Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion

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Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion Current Rectification at Polyimidazolium-Modified Micropipettes Kailin Zhang, Xiulan He, Yang Liu, Ping Yu, Junjie FEI, and Lanqun Mao Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 18, 2017

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Analytical Chemistry

Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion Current Rectification at Polyimidazolium-Modified Micropipettes Kailin Zhang,1,2 Xiulan He,2,3 Yang Liu,2 Ping Yu,2,3,* Junjie Fei,1,* Lanqun Mao2,3,* 1

Key Laboratory of Environmental Friendly Chemistry and Applications of Ministry of Education, College of

Chemistry, Xiangtan University, Xiangtan, Hunan 411105, China. 2

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. 3

University of Chinese Academy of Sciences, Beijing 100049, China

*

Corresponding Authors. E-mails: [email protected], [email protected], [email protected].

Fax: +86-10-62559373

ABSTRACT Development of new principles and methods for cerebral ATP assay is highly imperative not only for determining ATP dynamics in brain but also for understanding physiological and pathological processes related to ATP. Herein, we for the first time demonstrate that micrometer scale ion current rectification (MICR) at a polyimidazolium brush-modified micropipette can be used as the signal transduction output for the cerebral ATP assay with a high selectivity. The rationale for ATP assay is essentially based on the competitive binding ability between positively charged polyimidazolium and ATP toward negatively charged ATP aptamer. The method is well responsive to ATP with a good linearity within a concentration range from 5 nM to 100 nM, and high selectivity towards ATP. These properties essentially enable the method to determine the cerebral ATP by combining in vivo microdialysis. The basal dialysate level of ATP in rat brain cortex is determined to be 11.32 ± 2.36 nM (n = 3). This study demonstrates that the MICR-based sensors could be potentially used for monitoring neurochemicals in cerebral systems.

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INTRODUCTION Development of new sensing principle and technique for monitoring cerebral biological molecules have attracted even increasing interests because of imperative requirements towards selective, sensitive and simple analytical methods from neuroscience research communities.1-4 Adenosine triphosphate (ATP) is one of the most important chemical signaling agents both in energy metabolism and signal transduction.5-8 Although some elegant methods including liquid chromatography, fluorescence, chemiluminescence, bioluminescence and amperometric biosensors, have been employed for ATP sensing,9-14 the complexity of cerebral system presents a great challenge to the existing methods for direct, selective and sensitive determination of ATP in rat brain, especially for the basal level of ATP because of its relatively low concentration.15 In this case, electrochemical methods are particularly attractive because of their good analytical properties and less technical demands.16,17 However, the complexity of cerebral systems presents a great challenge for the electrochemists to develop electrochemical methods that are capable of directly and selectively recording neurochemical signal, especially for the electrochemical inactive molecules such as ATP, in the cerebral system. With the development of the microfabrication and micro/nano manufacture technique, the new sensing principles based on nanochannel have attracted more and more attraction for single molecule counting and biosensing. In this case, two mainly signal output models have been conducted, one is based on the resistance-pulse technique (i.e., i - t trace),18,19 the other is based on the ionic current rectification (i.e., i - v curve).20,21 The former one is essentially developed from the Coulter-counter principle, which is based on the decrease of ion current originated from the size blockade of the target when the target passes through the pore.22,23 Although this technique has been widely used for sensing single entity (e.g., molecules, protein and particles), 19,24-26 it remains challenging for complex sample analysis since the tiny pore is easily blocked by the non-target. The later one is based on a physical phenomenon of ion current rectification (i.e., the current at a specific potential is greater than that in the opposite potential, ICR), which is susceptible to the surface chemistry (e.g., charge) of channel. When the target is passing through the channel, it would influence the surface chemistry and ion distribution in channel and further change the current ratio in the opposite potential (i.e., rectification ratio, RR). This technique has been developed in the recent ten years and used for sensing 2


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various targets, such as metal ions, biomolecules and proteins.27-29 Recently, Pourmand's group has developed a nanosensor for intracellular pH and glucose sensing, demonstrating that the nanochannel sensor based on ICR could be used for sample analysis in a complex system.30,31 However, the relatively tiny and soft tip of nanopipettes renders difficulties in applying this method for real sample analysis in a simple way. Recently, the ICR in microscale has been reported by using asymmetric electrolyte solution, PEI-coated or biconical pipets.32-34 Very recently, we have found that the ICR could be obtained at a micrometer scale (i.e., MICR) in polyelectrolyte-modified micropipette (e.g., 5 µm radius) in symmetric electrolyte solution,35 which provides a new platform for cerebral biological molecular monitoring in terms of its unique property in ease-in-operation and relatively robust tip. Moreover, we have developed an effective approach to selectively sensing ATP based on dual-recognition-units strategy.36 Based on these studies, we demonstrate here a new strategy to selectively sensing ATP in the cerebral system with MICR. To accomplish this purpose, polyimidazolium brush is first modified onto the inner wall of micropipette by automatic transfer radical polymerization (ATRP) (Scheme 1) to enable the occurrence of MICR at the micropipette

