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Electrochemical impedance spectroscopy for real-time detection of lipid membrane damage based on a porous self-assembly monolayer support Meng Zhang, Qingyu Zhai, Liping Wan, Li Chen, Yu Peng, Chunyan Deng, Juan Xiang, and Jiawei Yan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00884 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 22, 2018
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Analytical Chemistry
Electrochemical impedance spectroscopy for real-time detection of lipid membrane damage based on a porous self-assembly monolayer support Meng Zhang,ϯ, § Qingyu Zhai,ϯ, § Liping Wan,ϯ Li Chen,‡ Yu Peng,‡ Chunyan Deng,ϯ Juan Xiang,*,ϯ Jiawei Yan*,‡ ϯ
College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, P. R. China State Key Laboratory of Physical Chemistry of the Solid Surfaces, Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China ‡
Tel.: +86-731-88876490; E-mail:
[email protected];
[email protected] ABSTRACT: Layer-by-layer dissolution and permeable pore formation are two typical membrane damage pathways, which induce membrane function disorder and result in serious disease, such as Alzheimer’s disease, Keshan disease, Sickle-cell disease and so on. To effectively distinguish and sensitively monitor these two typical membrane damage pathways, a facile electrochemical impedance strategy was developed on a porous self-assembly monolayer (pSAM) supported bilayer lipid membrane (BLM). The pSAM was prepared by selectively electrochemical reductive desorption of the mercaptopropionic acid in a mixed mercaptopropionic acid/11-mercaptoundecanoic acid self-assembled monolayer, which created plenty of nanopores with tens of nanometers in diameter and several nanometers in height (defined as inner-pores). The ultra-low aspect ratio of the inner-pores was advantageous to the mass transfer of electrochemical probe [Fe(CN)6]3-/4-, simplifying the equivalent electric circuit for electrochemical impedance spectroscopy analysis at the electrode/membrane interface. [Fe(CN)6]3-/4- transferring from the bulk solution into the innerpore induce significant changes of the interfacial impedance properties, improving the detection sensitivity. Based on these, the different membrane damage pathways were effectively distinguished and sensitively monitored with the normalized resistancecapacitance changes of inner-pore-related parameters including the electrolyte resistance within the pore length (Rpore), and the metal/inner-pore interfacial capacitance (Cpore) and the charge-transfer resistance (Rct-in) at the metal/inner-pore interface.
Biological membranes, including the external boundaries of cells as well as separate compartments within cells, are essential components of all living cells. Many chemical toxins and abnormally expressed proteins can interact with biomembranes and induce membrane damage, which may change the membrane permeability, disorder the membrane function, finally result in serious disease, such as Alzheimer’s disease, Keshan disease, Sickle-cell disease, and so on.1-5 Different reagents can induce membrane function disorder via different membrane damage pathways. For example, islet amyloid polypeptide, Melittin and Defensins damage membrane via layerby-layer dissolution (defined as detergent effect).6-10 On the other hand, some overexpressed proteins such as amyloid β peptide (Aβ) and α-synuclein can directly interact with lipid membrane, inducing the formation of membrane pores (defined as pore effect).11-14 Unveiling the definitive membrane damage pathway is critical for understanding the role of reagent in the disease mechanism and the related drug discovery. To detect the membrane damage, three lipid membrane structures including planar bilayer lipid membrane (BLM), black lipid membrane and vesicles have been used as typical models. In which, the BLM model system was mostly used due to its good stability, a long lifespan, a short formation time and thickness comparable to biomembranes. With the addition of chemical toxins or biomolecules into the system, the final structure and permeability changes of BLMs can be detected by electrochemical impedance spectroscopy (EIS), fluorescence, and potential clamp etc.15-18 However, it is still chal-
