Spectrophotometric determination of various polyanions with polymeric film optodes using microtiter plate reader

Analytica Chimica Acta 699 (2011) 107–112

Contents lists available at ScienceDirect

Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

Spectrophotometric determination of various polyanions with polymeric film optodes using microtiter plate reader Nedime Dürüst a,∗ , Mark E. Meyerhoff b , Nazangül Ünal a , Sibel Nac¸ a a b

Department of Chemistry, Abant Izzet Baysal University, Gölköy, Bolu 14280, Turkey Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA

a r t i c l e

i n f o

Article history: Received 27 March 2011 Received in revised form 1 May 2011 Accepted 8 May 2011 Available online 14 May 2011 Keywords: Polyanion Thickening agent Polymeric film sensor Chromoionophore Microtiter plate-format optode

a b s t r a c t Polycation-sensitive membrane optodes based on the chromoionophore 2 ,7 -dichlorofluorescein octadecylester (DCFOE) have previously been developed and used for determination of heparin via a titrimetric method. In this study, it is shown that some other important polyanions such as PPS (pentosan polysulfate), DNA, xanthan, Na-alginate, and carrageenan (food additive) can also be readily determined by using DCFOE-based microtiter plate-format optodes (MPOs) and polycationic titrants that bind these polyanionic species. The optical sensors are prepared with poly(vinyl chloride) (PVC), polyurethane (PU), bis(2-ethylhexyl)sebacate (DOS), and 2 ,7 -dichlorofluorescein octadecylester (DCFOE) and exhibit reproducible and sensitive absorbance changes in response to the varying polycationic titrant concentrations. Three different polycations; protamine, poly-l-lysine and poly-l-arginine, are employed as titrants. The method has a detection limit of 1 ␮g mL−1 , and a dynamic range of 1–40 ␮g mL−1 . After the quantitative determinations are successfully demonstrated in buffered solutions, similar titrations are also performed in real samples. The method is validated by recovery studies in these samples. The average polyanion recoveries were quantitative [99.7(±1.3) % for pastry cream with vanillin (protamine titrant); 100.4 (±3.3) % for pastry gel with strawberry(PLA titrant), and 102.9(±2.0) % for pastry gel with strawberry (PLL titrant)]. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Multiple polyionic species can be frequently found in various areas of science and technology, and a great number of these species play meaningful roles (e.g., heparin, a widely used anticoagulant; carrageenen, a food additive for thickening; pentosan polysulfate, a potential anti-inflammatory drug, etc.). In 1992, it was found that polyionic species can be detected readily with polyion-sensitive polymeric membrane electrodes. The pioneering work to develop electrochemical polyion-sensitive electrodes (PSEs) into a practical analytical tool was initiated by Ma et al. [1]. They showed that typical polymeric membranes doped with a lipophilic ionexchanger, e.g. tridodecylmethylammonium chloride (TDMAC), can display significant EMF response to heparin in physiological buffers and in blood. Subsequently, polymeric membranes doped with TDMAC or dinonylnaphthalene sulfonate (DNNS) as ionexchangers have been shown to elicit significant non-equilibrium potentiometric responses to low concentrations (∼ 0.2 ␮M) of biologically important polyanions and polycations, respectively, such as the anticoagulant heparin and its polycationic antidote

∗ Corresponding author. Tel.: +90 374 254 1262; fax: +90 374 253 4642. E-mail address: durust [email protected] (N. Dürüst). 0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2011.05.007

protamine [2–5]. A significant number of additional publications further focused on better characterizing these unique electrochemical sensors, and applying them for various applications [6–27]. Over the last 15 years, the films used to prepare polymer membrane-type ion-selective electrodes (ISEs) have also been adapted successfully to develop new optical polyion-selective sensors. These optical sensors were developed from plasticized poly(vinyl chloride) films by casting on glass plates. It has been reported that an anion exchanger and a pH dye (ETH 2412) can be used in plasticized poly(vinyl chloride) films to detect heparin [28]. Wang et al. synthesized 2 ,7 -dichlorofluorescein octadecylester (DCFOE) as a chromoionophore, and found that polycation-sensitive optical films can be prepared without using both an ion-exchanger and a pH dye. They used this film for the optical determination of heparin via a titration approach [29]. Kim et al. [30] have adapted the PVC-based optode membranes containing a lipophilic anion-exchanger, TDMAC, and a lipophilic pH indicator, ETH 2412, to microtiter plate-format. They used this type of optode for quantitative determination of heparin levels in serum and compared the results from proposed methods and conventional enzymatic heparin assay [31]. Meyerhoff’s group have employed the DCFOE-based films within a microtiter plate-format for detection of heparin via a titrimetric determination. It was also possible to use the sensing films for monitoring given protease

