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Preparation of a novel colorimetric luminescence sensor strip for the detection of indole-3-acetic acid
Biosensors and Bioelectronics 25 (2010) 2375–2378
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Preparation of a novel colorimetric luminescence sensor strip for the detection of indole-3-acetic acid Yan Liu, Haitao Dong, Wenzhu Zhang, Zhiqiang Ye ∗ , Guilan Wang, Jingli Yuan ∗ State Key Laboratory of Fine Chemicals, Department of Chemistry, Dalian University of Technology, Dalian 116012, China
a r t i c l e
i n f o
Article history: Received 2 February 2010 Received in revised form 3 March 2010 Accepted 3 March 2010 Available online 10 March 2010 Keywords: Colorimetric luminescence sensor Europium Horseradish peroxidase Indole-3-acetic acid Plant auxin Quantum dots
a b s t r a c t A novel colorimetric luminescence sensor strip for the detection of indole-3-acetic acid (IAA) has been fabricated by using green emissive quantum dots of cadmium telluride (CdTe QDs) as a background layer and a red emissive europium chelate, [4 -(9-anthryl)-2,2 :6 ,2 -terpyridine-6,6 -diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu3+ (ATTA-Eu3+ ), as a specific sensing layer coated on the surface of glass slide, respectively. The luminescence response of the sensor strip is given by the dramatic changes in emission colors from green to red at different IAA concentrations. This approach provides a simple, rapid, sensitive and accurate method for the detection of IAA without using any special scientific instruments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction As a minor component of metabolome, phytohormones are regulators produced by plants themselves, which are of particular significance given their role in the regulation of germination, growth, reproduction, and the protective responses of plants against stress (Davies, 1995). It has been known that the phytohormone indole-3-acetic acid (IAA) is one of the most important native auxins that can induce cell elongation and division with all subsequent results for plant growth and development (Woodward and Bartel, 2005; Ding et al., 2008). Recently, some sensitive methods for IAA determination in biological samples, such as GC–MS, LC–MS, HPLC and immunoassay, have been established (Gao et al., 1999; Maldiney et al., 1986; Fernandez et al., 1995). However, the sophisticated scientific instruments with complicated data collecting and processing systems are required, and thus high costs and the requirement for a professional operator severely limit their usage. Lanthanide chelates have been widely used as luminescence probes in highly sensitive fluoroimmunoassay and DNA hybridization assay (Yuan and Wang, 2006). In the previous works, we reported that a Eu3+ chelate, [4 -(9-anthryl)-2,2 :6 ,2 terpyridine-6,6 -diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu3+
∗ Corresponding authors. Tel.: +86 411 84706293; fax: +86 411 84706293. E-mail addresses: [email protected] (Z. Ye), [email protected] (J. Yuan). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.03.010
(ATTA-Eu3+ ), could be used as an efficient and specific luminescence probe for singlet oxygen (1 O2 ) based on the formation of strongly luminescent endoperoxide of ATTA-Eu3+ (EP-ATTA-Eu3+ ) (Song et al., 2005a,b). Recently, this probe was further used for monitoring the real-time IAA generation during the horseradish peroxidase (HRP)-catalyzed aerobic oxidation of IAA in tobacco cells (Guo et al., 2009). In this communication, we report the preparation of a novel luminescence sensor strip for the detection of IAA based on the quantum dots of cadmium telluride (CdTe QDs) and ATTA-Eu3+ , which displays the colorimetric IAA determination with precise, distinct, and tunable luminescence colors.
2. Experimental 2.1. Materials and methods The ligand ATTA and the water-soluble CdTe QDs (maximum emission wavelength at 522 nm) solution were synthesized by using the previously reported methods (Song et al., 2005b; Liu et al., 2008). (3-Aminopropyl)triethoxylsilane (APTES, 99%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), tetraethyl orthosilicate (TEOS, 99%) and Nhydroxysulfosuccinimide (NHS, 98.5%) were obtained from Acros Organics. IAA (crystalline) was purchased from Sigma. Horseradish peroxidase (HRP, R.Z. = 3.0) was purchased from Bio Basic Inc. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification.
