Electrochemical Analysis of Enzyme Based on the Self-Assembly of

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Electrochemical analysis of enzyme based on the self-assembly of lipid bilayer on an electrode surface mediated by hydrazone chemistry Juan Zhang, Xiaonan Wang, Tingjun Chen, Chang Feng, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03197 • Publication Date (Web): 22 Nov 2017 Downloaded from http://pubs.acs.org on November 24, 2017

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Analytical Chemistry

Electrochemical analysis of enzyme based on the self-assembly of lipid bilayer on an electrode surface mediated by hydrazone chemistry Juan Zhanga, Xiaonan Wanga,b, Tingjun Chena, Chang Fengc, Genxi Lia,c,* a

Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China. b Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, P. R. China. c State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China. Tel.: +86-25-83593596, Fax: +86-2583592510, E-mail: [email protected]. ABSTRACT: In this work, a new strategy for electrochemical analysis of enzyme has been proposed based on a self-assembled lipid bilayer on an electrode surface mediated by hydrazone chemistry. Taking aldolase as an example, the enzyme can catalyze the formation of products containing carbonyl groups. These groups can react with hydrazine groups of the functional lipid derivative, resulting in the self-assembly of lipid bilayer on a guanidinium modified electrode surface. The lipid bilayer will then prevent the movement of hydrophilic electrochemical probes. Consequently, the catalytic reaction of the enzyme may result in the change of the obtained electrochemical peak current. Experimental results reveal that aldolase activity can be analyzed over a widely linear detection range from 5 mU/L to 100 U/L with a low detection limit of 1 mU/L. Meanwhile, the method can exhibit good precision and reproducibility, and it can be applied for real sample analysis. What is more, since lipid bilayer is the universal basis for cellmembrane structure, while hydrazone chemistry is popular in nature, this work may also provide a new insight for the development of electrochemical analysis and electrochemical biosensors.

A lipid bilayer is a kind of self-assembled structure in living nature and the universal basis for living cell-membrane structure. Up to now, some membrane systems, such as black lipid membranes1, solid supported lipid bilayers2,3, hybrid lipid bilayers4,5, polymer cushioned lipid bilayer6,7, tethered lipid bilayer8, and supported vesicular layer9,10 have been artificially constructed. Lipid bilayers have also been explored for lab-ona-chip based systems using microfluidic device11. A few conducting substrate electrodes capable of supporting bilayers include indium-tin-oxide12, gold5, glassy carbon13, and pyrolyticgraphic14. Up to now, lipid bilayers have been grafted onto solid surfaces using silane-functionalized-poly(ethylene-oxide) tethers8, benzophenone derivatives by a photo-crosslinking reaction15, and alkanethiol5,16. It is of importance to explore new means for grafting lipid bilayers onto the solid surfaces. As a kind of chemoselective ligation, hydrazone chemistry is pervasivene and versatile in view of its modularity and unique structural property. Hydrazone chemistry has been used in many research fields, for instance, metal and covalent organic frameworks17,18, dynamic combinatorial chemistry19,20, holetransporting materials21, molecular switches22,23, metalloassemblies24,25, and sensors26,27. In this work, we propose that hydrazone ligation can also serve for the self-assembly of lipid bilayer onto the solid surface, because the carbonyl groups contained in the catalytic reaction related to enzymes can react with the hydrazine group to form a hydrazone bond so as to mediate the self-assembly of lipid bilayer. On the other hand,

it is well known that a lipid bilayer is impermeable to hydrophilic molecules. The presence of the lipid bilayer may effectively exclude hydrophilic electroactive compounds from reaching the electrode surface, resulting in the change of electrochemical signal. So, new strategy for the design of electrochemical method for enzyme analysis should be possible, based on hydrazone chemistry assisted self-assembly of lipid bilayer on an electrode surface. Enzymes have been extensively analyzed through electrochemical means. As far as signal output is concerned, enzymatic catalyzed products play a crucial role. The direct electrochemical signal of immobilized product such as a quinone derivative has been utilized to analyze NAD(P)H: Quinone oxidoreductase 128. Except that, the electrochemical signals of free products like p-nitrophenol 29 and p-aminophenol30 have also been explored for the detection of cellulase and β-D glucuronidase activities. Meanwhile, the decreasing resistance properties of immobilized catalyzed products compared to those of enzymatic substrates, have served for analysis of αamylase31 and cellulase32. Moreover, immobilized and free products can mediate the formation of signal substances for electrochemical assay. For instance, terminal deoxynucleotidyl transferase-generated C-rich DNA sequence on the electrode surface was employed for the synthetic template of AgNCs which possess high electro-catalytic activity to H2O2 reduction33; phosphate ions released by catalysis of both protein kinase and alkaline phosphatase, can induce deposition of redox precipitates34. Furthermore, electrochemical method for

