Design of Cyclic Peptide Based Glucose Receptors and Their

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Design of Cyclic Peptides Based Glucose Receptors and Their Application in Glucose Sensing Chao Li, Xin Chen, Fuyuan Zhang, Xingxing He, Guozhen Fang, Jifeng Liu, and Shuo Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02430 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017

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Analytical Chemistry

Design of Cyclic Peptides Based Glucose Receptors and Their Application in Glucose Sensing Chao Li,a Xin Chen,a Fuyuan Zhang,a Xingxing He,a Guozhen Fang,a Jifeng Liu,*a Shuo Wang*a a

:Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, Tianjin University of Science and

Technology, Tianjin 300457, China. Jifeng Liu, Email: [email protected], Shuo Wang, Email: [email protected].

ABSTRACT Glucose assay is of great scientific significance in clinical diagnostics and bioprocess monitoring and to design new glucose receptor is necessary for development of more sensitive, selective and robust glucose detection techniques. Herein, a series of cyclic peptides (CPs) glucose receptor were designed to mimic the binding sites of glucose binding protein (GBP) and CPs’ sequence contained amino acid sites Asp, Asn, His, Asp, Arg, which constituted the first layer interactions of GBP. The properties of these CPs used as a glucose receptor or substitute for the GBP were studied by using quartz crystal microbalance (QCM) technique. It was found that CPs can be formed a self-assembled monolayer at Au quartz electrode surface and the monolayer’s properties was characterized by using cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy. The CPs’ binding affinity to saccharide (i.e., galactose, fructose, lactose, sucrose, and maltose) was investigated and the CPs’ sensitivity and selectivity toward glucose were found to be dependent upon the

configuration,

amino

acids

sequence

of

the

CPs.

The

cyclic

unit

with

a

cyclo[-CNDNHCRDNDC-] sequence gave the highest selectivity and sensitivity for glucose sensing. This work suggested that a synthetic peptide bearing a particular functional sequence could be 1

ACS Paragon Plus Environment


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applied for developing a new generation of glucose receptor and would find huge application in biological, life science and clinical diagnostics field.

INTRODUCTION Glucose is the most important dietary saccharide source of energy from natural carbohydrate.1 In clinical or healthcare perspective, the measurement and control of glucose level in blood is of particular importance for diabetic patients to regularly monitor the concentrations and make fast and accurate determination of glucose levels.2 There have been many existing methods to measure the concentration of glucose including electrochemical and optical approaches.3-5 And now, electrochemical sensors have been the most widely used for glucose monitoring in food analysis, environmental monitoring, medical diagnosis etc.6 Usually, glucose oxidase, hexokinase or glucose dehydrogenase is used in such electrochemical glucose sensors to generate redox mediator species, which are thereafter detected electrochemically.7,8 However these enzymatic sensors require the enzyme to keep a close proximity to the electrode surface to ensure enough sensitivity. Additionally, these enzymatic sensors are susceptible to environmental factors including temperature, humidity, pH, ionic detergents and toxic chemicals, which make electrochemical glucose sensors poor performance.9 In the presence of redox actively pharmaceutic compounds or ascorbic acid molecules commonly found in blood, the selectivity of electrochemical methods would be deteriorated.10-12 More than 50 years have passed since the appearance of the first glucose enzyme electrodes by Clark and Lyons in 1962. Currently, glucose sensors occupy about 85% of the biosensor market, and novel glucose sensors related to this topic have been constantly reported by more than 1000 publications every year.13 Because the increasing importance of blood-glucose control for medical diagnosis and healthcare continues, there is a great need for more advanced glucose-sensing technologies. Simple enzyme-free glucose sensing is highly desirable and much research has been devoted to develop robust, enzyme-free glucose sensors. And one approach towards enzyme-free 2


