Chapter 2 Synthesis the water- soluble CdSe/ZnS quantum dots
2.3 Results and discussion
2.3.5 Photoluminescent spectroscopy of the detection of glucose
Since the PL intensity of QDs was found to be sensitively influenced by the acidity (or basicity) of the environment, we proposed that the MSA-CdSe/ZnS QDs should be potentially useful for assays that produce acidic or basic product. To examine the feasibility of such proposal, we designed an assay system specifically for glucose analysis.
In this system, GOD was used to catalyze the oxidation of glucose to release H2O2 and gluconolactone. The latter was then rapidly hydrolyzed to form D-gluconic acid and, consequently, lowered the pH value of the assay system. The catalytic reactions can be depicted in equations (1) and (2) as represented below
D-glucose +O2 ⎯⎯ →GOD⎯ D-gluconolactone + H2O2 (1) D-gluconolactone + H2O D-gluconic acid (2) →
Figure 2.9 exhibited the variation of fluorescence spectra of various assay reactions as a function of glucose concentration (0.2 to 10 mM). Note that, the fluorescence spectra, with excitation at 365 nm, were taken at the end-point of the enzymatic reaction that occurred in 10 mM of phosphate buffer, pH 8.0. The quenching of PL intensity of MSA-CdSe/ZnS QDs was observed with increasing glucose concentration. The catalytic oxidation of glucose also resulted in spectral shifts toward shorter wavelengths in the PL spectra. Since acids were found to be able to slowly etch QDs [49], the blue-shifted emission spectra were presumably attributed to the particle size reduction as more acid was produced.
Figure 2.9 The emission spectra of the assay samples at the end-point of the reaction. The assay system contained GOD, QDs, and the tested glucose with concentration 0, 0.2, 0.6, 2, 6, 8, 10 mM. Reactions were performed at 10 mM phosphate buffer, pH 8.0 for 30 min. The emission spectra were recorded under excitation with 365 nm lights.
Figure 2.10 represented the experiments of PL quenching and the pH values as a function of glucose concentrations oxidized in two different buffer systems, 10 mM and 30 mM phosphate, pH 8.0. The data obtained from the assay system of 10 mM phosphate, pH 8.0 were directly derived from Figure 2.7. The quench ratio of PL intensity was defined as 100 (Io-I)/Io, where Io and I represented the PL intensity of QDs observed at 586 nm with the reaction at zero time and at end-point, respectively. When the assays were performed in 10 mM phosphate, the quench ratio of PL intensity, [(Io-I)/Io], was found to be proportional to glucose concentration in the range of 0.2 to 10 mM, while at higher glucose concentration (>15 mM) the quenching effect was leveled-off
(data not shown), suggesting that the capacity of QDs be insufficient to reveal the existing acid.
Figure 2.10 Correlation between the quench ratio of PL intensity, pH perturbation and the tested glucose concentration. Two assay system, 10 mM and 30 mM phosphate, pH 8.0, were employed. The data obtained from the assay system using 10 mM phosphate were directly derived from fig.6. The quench ration of PL intensity was defined as 100 (Io-I)/Io, where Io and I represented the PL intensity of QDs observed at 586 nm with the reaction at zero time and at end-point, respectively. The data shown by open circle (○) and open square (□) were the pH measurements of the reactions assayed in 10 mM and 30 mM phosphate, respectively. The data shown by filled circle (●) and filled square (■) were the quench ratio of PL intensity obtained in 10 mM and 30 mM phosphate system, respectively.
To solve such a problem, one could use higher concentration of QDs
a convenient way for the deter
. Alternatively, increasing buffer concentration was suggested.
For that, the gluconic acid produced at early stage of reaction will be consumed by the buffer and allow QDs to effectively function as an indicator to track and reveal the later stage of reaction. The assay system using 30 mM phosphate, pH 8.0 was found to be suitable for glucose concentration ranging from 2 to 30 mM. The pH value of each reaction in both the 10 mM and 30 mM buffer systems also consistently showed the correlation between the fluorescence quenching and the amount of glucose measurement.
Our assay system can also provide
mination of glucose concentration by visualizing color change of QDs fluorescence. Figure 2.11 exhibited series of fluorescence images for various glucose analytes obtained from 0 to 14 mM in the 10 mM buffer system. The fluorescence of the reaction samples, irradiated with 365 nm light, resulted in the color change from pale orange to bright, yellowish green when glucose concentration was increased from 0 to 14 mM. Two breaking points between 0.6 mM to 2 mM and 10 mM to 12 mM were clearly observed, respectively. With appropriate control of the buffer system, it is feasible to develop a simple assay kit for semi-quantitative determination of glucose without using expensive instrumental setup.
[Glucose] (mM)
Figure 2.11
2.4 Conclusions
utiliz
Fluorescent photos of the assay samples. Samples were excited by 365 nm sources. The assay conditions were identical to those described in Fig. 2.9.
We have demonstrated that the water-soluble MSA-QDs can be ed as a reliable agent for the enzymatic determination of urea and glucose concentration. The MSA-QDs exhibit linear response in PL intensity when urea with concentration ranging from 0.01 to 100 mM was analyzed. Additionally, with this new approach, glucose can be successfully analyzed with wide range of concentration. As compared with other urea and glucose sensors (indicator) reported in the literature, the MSA-QD-based biosensor exhibits several advantages, such as the ease of its fabrication, low cost, no enzyme immobilization process required, high flexibility, and good sensitivity. It offers a persuasive way to determine the urea and glucose concentration without using complicate instrumental application.
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