Construction of Dopamine-Releasing Gold Surfaces Mimicking

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Construction of Dopamine-releasing Gold Surfaces Mimicking Presynaptic Membrane by On-chip Electrochemistry Jun Li, Chun-Lin Sun, Pengrong An, Xiaoyan Liu, Ruihua Dong, Jinghong Sun, Xingyu Zhang, Yanbo Xie, Chuanguang Qin, Wenfu Zheng, Hao-Li Zhang, and Xingyu Jiang J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 22, 2019

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Journal of the American Chemical Society

Construction of Dopamine-releasing Gold Surfaces Mimicking Presynaptic Membrane by on-chip Electrochemistry Jun Li, †‡ Chun-Lin Sun,§‡ Pengrong An, † Xiaoyan Liu,⊥ Ruihua Dong,⊥ Jinghong Sun, † Xingyu Zhang, † Yanbo Xie, † Chuanguang Qin, † Wenfu Zheng, *⊥ Hao-Li Zhang, *§ Xingyu Jiang*∥⊥ MOE Key Laboratory of Space Applied Physics and Chemistry, Joint Lab of Nanofluidics and Interfaces (LONI), School of Natural and Applied Science, Northwestern Polytechnical University, Xi'an, 710072, P. R. China. †

∥Department

of Biomedical Engineering, Southern University of Science and Technology, No. 1088 Xueyuan Rd, Nanshan District, Shenzhen, Guangdong 518055, P. R. China. ⊥CAS

Key Lab for Biological Effects of Nanomaterials and Nanosafety, National Center for NanoScience and Technology, 100190, P. R. China. § State Key Laboratory of Applied Organic Chemistry (SKLAOC), College of Chemistry and Chemical Engineering, Lanzhou University, 730000, P. R. China. KEYWORDS: dopamine SAMs, microfluidics, electrochemistry, dopamine release, presynaptic membrane

ABSTRACT: We report a strategy to construct a dopamine-releasing gold surface mimicking presynaptic membrane on microfluidic chip to simulate in vivo neural signaling. We constructed dopamine self-assembled monolayers (DA SAMs) by electrochemical deprotection of methyl group-protected DA SAMs on gold surface. Electrochemically controllable release of DA SAMs can be realized by applying non-hydrolytic negative potential on the gold surface. Our method in constructing DA SAMs avoids the polymerization and protonation of DA molecules which may lead to the failure of the DA SAMs formation. By combining microfluidics, we realized spatial and temporal controllable release of DA by electrochemistry from the gold surface. Furthermore, by culturing neurons on the patterned DA SAMs, the interface between the DA SAMs and the neurons could serve as a presynaptic membrane and the spatiotemporal release of DA could modulate the neuron activity with high precision. Our study holds great promise in the fields of neurobiology research and drug screening.

INTRODUCTION Controllable release of small quantities of small molecules from solid surfaces is an indispensable and essential procedure in many fields, such as biosensors,1 microfluidics,2 controllable biomolecules or nanomaterials release,3-6 modulation of surface properties,7 as well as microreactors.8 Electrochemical desorption of selfassembled monolayers (SAMs) provides an efficient procedure for controllable release of immobilized molecules. Electrochemical desorption of thiols has been studied and utilized in many previous works.5-7,9,10 However, electrochemical desorption of SAMs that anchored on gold surface via amino group has barely been studied. Dopamine (DA) is an important neurotransmitter which plays critical role in modulating the activities of the central nervous system.11 Controllable release of DA from solid surfaces is realized mainly through the cleavage of covalent bonds in molecules under external stimulis.12-15 This method requires skilled and complicated design and synthesis of proper molecules with certain structure, which makes the whole procedure challenging and costly.

