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Detection of metoprolol in human biofluids and pharmaceuticals via ion-transfer voltammetry at the nanoscopic liquid-liquid interface array Xiao Huang, Lisiqi Xie, Xingyu Lin, and Bin Su Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04099 • Publication Date (Web): 29 Nov 2016 Downloaded from http://pubs.acs.org on November 30, 2016
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Analytical Chemistry
Detection of metoprolol in human biofluids and pharmaceuticals via ion-transfer voltammetry at the nanoscopic liquid-liquid interface array Xiao Huang, Lisiqi Xie, Xingyu Lin and Bin Su* Institute of Analytical Chemistry, Department of Chemistry, Zhejiang University, Hangzhou 310058, China ABSTRACT: Metoprolol (MTP) is one of the most widely used antihypertensive drugs yet banned to use in sport competition. Therefore there has been an increasing demand for developing simple, rapid and sensitive methods suited to the identification and quantification of MTP in human biofluids. In this work, ultrathin silica nanochannel membrane (SNM) with perforated channels was employed to support nanoscale liquid/liquid interface (nano-ITIES) array for investigation of the ion-transfer voltammetric behavior of MTP and for its detection in multiple human biofluids and pharmaceutical formulation. Several potential interfering substances, including small molecules, D-glucose, urea, ascorbic acid, glycine, magnesium chloride, sodium sulfate and large molecules, bovine serum albumin (BSA), were chosen as models of biological interferents to examine their influence on the ion-transfer current signal of MTP. The results confirmed that the steady-state current wave barely changed in the presence of small molecules. Although BSA displayed an apparent blockade on the transfer of MTP, the accurate determination of MTP in multiple human biofluids (i.e. urine, serum and whole blood) and pharmaceutical formulation were still feasible, thanks to the molecular sieving and antifouling abilities of SNM. A limit of detection (LOD) within the physiological level of MTP during therapy could be achieved for all cases, i.e. 0.5 and 1.1 M for 100 times diluted urine and serum, respectively, and 2.2 M for 1000 times diluted blood samples. These results demonstrated that the nano-ITIES array behaved as a simplified and integrated detection platform for ionisable drug analysis in complex media.
For more than a half century, -blockers (e.g. atenolol, metoprolol, esmolol, and propranolol) have been playing important roles in the therapy of various cardiovascular diseases, such as hypertension, cardiac arrhythmias, angina pectoris, myocardial infarction and heart neurosis.1-3 Among them, metoprolol (MTP) is one of the most widely used antihypertensive drugs. It was marketed in Sweden in 1974 and also the first systemic -blocker allowed in the United States.3 In comparison with atenolol and esmolol, MTP has an obvious advantage in decreasing the side effects, for instances, triggering bronchospasm and delaying hypoglycemia to recovery.1 On the other hand, -blockers have been forbidden to use in sport activities by the International Olympic Committee due to its misuse as doping agents. Thus, the determination of blockers has vital practical meanings, and it is important to develop simple, sensitive and selective analytical methods suited to human biofluids and pharmaceutical formulations. So far, numerous methods have been developed for the determination of MTP in different media, such as pharmaceutical preparations, human urine, serum, plasma and blood.4-8 Among them, chromatography is one of the core technologies, in conjunction with using different detection techniques, such as mass spectrometry (MS),5,7,8 ultraviolet spectrophotometry (UV),6,9,10 and
fluorescence.11 However, these techniques are usually expensive and require time-consuming pre-separation or pre-treatments. Modern electrochemical techniques have attracted more and more interests due to its low cost and high sensitivity. However, only a few electrochemical methods have been reported so far to determine – blocker.12-15 For examples, Armstrong et al. have used capillary electrophoresis (CE) together with macrocyclic antibiotic, rifamycin B, to separate and detect chiral amino alcohols.14 Wang et al. have coupled CE with electrochemiluminescence to detect three -blockers in pharmaceuticals and human urine, and the detection limit of MTP reached to 0.1 M.15 Srivastava et al. have reported that a nafion-carbon nanotube nanocomposite modified glassy carbon electrode can be utilized for the determination of MTP using cyclic voltammetry (CV) and adsorptive stripping differential pulse voltammetry.12 Electrochemistry at the liquid/liquid interface, or socalled the interface between two immiscible electrolyte solutions (ITIES), has received increasing attentions as a facile approach to detect a wide range of ions,16-27 such as dopamine,22,23 propranolol,24,25,28 daunorubicin,26 and topotecan.27 By miniaturizing the ITIES, the analytical performance can be remarkably improved with an enhanced analytical sensitivity and decreased limit of detection (LOD).16,29 In the case of a nanoscopic ITIES
