- Email: [email protected]
Direct immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor
Accepted Manuscript Directly immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor Xiangyang Zhang, Guangyu Shen, Youming Shen, Dan Yin, Chunxiang Zhang PII: DOI: Reference:
S0003-2697(15)00299-7 http://dx.doi.org/10.1016/j.ab.2015.06.007 YABIO 12105
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
20 March 2015 3 June 2015 4 June 2015
Please cite this article as: X. Zhang, G. Shen, Y. Shen, D. Yin, C. Zhang, Directly immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.06.007
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 proof before it is published in its final 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.
Immunosensor based on a new polymer film Electrochemical methods
Directly immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor Xiangyang Zhang∗ , Guangyu Shen, Youming Shen*, Dan Yin, Chunxiang Zhang College of Chemistry and Chemical Engineering, Hunan University of Arts and Science, Changde 415000, PR China
* Corresponding author.
TEL.: +86 736 7186165 E-mail address:[email protected] (X. Zhang); [email protected] (Y. Shen)
1
Immunosensor based on a new polymer film Abstract A new polymer with aldehyde groups was designed and synthesized by use of poly -(epichlorohydrin) and 4-pyridinecarboxaldehyde. Antibodies can be directly immobilized on the surface of the polymer film through the covalent bonding of aldehyde groups of the film with amino groups of antibodies. In this study, human IgG was used as a model analyte to fabricate an electrochemical impedance immunosensor. Using the proposed immunosensor, we detected IgG within the range from 0.1 to 80 ng mL-1 with a detection limit of 0.07 ng mL-1 (S / N = 3). In addition, the
electrochemical
impedance
immunosensor
had
a
good
stability and
reproducibility. Keywords: new matrix; convenient immobilization method; electrochemical impedance immunosensor; Human immunoglobulin G
1. Introduction In the past few years, many bioanalytical methods for clinical diagnosis have experienced unprecedented growth. Owing to the advantages of high sensitivity, rapid detection, simple instrumentation, miniaturization and low cost, considerable efforts have been devoted to electrochemical impedance immunosensors for detecting protein [1, 2]. One of the most common issues faced in the development of electrochemical impedance immunosensors with good performance is the method utilized for immobilization of antibodies. The immobilization of antibodies on sensing surfaces can be carried out either by simply physical adsorption or chemical cross-linking. Although physical adsorption is the simplest and fastest way to immobilize antibodies, it is very sensitive to environmental conditions and has problems in stability [3, 4]. Compared
with
physical
adsorption,
chemical
cross-linking
method
for
immobilization antibodies could minimize the interference from the environmental and provide higher analytical accuracy [5]. Among chemical cross-linking method, various support matrixes with functional groups such as carboxylic acid and amine have been used to immobilize antibodies [6, 7]. Traditionally, those support matrixes for immobilization antibodies procedures usually involve three separate steps. The 2
Immunosensor based on a new polymer film first step is to creating a film through coating method. The second step involves the activation
functional
groups
with
the
mixture
of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [8, 9], or glutaraldehyde [10, 11]. The third step is the reaction between amine groups of protein and the linkage agents. However, these methods are trivial and time-consuming and will greatly influence the reproducibility of electrochemical impedance immunosensors. Therefore, it is important to develop a convenient immobilization method based on some new and easily prepared matrixes for the fabrication of electrochemical impedance immunosensors. Recently, chlorine-containing polymers such as poly(vinylchloride) (PVC) [12], poly(p-chloromethylstyrene) (PCMS) [13], and poly(epichlorohydrin) (PECH) [14] have been used as starting materials for synthesizing novel functional polymers with a wide range of applications. Among those polymers mentioned above, PECH has received considerable attention because they are biocompatibility, commercially available, very cheap, and chlorine atoms can be efficiently substituted by many nucleophilic reagents such as aliphatic carboxylates, sodium methoxide and series of substituted phenolates under mild conditions [15]. Moreover, PECH have been used for synthesizing a series of well-defined DNA-mimicking brush polymers [16], new self-assembled brush glycopolymer [17], novel lipid-mimicking brush polymers [18], and so on. However, to the best of our knowledge, PECH have not been used for synthesizing a kind of polymer bearing aldehyde groups. Polymer bearing aldehyde groups is one of the most popular matrixes in electrochemical immunosensors because they can directly immobilization of proteins. Despite general superiority offered by polymer with aldehyde groups, they cannot be obtained from chemical reagent company. Therefore, several attempts have been made to synthesis of polymer with aldehyde groups. Under such a research background, we begin to consider designed and synthesized a polymer with aldehyde groups by use of poly(epichlorohydrin) and immobilize protein on surface of the polymer film. In this study, we report a simple and rapid procedure for the immobilization of antibodies to fabricate an electrochemical impedance immunosensor for the detection of human IgG as model 3
Immunosensor based on a new polymer film biomolecules. This strategy can open a new door to broaden the potential applications of modified poly(epichlorohydrin) in clinical research. The whole process of electrochemical impedance immunosensor fabrication was shown in Fig. 1. (Preferred position for Fig. 1) 2. Experimental 2.1. Reagents and apparatus Poly (epichlorohydrin) (PECH, average Mw~700,000), 4-pyridinecarboxaldehyde, dimethylformamide (DMF) used in this work were of analytical grade and purchased from Sigma-Aldrich company. Human immunoglobulin G (IgG), goat anti-human immunoglobulin G antibody (Ab), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. 0.1 M phosphate buffer solution (PBS, pH 7.2) was prepared using Na2 HPO4 and NaH2PO4. All aqueous solutions were prepared with doubly distilled water. All electrochemical measurements, including cyclic voltammetric measurement (CV) and electrochemical impedance spectroscopy (EIS) were carried out with a CHI 660E
electrochemistry workstation (Shanghai CH
Instruments,
China).