Hence, construction and release of DA SAMs from gold surface under electrochemical modulation can simplify the experimental process and lower the cost. Catechol headgroups of NH2-terminated DA SAMs, serving as anchoring groups on the surface of titanium dioxide, are commonly used as anchoring intermediates for many functional molecules.16-19 Inversely, the catecholterminated DA SAMs where the amino group serves as anchoring group provide a surface with explicit structure as well as electroactive and biocompatible properties. However, in spite of the ability of amino groups to form covalent bonds with gold surfaces,20-25 catechol terminated DA SAMs have not been reported so far. The main difficulties in direct construction of catechol-terminated DA SAMs on gold surface are as following: i) DA in alkaline solution can undergo auto-oxidation process and result in polymerized DA on gold surface; ii) amino group of DA will be protonated in acidic solution and cannot form covalent bond with gold surface.26 Hence, the construction of catechol-terminated DA SAMs on gold surface should be achieved by alternative methods instead of immobilizing DA molecules directly on gold surface.

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Organoamine-based SAMs were previously studied in the field of molecular electronics.20,26,27 The Lone pair electrons of amines can bind to gold surface and provide a well-defined electronic coupling between the N lone pair and the Au.23 In this work, we report an electrochemically controllable release of amine-bond SAMs, i.e. DA SAMs. The DA SAMs can be released under the application of a negative potential on gold surface, which is similar to the electrochemical reductive desorption of thiol-bond SAMs.5,9,28 Herein, we propose a method to construct and release catechol-terminated DA SAMs under electrochemical modulation on gold surfaces. We utilized protected dopamine molecules (abbreviated as p-DA) to construct SAMs on gold surfaces to avoid spontaneous polymerization of dopamine. For the p-DA SAMs, two hydroxyl groups of p-DA were initially protected by methyl groups, and then deprotected by electrochemical reaction, resulting in the formation of catechol-terminated DA SAMs. The DA molecules can be released from the gold surface under a negative potential. By using this method, we realized construction and release of the catechol terminated DA SAMs (abbreviated as DA SAMs in this work) under electrochemical modulation for the first time. Furthermore, we realized spatial and temporal modulation of neuron activity by electrochemically controllable release of DA SAMs combining microfluidic device.

EXPERIMENTAL SECTION Materials. The reagents and materials used in this experiment were from commercial suppliers at analytical grade. The electrodes used in electrochemical experiments were purchased from Tianjin AIDAhengsheng Science Technology Development Co., Ltd., China. The peptide CysArg-Gly-Asp-Lys-fluorescein-5-isothiocyanate (which was abbreviated as CRGDK-FITC peptide) was synthesized by Shanghai Bootech Bioscience and Technology Co., Ltd., China. All aqueous solutions were prepared using ultrapure water (Merck Millipore, Milli-Q, 18.2 MΩ·cm). All the experiments were performed at room temperature. Preparation of SAMs. The p-DA SAMs were prepared on gold-coated silicon wafer (5 nm Cr and 50 nm Au; the wafer was cut into slides of 5 mm × 20 mm size). Before incubation in the solution of p-DA, the slides were precleaned by incubation in ultrapure water and CH2Cl2 thrice, respectively. The p-DA SAMs were constructed by immersing clean gold substrates in 10−3 M solutions of pDA in CH2Cl2 for 48 h at room temperature. The samples of glutamic acid SAMs were prepared by incubation of gold coated silicon slides (5 mm × 5 mm size) in 10-3 M of glutamic acid solution in water for 48 h. The slides modified with p-DA SAMs were removed from the solution, further washed away physically adsorbed molecules with CH2Cl2, and dried with a N2 flow. Electrochemical Experiments. All the electrochemical experiments were conducted with a CHI660E (CHI, U.S.A.) electrochemistry workstation. In the electrochemical experiments, working electrode: gold-coated silicon slide (5 mm × 20 mm size); counter electrode: Pt wire; reference electrode: saturated calomel electrode (SCE).

