 μM SOS. (black line) (Cell 4) (C) AdSV of increasing SOS concentration (0.25-6.0 µM) (Cell 4). Adsorption potential: -0.425 V, pre-concentration time: 60 s, scan rate: 5 mV s-1.

An equivalent phenomenon can be expected in applications with real biological matrices. Nevertheless, prudent optimization of the adsorption potential as well as the chosen electrolyte might help to diminish this issue, as previously reported for insulin detection with interferents present.53 Overall, combining AdSV with TDMA+ in the organic phase produced a calculated detection limit of 0.162 μM in the synthetic urine matrix. Table 1 summarizes the analytical characteristics of the different voltammetric methods investigated together with the different organic phase cations employed in this work.

CONCLUSIONS The electrochemical characteristics of sulfated sucrose (SOS) was examined utilizing voltammetry at a liquidorganogel μITIES array. The recorded CV of SOS showed no response at low μM concentration when BTPPA+ was employed in the organic phase. However, a distinct peak was evident on the reverse (positive-going) CV scan at ca. -0.47 V

(1) Islam, T.; Linhardt, R. J. In Carbohydrate-Based Drug Discovery; Wiley-VCH Verlag GmbH & Co. KGaA, 2003, pp 407-439. (2) Gandhi, N. S.; Mancera, R. L. Chem. Biol. Drug Des. 2008, 72, 455-482. (3) Capila, I.; Linhardt, R. J. Angew. Chem. Int. Ed. 2002, 41, 390-412. (4) Ornitz David, M. Bioessays 2000, 22, 108-112. (5) Bourin, M.-C.; Lindahl, U. Biochem. J 1993, 289, 313. (6) Hiebert, L. M.; Wice, S. M.; Ping, T.; Hileman, R. E.; Polat, T.; Linhardt, R. J. Pharm. Res. 2002, 19, 838-844. (7) Hileman, R. E.; Siegel, M. M.; Tabei, K.; Balagurunathan, K.; Linhardt, R. J. Electrophoresis 1998, 19, 2677-2681. (8) Szabo, S.; Hollander, D. The American journal of medicine 1989, 86, 23-31. (9) Zhu, X.; Hsu, B. T.; Rees, D. C. Structure 1993, 1, 27-34. (10) Yeh, B. K.; Eliseenkova, A. V.; Plotnikov, A. N.; Green, D.; Pinnell, J.; Polat, T.; Gritli-Linde, A.; Linhardt, R. J.; Mohammadi, M. Mol. Cell. Biol. 2002, 22, 7184-7192. (11) Prestrelski, S. J.; Fox, G. M.; Arakawa, T. Arch. Biochem. Biophys. 1992, 293, 314-319. (12) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161-227. (13) Wolff, J. J.; Laremore, T. N.; Leach Iii, F. E.; Linhardt, R. J.; Amster, I. J. Eur. J. Mass Spectrom. 2009, 15, 275-281. (14) Zidkova, J.; Chmelik, J. Feedback 1997, 91. (15) Ruiz-Calero, V.; Puignou, L.; Galceran, M. T. J. Chromatogr. A 1998, 828, 497-508.