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enzyme analysis has been established by using substrate as a linker for the linkage of signal probe35. In this work, taking aldolase as an example, an electrochemical method for the assay of enzyme activity has been explored by using hydrazone ligation assisted self-assembly of lipid bilayer on electrode surface. As a disease marker for diagnosis and treatment of multiple cancers including lung squamous cell carcinoma36, renal cancer37, and hepatocellular carcinoma38, the aldolase catalyzes the reversible conversion of fructose-1,6-bisphosphate (FBP) to produce glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). Different from the enzyme substrate, the two products own carbonyl group in their molecular structure, thus they can be utilized as functional linkers to mediate the fabrication of lipid bilayer through hydrazone chemistry. Therefore, an electrochemical method for the analysis of aldolase is proposed. The established method will be potentially applied for clinical analysis of aldolase in serum.

EXPERIMENTAL SECTION Materials and reagents. Aldolase (EC 4.1.2.13, 10 U/mg, from rabbit muscle), GAP, DHAP, α-amylase, lipase, trypsin, glucose oxidase, mercaptoethanol (MCH), and Tris(2carboxyethyl)phosphine (TCEP) were purchased from Sigma (Shanghai, China). FBP was obtained from Aladdin (Shanghai, China). The tripeptide, arginine-glycine-cysteine (RGC), was prepared by Top-peptide Co., Ltd (Shanghai, China). The polypeptide (CRLVSYNGIIFFLK) and DNA (GGTGGTCCTGGA) were separately prepared by KeTai Co., Ltd (Shanghai, China) and Sangon Biotech (Shanghai, China). The 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) conjugated polyethylene Glycol 10000 and hydrazine (DSPE-PEG-Hydrazine) was obtained from Peng Sheng Biological Co., Ltd (Shanghai, China). For the preparation of buffers and aqueous solutions, ultrapure water with a specific resistance of 18 MΩ•cm was purified with a Millipore Mill-Q water purification system (Barnstead, USA). The aldolase solution was prepared using 0.01 M Tris buffer solution adjusted the pH to 7.5 using 1 M HCl. The phosphate buffer (pH 7.0) was prepared using 0.005 M NaH2PO4, 0.005 M Na2HPO4, and 0.1 M NaCl. Method for modification of gold electrode. The gold electrode was pretreated according to our previous method35. The pretreated electrode was firstly immersed in 2.5 µM RGC which was solubilized in 10 mM Tris-HCl buffer (pH 7.5) containing 5 mM TCEP for 16 h, to give RGC modified gold electrode (RGC/AuE). Subsequently, the RGC/AuE was continuously incubated in 1 mM MCH aqueous solution for 1 h to block the inactive sites and to achieve a well aligned RGC monolayer. After that, the modified electrode was immersed in GAP dissolved in 10 mM Tris-HCl (pH 7.5) for 25 min, to obtain GAP functionalized gold electrode (GAP/RGC/AuE). Method for tethered lipid bilayer formation. In order to give lipid bilayer covered electrode, DSPE-PEG-hydrozide was dissolved in chloroform. Subsequently, a 5 µL aliquot of the solution was dropped onto the surface of GAP/RGC/AuE, followed by the immediately transfer of the electrode into the

