- Email: [email protected]
Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor
Accepted Manuscript Title: Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor Author: Xiangyang Zhang Guangyu Shen Shouyuan Sun Youming Shen Chunxiang Zhang Anguo Xiao PII: DOI: Reference:
S0925-4005(14)00431-6 http://dx.doi.org/doi:10.1016/j.snb.2014.04.030 SNB 16796
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
28-2-2014 9-4-2014 10-4-2014
Please cite this article as: X. Zhang, G. Shen, S. Sun, Y. Shen, C. Zhang, A. Xiao, Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.04.030 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.
Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor Xiangyang Zhanga∗, Guangyu Shena∗, Shouyuan Suna, Youming Shena, Chunxiang Zhanga, Anguo
a
ip t
Xiaoa
College of Chemistry and Chemical Engineering, Hunan University of Arts and Science,
cr
Changde 415000, PR China
us
Abstract
Antibodies can be directly immobilized on the surface of dialdehyde cellulose film
an
through the covalent bonding of the aldehyde groups of the dialdehyde cellulose with amino groups of antibodies. In this work, human IgG was used as a model analyte to
M
fabricate an electrochemical immunosensor. Using the proposed immunosensor, we detected IgG within the range from 0.5 to 45 ng/mL with a detection limit of
d
0.3 ng/mL (S/N = 3). The electrochemical immunosensor had a good specificity, stability and reproducibility. This strategy may pave a simple way to fabricate an
Ac ce p
te
electrochemical immunosensor in other applications.
Keywords: Dialdehyde cellulose film; Antibody immobilization; Electrochemical immunosensor; Human immunoglobulin G
1. Introduction
Recently, many techniques such as fluorescence [1, 2], chemiluminescence [3, 4],
quartz crystal microbalance [5, 6], surface plasmon resonance [7, 8], lateral flow [9], and electrochemistry [10, 11] have been developed for immunoassay. Among these techniques mentioned above, electrochemistry methods have received considerable attention for immunoassay due to their high sensitivity, inherent simplicity, rapid detection,
miniaturization and low cost. Electrochemical
E-mail address:[email protected]
1
Page 1 of 15
immunosensors are based on specific recognition between antigen and antibody. One of the most common issues faced in the development of electrochemical immunosensors with good performance is the method utilized for the immobilization of antibodies. The immobilization of antibodies on sensing surfaces can be carried out
ip t
by simply physical adsorption, chemical cross-linking, entrapment in conduction
polymer, binding to gold-thiol monolayers, utilizing avidin-biotin affinity methods
cr
and other methods. Physical adsorption is very sensitive to environmental conditions
and has problems in stability [12, 13]. In contrast to physical adsorption, covalent
us
binding method for immobilization is preferred. Chemical cross-linking is accomplished by derivatizing the surfaces using crosslinkers to covalently attach the
an
antibodies to the surface of immunosenser. Among chemical cross-linking method, various kinds of materials with functional groups (carboxylic acid, amine etc.) have
M
been used to immobilize antibodies [14, 15]. The immobilization procedure usually involves three separate steps. The first one is to creating a film containing functional
functional
groups
with
d
groups (carboxylic acid, amine etc.). The second step involves the activation the
mixture
of
1-ethyl-3-(3-dimethylaminopropyl)
te
-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [16, 17] or glutaraldehyde
Ac ce p
[18, 19]. The third step involves the reaction of the recognition element with the linkage agents. Those immobilization procedures work well but really cumbersome and time-consuming in practice. Moreover, another immobilization strategy mentioned above for immobilization of antibodies [20-22] suffer from some drawbacks, such as easily losing antibody, reducing antibody activity, fussy operation, and so on. Thus, it is still important to develop a convenient immobilization method and new matrixes for the fabrication of electrochemical immunosensors. Cellulose (CL) is consisting of aligned thread-like bundles of poly-β-(1, 4)-D-glucose molecules in extended-chain conformation. It can be further modified by various chemical derivatizations such as esterification [23], silylation [24] and oxidation [25] resulting in functionalized microfibers for various applications. In the past few years, cellulose derivatives have been widely used as carriers for immobilization of biomolecules including enzyme, cell and bacteria due to their good 2
Page 2 of 15
biocompatibility and stability [26-29]. However, to the best of our knowledge, there are few applications and studies of dialdehyde cellulose (DAC) in electrochemical immunosensors up to now. The properties of dialdehyde cellulose film inspired us to immobilize proteins on the electrode surface. Here, we report a simple and rapid
ip t
procedure for the immobilization of antibodies to fabricate an electrochemical
immunosensor. In addition, goat-anti human IgG antibody was used as model
cr
biomolecules. This strategy can open a new door to broaden the potential applications of dialdehyde cellulose in clinical research. The whole process of immunosensor
d
M
an
us
fabrication was shown in Fig. 1.
2. Experimental
te
Fig. 1. Schematic diagrams of preparation of the electrochemical immunosensor.
Ac ce p
2.1. Reagents and apparatus
Microcrystalline cellulose powder (particle size 90 μm), sodium metaperiodate,
sodium hydroxide and concentrated sulfuric acid used in this work were of analytical grade and purchased from Sinopharm chemical reagent 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 Na2HPO4 and NaH2PO4. All
aqueous solutions were prepared with doubly distilled water. All electrochemical measurements, including electrochemical impedance (EI) and differential pulse voltammetry (DPV) 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) 3
Page 3 of 15
modified with dialdehyde cellulose film as working electrode was used. IR spectra were recorded on Nicolet FT-170SX instrument using KBr discs in the 400-4000 cm-1 region. 2.2. Preparation of dialdehyde cellulose (DAC) CH 2OH H OH
O O
H
H
NaIO4
OH
H H
H H
ip t
CH 2OH
O H
O
H H
n
O
O
n
cr
DAC
Scheme 1 Synthetic route of dialdehyde cellulose
us
Microcrystalline cellulose powder was active by sodium hydroxide before use. An experimental procedure was performed as follows: 10 g of microcrystalline cellulose
an
was added to 100 mL of 14 wt% sodium hydroxide solution, soaking 24 h, and then filtered. The resin was washed with distilled water until neutral and dried under
M
vacuum at 50 oC for 24 h.
