Highly sensitive turn-on fluorescent detection of captopril based on energy transfer between fluorescein isothiocyanate and gold nanoparticles

Journal of Luminescence 134 (2013) 874–879

Contents lists available at SciVerse ScienceDirect

Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin

Highly sensitive turn-on fluorescent detection of captopril based on energy transfer between fluorescein isothiocyanate and gold nanoparticles M. Reza Hormozi-Nezhad a,b,n, H. Bagheri a, A. Bohloul a, N. Taheri a, H. Robatjazi a a b

Department of Chemistry, Sharif University of Technology, Tehran 11155-9516, Iran Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Tehran, PO Box 11365-9161, Iran

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 April 2012 Received in revised form 12 June 2012 Accepted 18 June 2012 Available online 7 July 2012

A novel approach for highly sensitive detection of captopril was developed based on the fluorescence resonance energy transfer (FRET) between gold nanoparticles (Au NPs) and fluorescein isothiocyanate (FITC), in which FITC acts as the donor and Au NPs as the acceptor. The fluorescence intensity of fluorescein isothiocyanate (FITC) was strictly quenched as a result of noncovalently adsorbed on Au NPs. Upon the addition of captopril, the fluorescence intensity of FITC ‘‘turn-on’’ due to the competition between captopril and FITC towards the surface of Au NPs. Under the optimum conditions, the fluorescence intensity of the released FITC displays a linear relationship in the range of 20 mg L  1 to 500 mg L  1 of captopril. Lower limit of detection for captopril, at the signal-to-noise ratio of 3 (3s), was 2.6 mg L  1. The developed methodology was successfully applied for the determination of captopril in human plasma. & 2012 Elsevier B.V. All rights reserved.

Keywords: Fluorescence resonance energy transfer (FRET) Gold nanoparticles (Au NPs) Captopril Fluorescein isothiocyanate (FITC)

1. Introduction As a specific inhibitor of angiotensin-converting enzyme (ACE inhibitor), Captopril, 1-[(2s)-3-mercapto-2-methylpropionyl]-lproline (Scheme 1), plays crucial biological role for the treatment of post myocardial infarction, congestive heart failure and hypertension on its own or in combination with other drugs [1,2]. Additionally, it is used for preventing kidney failure diseases associated with high blood pressure and diabetes [3,4]. Captopril has also been investigated for use in the treatment of cancer [5]. As knowledge of significance of captopril, it is very important to find a sensitive, accurate and simple method for measurement of captopril content in biological fluids as well as pharmaceutical samples. Several contributions have been proposed in literatures for the determination of captopril including iodimetric method [6], electroanalytical methods [7,8], FT-Raman [9], fluorimetry [10], spectrophotometry [11,12], plasma resonance light scattering [13], chemiluminescence [14,15], high performance liquid chromatography [16,17], and surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) [18]. Currently, the majority of the available methods, in spite of good sensitivity, require sophisticated instrumentation, expensive biological reagents and

n Corresponding author at: Department of Chemistry, Sharif University of Technology, Azadi Ave, Tehran 11155-9516, Iran. Tel.: þ98 2166165337; fax: þ 98 2166029165. E-mail address: [email protected] (M.R. Hormozi-Nezhad).

0022-2313/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jlumin.2012.06.032

cumbersome sample preparation. Thus, the development of new sensitive, fast and simple practical method for the determination of captopril is still in a great demand. Gold nanoparticles (Au NPs) have received great attention for potential biological analysis in recent years, because of exhibiting intense size dependent optical properties [19,20]. The colloidal form of Au NPs display very intense colors due to the surface plasmon resonance (SPR) phenomenon, a feature that can be attributed to the collective oscillation of the surface electrons as they interact with the incident photon at the resonance wavelengths [21]. The fascinating optical properties of these colloidal particles are composed from both absorbance and scattering of light, that together are known as the extinction. Having high order of magnitude for extinction coefficients, which is typically much greater than molecular chromophores [21], gold nanoparticle are emerging as promising platform for highly sensitive analysis of biologically active molecules through optical detection. As sensitivity is a key factor in the design of sensors and development of new analytical methods, significant efforts have been made for development of fluorescence-based sensing due to their high sensitivity and relative versatility. Recently, fluorescence resonance energy transfer (FRET) based assay through employing gold nanoparticles [22,23] have been developed for sensitive and selective detection of DNA [24–27], thrombin [28] iodide and iodate [29], acetylthiocholine and hydoquinone [30], thiol-containing molecules [31,32], melamine [33], metallic ions [34,35] and so on. Au NPs also, have been widely used for FRET based immunoassay [36–38]. These are due to the extremely high