A
conventional three-electrode cell, consisting of a Pt electrode as counter electrode, a saturated calomel electrode (SCE) as reference electrode, and a glassy carbon electrode (GCE) modified with modified poly(epichlorohydrin) film as working electrode was used. 2.2. Preparation of polymer 1 (Preferred position for scheme 1) As illustrated in Scheme 1, polymer 1 can be obtained according to the similar method reported in the literature [19]. Briefly, under the protection of argon, 1.24 g poly (epichlorohydrin) was dissolved in 20 mL anhydrous dimethylformamide (DMF) at 60 oC, and then 1.3 g 4-pyridinecarboxaldehyde was added. The mixture was stirred at 120 oC for 24 h. Most of the solvent was removed by constant rotation under vacuum. The crude polymer was dried at 50 oC in a vacuum overnight. The dried polymer was re-dissolved in dichloromethane and precipitated three times with ethanol. Finally a brown solid (0.5 g) was obtained after dying under vacuum at 50 oC for 24 h. 4
Immunosensor based on a new polymer film 2.3. Preparation of electrochemical immunosensor A GCE (3 mm in diameter) was polished repeatedly with 0.3 and 0.05 µm alumina slurries sequentially, followed by successive sonication in distilled water and ethanol, respectively. GCE was dried in air at room temperature and then 10 µL of 0.1% polymer 1 solution diluted in DMF was dropped on the electrode and dried in air at room temperature. Then, 10 µL of antibody solution (100 µg mL-1) was dropped on the modefied electrode and incubated for 1 h, followed by washing with ultrapure water to remove unspecific physically adsorption. In order to eliminate non-specific binding effect and block the remaining active sites, the electrode modified with antibody was incubated with 1.0 wt% BSA for 30 min at room temperature. The as-prepared immunosensor (designed as Ab/ polymer 1/GCE) was stored at 4 oC for further detection of IgG analyte. 2.4. Electrochemical measurements All
electrochemical
experiments
were
carried
out
in
a
conventional
electrochemical cell containing a three-electrode arrangement. CV and EIS measurements were performed in 10 mM K3Fe(CN)6/K4Fe(CN)6 solution. The CV measurements were taken at scanning rate 100 mVs -1 from -0.2 to 0.6 V relative to saturated calomel electrode. EIS measurements were carried out in the frequency range from 10 -1 to 105 Hz under an open potential. The amplitude of the alternative voltage was 5.0 mV. 3. Results and discussion 3.1. Characterization of polymer 1 (Preferred position for Fig. 2) To study the presence of aldehyde group, we measured IR-spectra of poly (epichlorohydrin) (PECH) and polymer 1 respectively. Compared with the spectrum of PECH, polymer 1 has an obvious aldehyde peak at 1730 cm−1 (Fig. 2). The result of spectrum showed that we have successfully synthesized the target product. 3.2. The electrochemical characterization of the modified electrodes (Preferred position for Fig. 3)
5
Immunosensor based on a new polymer film In order to evaluate the electron transfer property of the modified electrode, cyclic voltammograms were carried out using K3Fe(CN)6/K4Fe(CN)6 as a redox probe. Fig. 3 shows CVs of modified electrode in 10 mM K3Fe(CN)6/K4Fe(CN)6 solution at a scan rate of 100 mV s-1. Curve a is cyclic voltammograms of K3Fe(CN)6/K4Fe(CN)6 on the bare electrode, a couple of reversible redox peaks could be observed. When the electrode was stepwise modified with polymer 1 (curve b), antibody (curve c), BSA (curve d), and antigen (curve e), a decrease in peak current was observed, which indicates the decrease of peak current response corresponding to stepwise modifications consistent with the enhanced electron-transfer barriers introduced by these layers modified on the surface of the electrode. 3.3. The principle of detection The general electronic equivalent scheme is usually described on the basis of the model by Randles [20]. As shown in Fig. 6A, the circuit includes the ohmic resistance (Rs) of the electrolyte solution, the Warburg impedance (Zw) resulting from the diffusion of ions from the bulk electrolyte to the electrode interface, the double layer capacitance
(Cdl)
and
electron-transfer
resistance
(Ret).