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sensors involves using nanostructured materials modified electrodes as electrochemical sensor for direct oxidation of glucose. However, these nonenzymatic sensors and the nanostructured materials on electrode surface would be easily passivated or fouled by surface absorption.14 An alternative enzymeless approach for glucose detection involves the use of synthetic receptors, such as compounds or polymer with boronic acid.15,16 A boronic acid group binds reversibly with cis-1,2- and cis-1,3-diols to form five- and six-membered cyclic boronic esters, respectively. Receptors based boronic acid have been used successfully as fluorescence sensors, surface appended sensors, and potentiometric / amperometric sensors.17 It has been commonly believed that the lower pKa of boronic acid and the higher pH give higher binding affinity between boronic acid and saccharide molecules. However, this boronic acid receptor has low selectivity toward diol molecules, such as glucose, fructose, galactose, and D-mannose. Oligopeptides with designed sequence may be an effective receptor for glucose sensing with suitable selectivity and sensitivity. Oligopeptides have been designed to mimic protein-protein interactions and the epitopes of protein interfaces in living systems,18 and found applications in drug development,19 peptide libraries20 and biosensors21. This oligopeptides device offers significant advantages over other proteins for label-free biosensor design. Because proteins are susceptible to proteases ， nonspecific binding with other substances. Comparatively, small peptide can be immobilized at high density but still preserve their stability and specificity of binding. Recently, electrochemical modified sensors with oligopeptides have been used in the study of interaction between cancer marker CD20, human epidermal growth factor receptor and therapeutic monoclonal antibodies.22-25 Glucose binding protein (GBP) consists of 309 amino acids with a molecular weight of 33,310 Da26,27 and it has a high affinity for (D-) glucose or galactose, with dissociation constants (Kd) of 0.2 µM. There are two folded domains connected by three different peptide segments that serve as flexible hinge and produce a cleft between the domains, where the glucose-binding site is located. 3



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After glucose was engulfed in the deep cleft between the two domains, a hinge motion would occur for GBP and this result in the exclusion of solvent molecules from the binding site and enabling efficiently hydrogen-bonding interactions between the glucose and the GBP amino acids residues at the binding site.28 These binding sites involve Asp14, Asn91, His152, Asp154, Arg158, Asn211, Asp236, and Asn256, which comprised of the first-shell interactions with glucose.29 Herein, cyclic peptides (CPs) were designed as a glucose receptor to mimic the glucose binding sites of GBP. Usually, the cyclic unit of CPs adopts a flat conformation with the carbonyl and amino group of the amide backbone groups oriented perpendicular to the ring and all the side chains of amino acid pointing outwards. The cyclic unit can be tailored for specific applications in biosensors, biomaterials, electronic devices, and selective transmembrane transport channels etc.30 Cyclization often results in higher binding affinities toward target molecules and limits degradation by proteases.31 CPs will be a promising sensing device and their application in therapeutics, chemical probes, drug targeting, imaging and diagnostics have just been reviewed recently.32 In this work, the GBP’s first-shell interaction sites of Asp14, Asn91, His152, Asp154, Arg158, Asn211, Asp236, and Asn256 were comprised to form a cyclic unit by a S-S bond of two neighboring cysteines and the other extra cysteine site anchoring onto the Au film surface of quartz crystal microbalance (QCM) to form a self-assembled monolayer to detect minute changes in mass as glucose bind to the CPs on the QCM surface. The amino acid sequences of CPs were optimized toward

selectivity

and

sensitivity

of

glucose

binding

and

it

was

found

that

cyclo-[-CNDNHCRDNDC-] possesses high selectivity and sensitivity to glucose. We further used this system to detect glucose in human serum.

4


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EXPERIMENTAL Chemicals and Materials. Short peptides (CP-1, CP-2, CP-3, CP-4, CP-5, CP-6 and CS-7, C-HD 16 primary sequences as listed in Table 1) were chemically synthesized by Shanghai Bootech BioScience &Technology Co. Ltd.(Shanghai, China) and were received in lyophilized conditions. The quality of all of the peptides was assessed by HPLC and confirmed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI) analysis (purity > 98%). Human serum was supplied by a healthy voluteer.

QCM measurements. Peptide immobilization onto an Au transducer sensor surface was performed as described with some modifications.33-37 The Au quartz crystals used in this study were AT-cut, 7.995 MHz, unpolished, and coated with gold film of 1000 Å thickness on both sides (CHI Instruments, Shanghai). The geometric area of the coating area is about 0.196 cm2. The quartz crystal was cleaned with concentrated nitric and sulfuric acid mixture (1:3 v/v), ultrapure water (≥ 18 MΩ) and ethanol in series for three times to remove impurities and dried with nitrogen. QCM Au surface was immersed in peptide solution (1.5 mM in ultrapure water) at 4 °C for 12 h to allow peptide absorbed onto Au surface. After incubation, the electrode surface was washed with ultrapure water to minimize nonspecific adsorption，and placed in 1 mL of PBS. No significant difference was found for the amount of peptide adsorbed on gold surface as studied by QCM (∆F ~ 38.6Hz, ∆m ~ 51.7ng， 1.21×1014 mol cm-2). The quartz crystal was mounted in a custom-made Kel-f cell (CHI Instruments, Shanghai) sealed with two O-rings. Under flow injection conditions, the peptide-modified gold electrode was mounted in a custom-made flow cell (ALS Co., Japan) sealed with two O-rings, the volume of the flow cell is 70 µL, PBS (pH7.4, 20 mM) was used as the carrier solution with flow rate of 80 µL/min, the amount of the sample solution injected was 250 µL. The cell was put in 5



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thermostat box to keep the temperature at 298K.