M.R. Hormozi-Nezhad et al. / Journal of Luminescence 134 (2013) 874–879

extinction coefficient of Au NPs, which induces fluorescence quenching of the attached fluorophores as a result of fluorescence resonance energy transfer (FRET); a process which is inversely proportional to the sixth power of the distance between fluorophore and quencher [23]. Addition of the analytes with much stronger affinity for Au NPs results in detachment of the fluorophore molecules from the surface of the Au NPs, and consequently the fluorescence intensity increases [29]. In the present contribution, we have extended previous efforts of the determination of captopril by developing a simple, highly sensitive, and selective methodology for the quantitation of captopril in human plasma, using fluorescein isothiocyanate (FITC)-modified Au NPs (FITC-Au NPs). At first, through surface modification of the Au NPs with FITC, the fluorescence intensity of FITC molecule turns-off. However, the fluorescence of FITC switched to turn-on upon adding captopril (Scheme 2) due to the competition between captopril and FITC towards the surface of Au NPs. The recovered fluorescence intensity of released FITC molecules increases with an increase in captopril content, permitting one for the quantitative analysis of captopril.

875

(Captopril) 99.0% was purchased from ACROS. All other common laboratory chemicals were of the best grade available and were used without further purification. For the preparation of all samples, water purified with cartridges from Millipore (Milli-Q) to a resistively of 18.2 MO was used. All solutions were used within 1 h after preparation, and the experiments were performed at ambient temperature (2572 1C). Human blood plasma, used as a real sample, was taken from Iranian Blood Transfusion Organization (IBTO). 2.2. Apparatus UV–vis absorption spectra were obtained on a Lambda 25(PE). Varian Cary Eclipse fluorescence spectrophotometer and Motic AE 31 EF-INV-II inverted fluorescence microscope were used to collect the fluorescence spectra and images, respectively. Transmission electron microscopy (TEM) image was recorded with a PHILIPS MC 10TH microscope (USA) at an accelerating voltage of 80 kV. Measurements of pH were made with a Mettler Toledo (USA) pH meter. 2.3. Synthesis of colloidal Au NPs

2. Experimental 2.1. Reagents and solutions Hydrogen tetrachloroaurate (HAuCl4  3H2O, 99.9%) were purchased from Aldrich-Sigma. Trisodium citrate, FITC, NaH2PO4, Na2 HPO4, were obtained from Merck. L-3-(3,4-Dihydroxyphenyl)alanine

Citrate-capped gold nanoparticles with average size of 13.1 nm (see supporting information) were synthesized following the method pioneered by J. Turkevich et al. [39,40] in which a 50 mL solution containing 1 mM of HAuCl4 was prepared and heated under reflux. At the boiling point, 5 mL of 38.8 mM trisodium citrate was added to the this solution under vigorous stirring and the mixture was heated under reflux for an additional 30 min, during which the color of the solution changed to deep red indicating the formation of gold nanoparticles. The solution was set aside to cool to room temperature and stored at 4 1C for further utilization. The particle concentration of the Au NPs solutions was estimated to be 15 nM according to Beer’s law and the extinction coefficient (e) of 13.1 nm Au NPs at 520 nm [29,41]. 2.4. Preparation of FITC-Au NPs

Scheme 1. Structural formula of captopril.

A stock solution of 2.5  10  4 M FITC was prepared in 0.01 M NaH2PO4-Na2HPO4 buffer solution at pH 8. Then, 170 mL of the asprepared FITC solution was added to the 20 ml of synthesized Au

N

COOH

O

S S

COOH N

N

O

COOH

S

O

S

N

S

HOOC O

O HOOC

N

Free FITC

Bonded FITC Scheme 2. Schematic illustration of the captopril sensing mechanism based on Au-FITC.

