High Antifouling Property of Ion-Selective Membrane: toward In Vivo

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High Anti-Fouling Property of Ion-Selective Membrane: towards In Vivo Monitoring of pH Change in Live Brain of Rats with Membrane-Coated Carbon Fiber Electrodes Jie Hao, Tongfang Xiao, Fei Wu, Ping Yu, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03854 • Publication Date (Web): 14 Oct 2016 Downloaded from http://pubs.acs.org on October 17, 2016

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Analytical Chemistry

High Anti-Fouling Property of Ion-Selective Membrane: towards In Vivo Monitoring of pH Change in Live Brain of Rats with Membrane-Coated Carbon Fiber Electrodes Jie Hao, Tongfang Xiao, Fei Wu, Ping Yu, Lanqun Mao*

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, the Chinese Academy of Sciences (CAS), Beijing 100190, China, and University of Chinese Academy of Sciences, Beijing 100049, China

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Corresponding Author. E-mail: [email protected], Fax: (+86)-10-62559373.

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Abstract. In vivo monitoring of pH in live brain remains very essential to understanding acid-base chemistry in various physiological processes. This study demonstrates a potentiometric method for in vivo monitoring of pH in the central nervous system with carbon fiber-based proton-selective electrodes (CF-H+ISEs) with high anti-fouling property. The CF-H+ISEs are prepared by formation of a H+-selective membrane (H+ISM) with polyvinyl chloride polymeric matrixes containing plasticizer bis(2-ethylhexyl)sebacate, H+ ionophore tridodecylamine, and ion exchanger potassium tetrakis(4-chlorophenyl)borate onto carbon fiber electrodes (CFEs). Both in vitro and in vivo studies demonstrate that the H+ISM exhibits strong anti-fouling property against proteins, which enables the CF-H+ISEs to well maintain the sensitivity and reversibility for pH sensing after in vivo measurements. Moreover, the CF-H+ISEs exhibit a good response to pH changes within a narrow physiological pH range from 6.0 to 8.0 in quick response time with high reversibility and selectivity against species endogenously existing in the central nervous system. The applicability of the CF-H+ISEs is illustrated by real-time monitoring of pH changes during acid-base disturbances, in which the brain acidosis is induced by CO2 inhalation and brain alkalosis is induced by bicarbonate injections. The results demonstrate that brain pH value rapidly decreases in the amygdaloid nucleus by ca. 0.14 ± 0.01 (n = 5) when the rats breath in pure CO2 gas, while increases in the cortex by ca. 0.77 ± 0.12 (n = 3) following intraperitoneal injection of 5 mmol/kg NaHCO3. This study demonstrates a new potentiometric method for in vivo measurement of pH change in the live brain of rats with high reliability.

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INTRODUCTION In vivo monitoring of pH in the central nervous system (CNS) is of great physiological and pathological importance, because acid-base brain chemistry usually correlates closely with various brain activities and functions.1-4 Normally, brain pH fluctuation is limited around a pH value of 7.4 through the regulation by acid-base homeostasis.5 Once pH values fall out of the physiological range, the acid-base disturbance occurs.6 Such a disturbance normally relates to the occurrence of brain diseases such as epilepsy,7 ischemia8 and psychiatric disorders,9 and contributes significantly to morbidity and mortality through a number of proton-sensitive processes such as ion channel gating, synaptic transmission, cell-to-cell communication via gap junctions, and enzymatic activity in brain energy metabolism.10 Although in vitro pH measurements could be easily conducted with, for example pH electrodes, in vivo monitoring of pH in the CNS remains a challenge.11 Firstly, the pH probes should be reliable and stable enough to maintain the sensitivity and reversibility during in vivo measurements, because acid-base brain chemistry generally occurs as a slow process in the CNS,12 during which proteins readily adsorb on the surface of the electrodes.13-15 This has emerged as a big hurdle confronted by almost all in vivo methods, because irreversible and non-specific surface adsorption of proteins from brain tissues can inactivate the electrodes, resulting in gradual decrease in the sensing sensitivity and thus invalidating the as-formed method for in vivo measurements.13, 16-21 Secondly, the in vivo pH probe should have a high temporal/spatial resolution, in that pH in the brain varies with time and regions.11, 22-26 Thirdly, the chemical and physiological complexity of the CNS necessitates a high selectivity of the pH sensor in vivo.16,27-28 This is particularly essential for membrane-coated solid-state pH-sensitive electrodes with conducting substrates, because they are prone to interference from redox species such as ascorbic acid (AA) and 3



