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Apr 10, 2012 - Synthesis of fluorescent Ag nanoclusters and their application in α-l-fucosidase detection. Xiangling Ren , Zhenzhen Chen , Xianwei Me...
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Novel Fluorescence Method for Detection of α-L-Fucosidase Based on CdTe Quantum Dots Zhenzhen Chen, Xiangling Ren, Xianwei Meng, Yanqi Zhang, Dong Chen, and Fangqiong Tang* Laboratory of Controllable Preparation and Application of Nanomaterials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China S Supporting Information *

ABSTRACT: The enzyme α- L -fucosidase (AFu) plays an important role in the diagnosis of hepatocellular carcinoma (HCC) and fucosidosis. In this paper, a simple, sensitive and precise method based upon measuring the fluorescence quenching of CdTe semiconductor quantum dots (QDs) was developed for detecting the enzymatic activity of AFu. The detection limit of AFu was 0.01 U/L (n = 3) and the linear relationship was 0.01−4 U/L. The selectivity experiment indicated excellent selectivity for AFu over a number of interfering species. We have also studied the detection mechanism of AFu by X-ray photoelectron spectroscopy (XPS) and found that the quenching effect was caused by the oxidation of tellurium by 2-chloro-4-nitrophenol (2-CNP) which produced in AFu catalytic reaction. Moreover, the AFu sensor based on QDs was used satisfactorily for the assessment of AFu activity in serum samples. It will most probably be applicable in assembling diagnostic microdevice to realize the rapid clinic analysis of AFu. composition tunable fluorescence emission, large absorption cross sections, and exceptional brightness and photostability. In recent years, QDs have been commonly used in the sensing and biosensing due to their novel properties.16−23 It was proved that the fluorescence of QDs can be influenced by the reaction product of some enzyme catalytic processes.24−26 By analyzing the changes of fluorescence intensity, the enzyme activity can be measured. For example, the CdSe/ZnS QDs functionalized with methylene blue27 and the fluorescence quenching of CdTe/CdS QDs by H2O228 were both successfully used for glucose sensing. In our previous work, the influence of QDs by enzyme reaction product have also been successfully used for the fluorescent detection of glucose, choline, lactic dehydrogenase (LDH) and so on.24,29 But, the study of QDs enzyme sensors was most based on the quenching effect of H2O2 and the effect of other group on the fluorescence of QDs was merely studied. In addition, the quenching mechanism aslo exist discussing. Here we have used the CdTe QDs develop a system for the optical AFu detection based on the fluorescence quenching of QDs. This method was sensitive and provides a wide linear range of AFu concentrations under the optimal conditions. We have also studied the detection mechanism of AFu by X-ray photoelectron spectroscopy (XPS). Moreover, it successfully realized the AFu assay in serum with satisfactory results. The detection method described in this paper have many

he enzyme α-L-fucosidase (AFu) is present in all mammalian cells and involves in the catabolism of the fucose which contains glycocojugates. AFu can hydrolyze methyl α-L-fucoside and fucosidic linkages of fucoidan and blood-group substances.1 Most importantly, AFu have higher sensibility and particularity to the early diagnosis of hepatocellular carcinoma (HCC)2,3 and was used for the diagnosis of fucosidosis recognized in born disorder of metabolism and increases the sensitivity of detection to 95.5% in HCC patients.4 Different methods have been reported for the determination of AFu, such as, spectrofluorescence methods,5−7 chromatography,1 high performance liquid chromatography (HPLC)8 and electrophoresis technique.9 But, these methods still suffered from long detection time, complex process, and poor selectivity which arrested the application of AFu detection in early diagnosis of diseases. Previous studies have detect AFu by analyzing the absorbance of yellow colored species, such as 2-chloro-4-nitrophenol (2CNP) and 4-nitrophenol which produced in AFu catalystic reaction.10 This method suffered from a series of interference caused by the yellow color of the serum of patients. Moreover, many patients of HCC have high levels of bilirubin that influence the absorbance greatly.4,11 Therefore, improving the sensitivity of detecting AFu by effective and low cost method remains a challenge area of research. Hence, we have carried out attempts to overcome these disadvantages by testing the fluorescence quenching of novel nanomaterials. QDs are promising fluorescent nanometer-sized particles of intense research and broad applications.12−15 Comparied to organic dyes and fluorescent proteins, QDs have unique functional and structural properties, such as size and