108

N. Dürüst et al. / Analytica Chimica Acta 699 (2011) 107–112

enzymatic activities [32]. The utility of microtiter plate-format has also been reported for carrying out protamine/heparin titrations by using DNNS as a cation-exchanger and 9-(diethylamino)-5octadecanoylimino-5H-benzo[␣]-phenoxazine, ETH 5294, as a pH indicator dye [33]. Beyond heparin, there are a host of other polyanionic species for which the same optical sensing films may be useful for analytical purposes. Pentosan polysulfate (PPS), one of the polyanions targets which was selected in this work, is already being used as a new drug to treat bladder inflammation in medicine and as anti-arthritis agent [34–38]. DNA oligomers are known to be polyanion and can interact strongly with polycationic species. Hence, total DNA determination should be possible by titrimetry with polycationic species and measuring the first excess of titrant with polycation optical sensing films. Other polyanions are used routinely as thickening agents widely in foods such as dairy products, pudding, ice-cream, sauce, soup, jam and sausage and in the pharmaceutical industries. These agents are also frequently employed in tooth pastes, in drugs and in cosmetics as fillers, as well as homogenizing gel forming and strengthening materials in various products. Excess ingestion of these substances can be harmful to the digestive system. As mentioned above, pentosan polysulfate (PPS) is a highly sulfated polyionic species and an anti-osteoarthritis drug chemically derived from a natural polysaccharide [36]. It also has other numerous pharmacological activities. For example, it has been reported that PPS can inhibit the growth of tumors in animals and it can exhibit anticoagulant activity [39–41]. In addition, this drug has been found to show anti-HIV activity [42]. We have previously determined the PPS quantitatively in samples of plasma by polyionselective membrane electrodes [43]. However, to date, there is no report regarding the optical determination of PPS. The potential analytical utility of the potentiometric polyion sensors for detecting DNA, and for studying its interaction with protamine have been examined previously [44]. Buchanan et al. also examined the potentiometric response characteristics of polycation-sensitive membrane electrodes toward two classes of polycationic dendrimers; poly(amidoamine) (PAMAM) and poly (propylenimine) (PPI) [45]. The application of these potentiometric sensors for determining carrageenan via a potentiometric titration with polycations was also reported [46]. Herein, we report the quantitative determination of five different polyanionic analytes (DNA, PPS, carrageenan, xanthan, and alginate) using DCFOE-based microtiter plate-format optode and the reaction of these polyanions with given polycationic titrants. We also demonstrate the analytical utility of microtiter plate-format optodes (MPOs) for determination of some of these polyanions in food products. The mass ratios between the determined polyanions and titrant polycations are also assessed. We use the MPO method in this work, since it has the following advantages over other spectrophometric sensing systems. Each microtiter plate has 96 wells, and thus in comparison with a routine spectrophotometric analysis it is obvious that we can measure absorbances 96 times more rapidly. We can also consider this as time and sample savings since only 300 ␮L aliquots of sample solutions are placed in the wells. In addition, coating the uniform films on the glass slide is too difficult for normal spectrophometric studies. In the MPO method, the films can be prepared uniformly by casting 10 ␮L into each well.

2. Experimental 2.1. Chemicals and apparatus 2 ,7 -dichlorofluorescein (DCF), 1-iodooctadecane, bis(2ethylhexyl) sebacate (DOS), high-molecular weight poly(vinyl