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Electrochemical Surface Enhanced Raman Spectroscopy (EC-SERS). The SERS-active substrate (gold disk electrode) was prepared following a published procedure.29 The EC-SERS experiment setup has been reported in our previous work.30 EC-SERS analysis was carried out on an EC-SERS cell with three-electrode system, using a gold disk electrode (2 mm diameter) as working electrode, a Pt wire as counter electrode, and a silver wire as reference electrode. X-ray Photoelectron Spectroscopy (XPS). XPS spectra were acquired on a Kratos Axis Ultra DLD spectrometer. The samples for XPS measurements were prepared from the p-DA SAMs or DA SAMs on gold-coated silicon slides (5 mm × 5 mm size). Fabrication of Microfluidic Device for Hippocampal Neuron Culture. The microfluidic device for hippocampal neuron culture comprises a polydimethylsiloxane (PDMS) (Sylgard, Dow Corning) slab with an embedded microfluidic network, and a DA SAMs-modified gold surface (2 cm × 2 cm) in a polystyrene culture dish. There are three parallel channels (width, 1000 μm; depth, 100 μm) in the microfluidic chip. The PDMS slab was punched to have appropriately sized holes, and placed on the surface of the DA SAMs-modified gold surface (2 cm × 2 cm) to form enclosed microchannels. The DA SAMs in channel 2 were desorbed by electrochemistry (WE: gold surface; CE: Pt wire: RE: Ag wire). The cells were seeded into the channels and cultured in neurobasal medium, and the medium was replaced every 3 days. Calcium Imaging. The fluorescent calcium indicator Fluo4 AM (Molecular Probes) was used to characterize the calcium signals of rat hippocampal neurons. The cultures were rinsed with Hanks solution and treated with 2 µM Fluo-4 AM mixed with the same volume of 20% F-127 in DMSO. After incubation at 37 oC, in 5% CO2 for 45 min in darkness, the cultures were incubated in Hanks solution for 15 min in the same condition. Time-lapse fluorescence images were captured on a confocal microscope (Zeiss LSM 710). A potential of -1.0 V was applied on the gold surface for 400 s.

RESULTS AND DISCUSSIONS The construction and release process of DA SAMs on gold surfaces is illustrated in Scheme 1. The p-DA molecules were first immobilized onto the gold surface via amino groups by Au-N bond to form p-DA SAMs. To confirm that the amino group can form covalent Au-N bond with the gold surface, we performed control experiments by constructing SAMs using glutamic acid on gold surface. Xray photoelectric spectroscopy (XPS) analysis indicated that the C 1s, N 1s and O 1s signals of the glutamic acid provided the evidences that the glutamic acid can form covalent bond with the gold surface (Figure S1). Although glutamic acid possesses one amino and two carboxyl terminal groups, glutamic acid tend to form covalent bond with the gold surface via amino group, because the binding strength of amino group to gold is much higher than that for carboxyl terminal.31 Furthermore, the methyl group cannot form covalent bond with gold surface. From all these results, we can confirm that the p-DA molecules should bind to the gold surface via the amino group.


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Scheme 1: Illustration of the construction and release process of DA SAMs on gold surface under electrochemical modulation. We deprotected the p-DA molecules constructing the pDA SAMS on the gold surface by electrochemistry to form DA SAMs. With a dimethoxybenzene terminal group, p-DA molecule can be regarded as a protected DA molecule and is initially electrochemically inert. After an electrochemical deprotection process, p-DA molecule can turn into a 1,2benzoquinone structure (oxidation state of DA) which can be reversibly reduced to DA by a 2e−−2H+ reaction,32 i.e. the p-DA SAMs can transform to DA SAMs after the process of electrochemical deprotection (Scheme 1). The electrochemical deprotection reaction was reported in our previous work.33 Thus, we can obtain DA SAMs on gold surface by using electrochemical deprotection method. We monitored the construction process of DA SAMs by cyclic voltammetry (CV), XPS, and EC-SERS. In the CV analysis, the p-DA SAMs first underwent an electrochemical deprotection process, i.e. the p-DA SAMs scanned between 0 and +1.05 V in 0.5 mol/L H2SO4 at a scan rate of 100 mV/s (Figure 1a). There was no obvious redox peak in the first scan segment (from 0 to +1.05 V), nevertheless, a reduction peak at Epc = 0.505 V and an oxidation peak at Epa = 0.538 V were observed in the following successive scans. Further increase of the scan numbers led to gradual increase of the peak currents. With the increase of the scan numbers, more and more p-DA molecules immobilized on the gold surface were electrochemically deprotected, leading to more and more transformations from p-DA SAMs to DA SAMs. Moreover, the DA SAMs were electrochemically active. Hence, the peak current increased with the increase of scan number. After 10 continuous scans, the redox peak current was stabilized with the anodic and the cathodic peaks being symmetric. The electrochemical redox peaks after electrochemical deprotection process are consistent with our previous work.33 To confirm that the redox peaks are attributed to the electrochemical deprotection process of p-DA SAMs, we recorded the CVs of a bare gold electrode in 0.5 M H2SO4, which had no redox peaks (Figure S2). We characterized the resulting DA SAMs after the electrochemical deprotection of p-DA SAMs. We recorded CVs of the DA SAMs in PBS with different pH values with