lenging to distinguish the membrane damage pathway by the techniques. So far the only approach to provide the information about membrane damage pathways is real time atomic force microscopy (AFM) or scanning tunneling microscopy (STM), which is high-cost, poor-reproducibility, and inconvenient. Therefore, establishing a facile, low-cost, sensitive and effective method to reveal the membrane damage pathway is necessary and urgent. EIS is one of the commonly employed techniques to investigate the electrical properties of membrane.15,16,19 With an appropriate equivalent electric circuit (EEC) analysis, some parameters related with the electric and electron transfer properties on and cross the membrane can be extracted. However, to ensure the structure stability and the biocompatibility of BLM, a self-assembly monolayer (SAM) was commonly used to separate the BLM and the conductive substrate, which greatly decrease the sensitivity of EIS. As a result, those slight changes on the membrane structures are difficult to be detected. Meanwhile, due to the inevitable poor reproducibility, BLMs made under the same conditions, usually have a number of individual differences, such as resistance, capacitance, breakdown voltage, and stability.20,21 The significant difference on the structure-related parameter make the further analysis on membrane structure changes difficult. To solve the problem, some traditional ordered porous materials such as anodized porous alumina22,23, glass nanopores24,25, and silicon nitride16 were introduced to support BLMs. However, the high aspect ratio of the material nanopores is disadvantageous to mass
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transfer of matters, which decreased the sensitivity and increased the response time. Moreover, if the system is limited by mass-transport, the EEC analysis for the interface will be too complex to obtain analytic solution for the general case.19 Herein, we present an EIS analysis for real-time BLM damage based on a porous SAM (pSAM) support. The construction of pSAM and the usage of appropriate EEC were the key points in the work. By selectively electrochemical reductive desorption of the mercaptopropionic acid in a mixed mercaptopropionic acid/11-mercaptoundecanoic acid SAM, the pSAM with plenty of nanopores with tens of nanometers in diameter and several nanometers in height (defined as innerpores) was prepared. The ultra-low aspect ratio of the innerpores was advantageous to the mass transfer of electrochemical probe [Fe(CN)6]3-, simplifying the EEC for EIS analysis at the electrode/membrane interface. [Fe(CN)6]3-/4- transferring from the bulk solution into the inner-pore induce significant changes of the interfacial impedance properties, improved the detection sensitivity. Based on these, the different membrane damage pathways were effectively distinguished and sensitively monitored with the normalized resistance-capacitance (RC) changes of inner-pore-related parameters including the electrolyte resistance within the pore length (Rpore), and the constantphase element (Qpore) and the charge-transfer resistance (Rct-in) at the metal/inner-pore interface.
the unmodified region of the gold surface. Each step in above fabrication process was characterized by CV and EIS to obtain reproducible results. Preparation of monomeric Aβ peptide. To obtain a homogeneous solution of monomeric form, lyophilized Aβ1-40 peptide was dissolved in 1 mg·mL-1 HFIP for 2 h, sonicated for 30 min to dissociate any preexisting aggregates and frozen with liquid nitrogen. The solvent HFIP was removed by a freeze dryer (LaboGene ScanVac CoolSafe 110-4 Pro Freeze Dryer, Denmark) at -100 oC. Prior to use, the Aβ1-40 (0.25 mg) powder was dissolved in 20 mM Tris-HCl (pH = 7.4) to a desired concentration.