876

M.R. Hormozi-Nezhad et al. / Journal of Luminescence 134 (2013) 874–879

NPs, and the final volume of mixture was adjusted to the 50 mL with Milli-Q water. The resulted mixture was equilibrated at the ambient temperature.

concentrations have been investigated in order to establish the optimum analytical conditions for detection of captopril. 3.1. Effect of FITC concentration

2.5. Determination of captopril Different concentrations of captopril were added to the solutions containing 3 mL of FITC-Au NPs and 1 mL of 0.01 M phosphate buffer (pH 8) with further increasing the final volume to 10 ml. The resulted solutions were equilibrated at room temperature for 0–30 min, and then fluorescence spectra were recorded at 518 nm with an excitation wavelength of 470 nm. 2.6. Real sample analysis Certain amounts of captopril were spiked into the test tubes containing 3 mL of the fresh human blood as a real sample, and its concentration was determined in each sample after pretreatment in which 5 mg of EDTA was added to the each tube with subsequently centrifuging at 6000 rpm for 10 min. The supernatant, containing proteins, amino acids and captopril, was collected as the source of plasma. In order to precipitate the proteins out, the plasma then was mixed with 10 ml of acetonitrile followed by centrifugation at 7000 rpm for 15 min. The resulted supernatant was used for further analysis under optimum condition.

3. Results and discussions As shown in Fig. 1, the FITC emission spectra largely overlap with the extinction spectra of Au NPs. With this in mind, along with the high binding affinity of FITC molecules to the surface of Au NPs [29], a FRET process could be induced in which FITC and Au NPs act as the donor and acceptor, respectively. By virtue of this phenomenon, the fluorescence intensity of the FITC is dramatically quenched. However, in the presence of a thiolcontaining molecule, such as Captopril, with much stronger affinity towards the surface of Au NPs, the fluorescence intensity rises again, as a result of FITC displacement with captopril. Fig. 2 shows in fluorescence emission spectra and fluorescence microscopy images of FITC-Au NPs before and upon the addition of certain quantity of captopril. The effect of critical parameters including pH, time needed for growth of fluorescence intensity, FITC, and FITC-Au NPs

The effect of FITC concentration varying from 2.5  10  7 to 7.5  10  6 M was investigated on the fluorescence intensity of Au NPs-FITC (Fig. 3). The optimal concentration of FITC is necessary to saturate the surface of Au NPs, where no free FITC will remain in solution. The obtained results have shown that the fluorescence intensity rises dramatically upon increasing concentration of FITC up to 5.00  10  6 M. This can be attributed to the presence of free FITC molecules more than what is required for the saturation of Au NPs. Additionally, the fluorescence intensity was quite quenched at concentrations below to the 1.25  10  6 M, indicating all of the FITC molecules were loaded on Au NPs surface. Thus, the concentration of 1.25  10  6 M was chosen as the optimum conditions for FITC. 3.2. Effect of pH To trace pH dependant fluorescence intensity of FITC, the effect of pH varying from 3.5 to 11.5 was investigated on the fluorescent intensity. According to the Fig. 4 the fluorescence intensity of released FITC highly depends on pH. Based on pKa 6.4 for FITC and pKa 9.8 for thiol group of captopril, we propose that the pH of the solution only could influenced the fluorescence intensity of the FITC, rather than the displacement capacity of captopril in its corresponding thiolate form. Hence, to obtain high fluorescence intensity, pH 8.0 of NaH2PO4-Na2HPO4 buffer was selected for the further experiments. 3.3. Effect of Au NPs concentration Controlling the concentration of Au NPs is another critical parameter that should be taken into the consideration in order to improve the sensitivity of the method. As shown in Fig. 5, the fluorescence intensity was gradually increased upon increasing the concentration of Au NPs, in the presence of captopril, indicating that the more acceptors are presented. However, it can be seen that by increasing the concentration of the Au NPs to more than 1.8 nM, the fluorescence enhancement efficiency starts to be decreased. The negative effect of the higher concentration of Au NPs is probably due to the increasing in collisional encounter that lead to increasing the possibility of collisional quenching of released FITC [42]. Therefore, the concentration of 1.8 nM Au NPs was chosen as the optimum concentration in which the maximum fluorescence intensity was achieved. 3.4. Effect of time Fig. 6 shows the variation of fluorescence intensity of the released FITC vs. time, after addition the captopril. It can be deduced that the FITC displacement with captopril is almost 90% completed within 30 min. Therefore, all the fluorescence measurements were performed 30 min after initiation of the reaction. 3.5. Analytical figures of merit

Fig. 1. Overlapping the emission spectra of FITC and extinction spectra of Au NPs.