Ret
controls
the
electron-transfer kinetics of the redox probe from the solution to the electrode interface. It is a suitable parameter for sensing the interfacial properties of the prepared immunosensor step by step. In Nyquist plot of Faradic impedance, the semicircle diameter of EIS equals the electro-transfer resistance (Ret). Ret increases when antigen is bound by antibody. Moreover, the relative change of the electron transfer resistance (%∆Ret) was monitored as a function of the antigen concentration. It is calculated by the following equation:
% ∆Ret = Ret(Ag − Ab ) − Ret(Ab ) × 100 Ret(Ab ) Where Ret (Ag–Ab) is the impedance of the electrode after antibody-antigen reaction, Ret (Ab) represents the impedance of the antibody-modified electrode blocked with BSA. 3.4. Optimization of experimental conditions 6
Immunosensor based on a new polymer film (Preferred position for Fig. 4) The amount of antibody immobilized on the electrode was optimized. As shown in Fig. 4A, when the antibody concentration was less than 100 µg mL−1, the Ret increased with the increasing of its concentration. Then the Ret reached a plateau at 100 µg mL−1. Therefore, a 100 µg mL-1 antibody concentration in PBS was chosen for this work. In addition, the effect of pH during the immuno-reaction was investigated from 6.0 to 8.0. As can be seen from Fig. 4B, the impedance increased with pH from 6.0 to 7.0, and then decreased from 7.0 to 8.5. Thus, phosphate buffer solution of pH 7.0 was selected as the buffer solution in the immuno-reaction. (Preferred position for Fig. 5) In addition, the incubation time of Ab-Ag was also investigated over the range from 20 to 80 min. Fig. 5 showed that the impedance increased with the incubation time increasing untill 40 min. Therefore, 40 min should be selected for the subsequent experiment. 3.5. The detection of IgG (Preferred position for Fig. 6) Fig. 6A described the principle of detection based on the basis of the model of Randles, which we have already discussed this principle in the title of 3.3. Under the optimal conditions, the responses of impedance immunosensor toward different IgG concentrations were recorded. Fig. 6B showed the Faradaic impedance spectra of the immunosensor. It was found that the diameter of the Nyquist circle increased with the the increasing concentration of IgG. This may be caused by more IgG bound to the electrode surface, which act as a kinetic barrier for the electron transfer. A linear relationship between the relative change of impedance and the IgG concentration was obtained in the range of 0.1 to 80 ng mL-1 with a detection limit of 0.07 ng mL-1 based on a signal-to-noise ratio of 3 (Fig. 6C). 3.6. Specificity, reproducibility and stability of immunosensor (Preferred position for Fig. 7) In order to confirm the binding specificity of the sensor for IgG detection, other interfering proteins, such as BSA, and IgM were chosen as reference substances. Fig. 7
Immunosensor based on a new polymer film 6 exhibited Ret of the proposed immunosensor incubated with 40 ng mL-1 IgG, the mixture of IgG and thrombin or IgM (the concentration of IgG, thrombin and IgM is 40 ng mL-1, 400 ng mL-1, 400 ng mL-1) under the same experimental conditions. As can be seen from Fig. 7, no significant decreasing was obtained after interfering proteins were added into analyte, which indicated that the developed strategy could be used to identify IgG with high specificity. The reproducibility of the immunosensor was investigated as well. A sample with a IgG concentration of 40 ng mL-1 was successively detected five times under the optimized conditions. Acceptable repeatability was observed with a relative standard deviation (R.S.D.) of 8.27%, which indicates that the proposed impedance immunosensor can be used for analysis with good reproducibility. Furthermore, after the electrode was stored at 4 oC for three weeks, the immunosensor retained 91.3% of its initial current for 40 ng mL-1 of IgG. This indicated that the immnnosensor possesses a good stability. 4. Conclusions In conclusion, we have described a new method to directly immobilize antibodies on modified poly (epichlorohydrin) film for convenient construction of an electrochemical impedance immunosensor. The proposed method can avoid the use of linkage agents such as EDC+NHS or glutaraldehyde and can improve the reproducibility and stability. Human IgG was used as a model analyte in this paper. The experimental results demonstrated that the electrochemical impedance immunosensor based on modified poly(epichlorohydrin) film possesses high sensitivity, good reproducibility and stability. This strategy may pave a simple way to fabricate an electrochemical immunosensor in other applications. Acknowledgments This work was supported by the construct program of the key discipline in Hunan province (Applied Chemistry), Hunan Provincial Natural Science Foundation of China (15JJ3094), Scientific Research Fund of Hunan Provincial Education Department (13C635) and Startup Foundation for Doctors of Hunan University of Arts and Science (10305005). 8
Immunosensor based on a new polymer film References [1] T. Yang, S. Wang, H. L. Jin, W. W. Bao, S. M. Huang, J. C. Wang, An electrochemical impedance sensor for the label-free ultrasensitive detection of interleukin-6 antigen, Sensors and Actuators B: Chemical 178 (2013)310-315. [2] R. Elshafey, A. C. Tavares, M. Siaj, M. Zourob, Electrochemical impedance immunosensor based on gold nanoparticles-protein G for the detection of cancer marker epidermal growth factor receptor in human plasma and brain tissue, Biosensors andBioelectronics 50 (2013) 143-149. [3] H. Wang, Y. L. Liu, Y. H. Yang, T. Deng, G. L. Shen, R. Q. Yu, A protein A-based orientation-controlled
