- Email: [email protected]
Quantum dot-based pH probe for quick study of enzyme reaction kinetics
Enzyme and Microbial Technology 41 (2007) 127–132
Quantum dot-based pH probe for quick study of enzyme reaction kinetics Dahai Yu a , Zhi Wang a , Yan Liu b , Li Jin b , Yueming Cheng a , Jianguang Zhou b,∗ , Shugui Cao a,∗ b
a Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China Institute for Miniature Instrumentation, College of Chemistry, Jilin Province Research Center for Engineering and Technology of Spectral Instruments, Jilin University, Changchun 130023, PR China
Received 2 June 2006; received in revised form 24 November 2006; accepted 15 December 2006
Abstract Water-soluble luminescent CdTe–ZnS quantum dots (QDs) modified by mercaptopropyl-acid was used to determinate H+ in aqueous solutions. The fluorescence (FL) of the water-soluble QDs could be irreversibly quenched by H+ and the fluorescence intensity of the water-soluble QDs decreased linearly as the pH decreased in the range of 8.0–5.0. Based on this phenomenon, a simple, rapid and specific method to quick determination of enzyme reaction kinetics was proposed. The modified QDs was successfully used as pH probe in monitoring the hydrolysis of glycidyl butyrate catalyzed by porcine pancreatic lipase (PPL). The proposed method demonstrated improved sensitivity, stability and a wider monitoring range for H+ determinations as compared to the already described analytical methods based on PNP. © 2006 Elsevier Inc. All rights reserved. Keywords: Quantum dot; Fluorescence intensity; pH probe; Quench; Enzyme; Hydrolysis
1. Introduction As an eminent representation of nanotechnology, QDs are currently under intensive studies in physics, chemistry, material science, and especially in biology [1,2]. Following the report of using QDs as fluorescence-based biological labeling by Chan and Nie [3], the number of QDs biological label or probe studies has increased exponentially due to their superior properties to the organic fluorescent dyes. Indeed, the molar extinction coefficients, two-photon absorption cross-sections, luminescence lifetimes, and photobleaching resistances of these inorganic nanoparticles are significantly greater than that of their organic counterparts. It has been estimated that CdSe quantum dots are about 20 times brighter and 100 times more stable than single rhodamine 6G molecules [3]. In addition, QDs exhibit size-dependent fluorescence emission spectra that span the visible spectrum. Their broad emission spectra allow simultaneous excitation of different particle sizes at a single wavelength with emission at multiple wavelengths [4]; in virtue of these unique characters, QDs labels have been successfully used for a variety of bioanalytical purposes, such as cell label [5], DNA probe ∗
Corresponding authors. Tel.: +86 431 88498972; fax: +86 431 88980440. E-mail address: [email protected] (S. Cao).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.12.012
[6], immunity label [7], and binding assays using fluorescence resonant energy transfer (FRET) to probe for target events [8]. Recently, valuable progress has been achieved in watersoluble QDs as ionic probe. For example, the use of modified CdSe QDs for the sensitive determination of cyanide ions has been proposed [9]. CdSe–ZnS QDs modified with bovine serum albumin are also investigated for the determination of copper ions [10]. Interestingly, QDs have been reported of pHdependence [11,12]. The sensitivity of QDs photoluminescence to pH, which is probably a function of surface modifications and effects on exciton trap sites [13,14], may lead to applications utilizing QDs as pH probes [15]. In the present study, the modified QDs are used as pH probe for quick detecting of reaction kinetic study of hydrolysis of glycidyl butyrate catalyzed by PPL. Advantages of the proposed methodology with respect to previously published methods based on PNP are also discussed. 2. Materials and methods 2.1. Materials and reagents Glycidyl butyrate was prepared according to a procedure previously described [16]. Porcine pancreatic lipase (PPL) was purchased from Sigma (USA). p-Nitrophenoxide (PNP), phosphate-buffered saline (PBS), Tris–HCl
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and acetonitrile were purchased from Shanghai Chemical Reagents Company (China) and used as received without further purification. QDs were kindly donated by College of Chemistry, Jilin University (Changchun, China). All experiments were done with ultra pure water.
2.2. Preparation of enzyme solution and substrate solution In order to remove the water-insoluble materials, crude PPL was dispersed in PBS (pH 7.0, 50 mmol) and stirred at 4 ◦ C for 2 h, the resultant solution was then centrifuged at 8000 rpm for 5 min and the supernatant was lyophilized. Enzyme solution (10 mg/ml) was prepared by dissolving the lyophilized PPL in phosphate buffer (pH 8.0, 100 mmol). Substrate solution (14 mmol/l) was prepared by dissolving glycidyl butyrate (20 l) in acetonitrile (10 ml).
2.3. Fluorescence detection of reaction by QDs Substrate solution (100 l), QDs solution (2.5 ml), and PBS (300 l, pH 7.0, 50 mmol) were mixed in a cuvette (4 ml). One minute later, enzyme solution (100 l) was added to the above mixture. The mixture was immediately detected using luminescence spectrometer. Fluorescence spectra were excited at 428 nm and were detected every 1 min with a RF-5301 (Shimadazu) luminescence spectrometer equipped with a 20 kW xenon discharge lamp as a light source. The initial rate of reaction was regarded as the slope of FL intensity decreasing.