Characterization Methods. The QCM Au sensor was then characterized using a CHI 430 electrochemical workstation (CH Instruments, Shanghai). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) was used to characterize the peptide-modified QCM gold surface using CHI 760 electrochemical workstation (CH Instruments, Shanghai). All experiments were carried out using a three-electrode system with the QCM gold surface as the working electrode, an Ag/AgCl reference electrode (3 M KCl) was used as a reference electrode and a platinum wire (0.5 mm diameter) as counter electrode. CVs of couple Fe(CN)63-/4- were obtained using a solution of 0.1 M NaCIO4, containing 1 mM K3Fe(CN)6/K4Fe(CN)6 (1:1), the CV potential was scanned from −0.2 to 0.6 V at a scan rate of 50 mV/s. The EIS measurements were obtained by applying a 5 mV amplitude sine wave under bias at open circuit potential within a frequency window range of 0.1 Hz to 100 kHz. Atomic force microscopy (AFM) was used to characterize the morphology of the peptide modified gold-plated (about 2nm in thickness) silicon wafer surface (sputtering coated with Au target using a coater (JEOL, JFC-1600) at 20mA for 20s) before and after peptide binding with glucose. These samples were thoroughly rinsed with deionized water and mounted on a MultiMode 8 AFM with a Nanoscope V controller (Bruker, USA). Commercially available sharpened Si3N4 probes (radius of curvature approximately 2 nm, Bruker) were used for imaging. All images were collected in ScanAsyst Mode (PeakForce Tapping).

Molecular dynamics simulations and Adsorption thermodynamic. All the structures including the peptide molecules and the glucose molecule were built in Discover Studio 2016 software. First, the initial configuration was set to be closest to the structure of the analog system. Molecular conformation obtained by molecular mechanics of all molecular 6


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simulations of prepolymerization mixtures was carried out by Hyperchem 8.0 software. The geometries of CP, Glucose, and CP-Glucose complexes have been optimized through molecular mechanics using Assisted Model Building and Energy Refinement 3 (AMBER3) in AMBERR. Then, Hyperchem package was used to conduct 1 ps of short molecular dynamics simulation of the title compounds (the stoichiometry of peptide-glucose complex was 1:1). Electronic energies were calculated using semiempirical methods with modified neglect of diatomic overlap (MNDO), PM3 (a reparameterization of Austin Model 1), complete neglect of differential overlap (CNDO), and typed neglect of differential overlap (TNDO) algorithms. The binding energy of peptide-glucose complexes, ∆E, was calculated through eq 1. ∆E = E(complex) − E(peptide) − E(glucose)

(1)

Where E(complex) is the binding energy between peptide and glucose, E(peptide) is the energy level of peptide, and E(glucose) is the accumulative energy level of glucose. The thermodynamic parameters for the adsorption process, the standard free energy were calculated using the following equations38 : ∆G = −RTlnK

(2)

Where ∆G was the standard free energy of adsorption, kJ mol−1; R was the universal gas constant, 8.314 J mol−1 K−1; T was the absolute solution temperature, K; K was the Langmuir equilibrium constant.39,40

RESULTS AND DISCUSSION Design of the mimic GBP Peptide and Immobilization on Au Surface. Glucose binds with native GBP at cleft site through a network of hydrogen bonding interactions involving Asp14, Asn91, His152, Asp154, Arg158, Asn211, Asp236, and Asn256. In order to maintain this binding activity and selectivity toward glucose as soon as possible, design of the mimic 7



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GBP peptide involved in hydrogen-bonding interaction sites and the sequences of the peptides tested were listed in Table 1. The well-designed peptides were engineered with each peptide contained three parts: (1) coupling amino acid to Au surface (cysteine); (2) disulfide bond amongst two cysteine; (3) the functional hydrogen bonding interaction sites (in bold). These amino acids formed a cyclic unit with two cysteine were enclosed to form S-S bond and CPs can be immobilized on Au surface with the extra Cys site and the side chains of the Asn, Asp, His and Arg sites were oriented outwards the cyclic. Beside of the two amino acid (His, Arg), all the three cyclic peptides (CP-2, CP-3, CP-4) contained the hydrogen bonding sites (Asp, Asn), while peptide CP-1 did not contain H and R, and peptides CP-5 and CP-6 did not contain the amino acid Arg and His, respectively. In view of spatial arrangement all the six cyclic peptides also differed in the number and arrangement of amino acids, only CP-1, CP-3, CP-5 and CP 6 contained the sites of Asp-Asn-Asp and Asn-Asp-Asn (Aspartic acid and asparagine alternated with each other), other peptides did not. For comparison, the amino acids sites were form a “Y” shape

