Author’s Accepted Manuscript Novel voltammetric and impedimetric sensor for femtomolar determination of lysozyme based on metal-chelate affinity immobilized onto gold nanoparticles Abbas Arabzadeh, Abdollah Salimi www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30187-1 http://dx.doi.org/10.1016/j.bios.2015.06.019 BIOS7755

To appear in: Biosensors and Bioelectronic Received date: 17 April 2015 Revised date: 28 May 2015 Accepted date: 9 June 2015 Cite this article as: Abbas Arabzadeh and Abdollah Salimi, Novel voltammetric and impedimetric sensor for femtomolar determination of lysozyme based on metal-chelate affinity immobilized onto gold nanoparticles, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.06.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel voltammetric and impedimetric sensor for femtomolar determination of lysozyme based on metal-chelate affinity immobilized onto gold nanoparticles Abbas Arabzadeha, Abdollah Salimia,b* a

b

Department of Chemistry, University of Kurdistan, 66177-15175 Sanandaj, Iran.

Research Center for Nanotechnology, University of Kurdistan, 66177-15175 Sanandaj, Iran.

*Corresponding author Tel.: +98 87 33624001; Fax: +98 87 33624001, E-mail addresses: [email protected] , [email protected] (A.Salimi).

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Abstract In this study, we reported iminodiacetic acid-copper ion complex (IDA-Cu) immobilized onto gold nanoparticles (GNPs)-modified glassy carbon electrode as a novel electrochemical platform for selective and sensitive determination of lysozyme (Lys). IDA-Cu complex acted as an efficient recognition element capable of capturing Lys molecules. GNPs acts as a substrate to immobilize IDA-Cu coordinative complex and its interaction with Lys leds to a great signal amplification through measuring changes in differential pulse voltammetric (DPV) peak current of [Fe(CN)6]3-/4-redox probe. Upon the recognition of the Lys to the IDA-Cu, the peak current decreased due to the hindered electron transfer reaction on the electrode surface. Under optimum condition, it was found that the proposed method could detect Lys at wide linear concentration range (0.1 pM-0.10 mM) with detection limit of 60 fM. Furthermore, electrochemical impedance spectroscopy (EIS) detection of Lys was demonstrated as a simple and rapid alternative analytical technique with detection limit of 80 fM at concentration range up to 0.1mM. In addition, the proposed sensor was satisfactorily applied to the determination of Lys in real samples such as hen egg white. The proposed modified electrode showing the high selectivity, good sensitivity and stability toward Lys detection may hold a great promise in developing other electrochemical sensors based on metal-chelate affinity complexes. Keywords: Lysozyme detection, Electrochemical sensor, Metal-Chelate affinity complex, Voltammetric and impedimetric, Gold nanoparticles.

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1. Introduction Lysozyme (Lys), a ubiquitous protein consisting of 129 amino acids that is widely distributed in body tissues and secretions, can break down bacteria by breaking the β1-4 bond found in peptidoglycan residues of bacterial cell walls, thus serving as a natural “drug” (Cheng et al., 2007; Liu et al., 2014; Huang et al., 2012). The abnormal concentration of Lys in serum and urine is related to many diseases, such as leukemia, renal diseases, and meningitis, and it has been used as an important biomarker (Levinson et al; 2002). Also, due to its lytic activation, clear structure and low molecular weight, it has a wide application in protein research (Riter et al., 2005), medical treatment (Huang et al., 1999), and food industry (Proctor and Cunningham, 1988). Hence, the detection of Lys is of considerable importance. To date, different methods have been used for the determination of Lys, such as resonance Rayleigh scattering method (Cai et al., 2011), spectrophotometry (Jiang and Luo, 2004), fluorescence (Liu et al., 2014), colorimetry (Chen et al., 2008), chemilumenesence (Li et al., 2011), localized surface plasmon resonance (Lie et al., 2014), turbidimetric (Mörsky, 1983), electrochemistry (Liu et al., 2011a) and high efficiency liquid chromatography (Pellegrino and Tirelli, 2000). Many of these methods mentioned above have poor sensitivity and specificity and also are expensive and need special equipment for performance, which limited their applications. Then, developing of novel and low cost protein detection strategies with high simplicity, sensitivity and selectivity is a challenge. Immobilized metal ion affinity chromatography (IMAC), first proposed by Porath (Porath et al., 1975), is a separation method based on the affinity adsorption between the metal-binding amino acids in the side chains of proteins (e.g. histidine, lysine, glutamic acid, tyrosine, aspartic acid, etc.) and the metal ions (e.g. Cu2+, Ni2+, Zn2+, Fe2+, etc) chelated by a ligand (e.g. iminodiacetic 3