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Scheme 1. Schematic Illustration of MICR-based Sensor for Cerebral ATP Assay Combined with Microdialysis Technique. and is used as one of the recognition unit for highly selective ATP sensing. The confinement of ATP aptamer onto the polyimidazolium brush modified onto the inner wall of the micropipette essentially decreases the rectification ratio because of the decrease of the net surface charge (Scheme 1, blue curve). The presence of ATP leads to the dissociation of the aptamer from the inner surface because it combines with the aptamer more strongly, resulting in the increase of net surface charge and thus the rectification ratio (Scheme 1, red curve). 3



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The constructed ATP sensor bears good linearity within a concentration range varying from 5 nM to 100 nM, reversible responsibility and high selectivity towards ATP. Moreover, the ATP sensor could be used for cerebral ATP assay by combining in vivo microdialysis. This study demonstrates that the MICR-based sensors could be used for in vivo sensing, which would be further developed for tissue-implantable in vivo analysis.

EXPERIMENTAL SECTION Materials and Reagents. Borosilicate capillaries (outer diameter ~1.50 mm, inner diameter ~0.86 mm, Sutter

Instrument

Co.,

USA),

2-bromoisobutyrylbromide,

copper(I)

bromide

(CuBr,

98%),

N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA, 99%) were purchased from J&K Chemical Company

(Tianjin,

China).

1-Vinyl-3-butylimidazolium

chloride

([Vbim][Cl])

and

2-bromo-2-methylN-(3-(triethoxysilyl)propyl) propanamide (BTPAm) were synthesized according to the previous report.35 The 30-mer ATP-binding aptamer (5′- ACC TGG GGG AGT ATT GCG GAG GAA GGT TTT-3′) was synthesized and purified by Invitrogen Biotech Co. Ltd. (Shanghai, China). The aptamer dry powder was dissolved in TE buffer (10 mM Tris; 0.10 mM EDTA; pH 8.0) to form stock solution with a concentration of 10 µM. ATP hydrolase (ATPase; EC 3.6.1.3), adenosine-5′-triphosphate (ATP), adenosine-5′-diphosphate (ADP), adenosine-5′-monophosphate (AMP), cytidine-5′-triphosphate (CTP), uridine-5′-triphosphate (UTP), guanosine-5′-triphosphate (GTP), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), uric acid (UA), ascorbic acid (AA), and 5-hydroxytryptamine (5-HT) were all purchased from Sigma-Aldrich (Shanghai, China). Artificial cerebrospinal fluid (aCSF) used as the perfusion solution for in vivo microdialysis was prepared by mixing NaCl (126 mM), KCl (2.4 mM), KH2PO4 (0.5 mM), MgCl2 (0.85 mM), NaHCO3 (27.5 mM), Na2SO4 (0.5 mM) and CaCl2 (1.1 mM) into water. Other chemicals were of at least analytical grade reagents and were used as received. All the solutions were prepared with Milli-Q water. Micropipettes were fabricated with a CO2-laser-based pipette puller (P-2000, Sutter Instrument) with the following parameters: Heat = 325, Filament = 4, Velocity = 50, Delay = 210, and Pull = 175. The Micropipettes were polished by a pipette micro forge (MF-900, Narishige Instrument). Each pipette was checked the consistence of the pore size and conical angle by optical microscopy. To remove organic contaminants of surface, borosilicate glass capillaries (0.86 mm (I.D.) and 1.5 mm (O.D.), Sutter Instrument) 4


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were pretreated in piranha solution (30% H2O2/H2SO4 = 3:7). The pipettes with the pretreatment were then immersed into a clean solution (30% H2O2/NH4OH/H2O = 1:1:5) to make silicon hydroxyl disintegrate completely.