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© 2012 American Chemical Society

Received: January 17, 2012 Accepted: April 10, 2012 Published: April 10, 2012 4077

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Figure 1. (A) Emission spectra of 10 μL QDs with 0.5 mM CNP-AFu and varying concentrations of AFu in water. a∼j 0, 0.01, 0.1, 0.2, 0.3, 0.5, 1, 3, 4, 5 U/L. The inset displays plots of the relative fluorescence intensity of QDs versus the concentration of AFu. (B) Emission spectra of 10 μL QDs with 0.5 mM CNP-AFu and varying concentrations of AFu in PBS (pH = 7). a−j: 0, 0.01, 0.1, 0.2, 0.3, 0.5, 1, 3, 4, 5 U/L. The inset displays plots of the relative fluorescence intensity of QDs versus the concentration of AFu. (C) The corresponding emission color of QDs with of varying concentrations of AFu (0.3 U/L, 1 U/L, 4 U/L) in water (a, b, c) and PBS (pH = 7) solution (d, e, f), respectively. (D) Plots of the fluorescence peak of QDs with 0.5 mM CNP-AFu and varying concentrations (0.3, 0.5, 1, 3, 4 U/L) of AFu in water.

Fluorescence measurements were carried out on a Cary Eclipse fluorescence spectrophotometer (Varian, Inc.). The emission spectra of QDs were recorded in the wavelength of 500−680 nm upon excitation at 480 nm. The exciting slit and the emission slit were 5 and 5 nm, respectively. The samples for the fluorescence measurements were placed in a 10 mm optical path length quartz fluorescence cuvette. Transmission electron microscopy (TEM) was performed on a JEOL JEM 2010F electron microscope operating at 200 kV. X-ray photoelectron spectroscopy (XPS) measurements were performed with a PHI Quantera SXM X-ray photoelectron spectrometer, using an Al Kamonochromator source. UV−vis spectra were recorded using a JASCO V-570 spectrophotometer at room temperature. Synthesis of CdTe Quantum Dots. CdTe QDs were obtained via a method described in the literature with minor modification.30 Briefly, the CdTe precursor solution was prepared by adding freshly prepared NaHTe solution to a nitrogen-saturated Cd(NO3)2 solution at pH 11.5 (adjusted by dropwise addition of 1 M solution of NaOH) in the presence of 3-mercaptopropionic acid (MPA) as stabilizer. The precursor

advantages, such as high sensitivity, little interference, simple and fast determination procedure, safe operation, little equipment investment, and accurate measuring result. It will play an important role in the clinical diagnosis, etiology, and prognosis.

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EXPERIMENTAL SECTION

Materials and Methods. 2-chloro-4-nitrophenyl-α-L-fucopyranoside (CNP-AFu, DZ082B-R1) and the enzyme α-Lfucosidase (AFu, DZ082B, EC 3.2.1.51) were purchased from Sigma. 3-Mercaptopropionic acid was purchased from Alfa Aesar, a Johnson Matthey company. Tellurium powder, cadmium nitrate and sodium hydroxide were purchased from Institute of Tianjin Jinke fine chemicals. Phosphate buffered saline (PBS) was prepared: 1.02 mM Na2HPO4, 6.45 mM KH2PO4, pH 7.01. The reagents such as Na2HPO4, KH2PO4 and cadmium nitrate were of analytical reagent grade. The ultrapure water (0.22 μm) was produced using a Millipore-Q water system. 4078