3.5.1. Linear range, detection limit, and reproducibility of the method The linear range for the determination of captopril was evaluated under the optimum conditions. The fluorescence spectra of the released FITC in presence of different concentrations of captopril are shown in Fig. 7. The fluorescence intensity of the

M.R. Hormozi-Nezhad et al. / Journal of Luminescence 134 (2013) 874–879

877

800

a 600

If /a.u.

c 400

200

b 0 500

550

600 Wavelength (nm)

650

700

Fig. 2. Fluorescence spectra and corresponding fluorescence microscopy images of: (a) FITC 1.0  10  6 M; (b) FITC (1.0  10  6 M)-Au NPs (1.5 nM) and (c) FITC (1.0  10  6 M)-Au NPs (1.5 nM) in the presence of 500 mg.L  1 captopril, and pH¼ 7.5.

600

800 700

500

600 400 If/a.u

If/a.u.

500 400 300

300 200

200 100

100 0

0 0

1

2

3 4 5 FITC Concentration(µM)

6

7

8

3.5

4.5

5.5

6.5

7.5 pH

8.5

9.5

10.5

11.5

Fig. 3. Effect of FITC concentration on fluorescence intensity in the presence of Au NPs (1.5 nM), and pH¼ 7.5.

Fig. 4. Effect of pH on the fluorescence intensity of FITC (1.25  10  6 M)-Au NPs (1.5 nM) in the presence of 500 mg L  1 captopril.

mixture was enhanced along with elevating the concentrations of analytes. It is found that the fluorescence intensity showed a linear relationship in the range of 20–500 mg L  1 of captopril. Lower limit of detection for the determination of captopril was 2.6 mg L  1 at the signal-to-noise ratio of 3 (3s) [43]. The study of precision, which was made with five independent experiments, has revealed the relative standard deviation (% RSD) of 4.3% and 2.1% for the determinations of 50 and 300 mg L  1 captopril, respectively.

3.6. Interferences study To assess the ability of the proposed method for the analysis of captopril in complex real samples, the interference of foreign metal ions, sugars and amino acids, was evaluated on selectivity of the method. This was conducted through the analysis of a standard solution of captopril (500 mg L  1) in the presence of the excess amounts of co-existing compounds. Among tested ions and molecules with possibility of interfering; Cl  , CH3COO  , SO24  ,

878

M.R. Hormozi-Nezhad et al. / Journal of Luminescence 134 (2013) 874–879

450

600

400

500

350 300 If/a.u.

If/a. u.

400 300

250 200 150

200

100 50

100 0

0

0

0.5

1

1.5 2 2.5 3 3.5 Au NP Concentration (nM)

4

4.5

5

Fig. 5. Effect of Au NP concentration on the detection of 500 mg L  1 of captopril in the presence of 1.25  10  6 M FITC, and pH¼ 8.0.

Fig. 8. Fluorescence intensity of FITC (1.25  10  6 M)-Au NPs (1.8 nM) in the presence of 500 mg L  1 of captopril, and 50 mg L  1 of the other specious (pH¼8.0).

600 Table 1 Determination of captopril in human plasma.

If/ a.u.

500 400

Sample

Added (mg L  1)

Found (mg L  1)

Recovery%

RSD% (n¼ 5)

300

1 2 3 4 5

50.0 100.0 200.0 300.0 400.0

48.0 104.0 215.0 320.0 422.0

96.0 104.0 107.7 106.6 105.5

4.1 3.5 2.9 2.7 3.8

200 100 0

0

10

20

30 40 Time (min)

50

60

70

Fig. 6. Kinetic curve for FITC displacement with captopril. Condition: 1.25  10  6 M FITC; 1.8 nM of Au NPs; pH 8.0; 500 mg L  1 captopril. 500 450 500

400 450

350 300 If/a.u.