acid, terpyridine, nitrilotriacetic acid) which immobilized on a solid support (Gutierrez et al., 2007; Block et al., 2009; Chakrabarti, 1990; Chen et al., 1997; Ke et al., 2010; Ali et al., 2011; Yan et al., 2013). The biospecific affinity adsorption applied in these separation methods could provide high selectivity for biological separation which led to their wide use applications in laboratory and industrial areas (Chen et al., 1997). A research has been done by Wu et al. to evaluate the performance of different chelating ligands toward protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes (Wu et al., 2003). The iminodiacetic acid (IDA) ligand has the highest chelating capacity in comparison to the other chelators including N,N,N-tris(carboxymethyl) ethylenediamine, Cibacron blue 3GA, and Cibacron red 3BA. On the other hand, due to attractive electronic, chemical and mechanical properties of nanomaterials, there have been tremendous efforts to apply various nanomaterials for immobilizing a large variety of species onto their surfaces. The high specific surface area of nanomaterials would offer higher capacity for loading of ligands and thus enhance the amounts of immobilized metal ions which would result in a larger capacity of biomolecules binding (Yan et al., 2013). In addition, decoration of nanomaterials with metal-chelating organic ligands would also provide another biosensing platform for the immobilization and detection of biomolecules that display affinity toward specific metal ions. Recently, a lactoferrin biosensor has been fabricated based on the immobilization of iron-terpyridine complexes inside the single conical nanopores fabricated in polymer membrane (Ali et al., 2011). Thus, novel biosensors for protein and enzyme assay can be developed through the combination of nanomaterials with metal chelating ligands. In the present study, we reported for the first time an electrochemical signal transduction method for protein biosensing through the use of an immobilized metal ion affinity (IMA) probe

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on gold nanoparticles (GNPs) modified electrode. A glassy carbon (GC) electrode was electrochemically modified with GNPs after which the IDA-Cu complex, as recognition element for capturing of lysozyme molecules, was covalently attached. Cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were used to investigate the modified electrode fabrication process and determination of lysozyme. The addition of Lys to the IDA-Cu complex on the electrode surface leads to changes in the electron transfer kinetics and diffusion of the electrochemical probe ([Fe(CN)6]3-/4-); which can be followed by both DPV and EIS techniques. The analytical characteristics of the sensor were studied and its applicability toward lysozyme detection in a real sample such as hen egg white was also evaluated. 2. Experimental methods 2. 1. Chemicals HAuCl4.3H2O,

2-Mercaptoethanol

(ME),

3-Chloropropyl

trimethoxysilan

(CPTMS),

iminodiacetic acid (IDA), Na2CO3, CuSO4.5H2O, K4[Fe(CN)6], K3[Fe(CN)6] , toluene and Lys from hen egg white were purchased from Merck (Germany) and Sigma-Aldrich (USA). Doubly deionized water was used throughout the experiments. Analytical grade chemicals were used for buffer preparation. All Lys solutions were prepared in phosphate buffer (50 mM, pH 7.0). 2. 2. Instruments and measurements Electrochemical measurements were performed with Micro-Autolab (μ3AUT-70751), potentiostat/galvanostat and Metrohm (Type 1.757.0010) instruments connected to a threeelectrode cell, linked to a computer (Pentium IV, 1200 MHz) and the cell linked to the instrument software. A conventional three-electrode cell was used for all experiments. Modified GC electrode used as working electrode, platinum wire and Ag/AgCl/KCl (sat'd) were used as 5