To 10 g of sodium metaperiodate in 100 mL of water, concentrated sulfuric acid was added until the pH value reaches 1.0. And then 5 g of cellulose suspended was
d
added. The mixture was stirred at 37 oC in the dark for 4 h. After the remaining
te
periodate was decomposed by adding of excess ethylene glycol, the product was washed in deionized water several times until the pH of the washing medium was
Ac ce p
equal to that of pure water. Finally the product was dried under vacuum at 50 oC for 24 h.
2.3. Determination of aldehyde content of DAC The aldehyde content of the DAC was determined by an oxime reaction [30]. The
suspension of dialdehyde cellulose, both 0.1 g solid in 30 mL water, was adjusted to pH 4.5 with HCl. Hydroxylamine hydrochloride solution (0.43 g in 20 mL water, adjusted to pH 4.5) was added to the DAC suspension solution. The mixture was stirred at room temperature for 24 h. The conversion of aldehyde to oxime was determined by recording the consumption of 0.1 M NaOH, performing the reaction at a constant pH of 4.5. Moreover, the same quality of DAC was carried out a blank titration. The result shows that the number of aldehyde groups containing in 100
glucose units is 81. 4
Page 4 of 15
2.4. Preparation of the electrochemical immunosensor DAC was added to dry DMF solution and under the protection of argon, the solution was stirred at 80oC for 2 h. Then, the solution was cooling down to room temperature and getting a true solution of DAC. A GCE (3 mm in diameter) was
ip t
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
cr
air at room temperature and then 10 μL of 0.1% DAC solution diluted in DMF was dropped on the electrode and dried in air at room temperature for 5h. Then, 10 μL of
us
antibody solution (150 μg/mL) was dropped on the modified electrode and incubated for 1 h, followed by washing with ultrapure water to remove unspecific physically
an
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
M
for 30 min at room temperature. The as-prepared immunosensor (designed as Ab/DAC/GCE) was stored at 4 oC for further detection of IgG analyte.
All
electrochemical
d
2.5. Electrochemical measurements
experiments
were
carried
out
in
a
conventional
te
electrochemical cell containing a three-electrode arrangement. EI and DPV
Ac ce p
measurements were performed in 10 mM K3Fe(CN)6/K4Fe(CN)6 solution. The DPV measurements were taken under the following: the potential range was from −0.6 to 0.1 V, pulse amplitude was 0.05 V, pulse width was 0.05 s, and sample width was 0.02 s.
3. Results and discussion
3.1. Characterization of dialdehyde cellulose
5
Page 5 of 15
ip t cr
us
Fig. 2. FTIR spectra of DAC (a) and microcrystalline cellulose powder (b). To study the presence of aldehyde group, we measured IR-spectra of cellulose
an
powder and DAC respectively. The spectrum showed typical characteristics of DAC with an aldehyde peak at 1721 cm−1 (Fig. 2). Owing to the aldehyde group of
M
dialdehyde cellulose rarely exists in the free form and hemiacetal linkages being
Ac ce p
te
d
formed, aldehyde peak of DAC was much smaller than expected [31].
6
Page 6 of 15
Fig. 3. SEM of cellulose powder,δ= 50 µm (A), SEM of cellulose powder active by sodium hydroxide solution,δ= 10 µm (B), SEM of dialdehyde cellulose powder, δ = 50 µm (C) and SEM of dialdehyde cellulose powder, δ = 5 µm (D).
ip t
Fig.3A shows the SEM of cellulose powder. It is seen that the cellulose is in the form of fairly long fibres, but the fibrous form of cellulose is not retained after treated
cr
by sodium hydroxide solution and there is a large crack (Fig.3B). This form of cellulose with large crack is a good for penetration, spread and swelling of the
us
reagents. Additionally, from the SEM images of DAC powder (Fig.3C, Fig.3D), it is clear that the form of DAC is microporous and the specific surface area is large. The
an
specific surface area indicate that DAC can be easily formed a film and fixed more antibodies.
Ac ce p
te
d
M
3.2. The electrochemical characterization of the modified electrodes
Fig. 4. Nyquist plot of Faradic impedance obtained in 10 mM K3Fe(CN)6/K4Fe(CN)6 for bare GCE (a), DAC-modified GCE (b), Ab-DAC-modified GCE (c), BSA-Ab-DAC-modified GCE (d) and IgG-BSA-Ab-DAC-modified GCE (e). The concentration of IgG is 30 ng/mL. Electrochemical impedance was used to characterize the interfacial properties of
surface-modified electrodes. Generally, the semicircle diameter of the Nyquist plot of electrochemical impedance spectroscopy represents the electron-transfer resistance [32]. Fig. 4 shows the electrochemical impedance spectroscopy for the modified gold electrode in 10 mM K3Fe (CN)6/K4Fe(CN)6 solution. As can be seen from Fig. 4, the electrochemical impedance spectroscopy of the bare gold electrode had a small 7
Page 7 of 15
semicircle diameter (curve a), implying low resistance to the redox probe dissolved in electrolyte solution. After GCE was modified with DAC film (curve b), the semicircle diameter of the electrochemical impedance spectroscopy was turned to be larger than that of the bare GCE. When the electrode was further modified with antibodies (curve
ip t
c), BSA (curve d) and antigens (curve e), the semicircle diameter increased step by
step. The results were consistent with the enhanced electron-transfer barriers
M
an
us
cr
introduced of these layers.