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phosphate buffer (pH 7.0). After 20 min, lipid bilayer (LB) was formed to give LB/GAP/RGC/AuE, followed by thoroughly washing using a 1% N, N-dimethyldodecylamine Noxide (LDAO) in water detergent solution to remove all the free lipids. Characterization of lipid bilayer. Lipid bilayer was characterized by Fourier transform infrared spectroscopy (FT-IR), impedance spectroscopy, fluorescent microscopy, fluorescent spectroscopy, and contact angle goniometer. Considering the feasibility of FT-IR measurement, gold nanoparticles (AuNPs) were used for the preparation of samples instead of AuE. RGC/AuNPs, GAP/RGC/AuNPs, and LB/GAP/RGC/AuNPs were prepared according to the method described in the Supporting Information. The FT-IR spectra of RGC/AuNPs, GAP/RGC/AuNPs, and LB/GAP/RGC/AuNPs were recorded using VERTEX 70 Fourier Transform Infrared spectrometer (Bruker Co. Ltd., Bergisch Gladbach, Germany). The impedance spectra of GAP/RGC/AuE and LB/GAP/RGC/AuE were performed in the frequency range from 0.01 Hz to 10 kHz with a signal amplitude of 10 mV using a CHI660C electrochemical workstation (CH Instruments, Shanghai, China) at room temperature. A 5 mM [Fe(CN)6]3-/4- containing 0.1 M KCl in 5 mM phosphate buffer (pH 7.0) was used as redox probe with Biasing potential of 0.224 V. In view of feasibility for measurement using fluorescence microscopy and fluorescence spectrometer as well as water contact angle goniometer, gold plaque (Au) was utilized instead of AuE for preparation of samples. RGC/Au, GAP/RGC/Au, and LB/GAP/RGC/Au were prepared by using the method described in the Supporting Information. After that, 0.001% (Molar ratio) of Dio molecule was mixed with 1 µM of DSPE-PEG-hydrazine in chloroform. Subsequently, a 5 µL aliquot of the mixture was dropped onto the surface of GAP/RGC/Au, followed by the immediately transfer of the gold plaque into the phosphate buffer (pH 7.0) for 20 min, to give LB+Dio/GAP/RGC/Au. Finally, the modified gold plaques were rinsed thoroughly with a 1% LDAO to remove all the free lipids and DiO molecules. The samples obtained were imaged through fluorescence microscopy (Nikon Eclipse 80I, Nikon Instruments Inc., Japan). For LB+Dio/GAP/RGC/Au with different GAP concentrations, quantitative fluorescence intensities were collected on an F-7000 fluorescence spectrometer (Hitachi, Ltd., Japan) using 488 nm of excitation wavelength and 505 nm - 525 nm of emission range. The fluorescence intensity was determined from the area of the emission peak minus the counts for a 1% LDAO control solution. Water contact angles were measured using an OCA15EC contact angle goniometer (Eastern-Dataphy, HK, China). During the measurements, the syringe tip remained in the water drop. Contact angles were determined using both sides of three drops for each sample. Aldolase assay. For enzyme analysis, 50 µL 5 µM FBP was incubated with 50 µL aldolase in 10 mM Tris-HCl buffer (pH


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7.5) with different concentrations (0.005 U/L to 100 U/L) at 25 oC for 10 min. Subsequently, RGC/AuE was immersed in the reacting solution for 20 min, to give product modified electrode (Pro/RGC/AuE). Then a 5 µL aliquot of DSPE-PEGhydrazide in chloroform was dropped onto Pro/RGC/AuE and the modified electrode was instantly transferred into the phosphate buffer (pH 7.0) for 20 min, to give LB/Pro/RGC/AuE. In contrast, FBP/RGC/AuE can be obtained in absence of aldolase. At last, the electrodes were washed thoroughly by 1% LDAO and used for electrochemical measurements.

LB/GAP/RGC/AuNPs (curve e), the absorbance band at 1639 cm-1 can belong to C=N stretching vibration, confirming the formation of hydrazone bond (Figure 1 (B)).

Specificity analysis and real sample detection. For specific analysis of enzyme products as linkers, different samples including peptide and DNA as well as the mixtures of these substances with the products were used instead of the products for enzyme assay. For specific analysis of aldolase detection, different samples including α-amylase, BSA, lipase, glucose oxidase, and trypsin as well as the mixtures of these substances with aldolase were used instead of aldolase for enzyme assay. For the electrochemical analysis in real samples, different amounts of aldolase were added into fetal calf serum to give biological fluid samples. The enzyme contents were detected using established electrochemical method. Electrochemical Measurement. Cyclic and linear sweep voltammetric measurements were carried out using a CHI660C electrochemical workstation (CH Instruments, Shanghai, China) with a conventional three electrode cell in which the modified electrode as the working electrode, saturated calomel electrode (SCE) as the reference electrode, and platinum wire as the counter electrode. Cyclic voltammetry was performed over the potential range from -0.2 V to 0.8 V at a scan rate of 100 mV/s using 5 mM [Fe(CN)6]3-/4- in 0.1 M KCl. The same redox probe and electrolyte were used for linear sweep voltammetry over the potential range from -0.2 V to 0.6 V with a pulse amplitude of 50 mV and a pulse width of 50 ms at a scan rate of 100 mV/s.