(C-HD 16), and

CGSGSGSK sequence was immobilized onto QCM Au electrode surface at the terminal cysteine as an arm to link the hydrogen bonding sites of Asn-Asp-Asn-His and Arg-Asp-Asn-Asp. On the basis of the previous writings, the CGSGSGS linker with its repeating units of glycine and serine residues which favor the α helix would likely cause the peptide to initially form the α helix, so peptides with the peptide linker CGSGSGS heavily favor the α helix conformation after immobilization onto gold.41 While CS-7 was only a short sequence CGSGSGS, without any mimic GBP hydrogen-bonding interaction sites. As shown in Figure 1, binding affinity of mimic GBP peptides immobilized QCM sensor surfaces for glucose varied and was apparently influenced by the peptides used. Negative control of GBP mimic peptides was conducted with the sequence of CGSGSGS (CS-7). The control CS-7 peptide did not bind with glucose or any other saccharrides (i.e., galactose, fructose, lactose, sucrose, maltose). Under the experimental conditions, six parallel QCM measurements were carried out to 8


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evaluate the binding capability of each peptide with glucose. As can be seen in Figure 1, the “Y” shaped C-HD 16 with an mimic open GBP peptide sequence had both lower sensitivity and selectivity towards saccharide investigated (i.e., galactose, fructose, lactose, sucrose, maltose). CP-1, CP-2 and CP-4 had poor performance in sensitivity and selectivity toward glucose interaction (∆F < 20 Hz). CP-5 and CP-6 could bind more saccharide than CP-1, CP-2 and CP-4, it proved to be of good sensitivity, but they failed to give obvious selectivity for glucose binding. Comparatively, the CP-3 modified QCM Au electrode exhibited obvious binding behavior with glucose in comparison to CP-1, CP-2, CP-4, CP-5, CP-6 modified QCM Au electrodes and showed high sensitivity with a large frequency shift for glucose binding (∆F > 116 Hz). These showed that mimic GBP peptides sequence adopting a close/cyclic unit was necessary for interacting with saccharide, and the sequence of cyclo[-CNDNHCRDNDC-] had the highest binding affinity of glucose.

Electrochemical Characterization of the Adsorption of the GBP CP-3 on Au Surface. The mimic GBP peptides modified Au surface and the CPs−glucose interactions were further examined to explain its binding affinity to glucose by CV and EIS. As shown in Figure 2a, CV of K4Fe(CN)6/K3Fe(CN)6 solution on the bare Au surface gave reversible redox peaks and well defined redox peaks could be observed with peak potential separation (∆Ep) of 98 mV. After the Au electrode adsorbed with CPs, the faradic current was dramatically decreased and peak potential separation was increased, indicating sluggish electron-transfer kinetics through the modified monolayer on Au surface. The self-assembled layer of CP absorption acts as an inert electron transfer blocking layer. But this self-assembled structure might be a monolayer, because current flow was reduced but not completely insulated. When the self-assembled layer of peptide (CP-3) bound with glucose, the peak current at Au surface decreased further and almost vanished (Figure 2c). And this is a typical character of CV at 9



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the insulating molecular multilayer, where the adsorbed substance would build up thickness until current flow would be completely impeded. It can be inferred that the glucose molecules bind with CP monolayer to from a multilayer structure. The EIS was also recorded for peptide coated Au surface before and after glucose binding (Figure 2b, 2d). In EIS, the semicircle diameter in the impedance spectrum equals the electron-transfer resistance, RCT. This resistance controls the electron transfer kinetics of the redox probe (K4Fe(CN)6/K3Fe(CN)6) at the electrode interface. Just as expected, the self-assembly of the peptide on the Au surface functioned as a barrier to the interfacial electron transfer and the RCT were around 9.2, 11.8, 12.2, 11.2, 12.6, 13.8 and 10.2 kΩ for CP-1,CP-2,CP-3,CP-4,CP-5,CP-6 and C-HD 16 modified Au surface, respectively. After the Au electrode immersed in glucose solutions with different concentrations (10nM, 100nM, 1µM, 10µM, 100µM, 1mM, 5mM), the RCT of CP-3 modified Au surface was increased respective from 13.4 to 13.5, 20.5, 25.9, 29.2, 34.1 and 39.9 kΩ for different glucose concentrations used (Figure 2d). These shown that the complexation of the peptide SAM with glucose changed the film characteristics, such as ion transfer resistance and the dielectric capacitance.