immobilization
strategy
for
antibodies
using
nanometer-sized gold particles and plasma-polymerized film, Analytical Biochemistry 324 (2004) 219-226. [4] L. Tedeschi, C. Domenici, A. Ahluwalia, F. Baldini, A. Mencaglia, Antibody immobilisation on fibre optic TIRF sensors, Biosens. Bioelectron. 19 (2003) 85-93. [5] Y. Huang, X. Nie, S. Gan, J. Jiang, G. Shen, R. Yu, Electrochemical immunosensor of platelet-derived growth factor with aptamer-primed polymerase amplification, Anal. Biochem. 382 (2008) 16-22. [6] H. Dong, X. Cao, C.M. Li, W. Hu, An in situ electrochemical surface plasmon resonance immunosensor with polypyrrole propylic acid film: comparison between SPR and electrochemical responses from polymer formation to protein immunosensing, Biosens. Bioelectron. 23 (2008) 1055-1062. [7] S. Dulay, P. Lozano-Sanchez, E. Iwuoha, I. Katakis, C.K. O’Sullivan, Electrochemical detection of celiac disease-related anti-tissue transglutaminase antibodies using thiol based surface chemistry, Biosens. and Bioelectron.26 (2011)3852-3856. [8] H. Xu, K. Gorgy, C. Gondran, A. L. Goff, N. Spinelli, C. Lopez, E. Defrancq, S. Cosnier,
Label-free
impedimetric
thrombin
sensor
based
on
poly(pyrrole-nitrilotriacetic acid)-aptamer film, Biosensors and Bioelectronics 41 (2013) 90-95. 9
Immunosensor based on a new polymer film [9] W.-J. Jin, G.-J. Yang, H.-X. Shao, A.-J. Qin, A novel label-free impedimetric immunosensor
for detection of semicarbazide residue based on gold
nanoparticles-functional chitosan composite membrane, Sensors and Actuators B: Chemical 188 (2013) 271-279. [10] V. Escamilla-Gómez, S. Campuzano, M. Pedrero, J.M. Pingarrón, Immunosensor for
the
determination
of
Staphylococcus
aureus
using
a
tyrosinase-mercaptopropionic acid modified electrode as an amperometric transducer, Analytical and Bioanalytical Chemistry 391 ( 2008) 837-845. [11] K.J. Feng, Y. Kang, J.-J. Zhao, Y.-L. Liu, J.-H. Jiang, G.-L. Shen, R.-Q. Yu, Electrochemical immunosensor with aptamer-based enzymatic amplification, Analytical Biochemistry 378 (2008) 38-42. [12] P. Tiemblo, J. Guzmán, E. Riande, C. Mijangos, H. Reinecke, The gas transport properties
of
PVC
functionalized
with
mercapto
pyridine
groups,
Macromolecules 35 (2002) 420-424. [13] G. Lisa, E. Avram, G. Paduraru, M. Irimia, N. Hurduc, N.Aelenei, Thermal behaviour of polystyrene, polysulfone and their substituted derivatives, Polym. Degrad. Stabil. 82 (2003) 73-79. [14] E.-H. Sohn, J. Ahn, B. G. Kim, J.-C. Lee, Effect of n-alkyl and aulfonyl groups on the wetting properties of comblike poly(oxyethylene)s and stick-slip behavior, Langmuir 27(2011), 1811-1820. [15] R. Navarro, M. Pérez, G. Rodriguez, H. Reinecke, Selective nucleophilic substitution reactions on poly(epichlorohydrin) using aromatic and aliphatic thiol compounds, European Polymer Journal 43 (2007) 4516-4522. [16] J. C. Kim, J. Jung, Y. Rho, M. Kim, W. Kwon, H. Kim, I. J. Kim, J. R. Kim, M. Ree, Well-defined DNA-mimic brush polymers bearing adenine moieties: synthesis,
layer-by-layer
self-assembly,
and
biocompatibility
Biomacromolecules, 12 (2011) 2822-2833. [17] J. C. Kim, Y. Rho, G. Kim, M. Kim, H. Kim, I. J. Kim, J. R. Kimc, M. Ree, New self-assembled brush glycopolymers: synthesis, structure and properties, Polym. Chem. 4 (2013) 2260-2271. 10
Immunosensor based on a new polymer film [18] J. Jung, H. Kim, M. Ree, Self-assembly of novel lipid-mimicking brush polymers in nanoscale thin films, Soft Matter 10 (2014) 701-708 [19] G. A. Lindsay, M. J. Roberts, A. P. Chafin, R. A. Hollins, L. H. Merwin, J. D. Stenger-Smith, R. Y. Yee, P. Zarras, Ordered films by alternating polyelectrolyte deposition of cationic side chain and anionic main chain chromophoric polymers, Chem. Mater. 11 (1999) 924-929. [20] F. Patolsky, M. Zayats, B. Katz, I. Willner, Precipitation of an insoluble product on enzyme monolayer electrodes for biosensor applications: characterization by Faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance analyses. Anal. Chem. 71 (1999) 3171-3180.
11
Immunosensor based on a new polymer film Figure captions: Fig. 1. Schematic diagrams of preparation of the electrochemical immunosensor. Scheme 1 Synthetic route for polymer 1 Fig. 2. FTIR spectra of PECH (a) and polymer 1 (b). Fig. 3. Cyclic voltammetry profiles of the different modified electrodes: bare Au electrode (curve a), polymer 1/Au electrode (curve b), anti-IgG/ polymer 1/Au electrode (curve c), BSA/anti-IgG/ polymer 1/Au electrode (curve d), and IgG/BSA/anti-IgG/ polymer 1/Au electrode (curve e). All measurements were processed in 10 mM K3Fe(CN)6/K4Fe(CN)6. The concentration of antigen is 40 ng mL-1. Fig. 4. (A) Effect of the antibody concentration on Ret. (B) Effect of pH of the immunoreaction time on Ret. The concentration of antigen is 40 ng mL-1. Fig. 5. Effect of the immuno-reaction time on Ret. The concentration of antigen is 40 ng mL-1. Fig. 6. (A) The principle of detection human IgG. (B) Faradaic impendence spectra of the immunosensor to different concentrations of human IgG from a to f: 0.0, 0.1, 10.0, 40.0, 60.0, 80.0 ng mL-1. (C) Calibration curve of the immunosensor to different concentrations of human IgG. Fig. 7. Specificity of the immunosensor to IgG, thrombin+IgG, and IgM+IgG, respectivity. The concentrations of thrombin and IgM are 400 ng mL-1. The concentration of IgG is 40 ng mL-1.