chloride) (PVC), protamine sulfate (from Herring, grade III), polyl-arginine hydrochloride, MW = 5000–15,000 (PLA), poly-l-lysine hydrochloride, MW = 15,000–30,000 (PLL), deoxyribonucleic acid (DNA) [Sodium salt, from Salmon testes, is a double-stranded (ds) molecule, contains 260 units/mg solid], HEPES [N-(2-hydroxyethyl) piperazine-N -(2-ethanesulfonic acid)], TRIS [tris[hydroxymethyl] aminomethane], and tetrahydrofuran (THF) were obtained from Sigma-Aldrich Chemical Co. ␭-carrageenan, xanthan, alginate, and polyurethane-SG-80A (PU) were purchased from Fluka. Carrageenan, xanthan, and alginate are polyanions and their exact molecular weight are not known. Condensed formulas are (C12 H17 O19 S3 )n , (C35 H49 O29 )n , and (C6 H7 O6 Na)n , respectively. The sodium salt of pentosan polysulfate, PPS (Molecular weight is 5700 Da) was kindly provided by Dr. Peter Ghosh (Royal North Shore Hospital, Sydney, Australia). 2 ,7 -Dichlorofluorescein octadecylester (DCFOE) was synthesized as described by Wang et al. [29]. The buffer solution used in all experiments was 50 mM Tris–HCl (or HEPES), pH 7.4, containing 120 mM NaCl. Other reagents were analytical grade. All solutions were prepared using 0.055 ␮S cm−1 deionized water produced by a Tka Smart 2 Pure water purification system. A UV–VIS double-beam spectrophotometer (Hitachi U-2900) was used for preliminary spectrophotometric studies. All titrations were performed by using a microtiter plate absorbance reader (Molecular Devices Versamax Model). A Thermo Scientific Orion 4 Star pH/ISE meter was used to adjust the pH of buffer solutions. 2.2. Preparation of film optodes Polycation-sensitive and microtiter plate-format films were prepared as previously described [29,32]. The film composition was 1 wt.% polycation-sensitive chromoionophore (DCFOE), 30 wt.% polymer (15 wt.% PVC + 15 wt.% PU), and 69 wt.% DOS (as plasticizer). The cocktail solution was prepared by dissolving a total amount of 200 mg of film components in 2 mL of freshly distilled THF. This solution was then uniformly dispensed (10 ␮L/well) into each U-bottomed microwell of microtiter plates by means of a multipette dispenser with combitips. The thickness of the membranes in the wells is ca. 20 ␮m. Microtiter plates used were polypropylene-based. The microtiter plate-format films were dried in a dust-free environment for 1 day. Lifetimes of the plates are at least one week. 2.3. Titration of target polyanions using microtiter plate-format optodes All the wells of microtiter plates were filled in with 300 ␮L buffer (Tris–HCl or HEPES, pH 7.4) and equilibrated in buffer until the polymer film-coated wells had a stable absorbance value. This waiting time was at least 20 min. Absorbance measurements of optode films in buffer were made at 540 and 620 nm. Titrant solutions were prepared from their stock solution by appropriate dilution. Titration of each analyte polyanion was carried out by adding given aliquots of polycationic standard solutions to a series of separate eppendorf plastic tubes containing a fixed volume of a given polyanion solutions. Each analyte-titrant mixture in plastic tubes were prepared as 1000 ␮L total volume, so that the final mixture can be assayed using three different wells of the polycation sensitive microtiter plate. Before these solutions were transferred to the wells, they were shaken thoroughly (for 3–5 min). Then, 300 ␮L aliquots of the polycationic titrant-target polyanion solutions were transferred into each well of a microtiter plate coated with the polycation-sensitive film optode to determine the unbound polycationic titrant level. After all the wells were filled in with these solution mixtures, they were incubated for at least 3 min (in the case of poly-l-arginine and protamine titrants),

N. Dürüst et al. / Analytica Chimica Acta 699 (2011) 107–112

109

or 20 min in the case of poly-l-lysine as the titrant. Then wells were emptied and filled in with plain buffer solution again and the absorbances were recorded in buffer solution at same wavelengths. All measurements and pre-equilibrations were carried out at ambient temperature (∼25 ◦ C). Using the method outlined above, each of the target polyanions (PPS, DNA, ␭-carrageenan, xanthan, and alginate) were titrated by using microtiter plateformat optodes. Related spectrophotometric titration curves were prepared by plotting the total absorbance change from the baseline value (A) vs. the concentration of the polycationic titrant solutions (i.e., protamine, poly-l-arginine, and poly-l-lysine). The proportional titration curves were obtained for various concentrations of all target polyanions using the three different polycationic titrants. The mass ratios between the five target polyanions and three polycationic titrants were estimated from the end-points of the titration curves. 2.4. Analysis of samples Several real samples containing target polyanions were analyzed. Known weights of the samples (0.50–5.00 g) were dissolved completely in 100 mL of ultrapure deionized water and titrated with the polycationic titrants using the DCFOE-based microtiter plate-format optodes as the endpoint detection system. Polyanion concentrations present in samples were found by using the endpoints of the titration curves and stoichiometries calculated from titration curves of standard polyanion solutions. Recovery studies were also carried out in samples containing the target polyanions. The ability of this method to detect the polyanions in some samples was assessed by spiking food product samples with varying levels of the polyanions. For recovery studies, two different food product sample were tested by using protamine, PLA, and PLL as titrants. 3. Results and discussion 3.1. Response of film to polycationic titrants DCFOE-based PU/PVC membrane film itself has maximal absorbance at 490 nm at pH 7.40 but this value shifts to longer wavelength, to ∼540 nm, in the presence of polycations. All preliminary experiments were carried out using the UV–VIS spectrophotometer and then all absorbance changes for titrations were measured at this wavelength using the microtiter plate reader. Figs. 1 and 2 show the maximum absorption shifts of the films in the presence and absence of titrant polycations. The colorless polycationic titrant which remained from the consumption by the polyanionic analyte converts the initial yellow color of film to pink. At that moment, the interaction between the polycationic titrant and polycation-sensitive dye (DCFOE) begins and therefore the absorbance values increase. This color change in the microplate wells is shown visually in Fig. 3.