2.4. UV detection of reaction by PNP PNP (230 l, 2 mmol/l), PBS (650 l, pH 7.0, 10 mmol/l), and substrate solution (70 l) were mixed in a quartz cuvette (1 ml). One minute later, enzyme solution (50 l) was added to the above mixture. The mixture was immediately detected using UV detector. UV absorption was detected every 1 min at 405 nm using UV-2550 (Shimadazu). The initial rate of reaction was regarded as the slope of adsorption value (OD) decreasing.
3. Results and discussion 3.1. Fluorescent feature of QDs The emission spectra of QDs modified with mercaptoaceticacid is shown in Fig. 1. The maximum emission wavelength is observed at 562 nm. The line width of the FL spectrum is narrow and symmetrical, showing that the QDs is nearly of monodisperse and uniform. This is much better than the emis-
Fig. 1. Fluorescence emission spectrum of QDs.
Fig. 2. The emission spectrum of QDs to various concentrations of QDs. (A–E) Concentration of QDs: 1.0 × 10−6 , 0.8 × 10−6 , 0.6 × 10−6 , 0.4 × 10−6 , and 0.2 × 10−6 mol/l. Inset shows the variation of the FL intensity with concentration of QDs at 562 nm. Conditions: QDs was diluted to various concentrations from 1 × 10−6 to 0.2 × 10−6 mol/l, respectively, and detected at 428 nm.
sion characteristics for typical organic dye species, which often have much broader and asymmetric emission profiles [17]. 3.2. Effect of the concentration on the FL intensity of QDs It is known that variations of the fluorescence intensity simply follow the concentration changes of the fluorescence probe [18]. As shown in Fig. 2, the FL intensity of QDs increases linearly with the concentration of QDs increasing, whereas the line width and maximum emission wavelength keeps unchanged under the experimental concentration range. 3.3. Effect of temperature on the FL intensity of QDs Temperature has great effect on the FL intensity and peak position of water-soluble core/shell QDs. As shown in Fig. 3, the FL intensity of QDs decreases about 25% with the temperature increasing from 20 to 50 ◦ C. Similar results have been reported by Knight et al. [19]. As temperature increases, most carriers that are in deep trap sites emit non-radiation. Thus, as for the water-soluble core/shell CdTe/ZnS, the increased temperature results in the decreased quantum yield of fluorescence, which contributes to the declining FL intensity when it excites with the same energy [20]. In addition, it is found that the fluorescence emission peak of QDs shifts towards the red at a rate of 0.42 nm/◦ C, when the temperature increases. Temperature effect on FL peak shift of QDs may attribute to the variation of particle size of QDs. It is known that the wavelength of fluorescence depends on the bandgap and thus on the size of the quantum dot [21,22]. The larger the particle size of QDs is, the higher the fluorescence emission peak of QDs will be [13,23]. As the temperature increases, the particle size of QDs may increase concomitantly. Further, because a single wavelength can be used for simultaneous excitation of all different-sized QDs, the variation of fluorescence emission peak of QDs under various temperatures can be observed. Temperature effect on FL peak shift of
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with the core. Therefore, part of QD particles may be destroyed by the added H+ . It is also found that Te is separated out from solution, when abundant H+ is added to the QDs solution. It is known that synthesis of CdTe QDs is used by NaHTe and CdCl2 [17]. Addition of H+ may induce reversible reaction, and Te is precipitated from the unstable NaHTe which is quickly oxidized by O2 . In addition, the emission spectrum of QDs exhibits a red shift in the emission peak with pH decreasing, although the intrinsic emission bandwidth is unchanged. The shift of the emission peak may result from the conjugation between CdTe nanoparticles and H+ , and the increased surface electric charge may increase the orientation polarization rate and the Stokes shift [25]. Although the fundamental mechanisms of these changes are still unknown, it is clear that the fluorescence signals of core/shell quantum dots are sensitive to H+ . Fig. 3. The emission spectrum of QDs to the variation of temperature. (A–G) 20, 25, 30, 35, 40, 45, and 50 ◦ C. Inset shows the variation of the FL intensity with temperature at the maximum emission wavelength. Conditions: QDs solution (3 ml) was incubated under various temperatures for 1 min and detected at 428 nm.
QDs clusters may also be attributed to inter-QDs dipole–dipole interactions [24]; however, the real reasons are still not clear. 3.4. Effect of buffer on the FL intensity of QDs 3.4.1. Effect of pH on the FL intensity of QDs It is known that QDs are pH sensitive. However, the effect of pH on FL intensity of QDs varies with different reports [11,14]. In the present study, it is observed that the FL intensity of QDs linearly decreases by 90% with pH deceasing from 8.0 to 5.0 (Fig. 4). The restoration of the FL intensity of QDs cannot be achieved by adding OH− . It may suggest that the fluorescence quenching by H+ is irreversibly. The possible explanation may be that not all particles of QDs are perfectly capped with the shell and the added H+ can pass through the shell layer and interact
Fig. 4. The emission spectrum of QDs to the variation of pH. (A–H) pH 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Inset shows the variation of the FL intensity with pH at 562 nm. Conditions: QDs solutions were incubated in PBS (100 mmol) with various pH, respectively, and detected at 428 nm.