12
Immunosensor based on a new polymer film
Fig. 1. Schematic diagrams of preparation of the electrochemical immunosensor.
Scheme 1 Synthetic route for polymer 1
Fig. 2. FTIR spectra of PECH (a) and polymer 1 (b).
13
Immunosensor based on a new polymer film
Fig. 3. Cyclic voltammetry profiles of the different modified electrodes: bare Au electrode (curve a), polymer 1/Au electrode (curve b), anti-IgG/ polymer 1/Au electrode (curve c), BSA/anti-IgG/ polymer 1/Au electrode (curve d), and IgG/BSA/anti-IgG/ polymer 1/Au electrode (curve e). All measurements were processed in 10 mM K3Fe(CN)6/K4Fe(CN)6. The concentration of antigen is 40 ng mL-1.
14
Immunosensor based on a new polymer film
Fig. 4. (A) Effect of the antibody concentration on Ret. (B) Effect of pH of the immunoreaction time on Ret. The concentration of antigen is 40 ng mL-1.
Fig. 5. Effect of the immuno-reaction time on Ret. The concentration of antigen is 40 ng mL-1.
15
Immunosensor based on a new polymer film
Fig. 6. (A) The principle of detection human IgG. (B) Faradaic impendence spectra of the immunosensor to different concentrations of human IgG from a to f: 0.0, 0.1, 10.0, 40.0, 60.0, 80.0 ng mL-1. (C) Calibration curve of the immunosensor to different concentrations of human IgG.
16
Immunosensor based on a new polymer film
Fig. 7. Specificity of the immunosensor to IgG, thrombin+IgG, and IgM+IgG, respectivity. The concentrations of thrombin and IgM are 400 ng mL-1. The concentration of IgG is 40 ng mL-1.
17
S0003-2697(15)00299-7 http://dx.doi.org/10.1016/j.ab.2015.06.007 YABIO 12105
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
20 March 2015 3 June 2015 4 June 2015
Please cite this article as: X. Zhang, G. Shen, Y. Shen, D. Yin, C. Zhang, Directly immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor, Analytical Biochemistry (2015), doi: http://dx.doi.org/10.1016/j.ab.2015.06.007
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 proof before it is published in its final 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.
Immunosensor based on a new polymer film Electrochemical methods
Directly immobilization of antibodies on a new polymer film for fabricating an electrochemical impedance immunosensor Xiangyang Zhang∗ , Guangyu Shen, Youming Shen*, Dan Yin, Chunxiang Zhang College of Chemistry and Chemical Engineering, Hunan University of Arts and Science, Changde 415000, PR China
* Corresponding author.
TEL.: +86 736 7186165 E-mail address:[email protected] (X. Zhang); [email protected] (Y. Shen)
1
Immunosensor based on a new polymer film Abstract A new polymer with aldehyde groups was designed and synthesized by use of poly -(epichlorohydrin) and 4-pyridinecarboxaldehyde. Antibodies can be directly immobilized on the surface of the polymer film through the covalent bonding of aldehyde groups of the film with amino groups of antibodies. In this study, human IgG was used as a model analyte to fabricate an electrochemical impedance immunosensor. Using the proposed immunosensor, we detected IgG within the range from 0.1 to 80 ng mL-1 with a detection limit of 0.07 ng mL-1 (S / N = 3). In addition, the
electrochemical
impedance
immunosensor
had
a
good
stability and
reproducibility. Keywords: new matrix; convenient immobilization method; electrochemical impedance immunosensor; Human immunoglobulin G
1. Introduction In the past few years, many bioanalytical methods for clinical diagnosis have experienced unprecedented growth. Owing to the advantages of high sensitivity, rapid detection, simple instrumentation, miniaturization and low cost, considerable efforts have been devoted to electrochemical impedance immunosensors for detecting protein [1, 2]. One of the most common issues faced in the development of electrochemical impedance immunosensors with good performance is the method utilized for immobilization of antibodies. The immobilization of antibodies on sensing surfaces can be carried out either by simply physical adsorption or chemical cross-linking. Although physical adsorption is the simplest and fastest way to immobilize antibodies, it is very sensitive to environmental conditions and has problems in stability [3, 4]. Compared
with
physical
adsorption,
chemical
cross-linking
method
for
immobilization antibodies could minimize the interference from the environmental and provide higher analytical accuracy [5]. Among chemical cross-linking method, various support matrixes with functional groups such as carboxylic acid and amine have been used to immobilize antibodies [6, 7]. Traditionally, those support matrixes for immobilization antibodies procedures usually involve three separate steps. The 2
Immunosensor based on a new polymer film first step is to creating a film through coating method. The second step involves the activation
functional
groups
with
the
mixture
of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [8, 9], or glutaraldehyde [10, 11]. The third step is the reaction between amine groups of protein and the linkage agents. However, these methods are trivial and time-consuming and will greatly influence the reproducibility of electrochemical impedance immunosensors. Therefore, it is important to develop a convenient immobilization method based on some new and easily prepared matrixes for the fabrication of electrochemical impedance immunosensors. Recently, chlorine-containing polymers such as poly(vinylchloride) (PVC) [12], poly(p-chloromethylstyrene) (PCMS) [13], and poly(epichlorohydrin) (PECH) [14] have been used as starting materials for synthesizing novel functional polymers with a wide range of applications. Among those polymers mentioned above, PECH has received considerable attention because they are biocompatibility, commercially available, very cheap, and chlorine atoms can be efficiently substituted by many nucleophilic reagents such as aliphatic carboxylates, sodium methoxide and series of substituted phenolates under mild conditions [15]. Moreover, PECH have been used for synthesizing a series of well-defined DNA-mimicking brush polymers [16], new self-assembled brush glycopolymer [17], novel lipid-mimicking brush polymers [18], and so on. However, to the best of our knowledge, PECH have not been used for synthesizing a kind of polymer bearing aldehyde groups. Polymer bearing aldehyde groups is one of the most popular matrixes in electrochemical immunosensors because they can directly immobilization of proteins. Despite general superiority offered by polymer with aldehyde groups, they cannot be obtained from chemical reagent company. Therefore, several attempts have been made to synthesis of polymer with aldehyde groups. Under such a research background, we begin to consider designed and synthesized a polymer with aldehyde groups by use of poly(epichlorohydrin) and immobilize protein on surface of the polymer film. In this study, we report a simple and rapid procedure for the immobilization of antibodies to fabricate an electrochemical impedance immunosensor for the detection of human IgG as model 3