RESULTS AND DISCUSSION
EXPERIMENTAL SECTION Reagents. Mercaptopropionic acid (MPA), 11mercaptoundecanoic acid (MUA), 1,2-Dioleoyl-sn-glycero-3phosphocholine (DOPC), Tritox X-100, 1,1,1,3,3,3hexafluoro-2-propanol (HFIP), NaCl, and KCl were purchased from Sigma (Shanghai, China). β-amyloid peptide (Aβ1-40) was from Wuhan Fine Peptide Co., Ltd. (Wuhan, China). Triton100, Na3[Fe(CN)6], and Na4[Fe(CN)6] were from Aladdin (Shanghai, China). All other reagents were of analytical grade and used as received. All solutions were prepared with deionized water with a resistivity of 18.2 MΩ·cm produced using a Millipore Simplicity 185 System (Millipore Co., Billerica, MA) and stored at 4°C as stock solution. Apparatus and measurements. Cyclic voltammetry (CV) and EIS were conducted with a Gamry Reference 600 electrochemical workstation (Gamry Instruments Co., Ltd., Warminster, PA, U.S.A.). A conventional three-electrode system was used in this work. Different modified Au(111) were used as the working electrodes, and a platinum wire electrode and an Ag/AgCl electrode were used as the counter and reference electrodes, respectively. All of the potentials in this study were with respect to the Ag/AgCl electrode. The electro-fabrication of pSAM was performed in N2-saturated 0.5 M NaOH solution to prevent the oxygen disturbance. AFM measurements were performed on an Agilent 5500 SPM (Keysight, USA) at room temperature. Fabrication of a porous SAM supported bilayer lipid membrane (pSAM-BLM). The bare Au electrode (2 mm in diameter) was dipped into a mixed MPA(1 mM)/MUA(4 mM) ethanol solution for 16 h at room temperature to form a mixed MUA/MPA SAM. Then MPA was stripped off from the Au substrate by a linear scanning from -0.1 to -1.0 V in 0.5 M NaOH solution (scan rate: 50 mV·s-1) to obtain a MUA pSAMs. After being thoroughly rinsed by deionized water, the pSAM was immersed into 150 µL DOPC giant unilamellar vesicles solution (GUVs, prepared by electroformation and stored at 4oC for later use26) for 2 h to form a pSAM-BLM. Finally, the electrode was immersed into an ethanol solution containing 4 mM MUA at room temperature for 2 h to block
Figure 1. Fabrication and characterization of pSAM-BLM. (A) Schematic representation of the pSAM-BLM fabrication. (B) Selectively desorption of the MPA from a mixed MPA/MUA SAM by a linear scanning from -0.1 to -1.0 V in a 0.5 M NaOH solution. The electrode was immersed in a χMPA/χMUA=1/4 solution overnight to form the mixed SAM. The scan rate was 50 mV·s-1. (C) Topographical AFM images of the mixed MPA/MUA SAM (top panel) and the pSAM after the MPA had been desorbed (bottom panel). (D) CV characterization of a pSAM in 1 mM Na3[Fe(CN)6]/Na4[Fe(CN)6] solution before and after painting BLM.
Fabrication and characterization of pSAM-BLM. Figure 1A shows schematically the steps involved in the fabrication of pSAMs via selective desorption of an alkanethiol, and the subsequent suspending of the lipid membrane. By choosing a stripping potential at a value between the reduction potential of the shorter alkanethiol MPA and that of the longer MUA, MPA can be stripped, and segregated bare gold regions with ultra-low aspect ratio can be created (defined as inner-pores). Due to the slow diffusion of alkanethiol molecules from a densely packed region to a bare Au region (e.g., 96 h or longer)27,28, the attachment of the lipid membrane onto the MUA SAMs (which takes only 1 h to densely cover the bare gold regions) will stabilize the porous structure. The effect of selectively desorbing an alkanethiol in a binary SAM was examined using linear scan voltammetry (LSV), as shown in Figure 1B. In the first potential scan, components of different chain lengths undergo reductive desorption at different potentials (MPA at -0.97 V, and MUA at -1.08 V). These potentials are essentially identical to the reduction potentials at SAMs of individual alkanethiols (Figure S-1 in supporting
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Analytical Chemistry information). This suggests that there is a high degree of phase separation in the binary SAM. Apparently desorption of the hydrophilic MPA is a fast process. Therefore, after a linear scanning from -0.1 to -1.0 V, in the second potential scan, the peak corresponding to the MPA reduction disappeared. On the basis of the amount of MPA desorbed and the surface density of a closely packed alkanethiol (7.6×10-10 mol·cm-2 expected for the (√3×√3)R30° packing29), the ratio of gold areas freshly exposed from the MPA/MUA films were calculated to be 32%. Electrochemical AFM experiments were performed to monitor the reductive desorption of MPA. Two AFM images of the mixed MPA/MUA SAM (top panel) and the porous SAM after the MPA had been desorbed (bottom panel) were shown in Figure 1C. It can be seen that reductive desorption of MPA resulted in the appearance of some small and isolated “dark spots”. The size of these spots, which corresponds to the dimension of the area originally occupied by the MPA molecules, range from 12 to 19 nm. On the basis of the consistency between the CV and AFM results, it can be expected that the MPA domains may be altered by varying the soaking solution composition, which facilitates to adjust the inner-pore size of fabricated pSAM. The successive fabrication of the pSAM-BLM was also characterized by CV. The peak currents in which can reflect the electron transfer of the redox probe [Fe(CN)6]3-/4- at the membrane/electrode interface. As displayed in Figure 1D, a pair of well-defined redox peaks can be observed for the bare Au electrode (black curve), indicating the fast electron transfer of the redox probe. With the loading of mixed SAM, the interfacial electron transfer was completely blocked, and the redox peak currents (red curve) disappeared. The subsequent desorption of MPA led to the formation of inner-pore, the redox peak currents (blue curve) was partly recovered. Afterword, the suspending of BLM blocked some inner-pores, which induced the peak current decrease again (purple curve). Finally, when MUA was filled into the uncovered inner-pores, the redox peak currents disappeared again (green curve).
