Chapter 3 Investigation of the photoactivated CdSe/ZnS quantum
3.3 Results and Discussion
3.3.2 Influence of pH on the photoluminescence of POD-QDs
Previous studies have indicated that the PL intensity of water-soluble MSA-QDs is sensitive to the acidity or basicity of a solution [21,22]. As anticipated, this feature was maintained in the case of POD-QDs. As the pH value varied from 8.0 to 5.0, the PL intensity of the QDs was quenched and the emission spectrum was blue-shifted for 10 nm (as shown in Figure 3.7). It is plausible to assume that the quenching phenomenon and the blue-shift of the emission spectra are due to the acidic etching process occurring on the surface of POD-QDs. Consequently, the number of hole-traps on the particle surface was increased and the PL intensity was diminished.
We also confirmed the quenching ratio of the PL intensity exhibited a linear response to the acidity of the solution (shown in Figure 3.7,
upper right inset)
Figure 3.7 Comparison of PL spectra for POD-QDs as a function of pH. The inset reveals correlation between PL quenching ratio of POD-QDs and pH, 100 (Io- I)/Io represents the ratio of PL quenching, I0 is the PL intensity at 570 nm with pH 8.0, and I is the intensity at emission maximum.
3.3.3 Photoluminescence spectroscopy for the detection of triglyceride
Since the PL intensity of QDs is highly sensitive to acidity, we proposed that the POD-QDs should be potentially useful for many biochemical assays that generate acidic or basic products. To examine the feasibility of this proposal, an assay system for triglyceride analysis was established. Tributyrin, a glycerol tributyrate, can be hydrolyzed by lipase to produce glycerol and butyric acid. The general underlying biocatalyzed chemical reaction on which this
operation is based can be described as the follows:
⎯lipase⎯ →⎯ + (2)
(Tributyrin) (Glycerol) (Butyric acid)
The pH of the solution changes with the fatty acid produced [23].
The POD-QDs are potentially useful in indicator design-as they are significantly responsive under fluorescence and have a high surface/volume ratio. Although the lipase-catalyzed hydrolysis of tributyrin releases glycerol and fatty acid as products (see equation 2), the pH value of the assayed sample gradually decreases with the degradation of tributyrin. By controlling the initial concentration of employed buffer, the pH condition of a solution can be continuously perturbed by the formation of fatty acid. As demonstrated in Figure 3.8, the variations in fluorescence spectra and the PL intensity of POD-QDs were observed as a function of tributyrin concentration catalyzed to the end point by lipase. The maximal emission peaks were gradually blue-shifted from 570 nm to 560 nm when 0 to 25 mM of tributyrin solution samples were tested. The PL intensity of POD-QDs was increasingly quenched as the concentration of the employed tributyrin was increased. Apparently, the fatty acid, produced as a result of the lipase catalysis, perturbed the acidity of the
H2C-OCOC4H9 H2C-OH H2C-OH
H2C-OCOC4H9 C4H9COOH
H2C-OCOC4H9 H2C-OH
assay system and thus influenced the luminescence property of QDs.
The blue-shifted PL spectra indicated a trend of decreasing particle size [7].
Figure 3.8 Variation of PL intensity of POD-QDs as a function of tributyrin concentration in 10 mM phosphate buffer. The PL spectra were recorded at excitation wavelength of 365 nm.
Additionally, Figure 3.9 shows the absorption spectra of lipase/POD-QDs with different tributyrin concentrations (0, 4 and 10 mM) in a 10 mM phosphate buffer with pH 8.0. The slight scattering phenomenon, which results from lipase, tributyrin, and solvent, influences the smoothing of the absorption spectra. It can also be observed that the lipase-catalyzed hydrolysis promoted spectral shifts toward shorter wavelengths in the case of both absorption and luminescence, which is consistent with a possible decrease in particle
size during fatty acid production [16].
Figure 3.9 Absorption spectra of lipase/POD-QDs with different tributyrin concentration (0, 4 and 10 mM) in 10 mM phosphate buffer with pH 8.0. The assay system obtained in 0 mM (—), 4 mM (---) and 10 mM (oooo) tributyrin with lipase/POD-QDs.