AFM Characterization of Peptide Immobilization and Binding with Glucose. The results of QCM, CV, and EIS clearly demonstrated that designated the mimic GBP peptides had different binding capability toward glucose. The self-assembly monolayer of the CPs on the Au surface was formed through the Au-S covalent bond interaction between the -SH groups in cysteine residues of the peptide with the Au surface. AFM was used to further characterize the immobilized peptides before and after bound with glucose at the Au surface. As seen from Figure 3, the AFM images shown that the bare Au surface was atomically smooth and roughness is about 0.16 nm. After Au surface was adsorbed with peptides (e.g. CP-1, CP-3 and CP-6), it could be observed that there 10


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were a lot of nanoclusters like domains and the roughness was increased to 0.25-0.3 nm. This indicated that SAM of CP molecules adsorbed onto Au surface were formed and thickness was about that of single layer cyclic peptide, indicating that CP molecules were adsorbed onto Au substrate to form a compact monolayer. To investigate the CPs-glucose interaction on Au, CPs modified Au-silica wafer was immersed into glucose solution (1 mM) for 1 h and then was washed several times with deionized water to remove nonspecifically adsorbed species and dried under Ar gas. And then AFM images of the surface were taken again to confirm the interaction and the dimensions of the CPs monolayer with glucose. After binding with glucose, the AFM image showed quite a different morphology from the pure CP monolayer. And bigger nanocluster appeared and the height was about 1-2 nm for CP-1 and CP-6 bound with glucose. The height increased to 2-3 nm for surface of CP-3 bound with glucose and a lot of island like domains could be observed and this might be due to the bound glucose molecules. As discussed above, the glucose binding capacity of CP-1 and CP-6 were less than CP-3, so the bound glucose molecules at CP-3 monolayer could aggregated to form nanocluster or even island domains as observed in AFM images (Figure 3), and the roughness of CP-3 bound with glucose was increased to about 0.6 nm.

Analytical Performance of the GBP Mimic Peptide CP-3 Modified QCM Au Electrode for Glucose Detection. The aforementioned results demonstrated that glucose bound to CP-3 on a QCM Au surface with high selectivity. This CP-3 modified Au may be used as glucose sensor and find its potential use in determining the glucose concentrations in different samples in real-time. Usually, the GBP may bind glucose as well as galactose,42 but at the sensor surface modified with CP-3, glucose had higher response than galactose. As shown in Figure 1, there was a nearly 10 times lower response (∆F ~ 10 Hz) when galactose were sequentially spiked into the QCM cell with CP-3 modified Au surface than 11



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that (∆F ~ 116 Hz) of glucose. The CP-3 peptide in this work showed quite lower binding affinity for galactose than glucose and this selectivity may originate from the lack of the second layer amino acids or even the more complicate sites that may interact with galactose. According to Sauerbrey’s equation,43 the interfacial changes in mass ( ∆m ) caused by the molecular deposition onto a sensor surface are directly related to the resonant frequency shifts ( ∆F ) on the QCM Au electrode surface, a fitted ∆F of 1 Hz corresponds to about 1.34 ng of ∆m for the 7.995 MHz quartz crystal which was employed in this work. Through this equation, we can roughly estimate that about 155.54 ng of glucose was bound to the CP-3 modified gold QCM electrode. This indicated that the CP-3 modified Au QCM electrode had high affinity for glucose. The responding of CP-3’s interaction with glucose was rapid and glucose could be detected within 30 min (Figure 4a). The frequency shift increased with increasing glucose concentration ranging from 10 nM to 20 mM, where the binding sites at CP-3 sensor surface was saturated with glucose. So a working curve of glucose detection was obtained (Figure 4b). Furthermore, in order to optimize the binding capacity of the CP-3 sensor, the effects of pH and ionic strength were also investigated on the interaction of CP-3 with glucose, the optimal pH was set at 7.4, and the optimal PBS concentration was 20 mM (Figure S1, Supporting Information).