3.4.2. Effect of various buffers on the FL intensity of QDs For enzymatic reaction, buffers are widely used to hold the activity and stability of the enzyme. However, it has been reported that cations have effects on QDs [10], and some ionic may quench the FL intensity of QDs. So finding a proper buffer seems important when QDs is used as probe. Effect of various buffers on the FL intensity of QDs is investigated by adding the same amount of buffers in the QDs, respectively. As shown in Fig. 5, the FL intensity of QDs decreases 40% and 13% in the PBS and Tris–HCl, respectively, compared with that of the same amount of water is added. The decrease of FL intensity of QDs in buffers can be interpreted in view of an interaction between particle surfaces of QDs. Either the cation or anion of buffers may have effect on the QDs structure and decrease the FL intensity of QDs [26]. 3.4.3. Effect of ionic strength on the FL intensity of QDs Since ionic strength may influence the surface electrostatics of the QDs, fluorescence spectrum of QDs is detected by changing the ionic strength of buffers. For both of the two buffers,
Fig. 5. The emission spectrum of QDs to the addition of various buffers. (A–C) Water (pH 7.0); Tris–HCl (pH 7.0); PBS (pH 7.0). Conditions: QDs solution (1 ml) was added to ultra pure water (2 ml), Tris–HCl (2 ml, 100 mmol) and PBS solution (2 ml, 100 mmol) with the same pH value (7.0), respectively, and detected at 428 nm.
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Fig. 6. The emission spectrum of QDs to addition of various ionic strength of (a) PBS and (b) Tris–HCl. (A–E) Ionic strengths: 100, 200, 300, 400, and 500 mmol/l. Inset shows the variation of the FL intensity with ionic strengths of PBS or Tris–HCl at 562 nm. Conditions: PBS or Tris–HCl buffer (2 ml) with various ionic strengths was added to QDs solution (1 ml), respectively, and detected at 428 nm.
Scheme 1. Lipase catalyzed hydrolysis of glycidyl butyrate.
Scheme 2. Principle of PNP as pH probe.
charged particles tend to aggregate. The higher the ionic strength is, the higher the QDs flocculate. Qualitatively, this phenomenon is explained by the reduction of the thickness of the so-called diffuse layer of a counter-ion cloud surrounding a charged particle with increasing ionic strength. As a consequence, in buffercontaining solutions charged particles can approach each other closely enough during thermal collisions so that the repulsive
Fig. 7. The emission spectrum of QDs to hydrolysis of glycidyl butyrate at various time. (A–H) 1, 2, 3, 4, 5, 6, 7, and 8 min. Inset shows the variation of the FL intensity with time of hydrolysis at 562 nm.
the FL intensity of QDs decreases with ionic strength increasing (Fig. 6). In this context, QDs is modified by mercaptopropyl-acid for mediating solubility in water. Coulomb repulsion between nanocrystals with surface charge of the same polarity prevents aggregation in water. However, in buffer-containing solutions Table 1 Influence of additives on the FL intensity of QDs Additive
FL intensity
Ultra pure water Substrate Acetonitrile Enzyme solution Glycidol
188.74 187.81 188.25 188.66 187.37
Reaction conditions: Additives (100 l) were added to QDs solution (2.9 ml), respectively. Temperature: 30 ◦ C. The FL intensity of QDs was 196 at maximum emission wavelength without any reagents or products.
Fig. 8. Stability of QDs and PNP. (䊉) The variation of QDs, () the variation of PNP without buffer, and () the variation of PNP with high ionic strength of buffer (200 mmol). The pH of all the detected solutions were 7.0.
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Fig. 9. Absorption decays with different substrate concentration of PNP (a) and QDs (b). Substrate concentration: (䊉) 1.0 × 10−3 mol/l, () 2.0 × 10−3 mol/l, () 4.0 × 10−3 mol/l, and (+) 8.0 × 10−3 mol/l. The symbols represent experimental data and the solid lines were calculated by fitting the data. Conditions: The reaction was performed under the standard method described in Sections 2.3 and 2.4 except that the substrate concentration varied from 1.0 × 10−3 to 8.0 × 10−3 mol/l.