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optical properties (fluorescence intensity and fluorescence peak) of QDs and offered increased accuracy. Effect of pH on the Detection of AFu by QDs Sensor. Investigation of the effect of pH value on the performance of the biosensor is of great importance, because the activity of AFu is pH dependent33 and the surface of QDs can also be affected by pH. So, we have detected AFu in the solution with different pH value, the result was shown in Figure 2. From the Figure 2,

concentrations were [Cd] = 10 mM, [MPA] = 14 mM, [Te] = 5 mM, respectively. The CdTe precursor solution was heated at 90 °C for 8 h. With the reacting time prolonged, the color of CdTe precursor solution gradually changed from wine red to orange and then bright red. Then the QDs used in this paper were obtained. As shown in Figure S1A (Supporting Information), the obvious UV−vis absorption peak indicated that the CdTe QDs were closed to monodispersed. The fluorescence emission maximum appeared at 585 nm. The photoluminescence full width at half-maximum was about 51 nm for QDs. The concentration of the QDs was 0.58 × 10−6 M.31 The X-ray Diffraction (XRD) patterns of the CdTe QDs are shown in Figure S1B (Supporting Information). The peak in the (111) direction is at 24.4°. The XRD results indicate that QDs have fine nanocrystal structure which is consistent with the reported results.32 As shown in Figure S1C (Supporting Information), Transmission electron microscope (TEM) images also suggest that the CdTe QDs have a narrow size distribution. The average size of the CdTe QDs was 4.5 nm. Fluorescence Measurements. The AFu detection procedure by QDs was described as follows: 10 μL of QDs was diluted into 400 μL, and then reacted with CNP-AFu and different concentrations of AFu solution (concentration from 0.01 to 5 U/L in water, 0.01−5 U/L in PBS (pH = 7) and 0.01−5 U/L in serum solution) for 5 min (see Figure S2 in the Supporting Information). The time of the sample preparation was 1 min.

Figure 2. Detection of AFu (10 μL QDs with 0.5 mM CNP-AFu and 3 U/L AFu) used QDs biosensor in different conditions: pH5, water (pH6), pH7, pH10.

we can see that the pH value can have an impact on the enzyme reaction system. The fluorescence intensity of AFu detection system in the PBS solution (pH = 7) was the most remarkably quenched in the same enzyme reaction system. A higher sensitivity of QDs sensor will be obtained when the quenching effect of QDs was greater. Hence, the optimum response is achieved at pH 7. Moreover, most clinic analysis of AFu was in bodily fluids (the pH value of most bodily fluids was 7). So the QDs AFu sensor was very suit to the serum environment. Study the Detection Mechanism of AFu by QDs. Recently, many researchers have focused on the application of QDs for the development of sensory systems based on the changes of fluorescence intensity because of the fluorescence resonance energy transfer (FRET), electron transfer (ET), or other interactions occurring at the QDs surface.34,35 In this paper, AFu under test can catalyze CNP-AFu to generate 2CNP. The catalytic reaction of AFu was shown in the following reaction.

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RESULTS AND DISCUSSION Fluorescence Detection of AFu Based on QDs. Figure 1A shows the fluorescence quenching of 10 μL QDs upon interaction with 0.5 mM CNP-AFu and different concentrations of AFu (concentration from 0.01 to 5 U/L) for a fixed interval time of 5 min. As the concentration of AFu increased, the quenching of the QDs was enhanced. Calibration curve in the inset were the plots of the optical analysis of different concentrations of AFu by the quenching of the emission maximum of QDs. The results exhibited a good linear relationship in the range of 0.01 to 4 U/L (see inset in Figure 1A). The detection limit was 0.01 U/L (n = 3). These values were much more sensitive than what observed in some other detection systems.5,9 The table of detection limits comparison to some other methods was shown in the Supporting Information (see Table S1 in the Supporting Information). We also tested AFu in PBS (pH = 7), see in Figure 1B. The solution samples were mixed with 10 μL QDs, 0.5 mM CNPAFu and different concentration of AFu (concentration from 0.01 to 5 U/L). As AFu was added gradually, the quenching effect of the QDs enhanced (show in Figure 1B) and the linear relationship was 0.01−5 U/L (see inset in Figure 1B). The Figure 1C shows the corresponding emission color of QDs with of varying concentrations of AFu (0.3 U/L, 1 U/L, 4 U/L) in water and PBS (pH = 7) solution, respectively. An interesting phenomenon was observed during the fluorescence detection of AFu based on QDs. As the adding amount of AFu increased, the fluorescence peak of QDs showed a gradual red shift (show in Figure 1A and Figure 1C) with a linear relationship of 0.3−4 U/L (show in Figure 1D). Curiously, this phenomenon did not exist in PBS (pH = 7) solution (see in Figure 1B and Figure 1C). Hence, the method presented in this paper can achieve a multidimensional sensing of AFu in water sample based on simultaneous utilization of the double-channel