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All luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer. A popular 365 nm UV-lamp was used as an excitation source for the sensor strip. A family use digital camera (IXUS-75, Canon Comp.) was used for recording the luminescence color images of the sensor strip. 2.2. Preparation of the sensor strip After a mixture of 100 L TEOS and 10 mL APTES was stirred in 1.0 mL methanol for 1 h, 1.0 mL of distilled water was introduced to the mixture for promotion of hydrolysis. After stirring for another 2 h, 1.0 mL of the CdTe QDs solution was added to the mixture with stirring, and then the mixture was stored in an incubator at 30 ± 0.2 ◦ C for 4 h. The mixture was dropped onto the surface of a glass slide (48 mm × 12.4 mm × 0.9 mm) that had been treated with 4 M NaOH, and then the slide was dried horizontally in darkness at 30 ± 0.2 ◦ C for more than 8 h. After 500 L of APTES was further dropped onto the slide surface, the slide was kept under darkness for another 8 h. To 20 L of 0.05 M carbonate buffer of pH 9.5 containing 0.71 mg of ATTA was added 80 L of ethanol containing 4.0 mg of EDC and 1.0 mg of NHS. After stirring for 2 h at room temperature, the solution was introduced to 1.0 mL of 0.05 M acetate buffer solution of pH 4 containing 1.0 mM of EuCl3 . The solution was stirred for another 2 h, and then was dip-coated onto the surface of CdTe QDs layer to form the sensing layer. 2.3. Reactions of the sensor strip with reactive oxygen species All the reactions were carried out in 0.05 M carbonate buffer of pH 10.5 on the sensor surface. Superoxide solution was prepared by adding KO2 to dry dimethyl sulfoxide and stirring vigorously for 10 min (Zhao et al., 2003). Hydroxyl radical (• OH) was generated through the Fenton reaction of ferrous ammonium sulfate and hydrogen peroxide (Zhao et al., 2003). Singlet oxygen was chemically generated from the MoO4 2− –H2 O2 system in alkaline media (Aubry and Cazin, 1988).
Fig. 1. (A) Emission spectra of ATTA-Eu3+ (ex = 335 nm) in the reaction with 1 O2 generated from the MoO4 2− –H2 O2 system (the inset shows the correlation of 1 O2 concentration and luminescence intensity). A series of H2 O2 solutions with the concentrations of 100, 50, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.01 and 0.0 mM were added to the 0.1 M carbonate buffer solution of pH 10.5 containing 100 M ATTAEu3+ and 10 mM Na2 MoO4 for the measurements, respectively. (B) Construction of the luminescence sensor strip for IAA.
2.4. Detection of IAA using the sensor strip Five microliters of 0.25 mM HRP was dropped onto the surface of the sensor strip, and dried at room temperature. Then 5.0 L of 0.05 M acetate buffer solutions of pH 4.0 containing different concentrations of IAA were dropped onto the surface of the HRP film. After incubating for 10 min at room temperature, the luminescence image of the sensor strip was recorded by the digital camera. 3. Results and discussion 3.1. Luminescence response of ATTA-Eu3+ to 1 O2 and IAA Ever since 1955, the aerobic oxidation of IAA catalyzed by HRP has been studied extensively, because this reaction may provide an evidence to explain the action mechanism of IAA as a plant growth regulator at molecular level (Kenten, 1955). In the previous reports, 1 O was demonstrated to be one of main products in the aerobic 2 oxidation of IAA catalyzed by HRP at lower pH (Kanofsky, 1988), and ATTA-Eu3+ was successfully used as a luminescence probe to monitor the 1 O2 generation in such a biochemical system (the most optimal condition of pH for this reaction is 4.0) (Song et al., 2005b). Therefore, the highly specific luminescence enhancement of ATTAEu3+ in the IAA-HRP system also allows the system available for the highly sensitive detection (a detection limit of 7.8 × 10−8 M) of IAA (Song et al., 2005b). In this work, ATTA-Eu3+ was used for the preparation of a luminescence sensor strip for the detection of IAA because of its excellent stability, selectivity and sensitivity