Preparation of PimB-Modified Micropipettes. PimB-modified micropipettes were fabricated according to our previous report.35 Typically, 2-bromo-2-methyl-N-(3-(triethoxysilyl) Propanamide) (BTPAm) and 1-vinyl-3-butylimidazolium chloride ([Vbim][Cl]) were synthesized according to previous reports.37 The tip of micropipettes was backfilled with BTPAm diluent (5%, v/v in acetonitrile) and placed in pure acetonitrile atmosphere overnight, followed by washing with acetonitrile, ethanol and deionized water, each for 3 times, to obtained initiator-modified micropipettes. A mixture containing [Vbim][Cl] monomer and CuBr in deionized water was added in a three-neck flask and the initiator-modified micropipettes backfilled with [Vbim][Cl] monomer solution were immersed into the mixture that was pre-degassed by purging with N2 for 15 min to remove dissolved O2. PMEDTA was then added to the monomer solution at a [Vbim][Cl]/CuBr/PMEDTA molar ratio of 50:5:15 (dissolving in H2O). The resulting mixture was degassed for another 15 min and was kept under an N2 atmosphere. The polymerization started when the reaction tube was immersed in an oil bath at 60 °C and stopped by removing the reaction vessel and cooling down to room temperature. The obtained PimB-modified micropipettes were then washed by deionized water for 3 times.

ATP Microsensor Based on MICR. MICR-based microsensors were prepared by immersing the PimB-modified micropipettes into TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 4 µM ATP aptamer at room temperature for 2 h. After that, the micropipettes were taken out of the solution and the inner and outer of the micropipettes were rinsed with deionized water and 10 mM KCl solution. The obtained aptamer/PimB-modified micropipettes were used as the sensors for ATP sensing. The regeneration of the microsensors was performed by immersing the micropipettes previously challenged to the ATP solution into TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 4 µM ATP aptamer at room temperature for 2 h. The regenerated aptamer/PimB modified micropipettes were finally rinsed with Milli-Q water.

Electrochemical Measurements and Current-Voltage Recording. An Ag/AgCl electrode (0.6 mm, in diameter) was inserted into the glass capillary and served as working electrode, and the other Ag/AgCl electrode (0.6 mm, in diameter) was placed in bulk solution as auxiliary/reference electrode. In all cases, the micropipettes were filled with the same electrolyte as the bulk solution. Current-voltage curves were recorded 5



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with a CHI electrochemical workstation (CHI Instrument Co. Ltd., China) by scanning the voltage from -1.0 V to +1.0 V at a scan rate of 50 mV/s. The error bars in each figure represent the measuring error of at least 3 independent measurements with the same micropipettes. All measurements were performed at room temperature.

In Vivo Microdialysis and Cerebral ATP Sensing. In vivo microdialysis were carried out with procedures reported in our earlier works.38,39 All animal procedures were approved by the Animal Care and Use Committee at National Center for Nanoscience and Technology of China. Briefly, adult male Sprague-Dawley rats (250-300 g) were housed on a 12:12 h light-dark schedule with food and water ad libitum. A microdialysis guide cannula (BSA/MD-2250) was implanted into the brain cortex (AP = 0 mm, L = 2.58 mm from bregma, V = 0.25 mm from dura) using standard stereotaxic procedures. The guide cannula was kept in place with three skull screws and dental cement. Stainless steel dummy blockers were inserted into the guide cannula and fixed until the insertion of the microdialysis probe. Throughout the surgery, the body temperature of the animals was maintained at 37 °C with a heating pad. After the rats were allowed to recover for at least 24 h, a microdialysis probe (BAS: dialysis length 4 mm; diameter, 0.24 mm, cutoff, 20 kDa) was first implanted into the brain cortex. The microdialysate was collected for analysis after the probe was continuously perfused with aCSF at 0.5 µL/min for at least 90 min for equilibration. To determine ATP in brain cortex microdialysates, the microsensor was first immersed into the microdialysates for 10 min and then current-voltage curve was recorded in 10 mM KCl solution.

RESULT AND DISSCUSSION Competitive Binding of Aptamer with ATP and Polyimidazolium Based on MICR. To verify the applicability of the MICR as the signal output for ATP sensing, the polyimidazolium was first confined onto the inner surface of the micropipette (e.g., 10 µm) by surface initiated atom transfer radical polymerization (SI-ATRP). As shown in Figure 1A, a linear current-voltage curve was obtained at bare micropipette (black curve) in 10 mM KCl solution, which was consistent with previous report, since the radius of pipette was much larger than the thickness of double layer (ca. 3 nm).35 For the PimB-modified micropipette, a typical nonlinear current-voltage curve (blue curve) was observed, essentially demonstrating that MICR was 6


