In Situ Potentiometry and Ellipsometry: A Promising Tool to Study

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Technical Note

In-situ potentiometry and ellipsometry: a promising tool to study biofouling of potentiometric sensors Grzegorz Lisak, Thomas Arnebrant, Andrzej Lewenstam, Johan Bobacka, and Tautgirdas Ruzgas Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04364 • Publication Date (Web): 11 Feb 2016 Downloaded from http://pubs.acs.org on February 15, 2016

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Analytical Chemistry

In-situ potentiometry and ellipsometry: a promising tool to study biofouling of potentiometric sensors Grzegorz Lisak1,2,3,*, Thomas Arnebrant2,3, Andrzej Lewenstam1,4, Johan Bobacka1, Tautgirdas Ruzgas2,3 1

Johan Gadolin Process Chemistry Centre, c/o Laboratory of Analytical Chemistry, Åbo Akademi University, FIN-20500 Åbo-Turku, Finland

2

Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, SE-205 06 Malmö, Sweden

3

Biofilms - Research Center for Biointerfaces, Malmö University, SE-205 06 Malmö, Sweden 4

Faculty of Materials Science and Ceramics, AGH – University of Science and Technology, 30059 Krakow, Poland *corresponding author: [email protected] Tel: +35822153247

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Abstract In-situ potentiometry and null ellipsometry was combined and used as a tool to follow the kinetics of biofouling of ion-selective electrodes (ISEs). The study was performed using custom made

solid-contact

K+-ISEs

consisting

of

a

gold

surface

with

immobilized

6-

(ferrocenyl)hexanethiol as ion-to-electron transducer that was coated with a potassium-selective plasticized polymer membrane. The electrode potential and the ellipsometric signal (corresponding to the amount of adsorbed protein) were recorded simultaneously during adsorption of bovine serum albumin (BSA) at the surface of the K+-ISEs. This in-situ method may become useful in developing sensors with minimized biofouling.


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INTRODUCTION Ion-selective electrodes (ISEs) are massively implemented in routine clinical analysis of electrolytes in biological samples, such as whole blood, serum and urine.

The reliable

determination of ions in such sample types, however, is possible only by applying flow-through sensors and strict measurement protocols.1 Owing to these precautions fouling of ISEs by blood plasma proteins and lipids, e.g. substances adsorbed at the surface of the electrodes, is minimized.2 The routine measurements in most environmental, clinical, and foodstuff samples are not straightforward.3, 4 Fouling of ISEs is time dependent, which influences the time needed for equilibration of an electrochemical cell and may result in errors in the ion determination.5 The fouling of electrochemical sensors may be of different nature, namely physical, chemical and/or biological (e.g. physical and chemical adsorption of fouling components at the sensing element, presence of interfering ions and biofilm formation). In fact the equilibration of the potential, due to the fouling of ISEs, may last for hours or may even never be attained.5-10 There are ways to reduce the effects of fouling of potentiometric sensors. This can be done e.g. by the use of different surfactants contained in electrode washing solutions to clean the surface of ISEs.1,2 Another way is to modify the ion-selective membrane (ISM) to obtain antifouling properties, e.g. by the use of poly(ethylene glycol)11 or by applying a protective coating.12 Poly(vinyl chloride) (PVC), the most commonly used polymer matrix of the ISM, can be modified by various species in order to obtain specific properties of the membrane.13 Furthermore fouling components consisting of bigger particles may be removed before the actual measurement, e.g. by application of paper- and textile-based sampling.14-16 Interestingly, red cells can be filtered out from whole blood using paper substrates having a pore size smaller than 2.5 µm.17 Finally, by understanding the kinetics of the fouling process and its influence on the ISM a reliable potentiometric signal may be obtained e.g. by applying non-equilibrium, time-dependent measurement protocols.5

The fouling process can be studied by potentiometry,18,19 by

electrochemical impedance spectroscopy (EIS)20-22, by quartz crystal microbalance23, by scanning electron microscopy (SEM)11,24 or by measuring radioactivity of labeled fouling component.25 Among these experimental methods, potentiometry is a powerful technique to follow the influence of biofouling on the measured potential in real time.26 However, in order to really quantify the amount of fouling compounds that are adsorbed at the electrode surface, a complementary analytical technique, independent of potentiometry, has to be used. This can be 3 ACS Paragon Plus Environment