RESULTS AND DISCUSSIONS Formation of lipid bilayer on electrode surface. The mechanism for the formation of lipid bilayer mediated by GAP on the surface of gold electrode through hydrazone chemistry is given in Figure 1 (A). RGC containing thiol group can coordinate with gold electrode through Au-S bond, to give RGC/AuE (Figure 1 (A)). As exhibited in Figure 1 (B), the absorbance band at 2450-2550 cm-1 assigned to the S-H bond, disappears in the spectrum of RGC/AuNPs (curve b)39. As the product of aldolase catalytic reaction, GAP can combine with RGC through interaction between its phosphate acid group and guanidinium group of the latter (Figure 1 (A)). It has been reported that the arginine-phosphate electrostatic interaction possesses a “covalent like” stability40. Compared GAP (curve c) with GAP/RGC/AuNPs (curve d), P=O stretching absorption band shifts from 1058 cm−1 to 1045 cm−1, suggesting the formation of hydrogen bonds (Figure 1(B)). Subsequently, the carbonyl group of GAP can chemoselectively react with hydrazine group of DSPE-PEG-Hydrazine to form hydrazone bond, resulting in the grafting of lipid bilayer on the surface of gold electrode (Figure 1 (A)). For

Figure 1. (A) Schematic illustration for mechanism of lipid bilayer formation on the surface of gold electrode through hydrazone chemistry. (B) FT-IR spectra of (a) RGC, (b) RGC/AuNPs, (c) GAP, (d) GAP/RGC/AuNPs, and (e) LB/GAP/RGC/AuNPs. (C) Complex plane plot for the electrochemical impedance measurements of the gold electrode at different modification stages: (a) GAP/RGC/AuE and (b) LB/GAP/RGC/AuE. Electrochemical species: 5 mM [Fe(CN)6]3-/4- containing 0.1 M KCl, Electrolyte: 5 mM phosphate buffer (pH 7.0), Biasing potential: 0.224 V, Amplitude: 10 mV, and Frequency range: 0.01 Hz - 10 kHz. (D) Fluorescence images of LB/GAP/RGC/Au with different concentrations of GAP ((a) 0, (b) 0.5, (c) 1, (d) 1.5, (e) 2, (f) 2.5, (g) 3, (h) 3.5, (i) 4, and (j) 4.5 µM) using Dio as molecular probe. (E) Fluorescence spectra of LB/GAP/RGC/Au with different GAP concentrations (0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 µM) using Dio as molecular probe. (F) Contact angles of LB/GAP/RGC/Au with different GAP concentrations (0, 0.5, 1, 2, 3, 4, and 5 µM). In order to verify the formation of lipid bilayer, the impedance spectroscopy has been used for probing the feature of surface-modified electrode. As shown in Figure 1 (C),



GAP/RGC/AuE exhibits an almost straight line, the characteristic of a diffusional limiting step of the electrochemical process. LB/GAP/RGC/AuE shows the presence of a single semicircle in the high frequency domain, indicating that a single mechanism of electron transfer dominates the process16. From these plots using a model of electrical equivalent circuit, resistance of LB/GAP/RGC/AuE has been determined to be 3.4 × 104 Ω, in accordance with the value of lipid bilayer reported previously16. Meanwhile, the capacitance values Cm and the electrode double layer Cdl are identical to those in the previous report, confirming the model of a lipid bilayer decoupled from the electrode by a hydrate cushion5,41,42. The thickness of lipid bilayer is further calculated using the capacitance values Cm, giving a value of about 4.3 nm, in well agreement with that of lipid bilayer11. Fluorescence microscopy and spectroscopy have been further utilized to confirm the formation of lipid bilayer on the gold electrode surface. As a lipophilic trace, Dio is weakly fluorescent in water but highly fluorescent and quite photostable when incorporated into lipid membranes43. As exhibited in Figure 1 (D), no fluorescence can be observed without GAP, due to no loading of DSPE-PEG-Hydrazine on the electrode surface. With the increase of GAP concentrations from 0.5 µM to 5 µM, the fluorescence gradually become bright and then keep unchanged, suggesting the stepwise self-assembly of lipid bilayer and eventual formation at 4.5 µM of GAP concentration. At the same time, fluorescence intensities show the corresponding changes along with the increasing GAP concentrations (Figure 1(E)). These results well indicate the successful self-assembly of GAP mediated lipid bilayer on the surface of gold electrode. Furthermore, we further confirm the existence of lipid bilayer by testing wettability of solid surface through contact angle measurement44. As shown in Figure 1 (F), the static contact angles decrease along with gradual modification of RGC and GAP, the hydrophilicity of the surface of gold plaque increases. However, the angles increase for LB/GAP/RGC/Au, suggesting the enhanced hydrophobicity and the corresponding formation of lipid bilayer covering the surface of gold plaque. Meanwhile, the angles increase accompanying with the increase of GAP concentrations, indicating the rising amount of lipid bilayer on the electrode surface. The angles are comparable to those previously published45. Preventing effect of lipid bilayer on the transport of hydrophilic electroactive probes. Due to hydrophobic property of lipid component, lipid bilayer is impermeable to hydrophilic compounds and can efficiently prevent hydrophilic electroactive probes from reaching the electrode surface (Figure 2 (A)). As shown in Figure 2 (B), the reduction and oxidation of hydrophilic electroactive probe K3[Fe(CN)6] are greatly attenuated for LB/GAP/RGC/AuE (curve d) compared with the bare AuE (curve a), RGC/AuE (curve b), and GAP/RGC/AuE (curve c). At the bare electrode, reduction and oxidation of the ion are facile, reversible, and one-electron processes. After modified with RGC (Figure 2 (B), curve b), the current values almost keep unchangeable in comparison with that of bare AuE (Figure 2 (B), curve a). With the further addition of GAP (Figure 2 (B), curve c), the value decreases owing to the insulating characteristic of GAP. After formation of the bilayer

