hemoglobin multilayer films for electrochemical and electrochemiluminescent biosensing

Sensors and Actuators B 174 (2012) 421–426

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Multiwall carbon nanotube–CdS/hemoglobin multilayer films for electrochemical and electrochemiluminescent biosensing Zhong-Qin Pan a,b , Chuan-Guo Shi c , Hong Fan a , Ning Bao a,b , Chun-Mei Yu a,b , Yang Liu a,b , Rong Lu d , Qin-Hui Zhang d , Hai-Ying Gu a,b,c,∗ a

Institute of Analytical Chemistry for Life Science, School of Public Health, 9 Seyuan Road, Nantong, Jiangsu 226019, PR China School of Public Health, Nantong University, Nantong 226019, PR China c College of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, PR China d Affiliated Hospital, Nantong University, Nantong 226021, PR China b

a r t i c l e

i n f o

Article history: Received 28 June 2012 Received in revised form 23 August 2012 Accepted 28 August 2012 Available online 4 September 2012 Keywords: Multiwall carbon nanotubes CdS quantum dots Hemoglobin Layer-by-layer assembly Electrochemiluminescence Direct electrochemistry

a b s t r a c t This paper presents both electrochemical and electrochemiluminescence (ECL) detections based on the layer-by-layer (LBL) films fabricated with hemoglobin (Hb) and the optimal nanocomposite containing multiwall carbon nanotubes (MWCNTs) and CdS quantum dots (QDs). Hb and the MWCNT–CdS nanocomposite were electrostatically assembled on the chitosan (CS) modified glassy carbon electrode (GCE) forming with up to six bilayer films. Our results on cyclic voltammograms (CVs) revealed that the redox peak currents of Hb at the six-bilayer film ({MWCNT–CdS/Hb}6 ) modified GCE were the largest. Hydrogen peroxide (H2 O2 ) could be stably detected using the six bilayer films modified GCE with a wide linear range from 0.125 ␮M to 1.20 mM. Nitrite (NO2 − ) could also be determined through CdS QDs of the multilayer films based on stable ECL intensities with a linear range from 0.60 ␮M to 0.80 mM. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Biosensors have far-reaching influences in versatile applications in the field of medical research [1,2], information technology [3,4], material science [5,6], food safety, and environmental protection [7–9]. And the performance of biosensors was normally characterized by their stability, reproducibility, selectivity, and sensitivity. As a result, a number of approaches have been developed for the improvement of biosensors, especially of the protein-based biosensors [10,11]. Layer-by-layer (LBL) assembly, as a technique for the construction of thin and controllable films, has attracted tremendous interests since developed by Decher in 1997 [12]. Afterwards, it has been widely used in the fabrication of electrochemical biosensors on the basis of alternative electrostatic adsorption of charged components: proteins, nanoparticles (NPs), polyions, etc. [13–16]. However, stable and reproducible LBL-based

Abbreviations: ECL, electrochemiluminescence; MWCNTs, multiwall carbon nanotubes; CS, chitosan; Hb, hemoglobin; GCE, glassy carbon electrode; CVs, cyclic voltammograms; PBS, phosphate buffer solution; LBL, layer by layer; QDs, quantum dots; TGA, thioglycolicacid. ∗ Corresponding author at: Institute of Analytical Chemistry for Life Science, School of Public Health, 9 Seyuan Road, Nantong, Jiangsu 226019, PR China. Tel.: +86 513 8501 2916; fax: +86 513 8501 2916. E-mail addresses: [email protected], [email protected] (H.-Y. Gu). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.08.069