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(nano-ITIES), the sensing capacity can be further improved.30 At a sufficiently small nano-ITIES, ion transfer will be dominated by the molecular properties, such as size and charge. Recently, we have developed a novel method to build nano-ITIES array with the support of ultrathin silica nanochannel membrane (SNM).31,32,33 In this work, we demonstrated this nano-ITIES array was capable of detecting MTP (MTP has a pKa of 9.5 and is a cation at the physiological pH,6 see its chemical structure in Figure S1) in complex samples. The ion-transfer voltammetric behavior of MTP was firstly investigated by CV. Then we studied the interference of various substances, including D-glucose, urea, ascorbic acid (AA), glycine, MgCl2, Na2SO4 and albumin from bovine serum (BSA), on the sensing signals of MTP. Finally, on the basis of the size-exclusion effect, excellent anti-fouling and anti-interference ability of SNM arising from its high density of perforated nanochannels (2 3 nm in diameter),32,34 the nano-ITIES array was utilized for the determination of MTP in human biofluids (i.e. urine, serum and blood) and pharmaceutical formulations without pre-treatments.
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as shown in Scheme 1a. A U-tube cell was used (see details in Figure S2a) and the electrolyte compositions are illustrated in Scheme 1b. The Galvani potential difference ( ow ) across the water/1,2-dichloroethane (DCE) interface was calibrated by considering the formal transfer potential of TMA+ as 0.16 V.37 The CV recorded during the second potential cycling scan was shown and the scan rate was 0.02 V s1, unless otherwise specified. Scheme 1. Illustration of the SNM/p-SiNF supported nano-ITIES (a) and the electrochemical cell composition (b).
EXPERIMENTAL SECTION Chemicals and Reagents. All chemicals were used as received without further purification. MTP tartrate salt (99%) was bought from Meyer. The artificial serum was composed of 1.5 mM KCl, 5.0 mM CaCl2, 1.6 mM MgCl2, 1.0 mM NaH2PO4, 1.0 mM KH2PO4, 4.7 mM D-glucose, 2.5 mM urea and 0.6 mM BSA.35 Betaloc-25 tablets containing MTP tartrate (25 mg/tablet) were purchased from GuoDa Drugstore. Normal human serum and blood (stored in heparin sodium salt-anticoagulant tubes) were obtained from the Zhejiang University Hospital. Urine was donated by normal volunteers. Urine and human serum (or blood) samples were diluted by the aqueous solution containing 10 mM LiCl and the BSA-free artificial serum, respectively, prior to electrochemical analysis. Bis(triphenylphosphor anylidene)ammonium tetrakis(pentafluorophenyl)borate (BATB) was prepared as reported previously.36 Indium tin oxide (ITO) coated glass ( 70%) are ionisable under the physiological condition, iontransfer voltammetry at the liquid/liquid interface has manifested itself to be a facile approach to determine the partition coefficient.40 The partition coefficient of MTP between water and DCE, namely log PMTP , is given by,
log PMTP
w o Gtr,0 ,MTP
2.3RT
(2)
where R is the gas constant and T the absolute temperature. The calculated value of log PMTP is 2.37. Considering the physical structure of SNM/p-SiNF and the interface position, the overall ITIES can be equivalent to an array of micro-ITIES, each of which further consists of an array of nano-ITIES. Due to the high density of nanochannels (i.e. 4.0 104 m2) in the SNM, the radial diffusion fields at the adjacent nanochannel orifices strongly overlap. However, the linear diffusion induced by this overlap (that leads to a peak-shaped voltammogram) was not reflected in Figure 2, because of the dominance of radial diffusion at the edge of each SiN micropores.41 Thanks to a large ratio of pore-to-pore separation versus radius of SiN micropores (d/ra = 75/2.5 = 30), the molecular diffusion to each micropores occurs independently. Note that the linear diffusion of molecules inside SiN micropores and silica nanochannels are negligible, given both p-SiNF and SNM are ultrathin (150 nm and 80 nm, respectively).31,32 In this case, the overall ITIES behaves as an inlaid microdisc electrode.42 The steady-state current is given by,43
Figure 2. (a) CVs at the nano-ITIES array using Cell 1 (y = 0) illustrated in Scheme 1b: x = 0 (black curve), x = 100 (red curve). (b) Background-subtracted CVs (forward scan only) at different concentrations of MTP: x = 1, 3, 5, 10, 30, 50, 100, 200, 300, 400, 500 and 1000 (from bottom to top), using Cell 1 (y = 0) in Scheme 1b. The inset is the linear fitting of the steady-state current (+0.23 V) versus the MTP concentration.