Fig. 1. Absorption spectra of the film optode before (left) and after (right) exposed to titrant poly-l-arginine (500 ␮g mL−1 ) at pH 7.4.

There are no reported data regarding the stoichiometries for the polyanions xanthan and alginate. 3.3. Analysis of some food products containing polyanions as additive After having been obtained reproducible results for each target polyanion in the buffered solutions, efforts were made to assess the ability to determine certain target polyanions in the real samples using the titrimetric approach with optical detection of the polycation/polyanion endpoint. The amount of polyanions present in the some food product samples was determined using this approach. Solutions of food product samples containing polyanions were prepared at given concentrations (0.50–5.00 g sample/100 mL) by shaking well and filtering under vacuum. These sample solutions were then titrated with the different polycationic titrants. Varying polyanion concentrations were calculated from the end-point break of the titration curves. Eight different product samples;

3.2. Titration of target polyanions The target polyanions were determined by using DCFOE-based polycation-sensitive optodes with the titrants protamine, poly-larginine, and poly-l-lysine. Three of the typical spectrophotometric titration curves obtained for varying levels of PPS, DNA, and Alginate in the buffered saline are shown in Figs. 4–6, respectively. Proportional end-points were obtained from titration curves for each analyte. The reaction stoichiometries between the analytes and the titrants were obtained from end-points of titration curves and are summarized in the Table 1. The stoichiometries for DNA, PPS and carrageenan are in good agreement with the reported values [43,44,46] that were obtained by the prior potentiometric membrane electrode-based method.

Fig. 2. Absorption spectra of the film optode before (left) and after (right) exposed to titrant poly-l-lysine (100 ␮g mL−1 ) at pH 7.4.

110

N. Dürüst et al. / Analytica Chimica Acta 699 (2011) 107–112

Table 1 The mass ratios obtained from titration curves of different levels of polyanions using polycation-sensitive film optodes. Polycations

Polyanions

Protamine (␮g) Poly-l-arginine (␮g) Poly-l-lysine (␮g) a

PPS (␮g) [Polycation/polyanion]a

DNA (␮g)

Carr. (␮g)

Xanthan (␮g)

Alginate (␮g)

1.41 (±0.09) 1.10 (±0.08) 0.96 (±0.04)

1.18 (±0.03) 0.62 (±0.02) 0.44 (±0.02)

1.12 (±0.05) 0.93 (±0.06) 0.70 (±0.05)

0.42 (±0.03) 0.34 (±0.04) 0.32 (±0.04)

1.22 (±0.04) 0.94 (±0.05) 0.82 (±0.05)

Average of six measurements.

0,35 0,3

∆A

0,25 0,2 0,15

0 μg/mL DNA 5 μg/mL DNA

0,1

10 μg/mLDNA 20 μg/mL DNA

0,05

40 μg/mL DNA

0 Fig. 3. Wiew of the microplate after the titrations of 0 ␮g mL−1 Carr. (first half of the plate) and 10 ␮g mL−1 Carr. (second half of the plate) with protamine at 540 nm. Yellow wells: polycationic titrant is interacting with polyanionic analyte. Orange wells: interaction between polycationic titrant and polyanionic analyte has already finished and excess of the titrant is now beginning to interact with polycationsensitive film. Pink wells: the higher concentrations of the titrant is interacting with much more amount of dye. (Each group of three wells contain same concentration of titrant.). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

coconut pudding, hot chocolate, chocolate sauce, pastry cream with cocoa, banana milk, banana pudding, pastry cream with vanilla, and pastry gel with strawberry were purchased from the local markets and were tested. Total polyanions amounts in these samples were measured by the spectrophotometric titration method using polycation-sensitive DCFOE-based microtiter plate-format optodes. The results are expressed as % m m−1 (g polyanion/100 g product sample) and are shown in Table 2. The samples assayed