electrostatic forces are overcome by the short range attractive van der Waals forces [5]. 3.5. QDs as pH probe for enzyme-catalyzed hydrolysis It is well known that H+ will be released when an ester is hydrolyzed by lipases [27,28]. The releasing rate of H+ that represents the activity of lipase is detected by monitoring the hydrolysis. Herein, QDs is used as fluorescence pH probe to monitor the hydrolysis of glycidyl butyrate catalyzed by lipase (Scheme 1). The initial reaction rate of hydrolysis of glycidyl butyrate catalyzed by PPL is shown in Fig. 7. It is observed that the FL intensity of QDs linearly decreases with time increasing. It indicates that QDs has been successfully used as pH probe for quick enzyme reaction kinetic study. 3.5.1. Effect of external environment on the FL intensity of QDs Since QDs are sensitive to the external environment, the effect of substrate, solvent, enzyme solution and products on the FL intensity of QDs in enzyme-catalyzed reaction was investigated. No significant influence of all these materials on the FL intensity of QDs is observed (Table 1). It suggests that the decreasing of FL intensity during the reaction is caused by the released acid. 3.5.2. Comparison of PNP and QDs as pH probe PNP is widely used as pH probe for monitoring the releasing of protons during the ester hydrolysis [29]. The yellow color of the phenoxide fades as the reaction proceeds and reveals quantitatively the amount of ester hydrolyzed (Scheme 2). However, there are some disadvantages for PNP as pH probe: firstly, it is unstable. When PNP is incubated in a buffer with a fixed ionic strength, the OD value of PNP persistent varies at either low or high ionic strength (Fig. 8). It seems important to select a buffer with proper ionic strength to inhibit the variation of OD value of PNP solution. In contrast, this phenomenon is not observed for QDs, and the FL intensity of QDs does not fluctuate with the variation of time at a fixed ionic strength. Secondly, the reaction rate could only be correctly monitored for UV detection when OD value is
lower than 1. However, no such limit is observed for QDs. Thirdly, the sensitivity of fluorescence analysis is 10–100 multiple than spectroscopic method, leading detection limit of QDs to be much lower than PNP. Finally, in order to compare the monitoring range of QDs and PNP, the same probe concentration (10−4 mol/l) is used in the same reaction. As shown in Fig. 9a, the OD value of PNP decreases very fast with time increasing, especially at high substrate concentration. When exceeding 10 min, PNP is almost depleted and the reaction rate could not be detected correctly. On the other hand, QDs can be used to detect the reaction rate correctly even exceeds 15 min with the high concentration of substrate. It suggests that ODs has a wider monitoring range than PNP (Fig. 9b). 4. Conclusion Water-soluble CdTe/ZnS QDs are sensitive to environmental factors and found to be a satisfactory pH probe that could have potential applications in chemical and biochemical sensing. The probable H+ quenching mechanism can be explained as the addition of H+ may cause reversible reaction and Te is oxidized by oxygen and precipitated from the solution. The modified QDs is successfully used as pH probe in monitoring the hydrolysis of glycidyl butyrate catalyzed by PPL. In addition, we attempt to use QDs in culture medium and detect the released acid during the cultivation of bacterium. Clearly, the potential of QDs as pH probe has just begun to be realized; therefore, one is likely to see more work in this area in the coming years. Acknowledgements The authors are grateful for the financial support from National Natural Science Foundation of China (No. 20272017) and the Foundation of Research Program of Jilin University, China. References [1] Alivisatos AP. Semiconductor clusters, nanocrystals, and quantum dots. Science 1996;271:933–7.
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[2] Bruchez M, Moronne M, Gin P, Weiss S, Alivisatos AP. Semiconductor nanocrystals as fluorescent biological labels. Science 1998;281: 2013–6. [3] Chan WCW, Nie SM. Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 1998;281:2016–8. [4] Han M, Gao X, Su JZ, Nie S. Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 2001;19: 631–5. [5] Parak WJ, Pellegrino T, Plank C. Labelling of cells with quantum dots. Nanotechnology 2005;16:R9–25. [6] Huang ZL, Zhao YD, Luo QM. Quantum-dot-tagged microbeads and their use as fluorescent biological probes. Curr Anal Chem 2006;2:59– 66. [7] Goldman ER, Balighian ED, Mattoussi H, Kuno MK, Mauro JM, Tran PT, et al. Avidin: a natural bridge for quantum dot-antibody conjugates. J Am Chem Soc 2002;124:6378–82. [8] Willard DM, Carillo LL, Jung J, van Orden A. CdSe–ZnS quantum dots as resonance energy transfer donors in a model protein–protein binding assay. NanoLett 2001;1:469–74. [9] Jin WJ, Fernandez-Arguelles MT, Costa-Fernandez JM, Pereiro R, SanzMedel A. Photoactivated luminescent CdSe quantum dots as sensitive cyanide probes in aqueous solutions. Chem Commun 2005;7:883–5. [10] Xie HY, Liang HG, Zhang ZL, Liu Y, He ZK, Pang DW. Luminescent CdSe–ZnS quantum dots as selective Cu2+ probe. Spectrochim Acta A 2004;60:2527–30. [11] Tomasulo M, Yildiz I, Raymo FMJ. pH-sensitive quantum dots. J Phys Chem B 2006;110:3853–5. [12] Sun YH, Liu YS, Vernier PT, Liang CH, Chong SY, Marcu L, et al. Photostability and pH sensitivity of CdSe/ZnSe/ZnS quantum dots in living cells. Nanotechnology 2006;17:4469–76. [13] Gao MY, Kirstein S, Mohwald H, Rogach A, Kornowski A, Eychmuller A, et al. Strongly photoluminescent CdTe nanocrystals by proper surface modification. J Phys Chem B 1998;102:8360–3. [14] Zhang H, Zhou Z, Yang B, Gao MY. The influence of carboxyl groups on the photoluminescence of mercaptocarboxylic acid-stabilized CdTe nanoparticles. J Phys Chem B 2003;107:8–13. [15] Chen YF, Rosenzweig Z. Luminescent CdS quantum dots as selective ion probes. Anal Chem 2002;74:5132–8.