auxiliary and reference electrode, respectively. EIS measurements were carried out in a conventional three-electrode cell, powered by an electrochemical system comprising the ZAHNER (model IM6ex, Germany). The system was run on a PC using THALES USB software. For impedance measurements, a frequency range of 0.1 Hz to 10 kHz was employed. The AC voltage amplitude used was 5 mV. Scanning electron microscopy (SEM) image was obtained with a MIRA3 TESCAN HV: 20.0 KV from Czech Republic. 2.3. Preparation of GNPs modified GC electrode Prior to coating, GC electrode was subsequently polished with 0.05 μm alumina powder on polishing cloth and sonicated successively in water/ethanol (9/1 v/v) in order to remove adsorbed particles. The procedure for the deposition of GNPs onto GC electrode was adapted based on the previously published reports (Dai et al., 2004; El-Deab et al., 2003; Finot et al., 1999; Ryoo et al., 2006). The polished GC electrode was immersed into 0.5 M H2SO4 solution containing 1.0 mM HAuCl4. GNPs were electrodeposited on the GC electrode by applying a potential step from 1.1 to 0.0 V during 600 s (Ryoo et al., 2006). The formation of GNPs on the GC surface was characterized by taking SEM images. 2. 4. Modification of GCE/GNPs with IDA-Cu complex GC/GNPs modified electrode was rinsed several times with deionized water. Then, 10 μl of ME was placed on the surface of the GCE/GNPs and after 18 h, a self-assembled layer of ME was attached on the surface of GNPs via Au-S binding (GCE/GNPs/ME). In the next step, after resigning of GCE/GNPs/ME several times with deionized water, it was immersed in the 1.5 ml of dry toluene containing 0.10 ml CPTMS and maintained in 65

with magnetic stirring for 18

h or in ambient temperature for 24 h. As shown in Scheme 1, the reaction between alcohol group of ME (R΄OH) and CPTMS (R''O)3SiR was carried out to form R΄-O-Si-R (Khan and Siddiqui, 6

2014). GCE/GNPs/ME/CPTMS was rinsed several times with acetone and it was dried and immersed in the 1.0 ml of deionized water containing 0.20 M IDA, 1.0 M Na2CO3 and maintained in 80

with magnetic stirring overnight. Reaction between R΄Cl and amines

(NHR2) for formation of R΄NR2 has been previously reported (Wu et al., 2003). The primary amine on one terminus of the ligand was exploited for the covalent linkage with surface -Cl groups while diacetic acid -(COOH)2 moieties on the other terminus were used for metal ion complexation. Finally, to form a complex between the immobilized chelating groups and metal ions, the IDA functionalized modified electrode was immersed into 25 mM CuSO4 (in deionized water with pH = 5.5-6.0) solution at room temperature for 2 h according to reported procedure in the literature (Ke et al., 2010; Wu et al., 2003). Subsequently, the modified electrode was washed several times with deionized water and placed in a solution containing 3 mM [Fe(CN)6]3-/4- and 0.1 M KCl and potential cycling was applied in the range of -0.50 and 1.0 V at a scan rate of 0.10 Vs-1 for 20 times to eliminate untreated compounds on the modified electrode surface and to increase its reproducibility and stability. Then, the as-prepared GCE/GNPs/ME/CPTMS/IDA-Cu modified electrode with metal ion affinity system was washed with deionized water and used for determination of Lys. 2. 5. Real sample preparation and analysis In this study, two different hen egg whites were used as real samples for analysis. For each sample, three fresh hen eggs whites were separated from yolk and homogenized in an ice bath for 30 min. Then 10 g of the homogenized hen egg white was removed and was brought with phosphate buffer solution (50 mM, pH 7.0) to volume 50 mL and homogenized in an ice bath for 6 h. The resulting solution was further centrifuged (15 000 rpm) at 4 °C for 30 min, and the supernatant was used as the Lys resource (Zhu and Sun, 2014). 10 L of the Lys sample dropped 7