0

10

20

30

40

50

60

70

Poly-L-Lysine (μg/mL) Fig. 5. Spectrophotometric titration curves of 0.0 (䊉), 5 (), 10 (), 20 (), and 40 () ␮g mL−1 of DNA with standard poly-l-lysine.

contained either carrageenan, xanthan or alginate and this was indicated on the label, so that assessing the concentration of polyanion was possible since the stoichiometry for a given titrant with that polyanion in the food product was known. A representative graph related to the titration of coconut pudding containing xanthane is presented in Fig. 7. In order to investigate whether the sample matrix would have an effect on the determination, we spiked some real food product samples as well. The accuracy of the method was confirmed by these recovery experiments. For this purpose, the proportional amounts of related polyanion were added to the solutions of these food product samples. Two of the studied food product samples, pastry cream with vanillin containing alginate and pastry gel with strawberry

0,25

0,35 0,3 0,25

0,15 0 μg/mL PPS

0,1

5 μg/mL PPS 10 μg/mL PPS

0,05

20 μg/mL PPS 40 μg/mL PPS

0

ΔA

ΔA

0,2

0,2 0,15

0 μg/mL Alginate 5 μg/mL Alginate

0,1

10 μg/mL Alginate 20 μg/mL Alginate

0,05

40 μg/mL Alginate

0

20

40

60

80

100

120

140

Protamine (μg/mL) Fig. 4. Spectrophotometric titration curves of 0.0 (䊉), 5 (), 10 (), 20 (), and 40 () ␮g mL−1 of PPS with standard protamine solution monitoring the change in absorbance at 540 nm.

0 0

20

40

60

80

100

120

Poly-L-Arginine (μg/mL) Fig. 6. Spectrophotometric titration curves of 0.0 (䊉), 5 (), 10 (), 20 (), and 40 () ␮g mL−1 of Alginate with standard poly-l-arginine.

N. Dürüst et al. / Analytica Chimica Acta 699 (2011) 107–112 Table 2 Thickening agents concentration levels measured in some food products using polycation- sensitive film optodes.

Carrageenan

Xanthan

b

Coconut pudding Hot chocolateb Chocolate sauceb Pastry cream with cocoab Banana milkb Banana puddingb Pastry cream with vanillinb Pastry gel with strawberryc a b c

1.52 (±0.08)

samples tested are in good agreement with the proportional amounts added of polyanions. The results obtained show average polyanion recoveries of 99.7 (±1.3) %, 100,4 (±3,3)%, and 102,9 (±2,0) %, for pastry cream with vanillin (titrated with protamine), pastry gel with strawberry (titrated with PLA), and pastry gel with strawberry (titrated with PLL) samples, respectively. The recovery of polyanions added to some real samples is quantitative. In this regard, we can conclude that the effect of the sample matrix is negligible based on the high recovery percentages.

1.72 (±0.22)

4. Conclusion

% Analyte (w w−1 )a

Food products

111

Alginate

0.08 (±0.00) 2.98(±0.20) 2.46 (±0.14) 0.40 (±0.02) 0.02 (±0.00) 2.63 (±0.19)

In summary, in this study clearly demonstrate the potential analytical applications of polyanion-sensitive microtiter plate-formate optodes (MPOs) for the measurement of polyanion analytes like PPS, DNA, carrageenan, xanthate, and alginate via a simple titrimetric approach. Microtiter plates and readers are essential equipment in all life sciences and analytical laboratories, and since microtiter plate-format optodes can provide high sample throughput (96 samples in less than 5 min), this new method should be attractive to many researchers and quality control chemists who need to measure levels of polyanions. In addition, relatively rapid titrations can be done by means of MPOs, and hence they are very appropriate for the routine analysis. The new method also provides good detection limits (around 1 ppm) for all the polyanion species tested.

Average of six measurements. Used titrant: protamine. Used titrant: poly-l-lysine.