Immunosensor based on a new polymer film biomolecules. This strategy can open a new door to broaden the potential applications of modified poly(epichlorohydrin) in clinical research. The whole process of electrochemical impedance immunosensor fabrication was shown in Fig. 1. (Preferred position for Fig. 1) 2. Experimental 2.1. Reagents and apparatus Poly (epichlorohydrin) (PECH, average Mw~700,000), 4-pyridinecarboxaldehyde, dimethylformamide (DMF) used in this work were of analytical grade and purchased from Sigma-Aldrich company. Human immunoglobulin G (IgG), goat anti-human immunoglobulin G antibody (Ab), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. 0.1 M phosphate buffer solution (PBS, pH 7.2) was prepared using Na2 HPO4 and NaH2PO4. All aqueous solutions were prepared with doubly distilled water. All electrochemical measurements, including cyclic voltammetric measurement (CV) and electrochemical impedance spectroscopy (EIS) were carried out with a CHI 660E
electrochemistry workstation (Shanghai CH
Instruments,
China).
A
conventional three-electrode cell, consisting of a Pt electrode as counter electrode, a saturated calomel electrode (SCE) as reference electrode, and a glassy carbon electrode (GCE) modified with modified poly(epichlorohydrin) film as working electrode was used. 2.2. Preparation of polymer 1 (Preferred position for scheme 1) As illustrated in Scheme 1, polymer 1 can be obtained according to the similar method reported in the literature [19]. Briefly, under the protection of argon, 1.24 g poly (epichlorohydrin) was dissolved in 20 mL anhydrous dimethylformamide (DMF) at 60 oC, and then 1.3 g 4-pyridinecarboxaldehyde was added. The mixture was stirred at 120 oC for 24 h. Most of the solvent was removed by constant rotation under vacuum. The crude polymer was dried at 50 oC in a vacuum overnight. The dried polymer was re-dissolved in dichloromethane and precipitated three times with ethanol. Finally a brown solid (0.5 g) was obtained after dying under vacuum at 50 oC for 24 h. 4
Immunosensor based on a new polymer film 2.3. Preparation of electrochemical immunosensor A GCE (3 mm in diameter) was polished repeatedly with 0.3 and 0.05 µm alumina slurries sequentially, followed by successive sonication in distilled water and ethanol, respectively. GCE was dried in air at room temperature and then 10 µL of 0.1% polymer 1 solution diluted in DMF was dropped on the electrode and dried in air at room temperature. Then, 10 µL of antibody solution (100 µg mL-1) was dropped on the modefied electrode and incubated for 1 h, followed by washing with ultrapure water to remove unspecific physically adsorption. In order to eliminate non-specific binding effect and block the remaining active sites, the electrode modified with antibody was incubated with 1.0 wt% BSA for 30 min at room temperature. The as-prepared immunosensor (designed as Ab/ polymer 1/GCE) was stored at 4 oC for further detection of IgG analyte. 2.4. Electrochemical measurements All
electrochemical
experiments
were
carried
out
in
a
conventional
electrochemical cell containing a three-electrode arrangement. CV and EIS measurements were performed in 10 mM K3Fe(CN)6/K4Fe(CN)6 solution. The CV measurements were taken at scanning rate 100 mVs -1 from -0.2 to 0.6 V relative to saturated calomel electrode. EIS measurements were carried out in the frequency range from 10 -1 to 105 Hz under an open potential. The amplitude of the alternative voltage was 5.0 mV. 3. Results and discussion 3.1. Characterization of polymer 1 (Preferred position for Fig. 2) To study the presence of aldehyde group, we measured IR-spectra of poly (epichlorohydrin) (PECH) and polymer 1 respectively. Compared with the spectrum of PECH, polymer 1 has an obvious aldehyde peak at 1730 cm−1 (Fig. 2). The result of spectrum showed that we have successfully synthesized the target product. 3.2. The electrochemical characterization of the modified electrodes (Preferred position for Fig. 3)
5
Immunosensor based on a new polymer film In order to evaluate the electron transfer property of the modified electrode, cyclic voltammograms were carried out using K3Fe(CN)6/K4Fe(CN)6 as a redox probe. Fig. 3 shows CVs of modified electrode in 10 mM K3Fe(CN)6/K4Fe(CN)6 solution at a scan rate of 100 mV s-1. Curve a is cyclic voltammograms of K3Fe(CN)6/K4Fe(CN)6 on the bare electrode, a couple of reversible redox peaks could be observed. When the electrode was stepwise modified with polymer 1 (curve b), antibody (curve c), BSA (curve d), and antigen (curve e), a decrease in peak current was observed, which indicates the decrease of peak current response corresponding to stepwise modifications consistent with the enhanced electron-transfer barriers introduced by these layers modified on the surface of the electrode. 3.3. The principle of detection The general electronic equivalent scheme is usually described on the basis of the model by Randles [20]. As shown in Fig. 6A, the circuit includes the ohmic resistance (Rs) of the electrolyte solution, the Warburg impedance (Zw) resulting from the diffusion of ions from the bulk electrolyte to the electrode interface, the double layer capacitance
(Cdl)
and
electron-transfer
resistance
(Ret).