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offered by ellipsometry, which proved to be a useful technique in studying protein adsorption on various surfaces, including that of PVC.27,28 Moreover, ellipsometry was previously successfully coupled with electrochemical methods (cyclic voltammetry) to study e.g. polyelectrolyte multilayer films on gold surfaces.29 In this work, in-situ potentiometry and null ellipsometry were combined and applied to follow kinetics of biofouling and to quantify the amount of bovine serum albumin (BSA) adsorbed on potassium selective potentiometric sensors (K+-ISEs) in 0.01 mol dm–3 phosphate buffered saline (PBS) solution (pH = 7.4). The combined technique was used here for the very first time in order to continuously follow biofouling of ISEs. MATERIALS AND METHODS Reagents. Sodium chloride (NaCl), potassium ionophore I (valinomycin), potassium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (KTFPB), bis(2-ethylhexyl) sebacate (DOS), poly(vinyl chloride) high molecular weight (PVC) and tetrahydrofuran (THF) were purchased from Fluka (Buchs, Switzerland). Nitrogen gas was purchased from Aga, part of Linde Group (Munich, Germany), acetonitrile (ACN) was purchased from VWR Chemicals (Radnor, USA), 99.5% ethanol was purchased from Kemetyl (Jordbro, Sweden), while phosphate buffered saline (PBS,

tablets),

6-(ferrocenyl)hexanethiol

(FT),

bovine

serum

albumin

(BSA),

3,4-

ethylenedioxythiophene and poly(sodium 4-styrenesulfonate) were purchased from SigmaAldrich (Steinheim, Germany). Aqueous solutions were prepared with freshly deionized water of 18.2 MΩ cm resistivity obtained with the ELGA purelab ultra water system (High Wycombe, United Kingdom). Phosphate buffer saline (PBS) at 0.01 mol dm–3 was prepared by dissolving readymade tablets in deionized water to obtain 0.01 mol dm–3 Na2HPO4, 0.0018 mol dm–3 KH2PO4, 0.0027 mol dm–3 KCl and 0.137 mol dm–3 NaCl. Potentiometry. The design of K+-ISEs used in in-situ potentiometry and null ellipsometry is presented in Figure 1. The substrates were manufactured in a Balzers UMS 500 P system by electron-beam deposition of 200 nm gold onto silicon (100) wafers, pre-coated with a 2.5 nm thick titanium adhesion layer (Laboratory of Applied Physics, Linköping University, Linköping, Sweden). The substrates were chemically cleaned with 99.5% ethanol and deionized water (repeating the step three times), then were dried under nitrogen and plasma cleaned (Harrick Plasma Cleaner PDC-32G, Harrick Scientific Corporation, Ossining, USA) in residual 4 ACS Paragon Plus Environment

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air for 600 s. Subsequently, a monolayer, consisting of 6-(ferrocenyl)hexanethiol was allowed to self-assemble on the gold surface by soaking the gold electrodes at room temperature in a 10–3 mol dm–3 solution of 6-(ferrocenyl)hexanethiol in ACN for 20 h. After that, the substrates were washed with ACN and the presence of 6-(ferrocenyl)hexanethiol at the gold surfaces were confirmed by means of cyclic voltammetry.30 Substrates were left for evaporation of the solvent for 1 h and then they were dip coated with the K+-selective membrane cocktail. The K+-ISM cocktail was prepared by dissolving the membrane components (1% (w/w) potassium ionophore I, 0.5% KTFPB, 65.2% DOS and 33.3% PVC) in THF. Dip-coating was made from a dilute membrane cocktail solution (dry mass of 0.5 %). The electrodes (Au/FT/K+-ISM) were left for evaporation of the solvent for approx. 15 h. Then the ISM (approx. 0.5 × 1.2 cm2 area) covering the 0.5 × 1 cm2 area of thiolated gold surface was left while the rest of ISM was removed using tissue paper. The ISM-free surface (except of most top part of the electrode) was painted with nail polish (Cosmo, Poland) in order to block the electroactive surface (gold) other than ISM from direct contact with the solution. The prepared K+-ISEs were used as indicator electrodes, while a single junction Ag/AgCl/3 M NaCl electrode was used as reference electrode. The open circuit potential of the potentiometric cell was measured by a potentiostat from Ivium (CompactStat, Ivium (Eindhoven, The Netherlands). Null ellipsometry. An automated ellipsometer, type 43603-200E, Rudolph Research (Fairfield, USA) equipped with a xenon lamp of a fixed angle of incidence (67.75o) and a light detector (442.9 nm) with an interference filter, ultraviolet and infrared blocking (Mells Griot, The Netherlands) was used together with potentiometry to in-situ monitor the adsorption of BSA at the surface of Au, Au/FT and Au/FT/K+-ISM electrodes. In-situ potentiometry and ellipsometry. The bare gold (Au), gold covered with a 6(ferrocenyl)hexanethiol self-assembled monolayer (Au/FT) and K+-ISE were investigated by means of in-situ potentiometry and ellipsometry. This study was performed not only on K+-ISE (Figure 1.) but also onto intermediate layers in the design of ISEs, namely bare Au and 6(ferrocenyl)hexanethiol self-assembled monolayer (Au/FT). It was done in order to identify different adsorption characteristics of BSA at different surfaces in the design of the ISEs, thus to identify the presence of 6-(ferrocenyl)hexanethiol on gold electrode in respect to the BSA adsorption characteristics on bare gold and furthermore to identify the presence of PVC-based membrane on top of the 6-(ferrocenyl)hexanethiol/Au. In the ellipsometric setup, the single 5 ACS Paragon Plus Environment


