Monitoring leaching of Cd2+ from cadmium-based quantum dots by an Cd aptamer fluorescence sensor

Abstract

Quantum Dots (QDs) have been demonstrated with outstanding optical properties and thus been widely used in many biological and biomedical studies. However, previous studies have shown that QDs can cause cell toxicity, mainly attributable to the leached Cd²⁺. Therefore, identifying the leaching kinetics is very important to understand QD biosafety and cytotoxicity. Toward this goal, instrumental analyses such as inductively coupled plasma mass spectrometry (ICP-MS) have been used, which are time-consuming, costly and do not provide real-time or spatial information. To overcome these limitations, we report herein a fast and cost-effective fluorescence sensor based a Cd²⁺-specific aptamer for real-time monitoring the rapid leaching kinetics of QDs in vitro and in living cells. The sensor shows high specificity towards Cd²⁺ and is able to measure the Cd²⁺ leached either from water-dispersed CdTe QDs or two-layered CdSe/CdS QDs. The sensor is then used to study the stability of these two types of QDs under conditions to mimic cellular pH and temperature and the results from the sensor are similar to those obtained from ICP-MS. Finally, the sensor is able to monitor the leaching of Cd²⁺ from QDs in HeLa cells. The fluorescence aptamer sensor described in this study may find many applications as a tool for understanding biosafety of numerous other Cd-based QDs, including leaching kinetics and toxicity mechanisms in living systems.

Keywords: Aptamer sensor; Cadmium-based quantum dots; Real-time monitoring.

Figure 1.

(A) Design of aptamer sensor (Cd-Apt) for detection of Cd²⁺. (B) Design of control aptamer sensor (Cd-Apt control).

Figure 2.

In vitro characterization of Cd-Apt aptamer sensors. (A) Fluorescence quenching efficiency of different molar ration of quencher strand with 200 nM aptamer strand. F represents aptamer strand; Q represents quencher strand. λ_Ex: 633 nm, λ_Em: 665 nm. Data are shown as the mean ± SD. (B) Fluorescence spectra of the 200 nM Cd-Apt in response to different concentration of CdCl₂ (0, 0.05, 0.1, 1, 5, 10, 20, 50, and 100 μM). λ_Ex: 633 nm, λ_Em: 650 – 800 nm. (C) Linear relationship between fluorescence and various concentration of CdCl₂ in Tris-HAc buffer. Inset shows corresponding response of Cd-Apt. (D) Selectivity of the Cd-Apt aptamer sensors when interacting with different metal ions. The fluorescence changes of Cd-Apt were measured in presence of 100 μM metal ions with the incubation of 20 min. F₀ denotes the fluorescence intensity in absence of Cd²⁺, F means the fluorescence intensity in presence of Cd²⁺. λ_Ex: 633 nm, λ_Em: 665 nm. Error bars represent standard deviations from three experiments. The sensor concentration was set at 200 nM, which was composed of 200 nM Cd-Apt-F and 400 nM Cd-Apt-S. 200 nM Cd-Apt control sensors was composed of 200 nM Cd-Apt-C and 400 nM Cd-Apt-S. Data are shown as the mean ± SD.

Figure 3.

Amount of Cd leached from CdTe QDs and CdSe/CdS QDs in a buffer mimicking conditions in living cells (1 × PBS buffer at pH = 7.4, 37 °C) (A) Time-dependent fluorescent changes of Cd-Apt sensor after being incubated with the filtrates from the dialysis of CdTe (black line) and CdSe/CdS QDs (red line). The amount of Cd determined by the fluorescent sensor are compared with those measured by ICP-MS (blue and green line). Data are shown as the mean ± SD. (B) Calibration curve of fluorescent intensity of the Cd-Apt sensor vs. Cd²⁺ concentration in PBS buffer. F₀ denotes the fluorescence intensity Cd-Apt sensors without any treatment, F means the fluorescence intensity treated with CdTe or CdSe/CdS filtrates. λ_Ex: 633 nm, λ_Em: 665 nm. Error bars represent standard deviations from three experiments. 200 nM of Cd-Apt is composed of 200 nM Cd-Apt-F and 400 nM Cd-Apt-S. Data are shown as the mean ± SD.

Figure 4.

Schematic of Cd-Apt aptamer sensor for the detection of free Cd²⁺ leached from Cd-QDs in cells.

Figure 5.

(A) Confocal microscopy of HeLa cells incubated with 200nM Cd-Apt sensors for 4 h, then incubated with different concentration of CdCl₂, CdTe QDs and CdSe/CdS QDs for another 4 h. (B) Quantitative analysis of fluorescence images. Data are shown as the mean ± SD. (C) Calibration curve for Cd-Apt-transfected cells response to different concentration of CdCl₂. F₀ denotes the fluorescence intensity of HeLa cells transfected with Cd-Apt sensors only, F means the fluorescence intensity of HeLa cells treated with Cd-Apt sensors and CdCl₂, CdTe or CdSe/CdS QDs. λ_Ex: 633 nm, λ_Em: 650 – 700 nm. Error bars represent standard deviations from three experiments. The sensor concentration was set at 200 nM, which was composed of 200 nM Cd-Apt-F and 400 nM Cd-Apt-S. 200 nM Cd-Apt control sensors was composed of 200 nM Cd-Apt-C and 400 nM Cd-Apt-S. Data are shown as the mean ± SD.

Figure 6.

(A) Confocal microscopic images of HeLa cells incubated with 200 nM Cd-Apt or Cd-Apt control sensor for 4 h, then incubated with 5 μM CdCl2, CdTe QDs and CdSe/CdS QDs for different time points. (B) Quantitative analysis of fluorescence images shown in (A). F0 denotes the fluorescence intensity of HeLa cells transfected with Cd-Apt sensors only, F means the fluorescence intensity of HeLa cells treated with Cd-Apt sensors and CdCl2, CdTe QDs and CdSe/CdS QDs. λEx: 633 nm, λEm: 650 – 700 nm. Error bars represent standard deviations from three experiments. 200 nM Cd-Apt was composed of 200 nM Cd-Apt-F and 400 nM Cd-Apt-S. 200 nM Cd-Apt control was composed of 200 nM Cd-Apt-C and 400 nM Cd-Apt-S. Data are shown as the mean ± SD.