Fig. 5. DPVs of the different electrode obtained in 10 mM K3Fe(CN)6/K4Fe(CN)6 for
d
bare GCE (a), DAC-modified GCE (b), Ab-DAC-modified GCE (c) and
te
IgG-BSA-Ab-DAC-modified GCE (e). The concentration of IgG is 30 ng/mL. DPV was also used for monitoring the process of the modification of electrode. As
Ac ce p
can be seen from Fig. 5, the bare GCE (curve a) had larger DPV current than the DAC/GCE (curve b). A current decrease appeared after antibodies were immobilized on the surface of DAC/GCE (curve c). When antigens were immobilized on the surface of the electrode modified with antibodies, the peak current further decreased (curve d). This indicates that the peak current decreased is consistent with the increasing of the electron-transfer blocking layers. 3.3. Optimization of experimental conditions
8
Page 8 of 15
ip t cr
Fig. 6. (A) Effect of antibody concentration on the peak current response of the
us
immunosensor. (B) The influence of reaction time of antibody with DAC on the peak current response of the immunosensor to 30 ng/mL IgG in 10 mM
an
K3Fe(CN)6/K4Fe(CN)6
The effect of the concentration of antibody on the response of the immunosensor
M
was investigated. When it changes from 50 to 200 μg/mL, experimental data show that the peak current decreases with the increasing antibody concentration till the peak current reach a plateau at 150 μg/mL (Fig. 6A). Therefore, a 150 μg/mL antibody
d
solution in PBS was chosen for this work.
te
The influence of reaction time of antibody with DAC on the performance of the immunosensor was also investigated over the range from 20 to 80 min. At room
Ac ce p
temperature, the DPV response of the immunosensor decreased with the immobilization time up to 60 min (Fig. 6B), Therefore, a reaction time of 60 min was selected for this work.
3.4. The detection of thrombin based on DPV Under the optimal conditions, the responses of the DAC-based immunosensor
toward different IgG concentrations were recorded. From Fig. 7A, we could see that the peak current gradually decreased with the increasing concentration of IgG. The dose response curve of the proposed immunosensor for IgG showed a linear range from 0.5 to 45 ng/mL (Fig. 7B) with a detection limit of 0.3 ng/mL based on a signal-to-noise ratio of 3.
9
Page 9 of 15
ip t cr
Fig. 7. (A) DPVs of the immunosensor for the detection of different concentrations of
IgG. Error bars represent standard deviation, n=3.
us
IgG. (B) The linear relationship between the peak current and the concentration of
te
d
M
an
3.5. Specificity, reproducibility and stability of the immunosensor
Fig. 8. Specificity of the immunosensor to IgG, IgG+BSA, IgG+HSA, respectivity.
Ac ce p
The concentration of IgG, BSA and HSA is 30 ng/mL, 3 μg/mL, 3 μg/mL. Error bars represent standard deviation, n=3. In order to confirm the binding specificity of the sensor for IgG detection, other
interfering proteins, such as BSA and HSA were chosen as reference substances. Fig. 8 exhibited peak current signals of the proposed immunosensor incubated with 30 ng/mL IgG, the mixture of IgG and BSA or HSA (the concentration of IgG, BSA and HSA is 30 ng/mL, 3 μg/mL, 3 μg/mL) under the same experimental conditions. As can be seen From Fig. 8, 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 30 ng/mL was successively detected 5 times under the optimized
10
Page 10 of 15
conditions. Acceptable repeatability was observed with a relative standard deviation (R.S.D.) of 7.13%, which indicates that the proposed antibody sensor can be used for analysis with good reproducibility. Furthermore, after the electrode was stored at 4 for three weeks, the immunosensor retained 90.6% of its initial current for 30 ng/mL
ip t
of IgG. This indicated that the immnnosensor possesses a good stability. 4. Conclusions
cr
In this report, we have described a new method to directly immobilize antibodies
on dialdehyde cellulose film for the fabrication of an electrochemical immunosensor.
us
The proposed method can avoid the use of linkage agents such as EDC+NHS or glutaraldehyde. Human IgG was used as a model analyte in this paper. The
an
experimental results demonstrated that the electrochemical immunosensor based on DAC film possesses high sensitivity, good reproducibility and stability. It has a huge
M
potential in immobilization of different biomolecules intended for diverse applications in areas like diagnostics, affinity chromatography and in enzyme based industries.
d
Acknowledgments
This work was supported by the construct program of the key discipline in Hunan
te
province (Applied Chemistry), Scientific Research Fund of Hunan Provincial
Ac ce p
Education Department (13C635) and Startup Foundation for Doctors of Hunan University of Arts and Science. References
[1] H.L. Guo, X.H. Zhou, Y. Zhang, B.D. Song, L.H. Liu, J.X. Zhang, H.C. Shi, Highly sensitive and rapid detection of melamine in milk products by planar waveguide fluorescence immunosensor (PWFI) ， Sensors and Actuators B:
Chemical 194 (2014) 114-119.
[2] H.A. Engström, P.O. Andersson, S. Ohlson, A label-free continuous total-internal -reflection-fluorescence-based immunosensor, Analytical Biochemistry 357 (2006) 159-166. [3] L.D. Wu, M. Li, M. Zhang, M. Yan, S.G. Ge, J.H. Yu, Ultrasensitive electrochemiluminescence immunosensor for tumor marker detection based on nanoporous sliver@carbon dots as labels, Sensors and Actuators B: Chemical 186 11
Page 11 of 15
(2013)761-767. [4] S.R. Jain, E. Borowska, R. Davidsson, M. Tudorache, E. Pontén, J. Emnéus, A chemiluminescence flow immunosensor based on a porous monolithic metacrylate and polyethylene composite disc modified with protein G, Biosens Bioelectron 19
ip t
(2004) 795-803.
[5] C. Crosson, C. Rossi, Quartz crystal microbalance immunosensor for the
cr
quantification of immunoglobulin G in bovinemilk, Biosensors and Bioelectronics 42 (2013) 453-459.
us
[6] M. Salmain, M. Ghasemi, S. Boujday, C.-M. Pradier, Elaboration of a reusable immunosensor for the detection of staphylococcal enterotoxin A (SEA) in milk
an
with a quartz crystal microbalance, Sensors and Actuators B: Chemical 173 (2012)148-156.
M
[7] H. Dong, X. Cao, C.M. Li, W. Hu, An in situ electrochemical surface plasmon resonance immunosensor with polypyrrole propylic acid film: comparison
d
between SPR and electrochemical responses from polymer formation to protein immunosensing, Biosens Bioelectron 23 (2008) 1055-1062.
te
[8] J.Y. Jyoung, S. Hong, W. Lee, J.W. Choi, Immunosensor for the detection of
Ac ce p
Vibrio cholerae O1 using surface plasmon resonance, Biosens Bioelectron 21 (2006) 2315-2319.
[9] A. Marzo, J. Pons, D.A. Blake, A. Merkoçi, High sensitive gold-nanoparticle based lateral flow Immunodevice for Cd2+ detection in drinking waters, Biosens Bioelectron., 47 (2013) 190-198.