Ret
controls
the
electron-transfer kinetics of the redox probe from the solution to the electrode interface. It is a suitable parameter for sensing the interfacial properties of the prepared immunosensor step by step. In Nyquist plot of Faradic impedance, the semicircle diameter of EIS equals the electro-transfer resistance (Ret). Ret increases when antigen is bound by antibody. Moreover, the relative change of the electron transfer resistance (%∆Ret) was monitored as a function of the antigen concentration. It is calculated by the following equation:
% ∆Ret = Ret(Ag − Ab ) − Ret(Ab ) × 100 Ret(Ab ) Where Ret (Ag–Ab) is the impedance of the electrode after antibody-antigen reaction, Ret (Ab) represents the impedance of the antibody-modified electrode blocked with BSA. 3.4. Optimization of experimental conditions 6
Immunosensor based on a new polymer film (Preferred position for Fig. 4) The amount of antibody immobilized on the electrode was optimized. As shown in Fig. 4A, when the antibody concentration was less than 100 µg mL−1, the Ret increased with the increasing of its concentration. Then the Ret reached a plateau at 100 µg mL−1. Therefore, a 100 µg mL-1 antibody concentration in PBS was chosen for this work. In addition, the effect of pH during the immuno-reaction was investigated from 6.0 to 8.0. As can be seen from Fig. 4B, the impedance increased with pH from 6.0 to 7.0, and then decreased from 7.0 to 8.5. Thus, phosphate buffer solution of pH 7.0 was selected as the buffer solution in the immuno-reaction. (Preferred position for Fig. 5) In addition, the incubation time of Ab-Ag was also investigated over the range from 20 to 80 min. Fig. 5 showed that the impedance increased with the incubation time increasing untill 40 min. Therefore, 40 min should be selected for the subsequent experiment. 3.5. The detection of IgG (Preferred position for Fig. 6) Fig. 6A described the principle of detection based on the basis of the model of Randles, which we have already discussed this principle in the title of 3.3. Under the optimal conditions, the responses of impedance immunosensor toward different IgG concentrations were recorded. Fig. 6B showed the Faradaic impedance spectra of the immunosensor. It was found that the diameter of the Nyquist circle increased with the the increasing concentration of IgG. This may be caused by more IgG bound to the electrode surface, which act as a kinetic barrier for the electron transfer. A linear relationship between the relative change of impedance and the IgG concentration was obtained in the range of 0.1 to 80 ng mL-1 with a detection limit of 0.07 ng mL-1 based on a signal-to-noise ratio of 3 (Fig. 6C). 3.6. Specificity, reproducibility and stability of immunosensor (Preferred position for Fig. 7) In order to confirm the binding specificity of the sensor for IgG detection, other interfering proteins, such as BSA, and IgM were chosen as reference substances. Fig. 7
Immunosensor based on a new polymer film 6 exhibited Ret of the proposed immunosensor incubated with 40 ng mL-1 IgG, the mixture of IgG and thrombin or IgM (the concentration of IgG, thrombin and IgM is 40 ng mL-1, 400 ng mL-1, 400 ng mL-1) under the same experimental conditions. As can be seen from Fig. 7, no significant decreasing was obtained after interfering proteins were added into analyte, which indicated that the developed strategy could be used to identify IgG with high specificity. The reproducibility of the immunosensor was investigated as well. A sample with a IgG concentration of 40 ng mL-1 was successively detected five times under the optimized conditions. Acceptable repeatability was observed with a relative standard deviation (R.S.D.) of 8.27%, which indicates that the proposed impedance immunosensor can be used for analysis with good reproducibility. Furthermore, after the electrode was stored at 4 oC for three weeks, the immunosensor retained 91.3% of its initial current for 40 ng mL-1 of IgG. This indicated that the immnnosensor possesses a good stability. 4. Conclusions In conclusion, we have described a new method to directly immobilize antibodies on modified poly (epichlorohydrin) film for convenient construction of an electrochemical impedance immunosensor. The proposed method can avoid the use of linkage agents such as EDC+NHS or glutaraldehyde and can improve the reproducibility and stability. Human IgG was used as a model analyte in this paper. The experimental results demonstrated that the electrochemical impedance immunosensor based on modified poly(epichlorohydrin) film possesses high sensitivity, good reproducibility and stability. This strategy may pave a simple way to fabricate an electrochemical immunosensor in other applications. Acknowledgments This work was supported by the construct program of the key discipline in Hunan province (Applied Chemistry), Hunan Provincial Natural Science Foundation of China (15JJ3094), Scientific Research Fund of Hunan Provincial Education Department (13C635) and Startup Foundation for Doctors of Hunan University of Arts and Science (10305005). 8