Figure 2. (A) EIS in Bode plots of a pSAM-BLM in 1 mM Na3[Fe(CN)6]/Na4[Fe(CN)6] solution at zero bias in a frequency range of 10-3−106 Hz. (B) Two equivalent circuits used to simulate the impedance data. Solid red line and black line in Panel A are fittings for lipid bilayers using EEC 1 and EEC 2, respectively. (C) The overall interfacial structure of the pSAM-BLM model.
frequency range of 10-3−106 Hz (Figure 2A). The successful formation of pSAM-BLMs is approved by a value of the impedance magnitude above 1 MΩ as frequency toward zero.30 However, the non-typical impedance responses in Bode representations provide clear indication that more than one time constant are required to describe the process. To extract the electrical properties including the membrane resistance and the specific membrane capacitance, an appropriate EEC that can describe all EIS behaviors of the system is necessary. The EEC 1 as depicted in Figure 2B is commonly used in the literature19 for electrode coated with two inert porous layers, wherein the parallel combination of Rct-in (charge-transfer resistance) and Cpore (double-layer capacitance) corresponds to the impedance at the inner-pore/metal interface. Rpore is the electrolyte resistance within the pore length, and the insulating part of the inner MUA coating is considered to be a capacitor CMUA, which is in parallel with the impedance in the innerpore. The effect of the outer BLM layer is taken into account with an additional series RBLMCBLM circuit. The resistance of the electrolyte solution Rs is added in series with the previous impedance. However, obvious fitting deviations were seen for Bode plots (red lines in Figure 2A), indicating that the EEC 1 is too simple to describe the structure of the pSAM-BLMs and their surroundings. More careful refinement of the EEC for fitting to all types of impedance representations of the pSAMBLM system is required. Figure 2C illustrated the complex situation of the pSAMBLM system in practice, including the pSAM-supported BLM (zone 3), the compact MUA SAM uncovered by BLM (zone 1) and the pinholes in MUA (zone 2). Zones 1 and 2 usually act as background and are irreproducible in each fabrication, could influence the impedance response. Considering that the total current is the sum of the above three individual current contributions, we propose the EEC 2, also depicted in Figure 2B, wherein two RC modules were added in parallel with the EEC 1. The CMUA-uc is the capacitance of the compact MUA SAM uncovered by BLM (Zone 1). The parallel Rct-hole and Qhole correspond to the charge-transfer resistance and doublelayer capacitance at the pinhole/metal interface, which is in series with Rhole (the electrolyte resistance within the pinhole length), as a circuit analog for zone 2. Here, the ideal capacitances Cpore and Chole, corresponding to the metal/inner-pore and metal/pinhole interfacial capacitance, respectively, are replaced by two constant-phase elements (CPE) Qpore and Qhole, taking into account the frequency dispersion of the capacitance due to factors such as inhomogeneity of the surface.31-33 The constant-phase element can be expressed as YCPE(ω)=Y0(iω)n, where Y0 and n (0 < n