(LB/GAP/RGC/AuE), the electron value decreases apparently and the process is no longer readily reversible (Figure 2 (B), curve d). The presence of the bilayer reduces the rate of electron transfer in comparison with the bare AuE and GAP/RGC/AuE, respectively.

Figure 2. (A) Schematic illustration for inhibition of transport of hydrophilic electroactive probes by GAP assisted lipid bilayer on the surface of gold electrode. (B) Cyclic voltammograms of 5 mM [Fe(CN)6]3-/4- in 0.1 M KCl at (a) bare AuE, (b) RGC/AuE, (c) GAP/RGC/AuE, and (d) LB/GAP/RGC/AuE. The scan rate was 100 mV/s. (C) Complex plane plot for the electrochemical impedance measurements of the gold electrode at different modification stages: (a) bare AuE, (b) RGC/AuE, (c) GAP/RGC/AuE, and (d) LB/GAP/RGC/AuE. Electrochemical species: 5 mM [Fe(CN)6]3-/4- containing 0.1 M KCl, Electrolyte: 5 mM phosphate buffer (pH 7.0), Biasing potential: 0.224 V, Amplitude: 10 mV, and Frequency range: 0.01 Hz - 10 kHz. After modification of RGC (Figure 2 (C), curve b), the resistance reduces in comparison with that of bare AuE (Figure 2 (C), curve a), due to electrostatic attraction between positively charged surface of RGC/AuE and negatively charged electroactive probe, [Fe(CN)6]3-/4-. Along with further addition of GAP (Figure 2 (C), curve c), the resistance increases, signifying that GAP inhibits electron transfer from solution to electrode surface due to the insulating characteristics. After the formation of lipid bilayer, the resistance increases extremely obviously for LB/GAP/RGC/AuE (curve d), as a result of the inhibiting effect of lipid bilayer on transport of K3[Fe(CN)6]. Mechanism investigation for the assay of aldolase activity. Using aldolase as an example, we develop enzymatic electrochemical method based on hydrazone chemistry mediated lipid bilayer. The mechanism for electrochemical analysis of aldolase is exhibited in Figure 3 (A). In the presence of aldolase, FBP can reversibly convert to products (Pro) including GAP and DHAP. Different from FBP only with phosphate acid group, both GAP and DHAP contain two functional groups, i.e. phosphate group and carbonyl group. Phosphate group can strongly interact with guanidinium group exposed on the surface of RGC/AuE40, resulting in the formation of Pro/RGC/AuE and the corresponding exposure of carbonyl


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group on the surface of modified electrode. Subsequently, carbonyl group can chemoselectively react with hydrazine group of DSPE-PEG-Hydrazine to form hydrazone bond, leading to self-assembly of lipid bilayer covering electrode surface. In view of preventing effect of lipid bilayer on hydrophilic electrochemical probe, a low electrical current value can be obtained. On contrary, DSPE-PEG-Hydrazine can not link to FBP/RGC/AuE and the corresponding lipid bilayer can not form without enzyme, owing to the shortage of carbonyl group on the surface of FBP modified electrode. Therefore, a relatively high current value can be given in absence of aldolase.