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electrode was vertically mounted into a glass trapezoid cuvette (Hellma, Germany) containing 5 ml of 10–2 mol dm–3 PBS that was thermostated at 25oC. Specifications of the ellipsometer setup can be found elsewhere.31 To avoid the influence of swelling of the PVC-based ISM on the determination of BSA adsorption kinetics during the ellipsometric measurement32 and to avoid potential drift due to the conditioning of the electrodes19, the K+-ISEs were left to equilibrate with PBS (containing 4.5 × 10–3 mol dm–3 K+) for 2 h before measurements. Then, the PBS solution was changed to a fresh one, the reference electrode was added to the cuvette and a four zone ellipsometric measurement was conducted to determine the effective refractive index of the electrode surface. The potentiometric and ellipsometric measurements where then started and a baseline was recorded for 200 s, followed by the addition of an aliquot of BSA in 10–2 mol dm–3 PBS solution resulting in a BSA concentration of 1 mg ml–1 in the cuvette. The concentration of 1 mg ml–1 BSA was chosen in order to reach full monolayer coverage of the protein on the electrode surface.33 The measurement was run for 1800 s and then the cuvette was rinsed (using a peristaltic pump) with pure 10–2 mol dm–3 PBS solution in order to remove unbound and loosely bound BSA from the surface of the K+-ISEs. After that the potentiometric and ellipsometric measurements were continued for another 1800 s. For the duration of the whole experiment the ellipsometric signal (∆ and Ψ values) was recorded approximately every 12 s, while the potentiometric signal (EMF) was recorded every 10 s. Optical thickness ( ) and the refractive index ( ) of the adsorbed BSA layer was calculated from ∆ and Ψ values by using software based on algorithm first proposed by McCrackin.34 The adsorbed amount (Г) of BSA on the surface of K+-ISEs was calculated from the mean values of and of the layer, according to the Feijter equation (Eq. 1.), where is the refractive index of the medium, e. g., of PBS solution, = 1.334: Г =

(Eq. 1)

A refractive index increment for the protein ( = 0.18

) was used.35,36 Each K+-ISEs was

used only once. Every time a newly produced K+-ISE was used and the in-situ potentiometric and ellipsometric measurements were repeated three times, n= 3. For the data presentation in the potentiometric mode, the potential of ISEs when equilibrated with PBS solution (equilibration potential obtained during first 200 s measurement time) was brought to zero.