[10] X. Sun, Z. Ma, Electrochemical immunosensor based on nanoporpus gold loading thionine for carcinoembryonic antigen, Analytica Chimica Acta, 780 (2013) 95-100. [11] Y. Xie, A. Chen, D. Du, Y. Lin, Graphene-based immunosensor for electro -chemical quantification of phosphorylated p53 (S15), Analytica Chimica Acta, 699 (2011) 44-48. [12] 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
12
Page 12 of 15
nanometer-sized gold particles and plasma-polymerized film, Analytical Biochemistry 324 (2004) 219-226. [13] L. Tedeschi, C. Domenici, A. Ahluwalia, F. Baldini, A. Mencaglia, Antibody immobilisation on fibre optic TIRF sensors, Biosens Bioelectron 19 (2003)
ip t
85-93.
[14] S. Dulay, P. Lozano-Sanchez, E. Iwuoha, I. Katakis, C.K. O’Sullivan,
cr
Electrochemical detection of celiac disease-related anti-tissue transglutaminase
26 (2011)3852-3856.
us
antibodies using thiol based surface chemistry, Biosensors and Bioelectronics
[15] Y. Huang, X.-M. Nie, S.-L. Gan, J. Jiang, G.-L. Shen, R.-Q. Yu, Electrochemical of
platelet-derived
growth
factor
an
immunosensor
with
aptamer-primed
polymerase amplification, Analytical Biochemistry 382 (2008) 16-22.
M
[16] R. Genc, D. Murphy, A. Fragoso, M. Ortiz, C.K. O’Sullivan, Signal-enhancing thermosensitive liposomes for highly sensitive immunosensor development,
d
Analytical Chemistry 83 (2011) 563-570.
[17] H.M. Nassef, M.C. Bermudo Redondo, P.J. Ciclitira, H.J. Ellis, A. Fragoso, C.K.
te
O’Sullivan, Electrochemical immunosensor for detection of celiac disease toxic
Ac ce p
gliadin in foodstuff, Analytical Chemistry 80 (2008) 9265-9271. [18]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.
[19] 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. [20] S. Cosnier, Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review, Biosensors and Bioelectronics 14 (1999) 443-456. [21] H.X. Chen, F. Liu, F.J. Qi, K. Koh, K.M. Wang, Fabrication of calix[4]arene derivative monolayers to control orientation of antibody immobilization,
13
Page 13 of 15
International Journal of Molecular Sciences 15 (2014) 5496-5507. [22] E. Zaccoa, R. Galvea, M.P. Marcob, S. Alegret, M.I. Pividoria, Electrochemical biosensing of pesticide residues based
on affinity biocomposite platforms,
Biosensors and Bioelectronics 22 (2007) 1707-1715.
ip t
[23] S. Berlioz, S. Molina-Boisseau, Y. Nishiyama, L. Heux, Gas-phase surface esterification of cellulose microfibrils and whiskers, Biomacromolecules 10
cr
(2009) 2144-2151.
[24] M. Andresen, L.-S. Johansson, B. S. Tanem, P. Stenius, Properties and
us
characterization of hydrophobized microfibrillated cellulose, Cellulose 13 (2006) 665-677.
an
[25] T. Saito, Y. Nishiyama, J.-L. Putaux, M. Vignon, A. Isogai, Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of
M
native cellulose, Biomacromolecules 7 (2006) 1687-1691. [26] V. Vamvakaki, K. Tsagaraki, N. Chaniotakis, Carbon nanofiber-based glucose
d
biosensor, Analytical Chemistry 78 (2006) 5538-5542. [27] P. Paul, C. Seok-Youl, S. Bongsup, L. Jungwoo, C. Meghan, A.K. Nicholas,
te
Molecularly engineered nanocomposites: layer-by-layer assembly of cellulose
Ac ce p
nanocrystals, Biomacromolecules 6 (2005) 2914-2918. [28] M.M. de Souza Lima, R. Borsali, Rodlike cellulose microcrystals: structure, properties, and applications, Macromolecular Rapid Communications 25 (2004) 771-787.
[29] R. Sternberg, D.S. Bindra, G.S. Wilson, D.R. Thevenot, Covalent enzyme coupling on cellulose acetate membranes for glucose sensor development, Analytical Chemistry 60 (1988) 2781-2786.
[30] U.-J. Kim, M. Wada, S. Kuga, Solubilization of dialdehyde cellulose by hot water, Carbohydrate Polymers 56 (2004) 7-10. [31] A. J. Varmat, V. B. Chavan, A study of crystallinity changes in oxidized celluloses, Polymer Degradation and Stability 49 (1995) 245-250.
14
Page 14 of 15
[32] X.M. Ren, P.G. Pickup, An impedance study of electron transport and electron transfer in composite polypyrrole + polystyrenesulphonate films, Journal of Electroanalytical Chemistry 420 (1997) 251-257.
ip t
Biographies
Xiangyang Zhang received his Ph.D. in organic chemistry in 2011 at Chengdu
cr
Institute of Organic Chemistry, Chinese Academy of Science, China. He is currently working as a lecturer at Hunan University of Arts and Science. His research interests
us
are organic functional materials and their applications in sensor.
Guangyu Shen received her Ph.D. in analytical chemistry at Hunan University, China.
an
She is currently working as an associate professor at Hunan University of Arts and Science. Her current research interest is focused on electrochemical sensors.
M
Shouyuan Sun is currently a student majoring in applied chemistry. He is working for his bachelor in Hunan University of Arts and Science., China. His research interests
d
are the synthesis of functional materials.
Youming Shen received his Ph.D. in organic chemistry in 2009 at Hunan University,
te
China. He is currently working as a lecturer at Hunan University of Arts and Science.
Ac ce p
His research interests are functional nanomaterials and their applications in sensor. Chunxiang Zhang received her master's degree in organic chemistry from Hunan University, China. She is currently working as a teaching assistant at Hunan University of Arts and Science. Her current research interests focus on biosensors. Anguo Xiao received his Ph.D. in polymer chemistry and physics from Zhejiang University, China. He is currently working as an associate professor at Hunan University of Arts and Science. His current research interests focus on the synthesis of functional polymer materials.