biosensors can hardly be obtained due to repeatable and delicate steps during the assembly process. Therefore, one major task in the research work on developing LBL-based biosensors is to choose perfect materials. Carbon nanotubes (CNTs), one of the current inspiring materials, have been applied in biosensing due to their unique physical, mechanical, and electronic properties [17–20]. For example, Zhang et al. exploited sulphydryl-functionalized multi-walled carbon nanotubes (MWCNTs) and phosphate-functionalized gold nanoparticles (NPs) for the detection of hydrazine [17]. Chen et al. utilized various CNTs to support Pt nano-particles for electrooxidation of methanol [20]. Quantum dots (QDs) are recently typical for LBL assembly approach because of their unique macroscopic quantum tunneling effect. They can incredibly increase the amount of immobilized proteins on the electrodes and enhance the response of protein-based biosensors [21]. Furthermore, QDs have optical and electronic properties for the application on electrochemiluminescence (ECL) detections [22–25]. In addition, Shi et al. reported the construction of {CdS/Hb}n film via the LBL technique and hemoglobin (Hb) could increase the stability of LBL-based ECL biosensors [21]. However, ECL applications on QDs were limited by unstable ECL responses owing to the properties of water solubility and easy aggregation of QDs. To address this problem, Wang et al. have exploited MWCNTs NPs to improve the emission intensity of CdS QDs [26,27].

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Therefore, in this work, we synthesized different MWCNT–CdS nanocomposites by hydrothermal precipitation method and preferred the MWCNT–CdS nanocomposite of best fluorescence intensity. The optimal nanocomposite and Hb were LBL assembled on chitosan (CS) modified glassy carbon electrode (GCE) to form {MWCNT–CdS/Hb}n films for the fabrication and application of stable and sensitive LBL-based biosensors. The modified GCE with the {MWCNT–CdS/Hb}n –CS film was used for both electrochemical detection of hydrogen peroxide (H2 O2 ) and ECL determination of nitrite (NO2 − ).

increased with the increase of bilayer number (n = 1–6). And their potentials were almost the same. The peak currents did not grew any more at n > 6. Fig. 1B shows the redox peak currents obtained from CVs with the modified electrodes with the number of bilayers (MWCNT–CdS/Hb) from 1 to 6 in 0.10 M pH 7.0 PBS. Therefore, in our following investigations the electrodes modified with the six-bilayer film ({MWCNT–CdS/Hb}6 ) should be used for the electrochemical measurement. The electron transfer number (n) was calculated according to the Faraday’s law:

2. Experimental

IP =

2.1. Materials and methods Bovine Hb (64.5 kDa) was purchased from Sigma and used without further purification. MWCNTs (95%, ∼20–40 nm) were purchased from Shenzhen Nanotech. Port. Co. Ltd. All other reagents were of analytical reagent grade. Double-distilled water was used in all experiments. Electrochemical measurements were performed with a CHI 660B electrochemical working station (CH Instruments Co., USA) equipping with conventional three-electrode system. The optimal MWCNT–CdS nanocomposite was characterized by transmission electron microscopy (TEM) (Tecnai 12, Netherland). The morphology of the films was studied with scanning electron microscopy (SEM) (S-4800, Japan). The ultraviolet–visible (UV–vis) absorption spectra were recorded with a UV-2450 spectrophotometer (Shimadzu, Japan), and indium-tin oxide (ITO) substrates were cleaned for measurements. The ECL emissions were recorded using a Model MPI-E electrochemiluminescence analyzer system (Xi’An Remax Electronic Science & Technology Co. Ltd., China) with Ag/AgCl electrode as the reference electrode. 2.2. Synthesis of MWCNT–CdS nanocomposites The preparation process of the MWCNT–CdS nanocomposite was as follows: 0.50 mL of 2.5 mg mL−1 CdS solution and 0.50 mL double-distilled water were mixed, followed by adding 0.03 mg mL−1 MWCNTs dispersions with different volumes. And then the mixtures were ultrasonically synthesized for 30 min at air atmosphere, respectively. Finally, different MWCNT–CdS nanocomposites were kept at 4 ◦ C for comparison. 2.3. The preparation of electrochemical and ECL biosensors The CS modified GCE (CS–GCE) was prepared according to our previous work [28]. The positive-charged electrode was then dipped into MWCNT–CdS dispersion at 4 ◦ C for 20 min, washed with double-distilled water and dried in air. Afterwards, the obtained MWCNT–CdS–CS–GCE was incubated in 3.0 mg mL−1 Hb solution at 4 ◦ C for 20 min, washed with twice-distilled water and dried in air. The procedure was repeated for the desired bilayer number of {MWCNT–CdS/Hb}n . The final electrode was denoted as {MWCNT–CdS/Hb}n –CS–GCE. And the LBL assembly process was shown in Supplemental Fig. S1. 3. Results and discussion 3.1. The CV study and characterization of the {MWCNT–CdS/Hb}n multilayer film It is well known that the films with different bilayers have different characteristics. Fig. 1A illustrates that the peak currents (Ip ) at the {MWCNT–CdS/Hb}n films on the underlying electrode