Voltammetric Behavior of MTP at the Nano-ITIES Array. Figure 2a compares CVs obtained using Cell 1 in Scheme 1b in the absence and presence of 100 M MTP in the aqueous phase containing 10 mM LiCl. The transfer of Li+ and Cl from aqueous phase to DCE, respectively, at the positive and negative limits determined a background potential window, ranging from 0.28 to +0.42 V (see the black curve). After addition of MTP in the aqueous solution, both the forward transfer of MTP from water to DCE and the reverse one produced well-defined sigmoidal responses or the so-called symmetric steady-state limiting current wave. The shape of voltammetric wave depends on the geometry of diffusion field at the interface, which is primarily determined by the interface position within the channels/pores and the pore-to-pore separation. As we have reported recently,31 it is likely that the aqueous phase permeates silica nanochannels to get contact with the organic phase at the orifices of silica nanochannels on the other end, forming nano-ITIES array at the boundary between SNM and p-SiNF (see Scheme 1a). The standard
Iss 4nNm FDcr
(3)
where D and c are the molecular diffusion coefficient and the bulk concentration in the aqueous phase. r is the radius of micropore. Nm is the number of micropores and equal to 16 in the present case. Figure 2b showed that the steady-state current after background subtraction increased with increasing the concentration of MTP. From the linear fitting (see the inset of Figure 2b), the apparent diffusion coefficient of MTP was estimated to be 3.24 105 cm2 s1 according to eq. 1. This value is close to that reported previously, i.e. 2.26 105 cm2 s1.38 In addition, the linear fitting also yielded the analytical sensitivity and limit of detection (LOD), namely 6.5 105 nA M1 m2 and 0.5 M, as shown in Table 1. Note that the LOD here is described as the analyte concentration that gives a Faradaic current signal significantly different from the background (3 times the standard deviation of
transfer potential of MTP ( o MTP ) at the water/DCE interface obtained from the CVs in Figure 2a is +0.14 V, which is in good agreement with that reported previously.38 The formal Gibbs transfer energy of MTP w
w o Gtr,0 ,MTP
0
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the blank different). The analytical sensitivity is defined as the slope of the plot of current density, namely the current normalized by the geometric area of the electrified interface, versus the analyte concentration. Influence of Different Interferents on the MTP Transfer. Different substances, including D-glucose, urea, ascorbic acid (AA), glycine, MgCl2 and Na2SO4, were selected as models of interferents to examine their effect on the detection of MTP in biological samples. Figure S3 compares CVs of the background and of 100 M MTP at the nano-ITIES array in the absence and presence of respective interferents at a concentration of 5.0 mM (using Cell 1 in Scheme 1b, x = 100, y = 5). D-glucose was chosen as a model of sugar interferents. Since it is neutral and hence does not influence the transfer of MTP (see Figure S3a). As the main organics in urine, urea was also investigated and displayed no influence on the signal of MTP (see Figure S3b), because it is also a neutral compound.44 With a pKa of 4.17,26 AA was deprotonated in the aqueous phase, however no transfer signal of AA was observed (see Figure S3c) since it is very hydrophilic. Glycine was used as a model of amino acid interferents and has been widely reported in interference studies.26 At the physiological pH, glycine is also neutral, hence it does not influence the transfer of MTP either (see Figure S3d). Note that amino acids can be protonated a low pH (pH 1.0)45 and then transfer across the ITIES in the presence of an ionophore. The possible interference of metal ions, such as Mg2+ and Na+, were also evaluated. The addition of MgCl2 and Na2SO4 did not introduce any new current signal but a decrease of the potential window, in agreement with previous studies.26,46 The changes of steady-state current magnitude in the presence of different interferents, defined as the relative difference (see Table 2 footnote), were in the range of 0.6 to 7% (as