different scan rates. The E0 as a function of pH value at 100 mV/s was plotted, with a slope equaling to -65 mV/pH (Figure 1b). This slope value is quite close to the theoretical value of −59 mV/pH for a 2e−−2H+ reaction.34 It is obvious that the increase of pH value can lead to the decrease of E0. We recorded a series of CVs of the DA SAMs in PBS (pH: 7.4) at different scan rates (Figure 1c), which indicated that the peak currents increased with the increase of the scan rate, showing no significant shift of the peak positions. We analyzed the peak currents (both anodic and cathodic peak currents) as a function of scan rate (Figure 1d), which showed that both the anodic and cathodic peak currents varied linearly with the scan rates, indicating that the redox behavior of the catechol headgroup of DA SAMs was a surface-controlled reaction. The surface coverage (Γ, mol/cm2) for the DA SAMs was 1.16 × 10−10 mol/cm2, which was calculated by integrating the area of the anodic or cathodic peak in PBS (pH: 7.4). The Γ for the DA SAMs was relatively lower compared with that of the SAMs bound to the gold surface with thiol group (2.82 × 10-10 mol/cm2),30,35 which may be assigned to the lower binding strength of amino group compared with thiol group.22 To further confirm the formation of DA SAMs after the electrochemical deprotection process of the p-DA SAMs, we analyzed the p-DA SAMs before and after electrochemical deprotection process by XPS. Two C peaks at 286.1 eV and 284.6 eV appeared in the spectrum before the electrochemical deprotection process (Figure 2a), which can be attributed to C 1s and C-O bond, respectively. By comparison, a new peak (288.8 eV) appeared in the spectrum after the electrochemical deprotection, which can be assigned to the C=O bond of oxidized DA molecules in the DA SAMs. (Figure 2b) Scanning electron microscopy combined with energy dispersive spectroscopy (SEM-EDS) and contact angle analysis further confirmed the successful DA-SAM formation on gold surface (Figure S3, S4). Ellipsometric measurement indicated that the thickness of the DA SAMs on gold surface is about 10.2 ± 2.1 Å. These results all verified that after the electrochemical deprotection process, DA SAMs were successfully constructed on the gold surface.



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Figure 1. The electrochemical deprotection process of p-DA SAMs and characterization of DA SAMs by electrochemistry. (a) Sequence of cyclic voltammograms (CVs) of the p-DA SAMs in 0.5 mol/L H2SO4. Scan number,10. (b) Plots of E0 vs. pH. The E0 was obtained from the midpoint of reduction and oxidation peak potentials from the CVs. (c) CVs of the DA SAMs after electrochemical deprotection in PBS (pH: 7.4) for different scan rates (from 0.1 to 0.9 V/s). (d) The linear dependence of the peak current on the potential scan rate to highlight the presence of a surface-confined redox species.

Figure 2. XPS characterization of the SAMs before and after electrochemical deprotection process, and EC-SERS characterization to confirm the two states of DA SAMs under different potentials. XPS spectra of p-DA SAMs (a) and DA SAMs (b), respectively. (c) CVs of the DA SAMs in 0.2 mol/L PBS (pH: 7.4) after the electrochemical deprotection process. (d) EC-SERS spectra of the DA SAMs in PBS (pH: 7.4) under different potentials vs. silver wire.