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Figure 1. Schematic illustration of K+-ISEs used in in-situ potentiometry and null ellipsometry. RESULTS AND DISCUSSION The in-situ potentiometry and ellipsometry was performed simultaneously in order to study the kinetics of biofouling and to quantify the amount of BSA adsorbed as well as to measure the influence of the adsorbed BSA on the potentiometric response of the K+-ISEs. The ISEs used in this study consisted of a gold substrate covered with 6-(ferrocenyl)hexanethiol (FT) and coated with a K+-ISM. Thus, before the actual study devoted to biofouling of ISEs, the adsorption of BSA onto intermediate layers in the design of ISEs, namely bare gold (Au) and gold covered with a 6-(ferrocenyl)hexanethiol self-assembled monolayer (Au/FT) were investigated by means of insitu potentiometry and ellipsometry. The results are presented in Figure 2. In the first step (200 s) the potential of the bare Au electrode (equilibration potential approx. 70 mV) showed a drift to lower potentials (0.5 mV min–1) while the Au/FT electrode (equilibration potential approx. 55 mV) exhibited a stable potential (0.05 mV min–1). The potential of bare Au electrodes are usually unstable in the absence of a redox couple in the solution. Thus the complete coverage of the gold electrode by 6-(ferrocenyl)hexanethiol, in case of Au/FT, was confirmed by decreasing ten times the potential drift compared to the drift in case of bare Au electrode. The fairly polar 6(ferrocenyl)hexanethiol was found to have a strong affinity toward gold surfaces forming a stable monolayer.30 During the same period of time (0–200 s) the ellipsometric signals, e.g. ∆ and Ψ angles, were constant for both electrodes indicating that the interfaces had stabilized. Once the BSA was added to the measuring solution, the potentiometric signal dropped down while the calculated amount of adsorbed BSA at the surface of the electrodes from the ellipsometric measurement instantly increased. After 1800 s measurement time, the potential drop due to biofouling of the electrodes was 42.5 mV (when correction for the downward drift was included that potential drop was ca 16 mV) for the Au electrode and 26.5 mV for the Au/FT electrode. The 7 ACS Paragon Plus Environment


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adsorbed amount of BSA at the surface was 2.22 mg m–2 for Au and 1.63 mg m–2 for Au/FT. Moreover, from the obtained profiles of adsorbed amount of BSA vs. time it can be concluded that the biofouling of both surfaces occurred fast as 75% of the maximum adsorbed amount was reached already after 30 s for Au and after 80 s for Au/FT (from the time of injection of 1 mg ml– 1

BSA to the measuring solution).

The potentiometric signal clearly correlates with the

ellipsometric data as the potential drop of the electrodes clearly correlates with the amount of BSA adsorbed at the surface of the electrodes (Figure 2). After washing off unbound and loosely bound BSA from the measuring solution, the adsorbed amount of BSA at the surface of electrodes decreased slightly to 2.19 and 1.47 mg m–2 for Au and Au/FT electrodes, respectively.

Figure 2. In-situ potentiometry and ellipsometry of 1 mg ml–1 BSA adsorption (biofouling) in 10– 2

mol dm–3 PBS at bare Au (■) and Au/FT (○) electrodes, where the potentiometric signal is

marked with blue color and the ellipsometric signal is marked with black color. In-situ potentiometry and ellipsometry of Au/FT/K+-ISM electrodes during biofouling by BSA is presented in Figure 3. In the first step (200 s) the potential of the Au/FT/K+-ISM electrodes (equilibration potential 115 ± 5 mV) drifted upwards (0.4 mV min–1). The PVC-based ion selective membranes are vulnerable towards the uptake of the electrolyte. In case of very thin membranes (101 ± 23 nm, estimated from cross section of ISEs in SEM), water may reach the solid contact layer within short periods of time.37,38 The water penetration through the membrane occurs relatively fast compared to thicker membranes (see supporting information). The accumulating aqueous layer between the ion-selective membrane and the self-assembled monolayer may be responsible for the observed potential drift. Moreover, the 6(ferrocenyl)hexanethiol layer is more hydrophilic (θc= 70.0 ± 4.1 degrees) than the ion-selective 8 ACS Paragon Plus Environment