nFQ n2 F 2 A = 4RT 4RT

(1)

where Q is the total amount of charge integrated from the cyclic voltammetric peak; n is the electron transfer number; T is the temperature in Kelvin (T = 298 K), R is the gas constant (R = 8.314 J mol−1 K−1 ); and F is the Faraday constant (F = 96493 C mol−1 ). The transfer number n was calculated to be 0.97 according to the slope of the Ip ∝ v obtained from Eq. (1). Therefore, it could be regarded as a single electron transfer reaction. On another note, when nEp ≤ 200 mV, the heterogeneous electron transfer rate constant ks could be estimated according to the Laviron’s equation as following [29,30]: log ks = ˛ log(1 − ˛) + (1 − ˛) log ˛ − log

˛(1 − ˛)nFEp RT − nF 2.3RT (2)

where ˛ is the charge transfer coefficient, and it was set to be 0.5 here; Ep is the potential differences, which was 78 mV; n is the electron transfer number, which was 0.97; and ks can be calculated to be 2.26 s−1 at 250 mV s−1 . The value of ks was in the controlled range of surface-controlled quasi-reversible process [31], and was larger than that obtained for Hb on polymer–MWCNT–GCE (0.4 s−1 ), indicating faster heterogeneous electron transfer rate between Hb and the underlying GCE [32]. Thus, the optimal MWCNT–CdS nanocomposite could provide a suitable support for direct electrochemistry of Hb. UV–vis spectroscopy was used to examine the denaturation of Hb (see Fig. 1C). It could be observed that {MWCNT–CdS/Hb}n films (n = 1–6) modified on ITO substrates all showed Soret bands at 407 nm, which was very close to that of Hb in pH 6.0 PBS (406 nm). Such results demonstrated that Hb in the multilayers was not denatured and the multilayer films could be used for further study. SEM was employed to characterize the morphology of the {MWCNT–CdS/Hb}n multilayer films. Fig. 1D represents the SEM image of the outmost Hb-layer, it could be easily found that Hb has been homogeneously assembled on the MWCNT–CdS nanocomposite. All the results showed that the optimal MWCNT–CdS nanocomposite was excellent to assemble Hb. 3.2. The characteristics of the {MWCNT–CdS/Hb}6 multilayer film on the GCE Fig. 2A shows CVs of the {MWCNT–CdS/Hb}6 film with the scan rate from 100 mV s−1 to 1000 mV s−1 in 0.10 M pH 7.0 PBS. The CV redox peak heights at the {MWCNT–CdS/Hb}6 film were linearly increased with the increase of the scan rate. Such relationship indicated that the electrochemical process was limited by the surface transfer procedure. Meanwhile, as shown in Fig. 2B, the CV redox peak potentials at the film were influenced remarkably by the pH levels of the solution. The formal potential was gradually and linearly decreased with the increase of pH values (inset of Fig. 2B). Based on the average of CV redox peak potentials, the formal potentials (E  0 ) was proportional to the pH values (pH = 3.0–9.0) with

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Fig. 1. (A) Cyclic voltammograms of the {MWCNT–CdS/Hb}6 –CS–GCE in pH 7.0 PBS at 100 mV s−1 with different bilayer numbers (n): n = 1–6. (B) The redox peak currents varied with the bilayer number n (n = 1–6). (C) Ultraviolet-visible absorption spectra of the {MWCNT–CdS/Hb}6 film on the ITO substrates with bilayer number (n) from 1–6. Inset: ultraviolet–visible absorption spectroscopy of Hb in pH 6.0 PBS. (D) The SEM image of the Hb layer on the {MWCNT–CdS/Hb}6 –CS–GCE.