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catecholamine in the extracellular fluid. Finally, the pH probes should be fabricated in small sizes with yet enough robustness to ensure a fast response time and ease of being implanted into the brain tissue. Traditional glassy membrane-coated pH-sensitive microelectrodes can be made as small as several micrometers in tip diameter.29, 30 However, these electrodes not only require routine maintenance for potentiometric measurements but also risk being broken during tissue implantation.11, 22 In this study, we demonstrate an in vivo potentiometric method for quantitative monitoring of pH changes in live brain of rats with carbon fiber-based proton-selective electrodes (CF-H+ISEs). This method essentially benefits from a combination of the high anti-fouling property of a proton-selective membrane (H+ISM) against protein adsorption and advantages of carbon fiber microelectrodes for in vivo measurements in their mechanical robustness, small sizes, and good electrochemical properties. The resulting CF-H+ISEs display remarkably stable sensitivity after in vivo implantation, quick and reversible response to pH within a physiologically relevant narrow range, and high selectivity in the complex brain environment, which together well enable them for real-time and in vivo analysis. Therefore, our study provides a novel and effective platform for in vivo pH sensing, which is envisaged to facilitate future researches on physiological and pathological processes associated with pH changes.

EXPERIMENTAL SECTION Reagents and Solutions. Hydrogen ionophore I, bis(2-ethylhexyl)sebacate, potassium tetrakis (4-chlorophenyl), poly(vinyl chloride), AA, dopamine (DA), (±)-epinephrine (E), 5-hydroxytryptamine (5-HT), noradrenaline (NE), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and uric acid (UA) were all purchased from Sigma and used as supplied. Tetrahydrofuran (THF), NaOH, KH2PO4, NaCl, KCl, MgCl2, Na2SO4 and CaCl2 4


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were purchased from Beijing Chemical Co. (Beijing, China). A stock solution of NaOH (0.2 M) was prepared before use. In order to simulate the cerebrospinal fluid to some extent, an artificial cerebral fluid (aCSF)-mimicking solution of KH2PO4 (0.2 M) with NaCl (126 mM), KCl (2.4 mM), MgCl2 (0.85 mM), Na2SO4 (0.5 mM) and CaCl2 (1.1 mM) was prepared. Other chemicals were of at least analytical grade reagents and used as received. All aqueous solutions were prepared with Milli-Q water (18.2 MΩ⋅cm). To study protein adsorption onto the ion-selective membrane, bovine serum albumin (BSA) was used as the model protein and labeled with fluorescein isothiocyanate (FITC). Briefly, equal volumes of BSA solution (10 mg/mL) and FITC solution (0.2 mg/mL), both prepared in 0.1 M carbonate-bicarbonate buffer at pH 9, were mixed and continuously shaken for 4 hours at 4 °C. The resulting FITC-labeled BSA (FITC-BSA) was purified by ultrafiltration (Spectrapor, cut-off 3 kDa) at 5000 x g, 4 °C for 5 times, with aCSF as the washing buffer. Fluorescence of the solution filtered out was monitored at 450 nm to ensure complete removal of unreacted FITC. Finally, the purified FITC-BSA solution was concentrated to 40 mg/mL and stored in the freezer before use. Preparation of CF-H+ISEs. Fabrication and pretreatment of carbon fiber microelectrodes (CFEs) were performed as described previously.13 To prepare CF-H+ISEs, hydrogen ionophore I (1.8 µL), bis (2-ethylhexyl) sebacate (100.0 mg), poly(vinyl chloride) (50.0 mg), and potassium tetrakis (4-chlorophenyl) (1.0 mg) were mixed into THF (1.5 mL). One drop of the resulting solution was applied onto a smooth glassy plate, and the CF-H+ISEs were prepared by carefully immersing and rolling the CFEs into the droplet until the solvent evaporates. The as-prepared electrodes were taken out from the droplet, air-dried and conditioned in 1.0×10−3 M HCl solution for 12 hours before in vivo and in vitro measurements. Apparatus and Measurements. Electrochemical measurements were performed with a 5