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membrane (θc= 77.7 ± 1.9), which creates the condition in which the water present in the ISM is easily transported to the self-assembled monolayer │ ISM interface (see supporting information). During the same period of time (0–200 s) the ellipsometric signal from the Au/FT/K+-ISM electrodes was not changing showing that the interface had stabilized. Again, once the 1 mg ml–1 BSA was added to the measuring solution, the potentiometric signal immediately decreased while the calculated amount of adsorbed BSA at the surface of the ISEs (from ellipsometry) instantly increased. The potential drop related to the adsorption of BSA at the surface of the ISE was determined to be approx. 16 mV (taking into account the upward drift of the ISE potential). After 1800s measurement time, the adsorbed amount of BSA at the surface of the Au/FT/K+-ISM electrodes was 2.60 ± 0.04 mg m–2. After washing off unbound and loosely bound BSA (another 1800 s) the adsorbed amount of BSA at the surface of Au/FT/K+-ISM electrodes was 2.45 ± 0.29 mg m–2 while the EMF (after an initial jump of potential which was related to the removal of loosely bound BSA33) retained the upward drift (not related to the surface adsorption). The washing with PBS buffer did not result in removal of any significant amount of BSA due to its irreversible adsorption at the surface. Again, the adsorption of BSA occurred rapidly as 75% of the total adsorbed amount of BSA was reached already within 200 s of biofouling time. The standard deviation was calculated from three consecutive measurements using each time a newly prepared K+-ISE.

Figure 3. In-situ potentiometry and ellipsometry of 1 mg ml–1 BSA adsorption (biofouling) in 10– 2

mol dm–3 PBS at Au/FT/K+-ISM electrodes, where the potentiometric signal is marked with

blue color and the ellipsometric signal is marked with black color. Standard deviation is marked from three consecutive measurements, each time with a new Au/FT/K+-ISM electrode.



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From all in-situ potentiometry and ellipsometry measurements it can be concluded that the amount of adsorbed BSA depends on the type of the surface, while the kinetics of the adsorption of BSA is rather independent of the applied surface. The adsorption of BSA on the FT-modified gold surface is lower than on clean bare gold. This indicates that the FT layer reduces BSA-gold interaction, e.g., electrostatic and image charge interaction. The FT-modified gold interface has also a lower value of the surface potential (Stern potential), thus adsorption of BSA on this surface results in a lower absolute change in the potential. Moreover, in general the results from simultaneous measurements of the potential of K+-ISEs and of the adsorbed amount of BSA at the K+-ISEs are complementary to each other. The mechanism of the irreversible adsorption of the protein at the surface is related to occurrence of multiple processes and interactions when BSA is in contact with the surface of the electrode. Such interactions include electrostatic and van der Waals forces, hydrogen bonding and hydrophobic interactions which finally lead to protein conformational changes during the adsorption process resulting in the formation of the gel-like regions of denatured protein at the ion-selective membrane.33,39 Such features present at the membrane surface influence the electroanalytical performance of the sensor as the mass transfer of the analyte to the surface is disturbed, delaying equilibration of the sensor and leading to the local activity of ions being different from the one in the bulk of the solution.40,41,42 Furthermore, the isoelectric point of BSA is 4.7, which means that the protein carries a negative charge. The shifts of the open circuit potential of K+-ISEs to negative values can then be explained by the adsorption of the negatively charged BSA on the surface of K+-ISEs and the resulting concurrent redistribution of potassium ions at the K+-ISM interface, i.e. the concentration decrease at the solution side and increase at the membrane side

41

. The adsorbed

BSA did not block the entire active surface of the sensor and can be assumed to be uniformly distributed along the membrane according to the concept of random sequential adsorption of proteins at the surface.43 The

reason

for

using

6-(ferrocenyl)hexanethiol

instead

of

e.g.

poly(3,4-

ethylenedioxythiophene) polystyrene sulfonate (PEDOT(PSS–) that is a commonly used solid contact in ISEs44,45, was that the investigated adsorbed amount of BSA on PEDOT(PSS–) was unreliable. Various thicknesses of PEDOT(PSS–), namely 20, 50 and 100 nm were electropolymerised according to the procedure described elsewhere46 and were investigated in the simultaneous measurement of potential and adsorption of BSA (results not shown here). The 10 ACS Paragon Plus Environment