on the electrode surface and after immersing of the working electrode in electrochemical cell, the standard addition method was adopted to measure the Lys concentration with DPV as the measuring technique. 3. Results and discussion 3.1. The fabrication and characterization of the fabricated electrochemical Lys sensor Scheme 1 represents the fabrication process of the proposed electrochemical biosensor for detection of Lys based on the immobilized IDA-Cu metal-chelate affinity complex. At first, a layer of GNPs was electrodeposited on the GC electrode surface. Fig. 1 shows the SEM image of GCE/GNPs modified electrode surface. As can be seen, the GNPs were homogeneously deposited on the surface of GCE, and they were well dispersed and remained relatively stable without aggregation with sizes ranging from 20 to 65 nm and an average of 36 nm. The GNPs on the surface of the electrode not only led to enhance the rate of electron transfer reactions, but it also was beneficial for immobilizing molecules with specific functional groups such as thiols. After this step, we self-assembled a layer of ME on GCE/GNPs followed by the covalent attachment of CPTMS molecules through hydrolysis of silicon alkoxide molecules. Then, IDA chelating ligand covalently binds to the electrode surface by performing a nucleophilic attack of secondary amine group on the IDA to the chloride-bearing carbon at CPTMS molecule. The addition of Cu2+ ion in the next step results in the formation of IDA-Cu complex which can provide a quite suitable interface with high affinity for the subsequent conjugation of Lys protein molecules. Through the incubating various concentrations of Lys with the biosensor and following DPV or EIS signals of ([Fe(CN)6]3-/4-as redox indicator, a sensitive and specific electrochemical biosensor was successfully presented.

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The Lys biosensor fabrication process was investigated by recording CVs of the modified electrode in ([Fe(CN)6]3-/4- solution during preparation steps (Fig. 2A). As shown, a reversible redox response at the bare GCE can be clearly observed (Fig. 2A voltammogram “a”). In the case of GCE/GNPs modified electrode, the peak current increased greatly accompanied by a decrease in peak potential separation which can be ascribed to the large electron transfer capability and high specific surface area of GNPs (Fig. 2A, voltammogram “b”). As expected, during sequent immobilization of ME, CPTMS and IDA at the GCE/GNPs electrode surface on the next steps, (Fig. 2A voltammograms “c, d and e”), the peak current clearly decreased and the peak potential separations consequently increased. Further decrease in peak current and increase in peak potential separations occurred after covalent attachment of IDA, indicating the successful attachment of IDA ligand to the surface of electrode. The decrease in peak current can be attributed to the blocking the electron-transfer efficiency of ([Fe(CN)6]3-/4-redox probe at solid/liquid interface as a result of coverage the surface with IDA on one hand and the electrostatic repulsion between anionic ([Fe(CN)6]3-/4-and the surface bound carboxylate groups of IDA with negative charges on the other hand. At the next step, after interaction of IDA with copper ions, the peak current was slightly increased along with a decrease in peak potential separation which may be ascribed to a strong electrostatic attractive interaction between the Cu2+ ions and ([Fe(CN)6]3-/4-or decreasing of the negative charge of the electrode surface during complex formation between IDA and Cu2+. This observation confirms the formation IDA-Cu complex at the electrode surface (Fig. 2A voltammogram “f”). When the modified electrode was incubated with 100 nM and 0.10 mM solutions of Lys for 90 min (Fig. 2A voltammograms “k and l”), a dramatic decrease in peak current and increase in peak potential separation has been observed which is proportional to the Lys concentration. It demonstrates that Lys adsorption has