Immunosensor based on a new polymer film References [1] T. Yang, S. Wang, H. L. Jin, W. W. Bao, S. M. Huang, J. C. Wang, An electrochemical impedance sensor for the label-free ultrasensitive detection of interleukin-6 antigen, Sensors and Actuators B: Chemical 178 (2013)310-315. [2] R. Elshafey, A. C. Tavares, M. Siaj, M. Zourob, Electrochemical impedance immunosensor based on gold nanoparticles-protein G for the detection of cancer marker epidermal growth factor receptor in human plasma and brain tissue, Biosensors andBioelectronics 50 (2013) 143-149. [3] H. Wang, Y. L. Liu, Y. H. Yang, T. Deng, G. L. Shen, R. Q. Yu, A protein A-based orientation-controlled
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Immunosensor based on a new polymer film [9] W.-J. Jin, G.-J. Yang, H.-X. Shao, A.-J. Qin, A novel label-free impedimetric immunosensor
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Immunosensor based on a new polymer film [18] J. Jung, H. Kim, M. Ree, Self-assembly of novel lipid-mimicking brush polymers in nanoscale thin films, Soft Matter 10 (2014) 701-708 [19] G. A. Lindsay, M. J. Roberts, A. P. Chafin, R. A. Hollins, L. H. Merwin, J. D. Stenger-Smith, R. Y. Yee, P. Zarras, Ordered films by alternating polyelectrolyte deposition of cationic side chain and anionic main chain chromophoric polymers, Chem. Mater. 11 (1999) 924-929. [20] F. Patolsky, M. Zayats, B. Katz, I. Willner, Precipitation of an insoluble product on enzyme monolayer electrodes for biosensor applications: characterization by Faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance analyses. Anal. Chem. 71 (1999) 3171-3180.
11
Immunosensor based on a new polymer film Figure captions: Fig. 1. Schematic diagrams of preparation of the electrochemical immunosensor. Scheme 1 Synthetic route for polymer 1 Fig. 2. FTIR spectra of PECH (a) and polymer 1 (b). Fig. 3. Cyclic voltammetry profiles of the different modified electrodes: bare Au electrode (curve a), polymer 1/Au electrode (curve b), anti-IgG/ polymer 1/Au electrode (curve c), BSA/anti-IgG/ polymer 1/Au electrode (curve d), and IgG/BSA/anti-IgG/ polymer 1/Au electrode (curve e). All measurements were processed in 10 mM K3Fe(CN)6/K4Fe(CN)6. The concentration of antigen is 40 ng mL-1. Fig. 4. (A) Effect of the antibody concentration on Ret. (B) Effect of pH of the immunoreaction time on Ret. The concentration of antigen is 40 ng mL-1. Fig. 5. Effect of the immuno-reaction time on Ret. The concentration of antigen is 40 ng mL-1. Fig. 6. (A) The principle of detection human IgG. (B) Faradaic impendence spectra of the immunosensor to different concentrations of human IgG from a to f: 0.0, 0.1, 10.0, 40.0, 60.0, 80.0 ng mL-1. (C) Calibration curve of the immunosensor to different concentrations of human IgG. Fig. 7. Specificity of the immunosensor to IgG, thrombin+IgG, and IgM+IgG, respectivity. The concentrations of thrombin and IgM are 400 ng mL-1. The concentration of IgG is 40 ng mL-1.
12
Immunosensor based on a new polymer film
Fig. 1. Schematic diagrams of preparation of the electrochemical immunosensor.
Scheme 1 Synthetic route for polymer 1
Fig. 2. FTIR spectra of PECH (a) and polymer 1 (b).
13
Immunosensor based on a new polymer film
Fig. 3. Cyclic voltammetry profiles of the different modified electrodes: bare Au electrode (curve a), polymer 1/Au electrode (curve b), anti-IgG/ polymer 1/Au electrode (curve c), BSA/anti-IgG/ polymer 1/Au electrode (curve d), and IgG/BSA/anti-IgG/ polymer 1/Au electrode (curve e). All measurements were processed in 10 mM K3Fe(CN)6/K4Fe(CN)6. The concentration of antigen is 40 ng mL-1.
14
Immunosensor based on a new polymer film
Fig. 4. (A) Effect of the antibody concentration on Ret. (B) Effect of pH of the immunoreaction time on Ret. The concentration of antigen is 40 ng mL-1.
Fig. 5. Effect of the immuno-reaction time on Ret. The concentration of antigen is 40 ng mL-1.
15
Immunosensor based on a new polymer film
Fig. 6. (A) The principle of detection human IgG. (B) Faradaic impendence spectra of the immunosensor to different concentrations of human IgG from a to f: 0.0, 0.1, 10.0, 40.0, 60.0, 80.0 ng mL-1. (C) Calibration curve of the immunosensor to different concentrations of human IgG.
16
Immunosensor based on a new polymer film
Fig. 7. Specificity of the immunosensor to IgG, thrombin+IgG, and IgM+IgG, respectivity. The concentrations of thrombin and IgM are 400 ng mL-1. The concentration of IgG is 40 ng mL-1.
17