15
Page 15 of 15
S0925-4005(14)00431-6 http://dx.doi.org/doi:10.1016/j.snb.2014.04.030 SNB 16796
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
28-2-2014 9-4-2014 10-4-2014
Please cite this article as: X. Zhang, G. Shen, S. Sun, Y. Shen, C. Zhang, A. Xiao, Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.04.030 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.
Direct immobilization of antibodies on dialdehyde cellulose film for convenient construction of an electrochemical immunosensor Xiangyang Zhanga∗, Guangyu Shena∗, Shouyuan Suna, Youming Shena, Chunxiang Zhanga, Anguo
a
ip t
Xiaoa
College of Chemistry and Chemical Engineering, Hunan University of Arts and Science,
cr
Changde 415000, PR China
us
Abstract
Antibodies can be directly immobilized on the surface of dialdehyde cellulose film
an
through the covalent bonding of the aldehyde groups of the dialdehyde cellulose with amino groups of antibodies. In this work, human IgG was used as a model analyte to
M
fabricate an electrochemical immunosensor. Using the proposed immunosensor, we detected IgG within the range from 0.5 to 45 ng/mL with a detection limit of
d
0.3 ng/mL (S/N = 3). The electrochemical immunosensor had a good specificity, stability and reproducibility. This strategy may pave a simple way to fabricate an
Ac ce p
te
electrochemical immunosensor in other applications.
Keywords: Dialdehyde cellulose film; Antibody immobilization; Electrochemical immunosensor; Human immunoglobulin G
1. Introduction
Recently, many techniques such as fluorescence [1, 2], chemiluminescence [3, 4],
quartz crystal microbalance [5, 6], surface plasmon resonance [7, 8], lateral flow [9], and electrochemistry [10, 11] have been developed for immunoassay. Among these techniques mentioned above, electrochemistry methods have received considerable attention for immunoassay due to their high sensitivity, inherent simplicity, rapid detection,
miniaturization and low cost. Electrochemical
E-mail address:[email protected]
1
Page 1 of 15
immunosensors are based on specific recognition between antigen and antibody. One of the most common issues faced in the development of electrochemical immunosensors with good performance is the method utilized for the immobilization of antibodies. The immobilization of antibodies on sensing surfaces can be carried out
ip t
by simply physical adsorption, chemical cross-linking, entrapment in conduction
polymer, binding to gold-thiol monolayers, utilizing avidin-biotin affinity methods
cr
and other methods. Physical adsorption is very sensitive to environmental conditions
and has problems in stability [12, 13]. In contrast to physical adsorption, covalent
us
binding method for immobilization is preferred. Chemical cross-linking is accomplished by derivatizing the surfaces using crosslinkers to covalently attach the
an
antibodies to the surface of immunosenser. Among chemical cross-linking method, various kinds of materials with functional groups (carboxylic acid, amine etc.) have
M
been used to immobilize antibodies [14, 15]. The immobilization procedure usually involves three separate steps. The first one is to creating a film containing functional
functional
groups
with
d
groups (carboxylic acid, amine etc.). The second step involves the activation the
mixture
of
1-ethyl-3-(3-dimethylaminopropyl)
te
-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) [16, 17] or glutaraldehyde
Ac ce p
[18, 19]. The third step involves the reaction of the recognition element with the linkage agents. Those immobilization procedures work well but really cumbersome and time-consuming in practice. Moreover, another immobilization strategy mentioned above for immobilization of antibodies [20-22] suffer from some drawbacks, such as easily losing antibody, reducing antibody activity, fussy operation, and so on. Thus, it is still important to develop a convenient immobilization method and new matrixes for the fabrication of electrochemical immunosensors. Cellulose (CL) is consisting of aligned thread-like bundles of poly-β-(1, 4)-D-glucose molecules in extended-chain conformation. It can be further modified by various chemical derivatizations such as esterification [23], silylation [24] and oxidation [25] resulting in functionalized microfibers for various applications. In the past few years, cellulose derivatives have been widely used as carriers for immobilization of biomolecules including enzyme, cell and bacteria due to their good 2
Page 2 of 15
biocompatibility and stability [26-29]. However, to the best of our knowledge, there are few applications and studies of dialdehyde cellulose (DAC) in electrochemical immunosensors up to now. The properties of dialdehyde cellulose film inspired us to immobilize proteins on the electrode surface. Here, we report a simple and rapid
ip t
procedure for the immobilization of antibodies to fabricate an electrochemical
immunosensor. In addition, goat-anti human IgG antibody was used as model
cr
biomolecules. This strategy can open a new door to broaden the potential applications of dialdehyde cellulose in clinical research. The whole process of immunosensor
d
M
an
us
fabrication was shown in Fig. 1.
2. Experimental
te
Fig. 1. Schematic diagrams of preparation of the electrochemical immunosensor.
Ac ce p
2.1. Reagents and apparatus
Microcrystalline cellulose powder (particle size 90 μm), sodium metaperiodate,
sodium hydroxide and concentrated sulfuric acid used in this work were of analytical grade and purchased from Sinopharm chemical reagent 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 Na2HPO4 and NaH2PO4. All
aqueous solutions were prepared with doubly distilled water. All electrochemical measurements, including electrochemical impedance (EI) and differential pulse voltammetry (DPV) 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) 3
Page 3 of 15
modified with dialdehyde cellulose film as working electrode was used. IR spectra were recorded on Nicolet FT-170SX instrument using KBr discs in the 400-4000 cm-1 region. 2.2. Preparation of dialdehyde cellulose (DAC) CH 2OH H OH
O O
H
H
NaIO4
OH
H H
H H
ip t
CH 2OH
O H
O
H H
n
O
O
n
cr
DAC
Scheme 1 Synthetic route of dialdehyde cellulose
us
Microcrystalline cellulose powder was active by sodium hydroxide before use. An experimental procedure was performed as follows: 10 g of microcrystalline cellulose
an
was added to 100 mL of 14 wt% sodium hydroxide solution, soaking 24 h, and then filtered. The resin was washed with distilled water until neutral and dried under
M
vacuum at 50 oC for 24 h.