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been successfully achieved onto the electrode surface with the result of substantially hindering the electron transfer between the electrode surface and ([Fe(CN)6]3-/4-redox couple in solution due to this fact that the Lys can act as a mass transfer blocking layer making the diffusion of ferricyanide toward the electrode surface difficult. The stepwise sensor fabrication process and the formation of IDA-Cu/Lys complex were also investigated by recording EIS plots. Fig. 2B shows the obtained Nyquest plots recorded in a [Fe(CN)6]3-/4-solution at frequency range 0.1 Hz to 10 kHz during sensor fabrication process. The insert of Fig. 2B shows a general equivalent circuit which has been used as a model for impedance spectra of the current system. The equivalent circuit contains the resistance of solution (Rs), the charge transfer resistance (Rct), the Warburg element (W), and the double layer capacitance (Cdl). The Rct value of [Fe(CN)6]3-/4-as redox probe can be determined by fitting the experimental data to the model circuit. For GCE/GNPs modified electrode, the calculated value for Rct was 0.39 KΩ (Fig. 2B, plot a). Low charge transfer resistance for GNPs modified electrode compared to the other plots can be ascribed to the increased effective surface area and large electron transfer capability of GNPs. Subsequently, when ME, CPTMS and IDA were loaded on the GCE/GNPs, proportionate with each stage, Rct increased, suggesting that these compounds immobilized on the electrode surface (Fig. 2B, plot b, c and d). After immobilization of copper ions on the modified electrode surface (Fig. 2A voltammograms “e”), the R ct was slightly decreased due to the decreasing of charge transfer repulsion between [Fe(CN)6]3-/4- and IDA. The Rct after formation of complex between IDA chelate group and copper ions on the surface of modified electrode was calculated to be 1.72 KΩ. When the biosensor based on immobilized copper ions-IDA affinity complex was incubated in the 100 nM and 0.10 mM Lys solutions for 90min, a substantial increase in Rct was observed (4.84 and 9.51 KΩ, respectively)

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implying the formation of IDA-Cu/Lys complex on the electrode surface (Fig. 2A voltammogram “f and k”). The result was consistent with the fact that the protein can decrease the conductivity and hinders the interfacial electron transfer kinetics. Thus, proportionate to the Lys concentration on the modified electrode surface, the electron transfer kinetics decreases and Rct increases, indicating that the proposed metal ion affinity-based modified electrode can be applied as an efficient impedimetric biosensor for Lys detection. The herein obtained impedance data are in accordance with the preceding results obtained by CV, all demonstrate that the sensing interface is constructed successfully. The fabrication reproducibility of the sensore was also testified by preparing independently five GCE/GNPs/ME/CPTMS/IDA-Cu electrodes and measuring the peak current of recorded DPV after incubation with 100 nM of lysozyme. Almost same results were found for all electrodes and the RSD value of about 4.5% was obtained, indicating good repeatability of the modification process. 3. 2. Voltammetric and impedimetric detection of Lys To evaluate the application of the prepared assay platform toward the quantification of Lys, DPV was used as measuring technique. Under optimal experimental conditions (phosphate buffer 50 mM, pH 7.0 and incubation time 90 min), we examined the performance of the electrochemical Lys biosensor to detect different concentrations of Lys. Fig. 3 shows typical DPVs of Lys biosensor in the presence of different concentrations of Lys (0.10 pM-0.10 mM, from “a” to “k”). As illustrated, a well-defined voltammetric response was observed which decrease with increasing of Lys concentrations. The interactions between the IDA-Cu complex and Lys on the electrode, enhanced hindering of electron transfer reaction of redox probe on the biosensor surface. The dependence of the DPV current response on the Lys concentration is also shown (inset of Figure 3). As can be seen, a linear relation between the current response and the Lys 11