We monitored the changes of the headgroups of the SAMs under different potentials to verify the hypothesis proposed in Scheme 1, i.e., the headgroups of the DA SAMs present as the catechol form under negative potentials and

the 1,2-benzoquinone form under positive potentials. The CV of the DA SAMs in 0.2 mol/L PBS (pH: 7.4) showed that the catechol headgroups of DA SAMs can be oxidized at 0.112 V (Figure 2c), and the resulting 1,2-benzoquinone


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headgroups can be reduced at -0.15 V, and this electrochemical response is reversible. Thus, the headgroups of the DA SAMs exist as catechol state under 0.3 V and as 1,2-benzoquinone state under +0.1 V, respectively. We carried out EC-SERS analysis, i.e. acquiring of SERS spectra under two different potentials in PBS (pH: 7.4) on a SERS-active gold electrode (Figure 2d). When we applied -0.3 V on the gold surface, the SERS bands showed two obvious peaks at the position of 1602 cm-1 and 1441 cm-1, which can be attributed to the stretching mode of benzene ring and bending mode of C-H in benzene ring, respectively.36,37 When the applied potential changed to +0.1 V, the SERS bands showed a new arising peak at the position of 1663 cm-1 due to the carbonyl group, indicating the conversion of the headgroup from catechol to 1,2-benzoquinone headgroup. When we alternately applied -0.3 V and +0.1 V on the electrode surface, the SERS spectrum changed correspondingly. These results implied that the conversion between catechol and 1,2-benzoquinone states under electrochemical modulation is highly reversible. Both spectra under +0.1 V and -0.3 V showed intense peak at the position of 1602 cm-1, which is consistent with the aromatic C=C stretching band of p-DA SAMs at 1597 cm-1 (Figure S5) and the anthraquinone derivative reported in literature.36 The EC-SERS results further affirmed the conversion of p-DA SAMs to DA SAMs after the electrochemical deprotection process.

Figure 3. Characterization of the release of DA SAMs under electrochemical conditions. (a) Illustration of the release process of DA SAMs under electrochemical modulation. (b) CVs of the DA SAMs before (black line) and after (red line) the application of negative potential (-1.0 V for 80 s).

After successful construction of DA SAMs on gold surface, we realized the release of dopamine from gold surface into solutions by electrochemical modulation. Thiols can adsorb onto gold surface and can be released by a reductive desorption process.7,28,38 We assume that the release of amino-bonded SAMs (such as DA SAMs) is similar to that of thiol SAMs. Under this hypothesis, the release process of DA SAMs can be realized by the application of negative potential (-1.0 V in this work) as shown in Figure 3a. To verify this hypothesis, we carried out electrochemical and fluorescent analyses. The CV of the DA SAMs exhibited a cathodic peak before the application