To 10 g of sodium metaperiodate in 100 mL of water, concentrated sulfuric acid was added until the pH value reaches 1.0. And then 5 g of cellulose suspended was
d
added. The mixture was stirred at 37 oC in the dark for 4 h. After the remaining
te
periodate was decomposed by adding of excess ethylene glycol, the product was washed in deionized water several times until the pH of the washing medium was
Ac ce p
equal to that of pure water. Finally the product was dried under vacuum at 50 oC for 24 h.
2.3. Determination of aldehyde content of DAC The aldehyde content of the DAC was determined by an oxime reaction [30]. The
suspension of dialdehyde cellulose, both 0.1 g solid in 30 mL water, was adjusted to pH 4.5 with HCl. Hydroxylamine hydrochloride solution (0.43 g in 20 mL water, adjusted to pH 4.5) was added to the DAC suspension solution. The mixture was stirred at room temperature for 24 h. The conversion of aldehyde to oxime was determined by recording the consumption of 0.1 M NaOH, performing the reaction at a constant pH of 4.5. Moreover, the same quality of DAC was carried out a blank titration. The result shows that the number of aldehyde groups containing in 100
glucose units is 81. 4
Page 4 of 15
2.4. Preparation of the electrochemical immunosensor DAC was added to dry DMF solution and under the protection of argon, the solution was stirred at 80oC for 2 h. Then, the solution was cooling down to room temperature and getting a true solution of DAC. A GCE (3 mm in diameter) was
ip t
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
cr
air at room temperature and then 10 μL of 0.1% DAC solution diluted in DMF was dropped on the electrode and dried in air at room temperature for 5h. Then, 10 μL of
us
antibody solution (150 μg/mL) was dropped on the modified electrode and incubated for 1 h, followed by washing with ultrapure water to remove unspecific physically
an
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
M
for 30 min at room temperature. The as-prepared immunosensor (designed as Ab/DAC/GCE) was stored at 4 oC for further detection of IgG analyte.
All
electrochemical
d
2.5. Electrochemical measurements
experiments
were
carried
out
in
a
conventional
te
electrochemical cell containing a three-electrode arrangement. EI and DPV
Ac ce p
measurements were performed in 10 mM K3Fe(CN)6/K4Fe(CN)6 solution. The DPV measurements were taken under the following: the potential range was from −0.6 to 0.1 V, pulse amplitude was 0.05 V, pulse width was 0.05 s, and sample width was 0.02 s.
3. Results and discussion
3.1. Characterization of dialdehyde cellulose
5
Page 5 of 15
ip t cr
us
Fig. 2. FTIR spectra of DAC (a) and microcrystalline cellulose powder (b). To study the presence of aldehyde group, we measured IR-spectra of cellulose
an
powder and DAC respectively. The spectrum showed typical characteristics of DAC with an aldehyde peak at 1721 cm−1 (Fig. 2). Owing to the aldehyde group of
M
dialdehyde cellulose rarely exists in the free form and hemiacetal linkages being
Ac ce p
te
d
formed, aldehyde peak of DAC was much smaller than expected [31].
6
Page 6 of 15
Fig. 3. SEM of cellulose powder,δ= 50 µm (A), SEM of cellulose powder active by sodium hydroxide solution,δ= 10 µm (B), SEM of dialdehyde cellulose powder, δ = 50 µm (C) and SEM of dialdehyde cellulose powder, δ = 5 µm (D).
ip t
Fig.3A shows the SEM of cellulose powder. It is seen that the cellulose is in the form of fairly long fibres, but the fibrous form of cellulose is not retained after treated
cr
by sodium hydroxide solution and there is a large crack (Fig.3B). This form of cellulose with large crack is a good for penetration, spread and swelling of the
us
reagents. Additionally, from the SEM images of DAC powder (Fig.3C, Fig.3D), it is clear that the form of DAC is microporous and the specific surface area is large. The
an
specific surface area indicate that DAC can be easily formed a film and fixed more antibodies.
Ac ce p
te
d
M
3.2. The electrochemical characterization of the modified electrodes
Fig. 4. Nyquist plot of Faradic impedance obtained in 10 mM K3Fe(CN)6/K4Fe(CN)6 for bare GCE (a), DAC-modified GCE (b), Ab-DAC-modified GCE (c), BSA-Ab-DAC-modified GCE (d) and IgG-BSA-Ab-DAC-modified GCE (e). The concentration of IgG is 30 ng/mL. Electrochemical impedance was used to characterize the interfacial properties of
surface-modified electrodes. Generally, the semicircle diameter of the Nyquist plot of electrochemical impedance spectroscopy represents the electron-transfer resistance [32]. Fig. 4 shows the electrochemical impedance spectroscopy for the modified gold electrode in 10 mM K3Fe (CN)6/K4Fe(CN)6 solution. As can be seen from Fig. 4, the electrochemical impedance spectroscopy of the bare gold electrode had a small 7
Page 7 of 15
semicircle diameter (curve a), implying low resistance to the redox probe dissolved in electrolyte solution. After GCE was modified with DAC film (curve b), the semicircle diameter of the electrochemical impedance spectroscopy was turned to be larger than that of the bare GCE. When the electrode was further modified with antibodies (curve
ip t
c), BSA (curve d) and antigens (curve e), the semicircle diameter increased step by
step. The results were consistent with the enhanced electron-transfer barriers
M
an
us
cr
introduced of these layers.
Fig. 5. DPVs of the different electrode obtained in 10 mM K3Fe(CN)6/K4Fe(CN)6 for
d
bare GCE (a), DAC-modified GCE (b), Ab-DAC-modified GCE (c) and
te
IgG-BSA-Ab-DAC-modified GCE (e). The concentration of IgG is 30 ng/mL. DPV was also used for monitoring the process of the modification of electrode. As
Ac ce p
can be seen from Fig. 5, the bare GCE (curve a) had larger DPV current than the DAC/GCE (curve b). A current decrease appeared after antibodies were immobilized on the surface of DAC/GCE (curve c). When antigens were immobilized on the surface of the electrode modified with antibodies, the peak current further decreased (curve d). This indicates that the peak current decreased is consistent with the increasing of the electron-transfer blocking layers. 3.3. Optimization of experimental conditions
8
Page 8 of 15
ip t cr
Fig. 6. (A) Effect of antibody concentration on the peak current response of the
us
immunosensor. (B) The influence of reaction time of antibody with DAC on the peak current response of the immunosensor to 30 ng/mL IgG in 10 mM
an
K3Fe(CN)6/K4Fe(CN)6
The effect of the concentration of antibody on the response of the immunosensor
M
was investigated. When it changes from 50 to 200 μg/mL, experimental data show that the peak current decreases with the increasing antibody concentration till the peak current reach a plateau at 150 μg/mL (Fig. 6A). Therefore, a 150 μg/mL antibody
d
solution in PBS was chosen for this work.
te
The influence of reaction time of antibody with DAC on the performance of the immunosensor was also investigated over the range from 20 to 80 min. At room
Ac ce p
temperature, the DPV response of the immunosensor decreased with the immobilization time up to 60 min (Fig. 6B), Therefore, a reaction time of 60 min was selected for this work.