to 1 O2 . As shown in Fig. 1A, when almost non-luminescent ATTAEu3+ was reacted with 1 O2 , the emission intensity at 612 nm could be remarkably increased, and the luminescence response showed a good linearity in a wide 1 O2 concentration range. In addition, it has been demonstrated that the reaction between ATTA-Eu3+ and IAA in the presence of HRP and O2 is a first-order reaction with a large reaction rate constant at 109 M−1 s−1 level (Song et al., 2005b), which indicates that IAA can be rapidly traced by ATTA-Eu3+ to yield EP-ATTA-Eu3+ , accompanied by the remarkable increase of luminescence intensity. 3.2. Preparation and characterization of the luminescence sensor strip for IAA Fig. 1B shows the structure diagram of the QDs-ATTA-Eu3+ colorimetric luminescence sensor strip prepared in this work. The sensor is constructed by coating a green emissive CdTe QDs layer and a red emissive ATTA-Eu3+ layer on the surface of a glass slide, respectively. The use of the green emissive QDs for the sensor preparation is based on the following considerations: (1) both the QDs and the Eu3+ chelate probe can be simultaneously excited at a single wavelength; (2) the luminescence of the QDs is more stable against photobleaching than that of other organic luminescence dyes (Alivisatos, 2004); (3) the QDs can provide a clear and stable background luminescence that dose not interfere the observation of the Eu3+ probe luminescence, which enables the sensor strip to
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oxygen species. Another important factor of the sensor strip is the uniform distribution and adhesion of ATTA-Eu3+ molecules on the surface of the QDs layer. As shown in Fig. 1B, the rich amino groups on the QDs layer surface enable the EDC-NHS activated (Zhang et al., 2007) ATTA-Eu3+ molecules to be stably and homogeneously bound on the surface to form a specific sensing layer for IAA. Fig. 2B shows the emission spectra of the CdTe QDs-ATTA-Eu3+ HRP system in 0.05 M acetate buffer of pH 4.0 in the presence of different concentrations of IAA. It is obvious that the luminescence of the CdTe QDs at 522 nm is highly stable, and that of the Eu3+ probe at 612 nm is regularly increased with the increase of the IAA concentration. By plotting the luminescence intensity of the Eu3+ probe against the IAA concentration, a good straight line calibration curve that can be expressed as log(signal) = 0.191 log[IAA] + 1.926 (R = 0.997) was obtained in the IAA concentration range of 1.0–100 mM. Such a stable background color intensity and the linear relationship between the IAA concentration and the luminescence intensity of the Eu3+ probe assure a precise and quantitative determination of IAA.
3.3. Detection of IAA by the sensor strip
Fig. 2. (A) Emission spectra of four samples of the water-soluble CdTe QDs solutions. (B) Luminescence spectroscopic response of the CdTe QDs-ATTA-Eu3+ -HRP system towards various concentrations of IAA (0.0, 0.08, 0.2, 0.8, 4.0, 20.0, and 100 mM, ex = 335 nm). The inset shows the calibration curve for IAA (1.0–100 mM, the points at low IAA concentration level are deviated from the calibration curve).