Figure 3. (A) Schematic illustration of mechanism for electrochemical analysis of aldolase based on inhibition of the transport of hydrophilic electroactive probe by hydrazone ligation assisted lipid bilayer. (B) Cyclic voltammograms obtained using (a) FBP/RGC/AuE, and (b) DSPE-PEG-Hydrazine + FBP/RGC/AuE. (C) Cyclic voltammograms with different aldolase concentrations at (a) 0 U/L and (b) 100 U/L in buffer solution (2.5 M (NH4)2SO4, 0.01 M Tris, 0.001 M EDTA, pH 7.5). Scanning rate: 100 mV/s. Electrolyte: 5 mM [Fe(CN)6]3/4in 0.1 M KCl. As exhibited in Figure 3 (B), after adding DSPE-PEGHydrazine (curve b), little electrical current change can be found compared to that of FBP/RGC/AuE (curve a). At the same time, high current value can be obtained (Figure 3 (B), curve b). In absence of aldolase (Figure 3 (C), curve a), almost same cyclic voltammogram and current value are detected in comparison with those for DSPE-PEG-Hydrazine + FBP/RGC/AuE (Figure 3 (B), curve b). It suggests no formation of lipid bilayer on the surface of modified electrode, since that FBP are not cleaved into the product without enzyme. In contrast, the current value dramatically decreases in the presence of aldolase (Figure 3 (C), curve b). It can be explained for the self-assembly of lipid bilayer assisted by hydrazone ligation originated from the reaction between hydrazine group of DSPE-PEG-Hydrazine and carbonyl group on the surface of Pro/RGC/AuE. Specificity investigation for aldolase assay. The specific linkage of products between RGC/AuE and DSPE-PEGHydrazine is vital for the fabrication of aldolase electrochemi-

cal biosensor. It is well known that phosphate group generally exists in a variety of biomolecules such as peptide and DNA. So these substances have been chosen to evaluate the specificity of the products. As exhibited in Figure 4 (A), instead of Pro, the separate addition of peptide and DNA can not render the decreases of current values as a result of no self-assembly of DSPE-PEG-Hydrazine on the modified electrode surface. Inversely, low current value can be observed in the presence of Pro, resulting from the formation of lipid bilayer. Meanwhile, similar low current values can be given when Pro is separately mixed peptide and DNA with equal molar. The result confirms the priority of Pro to bind with guanidinium group on the surface of RGC/AuE. These well suggest the specificity of product as a linker.

Figure 4. (A) The specificity investigation of enzyme products, i.e. GAP and DHAP, as linkers against the different compounds: Pro (2.5 µM, molar ratio of GAP and DHAP = 1 : 1), peptide (2.5 µM), Pro (2.5 µM) + peptide (2.5 µM), DNA (2.5 µM), and Pro (2.5 µM) + DNA (2.5 µM). (B) The specificity investigation of aldolase analysis against the different samples: aldolase (0.1 U/L), α-amylase (10000 U/L), aldolase (0.1 U/L) + α-amylase (10000 U/L), BSA (0.5 mg/mL), aldolase (0.1 U/L) + BSA (0.5 mg/mL), lipase (10000 U/L), aldolase (0.1 U/L) + lipase (10000 U/L), glucose oxidase (10000 U/L), aldolase (0.1 U/L) + glucose oxidase (10000 U/L), trypsin (10000 U/L), and aldolase (0.1 U/L) + trypsin (10000 U/L). Error bars are obtained based on three independent measurements. We further investigate the specificity of the established electrochemical method for aldolase analysis and the results are shown in Figure 4 (B). Different from that of aldolase, the separate usages of α-amylase, BSA, lipase, glucose oxidase, and trypsin can do not result in the appearance of low current values, due to no cleavage of FBP. On contrary, low current values can be observed for the mixtures of these substances and aldolase, suggesting that these interfering proteins have negligible impacts on aldolase analysis and the method owns high specificity. Electrochemical detection of aldolase. Linearsweep voltammograms upon analyzing different concentrations of aldolase are shown in Figure 5 (A). The highest current value can be achieved in absence of aldolase. Enzyme substrate, FBP, is captured onto the surface of RGC/AuE to give FBP modified electrode, resulting in the exposure of phosphate acid group on the surface of FBP/RGC/AuE. The phosphate acid group can not react with hydrazine group of DSPE-PEGHydrazine, so the self-assembly of lipid bilayer can not occur and high current value can be observed. With the increase of



enzyme concentrations from 0 to 100 U/L, the current values gradually decline. It implies the stepwise cleavage of FBP into GAP and DHAP, and the two products are loaded onto RGC/AuE, leading to the happening of carbonyl groups on the surface of modified electrode. The carbonyl group can chemoselectively react with hydrazine group of DSPE-PEGHydrazine so as to realize the self-assembly of lipid bilayer which inhibits hydrophilic electrochemical probe from reaching the electrode surface.