of -1.0 V (Figure 3b), which agrees with the behavior for the desorption of a thiolate monolayer,39 in which the main cathodic peak can be assigned to the break of the sulfurgold bond, a one-electron reduction process.40 The onset of reductive desorption was seen at V1 = -0.6 V, indicating the starting release of the DA SAMs, the release of the DA SAMs was complete at V2 = -0.85 V, and the peak current occurred at -0.75 V. At the potential of -0.85 V, the DA SAMs were desorbed from the surface and the released DA could diffuse into the surrounding solution. In this work, we chose -1.0 V as the desorption potential. The red line in Figure 3b presented nearly no cathodic peak at the peak potential position (-0.75 V), demonstrating the total release of the DA SAMs into solution after application of 1.0 V for 80s. The electrochemical analysis revealed that the amine-bonded DA SAMs can be desorbed from gold surface under application of certain negative potentials. To confirm that the formation of amine-Au bond is reversible, p-DA SAMs was constructed on the same gold surface for the second time after electrochemical desorption. XPS analysis indicated that an N 1s peak at 400.5 eV emerged again, implying the formation of amine-Au bond again, thus, the formation of amine-Au bond is reversible (Figure S6). We further conducted fluorescence characterization to visually verify the release of DA SAMs under electrochemical modulation from gold surface. As reported, the catechol headgroups in SAMs can react with L-cysteine tailored peptide in solution.33 We designed a Lcysteine tailored peptide with a FITC luminophore, i.e. CRGDK-FITC for monitoring the catechol headgroup of the DA SAMs (Figure 4a). The surface reaction is presented in Figure 4a, resulting in FITC-terminated SAMs on the gold surface. Sequence of the CVs of the DA SAMs in 10-3 M CRGDK-FITC peptide solution in 0.2 M PBS (pH=7.4) showed successive decrease of both the anodic and cathodic peak currents (Figure 4b), which can be solely ascribed to the reaction taking place between the DA SAMs and the peptide. The decrease of peak current revealed that the catechol headgroups in DA SAMs decreased with the gradual reaction with the CRGDK-FITC peptide in solution. As illustrated in Figure 4a, after the reaction with CRGDK-FITC peptide, the electrochemically active catechol headgroups transformed into electrochemically inert structure, leading to the gradual decrease of the peak current. The CVs recorded before and after the reaction with CRGDK-FITC peptide indicated that the redox peak currents almost disappeared after the reaction with CRGDK-FITC peptide compared with that recorded before the taking place of the reaction (Figure 4c). We captured the fluorescence images of the gold surface before (left) and after (right) electrochemical release of the DA SAMs after reaction with CRGDK-FITC peptide (Figure 4d). The initial fluorescent image before electrochemical release indicated that the gold surface was functionalized with CRGDK-FITC (left one in Figure 4d). The well-distributed fluorescence on the surface was consistent with the uniformity of the DA SAMs. The CV of DA SAMs after reaction with CRGDK-FITC peptide in PBS (pH=7.4) was recorded (Figure S7), from which we can see that the desorption of the SAMs was completed at -1.1 V. The CV



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showed a desorption peak, which is not obvious. This may be attributed to the lower desorption efficiency of SAMs with longer chain.41 Keeping the gold electrode at -1.3 V (vs. SCE) for 100 s in PBS (pH 7.4) resulted in the release of

Figure 4. Fluorescent characterization of the release of the DA SAMs. (a) The structure of the CRGDK-FITC peptide, and the surface reaction between the DA SAMs and the CRGDK-FITC peptide in solution. (b) Sequence of the CVs of the DA SAMs in 10-3 M CRGDKFITC peptide solution in 0.2 M PBS (pH=7.4), scan number, 20. (c) CVs recorded before (black line) and after (red line) DA SAMs react with CRGDK-FITC peptide. (d) The fluorescence images of the gold surface before (left, FITC) and after (right) release of the DA SAMs, the scale bar, 500 m.

Figure 5. Construction and demonstration of a dopamine-releasing gold surface mimicking presynaptic membrane based on electrochemistry and microfluidic chip. (a) Schematic process presenting the construction of presynaptic membrane by on-chip electrochemistry. FL, fluorescent. (b) The correlation of the relative changes in Fluo-4 emission (ΔF/F) versus time after application of -1.0 V on the gold surface to release DA SAMs. Curves 1, 2, 3, and 4 show the signals from 4 different cells, which were pointed out in Figure 5c. The arrows indicated the starting time (0 s) of application of -1.0 V on the channels. (c) The correlation of the relative changes in fluo-4 emission versus time after application of -1.0 V on the gold surface to release DA SAMs. The representative calcium signal images of hippocampal neurons were captured at different time points.