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successfully obtained at the PimB-modified micropipette, as reported in our previous work.13 The rectification ratio, which is defined as the current ratio at opposite potential (RR = I+/I-), has been widely used as the quantitative parameters at ICR-based sensors. After the PimB-modified micropipette was immersed in TE buffer containing 4 µM ATP aptamer for 2 h, a similar nonlinear current-voltage curve (red curve) with a low RR was obtained, essentially indicating the confinement of aptamer onto the inner surface of the micropipettes due to the electrostatic assembly between aptamer and polyimidazolium. The decrease in RR was originated from the negative charge of the aptamer, which would neutralize the positive charge of PimB, and thus results in the lower surface charge (i.e., Qs) at the aptamer/PimB-modified micropipette than that at the PimB-modified micropipette. According to the three-layer model proposed in our previous study,35 the lower Qs gives rise to the smaller RR for the polyelectrolyte-modified micropipettes in symmetric electrolyte solution. This result essentially indicates that the RR for MICR could be tuned by changing the surface charge

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Figure 1. A) Typical I - V curves obtained at bare (black curve), PimB-modified (blue curve), and aptamer/PimB-modified (red curve) micropipettes in 10 mM KCl solution. B) I - V curves obtained at the aptamer/PimB-modified micropipettes in 10 mM KCl solution before (red curve) and after (blue curve) the aptamer/PimB-modified micropipettes were treated with 100 nM ATP for 10 min. Scan rate, 50 mV s-1.

To further demonstrate the possibility of MICR for sensing, the aptamer/PimB-modified micropipette was further immersed in 10 mM KCl solution containing 100 nM ATP for 10 min. As shown in Figure 1B, a nonlinear current-voltage curve (red curve) with a higher RR was obtained after the aptamer/PimB-modified

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micropipette was immersed into ATP solution compared with that at the aptamer/PimB-modified micropipette without ATP immersion, indicating that the immersion of ATP increases the Qs. This increase was considered to result from the competitive binding toward aptamer between ATP and PimB since ATP bears stronger binding constant with aptamer than polyimidazolium. The binding of ATP toward the aptamer could form a new structure that dissociates into solution from the inner surface of the micropipettes, giving rise to the increase of the Qs. This change in RR essentially demonstrated the possibility to develop ATP sensors based on MCIR, as demonstrated below.

Linearity, Selectivity and Reversibility of the Microsensor based on MICR. To investigate the applicability of the MICR for ATP sensing, the responses of the aptamer/PimB-modified micropipettes toward different concentrations of ATP were studied. As shown in Figure 2A, with increasing ATP concentration (e.g., from 5 nM to 100 nM), the current ratio at opposite potential (i.e., rectification ratio, RR) increased obviously, demonstrating the potential application of the present principle for ATP sensing.

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Figure 2. A) Typical I - V curves obtained at the aptamer/PimB-modified micropipettes treated with different ATP concentrations: (a) 0 nM, (b) 5 nM, (c) 20 nM, (d) 40 nM, (e) 60 nM, (f) 80 nM, and (g) 100 nM. Scan rate, 50 mV s-1. Electrolyte, 10 mM KCl solution. B) Plot of the change of rectification ratio (∆R = R − R0) against the ATP concentration. Error bars showing the standard deviation of the measurements taken from three independent experiments.

To eliminate the electrode-to-electrode variation in the background signal, we used the change of the rectification ratio (∆R = R − R0) for ATP quantification. Where, R0 was the initial rectification ratio at 1 V 8


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recorded at the freshly prepared aptamer/PimB-modified micropipettes. R was the rectification ratio at the aptamer/PimB-modified micropipettes after the micropipettes were treated in the different concentrations of ATP. As shown in Figure 2B, the ∆R shows a good linearity with the concentration of ATP within a concentration range from 5 to 100 nM (∆R = 0.281CATP (nM) + 11.394, r2 = 0.982).

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UA DA AA PAC Glu ascf 5-HT DO

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P UT

P CT

P AM

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Figure 3. (A) and (B) Histogram of signal intensity for different targets as indicated in the figure. Insets, typical I - V curves obtained at the microsensor in 10 mM KCl solution treated with various biological molecules: (A) blank (black), 500 nM UA (olive), 500 nM DA (dark cyan), 500 µM AA (magenta), 500 nM DOPAC (dark yellow), 500 nM Glu (navy), aCSF (blue), 500 nM 5-HT (cyan), 500 nM E (orange), 500 nM NE (purple) and 20 nM ATP (red), and (B) blank (black), GTP (100 nM) (blue), UTP (100 nM) (green), CTP (100 nM) (pink), AMP (100 nM) (cyan), ADP (100 nM) (violet) and ATP (20 nM) (red).