0,6 0,5 0,4

∆A

0,3 0,2

Acknowledgements 0 μg/mL Coconut Pudding

0,1

40 mg/mL Coconut Pudding

0 0

20

40

60

80

100

120

140

160

We are extremely grateful to the TUBITAK (The Scientific and Technological Research Council of Turkey, Grant 108T109) and Abant Izzet Baysal University, Directorate of Research Projects Commission (BAP Grant 2007.03.03.258) for financial support.

Protamine (μg/mL) Fig. 7. A typical spectrophotometric titration curve for coconut pudding containing xanthan with standard protamine: blank (䊉), and sample ().

containing carrageenan, were examined. The former product was titrated with protamine and the latter product with poly-l-lysine and poly-l-arginine. Carrageenan percentages calculated from PLL and PLA titration curves of the related product have been found as 2.61 and 2.65, respectively and we were able to obtain almost the same values using the different titrants. For example, titration curves of recovery studies with PLL for the food product mentioned above are shown in Fig. 8. The data obtained for food product 0,35 0,3

∆A

0,25 0,2 0 μg/mL Sample

0,15

500 μg/mL Sample

0,1

+10 μg/mL Carr. +20 μg/mL Carr.

0,05

+40 μg/mL Carr.

0 0

10

20

30

40

50

60

70

80

90

Poly-L-Lysine (μg/mL) Fig. 8. Spectrophotometric titration curves obtained from recovery studies of pastry gel with strawberry containing polyanion 0.0 (䊉), sample (), +10 (), +20 (), and 40 () ␮g mL−1 of carrageenan with standard poly-l-lysine.

References [1] S.C. Ma, V.C. Yang, M.E. Meyerhoff, Anal. Chem. 64 (1992) 694–697. [2] M.E. Meyerhoff, V.C. Yang, J.A. Wahr, L. Lee, J.H. Yun, B. Fu, E. Bakker, Clin. Chem. 41 (1995) 1355–1356. [3] M.E. Meyerhoff, B. Fu, E. Bakker, V.C. Yang, J.H. Yun, Anal. Chem. 68 (1996) 168A–175A. [4] N. Ramamurthy, N. Baliga, J.A. Wahr, U. Schaller, V.C. Yang, M.E. Meyerhoff, Clin. Chem. 44 (1998) 606–613. [5] S. Dai, J.M. Esson, O. Lutze, N. Ramamurthy, V.C. Yang, M.E. Meyerhoff, J. Pharm. Biomed. Anal. 19 (1999) 1–14. [6] J.H. Yun, S.C. Ma, B. Fu, V.C. Yang, M.E. Meyerhoff, Electroanalysis 5 (1993) 719–724. [7] S.C. Ma, V.C. Yang, B. Fu, M.E. Meyerhoff, Anal. Chem. 65 (1993) 2078–2084. [8] B. Fu, E. Bakker, J.H. Yun, V.C. Yang, M.E. Meyerhoff, Anal. Chem. 66 (1994) 2250–2259. [9] J.A. Wahr, M.E. Meyerhoff, V.C. Yang, J.H. Yun, L.M. Lee, X.C. Lou, Anesthesiology 83 (1995) A425–A1425. [10] B. Fu, E. Bakker, J.H. Yun, E.J. Wang, V.C. Yang, M.E. Meyerhoff, Electroanalysis 7 (1995) 823–829. [11] J.H. Yun, M.E. Meyerhoff, V.C. Yang, Anal. Biochem. 224 (1995) 212–220. [12] J.A. Wahr, J.H. Yun, V.C. Yang, L.M. Lee, B. Fu, M.E. Meyerhoff, J. Cardiothorac. Vasc. Anesth. 10 (1996) 447–450. [13] B. Fu, J.H. Yun, J.A. Wahr, M.E. Meyerhoff, V.C. Yang, Adv. Drug Deliver. Rev. 21 (1996) 215–223. [14] I.H.A. Badr, N. Ramamurthy, V.C. Yang, M.E. Meyerhoff, Anal. Biochem. 250 (1997) 74–81. [15] M. Dror, R.F. Baugh, A. Armstrong, V.C. Yang, M.E. Meyerhoff, Thromb. Haemost. (1997) P1156. [16] J.M. Esson, M.E. Meyerhoff, Electroanalysis 9 (1997) 1325–1330. [17] N. Baliga, V.C. Yang, M.E. Meyerhoff, Clin. Chem. 44 (1998) A52–A53. [18] N. Ramamurthy, N. Baliga, T.W. Wakefield, P.C. Andrews, V.C. Yang, M.E. Meyerhoff, Anal. Biochem. 266 (1999) 116–124. [19] E. Malinowska, A. Manzoni, M.E. Meyerhoff, Anal. Chim. Acta 382 (1999) 265–275. [20] J.H. Yun, I.S. Han, L.C. Chang, N. Ramamurthy, M.E. Meyerhoff, V.C. Yang, Pharm. Sci. Technol. Today 2 (1999) 102–110. [21] J.M. Esson, N. Ramamurthy, M.E. Meyerhoff, Anal. Chim. Acta 404 (2000) 83–94. [22] Q.S. Ye, M.E. Meyerhoff, Anal. Chem. 73 (2001) 332–336. [23] H.S.M. Abd-Rabboh, S.A. Nevins, N. Durust, M.E. Meyerhoff, Biosens. Bioelectron. 18 (2003) 229–236.