[16] Yu DH, Wang Z, Zheng LY, Zhang GR, Cao SG. Screening the reaction system for enzymatic resolution of glycidyl butyrate. Chinese J Bioprocess Eng 2004;2:40–3. [17] Rosenthal SJ. Bar-coding biomolecules with fluorescent nanocrystals. Nat Biotechnol 2001;19:621–2. [18] Marcotte N, Brouwer AM. Carboxy SNARF-4F as a fluorescent pH probe for ensemble and fluorescence correlation spectroscopies. J Phys Chem B 2005;109:11819–28. [19] Knight A, Gaunt J, Davidson T, Chechik V, Windsor S. Evaluation of the suitability of quantum dots as fluorescence standards. Npl Report Dql-As 2004:007. [20] Liu TC, Huang ZL, Wang HQ, Wang JH, Li XQ, Zhao YD, et al. Temperature-dependent photoluminescence of water-soluble quantum dots for a bioprobe. Anal Chim Acta 2006;559:120–3. [21] Murray CB, Kagan CR, Bawendi MG. Synthesis and characterization of monodisperse nanocrystals and close-packed nanocrystal assemblies. Annu Rev Mater Sci 2000;30:545–610. [22] Qu L, Peng X. Control of photoluminescence properties of CdSe nanocrystals in growth. J Am Chem Soc 2002;124:2049–55. [23] Costa-Fernandez JM. Optical sensors based on luminescent quantum dots. Anal Bioanal Chem 2006;384:37–40. [24] Biju V, Makita Y, Sonoda A, Yokoyama H, Baba Y, Ishikawa M. Temperature-sensitive photoluminescence of CdSe quantum dot clusters. J Phys Chem B 2005;109:13899–905. [25] Lakowicz JR. Principles of Fluorescence Spectroscopy. New York: Plenum Press; 1983. pp. 187–256. [26] Boldt K, Bruns OT, Gaponik N, Eychmuller A. Comparative examination of the stability of semiconductor quantum dots in various biochemical buffers. J Phys Chem B 2006;110:1959–63. [27] Long WS, Kow PC, Kamaruddin AH, Bhatia S. Comparison of kinetic resolution between two racemic ibuprofen esters in an enzymic membrane reactor. Process Biochem 2005;40:2417–25. [28] Chen CC, Tsai SW. Carica papaya lipase: a novel biocatalyst for the enantioselective hydrolysis of (R,S)-naproxen 2,2,2-trifluoroethyl ester. Enzyme Microb Technol 2005;36:127–32. [29] Somers NA, Kazlauskas RJ. Mapping the substrate selectivity and enantioselectivity of esterases from thermophiles. Tetrahedron: Asymmetry 2004;15:2991–3004.
Quantum dot-based pH probe for quick study of enzyme reaction kinetics Dahai Yu a , Zhi Wang a , Yan Liu b , Li Jin b , Yueming Cheng a , Jianguang Zhou b,∗ , Shugui Cao a,∗ b
a Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023, PR China Institute for Miniature Instrumentation, College of Chemistry, Jilin Province Research Center for Engineering and Technology of Spectral Instruments, Jilin University, Changchun 130023, PR China
Received 2 June 2006; received in revised form 24 November 2006; accepted 15 December 2006
Abstract Water-soluble luminescent CdTe–ZnS quantum dots (QDs) modified by mercaptopropyl-acid was used to determinate H+ in aqueous solutions. The fluorescence (FL) of the water-soluble QDs could be irreversibly quenched by H+ and the fluorescence intensity of the water-soluble QDs decreased linearly as the pH decreased in the range of 8.0–5.0. Based on this phenomenon, a simple, rapid and specific method to quick determination of enzyme reaction kinetics was proposed. The modified QDs was successfully used as pH probe in monitoring the hydrolysis of glycidyl butyrate catalyzed by porcine pancreatic lipase (PPL). The proposed method demonstrated improved sensitivity, stability and a wider monitoring range for H+ determinations as compared to the already described analytical methods based on PNP. © 2006 Elsevier Inc. All rights reserved. Keywords: Quantum dot; Fluorescence intensity; pH probe; Quench; Enzyme; Hydrolysis
1. Introduction As an eminent representation of nanotechnology, QDs are currently under intensive studies in physics, chemistry, material science, and especially in biology [1,2]. Following the report of using QDs as fluorescence-based biological labeling by Chan and Nie [3], the number of QDs biological label or probe studies has increased exponentially due to their superior properties to the organic fluorescent dyes. Indeed, the molar extinction coefficients, two-photon absorption cross-sections, luminescence lifetimes, and photobleaching resistances of these inorganic nanoparticles are significantly greater than that of their organic counterparts. It has been estimated that CdSe quantum dots are about 20 times brighter and 100 times more stable than single rhodamine 6G molecules [3]. In addition, QDs exhibit size-dependent fluorescence emission spectra that span the visible spectrum. Their broad emission spectra allow simultaneous excitation of different particle sizes at a single wavelength with emission at multiple wavelengths [4]; in virtue of these unique characters, QDs labels have been successfully used for a variety of bioanalytical purposes, such as cell label [5], DNA probe ∗
Corresponding authors. Tel.: +86 431 88498972; fax: +86 431 88980440. E-mail address: [email protected] (S. Cao).