Binding Affinity. The QCM data supported the following assumptions and formulas that were subsequently used to determine the Ka for the peptide monolayer/glucose interactions. The affinity constant Ka can be determined on the basis of the following reactions. The binding between glucose and the peptide monolayer was described by eq 3. [Glucose] + [CP-3 SAM] → [Glucose-CP-3 SAM complex]

(3)

Based on langmuir adsorption isotherm, association constant (Ka) for the binding between glucose and CP-3 monolayer can be evaluated by eq 4, 12


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[Glucose] / ∆M = [Glucose] / ∆Mmax + 1 / ∆Mmax Ka

(4)

Where ∆Mmax is the maximum binding amount, ∆M is the measured binding amount, and [glucose] is the original concentration of glucose. Figure 5 show the plot of [Glucose]/∆M versus [Glucose] based on the Figure 4. According to eq 2, the ratio of the slope to the intercept gives the association constant (Ka) as 8.14 × 107 M-1 (Table 2 and Figure 5).

Quantification of Glucose in Human Serum Sample. In order to quantify glucose in real samples, diluted human serum samples (1:10 in PBS) was used as carrier solution into flow cell mounted with the CP-3 modified QCM Au electrode. Six calibrators containing 0.01, 0.1, 0.5, 1, 2 mM of glucose in PBS buffer were sequentially spiked into the QCM cuvette bearing the CP-3 peptide and there was obvious response (∆F ~ 34.99 Hz) when glucose was spiked into the QCM cuvette. A working curve of glucose (up to 2 mM) in real serum samples can be obtained and it showed that GBP mimic peptide, CP-3 on a QCM Au surface can be applied in glucose sensing in real samples with the recovery were around 82-86%. However, fats, amino acids, urea, and proteins, especially sugars in human serum may bind nonspecifically and interfere with or inhibit CP-3/glucose interactions to produce false positive assay results. For this concern, nonspecific binding effects within the human serum sample particularly on the surface of the CP-3 modified Au surface was evaluated and determined by comparing frequency changes upon human serum sample addition followed by glucose addition. As shown in Figure S2, the glucose could be detected by the CP-3 sensor in PBS buffer (1mM, glucose, ∆F ~ 90Hz). However, when an undiluted serum was applied prior to the sensor surface, the frequency responds decreased by 20 times (1mM, glucose, ∆F

~ 4 Hz) due the nonspecific adsorption of the proteins in serum. Instead, when a diluted serum (10 × in PBS) replaced the undiluted serum, nonspecific binding was reduced and glucose binding increased significantly (1mM, glucose, ∆F ~ 40 Hz). 13



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Molecular Dynamic Simulation of Interaction of CP with Glucose. CP-3 structure was optimized by molecular dynamics simulation. At the most stable state, the size of CP-3 cavity was about 1.0 – 1.2 nm in diameter，the diameter of glucose molecule was 0.36 nm. CP-3 enabled efficient hydrogen-bonding interactions between the glucose, not simply filled the cavity of the CP. Moreover, by decreasing the conformational freedom of peptides, cyclization often results in higher binding affinities toward target molecules. When the molar ratio of peptide-glucose was 1:1, the hydrogen bond was formed between the hydroxyl of glucose and the groups (carboxyl, epoxy, amino) of CPs (Figure 7 and Figure S3)， structural characterization of these peptide-glucose revealed interactions with key residues, thus it suggested that peptides could accommodate glucose. In order to provide more precise quantitative interpretation of binding, we computed both the binding affinity and the kinetics of the overall process of binding, according to eq 1, the molar ratio was 1:1, the binding energies (E) calculated were shown in Table 2. These binding energy obtained from MDS calculation were correlated well with those obtained from QCM measurements (∆G = –RT lnK (R=8.314 J/mol • K, T=298 K) ). All these results showed that CP-3 bound with glucose had highest K among the CPs-glucose complexes investigated. The glucose sensors should be fabricated as easily useful as possible. In this work, QCM technique was used to study the interaction between the CP receptor and glucose and try to construct a model for this interaction. And the high cost of the gold coated quartz crystals is hard to allow it to be a disposable glucose detection device. However, the CP based glucose receptor studied in this work showed high selectivity and sensibility even in real samples’ analysis, and the synthetic cost of oligopeptides (short 7- to 20-mers) was not high. So if CP based glucose sensor combined with more cheap detection techniques including electrochemistry or fluorescence, it will find huge commercial potential in the glucose sensor markets. 14


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CONCLUSION The mimic GBP peptide were designed for glucose sensing and a series of CPs with different amino acid sequence mimic the first layer (Asp14, Asn91, His152, Asp154, Arg158, Asn211, Asp236, and Asn256) interactions of GBP can be used as a glucose receptor. The interaction between CPs monolayer and glucose were studied through QCM technique. CPs adsorbed at Au surface was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and atomic force microscopy and it was showed that CPs self-assembled into monolayer at the Au surface. Peptide with a cyclic unit is necessary to have binding affinity for glucose and the amino acids

sequence

have

obvious

effects

on

such

affinity.