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the DA SAMs and the diffusion of the DA molecules into the solution, as evidenced by the complete loss of fluorescence of the gold surface (right one in Figure 4d). The CVs of the DA SAMs after reaction with CRGDK-FITC peptide before and after application of negative potential were presented in Figure S8. After electrochemical desorption of the SAMs, the surface recovered to its initial state, i.e. bare gold surface. The hydrogen evolution reaction took place on the gold surface. This result can confirm the release of the SAMs. The fluorescent results also noted that the DA SAMs can be released from gold surface under electrochemical modulation. We combined DA SAMs with microfluidics as a presynaptic membrane and regarded the basal membrane of rat hippocampal neurons as postsynaptic membrane to study the effects of electrochemically released DA on the neurons. Microfluidics, used in this study, is a convenient platform for investigating the behaviors of neurons42 and in vitro construction of nerve networks43,44. Calcium, which is a universal second messenger, is of critical importance to neurons as it mediates neurotransmitter release and synaptic transmission.45 Application of DA induces calcium release from internal stores in cells.46 Thus, the calcium release in hippocampal neurons was set as a readout of the synapse activation. On the microfluidics, we attempt to simulate synaptic activities by spatiotemporal electrochemically controllable release of DA from the gold surface. We first dipped Au substrate in p-DA solution to form p-DA SAMs on the gold surface, and placed the PDMS slab carrying the microfluidic network onto the surface of the p-DA SAMs (Figure 5a). We can apply potentials onto the gold surfaces in the three channels separately. In channel 1 and 3, the p-DA SAMs underwent electrochemical deprotection process, resulting in the formation of DA SAMs. While in channel 2, the p-DA SAMs were desorbed by applying -1.0 V for 80 s, leading to the formation of bare gold surface (Figure 5a). After that, hippocampal neurons were seeded into the three channels and cultured for 1 week. The combination of the DAreleasing surface with microfluidic device can safeguard the same external conditions for all the control groups and the experimental groups. We used calcium signal change to indicate if the neurons can be activated by the electrochemical controllable release of DA from the gold surfaces in different channels (Figure 5a). In channel 1, the bottom of which was DA SAMs, the onset of calcium signals was observed at about 80 s and the signal lasted for nearly 30 s (Figure 5b). Different cells showed similar calcium signals (Figure 5b). Curves 1, 2, 3, and 4 show the signals from 4 different cells, which were pointed out in Figure 5c. We captured the representative calcium signal of hippocampal neurons at different time points (Figure 5c). The calcium signal remained unchanged until 80 s, after this time point, the signal enhanced rapidly. This may be attributed to the threshold of the calcium ion channel, only when the accumulation of released DA at the interface between the gold surface and the basal cell membrane to certain extent, can the open of the calcium channel be triggered. The surface coverage (Γ, mol/cm2) for the DA SAMs was 1.16 × 10−10 mol/cm2. Besides, the surface of each channel is about 0.05 cm2, and the height of each channel is 100 µm. Hence, we can calculate the density of

DA in channel is about 1.16 × 10-5 mol/L, which can induce hippocampal neuronal calcium release.45 After that, the calcium signal began to decrease at 86 s, and recover to the initial state after 100 s. To verify that the enhancement of calcium signal was due to the release of DA SAMs, we carried out two control experiments in the other two channels. In channel 2, in which the bottom was bare gold surface, we could not observe obvious change in calcium signal images after application of -1.0 V for 200 s (Figure 5b). In channel 3, the bottom of which was DA SAMs, the calcium signal image showed no obvious change when there was no potential applied to the surface in a duration of 200 s (Figure 5b). On the contrary, the calcium signal in both channel 2 and 3 decreased to a certain extent because of the photobleaching of the dyes. The two control experiments all confirm that the increase of the calcium signal of the hippocampal neurons was assigned to the electrochemically controllable release of DA SAMs. Therefore, by combining electrochemistry, DA SAMs on microfluidics, and neuron cells, we successfully constructed a DA-releasing gold surface that can mimick presynaptic membrane by on-chip electrochemistry. To further verify the synaptic response of neurons to the electrochemically triggered DA, we characterized the variation of membrane potential of rat hippocampal neurons by staining them with FluoVolt (Invitrogen), a membrane potential probe. If there is a potential variation on the postsynaptic membrane, the fluorescence intensity of the probe will change accordingly. The fluorescence intensity of the stained cells on the DA-releasing gold surface increased sharply and subsequently decreased after applying a potential (-1.0 V) (Figure S9), indicating a change in the membrane potential of the postsynaptic membrane. This result confirms the electrical response of the postsynaptic neuron in the variation of calcium signals (Figure 5c) under the same condition. As a control, the fluorescence intensity of neurons on bare gold surface showed no detectable change under the same electrochemical condition (Figure S9). These experiments confirmed that the response of the neurons is initiated by the electrochemically released DA from the gold surface. Thus, the presynaptic membrane-mimicking gold surface constructed in this study is successful. Even though release of DA from solid surfaces has been realized previously, none has allowed for a controlled release with high spatial and temporal resolution. For example, DA could be released through cleavage of covalent bonds in some molecules under external stimulis.12-14 The release of DA could also be realized by the adsorption and desorption of DA from SAMs, polymers, or nanomaterials.15,47,48,45 However, none of these works have applications in the activation of real neurons. Although a recent work presented a artificial synapse chip that can stimulate cells with microfluidic channel delivered bioactive molecules, no transmitter was immobilized or released.49 Moreover, these approaches require skilled and complicated designs, which make the whole procedures challenging and costly. In comparison, by combining microfluidics and desorption of DA SAMs, our method can realize spatiotemporal release of DA with high precision, lower cost, and facile operation. Before the release of DA, the DA SAMs surface are initially inert to the neurons