concentration was observed in two concentration ranges. For lower concentration range (i.e., 0.10 pM-10.0 nM), the regression equation was ∆I (Iblank-IsampleA)) = -4.7743 logC/M + 64.047 A and the correlation coefficient was 0.9925, while for higher concentration range (10.0 nM-0.10 mM), the corresponding regression equation was ∆I (Iblank-IsampleA)) = -1.628 log C/M + 38.824 µA with a correlation factor of 0.9911. The reason that calibration curve present two linear concentration ranges with different slopes is that the amount of IDA-Cu complex immobilized on the GNPs-modified surface is some certain. When the sensor is used to detect analyte, Lys can bind with abundant active sites of IDA-Cu on the electrode surface. With increasing of Lys concentration, the number of active sites of IDA-Cu on the sensor became fewer. Therefore, the sensitivity declines when the sensor is used to determine higher concentrations of Lys and calibration curves with different slopes were observed. This phenomenon has also been observed for some other sensors (Khezrian et al., 2013). The detection limit of the system was calculated as 60 fM (based on S/N=3). Due to the high ability of EIS technique for probing the interfacial properties at the electrode surface and the possibility of performing label-free detections (Kavosi et al., 2014), EIS measurement was also applied as an efficient alternative detection system to further confirm the analytical performance of the proposed system. Fig. 4 shows the recorded Nyquist plots obtained at the GCE/GNPs/ME/CPTMS/IDA-Cu electrode after being incubated with different concentrations of Lys in 0.1M KCl solution containing 3mM [Fe(CN)6]3-/4-. As shown, the Rct value increased with increasing the concentration of Lys, due to the hindrance of electron transfer process of [Fe(CN)6]3-/4- at electrode surface. The Rct was proportional to the Lys concentration at two concentration ranges. For lower concentration range ( 0.10 pM-10 nM), the linear equation could be depicted as ∆R (Rsample-Rblank (kΩ)) = -0.6309 log C/M + 8.6073 kΩ and

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the correlation coefficient was 0.9979, while for higher concentration of Lys ( 10 nM-0.10 mM) the corresponding regression equation was ∆Rct (Rsample-Rblank (kΩ))= -1.125 log C/M + 12.51 kΩ with a correlation coefficient of 0.9985. The relative standard deviations (RSD) for the measurement of each data point were less than 5.0%. The detection limit based on S/N=3 was 80 fM. The analytical characteristics of the proposed Lys biosensor was compared with previously reported Lys sensors (Table1A). As can be seen, the detection limit, sensitivity and linear concentration range of the proposed strategy for Lys biosensing are better or comparable with the other sensors reported so far. High lysozyme adsorption capacity on the copper ion immobilized onto IDA is attributed to the specific affinity between the immobilized metal ions and lysozyme molecules. Furthermore, lysozyme is a large molecule, acts as mass transfer blocking layer and it hinders the diffusion of ferricyanide toward the electrode surface, so the response of the proposed sensor toward trace concentration of lysozyme is recognizable. 3. 3. Interference study and analytical application The successful use of IDA-Cu affinity complex system for selective separation of Lys from hen egg white has been previously reported (Zhu and Sun, 2014), indicating the negligible interference effect of the major protein components in the fresh hen egg white, such as conalbumin, ovalbumin and ovomucoid on the separation of Lys. The practical applicability of our proposed biosensor toward the Lys determination in hen egg white as a complex difficult to analyze real sample was evaluated. For this purpose, the standard addition method was adopted to demonstrate the possibility of Lys detection in real samples. The calculated values for concentrations of Lys in the two samples were 3.66 (±0.19) and 4.0 (±0.20) mg mL-1 which are in accordance with those reported in the literature (Ma et al., 2014; Zheng et al., 2013; Kvasnička, 2003). Additionally, recoveries of Lys from egg white were investigated using the 13