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computer-controlled electrochemical analyzer (CHI 832B, Shanghai, China). For both in vitro and in vivo electrochemical measurements, a tissue-implantable microsized Ag/AgCl electrode was prepared as described previously and used as reference electrode.13 For electrode calibration, solution pH was measured with a pH meter (FE20-Five Easy Plus, Mettler Toledo, Switzerland). X-ray photoelectron spectroscopy (XPS) was performed on an ESCALab220i-XL electron spectrometer from VG Scientific using 300W Al Kα radiation (Thermo Scientific, USA). Surfaces were visualized by a tapping mode atomic force microscope (AFM) (Vecco Nanoscope III) by spin-coating the samples onto the surface of silicon substrates. Surface wettability was determined using a contact angle measuring analyzer (JC2000D, Powereach, China) at ambient temperature. Drop angles were recorded and determined with the analysis plugin. A polynomial was fitted to the edge of the droplet to determine the contact angles for at least five times at different positions of each sample. Scanning electron microscopy of CF-H+ISEs was performed on a Model S4300-F microscope (Hitachi, Japan). In Vivo Experiments. Adult male Sprague-Dawley rats (weighing 300-350 g) purchased from Health Science Center, Peking University, were housed on a 12:12 h light-dark schedule with food and water ad libitum. All procedures were approved by the Beijing Association on Laboratory Animal Care and the Association for Assessment and Accreditation of Laboratory Animal Care, and performed according to their guidelines. The animals were anaesthetized with chloral hydrate (345 mg/kg, i.p.) and positioned onto the stereotaxic frame via the ear rods, when the brain area confirmed accurately. The CF-H+ISEs were implanted into the cerebral cortex and amygdaloid nucleus using standard stereotaxic procedures. The microsized Ag/AgCl electrode was implanted into the dura of the brain. Brain acidosis was induced by exposing the animal for a short time (ca. 30 s) to CO2, which was contained in rubber bladders and delivered from a tube placed under the nose of the rat. 6


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Brain alkalosis was induced by intraperitoneal injection of 5 mmol/kg NaHCO3. Open circuit potential was recorded and employed for in vivo measurement of pH changes in live brain of rats.

RESULTS AND DISCUSSION Anti-Fouling Property of H+ISM against BSA. Ion-selective membranes (ISMs) based electrodes are particularly attractive for in vivo monitoring of physiologically important ions such as proton, potassium ion, sodium ion, and calcium ion. This is because that the ISM-based electrodes produce signal response (e.g., electromotive force and redox potential) without any external triggers, and that the current flow through the electric circuit is minimal, both of which make the potentiometric method extremely useful for in vivo studies with good physiological relevance.31 However, as mentioned above, ISM-based electrodes may suffer from the risk of electrode fouling caused by the strong adsorption of proteins from biological samples. Although previous attempts seem to suggest that protein adsorption may not be favored on the surface of ISMs,32-35 it remains controversial on the anti-fouling property of ISM-based electrodes when applied for in vivo analysis, especially for live brain monitoring.34-36 To study the anti-fouling property of proton-selective membrane (H+ISM) against proteins, we first performed in vitro studies with BSA as the model protein. As a starting point, we applied H+ISM onto CFEs to form the CF-H+ISEs, and continuously recorded the potential response of the CF-H+ISEs following addition of BSA into solution. As depicted in Figure 1, the CF-H+ISEs responded quickly to pH change, but stayed insensitive to a series of BSA addition, even though the concentration of BSA reached as high as 70 mg/mL. Furthermore, we also investigated responses of the CF-H+ISEs toward pH after the electrodes were immersed in aCSF containing 40 mg/mL BSA for 2 hours, taken out of the 7