be measured with diverse changes in luminescence colors for the colorimetric determination of IAA. Fig. 2A shows the emission spectra of four samples of the watersoluble CdTe QDs prepared in this work using a previous method (Liu et al., 2008). These QDs with narrow and symmetric emission peaks and the size-dependent emission wavelengths provide us a good opportunity to select the most suitable one for the preparation of the sensor’s background layer. In addition, the stable net structure of silicon–oxygen bonds in the sensor further provides a good environment to prevent QDs from the damage of reactive
In order to examine the selectivity of the sensor strip to 1 O2 (or IAA), the reactions of the sensor strip with different reactive oxygen species (1 O2 , • OH, O2 •− , H2 O2 ) were investigated under the same conditions. As shown in Fig. 3A, there was no notable change of the luminescence color after the sensor strip was reacted with • OH, O2 •− or H2 O2 , while a remarkable luminescence color change was observed after the sensor strip was reacted with 1 O2 . This result indicates that the sensor strip does not react with other reactive oxygen species except for 1 O2 . When a quencher of 1 O2 (azide, 1.0 mM) was dropped onto the sensor strip, the luminescence color change of the sensor strip was not observed. The above results demonstrate that the sensor strip prepared in this work is highly specific for 1 O2 , which can be attributed to the specific reactivity of anthracene unit in the sensing probe towards 1 O2 (Song et al., 2005b). Fig. 3B presents the luminescence colors of the sensor strip reacted with different concentrations of IAA (0.0–100 mM) under a 365 nm UV-lamp. When the HRP and IAA solutions were dropped onto the surface of the sensor strip, respectively, due to the luminescence enhancement of the sensing layer, the luminescence response of the sensor strip was given by a series of green-orangered “traffic light” color changes at different IAA concentrations, which was easier to be distinguished by the human eyes. Ordinarily, the naked human eyes have limited resolving power towards intensity changes in homochromatism (ca. 64 grades), but have a higher sensitivity to identify the color changes (ca. 10 million color
Fig. 3. (A) Luminescence colors of the sensor strip reacted with different reactive oxygen species. 1 O2 : 10 mM H2 O2 + 10 mM Na2 MoO4 ; • OH: 10 mM H2 O2 + 10 mM ferrous ammonium sulfate; O2 • − : 10 mM KO2 ; H2 O2 : 10 mM H2 O2 . (B) Luminescence color response of the sensor strip with (top) and without (bottom) a layer of the CdTe QDs towards various concentrations of IAA (from left to right: 100, 80, 50, 20, 10, 4.0, 2.0, 0.8, 0.2, 0.08, 0.02 and 0 mM).
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types). Without the green background emitted by the QDs layer, the naked human eyes have only lower resolution to identify the luminescence intensity change of ATTA-Eu3+ (Fig. 3B). Furthermore, different from traditional oxygen (O2 ) sensors (Wang et al., 2008; Evans et al., 2006), no luminescence quenching was observed during the experiment, indicating that the sensor strip could be used for the long-term continuous imaging detection of IAA. The limitation of the present method is that the sensor strip can only be used for the semi-quantitative detection of IAA. As shown in Fig. 3B, to distinguish the color difference by the naked human eyes when the concentration of IAA is more than 50 mM is still difficult. To solve this problem, a suitable instrument or device that can be used for the quantitative detection of the sensor strip is highly desirable. 4. Conclusion By using the green emissive CdTe QDs as a background layer and a red emissive Eu3+ chelate as a specific sensing layer coated on the surface of glass slide, respectively, a colorimetric luminescence sensor strip with green-yellow-red “traffic light” luminescence response for the detection of IAA was successfully prepared. The new sensor strip provides a simple and rapid method for both the qualitative and quantitative detection of IAA without using any special scientific instruments. Although the relative experiment was not carried out in this work, the sensor strip is also doubtless useful for the detection of IAA-related peroxidases. It is expected that a portable device with outstanding function for the quantitative detections of IAA and IAA-related peroxidase could be developed based on the present work.