(16) Skidmore, M. A.; Guimond, S. E.; Dumax-Vorzet, A. F.; Atrih, A.; Yates, E. A.; Turnbull, J. E. J. Chromatogr. A 2006, 1135, 52-56. (17) Deakin, J. A.; Lyon, M. Glycobiology 2008, 18, 483-491. (18) Sinnis, P.; Coppi, A.; Toida, T.; Toyoda, H.; KinoshitaToyoda, A.; Xie, J.; Kemp, M. M.; Linhardt, R. J. Journal of Biological Chemistry 2007, 282, 25376-25384. (19) Chuang, W.-L.; McAllister, H.; Rabenstein, D. L. Carbohydr. Res. 2002, 337, 935-945. (20) Guerrini, M.; Naggi, A.; Guglieri, S.; Santarsiero, R.; Torri, G. Anal. Biochem. 2005, 337, 35-47. (21) Gunay, N. S.; Tadano-Aritomi, K.; Toida, T.; Ishizuka, I.; Linhardt, R. J. Anal. Chem. 2003, 75, 3226-3231. (22) Ke, Y.; Li, S. L.; Chang, L. D.; Kapanadze, T. J. Chromatogr. B 2015, 978-979, 151-156. (23) Dürüst, N.; Meyerhoff, M. E. Anal. Chim. Acta 2001, 432, 253-260. (24) Fu, B.; Bakker, E.; Yang, V. C.; Meyerhoff, M. E. Macromolecules 1995, 28, 5834-5840. (25) Langmaier, J.; Samec, Z.; Samcová, E.; Tůma, P. Electrochem. Commun. 2012, 24, 25-27. (26) Hassan, S. S. M.; Meyerhoff, M. E.; Badr, I. H. A.; AbdRabboh, H. S. M. Electroanalysis 2002, 14, 439. (27) Kim, J. M.; Nguyen, L.; Barr, M. F.; Morabito, M.; Stringer, D.; Fitton, J. H.; Mowery, K. A. Anal. Chim. Acta 2015, 877, 1-8. (28) Herzog, G. Analyst 2015, 140, 3888-3896. (29) Peljo, P.; Girault, H. H. In Encyclopedia of Analytical Chemistry; John Wiley & Sons, Ltd, 2012. (30) Liu, S.; Li, Q.; Shao, Y. Chem. Soc. Rev. 2011, 40, 22362253. (31) Osakai, T.; Yuguchi, Y.; Gohara, E.; Katano, H. Langmuir 2010, 26, 11530-11537. (32) Felisilda, B. M. B.; Alvarez de Eulate, E.; Arrigan, D. W. M. Anal. Chim. Acta 2015, 893, 34-40. (33) Colombo, M. L.; Sweedler, J. V.; Shen, M. Anal. Chem. 2015, 87, 5095-5100. (34) Ortuño, J. A.; Olmos, J. M.; Torralba, E.; Molina, A. Sensors and Actuators B: Chemical 2016, 222, 930-936. (35) Santos, H. A.; García-Morales, V.; Roozeman, R.-J.; Manzanares, J. A.; Kontturi, K. Langmuir 2005, 21, 54755484.

(36) Amemiya, S.; Kim, Y.; Ishimatsu, R.; Kabagambe, B. Anal Bioanal Chem 2011, 399, 571-579. (37) Samec, Z.; Trojánek, A.; Langmaier, J.; Samcová, E. Electrochem. Commun. 2003, 5, 867-870. (38) Guo, J.; Yuan, Y.; Amemiya, S. Anal. Chem. 2005, 77, 5711-5719. (39) Jing, P.; Kim, Y.; Amemiya, S. Langmuir 2009, 25, 13653-13660. (40) Scanlon, M. D.; Strutwolf, J.; Arrigan, D. W. M. PCCP 2010, 12, 10040-10047. (41) Collins, C. J.; Berduque, A.; Arrigan, D. W. M. Anal. Chem. 2008, 80, 8102-8108. (42) Zazpe, R.; Hibert, C.; O'Brien, J.; Lanyon, Y. H.; Arrigan, D. W. M. Lab on a Chip 2007, 7, 1732-1737. (43) Katano, H.; Senda, M. Anal. Sci. 2001, 17, 1027-1029. (44) Samec, Z. Pure and Applied Chemistry 2004, 76, 21472180. (45) Felisilda, B. M. B.; Alvarez de Eulate, E.; Stringer, D. N.; Fitton, J. H.; Arrigan, D. W. M. Analyst 2017, 142, 31943202. (46) Wilke, S.; Zerihun, T. J. Electroanal. Chem. 2001, 515, 52-60. (47) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications; John Wiley & Sons, Inc.: New York, 2001. (48) Riva, J. S.; Bierbrauer, K.; Beltramo, D. M.; Yudi, L. M. Electrochim. Acta 2012, 85, 659-664. (49) Riva, J. S.; Iglesias, R.; Yudi, L. M. Electrochim. Acta 2013, 107, 584-591. (50) Trojánek, A.; Langmaier, J.; Samcová, E.; Samec, Z. J. Electroanal. Chem. 2007, 603, 235-242. (51) Amemiya, S.; Yang, X.; Wazenegger, T. L. J. Am. Chem. Soc. 2003, 125, 11832-11833. (52) Sobolev, V. E.; Jenkins, R. O.; Goncharov, N. V. Exp. Toxicol. Pathol. 2017. (53) O'Sullivan, S.; Alvarez de Eulate, E.; Yuen, Y. H.; Helmerhorst, E.; Arrigan, D. W. M. Analyst 2013, 138, 61926196.

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Electrochemical Behavior and Detection of Sulfated Sucrose at a

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