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solution and rinsed with water. We found that pre-treatment of the CF-H+ISEs with BSA did not affect the electrode response toward pH, without losing the sensitivity or reversibility (data not shown). These results strongly demonstrate that the presence of BSA does not influence the electrode performance in pH sensing. This makes it distinguished from in vivo amperometric/voltammetric methods, in which the presence of BSA greatly inactivates the electrodes and thereby largely decreases the sensing sensitivity. This difference could be explained by the less adsorption of BSA onto the ISM. To assess the possible adsorption of BSA onto the H+ISM, the H+ISM spin-coated on silicon wafers were immersed into 40 mg/mL BSA solution for 2 hours, taken out of the solution and washed with water for several times. For comparison, bare silicon wafer without H+ISM coating was also treated with BSA following the same procedure. Surface characterization of bare and H+ISM-coated silicon wafers before and after BSA treatment were then performed by XPS and tapping mode AFM imaging. As shown in Figure 2, pre-immersion of bare silicon wafer into BSA solution leads to the appearance of N atoms in the XPS, suggesting the absorption of BSA onto the wafer. In contrast, same treatment of the H+ISM-coated silicon wafer did not yield a significant change in the XPS, revealing that BSA adsorption onto H+SM was negligible or very weak. Figure 3 compares AFM images of the surfaces of bare (A, B) and H+ISM-coated (C, D) silicon wafers before (A, C) and after (B, D) being soaked in BSA solution for 2 hours. Upon BSA treatment, the surface of bare silicon wafer became rough (B), suggesting the formation of BSA aggregates on the wafer. In contrast, no BSA aggregates were spotted on the H+ISM after same BSA treatment (D), which coincides with the XPS result demonstrating that BSA does not adsorb and aggregate on the H+ISM. Protein adsorption on the H+ISM was also investigated by fluorescent microscopy with FITC-BSA. In this case, one drop of the H+ISM solution was casted onto a smooth glass 8


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slide and let dried in ambient air. After that, 20 µL of 40 mg/mL FITC-BSA was drop-coated on the H+ISM-coated glassy slide. After 15 hours, we found that FITC-BSA solution rolled /crept down from the H+ISM-coated glassy and finally solidified on the border of the glass slide (Figure 3 E, F), suggesting that the FITC-BSA prefers to adsorb onto bare surface without the H+ISM coating. This observation again indicates no strong adsorption of BSA onto the H+ISM, which was further confirmed by contact angle measurements displayed in Figure 3 (G, H). The contact angle of water on the H+ISM surfaces was about 90.7±1.5° (n = 5), which was in good agreement with the reported values.33 It did not change significantly (i.e., 88.4 ± 2.1° (n = 5) after the H+ISM-coated glassy slides were subject to same BSA (40 mg/mL) treatment for at least 12 hours. Since that BSA adsorption would definitely increase the surface hydrophilicity and lower the contact angle of water, this result then indicates little adsorption of BSA onto the H+ISM. As a step forward, we studied the electrode fouling of the H+ISM during short-term in vivo implantation in the live brain of rats. The as-prepared CF-H+ISEs were implanted into the live brain of rats, and their potential responses toward pH compared before and after several-hour (typically 3-hour) in vivo operation were shown in Figure 4. Interestingly, we observed that, in vivo implantation of the electrodes in the brain cortex for 3 hours did not reduce their sensitivity toward pH changes. Such a property endows the potentiometric method using CF-H+ISEs for in vivo monitoring of pH with a high reliability, simplifying the electrode calibration as no pre- and post-calibration are necessary. So far as we know, such a property of the CF-H+ISEs has not been observed previously and this observation would pave a new approach to in vivo measurements with ISM-based electrodes. Taken results above together, we proposed that the steady performance of the CF-H+ISEs during in vivo pH monitoring was due to the strong ability of the H+ISM against protein adsorption, whilst sensitivity loss of bare microelectrodes was attributed to inevitable 9