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potential drops due to biofouling of the electrodes were 37, 25 and 9 mV while the adsorbed amounts of BSA at the surface were 7.6, 5.5 and 185 mg m–2, respectively. Such amounts of BSA adsorbed on PEDOT(PSS–) were unrealistic and were the result of light adsorbing properties of PEDOT(PSS–) at the wavelength 442.9 nm (λ used in null ellipsometry) influencing the optical constants, which are correlated with the light-absorbing thickness of the film.47,48 From surface characterization (see supporting information) lack of uniformity for the thickness of the K+-ISM (ca 0.1 µm), which was prepared by dip-coating, was observed. The dip coating of the K+-ISM created pools of more diluted droplets of membrane cocktail at the surface of the electrode which later on resulted, after evaporation of THF, in an even thinner membrane. Also, the formation of nanodroplets of analyte in plasticized membranes did occur.49 Nonetheless, the K+-ISM which had been in contact with BSA exhibited different surface morphology, namely possessed additional morphological features, than the same membrane before adsorption of the protein. From experiments performed on conventional K+-ISEs (see supporting information) the magnitude of the potential drop of the ISEs after contact with BSA was clearly related to the thickness of the K+-ISM. The potential of the K+-ISEs with thinner membranes (3 µm) were more affected by the adsorption of BSA compared to the K+-ISEs with thicker membranes (60 µm). The potential drop owning to adsorption of BSA at the surface of conventional K+-ISEs was approx. 2.1-2.6 mV, for thinner membranes (3 µm) and 0.1-0.2 mV and for thicker membranes (60 µm), compared to approx. 16 mV for electrodes used in in-situ potentiometry and ellipsometry (0.1 µm thickness). This fact indicates that the thickness of the K-ISM plays a significant role in determining the potential stability during biofouling of the electrode and may be related to the water transport through the membrane to the solid contact membrane interface. This process occurs much faster for thinner membranes than for the thicker ones.49 The performance of the K+-ISEs in the clinically relevant concentration range for blood, 3-6 mmol L– 1

, was found to be rather stable but a small decrease in the slope was observed after contact with

protein (BSA). The K+-ISEs are less affected by the adsorbed BSA than voltammetric sensors as potentiometric sensor response is not influenced by the change in the active surface area of the electrode.50 However, even small changes in the potential of the potentiometric cell caused by occurring unfavorable processes, e.g. biofouling, may lead to considerable determination errors. Thus 1 mV error corresponds to 4%, 8%, and 12% error in the determination for mono-, di-, and 11 ACS Paragon Plus Environment


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trivalent ions, respectively.51 Finally, taking into account current ways to enhance the blood compatibility with ion-selective electrodes52, the results suggest that ISEs should be preconditioned, at optimized and controlled conditions, before the calibration and the actual measurement in a solution containing proteins (standard or the sample solutions). This would allow for sample proteins to adsorb at the electrode surface and, thus, to avoid a potential drop caused by the adsorption of proteins during the actual measurement. Interestingly a similar approach is applied in routine clinical analyzers.2 CONCLUSION The in-situ method that combines potentiometry with ellipsometry was developed and studied for the use in monitoring the biofouling of potentiometric sensors.

The results show that

ellipsometric measurements may be performed by the use of a null ellipsometer during potentiometric measurements on a K+-ISE which is made out of a gold substrate with immobilized 6-(ferrocenyl)hexanethiol self-assembled monolayer as ion-to-electron transducer and a K+-ISM. The adsorbed amount of BSA at the surface of the ISEs was determined to be 2.45 ± 0.29 mg m–2. 75% of the absorbed amount was reached within the first 200 s of biofouling, which suggests fast kinetics of BSA adsorption. The potential drop during the adsorption of BSA was approx. 16 mV. Moreover, electrodes based on PEDOT(PSS–) as ion-to-electron transducer exhibited better potentiometric performance than those based on the 6-(ferrocenyl)hexanethiol self-assembled monolayer. It was also found that for clinical applications it is recommended that the sensors are introduced/washed with the sample prior to the actual measurement. In this way the surface of the sensor is equilibrated with the sample and potential drop connected to adsorption/biofouling may be avoided. The developed method could be applied as a diagnostic tool for sensor performance during the experiments performed at biofouling conditions, e.g. at environmental analysis of water or clinical analysis of body fluids. In this way the signal caused by the fouling of the sensor surface could be better understood and possibly cancelled out from the overall sensor response. Assessing and controlling biofouling at the electrodes will enable the determination of analytes in real samples more reliably. At this stage, however, the method has some limitations, which must be addressed in further studies. The thickness of the K+-ISM must be optimized in order not to interfere with the ellipsometric measurements, while at the same time prevent the formation of 12 ACS Paragon Plus Environment