0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.12.012
[6], immunity label [7], and binding assays using fluorescence resonant energy transfer (FRET) to probe for target events [8]. Recently, valuable progress has been achieved in watersoluble QDs as ionic probe. For example, the use of modified CdSe QDs for the sensitive determination of cyanide ions has been proposed [9]. CdSe–ZnS QDs modified with bovine serum albumin are also investigated for the determination of copper ions [10]. Interestingly, QDs have been reported of pHdependence [11,12]. The sensitivity of QDs photoluminescence to pH, which is probably a function of surface modifications and effects on exciton trap sites [13,14], may lead to applications utilizing QDs as pH probes [15]. In the present study, the modified QDs are used as pH probe for quick detecting of reaction kinetic study of hydrolysis of glycidyl butyrate catalyzed by PPL. Advantages of the proposed methodology with respect to previously published methods based on PNP are also discussed. 2. Materials and methods 2.1. Materials and reagents Glycidyl butyrate was prepared according to a procedure previously described [16]. Porcine pancreatic lipase (PPL) was purchased from Sigma (USA). p-Nitrophenoxide (PNP), phosphate-buffered saline (PBS), Tris–HCl
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and acetonitrile were purchased from Shanghai Chemical Reagents Company (China) and used as received without further purification. QDs were kindly donated by College of Chemistry, Jilin University (Changchun, China). All experiments were done with ultra pure water.
2.2. Preparation of enzyme solution and substrate solution In order to remove the water-insoluble materials, crude PPL was dispersed in PBS (pH 7.0, 50 mmol) and stirred at 4 ◦ C for 2 h, the resultant solution was then centrifuged at 8000 rpm for 5 min and the supernatant was lyophilized. Enzyme solution (10 mg/ml) was prepared by dissolving the lyophilized PPL in phosphate buffer (pH 8.0, 100 mmol). Substrate solution (14 mmol/l) was prepared by dissolving glycidyl butyrate (20 l) in acetonitrile (10 ml).
2.3. Fluorescence detection of reaction by QDs Substrate solution (100 l), QDs solution (2.5 ml), and PBS (300 l, pH 7.0, 50 mmol) were mixed in a cuvette (4 ml). One minute later, enzyme solution (100 l) was added to the above mixture. The mixture was immediately detected using luminescence spectrometer. Fluorescence spectra were excited at 428 nm and were detected every 1 min with a RF-5301 (Shimadazu) luminescence spectrometer equipped with a 20 kW xenon discharge lamp as a light source. The initial rate of reaction was regarded as the slope of FL intensity decreasing.
2.4. UV detection of reaction by PNP PNP (230 l, 2 mmol/l), PBS (650 l, pH 7.0, 10 mmol/l), and substrate solution (70 l) were mixed in a quartz cuvette (1 ml). One minute later, enzyme solution (50 l) was added to the above mixture. The mixture was immediately detected using UV detector. UV absorption was detected every 1 min at 405 nm using UV-2550 (Shimadazu). The initial rate of reaction was regarded as the slope of adsorption value (OD) decreasing.
3. Results and discussion 3.1. Fluorescent feature of QDs The emission spectra of QDs modified with mercaptoaceticacid is shown in Fig. 1. The maximum emission wavelength is observed at 562 nm. The line width of the FL spectrum is narrow and symmetrical, showing that the QDs is nearly of monodisperse and uniform. This is much better than the emis-
Fig. 1. Fluorescence emission spectrum of QDs.
Fig. 2. The emission spectrum of QDs to various concentrations of QDs. (A–E) Concentration of QDs: 1.0 × 10−6 , 0.8 × 10−6 , 0.6 × 10−6 , 0.4 × 10−6 , and 0.2 × 10−6 mol/l. Inset shows the variation of the FL intensity with concentration of QDs at 562 nm. Conditions: QDs was diluted to various concentrations from 1 × 10−6 to 0.2 × 10−6 mol/l, respectively, and detected at 428 nm.