a slope of −47.5 mV pH−1 for the {MWCNT–CdS/Hb}6 film. This value was close to the theoretical value (−57.6 mV pH−1 ) at 18 ◦ C for reversible proton-coupled electron transfer with equal number of protons and electrons [28]. It should be emphasized that the redox

peak currents in pH 7.0 PBS were the largest by comparing with other redox peak currents in PBS with different pH levels. Therefore, all the following electrochemical experiments were conducted in a neutral environment. The reaction with one electron and one

Fig. 2. (A) Cyclic voltammograms of the {MWCNT–CdS/Hb}6 –CS–GCE in 0.10 M pH 7.0 PBS at vairous scan rates: 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 mV s−1 (a–j). Inset: plots of cathodic and anodic peak current vs. scan rate. (B) Cyclic voltammograms of the {MWCNT–CdS/Hb}6 –CS–GCE in 0.10 M PBS at 100 mV s−1 with various pH values of the solution (pH = 3.0–9.0) (a–j). Inset: plots of formal potential of Hb vs. pH.

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Fig. 3. (A) Amperometic responses of the {MWCNT–CdS/Hb}6 –CS–GCE upon successive additions of 0.125 ␮M H2 O2 to 8 mL 0.10 M pH 7.0 PBS with stir. Inset: amperometric response curve for H2 O2 . (B) Electrochemiluminescence emissions of the {MWCNT–CdS/Hb}6 –CS–GCE in 0.05 M K2 S2 O8 + 0.10 M pH 9.0 PBS containing 0.6 (a), 40 (b), 160 (c), and 400 (d) ␮M NO2 − on the modified GCE. Inset: the linear relationship between the concentration of NO2 − and ECL intensity in 0.05 M K2 S2 O8 + 0.10 M pH 9.0 PBS. Scan rate: 100 mV s−1 . Scan range: 0 to −1.7 V, the voltage of the PMT was biased at 600 V.

proton transfer between Hb and the electrode could be expressed as follows: HbhemeFe(III) + H+ + e−  HbhemeFe(II) 3.3. Electrochemical and ECL biosensing of the {MWCNT–CdS/Hb}6 film on the GCE Fig. 3A shows the amperometric current–time response to H2 O2 with the adding of 0.125 ␮M H2 O2 continuously at an applied potential of −0.40 V. The peak current grew linearly with the Table 1 Comparison of the analytical performance of Hb biosensors based on different schemes. Modified electrode {Hb/CdS}n –CS modified GCE Hb/CSNs–CS modified GCE Hb/AgNPs/MWNTs–CS modified GCE {Hb/GNPs}n –MWNTs/CS modified GCE {Hb/CdSe}n –CS modified GCE Hb/CdS/MWNTs modified GCE Hb/MCMs modified GCE {MWCNT–CdS/Hb}n –CS modified GCE

Linear range (␮M)

Detection limit (␮M)

Refs.

4. Conclusions

0.04–31

0.02

[21]

0.75–216

0.50

[29]

6.25–93

0.347

[33]

0.50–2000

0.21

[34]

0.04–4.8

0.02

[35]

0.30

[36]

2.0–7700 69–3000 0.125–1200

21 0.083

increase of the H2 O2 concentration from 0.125 ␮M to 1.20 mM. The range was wider than that in previous reports [29,33–35]. On the other hand, the detection limit of 0.083 ␮M (S/N = 3) was also comparable to those in previous reports [36,37]. Fig. 3B illustrates the dependence of ECL emissions on NO2 − concentration with the prepared {MWCNT–CdS/Hb}6 film. It could be clearly observed that ECL intensity decreased with the increase of the concentration of NO2 − . There was a linear relationship with the concentration of NO2 − from 0.60 ␮M to 0.80 mM, and the detection limit was of 0.40 ␮M (R2 = 0.9973). The linear range of the ECL biosensor was wider than that reported in the literature (from 1.0 ␮M to 0.50 mM) [38]. Comparison of the analytical performance of Hb biosensors based on different matrices was shown in Table 1.

[37] This work

CSNs, colloidal silver nanoparticles; GNPs, gold colloidal nanoparticles; AgNPs, silver nanoparticles; MWNT, multiwall carbon nanotube; MCMs, magnetic chitosan microsphere.