Acknowledgments The authors acknowledge financial supports from the National Natural Science Foundation of China (Nos. 20835001, 20975017), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 200801410003). References Alivisatos, A.P., 2004. Nat. Biotechnol. 22, 47–52. Aubry, J.M., Cazin, B., 1988. Inorg. Chem. 27, 2013-2014. Davies, P.J., 1995. Plant Hormones: Physiology. Biochemistry and Molecular Biology, second ed. Kluwer, Dordrecht, Netherlands, pp. 1–12. Ding, X.H., Cao, Y.L., Huang, L.L., Zhao, J., Xu, C.G., Li, X.H., Wang, S.P., 2008. Plant Cell 20, 228–240. Evans, R.C., Douglas, P., Williams, J.A.G., Rochester, D.L., 2006. J. Fluoresc. 16, 201–206. Fernandez, B., Centeno, M.L., Feito, I., Sanchez-Tames, R., Rodriguez, A., 1995. Phytochem. Anal. 6, 49–54. Gao, H.Y., Jiang, T.B., Heineman, W.R., Halsall, H.B., Caruso, J.L., 1999. Fresenius J. Anal. Chem. 364, 170–174. Guo, W.H., Ye, Z.Q., Wang, G.L., Zhao, X.M., Yuan, J.L., Du, Y.G., 2009. Talanta 78, 977–982. Kanofsky, J.R., 1988. J. Biol. Chem. 263, 14171–14175. Kenten, R.H., 1955. Biochem. J. 59, 110–121. Liu, Y., Shen, Q., Yu, D., Shi, W., Li, J., Zhou, J., Liu, X., 2008. Nanotechnology 19, 245601. Maldiney, R., Leroux, B., Sabbagh, I., Sotta, B., Sossountzov, L., Miginiac, E., 1986. J. Immunol. Methods 90, 151–158. Song, B., Wang, G.L., Yuan, J.L., 2005a. Chem. Commun., 3553–3555. Song, B., Wang, G.L., Tan, M.Q., Yuan, J.L., 2005b. New J. Chem. 29, 1431–1438. Wang, X.D., Chen, X., Xie, Z.X., Wang, X.R., 2008. Angew. Chem. Int. Ed. 47, 7450–7453. Woodward, A.W., Bartel, B., 2005. Ann. Bot. (Lond.) 95, 707–735. Yuan, J.L., Wang, G.L., 2006. Trends Anal. Chem. 25, 490–500. Zhang, H., Xu, Y., Yang, W., Li, Q.G., 2007. Chem. Mater. 19, 5875–5881. Zhao, H.T., Kalivendi, S., Zhang, H., Joseph, J., Nithipatikom, K., Vasquez-Vivar, J., Kalyanaraman, B., 2003. Free Radic. Biol. Med. 34, 1359–1368.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Preparation of a novel colorimetric luminescence sensor strip for the detection of indole-3-acetic acid Yan Liu, Haitao Dong, Wenzhu Zhang, Zhiqiang Ye ∗ , Guilan Wang, Jingli Yuan ∗ State Key Laboratory of Fine Chemicals, Department of Chemistry, Dalian University of Technology, Dalian 116012, China
a r t i c l e
i n f o
Article history: Received 2 February 2010 Received in revised form 3 March 2010 Accepted 3 March 2010 Available online 10 March 2010 Keywords: Colorimetric luminescence sensor Europium Horseradish peroxidase Indole-3-acetic acid Plant auxin Quantum dots
a b s t r a c t A novel colorimetric luminescence sensor strip for the detection of indole-3-acetic acid (IAA) has been fabricated by using green emissive quantum dots of cadmium telluride (CdTe QDs) as a background layer and a red emissive europium chelate, [4 -(9-anthryl)-2,2 :6 ,2 -terpyridine-6,6 -diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu3+ (ATTA-Eu3+ ), as a specific sensing layer coated on the surface of glass slide, respectively. The luminescence response of the sensor strip is given by the dramatic changes in emission colors from green to red at different IAA concentrations. This approach provides a simple, rapid, sensitive and accurate method for the detection of IAA without using any special scientific instruments. © 2010 Elsevier B.V. All rights reserved.
1. Introduction As a minor component of metabolome, phytohormones are regulators produced by plants themselves, which are of particular significance given their role in the regulation of germination, growth, reproduction, and the protective responses of plants against stress (Davies, 1995). It has been known that the phytohormone indole-3-acetic acid (IAA) is one of the most important native auxins that can induce cell elongation and division with all subsequent results for plant growth and development (Woodward and Bartel, 2005; Ding et al., 2008). Recently, some sensitive methods for IAA determination in biological samples, such as GC–MS, LC–MS, HPLC and immunoassay, have been established (Gao et al., 1999; Maldiney et al., 1986; Fernandez et al., 1995). However, the sophisticated scientific instruments with complicated data collecting and processing systems are required, and thus high costs and the requirement for a professional operator severely limit their usage. Lanthanide chelates have been widely used as luminescence probes in highly sensitive fluoroimmunoassay and DNA hybridization assay (Yuan and Wang, 2006). In the previous works, we reported that a Eu3+ chelate, [4 -(9-anthryl)-2,2 :6 ,2 terpyridine-6,6 -diyl]bis(methylenenitrilo) tetrakis(acetate)-Eu3+
∗ Corresponding authors. Tel.: +86 411 84706293; fax: +86 411 84706293. E-mail addresses: [email protected] (Z. Ye), [email protected] (J. Yuan). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.03.010
(ATTA-Eu3+ ), could be used as an efficient and specific luminescence probe for singlet oxygen (1 O2 ) based on the formation of strongly luminescent endoperoxide of ATTA-Eu3+ (EP-ATTA-Eu3+ ) (Song et al., 2005a,b). Recently, this probe was further used for monitoring the real-time IAA generation during the horseradish peroxidase (HRP)-catalyzed aerobic oxidation of IAA in tobacco cells (Guo et al., 2009). In this communication, we report the preparation of a novel luminescence sensor strip for the detection of IAA based on the quantum dots of cadmium telluride (CdTe QDs) and ATTA-Eu3+ , which displays the colorimetric IAA determination with precise, distinct, and tunable luminescence colors.
