Article pubs.acs.org/ac
Micro Electrochemical pH Sensor Applicable for Real-Time Ratiometric Monitoring of pH Values in Rat Brains Jie Zhou,† Limin Zhang,*,‡ and Yang Tian*,†,‡ †
Department of Chemistry, Tongji University, Siping Road 1239, Shanghai 200092, P. R. China Shanghai Key Laboratory of Green Chemistry and Chemical Processes, Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, North Zhoangshan Road 3663, Shanghai 200062, China
‡
S Supporting Information *
ABSTRACT: To develop in vivo monitoring meter for pH measurements is still the bottleneck for understanding the role of pH plays in the brain diseases. In this work, a selective and sensitive electrochemical pH meter was developed for realtime ratiometric monitoring of pH in different regions of rat brains upon ischemia. First, 1,2-naphthoquinone (1,2-NQ) was employed and optimized as a selective pH recognition element to establish a 2H+/2e− approach over a wide range of pH from 5.8 to 8.0. The pH meter demonstrated remarkable selectivity toward pH detection against metal ions, amino acids, reactive oxygen species, and other biological species in the brain. Meanwhile, an inner reference, 6-(ferrocenyl)hexanethiol (FcHT), was selected as a built-in correction to avoid the environmental effect through coimmobilization with 1,2-NQ. In addition, three-dimensional gold nanoleaves were electrodeposited onto the electrode surface to amplify the signal by ∼4.0-fold and the measurement was achieved down to 0.07 pH. Finally, combined with the microelectrode technique, the microelectrochemical pH meter was directly implanted into brain regions including the striatum, hippocampus, and cortex and successfully applied in real-time monitoring of pH values in these regions of brain followed by global cerebral ischemia. The results demonstrated that pH values were estimated to 7.21 ± 0.05, 7.13 ± 0.09, and 7.27 ± 0.06 in the striatum, hippocampus, and cortex in the rat brains, respectively, in normal conditions. However, pH decreased to 6.75 ± 0.07 and 6.52 ± 0.03 in the striatum and hippocampus, upon global cerebral ischemia, while a negligible pH change was obtained in the cortex.
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To date, a great number of excellent methods for pH determination have been developed, including nuclear magnetic resonance (NMR), fluorescent probes, and electrochemical methods.18−23 Although the NMR method can provide quantitative information on pH determination, it is rare to realize the real-time monitoring in living systems. Fluorescent probes have attracted more attention because they can be employed in bioimaging and biosensing in live cells and tissues with high sensitivity. We have developed a carbon dot-based two-photon fluorescent probe for monitoring pH gradients in live cells and tissues.19 Recently, we have also reported a ratiometric fluorescence probe for biosensing of pH and Cu2+ in live cells.20 However, those methods are hard to fill the requirements for real-time and in vivo measurement of pH in the live rat brain. Electrochemical approaches have several advantages as sensitive and simple methods, especially the realtime measurements and in vivo determination.24−27 Glass electrode currently is the most commonly used electrode for
n vivo analysis of chemical signals is a vital way to study brain functions and brain activity mapping. Inspired by the recent development of brain science, the critical role of pH for brain pathophysiology is attracting more and more attention.1−6 On one hand, it has been demonstrated that slight changes in intracellular or extracellular pH can produce conspicuous effects on the biochemical, ion-regulatory, or electrical machinery of nerve and glial cells; furthermore, brain acidosis will augment cell death under various pathophysiological conditions.7−13 On the other hand, pH is suggested to be related to many chronic degenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, renal failure, and ischemia, in which ischemia stroke is very fatal because it could give rise to disability and death.14−17 Global cerebral ischemia would induce selective neuronal cell death and thereafter disability and dementia. Even if the neurochemical changes a few seconds to minutes after the cerebral ischemia, the histological syndrome and functional symptoms of neural damage would occur from several hours to days. Thus, it is a pressing need to develop analytical methods for real-time monitoring of the pH in the live brain and further to under the exact role of pH plays in the ischemia process. © XXXX American Chemical Society
Received: September 24, 2015 Accepted: January 15, 2016
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DOI: 10.1021/acs.analchem.5b03634 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
change of pH was observed in the cortex of the rat brain even upon ischemia.
pH because of highly selective and reliable measurements in a wide pH range. However, it is easy to be broken and is very difficult to be miniaturized for in vivo measurement of pH.28,29 Despite many pH determination methods being reported,30−32 only a few works have realized in vivo monitoring of pH in live animals.33−38 Fast-scan cyclic voltammetry with carbon microelectrodes pioneered by Wightman’s group provided an elegant way to characterize the local pH changes in the brain.33−37 However, it is still challenging work to develop an accurate biosensor applicable for real-time monitoring of pH values upon global cerebral ischemia. Herein, it is the first time that a ratiometric micro electrochemical meter with high sensitivity and selectivity was developed for real-time monitoring of pH values in a rat brain followed by global cerebral ischemia (as shown in Scheme 1).