400 350

250

3.7. Analysis of real samples

200 150

300 IF/a.u

The criterion for interference is fixed at 75% variation of the calculated concentration of captopril. It should be noted that thiol-containing amino acids such as cysteine with the similar concentration of captopril had ability to recover the fluorescence intensity of FITC. However, most of aminothiols are bound to proteins or other amino acids in the form of disulfide [44] that could not interfere as long as they do not undergo hydrolysis [45,46]. Therefore, the resulted interferences from thiol-containing amino acids in real samples were eliminated through procedure discussed in Section 2.6.

100 250

50 200

0

0

100

200

300

400

500

150

The optimized method was applied for the determination of captopril in human serum and blood plasma based on the procedure described in Section 2.6. The results, are given in Table 1, show the potential and feasibility of the developed method for the determination of captopril in real samples.

100

4. Conclusions

50 0 500

550

600

650

700

Wavelength/nm

Fig. 7. Fluorescence emission spectra of FITC-Au NPs upon addition of different concentration of captopril in the range of 20–500 mg L  1 (inset is calibration curve for captopril) at optimum condition.

NO3 , L-Threonine, Glycince, L-Valine, L-Alanine, Phenyl alanine, L-Leucine, Glucose, and Fructose did not interfere at the concentrations 100 times higher than those of the analyte (Fig. 8).

At present, a simple assay based on FRET has been developed for the highly sensitive determination of captopril. The developed methodology could achieve quantification limit at low level, good linearity accompanied with acceptable accuracy, and reproducibility. In addition, this method was successfully applied to the quantitation of trace amounts of captoperil in human plasma, and is continuing to further development in order to investigation of its clinical potential for determination of trace amounts of other targets of interest in biological samples.

M.R. Hormozi-Nezhad et al. / Journal of Luminescence 134 (2013) 874–879

Acknowledgment The authors wish to express their gratitude to Sharif University of Technology Research Council for the support of this work.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jlumin.2012.06.032.

References [1] The European Pharmacopoeia, fifth ed., Council of Europe, Strasbourg, 2004, p. 1176, p. 2298. [2] The United States Pharmacopeia, 28th ed., USP Convention Inc., Rockville, 2004, p. 338, p. 1608. [3] E. Hommel, H-H. Parving, E. Mathiesen, B. Edsberg, M. Nielsen, J. Giese, British Medicinal J. 293 (1986) 467. [4] L-H. Tan, L-Zh. Du, M.R. Carr, J.K. Kuzin, B.S. Moffett, A.C. Chang, World J. Pediatr. 7 (2011) 89. [5] S. Attoub, A.M. Gaben, S. Al-Salam, M.A.H. Al Sultan, A. John, M.G. Nicholls, J. Mester, G. Petroianu, Ann. N.Y. Acad. Sci. 1138 (2008) 65. [6] E. Schmidt, W.R. Melchert, F.R.P. Rocha, J. Braz. Chem. Soc. 20 (2009) 236. [7] S. Shahrokhian, M. Karimi, H. Khajehsharif, Sens. Actuators B: Chem. 109 (2005) 278. [8] H. Karimi-Maleh, A.A. Ensafi, A.R. Allafchian, J. Solid State Electrochem. 14 (2010) 9. [9] S. Mazurek, R. Szostak, J. Pharm. Biomed. Anal. 40 (2006) 1225. [10] L. Wang, X.F. Yang, M. Zhao, J. Fluoresc. 19 (2009) 593. [11] P.D. Tzanavaras, D.G. Themelis, A. Economou, G. Theodoridis, Microchim. Acta 142 (2003) 55. [12] W.T. Suarez, A.A. Madi, L.C.S. de Figueiredo-Filho, O. Fatibello-Filho, J. Braz. Chem. Soc. 18 (2007) 1215. [13] Z. De Liu, C.Z. Huang, Y.F. Li, Y.F. Long, Anal. Chim. Acta 577 (2006) 244. [14] B. Li, Z. Zhang, M. Wu, Microchem. J. 70 (2001) 85. [15] J.A. Murillo Pulgarın, L.F.G. Bermejo, P.F. Lopez, Anal. Chim. Acta 546 (2005) 60.