2. Experimental 2.1. Materials and methods The ligand ATTA and the water-soluble CdTe QDs (maximum emission wavelength at 522 nm) solution were synthesized by using the previously reported methods (Song et al., 2005b; Liu et al., 2008). (3-Aminopropyl)triethoxylsilane (APTES, 99%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), tetraethyl orthosilicate (TEOS, 99%) and Nhydroxysulfosuccinimide (NHS, 98.5%) were obtained from Acros Organics. IAA (crystalline) was purchased from Sigma. Horseradish peroxidase (HRP, R.Z. = 3.0) was purchased from Bio Basic Inc. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification.
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All luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer. A popular 365 nm UV-lamp was used as an excitation source for the sensor strip. A family use digital camera (IXUS-75, Canon Comp.) was used for recording the luminescence color images of the sensor strip. 2.2. Preparation of the sensor strip After a mixture of 100 L TEOS and 10 mL APTES was stirred in 1.0 mL methanol for 1 h, 1.0 mL of distilled water was introduced to the mixture for promotion of hydrolysis. After stirring for another 2 h, 1.0 mL of the CdTe QDs solution was added to the mixture with stirring, and then the mixture was stored in an incubator at 30 ± 0.2 ◦ C for 4 h. The mixture was dropped onto the surface of a glass slide (48 mm × 12.4 mm × 0.9 mm) that had been treated with 4 M NaOH, and then the slide was dried horizontally in darkness at 30 ± 0.2 ◦ C for more than 8 h. After 500 L of APTES was further dropped onto the slide surface, the slide was kept under darkness for another 8 h. To 20 L of 0.05 M carbonate buffer of pH 9.5 containing 0.71 mg of ATTA was added 80 L of ethanol containing 4.0 mg of EDC and 1.0 mg of NHS. After stirring for 2 h at room temperature, the solution was introduced to 1.0 mL of 0.05 M acetate buffer solution of pH 4 containing 1.0 mM of EuCl3 . The solution was stirred for another 2 h, and then was dip-coated onto the surface of CdTe QDs layer to form the sensing layer. 2.3. Reactions of the sensor strip with reactive oxygen species All the reactions were carried out in 0.05 M carbonate buffer of pH 10.5 on the sensor surface. Superoxide solution was prepared by adding KO2 to dry dimethyl sulfoxide and stirring vigorously for 10 min (Zhao et al., 2003). Hydroxyl radical (• OH) was generated through the Fenton reaction of ferrous ammonium sulfate and hydrogen peroxide (Zhao et al., 2003). Singlet oxygen was chemically generated from the MoO4 2− –H2 O2 system in alkaline media (Aubry and Cazin, 1988).
Fig. 1. (A) Emission spectra of ATTA-Eu3+ (ex = 335 nm) in the reaction with 1 O2 generated from the MoO4 2− –H2 O2 system (the inset shows the correlation of 1 O2 concentration and luminescence intensity). A series of H2 O2 solutions with the concentrations of 100, 50, 25.6, 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.01 and 0.0 mM were added to the 0.1 M carbonate buffer solution of pH 10.5 containing 100 M ATTAEu3+ and 10 mM Na2 MoO4 for the measurements, respectively. (B) Construction of the luminescence sensor strip for IAA.