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EXPERIMENTAL SECTION Reagents and Chemicals. 1,2-Naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), 6-(ferrocenyl)hexanethiol (FcHT), 1,6-hexanedithiol (HDT), HAuCl4·3H2O, dopamine (DA), L-arginine (Arg), L-cysteine (Cys), L-glutamine (Glu), glycine (Gly), L-histidine (His), L-isoleucine (Iso), L-leucine (Leu), L-lysine (Lys), L-methionine (Met), L-phenylalanine (Phe), L-serine (Ser), L-threonine (Thr), L-valine (Val), D(+)glucose, 3-methoxytyramine hydrochloride (3-MT), 5-hydroxyindole-3-acetic acid (5-HIAA), adenosine 5′-triphosphate disodium salt hydrate (ATP), homovanillic acid (HVA), DLlactic acid, L-tyrosine, and tyramine were purchased from Sigma-Aldrich. Uric acid (UA) was obtained from Alfa Aesar. 2,2′-Azobis(2-methylpropionamidine)-dihydrochloride (AAPH), NaClO, and 30% hydrogen peroxide (H2O2) were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). 3,4-Dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), NaOH, HCl, KH2PO4, K2HPO4·3H2O, NaCl, KCl, CaCl2, MgCl2·6H2O, MnCl2·4H2O, CoCl2·6H2O, CdCl2·2.5H2O, NiCl2·6H2O, ZnCl2, FeCl2·4H2O, FeCl3·6H2O, CuCl2·2H2O, KO2, H2SO4, ethanol, and dimethyl sulfoxide (DMSO) were purchased from Sinopharm Chemical Reagent Co. Ltd. Artificial cerebrospinal fluid (aCSF) was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), CaCl2 (1.1 mM), NaHCO3 (27.5 mM), and Na2SO4 (0.5 mM) into doubly distilled water. The pH of aCSF was pH 7.4. All chemicals were analytical grade and commercially available. Milli-Q water (18.2 MΩ cm−1) was applied for preparation of all aqueous solutions. In selectivity test, hydroxyl radical (•OH) was generated by the reaction of Fe2+ and H2O2 (Fe2+/H2O2 = 1:6). Superoxide anion (O2•−) was derived from dissolved KO2 (10 μM) in the DMSO solution. Alkyl peroxyl radical (ROO•) was generated by thermolysis of AAPH (10 μM) in air-saturated aqueous solution at 310 K. Hypochlorite anion (ClO−) was provided by NaClO (10 μM). Singlet oxygen (1O2) was generated by the reaction of H2O2 (10 μM) and NaClO (10 μM). Peroxynitrite (ONOO−) was generated by the reaction of H2O2 (10 μM) and NaNO2 (10 μM). Preparation and Modification of Electrodes. The carbon fibers were purchased from Tokai Carbon Co. (Tokai, Japan). A copper wire was used to attach the carbon fiber with a diameter of 10 μm. Then, the copper wire with carbon fiber carefully traversed the capillary. The carbon fiber was exposed to the fine open end of the capillary and Cu wire was exposed to the other end of the capillary. Both open ends of the capillary were sealed taking epoxy resin with 1:1 ethylendiamine as the harder. The excess epoxy on the fiber was wiped off using acetone. After that, the carbon fiber microelectrode (CFME) was dried at 100 °C for 2 h. The length of the carbon fiber was tailored to 500 μm under a microscope. Before modification, the CFME was sequentially sonicated in acetone, 3 M HNO3, 1.0 M KOH, and distilled water each for 3 min. Then, the nanostructured Au leaves were electrochemically deposited on the pretreated CFMEs from 0.1 M HClO4 containing 4 mM HAuCl4 under a potential of −0.2 V vs Ag/AgCl for 150 s. The nanoleaves electrodeposited electrode were taken as CFME/Au. Then, the CFME/Au electrode was immerged in ethanol solution of HDT and FcHT saturated by N2 gas with an optimized concentration ratio for 24 h to
Scheme 1. Developed Ratiometric Electrochemical pH Meter for Real-Time Monitoring of pH in a Rat Brain Followed by Global Cerebral Ischemia
First, 1,2-naphthoquinone (1,2-NQ) was employed and optimized as a selective pH recognition element to establish a 2H+/2e− approach over a wide pH range from 5.8 to 8.0. Meanwhile, a pH insensitive molecule, 6-(ferrocenyl)-hexanethiol (FcHT) was used as a built-in correction because the oxidation of ferrocenyl group does not couple to proton transfer, resulting in the ratiometric determination of pH. Such ratiometric pH meter can greatly improve the accuracy of pH measurement in complex rat brain by the potential difference in the voltammetry of 1,2-NQ and FcHT. Second, gold nanoleaves were electrodeposited on carbon fiber microelectrode (CFME). The current signal was greatly improved by ∼4-fold and measurement was achieved down to 0.07 pH. Finally, the significant analytical performance of the present biosensor, as well as the properties of CFME including small size of 10 μm, easy to insert, and good biocompatibility, established a direct and reliable approach to determine pH in the rat brain followed by cerebral ischemia. The results demonstrated that the microenvironment in the striatum and hippocampus of the rat brain turned more acidic, and the pH value decreased by ∼0.46 pH and ∼0.61 pH, respectively, when the rat was aroused by cerebral ischemia. However, no obvious B
DOI: 10.1021/acs.analchem.5b03634 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