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summarized in Table 2), indicating that their influence on the voltammetric determination of MTP is negligible. Other important interfering substances are proteins, because they widely exist in human biofluids. Here, BSA was selected as the model, which is a water-soluble globular protein and has charges of 17 at the physiological pH (~7.4).47 The CVs were recorded using the Cell 2 in Scheme 1b. Two types of aqueous solutions, namely the artificial serum and BSA-free artificial serum, were employed. Figure S4 showed CVs in the presence of different concentrations of MTP in the BSA-free artificial serum. The obtained analytical sensitivity and LOD are comparable with that in the electrolyte solution (see Table 1). As shown in Figure S5, the presence of BSA in the artificial serum did not introduce additional Faradaic current (see the dotted lines). But the capacitive current apparently increased in the whole potential window, which most likely arose from the adsorption of BSA molecules on the SNM surface. As reported previously,48 the adsorption of BSA on silica surface is energetically favorable and kinetically fast. Although both BSA and silica (due to deprotonation of silanol groups with an isoelectric point of 2 3)49 are negatively charged at the physiological pH, the BSA-silica interaction still remains strong. The adsorbed BSA can physically block the nanopores, thus impeding the entrance of MTP to nanochannels and the subsequent transfer across the nano-ITIES. Indeed, the current due to MTP transfer was reduced by ~2 nA (~17%), as shown by the solid curves in Figure S5. Note that the current decrease may also arise from the binding of positively charged -blockers with adsorbed BSA.50 As reported previously,50 the -blockersBSA interaction is strong and the formation of -blockers -BSA complex is spontaneous.
Table 1. Analytical sensitivity and LOD derived from the linear fitting of concentration dependence of the steady-state current Analysis System LiCl BSA-free artificial serum a urine b serum b blood c blood
Linear Range (M) 1-1000 50-500 50-500 50-500 50-500 50-500
Sensitivity (nA M m ) 5 6.5 10 5 5.5 10 5 6.5 10 5 2.8 10 5 0.3 10 5 1.4 10 1
2
LOD (M) 0.5 0.5 0.5 1.1 9.5 2.2
Therapeutic range (M)
1
0.072-1.81
a
diluted by 100 times with the aqueous solution of 10 mM LiCl diluted by 100 times with BSA-free artificial serum c diluted by 1000 times with BSA-free artificial serum b
Table 2. Summary of the influence on the detection of MTP over different interfering substances.
Interfering substances a Relative difference (%) a
D-glucose 1.1
Urea 0.6
Ascorbic acid 2.8
Glycine 3.3
MgCl2 5.5
Na2SO4 6.6
BSA 31
Relative difference (%) = (I ssMTP+Y I ssMTP )/ I ssMTP
Determination of MTP in Biological Samples. In pharmacokinetic and biopharmaceutical measurements, separation of analytes from matrices and detection at a low concentration level are of urgent requirement. Here the nano-ITIES array supported by the SNM/p-SiNF was
utilized to detect MTP in human biofluids, including urine, serum and blood. After the oral administration, MTP undergoes extensive biotransformation and is excreted principally via the kidneys. Only about 3% of a dose is excreted as unchanged drug while about 10% after
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an intravenous dose.1 Urine of a healthy person contains trace concentration of proteins and a large amount of salts (e.g. Na+, Mg2+, NH4+, Cl), urea (20000 mg/L), creatinine (750 mg/L) and uric acid (500 mg/L).51 As demonstrated above, these substances do not affect significantly the transfer current of MTP. Figure 3a and 3b show CVs obtained at the nano-ITIES using the Cell 1 in Scheme 1b. Replacing 10 mM LiCl aqueous solution with urine led to a bit narrow potential window, in particular at the positive limit (see black dotted and solid curves), because of a higher electrolyte concentration in urine. After adding 0.1 mM MTP to urine, a steady-state current wave was displayed, similar to that observed in Figure 2a. The magnitude of this current wave apparently increased with the concentration of MTP (see Figure 3b). The sensitivity and LOD were also very close to those obtained in the pure electrolyte solution (see Table 1).