2‐chloro‐4‐nitrophenyl‐α ‐L‐fucopyranoside [CNP ‐ AFu] afuenzyme

XooooooooooY 2‐chloro‐4‐nitrophenol + α ‐L‐fucoside [2 ‐ CNP]

The fluorescence changes in the presence of variable concentrations of 2-CNP upon interaction with the QDs in water (see Figure 3A) or PBS (pH = 7) (see Figure 3B) for a fixed time interval of 5 min were shown in the Figure 3. As shown in Figure 3, the emission maximum of QDs was observed to be quenched by 2-CNP. With the increasing concentrations of 2-CNP, the emission of QDs decreased gradually. The obvious fluorescence influence of QDs indicated a strong interaction between QDs and 2-CNP. Furthermore, there was a curious phenomenon that 2-CNP can cause a red shift of the emission spectra of QDs in water solution (see Figure 3A) and the red shift did not happen in PBS (pH = 7) (see Figure 3B). Most importantly, the similar phenomenon was occurred during the detection of AFu based on QDs (see Figure 1). These results demonstrated that fluorescence 4079

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Te 3d spin−orbit doublet spectra for QDs. The peak intensity of Te 3d5/2 and Te 3d3/2 peaks observed at 572.4 and 583.2 eV (indicate the tellurium elemental state (Cd−Te) in QDs) decreased with the addition of 2-CNP in QDs, while the peak intensity of Te 3d5/2 and Te 3d3/2 peaks observed at 576.2 and 586.3 eV (corresponding to the tellurium oxide state (Te−O)) increased. This indicated that tellurium in QDs may be oxidized to the oxidation state after 5 min treatment in 2 μM 2-CNP. Moreover, other chemical elements changed little in the XPS spectrum (see the Supporting Information Figure S3). We put forward the hypothesis that the quenching mechanism of 2CNP was the oxidation of tellurium in QDs. The scheme 1 had shown mechanism of the AFu detection based on QDs. When AFu solution sample was added in the Scheme 1. Schematic Principle for Detection of AFu-Based on QDs

Figure 3. Emission spectra of QDs in the presence of varying concentrations (0, 0.5, 1, 2 μM) of 2-CNP in water solution (A) or PBS (pH = 7) (B).

detection system, AFu would catalyze CNP-AFu to produce 2CNP (see step 1 in Scheme 1). As electron/hole trapped on QDs was a good electron donor/acceptor, 2-CNP generated in the AFu enzyme reaction may cause oxidation reaction on the surface of the QDs (see step 2 in Scheme 1), which caused the quenching effect of QDs (see step 3 in Scheme 1). With the increasing of AFu, the quenching effect of the QDs has been enhanced, because the produce 2-CNP was increased. Hence, based on this principle, the AFu activity could be detected. Fluorescence Detection of AFu in Serum System Based on QDs. We know that serum AFu is a useful marker when the diagnosis of hepatocellular carcinoma (HCC), especially primary hepatic carcinoma. Here, we tried to achieve the detection of serum AFu based on QDs. First, we checked the selectivity of the detection of AFu by QDs. As shown in Figure 5A, we carried out studies with some active substances in serum under similar conditions that were used for the detection of AFu. The significant fluorescence decline was observed after the introduction of 0.5 mM CNP-AFu and 0.5 U/L AFu in the QDs solution (see “S” in Figure 5A). Then the additions of 10 mM glucose (see “S +glu” in Figure 5A), 20 mM L-lactic acid (see “S + LL” in Figure 5A), and 100 μM ascorbic acid (see “S + AA” in Figure 5A) did not cause any observable changes of the fluorescence intensities of the QDs in the mixture, respectively. This result indicated that the AFu sensor based on QDs showed good anti-interference performance. Then, we detected AFu in serum solution to study the application of QDs-based AFu sensor in clinical analysis. The solution samples were mixed with 10 μL QDs, 50 μL serum, 1 mM CNP-AFu, and different concentration AFu (concentration of 0.01−5 U/L). The phenomenon was similar with the