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water layer and resemble the design of conventional ISEs. The composition of the ion-selective membrane must also be optimized as it was found that adsorption of proteins widely depends on the chemical composition of the membrane.25 It is also recommended to use a more stable ion-toelectron transducer layer, e.g. PEDOT(PSS–) in the design of the ISEs. Study of biofouling on PEDOT(PSS-) will be possible by using spectroscopic ellipsometry instead of null ellipsometry thus ensuring in-situ potentiometry with ellipsometry measurements. Solving the mentioned limitations will increase the capabilities of the proposed electrochemical-ellipsometric method to study fouling of any potentiometric sensors under various conditions. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS GL acknowledges financial support from the Rector of Åbo Akademi University via Åbo Akademis Jubileumsfond 1968- forskningssamarbete till Sverige. TR and TA acknowledge financial support from the Swedish Research Council and the Gustaf Th Ohlsson Foundation. Supporting information. Surface characterization of conditioned K+-ISEs, used in in-situ potentiometry and ellipsometry and potentiometric characteristics (off ellipsometry) of conventional solid-contact potassium electrodes before and after fouling by BSA.



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REFERENCES (1) M.C. Rayana, R.W. Burnett, A.K. Covington, P. D'Orazio, N. Fogh-Andersen, E. Jacobs, R., R. Kataky, W.R. Külpmann, K. Kuwa, L. Larsson, A. Lewenstam, A.H. Maas, G. Mager, J.W. Naskalski, A.O. Okorodudu, C. Ritter, A.St John, Clin. Chem. Lab. Med. 2006, 44, 346352. (2) A. Lewenstam, Electroanal. 2014, 26, 1171-1181. (3) R. Byrne and D. Diamond, Nat. Mater. 2006, 5, 421-424. (4) D. Diamond, S. Coyle, S. Scarmagnani, J. Hayes, Chem. Rev. 2008, 108, 652-679. (5) G. Lisak, A. Ivaska, A. Lewenstam, J. Bobacka, Electrochim. Acta 2014, 140, 27-32. (6) G. Lisak, T. Sokalski, J. Bobacka, L. Harju, A. Lewenstam, Talanta 2010, 83, 436-440. (7) E. Pungor, E. Schmidt and K. Toth, Proc. IMEKO 1968, pp.121-134. (8) J. Buffle and N. Parthasarathy, Anal. Chim. Acta 1977, 93, 111-120. (9) A. Lewnstam, A. Hulanicki and E. Ghali, in A. Ivaska, A. Lewenstam, R. Sara (Eds.), Contemporary Electroanalytical Chemistry, Plenum Press, New York and London 1988, pp. 213-222. (10)

V. Young, J. Microchem. 1990, 42, 25-36.

(11)

M. Pawlak, E. Grygolowicz-Pawlak, G. A. Crespo, G. Mistlberger, E. Bakker,

Electroanal. 2013, 25, 1840–1846. (12)

M.J. Berrocal, R.D. Johnson, I.H.A. Bader, M. Liu, D. Gao, L.G. Bachas, Anal. Chem.

2002, 74, 3644-3648. (13)

M. Pawlak, E. Bakker, Electroanal. 2013, 26, 1121-1131.

(14)

J. Cui, G. Lisak, S. Strzalkowska, J. Bobacka, Analyst 2014, 139, 2133-2136.

(15)

G. Lisak, J. Cui, J. Bobacka, Sens.and Actuat. B 2015, 207, 933-939.

(16)

G. Lisak, T. Arnebrant, T. Ruzgas, J. Bobacka, Anal. Chim. Acta, 2015, 877, 71-79.

(17)

X. Yang, O. Forouzan, T.P. Brown, S.S. Shevkoplyas, Lab Chip 2012, 12, 274-280.

(18)

R. De Marco, D.J. Mackey, A. Zirino, Electroanal. 1997, 4, 330-334.

(19)

M. Wagner, G. Lisak, A. Ivaska, J. Bobacka, Sens. and Actuat. B 2013, 181, 694-701.

(20)

A.K. Covington, D. Zhou, J. Electroanal. Chem. 1992, 341, 77-84.