The

cyclic

unit

with

a

cyclo[-CNDNHCRDNDC-] sequence gave the high selectivity and sensitivity for glucose sensing. And it could be immobilized on the surface of Au and function as a substitute for the glucose-binding protein to detect glucose in serum samples. The immobilization of peptides onto gold is reproducible and reliable because of the use of a self-assembled monolayer. On the other hand, synthetic oligopeptides are generally more stable and can be more readily synthesized on a large scale under controlled conditions to ensure peptide quality. So the cost to produce a peptide-based glucose receptor can be significantly reduced. This work may be the first report on peptide based glucose receptor and this study on this QCM sensor is not just stayed at glucose sensing, because this glucose receptor may be used in new glucose sensor, real time tracking or imaging of glucose in vivo. And it is expected to establish a system of new glucose recognition or sensing in biological, life science and clinical diagnostics field.

ACKNOWLEDGEMENT This work was supported by the Natural Science Foundation of China (Funding, 21575102). Prof. Jianbo Jia (State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun) and Dr. Lei Wang and Dr. Wei Zhang 15



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(Department of Chemistry, Liaocheng University) are thanked for collaboration.

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Figure captions Figure 1. Frequency change vs time curve: bare gold electrode (black), CS-7 (yellow), C-HD 16 (dark yellow), CP 1 (dark cyan), CP 2 (pink), CP 3 (red), CP 4 (LT magenta), CP 5(navy), CP 6(wine) modified QCM Au electrodes were exposed to a small amount (70 µL) of various saccharides (Sucrose, Maltose, Lactose, Fructose, Galactose, Glucose, 1 mM) sequentially, carrier solution, PBS, pH 7.4, 20 mM. Figure 2. (a) Cyclic voltammograms and (b) Electrochemical Impedance Spectroscopy (Nyquist plots) of K4Fe(CN)6/K3Fe(CN)6 at peptides modified Au surface. (A, B) Peptides used are CP-1, CP-2, CP-3, CP-4, CP-5, CP-6 and C-HD 16 as indexed. (c) Cyclic voltammograms and (d) Electrochemical Impedance Spectroscopy (Nyquist plots) of K4Fe(CN)6/K3Fe(CN)6 at CP-3 modified Au surface. Rsolution, Cmonolayer, RCT, represented respective elements of solution resistance, capacitance of SAMs film, resistance of SAMs film. The concentrations of glucose immersed used were 10, 100 nM, 1, 10, 100µM, 1, 5mM as indexed. CV of 1 mM K4Fe(CN)6/K3Fe(CN)6 in 0.1 M NaClO4, scan rate, 50 mV/s; EIS, frequency range was 0.1-100 kHz, ac amplitude 5 mV. Figure 3. AFM images of bare AU substrate, peptide monolayer and peptide monolayer bound with glucose. Silica wafer coated with Au film was used as image substrate. The Au subtrate was incubated in peptide solution (CP-1, CP-3, CP-6, 1.5 mM, prepared in double distilled water) at 4 ℃ for overnight. Before AFM imaging, the Au coated wafer was thoroughly washed with deionized and then was dried under Ar flow. For AFM imaging of CP bound with glucose, the CP incubated substrate was immersed into glucose solution (1 mM) for 1 h. After washing with PBS and deionized water and dried under Ar flow. Figure 4. (a) The comparison of frequency change vs time curve: CP-3 modified Au QCM electrode was exposed to glucose with various concentrations. (b) The frequency change vs concentration of Glucose in PBS buffer. Figure 5. Plot of [Glucose]/∆M vs [Glucose] of Glucose binding with CP-3 SAM modified piezoimmunosensor to obtain the CP-3 SAM / Glucose binding association constant (Ka). Figure 6. (a) The comparison of frequency change vs time curve. (b) The frequency change (∆F) vs concentration curve of glucose binding capacity evaluation (human serum samples were diluted by 1:10 in PBS buffer). Figure 7. Detailed view of the interaction between different groups (carboxyl, epoxy, amino) of the mimic peptide (CP-3) with glucose (hydroxyl) through hydrogen bonds. Gray indicates a carbon atom, blue indicates nitrogen, red oxygen, yellow sulfur. Green ring indicates glucose molecule.