Figure 5. (A) Linear sweep voltammograms for aldolase at different concentrations: from 0 U/L to 100 U/L. (B) The linear relationship between peak current values and the logarithmic values of aldolase concentrations. The current values have been further utilized to quantitatively analyze aldolase and the corresponding results are shown in Figure 5(B). The current values linearly decrease with logarithmic values of increasing enzyme concentrations from 5 mU/L to 100 U/L, which is wider than previous reports35,46. The linear fitting equation of I=72.028-19.421lgC (U/L, R2=0.99897) can be obtained with detection limit of 1 mU/L (3 times signal to noise ratio), lower than the values in the previous report35,46,47. Meanwhile, the electrochemical experiments have been conducted three times and the detection precision has been evaluated using the slopes of three regression equations with enzyme concentrations from 5 mU/L to 100 U/L, giving 2.9% of RSD value. It well indicates good reproducibility and precision of the current electrochemical method. Aldolase assay in the biological fluids. In order to evaluate the practicable application of the established electrochemicalmethod in the biological sample, a certain amount of aldolase (10 U/L, 1 U/L, 0.1 U/L, and 0.01 U/L) is added into the fetal calf serum and the aldolase concentrations are determined using our current method (Table 1). The recovery ratio varies from 99.45% to 100.49% and the RSD values are basically within 5% with an average value of 3.325%. These results indicate that our method owns good anti-interfering capability and can well serve for aldolase analysis in the real sample such as serum. Table 1. Aldolase concentrations detected by our method and the comparison with the given concentrations in serum samples. Samples

Aldolase concentrations detected (U/L)

Standard concentration (U/L)

Recovery ratio (%)

Relative error (%)

1 2 3 4

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10.090±0.6088 0.959±0.0452 0.102±0.0030 0.010±0.0002

10 1 0.1 0.01

99.88 100.49 99.79 99.45

5.0 3.9 2.4 2.0

CONCLUSIONS In summary, a new strategy has been proposed to develop electrochemical method for enzyme activity analysis based on hydrazone ligation assisted self-assembly of lipid bilayer on an electrode surface. Using aldolase as an example, the new method shows wide detection range, low detection limit, good reproducibility and precision, as well as applicability. This work has also expanded not only the employment of hydrazone chemistry but also the application of lipid bilayer, which may contribute a new insight for the fabrication of electrochemical biosensors.

ASSOCIATED CONTENT Supporting Information Preparation of RGC/AuNPs, GAP/RGC/AuNPs, LB/GAP/RGC/AuNPs, RGC/Au, GAP/RGC/Au, and LB/GAP/RGC/Au, and optimization of experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author [email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 31671923 and 21235003).

REFERENCES (1) Winterhalter, M. Curr. Opin. Colloid. In. 2000, 5, 250-255. (2) Richter, R. P.; Bérat, R.; Brisson, A. R. Langmuir 2006, 22, 3497-3505. (3) Morigaki, K.; Kiyosue, K.; Taguchi, T. Langmuir 2004, 20, 7729-7735. (4) Zhou, X.; Moran-Mirabal, J. M.; Craighead, H. G.; Mceuen, P. L. Nat. Nanotechnol. 2007, 2, 185-190. (5) Terrettaz, S.; Mayer, M.; Vogel, H. Langmuir 2003, 19, 5567-5569. (6) Shao, J.; Wen, C.; Xuan, M.; Zhang, H.; Frueh, J.; Wan, M.; Gao, L.; He, Q. Phys. Chem. Chem. Phys. 2017, 19, 20082016. (7) Reddy, S. M. In Materials for Chemical Sensing; Springer, 2017, pp 75-103. (8) Purrucker, O.; Förtig, A.; Jordan, R.; Tanaka, M. Chem. Phys. Chem 2004, 5, 327-335. (9) Svedhem, S.; Pfeiffer, I.; Larsson, C.; Wingren, C.; Borrebaeck, C.; Höök, F. Chem. Bio. Chem 2003, 4, 339-343.


Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) Yoshina-Ishii, C.; Boxer, S. G. J. Am. Chem. Soc. 2003, 125, 3696-3697. (11) Castellana, E. T.; Cremer, P. S. Surf. Sci. Rep. 2006, 61, 429-444. (12) Cornell, B. A.; Braach-Maksvytis, V.; King, L.; Osman, P. Nature 1997, 387, 580-583. (13) Wu, Z.; Wang, B.; Cheng, Z.; Yang, X.; Dong, S.; Wang, E. Biosens. Bioelectron. 2001, 16, 47-52. (14) Yang, J.; Hu, N. Bioelectrochem. Bioenerg. 1999, 48, 117-127. (15) Naumann, C. A.; Prucker, O.; Lehmann, T.; Rühe, J.; Knoll, W.; Frank, C. W. Biomacromolecules 2002, 3, 27-35. (16) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126-1133. (17) Bunck, D. N.; Dichtel, W. R. J. Am. Chem. Soc. 2013, 135, 14952-14955. (18) Zhou, X. P.; Wu, Y.; Li, D. J. Am. Chem. Soc. 2013, 135, 16062-16065. (19) Corbett, P. T.; Leclaire, J.; Vial, L.; West, K. R.; Wietor, J. L.; Sanders, J. K. M.; Otto, S. Chem. Rev. 2006, 106, 36523711. (20) Jin, Y.; Yu, C.; Denman, R. J.; Zhang, W. Chem. Soc. Rev. 2013, 42, 6634-6654. (21) Lygaitis, R.; Getautis, V.; Grazulevicius, J. V. Chem. Soc. Rev. 2008, 37, 770-788. (22) Burdette, S. C. Nature Chem. 2012, 4, 695-696. (23) Ray, D.; Foy, J. T.; Hughes, R. P.; Aprahamian, I. Nature Chem. 2012, 4, 757-762. (24) Nitschke, J. R. Chem. Soc. Rev. 2014, 43, 1798-1799. (25) Reuven, D. G.; Li, H.; Harruna, I. I.; Wang, X. Q. J. Mater. Chem. 2012, 22, 15689-15694. (26) Su, X.; Aprahamian, I. Chem. Soc. Rev. 2014, 43, 19631981. (27) Zhang, J.; Liu, Y.; Lv, J.; Li, G. Nano Res. 2015, 8, 920930. (28) Chen, Y.; Liu, Y.; Chen, C.; Lv, J.; Zhang, J.; Li, G. Sensor Actuat B-Chem. 2015, 216, 343-348. (29) Cruys-Bagger, N.; Tatsumi, H.; Borch, K.; Westh, P. Anal. Biochem. 2014, 447, 162-168. (30) Rochelet, M.; Solanas, S.; Betelli, L.; Chantemesse, B.; Vienney, F.; Hartmann, A. Anal. Chim. Acta 2015, 892, 160166. (31) Zhang, J.; Cui, J.; Liu, Y.; Chen, Y.; Li, G. Analyst 2014, 139, 3429-3433. (32) Fapyane, D.; Ferapontova, E. E. Anal. Chem. 2017, 89, 3959-3965. (33) Hu, Y.; Zhang, Q.; Guo, Z.; Wang, S.; Du, C.; Zhai, C. Biosens. Bioelectron. 2017, 98, 91-99. (34) Shen, C.; Li, X.; Rasooly, A.; Guo, L.; Zhang, K.; Yang, M. Biosens. Bioelectron. 2016, 85, 220-225. (35) Wang, X.; Wang, M.; Zhang, Y.; Miao, X.; Huang, Y.; Zhang, J.; Sun, L. Biosens. Bioelectron. 2016, 83, 91-96. (36) Haraguchi, N.; Nishimura, J.; Hata, T.; Matsuda, C.; Yamamoto, H.; Doki, Y.; Mori, M. Int. J. Oncol. 2017, 50, 525-534. (37) Craven, R. A.; Stanley, A. J.; Hanrahan, S.; Dods, J.; Unwin, R.; Totty, N.; Harnden, P.; Eardley, I.; Selby, P. J.; Banks, R. E. Proteomics 2006, 6, 2853-2864. (38) Peng, S. Y.; Lai, P. L.; Pan, H. W.; Hsiao, L. P.; Hsu, H. C. Oncol. Rep. 2008, 19, 1045-1054.

(39) Zhao, S.; Zhang, J.; Zhu, M.; Zhang, Y.; Liu, Z.; Ma, Y.; Zhu, Y.; Zhang, C. J. Mater. Chem. B 2015, 3, 1612-1623. (40) Woods, A. S.; Ferré, S. J. Proteome. Res. 2005, 4, 13971402. (41) Naumann, R.; Baumgart, T.; Gräber, P.; Jonczyk, A.; Offenhäusser, A.; Knoll, W. Biosens. Bioelectron. 2002, 17, 25-34. (42) Terrettaz, S.; Ulrich, W. P.; Guerrini, R.; Verdini, A.; Vogel, H. Angew. Chem. Int. Ed. 2001, 40, 1740-1743. (43) Chen, H.; Kim, S.; Li, L.; Wang, S.; Park, K.; Cheng, J. X. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 6596-6601. (44) Kwok, D. Y.; Neumann, A. W. Adv. Colloid Interfac. 1999, 81, 167-249. (45) Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 1056710575. (46) Wang, X.; Liu, Y.; Zhang, J.; Li, G. Sens. Actuators B: Chem. 2017, 242, 687-693. (47) Shu, J. D.; Xie, H.; Chen, X. G. J. Clin. Oncol. 1996. 23, 704-706.



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Electrochemical Analysis of Enzyme Based on the Self-Assembly of

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