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protein adsorption during short-term in vivo recording. To further verify this hypothesis, we performed SEM imaging of the CF-H+ISEs as well as bare CFEs before and after being implanted in the live brain for 2 hours. As typically displayed in Figure 5, the surface of the CF-H+ISEs remained smooth after in vivo implantation, as compared to that of bare CFEs becoming rougher. This comparison demonstrates little adsorption and aggregation of proteins on the H+ISM, which forms a straightforward basis for in vivo monitoring of pH change in the live brain with the CF-H+ISEs, vide infra. Reversible and Sensitive Electrode Response toward pH. Having demonstrated the strong anti-fouling ability of the H+ISM against proteins, we next evaluated the analytical properties of the electrodes in terms of reversibility and linearity of the response as well as the selectivity toward pH. As described above, the electrodes used for in vivo pH measurements should have sensitivity as close to the theoretical value as possible so as to detect the tiny change of pH within a narrow pH range with good reversibility. Therefore, we tested the electrode response by varying pH back and forth between 6.0 to 8.0 (Figure S1). The CF-H+ISE was well responsive to pH changes, and the potential response was linear with pH values within the range from pH 6.0 to 8.0 (E (V) = 0.424 - 0.0584pH, R2 = 0.9998) at a slope of 58.4 mV/pH, which was quite close the Nernstian value (i.e. 59.1 mV at 25 °C). Moreover, the electrodes response to pH was completely reversible, because such linear relationship between the potential output and pH values was almost unaltered when pH changes in reversed directions. In addition, we examined the response time of the electrodes to changing pH by measuring the time required to achieve a 90% value of the steady potential. It was found that the CF-H+ISEs exhibited a quick response (i.e., less than 1 s) when the solution pH was increased from 7.24 to 7.61, which was markedly faster than commercial glassy membrane electrodes that normally have a response time of 5-15 s.37-39 Meanwhile, the CF-H+ISEs have a small tip size (i.e., ca. 20 µm), making them 10


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advantageous in monitoring pH changes during acid-base disturbance with a high spatial/temporal resolution. Selectivity. As reported previously,11 chemical environments in the CNS are quite complicated in terms of the temporal/spatial changes of chemical species during various brain activities. Change in the brain pH normally accompanies the change of inorganic salt ions like K+ and Na+.40, 41 To study the possible interference from both species to pH sensing, we added different concentrations of K+ and Na+ into the aCSF with a pH value of 7.30 and recorded the potential responses of the CF-H+ISEs upon these additions. As shown in Figure S2, addition of K+ (blue curve) and Na+ (red curve) at different concentrations did not produce obvious potential disturbance, demonstrating that these species do not interfere with pH measurement at the CF-H+ISEs. Compared with amperometric/voltammetric methods employed for in vivo monitoring of neurochemicals, potentiometric method with glassy membrane electrodes for pH monitoring encounters less interference from redox species because of the insulating nature of glass.42, 43 Different from the conventional glassy membrane electrodes, the use of conducting substrate (e.g., CFEs in this study) to form mechanically robust membrane-coated pH electrodes would allow redox species (such as AA) to produce potential interference to pH sensing. With the CF-H+ISEs, however, we found that addition of electroactive redox species endogenously existing in the CNS did not produce nontrivial potential disturbance, which ascertains the high selectivity of the CF-H+ISEs for pH measurements and further validates our method for selective pH sensing in live brain of rats. Toward In Vivo Monitoring of pH Change during Acid-base Disturbance. To demonstrate the capability of the CF-H+ISEs in monitoring pH changes during acid-base disturbance, animals were subject to CO2 inhalation and injection of sodium bicarbonate to evoke acid-base disturbance. CO2 inhalation has been hypothesized to activate neural 11



