CdSe/ZnS Quantum Dots Conjugated with a Fluorescein Derivative: a FRET-based pH Sensor for Physiological Alkaline Conditions

Abstract

Dual pH-dependent fluorescence peaks from a semiconductor quantum dot (QD) and a pH-dependent fluorescent dye can be measured by irradiating with a single wavelength light, and the pH can be estimated from the ratio of the fluorescent intensity of the two peaks. In this work, ratiometric pH sensing was achieved in an aqueous environment by a fluorescent CdSe/ZnS QD appended with a pH-sensitive organic dye, based on fluorescence resonance energy transfer (FRET). By functionalizing the CdSe/ZnS QD with 5-(and 6)-carboxynaphthofluorescein succinimidyl ester as a pH-dependent fluorescent dye, we succeeded in fabricating sensitive nanocomplexes with a linear response to a broad range of physiological pH levels (7.5 -9.5) when excited at 450 nm. We found that a purification process is important for increasing the high-fluorescence intensity ratio of a ratiometric fluorescence pH-sensor, and the fluorescence intensity ratio was improved up to 1.0 at pH 8.0 after the purification process to remove unreacted CdSe/ZnS QDs even though the fluorescence of the dye could not be observed without the purification process. The fluorescence intensity ratio corresponds to the fluorescence intensity of the dye, and this fluorescent dye exhibited pH-dependent fluorescence intensity changes. These facts indicate that the fluorescence intensity ratio linearly increased with increasing pH value of the buffer solution containing the QD and the dye. The FRET efficiencies changed from 0.3 (pH 7.5) to 6.2 (pH 9.5).

Precise Fractionation of CdSe/ZnS Quantum Dot-Organic-Dye Conjugates Using a Gel Filtration Column

“…14 The combination of CdSe/ZnS QDs and the pH-sensitive organic dye is nearly identical to a previously reported ratiometric pH sensor. [10][11][12][13] However, the most important part of this research is the precise fractionation method using the gel filtration column.…”

“…coefficient change of the fluorescein derivative, as described in our previous paper. 14 The extinction coefficient of the organic dye increased with increasing pH, and this caused an efficient FRET process from the CdSe/ZnS QDs to the organic dye, resulting in a relatively higher FL peak at high pH. In addition, the time-resolved FL lifetime is often used to confirm that the observed dual FL peaks are responsible for the FRET process.…”

“…The organic dye, 5-(and 6)-carboxynaphthofluorescein succinimidyl ester, was obtained from Life Technologies (C653); this dye shows pH-dependent changes in the FL intensity in alkaline solutions. 14 The organic dye was first dissolved in dimethyl sulfoxide (DMSO), and the resulting solution was then mixed with the buffer solution. The molar concentration of DMSO in the buffer solution was 10 mg/mL.…”

“…[10][11][12][13] This sensor uses fluorescence resonance energy transfer (FRET) from the semiconductor QDs to the dye, which exhibits pH-dependent changes in the FL profile. Specifically, in response to changes in the ionic conditions, specific organic dyes show pH-dependent molar absorption coefficients, 14 and the FRET efficiency increases as the molar absorption coefficient of the organic dye (acceptor) increases. 15 In addition, dual FL peaks appear when the concentrations of the organic dye and the semiconductor QD are optimized, then pH can be estimated from the ratio of the two peaks.…”

“…To solve this problem, we investigated a precise fractionation method using a gel filtration column for CdSe/ZnS QDs and the organic dye, 5-(and 6)-carboxynaphthofluorescein succinimidyl ester, which shows pH-dependent changes in FL profile. 14 In the filtration column, linear velocities of CdSe/ZnS QDs and the organic dye differ due to the difference in the hydrophilicities of their surfaces; this leads to precise fractionation based on the number of organic-dye molecules. Another merit of this method is that unreacted and/or aggregated raw materials (CdSe/ZnS QDs and the organic dye) are removed while passing through the column; otherwise, they would affect FL spectral changes owing to an unexpected chemical reaction between them.…”