Selectivity is another important issue for cerebral biomolecule analysis, we thus investigate the interference from the coexisting biomolecules such as DA, DOPAC, UA, AA, 5-HT, Glu, E, and NE. As shown in Figure 3A, there is almost no change in current after the aptamer/PimB-modified micropipettes was treated by the above-mentioned biomolecules, while the significant change was observed after the micropipettes were treated by 100 nM ATP (red curve), essentially demonstrating the high selectivity of the as-prepared MICR-based ATP microsensor. In addition to the biological molecules mentioned above, the as-prepared ATP microsensor based on MICR also exhibits a high selectivity towards the ATP analogues (i.e., UTP, GTP, CTP, ADP and AMP), as shown in Figure 3B. The high selectivity of the as-prepared ATP microsensor was considered to result from the recognition ability of the aptamer towards ATP and the different 9



affinity between PimB and ATP, ADP, AMP as described in our early report.36 In addition to the properties mentioned above, the re-generation ability of the MICR-based microsensor was also investigated. As shown in Figure 4, after being immersed in 100 nM ATP and regenerated for at least three cycles, the microsensor almost maintain its original response value, suggesting the prepared microsensor was easily re-generated. These properties of the MICR-based microsensor substantially enable it for cerebral ATP assay, as demonstrated below. 80

ATP

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aptamer

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aptamer

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Figure 4. Three regeneration cycles of the microsensor by alternatively immersing the microsensor in 100 nM ATP and 4 µM aptamer solution.

Cerebral ATP Assay. To investigate the validility of our microsensor prepared here for ATP analysis in complex matrix, we sampled the microdialysate from cortex of rat brain with in vivo microdialysis and then analyzed the ATP concentration with the microsensor. Figure 5A compares the I-V curves recorded with the microsensor in 10 mM KCl solution before (black curve) and after (red curve) the microsensor was pre-treated with the microdialysate from the rat brain cortex. The pretreatment in the microdialysate led to the increase in rectification ratio (red curve), showing the capability of our microsensor for sensing ATP in the brain microdialysate. To further confirm the specificity of the microsensor for sensing ATP in the brain microdialysate, we treated the microdialysate sample by adding ATPase, a class of enzymes specially catalyzing the decomposition of ATP into ADP and a free phosphate ion, into the sample. As shown in Figure 5B, no change was obtained for the I-V curves recorded in 10 mM KCl solution with the microsensor before 10


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(black curve) and after (red curve) the microsensor was pre-treated with the ATPase-treated microdialysate. This result well confirms the high specificity of our microsensor for ATP sensing. The basal dialysate level of ATP in the microdialysate of brain cortex was determined to be 11.32 ± 2.36 (n = 3). This value was almost consistent with that reported in the literature.40 These demonstrations suggest that the microsensor with MICR mechanism could find interesting applications in the investigations on the physiological and pathological processes associated with ATP.

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Figure 5. A) Typical I-V curves reocrded at the aptamer/PimB-modified micropipettes in 10 mM KCl before (black curve) and after (red curve) the micropipettes were pre-treated with the microdialysates from the rat brain cortex for 10 min. B) Typical I - V curves recorded at the aptamer/PimB-modified micropipettes in 10 mM KCl before (black curve) and after (red curve) the micropipettes were pre-treated with ATPase-treated brain microdialysate for 10 min.

CONCLUSIONS In summary, we have for the first time demonstrated that the MICR obtained at PimB-modified micropipettes could be used as a novel signal output for highly selective ATP in vivo sensing. The rational design by using PimB and aptamer essentially enables the as-prepared MICR-based microsensors bear good selectivity and linearity as well as reversibility. Compared with the traditional methods based on resistance-pulse method and nanoscale ICR, the as-prepared MICR-based microsensor shows good property in ease-in-operation and relatively robust tip. The present study not only opens a new way to MICR-based (bio)sensing but also provides a new avenue to in vivo analysis.

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ACKNOWLEDGEMENTS This work is financially supported by NSF of China (Grant Nos. 21475138 and 21322503 for P. Yu; 21621062, 296 21435007, and 21210007 for L. Mao; and 21475114, 21275123 for J. Fei), the National Basic Research Program of China (973 programs, 2013CB933704 and 2016YFA0200104), and the Chinese Academy of Sciences.

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I / nA

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240 180 120 Microdialysate

60 -1

0

E/V 1

PimB

ATP Aptamer

ATP

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Highly Selective Cerebral ATP Assay Based on Micrometer Scale Ion

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