We fabricated biosensors with functions of electrochemical and ECL detections based on the MWCNT–CdS nanocomposite and Hb. The proposed biosensor is superior at stable and reproducible electrochemical and ECL detections as well as simple fabrication. Specifically, the background current was rather low and stable for electrochemical detection of H2 O2 . Our investigations also revealed that MWCNTs with the optimal volume could enhance the ECL intensity and move the onset potential more positively of CdS QDs. All the results demonstrated that the optimal MWCNT–CdS nanocomposite based on LBL assembly technique and the CS supporting matrix is ideal for the improvement of direct electrochemistry of redox proteins and the construction of diverse biosensors, which may be beneficial to clinical diagnosis, food safety, and environmental protection.

Z.-Q. Pan et al. / Sensors and Actuators B 174 (2012) 421–426

Acknowledgements We are grateful to the National Natural Science Foundation of China (Grant nos. 20875051, 21075070, 81001263, 21175075), the Natural Science Foundation of Jiangsu Province (Grant nos. BK2009152, BK2011047), the Social Development Item of Nantong City (Grant nos. S2010017, S2010019, S2008008), the Natural Science Foundation of Nantong University (Grant no. 03041049), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) for their support of this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2012.08.069. References [1] N.M. Noah, S. Alam, O.A. Sadik, Detection of inducible nitric oxide synthase using a suite of electrochemical, fluorescence, and surface plasmon resonance biosensors, Analytical Biochemistry 413 (2011) 157–163. [2] A. Sandhu, H. Handa, M. Abe, Synthesis and applications of magnetic nanoparticles for biorecognition and point of care medical diagnostics, Nanotechnology 21 (2010) 442001. [3] J. Wang, E. Katz, Digital biosensors with built-in logic for biomedical applications-biosensors based on biocomputing concept, Analytical and Bioanalytical Chemistry 398 (2010) 1591–1603. [4] J.R. Siqueira, R.M. Maki, F.V. Paulovich, C.F. Werner, A. Poghossian, M.C.F. de Oliveira, V. Zucolotto, O.N. Oliveira, M.J. Schoning, Use of information visualization methods eliminating cross talk in multiple sensing units investigated for a light-addressable potentiometric sensor, Analytical Chemistry 82 (2010) 61–65. [5] H.Q. Yao, N.F. Hu, pH-controllable on–off bioelectrocatalysis of bienzyme layer-by-layer films assembled by concanavalin A and glucoenzymes with an electroactive mediator, Journal of Physical Chemistry B 114 (2010) 9926–9933. [6] S.L. Song, N.F. Hu, pH-controllable bioelectrocatalysis based on “on–off” switching redox property of electroactive probes for spin-assembled layer-by-layer films containing branched poly(ethyleneimine), Journal of Physical Chemistry B 114 (2010) 3648–3654. [7] S. Sankaran, S. Panigrahi, S. Mallik, Olfactory receptor based piezoelectric biosensors for detection of alcohols related to food safety applications, Sensors and Actuators, B 155 (2011) 8–18. [8] V. Scognamiglio, G. Pezzotti, I. Pezzotti, J. Cano, K. Buonasera, D. Giannini, M.T. Giardi, Biosensors for effective environmental and agrifood protection and commercialization: from research to market, Microchimica Acta 170 (2010) 215–225. [9] M. Badea, M. Romanca, C. Draghici, J.L. Marty, C.V.V.C.O. Marques, D.R. Mendes, O.P. Amarante, G.S. Nunes, Multidisciplinary collaboration for environmental protection using biosensors: detection of organophosphate insecticides in aqueous medium, Journal of the Brazilian Chemical Society 17 (2006) 807–811. [10] J.T. Zhu, C.G. Shi, J.J. Xu, H.Y. Chen, Direct electrochemistry and electrocatalysis of hemoglobin on undoped nanocrystalline diamond modified glassy carbon electrode, Bioelectrochemistry 71 (2007) 243–248. [11] W. Sun, Z.H. Zhu, X. Li, Y. Wang, Y. Zeng, X.T. Huang, Direct electrochemistry and electrocatalysis of horseradish peroxidase with hyaluronic acid–ionic liquid–cadmium sulfide nanorod composite material, Analytica Chimica Acta 670 (2010) 51–56. [12] G. Decher, Fuzzy nanoassemblies: toward layered polymeric multicomposites, Science 277 (1997) 1232–1237. [13] W. Zhao, J.J. Xu, H.Y. Chen, Electrochemical biosensors based on layer-by-layer assemblies, Electroanalysis 18 (2006) 1737–1748. [14] T. Osaka, S. Komaba, A. Amano, Y. Fujino, H. Mori, Electrochemical molecular sieving of the polyion complex film for designing highly sensitive biosensor for creatinine, Sensors and Actuators, B 65 (2000) 58–63. [15] Y. Chen, B. Jin, L.R. Guo, X.J. Yang, W. Chen, G. Gu, L.M. Zheng, X.H. Xia, Hemoglobin on phosphonic acid terminated self-assembled monolayers at a gold electrode: immobilization, direct electrochemistry, and electrocatalysis, Chemistry – A European Journal 14 (2008) 10727–10734. [16] Y. Chen, X.J. Yang, L.R. Guo, J. Li, X.H. Xia, L.M. Zheng, Direct electrochemistry and electrocatalysis of hemoglobin at three-dimensional gold film electrode modified with self-assembled monolayers of 3-mercaptopropylphosphonic acid, Analytica Chimica Acta 644 (2009) 83–89. [17] F. Zhang, L. Zhang, J.F. Xing, Y.W. Tang, Y. Chen, Y.M. Zhou, T.H. Lu, X.H. Xia, Layer-by-layer self-assembly of sulphydryl-functionalized multiwalled carbon nanotubes and phosphate-functionalized gold nanoparticles: detection of hydrazine, ChemplusChem (2012), http://dx.doi.org/10.1002/cplu.201200137. [18] P. Li, H.L. Liu, Y. Ding, Y. Wang, Y. Chen, Y.M. Zhou, Y.W. Tang, H.Y. Wei, C.X. Cai, T.H. Lu, Synthesis of water-soluble phosphonate functionalized single-walled carbon nanotubes and their applications in biosensing, Journal of Materials Chemistry 22 (2012) 15370–15378.