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systems similar to those underlying fear learning, especially those involving the amygdala. Figure 6 shows typical potential responses recorded with the CF-H+ISEs in the live brain of rats during CO2 inhalation (A) and injection of NaHCO3 injection (B). Compared to the pH level obtained with the animal under normal conditions (i.e., spontaneously breathing air), breathing pure CO2 gas rapidly decreased the pH level by ca. 0.14 ± 0.01 (n = 5) and induced respiratory acidosis, as reported previously.44, 45 As depicted in Figure 6B, the intraperitoneal injection of 5 mmol/kg NaHCO3 significantly increases the pH in rat cortex by 0.77 ± 0.12 (n = 3) (red curve). In contrast, the intraperitoneal injection of equal volume of saline did not induce obvious change (C). These results were consistent with those reported previously and demonstrate that the CF-H+ISEs could be used for in vivo monitoring of pH change during acid-base disturbance in certain physiological processes.

CONCLUSIONS We have observed that H+ISM exhibits a strong anti-fouling property against protein adsorption in vitro as well as in the live brain of rats, and, based on this, we have successfully developed an in vivo electrochemical method for real time monitoring of pH in the brain with the CF-H+ISEs. Compared with existing methods to detect pH in brain, the method developed here bears the advantages in terms of high reliability to well maintain its sensitivity during in vivo application, high temporal and spatial resolution, ease in the in vivo implantation, and capability of being applied to freely moving animals. This study essentially offers a new method to in vivo monitoring of pH in the brain of living biosystems with a high reliability and robustness. Moreover, ISMs with strong anti-fouling properties against protein adsorption would have their applications extended to in vivo monitoring of ions such as K+, Na+, Ca2+ in future physiological researches.

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ACKNOWLEDGEMENT We acknowledge financial support from the National Natural Science Foundation of China (21435007, 21321003, 21210007, 91413117 for L. Mao, 21475138, 91132708 for P. Yu) National Basic Research Program of China (2016YFA0200104, 2013CB933704), and Chinese Academy of Sciences.

ASSOCIATED CONTENT Supporting Information Linearity and reversibility as well as selectivity of the CF-H+ISE toward pH. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *

Prof. Lanqun Mao, [email protected]

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(16) Wilson, G. S.; Michael, A. C. Compendium of In Vivo Monitoring in Real-time Molecular Neuroscience World Scientific Publishing Co. Pte. Ltd., Singapore, 2014. (17) Cao, B.; Tang, Q.; Li, L.; Lee, C.; Wang, H.; Zhang, Y.; Castaneda, H.; Cheng, G. Chem. Sci. 2015, 6, 782-788. (18) Clark, J. J.; Sandberg, S. G.; Wanat, M. J.; Gan, J. O.; Horne, E. A.; Hart, A. S.; Akers, C. A.; Parker, J. G.; Willuhn, I.; Martinez, V.; Evans, S. B.; Stella N.; Phillips, P. E. Nat. Methods 2009, 7, 126-129. (19) Singh, Y. S.; Sawarynski, L. E.; Dabiri, P. D.; Choi W. R.; Andrews, A. M. Anal. Chem. 2011, 83, 6658-6666. (20) Vreeland, R. F.; Atcherley, C. W.; Russell, W. S.; Xie, J. Y.; Lu, D.; Laude, N. D.; Porreca, F.; Heien, M. L. Anal. Chem. 2015, 87, 2600-2607. (21) Harreither, W.; Trouillona, R.; Poulin, P.; Neri, W.; Ewing, A. G.; Safina, G.; Electrochim. Acta 2016, 210, 622-629. (22) Zhou, J.; Zhang, L.; Tian, Y. Anal. Chem. 2016, 88, 2113-2118. (23) Guo, Y.; Zhou, I. Y.; Chan, S. T.; Wang, Y.; Mandeville, E. T.; Igarashi, T.; Lo, E. H.; Ji, X.; Sun, P. Z. NeuroImage 2016, 141, 242-249. (24) Johnson, M. D.; Kao O. E.; Kipke, D. R. J. Neurosci. Methods 2007, 160, 276-287. (25) Andrade, C. S.; Otaduy, M. C.; Valente, K. D.; Park, E. J.; Kanas, A. F.; Filho, M. R. S.; Tsunemi, M. H.; Leite, C. C. Brain & Development 2014, 36, 899-906. (26) Zhao, F.; Zhang, L.; Zhu, A.; Shi, G.; Tian, Y. Chem. Commun. 2016, 52, 3717-3720. (27) Venton, B. J.; Michael, D. J.; Wightman, R. M. J. Neurochem. 2003, 84, 373-381. (28) Runnels, P. L.; Joseph, J. D.; Logman, M. J.; Wightman, R. M. Anal. Chem. 1999, 71, 2782-2789. (29) Theparambil, S. M.; Naoshin, Z.; Thyssen, A.; Deitmer, J. W. J. Physiol. 2015, 593, 3533-3547. 15