3.4. The detection of thrombin based on DPV Under the optimal conditions, the responses of the DAC-based immunosensor
toward different IgG concentrations were recorded. From Fig. 7A, we could see that the peak current gradually decreased with the increasing concentration of IgG. The dose response curve of the proposed immunosensor for IgG showed a linear range from 0.5 to 45 ng/mL (Fig. 7B) with a detection limit of 0.3 ng/mL based on a signal-to-noise ratio of 3.
9
Page 9 of 15
ip t cr
Fig. 7. (A) DPVs of the immunosensor for the detection of different concentrations of
IgG. Error bars represent standard deviation, n=3.
us
IgG. (B) The linear relationship between the peak current and the concentration of
te
d
M
an
3.5. Specificity, reproducibility and stability of the immunosensor
Fig. 8. Specificity of the immunosensor to IgG, IgG+BSA, IgG+HSA, respectivity.
Ac ce p
The concentration of IgG, BSA and HSA is 30 ng/mL, 3 μg/mL, 3 μg/mL. Error bars represent standard deviation, n=3. In order to confirm the binding specificity of the sensor for IgG detection, other
interfering proteins, such as BSA and HSA were chosen as reference substances. Fig. 8 exhibited peak current signals of the proposed immunosensor incubated with 30 ng/mL IgG, the mixture of IgG and BSA or HSA (the concentration of IgG, BSA and HSA is 30 ng/mL, 3 μg/mL, 3 μg/mL) under the same experimental conditions. As can be seen From Fig. 8, 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 30 ng/mL was successively detected 5 times under the optimized
10
Page 10 of 15
conditions. Acceptable repeatability was observed with a relative standard deviation (R.S.D.) of 7.13%, which indicates that the proposed antibody sensor can be used for analysis with good reproducibility. Furthermore, after the electrode was stored at 4 for three weeks, the immunosensor retained 90.6% of its initial current for 30 ng/mL
ip t
of IgG. This indicated that the immnnosensor possesses a good stability. 4. Conclusions
cr
In this report, we have described a new method to directly immobilize antibodies
on dialdehyde cellulose film for the fabrication of an electrochemical immunosensor.
us
The proposed method can avoid the use of linkage agents such as EDC+NHS or glutaraldehyde. Human IgG was used as a model analyte in this paper. The
an
experimental results demonstrated that the electrochemical immunosensor based on DAC film possesses high sensitivity, good reproducibility and stability. It has a huge
M
potential in immobilization of different biomolecules intended for diverse applications in areas like diagnostics, affinity chromatography and in enzyme based industries.
d
Acknowledgments
This work was supported by the construct program of the key discipline in Hunan
te
province (Applied Chemistry), Scientific Research Fund of Hunan Provincial
Ac ce p
Education Department (13C635) and Startup Foundation for Doctors of Hunan University of Arts and Science. References
[1] H.L. Guo, X.H. Zhou, Y. Zhang, B.D. Song, L.H. Liu, J.X. Zhang, H.C. Shi, Highly sensitive and rapid detection of melamine in milk products by planar waveguide fluorescence immunosensor (PWFI) ， Sensors and Actuators B:
Chemical 194 (2014) 114-119.
[2] H.A. Engström, P.O. Andersson, S. Ohlson, A label-free continuous total-internal -reflection-fluorescence-based immunosensor, Analytical Biochemistry 357 (2006) 159-166. [3] L.D. Wu, M. Li, M. Zhang, M. Yan, S.G. Ge, J.H. Yu, Ultrasensitive electrochemiluminescence immunosensor for tumor marker detection based on nanoporous sliver@carbon dots as labels, Sensors and Actuators B: Chemical 186 11
Page 11 of 15
(2013)761-767. [4] S.R. Jain, E. Borowska, R. Davidsson, M. Tudorache, E. Pontén, J. Emnéus, A chemiluminescence flow immunosensor based on a porous monolithic metacrylate and polyethylene composite disc modified with protein G, Biosens Bioelectron 19
ip t
(2004) 795-803.
[5] C. Crosson, C. Rossi, Quartz crystal microbalance immunosensor for the
cr
quantification of immunoglobulin G in bovinemilk, Biosensors and Bioelectronics 42 (2013) 453-459.
us
[6] M. Salmain, M. Ghasemi, S. Boujday, C.-M. Pradier, Elaboration of a reusable immunosensor for the detection of staphylococcal enterotoxin A (SEA) in milk
an
with a quartz crystal microbalance, Sensors and Actuators B: Chemical 173 (2012)148-156.
M
[7] H. Dong, X. Cao, C.M. Li, W. Hu, An in situ electrochemical surface plasmon resonance immunosensor with polypyrrole propylic acid film: comparison
d
between SPR and electrochemical responses from polymer formation to protein immunosensing, Biosens Bioelectron 23 (2008) 1055-1062.
te
[8] J.Y. Jyoung, S. Hong, W. Lee, J.W. Choi, Immunosensor for the detection of
Ac ce p
Vibrio cholerae O1 using surface plasmon resonance, Biosens Bioelectron 21 (2006) 2315-2319.
[9] A. Marzo, J. Pons, D.A. Blake, A. Merkoçi, High sensitive gold-nanoparticle based lateral flow Immunodevice for Cd2+ detection in drinking waters, Biosens Bioelectron., 47 (2013) 190-198.