proposed modified electrode. It could be seen in Table 1B that there is a good agreement between the spiked and found values for detection of Lys. So, these results indicate the interference effect of major protein components on the signal response of lysozyme is negligible. So, the methodology suggested in this work to be valid for the analysis of Lys in real samples. 4. Conclusion In this paper, a novel strategy was developed for the determination of Lys based on the synergistic effects of GNPs as a conductive support with high surface area and covalently immobilized coordinative complex of IDA-Cu as a recognition element to capture target proteins with electrochemical readout as a simple, sensitive and selective transduction method. The amount of affinity complex formed between immobilized metal-chelate and Lys as a measure of Lys concentration was monitored by following the changes in the redox response of [Fe(CN)6]3/4- as redox probe by using DPV and EIS techniques. Under optimized condition, the GCE/GNPs/ME/CPTMS/IDA-Cu modified electrode displayed a wide linear concentration range of 100fM-0.1 mM toward Lys. The calculated detection limits of the proposed sensor were 60 and 80 fM (S/N =3), when measuring techniques were DPV and EIS, respectively and RSD was less than 5% (n=5) for both measuring methods. This sensor was satisfactorily applied to the determination of Lys in hen egg white samples. The high sensitivity and selectivity, wide concentration range (10 orders of magnitude), reliability, simplicity and low cost preparation method were advantages of the proposed sensor compared to other Lys detection methods. The results indicate that the present protocol is quite promising in developing other electrochemical sensors based on metal-chelate affinity complexes. Acknowledgments

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The financial supports of the Iranian Nanotechnology Initiative and Research Office of University of Kurdistan are gratefully acknowledged. We thanks Dr. Hajhir Teymourian for his valuable corporations.

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Figure Captions Table 1A. Comparison of the reported assays for Lys detection, Table 1B. Analytical results of Lys added in egg white sample after dilution. Scheme 1. Representation of different steps for the Lys sensor fabrication. Fig. 1. High and low magnification SEM images of the electrochemically deposited GNPs on GCE. Fig. 2. (A) Recorded CVs and (B) Nyquist plots at different electrodes; GC, GC/GNPs/ME,

GC/GNPs/ME/CPTMS,

GC/GNPs/ME/CPTMS/IDA/Cu+2,

GC/GNPs,

GC/GNPs/ME/CPTMS/IDA,

GC/GNPs/ME/CPTMS/IDA/Cu+2/100

nM

Lys,

GC/GNPs/ME/CPTMS/IDA/Cu+2/0.10 mM Lys in solution containing 3 mM [Fe(CN)6]3-/4- and 0.1 M KCl, scan rate for recorded voltammograms was 0.10 V s-1; frequency range and applied potential for recorded Nyquist plots were 0.1 Hz to 10 kHz and 0.22 V, respectively. Inserts in Fig. 1B (C) shows an enlarged section indicated in Fig. 1B; and (D) is the equivalent circuit used to fit the experimental impedance data. Fig. 3. DPVs of the proposed biosensor after incubation with different concentration of Lys;(a) 0.0, (b)0.10 pM, (c)1.0 pM, (d)10.0 pM, (e)0.10 nM, (f)1.0 nM, (g)10.0 nM, (h)0.10 μM, (i)1.0 μM, (j)0.01 mM and (k) 0.10 mM in solution containing 3 mM [Fe(CN)6]3-/4- and 0.1 M KCl. Insert is plot of peak currents vs. Lys concentration. Fig. 4. EIS plots of the presented biosensor after incubation with different concentration of Lys;(a) 0.0, (b) 0.10 pM, (c) 1.0 pM, (d) 10.0 pM, (e) 0.10 nM, (f) 1.0 nM, (g) 10.0 nM, (h) 0.10 μM, (i) 1.0 μM, (j) 0.01 mM and (k) 0.10 mM in solution containing of 3 mM [Fe(CN)6]3-/4- and 0.1 M KCl. Insert is plot of Rct vs. Lys concentration.

18

Table 1A. Comparison of the reported assays for Lys detection Assay

Principle

Linear range

LOD

Ref.