2.4. Detection of IAA using the sensor strip Five microliters of 0.25 mM HRP was dropped onto the surface of the sensor strip, and dried at room temperature. Then 5.0 L of 0.05 M acetate buffer solutions of pH 4.0 containing different concentrations of IAA were dropped onto the surface of the HRP film. After incubating for 10 min at room temperature, the luminescence image of the sensor strip was recorded by the digital camera. 3. Results and discussion 3.1. Luminescence response of ATTA-Eu3+ to 1 O2 and IAA Ever since 1955, the aerobic oxidation of IAA catalyzed by HRP has been studied extensively, because this reaction may provide an evidence to explain the action mechanism of IAA as a plant growth regulator at molecular level (Kenten, 1955). In the previous reports, 1 O was demonstrated to be one of main products in the aerobic 2 oxidation of IAA catalyzed by HRP at lower pH (Kanofsky, 1988), and ATTA-Eu3+ was successfully used as a luminescence probe to monitor the 1 O2 generation in such a biochemical system (the most optimal condition of pH for this reaction is 4.0) (Song et al., 2005b). Therefore, the highly specific luminescence enhancement of ATTAEu3+ in the IAA-HRP system also allows the system available for the highly sensitive detection (a detection limit of 7.8 × 10−8 M) of IAA (Song et al., 2005b). In this work, ATTA-Eu3+ was used for the preparation of a luminescence sensor strip for the detection of IAA because of its excellent stability, selectivity and sensitivity
to 1 O2 . As shown in Fig. 1A, when almost non-luminescent ATTAEu3+ was reacted with 1 O2 , the emission intensity at 612 nm could be remarkably increased, and the luminescence response showed a good linearity in a wide 1 O2 concentration range. In addition, it has been demonstrated that the reaction between ATTA-Eu3+ and IAA in the presence of HRP and O2 is a first-order reaction with a large reaction rate constant at 109 M−1 s−1 level (Song et al., 2005b), which indicates that IAA can be rapidly traced by ATTA-Eu3+ to yield EP-ATTA-Eu3+ , accompanied by the remarkable increase of luminescence intensity. 3.2. Preparation and characterization of the luminescence sensor strip for IAA Fig. 1B shows the structure diagram of the QDs-ATTA-Eu3+ colorimetric luminescence sensor strip prepared in this work. The sensor is constructed by coating a green emissive CdTe QDs layer and a red emissive ATTA-Eu3+ layer on the surface of a glass slide, respectively. The use of the green emissive QDs for the sensor preparation is based on the following considerations: (1) both the QDs and the Eu3+ chelate probe can be simultaneously excited at a single wavelength; (2) the luminescence of the QDs is more stable against photobleaching than that of other organic luminescence dyes (Alivisatos, 2004); (3) the QDs can provide a clear and stable background luminescence that dose not interfere the observation of the Eu3+ probe luminescence, which enables the sensor strip to
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oxygen species. Another important factor of the sensor strip is the uniform distribution and adhesion of ATTA-Eu3+ molecules on the surface of the QDs layer. As shown in Fig. 1B, the rich amino groups on the QDs layer surface enable the EDC-NHS activated (Zhang et al., 2007) ATTA-Eu3+ molecules to be stably and homogeneously bound on the surface to form a specific sensing layer for IAA. Fig. 2B shows the emission spectra of the CdTe QDs-ATTA-Eu3+ HRP system in 0.05 M acetate buffer of pH 4.0 in the presence of different concentrations of IAA. It is obvious that the luminescence of the CdTe QDs at 522 nm is highly stable, and that of the Eu3+ probe at 612 nm is regularly increased with the increase of the IAA concentration. By plotting the luminescence intensity of the Eu3+ probe against the IAA concentration, a good straight line calibration curve that can be expressed as log(signal) = 0.191 log[IAA] + 1.926 (R = 0.997) was obtained in the IAA concentration range of 1.0–100 mM. Such a stable background color intensity and the linear relationship between the IAA concentration and the luminescence intensity of the Eu3+ probe assure a precise and quantitative determination of IAA.