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Biographies Zhong-Qin Pan obtained her BS degree in chemistry from Nanjing Xiaozhuang College, China, in 2008 and Master of Science degree from Nantong University, China, in 2011. Now she is a new teacher of Nantong University. Her research interests are in the areas of electroanalytical chemistry. Chuan-Guo Shi obtained his BS degree from Beijing Normal University in 1996 and PhD degree in analytical chemistry from Nanjing University, China, in 2008. Now he is a Professor of Nantong University, China. His research interests are in the areas of analytical chemistry. Hong Fan obtained her MBBS degree from Nantong University, China, in 2007 and master of epidemiological and statistical expertise degree in Nantong University, China in 2010. Now she is an assistant of Nantong University. Her research interests are in the areas of electroanalytical chemistry. Ning Bao obtained his BS degree on Chemistry in 1991 and his PhD on Analytical Chemistry in 2005 from Nanjing University, China. Currently he holds a position of full Professor in Nantong University, China. His research focuses on microfluidic biosensors.

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Chun-Mei Yu obtained her BS degree in chemistry from SuZhou University, China in 2000. Now she is a lecturer of Nantong University, China. Her research interests are in the areas of electroanalytical chemistry. Yang Liu obtained her MBBS degree from Nantong University, China, in 2006 and Science degree from Nantong University, China, in 2009. Now she is an assistant of Nantong University. Her research interests are in the areas of electroanalytical chemistry. Rong Lu obtained her MBBS degree from Nantong University China, in 1993 and Clinical degree from Nantong University, China, in 2004. Now she is an associate chief physician in Affiliated Hospital of Nantong University. Her research interests are in tissue engineering.

Qin-Hui Zhang obtained his MBBS degree from Nantong University, China, in 2005 and Science degree from Nantong University, China, in 2010. Now he is an associate chief technician in Affiliated Hospital of Nantong University. His research interests are in tissue engineering.

Hai-Ying Gu obtained his BS degree from Xuzhou Normal University in 1984 and PhD in analytical chemistry from Nanjing University, China, in 2002. Now he is a professor of Nantong University, (China). His research interests are in the areas of electroanalytical chemistry and tissue engineering.