intensity of QDs was selectively quenched by 2-CNP in the detection system. Thus, controlling the properties of QDs quenched by 2-CNP can realize the select detection of AFu. According to the previous studies, many factors may cause the fluorescence of QDs quenched. To study the quenching mechanism of QDs by 2-CNP, the XPS spectrum of QDs (Figure 4b) and QDs treated with 2 μM 2-CNP (Figure 4a) for 5 min were performed. The XPS is a powerful technique for providing qualitative information and chemical changes. Its detecting depth of sample is approximately 10 nm, far bigger than the size of QDs (4.5 nm). Figure 3 showed high resolution

Figure 4. Te (3d) photoeletron spectra for the sample of QDs. a) The QDs sample treated with 2 μM 2-CNP for 5 min b) The QDs sample without any treatment (blank control). 4080

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detection of AFu demonstrated the potential application in disease diagnosis. Moreover, this work presents a feasible approach for further research in detecting other kinds of substrate which can be catalyzed to generate phenol, such as, polyphenol oxidase, alkaline phosphatase and so on.

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ASSOCIATED CONTENT

* Supporting Information S

Details of CdTe QDs properties (Figure S1), time-dependent experiment (Figure S2), additional XPS figures (Figure S3), and the comparison of detection limit (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 10 82543521. Fax: +86 10 62554670. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge financial support from the National Natural Science Foundation of China (project No. 81171454, 61178035, 81000667) and China National Key Basic Research Program (973 Project) (project No. 2010CB933901).

Figure 5. (A) Fluorescence changes of QDs in the detection system (S), the coexistence of the detection system with 10 mM glucose (S + glu), 20 mM l-lactic acid (S + LL) or 100 μM ascorbic acid (S + AA), respectively. (B) Emission spectra of 10 μL QDs with 1 mM CNPAFu and varying concentrations of AFu in serum. a∼j: 0, 0.01, 0.1, 0.2, 0.3, 0.5, 1, 3, 4, 5 U/L. The inset displays plots of the relative fluorescence intensity of QDs versus the concentration of AFu.

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detection of AFu in PBS (pH = 7) and the linear relationship were 0.01−4.0 U/L (shown in Figure 5B). The recovery of the AFu in serum was also calculated and shown in table 1. From Table 1, we can see that the data Table 1. AFu Detection in Serum Sample serum sample

AFu added concentration (U/L)

AFu found concentration (U/L)

RSD (%, n = 5)

recovery (%)

sample 1 sample 2 sample 3

1.00 3.00 4.00

1.06 3.11 4.06

1.57 2.06 1.85

106.00 103.67 101.50

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determined by QDs sensor have a good agreement with the added concentration of AFu in the serum sample. It proved that the QDs-based sensor is appropriate for the practical application in clinical analysis of AFu.

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CONCLUSIONS In conclusion, the simple, fast, selectivity and sensitivity fluorescence method was developed for the detection of AFu based on QDs. We found that enzyme-catalytic product (2CNP) can quench the fluorescence of QDs efficiently. XPS studies have demonstrated that the oxidation of tellurium by 2CNP have a great influence on the fluorescence of 3mercaptoacetic acid-stabilized CdTe nanoparticles. Combining the unique property of QDs and the specificity of enzymatic reaction, we successfully detected AFu in serum. The detection limit was 0.01 U/L (n = 3) and the linear relationship was 0.01−4 U/L. The excellent performance of QDs in the 4081

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