3.3. Detection of IAA by the sensor strip
Fig. 2. (A) Emission spectra of four samples of the water-soluble CdTe QDs solutions. (B) Luminescence spectroscopic response of the CdTe QDs-ATTA-Eu3+ -HRP system towards various concentrations of IAA (0.0, 0.08, 0.2, 0.8, 4.0, 20.0, and 100 mM, ex = 335 nm). The inset shows the calibration curve for IAA (1.0–100 mM, the points at low IAA concentration level are deviated from the calibration curve).
be measured with diverse changes in luminescence colors for the colorimetric determination of IAA. Fig. 2A shows the emission spectra of four samples of the watersoluble CdTe QDs prepared in this work using a previous method (Liu et al., 2008). These QDs with narrow and symmetric emission peaks and the size-dependent emission wavelengths provide us a good opportunity to select the most suitable one for the preparation of the sensor’s background layer. In addition, the stable net structure of silicon–oxygen bonds in the sensor further provides a good environment to prevent QDs from the damage of reactive
In order to examine the selectivity of the sensor strip to 1 O2 (or IAA), the reactions of the sensor strip with different reactive oxygen species (1 O2 , • OH, O2 •− , H2 O2 ) were investigated under the same conditions. As shown in Fig. 3A, there was no notable change of the luminescence color after the sensor strip was reacted with • OH, O2 •− or H2 O2 , while a remarkable luminescence color change was observed after the sensor strip was reacted with 1 O2 . This result indicates that the sensor strip does not react with other reactive oxygen species except for 1 O2 . When a quencher of 1 O2 (azide, 1.0 mM) was dropped onto the sensor strip, the luminescence color change of the sensor strip was not observed. The above results demonstrate that the sensor strip prepared in this work is highly specific for 1 O2 , which can be attributed to the specific reactivity of anthracene unit in the sensing probe towards 1 O2 (Song et al., 2005b). Fig. 3B presents the luminescence colors of the sensor strip reacted with different concentrations of IAA (0.0–100 mM) under a 365 nm UV-lamp. When the HRP and IAA solutions were dropped onto the surface of the sensor strip, respectively, due to the luminescence enhancement of the sensing layer, the luminescence response of the sensor strip was given by a series of green-orangered “traffic light” color changes at different IAA concentrations, which was easier to be distinguished by the human eyes. Ordinarily, the naked human eyes have limited resolving power towards intensity changes in homochromatism (ca. 64 grades), but have a higher sensitivity to identify the color changes (ca. 10 million color
Fig. 3. (A) Luminescence colors of the sensor strip reacted with different reactive oxygen species. 1 O2 : 10 mM H2 O2 + 10 mM Na2 MoO4 ; • OH: 10 mM H2 O2 + 10 mM ferrous ammonium sulfate; O2 • − : 10 mM KO2 ; H2 O2 : 10 mM H2 O2 . (B) Luminescence color response of the sensor strip with (top) and without (bottom) a layer of the CdTe QDs towards various concentrations of IAA (from left to right: 100, 80, 50, 20, 10, 4.0, 2.0, 0.8, 0.2, 0.08, 0.02 and 0 mM).
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types). Without the green background emitted by the QDs layer, the naked human eyes have only lower resolution to identify the luminescence intensity change of ATTA-Eu3+ (Fig. 3B). Furthermore, different from traditional oxygen (O2 ) sensors (Wang et al., 2008; Evans et al., 2006), no luminescence quenching was observed during the experiment, indicating that the sensor strip could be used for the long-term continuous imaging detection of IAA. The limitation of the present method is that the sensor strip can only be used for the semi-quantitative detection of IAA. As shown in Fig. 3B, to distinguish the color difference by the naked human eyes when the concentration of IAA is more than 50 mM is still difficult. To solve this problem, a suitable instrument or device that can be used for the quantitative detection of the sensor strip is highly desirable. 4. Conclusion By using the green emissive CdTe QDs as a background layer and a red emissive Eu3+ chelate as a specific sensing layer coated on the surface of glass slide, respectively, a colorimetric luminescence sensor strip with green-yellow-red “traffic light” luminescence response for the detection of IAA was successfully prepared. The new sensor strip provides a simple and rapid method for both the qualitative and quantitative detection of IAA without using any special scientific instruments. Although the relative experiment was not carried out in this work, the sensor strip is also doubtless useful for the detection of IAA-related peroxidases. It is expected that a portable device with outstanding function for the quantitative detections of IAA and IAA-related peroxidase could be developed based on the present work.
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