19



30 Suc

Mal

Lac

Fru

Gal

Glc

60

90

120

150

0 -30

∆F (Hz)

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

Page 20 of 30

-60

Bare Au CS-7 C-HD16 CP-1 CP-4 CP-2 CP-5 CP-6 CP-3

-90

-120 -150 -180 0

30

180

Time (min)

Figure 1.

20


Page 21 of 30

40

Bare Au

a

6

Zim (KΩ)

I (µA)

b

8

20 0 C-HD 16 modified Au CP-1 modified Au CP-2 modified Au CP-3 modified Au CP-4 modified Au CP-5 modified Au CP-6 modified Au

-20 -40 -0.2

0.0

4

2

C-HD16 modified Au CP-1 modified Au CP-4 modified Au CP-2 modified Au CP-3 modified Au CP-5 modified Au CP-6 modified Au

Bare Au

0

0.2

0.4

0.6

0

4

E(V) vs Ag/AgCl

8

12

16

20

Zre (KΩ)

c

20

d

20

0

Zim (KΩ)

15

I (µA)

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

10 nM 100 nM 1 µM 10 µ M 100 µ M 1 mM 5 mM

-20 -0.2

10 nM 100 nM 1 µM 10 µM 100 µM 1 mM 5 mM

5 0

0.0

0.2

0.4

0

0.6

5

10 15 20 25 30 35 40 45 50 55

Zre (KΩ)

E(V) vs Ag/AgCl

Figure 2.

21



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

Figure 3.

22


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Page 23 of 30

30

Glucose

a

200

0 10 nM -30

150

-60

∆F (Hz)

∆F (Hz)

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


100

-90

-120

50

y = 4.5595x + 91.0149 2 R = 0.9679

-150 -180

20 mM

-210 -5

0 0

5

10

15

20

25

0

30

5

10

Cglucose (mM)

Time (min)

Figure 4.

23


15

20


70

[Glucose] / ∆M (nM/ng)

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

Page 24 of 30

60 50 40 30 20 y = 0.0619x + 0.7605 2 R = 0.9952

10 0 0

200

400

600

[Glucose] (nM)

800

1000

Figure 5.

24


Page 25 of 30

0

b

200

a

Diluted Serum Samples

180 CP-3 modified Au

Diluted serum+0.01mM Glc

160 Diluted serum+0.1mM Glc

-100

Diluted serum+0.5mM Glc

∆F (Hz)

-50

∆F (Hz)

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


140 120

-150

100

Diluted serum+1mM Glc Diluted serum+2mM Glc

-200 0

30

60

90

120

150

180

80 0

210

5

10

C (mM)

Time (min)

Figure 6.

25


15

20


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

Figure 7.

26


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Tables Table 1. List of Glucose-Binding Mimic Peptide Designs Designated name CP-1 CP-2 CP-3 CP-4 CP-5 CP-6 C-HD 16

Purity （％） 98 98 98 98 98 98 98

Primary sequencea cyclo[-CNDNCDNDC-] cyclo[-CDNNHCRDDNC-] cyclo[-CNDNHCRDNDC-] cyclo[-CNNNHCRDDDC-] cyclo[-CHNDNHCHDNDHC-] cyclo[-CRNDNRCRDNDRC-]

CS-7 98 CGSGSGS Hydrogen bonding sites in bold.

MW (g/mol) 977.03 1306.36 1306.36 1306.36 1580.64 1656.83 1662.63 553.55

a

27


PI 3.42 4.31 4.31 4.31 6.03 8.05 5.49


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Page 28 of 30

Table 2. Association Constants (Ka) for CP-Glucose Interactions Peptide on QCM

CP-1

CP-2

CP-3

CP-4

CP-5

CP-6

C-HD 16

Ka (M-1) ( ×105)

4.7

15.3

813.9

16.5

19.9

17.8

3.1

∆G= −RTlnKa (J/mol) ( ×10−4) ∆G (J/mol) calculated from eq 3 ( ×10−4)

-3.23

-3.53

-4.51

-3.54

-3.60

-3.56

-2.60

-1.87

-7.19

-9.93

-5.12

-8.31

-8.66

-1.60

28


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Table 3. The recovery of glucose detection in serum Frequence shift detected (∆F/Hz) 84.71

Recovery (％) 82.35

Concentration added (mM) 0.01

Frequence shift (∆F/Hz) 102.87

0.1

132.31

113.57

85.84

0.5

149.12

130.89

87.77

1

179.72

156.91

87.31

2

228.94

195.19

85.26

29



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Graphic abstract

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Design of Cyclic Peptide Based Glucose Receptors and Their

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