879

[16] T.M. Huang, Z. He, B. Yang, L.P. Shao, X.W. Zheng, G.L. Duan, J. Pharm. Biomed. Anal. 41 (2006) 644. [17] K. Kusmierek, E. Bald, Chromatographia 66 (2007) 71. [18] W.T. Chen, C.K. Chiang, Y.W. Lin, H.T. Chang, J. Am. Mass Spectrom. 21 (2010) 864. [19] H.C. Van de Hulst, Light Scattering by Small Particles, Dover, New York, 1981, p 397–400. [20] C. Burda, X. Chen, R. Narayanan, M.A. El-Sayed, Chem. Rev. 105 (2005) 1025. [21] Y. Sun, Y. Xia, Analyst 128 (2003) 686. [22] W. Zhong, Anal. Bioanal. Chem. 394 (2009) 47. [23] J. Ling, C.Zhi Huang, Anal. Methods 2 (2010) 1439. [24] B. Dubertret, M. Calame, A.J. Libchaber, Nat. Biotechnol. 19 (2001) 365. [25] Z. Gueroui, A. Libchaber, Phys. Rev. Lett. 93 (2004) 166108. [26] L. Dyadyusha, S.J.H. Yin, T. Brown, J.J. Baumberg, F.P. Booy, T. Melvin, Chem. Commun. (2005) 3201. [27] P.C. Ray, G.K. Darbha, A. Ray, J. Walker, W. Hardy, Plasmonic 2 (2007) 173. [28] W. Wang, C. Chen, M. Qian, X.S. Zhao, Anal. Biochem. 373 (2008) 213. [29] Y.M. Chen, T.L. Cheng, W.L. Tseng, Analyst 134 (2009) 2106. [30] S.Y. Lim, J.H. Kim, J.S. Lee, C.B. Park, Langmuir 25 (2009) 13302. [31] K.H. Lee, S.J. Chen, J.Y. Jeng, Y.C. Cheng, J.T. Shiea, H.T. Chang, J. Colloid Int. Sci. 307 (2007) 340. [32] S.J. Chen, H.T. Chang, Anal. Chem. 76 (2004) 3727. [33] L. Guo, J. Zhong, J. Wu, F. Fu, GG. Chen, Y. Chen, X. Zheng, S. Lin, Analyst 136 (2011) 1659. [34] C.C. Huang, H.T. Chang, Anal. Chem. 78 (2006) 8332. [35] X. Wang, X. Guo, Analyst 134 (2009) 1348. [36] N. Kato, F. Caruso, J. Phys. Chem. B 109 (2005) 19604. [37] J.-Q. Gu, J. Shen, L.-D. Sun, C.-H. Yan, J. Phys. Chem. C 112 (2008) 6589. [38] S. Mayilo, B. Ehlers, M. Wunderlich, T.A. Klar, H.-P. Josel, D. Heindl, A. Nichtl, K. Khurzinger, J. Feldmann, Anal. Chim. Acta 646 (2009) 119. [39] J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55. [40] J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech, J. Phys. Chem. B 110 (2006) 15700. [41] W. Zhao, W. Chiuman, J.C.F. Lam, S.A. MacManus, W. Chen, Y. Cui, R. Pelton, M.A. Brook, Y. Li, J. Am. Chem. Soc. 130 (2008) 3610. [42] P.P.H. Cheng, D. Silvester, G. Wang, G. Kalyuzhny, A. Douglas, R.W. Murray, J. Phys. Chem. B 110 (2006) 4637. [43] J.C. Miller, J.N. Miller, Statistics and Chemometrics for Analytical Chemistry, fourth ed., Prentice Hall, 2000. [44] J.V. Ros-Lis, B. Garci, D. Jimenez, R. Martinez-Manez, F. Sancenon, J. Soto, F. Gonzalvo, M.C. Valldecabres, J. Am. Chem. Soc. 126 (2004) 4064. [45] Y.V. Tcherkas, A.D. Denisenko, J. Chromatogr. A. 913 (2001) 309. [46] L. Shang, J. Yin, J. Li, S. Dong, Biosens. Bioelectron. 25 (2009) 269.