Figure 3.10 plots the quenching ratio of the PL intensity, 100[(Io- I)/Io], against the concentration of tributyrin. The assays were performed separately in 5, 10, 20 mM of phosphate buffer (pH 8.0), respectively. Since the PL intensity is perturbed by the presence of fatty acid, it is of interest to measure both the change in the PL quenching ratio and the final acidity of the reaction products simultaneously. The down right inset of Figure 3.10 exhibits the responses of pH change and the quenching ratio of PL intensity as a function of tributyrin concentrations hydrolyzed in 5 mM phosphate buffer. Though the pH response showed a good linear correlation at higher tributyrin concentration (0.6 ~ 6 mM), no significant pH change
was observed at lower concentration level (< 0.6 mM), which can be rationalized by considering that at [tributyrin] < 0.6mM the proton released can be effectively neutralized by the strong buffering capacity of phosphate buffer solution and thus the observed pH change is negligible.
Figure 3.10 Linear correlation between quenching percentage of PL intensity, [100 (I0 - I)/I0], and tributyrin concentration. Quenching ratio of PL intensity obtained in phosphate systems of 5-mM (○), 10-mM ( ) and 20-mM (∆). The inset exhibits the responses of pH change and the quenching ratio of PL intensity as a function of tributyrin concentrations hydrolyzed in 5 mM phosphate buffer.
However, the quenching ratio of PL intensity showed a linear response to triglyceride concentration in the range of 0.02 – 6 mM (correlation coefficient R = 0.986). As a matter of fact, the linearity was not preserved when 8 mM of tributyrin was examined under the same condition, suggesting that the capacity of QDs was insufficient to reflect the existing acid. To overcome and solve this problem, one
could use higher concentration of POD-QDs. Alternatively, the buffer concentration could be adjusted. In principle, the butyric acid produced at the early stage of the reaction will be consumed by the buffer and allow POD-QDs to effectively reveal the acidity of the reaction in late stage. By tuning the buffer capacity of the assay system, the wide-range concentration of triglyceride can be accurately measured. As shown in Figure 3.10, the 10-mM and 20-mM buffer systems are appropriate for the quantitative detection of tributyrin in the range of 0.2 – 10 (correlation coefficient R = 0.979) and 2 – 20 mM (correlation coefficient R = 0.988), respectively. The assay system also provides a convenient way to estimate triglyceride concentration by visualizing the color change of the QDs fluorescence with naked eyes.
When the sample with tributyrin concentration higher than 5 mM was assayed in the 10-mM phosphate system, the luminescence of the QDs became almost colorless; whereas, at low concentration level, the yellow fluorescence can be clearly observed (Figure 3.11). It is also feasible to determine tributyrin concentration in other ranges when phosphate buffer with different concentration is selected.
The designed system is promising for triglycerides analysis as compared with many other currently existing methods. Table 1 summarizes the detection limits of triglycerides with various methods including potentiometry, surface acoustic wave, optical spectroscopy and the present study. Our new approach exhibits great potential in triglycerides sensing not only for its simplicity of preparation but also the improvement in sensitivity of detection.
Figure 3.11 Evolution of fluorescence images taken at the end-point of the catalytic hydrolysis of tributyrin in 10 mM phosphate buffer under ultraviolet irradiation of 365 nm.
Table 3.1Comparison of performance of different methods for the detection of triglycerides.
Type Sensing Matrix Enzyme Linear range
Lipase/POD-QDs Lipase 0-25 0.02* This work
* The detection limit of this study was suggested on the basis of the lowest concentration of triglycerin yielding reliable measurements.
3.4 Conclusions
This investigation demonstrated an assay system containing POD-QDs and lipase for the quantitative analysis of triglyceride.
With this new approach, triglyceride in a wide range of concentration can be successfully analyzed. As compared to traditional triglyceride assay methods, the POD-QDs-based detection exhibits several advantages, such as ease of reagent preparation, low cost, no enzymatic immobilization, high flexibility, and good sensitivity. The newly developed method can quantitatively determine triglyceride without using multiple enzymes and/or complicated instrument setup.
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