Fluorescence

Lys reacted with single strand DNA binding (SSB)-Aptamer and releases SSB, binding of the free SSB to a molecule resulted in a turn-on fluorescence signal. The quenching of fluorescence during interaction lysozyme with CuInS2 quantum dot/cationic polyelectrolyte and aptamer The combination of Lys and aptamer led to the dissociation of doublestranded DNA, using the oxidation of tripropylamine probe FRET between an anionic conjugated polymer and a dyelabeled lysozyme aptamer Interaction between the aptamer and Lys on the surface of gold nanoparticles modified gold electrode increases the Rct Interaction of Apt-gold nanoprobe with Lys and release GNs, which aggregated to form large clusters with a RRS Based on competition between Lys and the Ru(bpy)32+ cation for the binding sites of Apt in the gold electrode. The hybridization lysozyme an ssDNA , leads to the dissociation of ds-DNA and the release of Ru(phen)3 2+ with a decreased ECL emission. The fluorescence of triazolylcoumarin molecules was quenched by Au nanoparticles (AuNPs), after the addition of lysozyme Based on Electrostatic Interaction with Human Serum AlbuminModified Gold Nanoparticles Cys-Ala-Leu-Asn-Asn (CALNN)capped gold nanoparticles are linked by lysozyme specifically to form aggregates Immobilized copper ions-IDA affinity complex was used for electrochemical determination of Lys 3-/4and [Fe(CN)6] ) was used as redox probe, the signals monitored by both DPV and EIS techniques.

0-15 nM

0.2 nM

Tang et al., 2011

40-500 nM

20 nM

Liu et al., 2014

0.001–1.1 nM

-

Liu et al., 2011a

Up to 2.7M

54 nM

Wang and Liu, 2009

0.1 pM-500 pM

0.1 pM

Chen et al., 2011

0.2-5.2 nM

0.05 nM

Ma et al., 2014

0.64-640 nM

0.12 nM

Li et al., 2011

0.9-45 pM

0.45 pM

Liu et al., 2011b

0.050-25μg mL-1 (Color.) 0.0035-1.75μM (Fluoro.)

23 ngmL-1 1.6 nM

Zheng et al., 2013

0.1-1M

50 nM

Chen et al., 2008

0.07-1.7 nM

5.5 pM

Huang et al., 2012

0.10pM-100 mM (DPV) 0.10 pM-100 mM (EIS)

0.06 pM 0.08 pM

This work

Fluorescence

Electrochemistry

Fluorescence resonance energy transfer(FRET) Electrochemical impedance spectroscopy

Resonance Rayleigh Scattering (RRS)

Chemiluminescence

Electrochemiluminescent

Colorimetric and Fluorometric

Colorimetric Detection

Colorimetric Detection

Electrochemical impedance spectroscopy and differential pulse voltammetry

19

Table 1B. Analytical results of Lys added in egg white sample after dilution Sample

Added lysozyme concentration (μM)

Concentration found (μM)

RSD%(n=4)

Sample 1*

0.0

0.510

8.0

0.100

0.627

8.5

0.0

0.055

9.0

0.010

0.0665

8.8

Sample 2*

*before determination samples 1 and 2 were diluted 100 and 1000 times, respectively.

20

Recovery%

117

115

Scheme 1

21

Fig. 1

22

Fig. 2

23

Fig. 3

24

Fig.4

25

- IDA chelate groups

- Copper ions

- Lysozyme

Lysozyme

Modified GCE After Interaction with Lys

Modified GCE

A

B

Graphical abstract

Highlights for review A novel voltammetric and impedimetric sensor for detection of lysozyme was developed▲ GCE/AuNPs used as electrochemical platform for iminodiacetic acid-Cu2+complex(IDA-Cu)▲ IDA-Cu2+ acted as metal-chelate affinity element capable of capturing Lys molecules▲ DPV and EIS methods were used for Lys detection at wide range,0.1 pM-0.10 mM(9 order)▲ LOD sensor was 60fM and it was applied for Lys detection in hen egg withe real sample▲

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