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seeded on, only when a negative potential is applied onto the surface can the neurons be activated. Furthermore, the activation zones of the neurons can be controlled in any shapes and areas and be triggered any time by combining microfluidics. Also, the activation of neurons can be observed conveniently by confocal microscope. Moreover, to verify that the DA SAMs are nontoxic, rat hippocampal neurons were grown on the surface of DA SAMs for a week, we assessed the cytotoxicity by staining the cells using a Live/Dead kit. The viability of the hippocampal neurons remains 97±3% (Figure S10), demonstrating the excellent biocompatibility of the DA SAMs. Together, our strategy shows unparalleled advantages as a facile platform for synapse research.

CONCLUSION We developed a strategy to construct DA SAMs on gold surface, and realized spatiotemporal and controllable release of DA into the interface of attached neurons and the gold surface and the activation of the neurons, mimicking the release of neurotransmitters from the presynaptic membrane to the postsynaptic membrane and the resulting neuron physiological activities in vivo. This presynaptic membranemimicking surface holds great promise for broad spectrum of applications such as the models for neuroscience research and the platforms for drug screening.

ASSOCIATED CONTENT Supporting Information. Details of experiment section, XPS, SERS and other CVs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] * [email protected]

Author Contributions ‡These

authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We acknowledge the financial support from the National Natural Science Foundation of China (NSFC 21602176, 21702085, 21572180, U1730133, U1732143, 21535001, 81730051, 21761142006, 81671784, 21505027, 31470911, 81673039), Natural Science Basic Research Plan in Shanxi Province of China (Program No. 2017JQ2039), the Minister of Science and Technology of China (2017YFA0205901), the Chinese Academy of Sciences (121D11KYSB20170026), Key Laboratory of Special Function Materials and Structure Design, Ministry of Education, Lanzhou University (lzujbky2018-kb06), Fundamental Research Funds for the Central Universities (3102017jc01001, 3102019ghxm020, 3102019PB006) and the support from Analytics and Testing center in NPU.

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Insert Table of Contents artwork here

We constructed a dopamine self-assembled monolayers (DA SAMs) by electrochemical deprotection of methyl groupprotected dopamine SAMs on gold surface, and realized electrochemically controllable release of DA SAMs by applying nonhydrolytic negative potential on the surface. By combining microfluidics, we realized spatial and temporal controllable release of DA by electrochemistry from the gold surface. Furthermore, by culturing neurons on the patterned DA SAMs, the DA SAMs could serve as a presynaptic membrane and the spatiotemporal release of DA could modulate the neuron activity with high precision.


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Construction of Dopamine-Releasing Gold Surfaces Mimicking

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