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Figure 1

50 mg/mL 20 mg/mL

30 mV 1 pH

0

1000

2000

3000

T/s

Figure 1. Potential responses of the CF-H+ISEs in phosphate buffer (pH 6.4) towards unit pH change and the successive addition of BSA (first 8 additions, each addition, 2.5 mg/mL; second 5 additions, each addition, 10 mg/mL).

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Figure 2

C1s

N1s

O1s

Si Si-H+ISM Si-H+ISM(BSA)

Si(BSA) 280

350

420

490

Binding Energy (eV)

Figure 2. XPS results of bare (Si, Si(BSA)) and H+ISM-coated (Si-H+ISM, Si-H+ISM(BSA)) silicon wafers before (Si, Si-H+ISM) and after (Si(BSA), Si-H+ISM(BSA)) BSA treatment.

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Figure 3

A

B

200 nm

C

D 200 nm

200 nm

200 nm

Bare

+

Bare

H ISM-coated

+

H ISM-coated

E

F

100 µm

100 µm

G

H

90.7±1.5°

88.4 ± 2.1°

Figure 3. Surface analysis of H+ISM. Top: AFM images of bare (A, B) and H+ISM-coated (C, D) silicon wafers before (A, C) and after (B, D) BSA treatment. Middle: Fluorescence microscopic images of FITC-BSA treated bare and H+ISM-coated glass slides. (E) bright field, and (F) dark field. Bottom: Contact angles of H+ISM before (G) and after (H) BSA treatment.

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Figure 4

30 mV 6.0

6.5

7.0

7.5

8.0

pH

50 mV 0

200

400

600

T/s

Figure 4. Potential responses of the CF-H+ISEs toward pH in phosphate buffer solution before (black curve) and after (red curve) the electrode was implanted in the brain cortex for 3 hours. Insert, pre- (black) and post- (red) calibration curves of the electrodes.

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Figure 5

A

B

5 µm

5 µm

C

20 µm

D

20 µm

Figure 5. SEM images of CFE (top lane) and CF-H+ISE (bottom lane), before (A or C) and after (B or D) being implanted in the brain for 2 hours.

21



Figure 6

5 mV

A

300 s CO2 Inhalation

30 mV

B 5 mmol / Kg NaHCO3

250 s

30 mV

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C

250 s

Saline

Figure 6. Potential responses recorded with the CF-H+ISEs in amygdaloid nucleus (A) and cerebral cortex (B, C) under different conditions of CO2 inhalation (A) and intraperitoneal injection of NaHCO3 (B) or saline (C).

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For TOC only

CF-H+ISE Ag/AgCl

20 µm

Before In Vivo After In Vivo

30 mV 6.0

6.5

7.0

pH

23


7.5

8.0

High Antifouling Property of Ion-Selective Membrane: toward In Vivo

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