As the clinical manifestation demonstrated, only 11% of metoprolol is bound to human serum protein from its large volume of distribution (5.6 L/kg) after the oral administration.1,51,52 Proteins (e.g. albumin) have been reported to be a major source of interference for serum and plasma measurements using the liquid/liquid interface electrochemistry.26,53 Although the MTP transfer current decreased in the presence of BSA (see Figure S5), its detection in human serum and blood are still possible. As shown in Figure 3c, the current wave due to the MTP transfer was clearly observed for the human serum sample. Note that here the human serum was diluted by 100 times with the BSA-free artificial serum prior to the analysis in order to lower the background. Furthermore, it can be seen from Figure 3d that the current magnitude linearly increased with the continuous addition of MTP to the sample, yielding an analytical sensitivity of 2.8 105 nA M1 m2. This value was about half of that obtained with the BSA-free artificial serum (as compared in Table 1). The LOD obtained for the serum sample was 1.1 M, which is within the physiological level of MTP during therapy. Human blood is one of the most complex biological samples because it contains ~45% (volume percentage) blood cells (erythrocytes, leukocytes, thrombocytes) and ~55% plasma with various kinds of electrolytes and proteins (60 80 g/L).1 Here the human blood (containing heparin sodium salt as anticoagulant) was also diluted with the BSA-free artificial serum prior to electrochemical analysis. As shown in Figures 3e and 3f, steady-state ion transfer current waves were also observed for the blood sample diluted by 100 times, similar to those in human serum. And the current magnitude linearly increased with the concentration of MTP. But an even low analytical sensitivity, namely 0.3 105 nA M1 m2, was obtained, which was much smaller than that obtained for serum sample by nearly 10 times and that for the BSA-free artificial serum by ca. 20 times. The LOD deduced from Figure 3f was 9.5 M. Figures S6 showed CVs obtained in the blood sample diluted by 1000 times for comparison. Both analytical sensitivity and LOD were improved (see Table 1). Above voltammetric results indicate that in human serum and blood there exist serious matrix effect on the MTP transfer across the nano-ITIES array. This effect could be mainly ascribed to the high amount of cells, proteins and other biological compounds. These species may adsorb on the surface of SNM from the aqueous solution side to impose a blockade on the transport of ions across the liquid/liquid interface.54 On the other hand, aforementioned large-sized biological species can be physically excluded by the SNM, preventing the nano-ITIES from fouling or contamination. Indeed, as we have reported recently, the SNM has excellent molecular sieving ability and anti-fouling capacity, thanks to the ultrasmall size of nanochannels (namely 2 3 nm in diameter).54 And the SNM-modified electrodes have been demonstrated to allow the direct amperometric detection of small redox molecules in diverse complex/real media (such as soil dispersions, milk, serum and whole blood).54-59 We do expect the similar
Figure 3. (a, c, e) CVs obtained at the nano-ITIES array for human urine, serum and blood samples in the absence (black solid curves) and presence of 0.1 mM MTP (solid red curves) using Cell 2 in Scheme 1b (x = 100). The urine was diluted by 100 times with the aqueous solution containing 10 mM LiCl. The serum and blood were diluted by 100 times with the BSA-free artificial serum. CVs obtained with the aqueous solution containing 10 mM LiCl are also given for comparison (black dotted curves) using Cell 1 in Scheme 1b (x = 100, y = 0). (b, d, f) Background-subtracted CVs (forward scan only) of different concentrations of MTP in urine (b), human serum (d) and blood (f). The insets show the linear fitting of the steady-state current (+0.18 V) versus the MTP concentration.
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effects at the nano-ITIES array supported by the SNM (as exemplified in Scheme 1a), which make the detection of MTP possible via the ion-transfer voltammetry in complex media without pre-treatment. This is advantageous in comparison with the conventional chromatographic techniques, which require strict sample pretreatment, for instance, removal of macromolecules and cells, to avoid damaging quite expensive chromatographic columns. Determination of MTP in Betaloc-25 Tablets. MTP in pharmaceutical formulations, namely tablets of Betaloc-25, was also analyzed. Twenty tablets of metoprolol tartrate were powdered and dissolved in 25 mL of 10 mM LiCl aqueous solution. Then it was filtered and the filtrate was further diluted by 200 times with 10 mM LiCl aqueous solution for detection. This solution is designated as the Betaloc solution and the theoretical concentration of MTP is 146 M. As shown in Figure 4a, the CV obtained with the Betaloc solution (red curve) displays a well-defined steady-state current wave, in comparison with the blank signal (black curve). In order to precisely determine its concentration, different amounts of standard MTP solution was added to the Betaloc solution and CVs were recorded. Clearly, the steady-state current increased continuously. The fitting of the steady-state current versus the MTP concentration yielded a straight line, as shown in Figure 4b. By extrapolating the line to the zero current point, we obtained the concentration of metoprolol in Betaloc-25, namely 145.5 0.3 M. This value was very close to the theoretic one, indicating that this approach is quite reliable and accurate for MTP determination.