required. In the whole surgery, the body of temperature of the animals was kept at 37 °C. Cerebral Ischemia/Reperfusion Models. In the twovessel occlusion ischemia and reperfusion models, the bilateral common carotids arteries were ligated and/or released with the methods reported previously.39,40 Briefly, the bilateral common carotid arteries were first separated via a ventral middle line cervical incision. Then, the atlantooccipital membrane was exposed from the left side by retraction of the trachea and esophagus to the right. A vascular clip (0.2 mm diameter) stainless steel, with a tapered blade tip, was utilized to make the basilar artery occluded. Blood flow in the basilar, after the cessation, could be visually confirmed. Occultation in both common carotid arteries was carried out using two Yasargil miniclips, and then removed the clips for 30 min of reperfusion. Ischemic duration was timed as soon as the last clip was used to the common carotid artery and the whole ischemic procedure usually lasted for about 15 min.
prepare a mixed monolayer at CFME/Au. Such electrode was taken as CFME/Au/HDT+FcHT. Finally, 1,2-NQ (or 1,4NQ) was assembled on the electrode surface by immersing CFME/Au/HDT+FcHT electrode in a solution of 5 mM 1,2NQ (or 1, 4-NQ) for 6 h. The nonchemisorbed quinone and FcHT were removed from the modified electrode using ethanol. The resulting electrode was taken as CFME/Au/1,2NQ+FcHT (or CFME/Au/1,4-NQ+FcHT). For comparison, the gold electrode modified with 1,2-NQ and FcHT was denoted as Au/1,2-NQ+FcHT. The cyclic voltammograms (CVs) of CFME/Au and Au electrodes were recorded in 0.5 M H2SO4 solution in the potential range from −0.2 to 1.5 V vs Ag/AgCl at 100 mV s−1. Then, the integration area of cathodic peak in CVs of CFME/Au and Au electrodes was used to estimate the real area of electrode surface. The surface coverage of 1,2-NQ and FcHT were estimated to be 0.78 × 10−11 mol cm−2 and 0.65 × 10−11 mol cm−2, respectively. Instruments and Measurements. X-ray diffraction (XRD) pattern was recorded by a D/max2550VB3+/PC Xray diffractometer using Cu (40 kV, 100 mA). X-ray photoelectron spectroscopy (XPS, AXIS Ultra DLD, Japan) equipped with an Al Kα (1486.6 eV photons) was used to characterize the Au nanoparticles. The scanning electron microscopy (SEM) image was obtained on a Hitachi S-4800 field emission scanning electron microscope and the energydispersive X-ray spectroscopic (EDX) was obtained on a Horiba silicon drift X-ray detector. Fourier transform-infrared spectroscopy (FT-IR, Nicolet iS10, Thermo Electron, America) was used to characterize the gold needlelike leaves. CHI 832 electrochemical workstation (CH Instrument) was employed in all electrochemical measurements with a three-electrode electrochemical cell. The reference electrode was a KClsaturated Ag|AgCl electrode, while the auxiliary electrode was a platinum wire. All electrochemical experiments were carried out at room temperature. In Vivo Detection. All procedures involving animals were conducted with approval of the Animal Ethics Committee in East China Normal University, China. The experimental method and surgery were performed as described previously.25 Briefly, the adult male Wistar rats (300−350 g) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China). The rats were anesthetized using chloral hydrate with an initial dose of 300 mg/kg (i.p.). During the surgical procedures, additional doses of 100 mg/kg (i.p.) to remain anesthesia after per hour. Meanwhile, the Wistar rat was wrapped in a heating pad to maintain the body temperature at 37 °C. The rats were placed in a stereotaxic frame (Beijing Tide-Gene Biotechnology Development Center) with the incisor bar set at 5 mm above the interaural line and appropriately placed holes were drilled through the skull. The CEME/Au/1,2-NQ+FcTH electrode was implanted in different regions in rat brain of the left striatum (AP = 0 mm, L = 2.5 mm anterior to the bregma, and V = 7.0 mm from the surface of skull), the cortex (AP = 0.2 mm, L = 5.6 mm from the bregma, V = 3.0 mm from the surface of skull), the dorsal hippocampus (AP = 5.0 mm, L = 5.0 mm from bregma, V = 2.5 mm from the surface of the skull), according to standard stereotaxic procedures. Another 2 mm plastical cannula was located at ∼5 mm far from working electrode, in which reference and counter electrodes were introduced. The surgical procedures for two-vessel occlusion ischemia were undertaken during in vivo measurements. Throughout the operation, supplements of chloral hydrate (100 mg/kg) were given as
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RESULTS AND DISCUSSION First of all, three-dimensional (3D) gold structures were electrodeposited onto CFME to form CFME/Au electrode. From the typical SEM images shown in Figure 1, we can see
Figure 1. SEM images of CFME coated by electrodeposited Au nanoleaves (A), lateral image of CFME/Au electrode (B), the enlarged Au nanoleaves (C), and EDX analysis of selected areas of Au nanoleaves (yellow) and carbon nanofiber (green) as shown in part D.