[10] X. Sun, Z. Ma, Electrochemical immunosensor based on nanoporpus gold loading thionine for carcinoembryonic antigen, Analytica Chimica Acta, 780 (2013) 95-100. [11] Y. Xie, A. Chen, D. Du, Y. Lin, Graphene-based immunosensor for electro -chemical quantification of phosphorylated p53 (S15), Analytica Chimica Acta, 699 (2011) 44-48. [12] 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
12
Page 12 of 15
nanometer-sized gold particles and plasma-polymerized film, Analytical Biochemistry 324 (2004) 219-226. [13] L. Tedeschi, C. Domenici, A. Ahluwalia, F. Baldini, A. Mencaglia, Antibody immobilisation on fibre optic TIRF sensors, Biosens Bioelectron 19 (2003)
ip t
85-93.
[14] S. Dulay, P. Lozano-Sanchez, E. Iwuoha, I. Katakis, C.K. O’Sullivan,
cr
Electrochemical detection of celiac disease-related anti-tissue transglutaminase
26 (2011)3852-3856.
us
antibodies using thiol based surface chemistry, Biosensors and Bioelectronics
[15] Y. Huang, X.-M. Nie, S.-L. Gan, J. Jiang, G.-L. Shen, R.-Q. Yu, Electrochemical of
platelet-derived
growth
factor
an
immunosensor
with
aptamer-primed
polymerase amplification, Analytical Biochemistry 382 (2008) 16-22.
M
[16] R. Genc, D. Murphy, A. Fragoso, M. Ortiz, C.K. O’Sullivan, Signal-enhancing thermosensitive liposomes for highly sensitive immunosensor development,
d
Analytical Chemistry 83 (2011) 563-570.
[17] H.M. Nassef, M.C. Bermudo Redondo, P.J. Ciclitira, H.J. Ellis, A. Fragoso, C.K.
te
O’Sullivan, Electrochemical immunosensor for detection of celiac disease toxic
Ac ce p
gliadin in foodstuff, Analytical Chemistry 80 (2008) 9265-9271. [18]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.
[19] 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. [20] S. Cosnier, Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized films. A review, Biosensors and Bioelectronics 14 (1999) 443-456. [21] H.X. Chen, F. Liu, F.J. Qi, K. Koh, K.M. Wang, Fabrication of calix[4]arene derivative monolayers to control orientation of antibody immobilization,
13
Page 13 of 15
International Journal of Molecular Sciences 15 (2014) 5496-5507. [22] E. Zaccoa, R. Galvea, M.P. Marcob, S. Alegret, M.I. Pividoria, Electrochemical biosensing of pesticide residues based
on affinity biocomposite platforms,
Biosensors and Bioelectronics 22 (2007) 1707-1715.
ip t
[23] S. Berlioz, S. Molina-Boisseau, Y. Nishiyama, L. Heux, Gas-phase surface esterification of cellulose microfibrils and whiskers, Biomacromolecules 10
cr
(2009) 2144-2151.
[24] M. Andresen, L.-S. Johansson, B. S. Tanem, P. Stenius, Properties and
us
characterization of hydrophobized microfibrillated cellulose, Cellulose 13 (2006) 665-677.
an
[25] T. Saito, Y. Nishiyama, J.-L. Putaux, M. Vignon, A. Isogai, Homogeneous suspensions of individualized microfibrils from TEMPO-catalyzed oxidation of
M
native cellulose, Biomacromolecules 7 (2006) 1687-1691. [26] V. Vamvakaki, K. Tsagaraki, N. Chaniotakis, Carbon nanofiber-based glucose
d
biosensor, Analytical Chemistry 78 (2006) 5538-5542. [27] P. Paul, C. Seok-Youl, S. Bongsup, L. Jungwoo, C. Meghan, A.K. Nicholas,
te
Molecularly engineered nanocomposites: layer-by-layer assembly of cellulose
Ac ce p
nanocrystals, Biomacromolecules 6 (2005) 2914-2918. [28] M.M. de Souza Lima, R. Borsali, Rodlike cellulose microcrystals: structure, properties, and applications, Macromolecular Rapid Communications 25 (2004) 771-787.
[29] R. Sternberg, D.S. Bindra, G.S. Wilson, D.R. Thevenot, Covalent enzyme coupling on cellulose acetate membranes for glucose sensor development, Analytical Chemistry 60 (1988) 2781-2786.
[30] U.-J. Kim, M. Wada, S. Kuga, Solubilization of dialdehyde cellulose by hot water, Carbohydrate Polymers 56 (2004) 7-10. [31] A. J. Varmat, V. B. Chavan, A study of crystallinity changes in oxidized celluloses, Polymer Degradation and Stability 49 (1995) 245-250.
14
Page 14 of 15
[32] X.M. Ren, P.G. Pickup, An impedance study of electron transport and electron transfer in composite polypyrrole + polystyrenesulphonate films, Journal of Electroanalytical Chemistry 420 (1997) 251-257.
ip t
Biographies
Xiangyang Zhang received his Ph.D. in organic chemistry in 2011 at Chengdu
cr
Institute of Organic Chemistry, Chinese Academy of Science, China. He is currently working as a lecturer at Hunan University of Arts and Science. His research interests
us
are organic functional materials and their applications in sensor.
Guangyu Shen received her Ph.D. in analytical chemistry at Hunan University, China.
an
She is currently working as an associate professor at Hunan University of Arts and Science. Her current research interest is focused on electrochemical sensors.
M
Shouyuan Sun is currently a student majoring in applied chemistry. He is working for his bachelor in Hunan University of Arts and Science., China. His research interests
d
are the synthesis of functional materials.
Youming Shen received his Ph.D. in organic chemistry in 2009 at Hunan University,
te
China. He is currently working as a lecturer at Hunan University of Arts and Science.
Ac ce p
His research interests are functional nanomaterials and their applications in sensor. Chunxiang Zhang received her master's degree in organic chemistry from Hunan University, China. She is currently working as a teaching assistant at Hunan University of Arts and Science. Her current research interests focus on biosensors. Anguo Xiao received his Ph.D. in polymer chemistry and physics from Zhejiang University, China. He is currently working as an associate professor at Hunan University of Arts and Science. His current research interests focus on the synthesis of functional polymer materials.
15
Page 15 of 15