112

N. Dürüst et al. / Analytica Chimica Acta 699 (2011) 107–112

[24] S.A. Buchanan, T.P. Kennedy, R.B. MacArthur, M.E. Meyerhoff, Anal. Biochem. 346 (2005) 241–245. [25] L. Wang, S. Buchanan, M.E. Meyerhoff, Anal. Chem. 80 (2008) 9845–9847. [26] L. Wang, M.E. Meyerhoff, Electroanalysis 22 (2010) 26–30. [27] K.L. Gemene, M.E. Meyerhoff, Anal. Chem. 82 (2010) 1612–1615. [28] E. Wang, M.E. Meyerhoff, V.C. Yang, Anal. Chem. 67 (1995) 522–527. [29] E. Wang, G. Wang, L. Ma, C.M. Stivanello, S. Lam, H. Patel, Anal. Chim. Acta 334 (1996) 139–147. [30] S.B. Kim, H.C. Cho, G.S. Cha, H. Nam, Anal. Chem. 70 (1998) 4860–4863. [31] S.B. Kim, T.Y. Kang, G.S. Cha, H. Nam, Anal. Chim. Acta 557 (2006) 117–122. [32] S. Dai, Q. Ye, E. Wang, M.E. Meyerhoff, Anal. Chem. 72 (2000) 3142–3149. [33] S.B. Kim, T.Y. Kang, H.C. Cho, M.H. Choi, G.S. Cha, H. Nam, Anal. Chim. Acta 439 (2001) 47–53. [34] V.R. Anderson, C.M. Perry, Drugs 66 (2006) 821–835. [35] M. Simon, R.H. McClanahan, J.F. Shah, T. Repko, N.B. Modi, Xenobiotica 35 (2005) 775–784.

[36] P. Ghosh, Sem. Arthritis Rheum. 28 (1999) 211–267. [37] P. Ghosh, J. Edelman, L. March, M. Smith, Curr. Ther. Res. Clin. E 66 (2005) 552–571. [38] S. Zaslau, S. Sparks, D. Riggs, B. Jackson, S.J. Kandzari, Am. J. Surg. 192 (2006) 640–643. [39] G. Zugmaier, M.E. Lippman, A. Wellstein, T. Vincent, J. Natl. Cancer Inst. 84 (1992) 1716–1724. [40] J.L. Marshall, A. Wellstein, J. Rae, R.J. Delap, K. Phipps, M.K. Hanfelt, J. Yunmbam, K.L. Xun, M.J. Duchin, Hawkins, Clin. Cancer Res. 3 (1997) 2347–2354. [41] P. Kongtawelert, P. Ghosh, J. Immunol. Methods 126 (1990) 39–49. [42] A.K. Srivastava, R.P. Sekaly, J.L. Chiasson, Mol Cell Biochem. 120 (1993) 127–133. [43] N. Dürüst, M.E. Meyerhoff, Anal. Chim. Acta 432 (2001) 253–260. [44] N. Dürüst, M.E. Meyerhoff, J. Electroanal. Chem. 602 (2007) 138–141. [45] S.A.N. Buchanan, L.P. Balogh, M.E. Meyerhoff, Anal. Chem. 76 (2004) 1474–1482. [46] S.S.M. Hassan, M.E. Meyerhoff, I.H.A. Badr, H.S.M. Abd-Rabbah, Electroanalysis 14 (2002) 439–444.