that gold nanostructures like leaves were uniformly deposited on the CFME surface (Figure 1A,B), and the diameter of CFME is ∼10 μm (Figure S1, Supporting Information). From a close observation for Au nanoleaves, it was found that the length of Au nanoleaves is ∼1−4 μm (Figure 1B,C). The crystalline orientation of nanostructured gold was investigated by recording X-ray diffraction (XRD) pattern (Figure S2, Supporting Information). The pronounced diffraction peaks of the gold nanostructures located at 38.1°, 44.3°, 65.6°, 77.6°, 81.7° in the 2θ range from 30° to 80° which corresponded to the (111), (200), (220), (311), (222) facets, respectively, indicating that the gold nanostructures exhibited a well-defined face-centered cubic crystal structure. The EDX analysis as shown in Figure 1D indicates more clearly that the Au element was uniformly distributed around the surface of carbon fiber. These observations were also in good agreement with the C
DOI: 10.1021/acs.analchem.5b03634 Anal. Chem. XXXX, XXX, XXX−XXX
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Analytical Chemistry
Au/1,2-NQ+FcHT electrode after the 1,2-NQ was coassembled with FcHT on the electrode surface. The other quinone molecule, e.g., 1,4-naphthoquinone (1,4-NQ), was also tried to construct ratiometric electrochemical biosensor. However, the oxidation peak for 1,4-NQ was observed too close to that of ferrocenyl group because of the large peak-topeak separation between the redox potential of 1,4-NQ (about 400 mV). Thus, 1,4-NQ is hard to be available for ratiometric determination in a wide range (Figure S5, Supporting Information) The CVs of CFME/Au/1,2-NQ+FcHT electrode were recorded in 10 mM PBS with different pH values. As demonstrated in Figure 3A, E1/2(1,2-NQ) ascribed to the 1,2NQ/1,2-NQ2− redox couple at about −20 mV was pH ratiometric determination of pH. The ratiometric potential sensitive, while E1/2(FcHT) located at 420 mV stayed constant with the change of pH, resulting in the separation (denoted as ΔE1/2 = E1/2(FcHT) − E1/2(1,2-NQ)) between E1/2(FcHT) and E1/2(1,2-NQ) exhibited a good linearity in the range of pH 5.8−8.0 with a detection limit of 0.07 pH change. The calibration plot of ΔE1/2 against varying pH showed a gradient of 58 mV/pH (Figure 3B). This gradient is close to the theoretically value calculated from the Nernst equation,41,42 indicating that the electrochemical process of 1,2-NQ is a 2H+/ 2e− Nernstian response. The current response obtained at GC/ Au/1,2-NQ+FcHT was ∼4.0-fold greater than that of at the Au/1,2-NQ+FcHT without electrodeposited Au nanoleaves due to the amplified property of the nanomaterials (Figure S6, Supporting Information). As demonstrated above, the developed strategy exhibited a sensitive and accurate method for pH determination. However, the complicated brain environment means a significant challenge to more particularly in selectivity. The selectivity of the present CFME/Au/1,2-NQ+FcHT electrodes was evaluated by determining the ΔE1/2 value in 10 mM PBS (pH 7.4) resulting from addition of other potential interferences including metal ions (Na+, K+, Ca2+, Mg2+, Cu2+, Zn2+, Co2+, Ni2+, Cd2+, Mn2+, Fe2+, and Fe3+), amino acids (Arg, Cys, Glu, Gly, His, Iso, Leu, Lys, Met, Phe, Ser, Thr, and Val), reactive oxygen species (•OH, 1O2, O2•−, ROO•, ClO−, ONOO− (ROS), and O2) and biological species (D(+)-glucose, 3-MT, 5-HIAA, ATP, DOPAC, HVA, DL-lactic acid, L-tyrosine, tyramine, UA, AA, and DA). No obvious shift of ΔE1/2 value was observed (