sion characteristics for typical organic dye species, which often have much broader and asymmetric emission profiles [17]. 3.2. Effect of the concentration on the FL intensity of QDs It is known that variations of the fluorescence intensity simply follow the concentration changes of the fluorescence probe [18]. As shown in Fig. 2, the FL intensity of QDs increases linearly with the concentration of QDs increasing, whereas the line width and maximum emission wavelength keeps unchanged under the experimental concentration range. 3.3. Effect of temperature on the FL intensity of QDs Temperature has great effect on the FL intensity and peak position of water-soluble core/shell QDs. As shown in Fig. 3, the FL intensity of QDs decreases about 25% with the temperature increasing from 20 to 50 ◦ C. Similar results have been reported by Knight et al. [19]. As temperature increases, most carriers that are in deep trap sites emit non-radiation. Thus, as for the water-soluble core/shell CdTe/ZnS, the increased temperature results in the decreased quantum yield of fluorescence, which contributes to the declining FL intensity when it excites with the same energy [20]. In addition, it is found that the fluorescence emission peak of QDs shifts towards the red at a rate of 0.42 nm/◦ C, when the temperature increases. Temperature effect on FL peak shift of QDs may attribute to the variation of particle size of QDs. It is known that the wavelength of fluorescence depends on the bandgap and thus on the size of the quantum dot [21,22]. The larger the particle size of QDs is, the higher the fluorescence emission peak of QDs will be [13,23]. As the temperature increases, the particle size of QDs may increase concomitantly. Further, because a single wavelength can be used for simultaneous excitation of all different-sized QDs, the variation of fluorescence emission peak of QDs under various temperatures can be observed. Temperature effect on FL peak shift of
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with the core. Therefore, part of QD particles may be destroyed by the added H+ . It is also found that Te is separated out from solution, when abundant H+ is added to the QDs solution. It is known that synthesis of CdTe QDs is used by NaHTe and CdCl2 [17]. Addition of H+ may induce reversible reaction, and Te is precipitated from the unstable NaHTe which is quickly oxidized by O2 . In addition, the emission spectrum of QDs exhibits a red shift in the emission peak with pH decreasing, although the intrinsic emission bandwidth is unchanged. The shift of the emission peak may result from the conjugation between CdTe nanoparticles and H+ , and the increased surface electric charge may increase the orientation polarization rate and the Stokes shift [25]. Although the fundamental mechanisms of these changes are still unknown, it is clear that the fluorescence signals of core/shell quantum dots are sensitive to H+ . Fig. 3. The emission spectrum of QDs to the variation of temperature. (A–G) 20, 25, 30, 35, 40, 45, and 50 ◦ C. Inset shows the variation of the FL intensity with temperature at the maximum emission wavelength. Conditions: QDs solution (3 ml) was incubated under various temperatures for 1 min and detected at 428 nm.
QDs clusters may also be attributed to inter-QDs dipole–dipole interactions [24]; however, the real reasons are still not clear. 3.4. Effect of buffer on the FL intensity of QDs 3.4.1. Effect of pH on the FL intensity of QDs It is known that QDs are pH sensitive. However, the effect of pH on FL intensity of QDs varies with different reports [11,14]. In the present study, it is observed that the FL intensity of QDs linearly decreases by 90% with pH deceasing from 8.0 to 5.0 (Fig. 4). The restoration of the FL intensity of QDs cannot be achieved by adding OH− . It may suggest that the fluorescence quenching by H+ is irreversibly. The possible explanation may be that not all particles of QDs are perfectly capped with the shell and the added H+ can pass through the shell layer and interact
Fig. 4. The emission spectrum of QDs to the variation of pH. (A–H) pH 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Inset shows the variation of the FL intensity with pH at 562 nm. Conditions: QDs solutions were incubated in PBS (100 mmol) with various pH, respectively, and detected at 428 nm.
3.4.2. Effect of various buffers on the FL intensity of QDs For enzymatic reaction, buffers are widely used to hold the activity and stability of the enzyme. However, it has been reported that cations have effects on QDs [10], and some ionic may quench the FL intensity of QDs. So finding a proper buffer seems important when QDs is used as probe. Effect of various buffers on the FL intensity of QDs is investigated by adding the same amount of buffers in the QDs, respectively. As shown in Fig. 5, the FL intensity of QDs decreases 40% and 13% in the PBS and Tris–HCl, respectively, compared with that of the same amount of water is added. The decrease of FL intensity of QDs in buffers can be interpreted in view of an interaction between particle surfaces of QDs. Either the cation or anion of buffers may have effect on the QDs structure and decrease the FL intensity of QDs [26]. 3.4.3. Effect of ionic strength on the FL intensity of QDs Since ionic strength may influence the surface electrostatics of the QDs, fluorescence spectrum of QDs is detected by changing the ionic strength of buffers. For both of the two buffers,
Fig. 5. The emission spectrum of QDs to the addition of various buffers. (A–C) Water (pH 7.0); Tris–HCl (pH 7.0); PBS (pH 7.0). Conditions: QDs solution (1 ml) was added to ultra pure water (2 ml), Tris–HCl (2 ml, 100 mmol) and PBS solution (2 ml, 100 mmol) with the same pH value (7.0), respectively, and detected at 428 nm.
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Fig. 6. The emission spectrum of QDs to addition of various ionic strength of (a) PBS and (b) Tris–HCl. (A–E) Ionic strengths: 100, 200, 300, 400, and 500 mmol/l. Inset shows the variation of the FL intensity with ionic strengths of PBS or Tris–HCl at 562 nm. Conditions: PBS or Tris–HCl buffer (2 ml) with various ionic strengths was added to QDs solution (1 ml), respectively, and detected at 428 nm.
Scheme 1. Lipase catalyzed hydrolysis of glycidyl butyrate.