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CONCLUSIONS In summary, the ion-transfer voltammetric behavior of antihypertensive drug, MTP, was studied at the nanoITIES array supported by the free-standing SNM consisting of ultrasmall channels (2 3 nm in diameter). Stable and reproducible steady-state current response due to heterogeneous transfer of MTP was obtained. The presence of small-sized interfering substances (i.e. Dglucose, urea, AA, glycine, MgCl2 and Na2SO4) did not change the current response of MTP, thus its accurate detection in urine and pharmaceutical formulation was achieved. Although large-sized biological species, such as BSA, displayed a strong blockade on the transfer of MTP, the detection of MTP in human serum and blood were still feasible, thanks to the excellent molecular sieving and anti-fouling ability of SNM. This approach is advantageous in terms of its simplicity, low cost, pretreatment-free and compatibility with portable or miniaturized devices, providing a novel and highly useful platform for ionisable drug analysis in biological and medical samples.
ASSOCIATED CONTENT Supporting Information Molecular structure of MTP, photographs of U-tube cell and SNM/p-SiNF chip, CVs of MTP in the presence of different interfering reagents, CVs of MTP in the artificial serum, CVs of MTP in the BSA-free artificial serum and CVs of MTP in 0.1% blood samples: This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * Dr. Bin Su Email:
[email protected] Homepage: http://mypage.zju.edu.cn/binsu
ACKNOWLEDGMENT The work is supported by the National Nature Science Foundation of China (21335001, 21575126) and the Nature Science Foundation of Zhejiang Province (LR14B050001).
REFERENCES
Figure 4. (a) CVs at the nano-ITIES array using Cell 1 (y = 0) illustrated in Scheme 1b for blank solution, Betaloc solution and Betaloc solution after addition of 50, 100, 200, 300, 400 M MTP. (b) The linear fitting of the steady-state current (+0.23 V) versus the MTP concentration.
(1) Brogden, R. N.; Heel, R. C.; Speight, T. M.; Avery, G. S. Drugs 1977, 14, 321. (2) Foody, J.; Farrell, M. H.; Krumholz, H. M. J. Am. Med. Assos. 2002, 287, 883. (3) Koch-Weser , J. New. Engl. J. Med. 1979, 301, 698. (4) Li, F.; Cooper, S. F.; Cote, M. J. Chromatogr. B 1995, 668, 67. (5) Ahmad, S.; Tucker, M.; Spooner, N.; Murnane, D.; Gerhard, U. Anal. Chem. 2015, 87, 754. (6) Jouyban, A.; Sorouraddin, M. H.; Farajzadeh, M. A.; Somi, M. H.; Fazeli-Bakhtiyari, R. Talanta 2015, 134, 681. (7) Santos, M. G.; Campos Tavares, I. M.; Boralli, V. B.; Figueiredo, E. C. Analyst 2015, 140, 2696. (8) Sarkar, A. K.; Ghosh, D.; Das, A.; Selvan, P. S.; Gowda, K. V.; Mandal, U.; Bose, A.; Agarwal, S.; Bhaumik, U.; Pal, T. K. J. Chromatogr. B 2008, 873, 77. (9) Alpdogan, G.; Sungur, S. Spectrochim. Acta 1999, 55, 2705. (10) Passos, M. L. C.; Saraiva, M.; Lima, J.; Korn, M. G. A. J. Brazil. Chem. Soc. 2008, 19, 563. (11) Cerqueira, P. M.; Boralli, V. B.; Coelho, E. B.; Lopes, N. P.; Guimaraes, L. F. L.; Bonato, P. S.; Lanchote, V. L. J. Chromatogr. B 2003, 783, 433.
Stability and Reuseability of SNM/p-SiNF. The stability and reuseability of the SNM/p-SiNF was tested by immersing the SNM/p-SiNF in 0.1 mM MTP aqueous solution containing 10 mM LiCl for five days. Then, the CVs were recorded and compared with the CVs obtained before immersion. The measured current magnitudes were very close to each other, indicating the SNM/p-SiNF chip has good stability and repeatability for building nano-ITIES array. Note that the used SNM/p-SiNF (after tens of electrochemical experiments) was usually kept in 0.1 M HCl ethanol solution in order to remove various surface bound materials.
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