(21)

Q. Ye, Z. Keresztes, G. Horvai, Electroanal. 1999, 10, 729-743.

(22)

A. Radu, S. Anastasova-Ivanova, B. Paczosa-Bator, M. Danielewski, J. Bobacka, A.

Lewenstam, D. Diamond, Anal. Methods 2010, 2, 1490-1498. 14 ACS Paragon Plus Environment

Page 15 of 17

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23)

Y. Zhang, Y. Fung, H. Sun, D. Zhu, S. Yao, Sen. and Actuat. B 2005, 108, 933-942.

(24)

C. Espadas-Torre, M.E. Meyerhoff, Anal. Chem. 1995, 67, 3108-3114.

(25)

C. Lim, S. Slack, S. Ufer, E. Lindner, Pure Appl. Chem. 2004, 76, 753–764.

(26)

J. Bobacka, A. Ivaska, A. Lewenstam, Chem. Rev. 2008, 108, 329-351.

(27)

R. Young, W.G. Pitt, S.L. Cooper, J. Colloid Interface Sci. 1988, 124, 28-43.

(28)

R.G.C. Oudshoorn, R.P.H. Kooyman, J. Greve, Thin Solid Films 1996, 284, 836-840.

(29)

K. Haberska, T. Ruzgas, Bioelectrochem. 2009, 76, 153-161.

(30)

S.E. Creager, G.K. Rowe, J. Electroanal. Chem. 1994, 370, 203-211.

(31)

M. Landgren, B. Joensson, J. Phys. Chem. 1993, 97, 1656-1660.

(32)

W. Ogieglo, H. Wormeester, K-J. Eichhorn, M. Wessling, N.E. Benes, Prog. Polym. Sci.

2015, 42, 42-78. (33)

T. Goda, E. Yamada, Y. Katayama, M. Tabata, A. Matsumoto, Biosens. Bioelectron.

2016, 77, 208-214. (34)

F.L. McCrackin, E. Passaglia, R.R. Stromberg, H.L. Steinberg, J. Res. Nat. Bur. Stand.

Sect. A 1963, 67a, 363-377. (35)

J. Vörös, Biophys. J. 2004, 84, 553-561.

(36)

D. Pankratov, J. Sotres, A. Barrantes, T. Arnebrant, S. Shleev, Langmuir 2014, 30, 2943-

2951. (37)

X. Li, S. Petrovic, D.J. Harrison, Sens. Actut. B 1990, 1, 275-280.

(38)

N. He, T. Lindfors, Anal. Chem. 2013, 85, 1006-1012.

(39)

A.J. Clark, L.A. Whitehead, C.A. Haynes, A. Kotlicki, Rev. Sci. Instrum. 2002, 73, 4339-

4346. (40)

C. Espadas-Torre, M.E. Meyerhoff, Anal. Chem. 1995, 67, 3108-3114.

(41)

A. Lewenstam, J. Solid State Electrochem. 2011, 15, 15-22.

(42)

X. Wang, G. Hertig, I.O. Wallinder, E. Blomberg, Langmuir 2014, 30, 13877-13889.

(43)

P. Katira, A. Agarwal, H. Hess, Adv. Mater. 2009, 21, 1599-1604.

(44)

J. Bobacka, Anal. Chem. 1999, 71, 4932-4937.

(45)

M. Guzinski, G. Lisak, T. Sokalski, J. Bobacka, A. Ivaska, M. Bochenska, A. Lewenstam,

Anal. Chem. 2013, 85 1555-1561. (46)

U. Abaci, H.Y. Guney, U. Kadiroglu, Electrochim. Acta 2013, 96, 214-224.

(47)

K.S. Kang, H.K. Lim, K.Y. Cho, K.J. Han, J. Kim, J. Phys. D: Appl. Phys. 2008, 41, 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 17

012003 (4pp). (48)

J.N. Hilfiker, R.A. Synowicki, J.A. Woollam, H.G. Tompkins, Proc. 51st Annual

Technical Conference, Society of Vacuum Coaters 2008, ISSN 0737-5921, 511-516. (49)

A.D.C. Chan, D.J. Harrison, Anal. Chem. Anal. Chem. 1993, 65, 32-36.

(50)

J. Kuhlmann, L.C. Dzugan, W.R. Heineman, Electroanal. 2012, 24, 1732-1738.

(51)

M. Guzinski, G. Lisak, J. Kupis, A. Jasinski, M. Bochenska, Anal. Chem. Acta 2013, 791,

1-12. (52)

V.G. Gavalas, M.J. Berrocal, L.G. Bachas, Anal. Bioanal. Chem. 2006, 384, 65-72.


Page 17 of 17

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