Scheme 2. Principle of PNP as pH probe.
charged particles tend to aggregate. The higher the ionic strength is, the higher the QDs flocculate. Qualitatively, this phenomenon is explained by the reduction of the thickness of the so-called diffuse layer of a counter-ion cloud surrounding a charged particle with increasing ionic strength. As a consequence, in buffercontaining solutions charged particles can approach each other closely enough during thermal collisions so that the repulsive
Fig. 7. The emission spectrum of QDs to hydrolysis of glycidyl butyrate at various time. (A–H) 1, 2, 3, 4, 5, 6, 7, and 8 min. Inset shows the variation of the FL intensity with time of hydrolysis at 562 nm.
the FL intensity of QDs decreases with ionic strength increasing (Fig. 6). In this context, QDs is modified by mercaptopropyl-acid for mediating solubility in water. Coulomb repulsion between nanocrystals with surface charge of the same polarity prevents aggregation in water. However, in buffer-containing solutions Table 1 Influence of additives on the FL intensity of QDs Additive
FL intensity
Ultra pure water Substrate Acetonitrile Enzyme solution Glycidol
188.74 187.81 188.25 188.66 187.37
Reaction conditions: Additives (100 l) were added to QDs solution (2.9 ml), respectively. Temperature: 30 ◦ C. The FL intensity of QDs was 196 at maximum emission wavelength without any reagents or products.
Fig. 8. Stability of QDs and PNP. (䊉) The variation of QDs, () the variation of PNP without buffer, and () the variation of PNP with high ionic strength of buffer (200 mmol). The pH of all the detected solutions were 7.0.
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Fig. 9. Absorption decays with different substrate concentration of PNP (a) and QDs (b). Substrate concentration: (䊉) 1.0 × 10−3 mol/l, () 2.0 × 10−3 mol/l, () 4.0 × 10−3 mol/l, and (+) 8.0 × 10−3 mol/l. The symbols represent experimental data and the solid lines were calculated by fitting the data. Conditions: The reaction was performed under the standard method described in Sections 2.3 and 2.4 except that the substrate concentration varied from 1.0 × 10−3 to 8.0 × 10−3 mol/l.
electrostatic forces are overcome by the short range attractive van der Waals forces [5]. 3.5. QDs as pH probe for enzyme-catalyzed hydrolysis It is well known that H+ will be released when an ester is hydrolyzed by lipases [27,28]. The releasing rate of H+ that represents the activity of lipase is detected by monitoring the hydrolysis. Herein, QDs is used as fluorescence pH probe to monitor the hydrolysis of glycidyl butyrate catalyzed by lipase (Scheme 1). The initial reaction rate of hydrolysis of glycidyl butyrate catalyzed by PPL is shown in Fig. 7. It is observed that the FL intensity of QDs linearly decreases with time increasing. It indicates that QDs has been successfully used as pH probe for quick enzyme reaction kinetic study. 3.5.1. Effect of external environment on the FL intensity of QDs Since QDs are sensitive to the external environment, the effect of substrate, solvent, enzyme solution and products on the FL intensity of QDs in enzyme-catalyzed reaction was investigated. No significant influence of all these materials on the FL intensity of QDs is observed (Table 1). It suggests that the decreasing of FL intensity during the reaction is caused by the released acid. 3.5.2. Comparison of PNP and QDs as pH probe PNP is widely used as pH probe for monitoring the releasing of protons during the ester hydrolysis [29]. The yellow color of the phenoxide fades as the reaction proceeds and reveals quantitatively the amount of ester hydrolyzed (Scheme 2). However, there are some disadvantages for PNP as pH probe: firstly, it is unstable. When PNP is incubated in a buffer with a fixed ionic strength, the OD value of PNP persistent varies at either low or high ionic strength (Fig. 8). It seems important to select a buffer with proper ionic strength to inhibit the variation of OD value of PNP solution. In contrast, this phenomenon is not observed for QDs, and the FL intensity of QDs does not fluctuate with the variation of time at a fixed ionic strength. Secondly, the reaction rate could only be correctly monitored for UV detection when OD value is
lower than 1. However, no such limit is observed for QDs. Thirdly, the sensitivity of fluorescence analysis is 10–100 multiple than spectroscopic method, leading detection limit of QDs to be much lower than PNP. Finally, in order to compare the monitoring range of QDs and PNP, the same probe concentration (10−4 mol/l) is used in the same reaction. As shown in Fig. 9a, the OD value of PNP decreases very fast with time increasing, especially at high substrate concentration. When exceeding 10 min, PNP is almost depleted and the reaction rate could not be detected correctly. On the other hand, QDs can be used to detect the reaction rate correctly even exceeds 15 min with the high concentration of substrate. It suggests that ODs has a wider monitoring range than PNP (Fig. 9b). 4. Conclusion Water-soluble CdTe/ZnS QDs are sensitive to environmental factors and found to be a satisfactory pH probe that could have potential applications in chemical and biochemical sensing. The probable H+ quenching mechanism can be explained as the addition of H+ may cause reversible reaction and Te is oxidized by oxygen and precipitated from the solution. The modified QDs is successfully used as pH probe in monitoring the hydrolysis of glycidyl butyrate catalyzed by PPL. In addition, we attempt to use QDs in culture medium and detect the released acid during the cultivation of bacterium. Clearly, the potential of QDs as pH probe has just begun to be realized; therefore, one is likely to see more work in this area in the coming years. Acknowledgements The authors are grateful for the financial support from National Natural Science Foundation of China (No. 20272017) and the Foundation of Research Program of Jilin University, China. References [1] Alivisatos AP. Semiconductor clusters, nanocrystals, and quantum dots. Science 1996;271:933–7.
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