A Graphene Oxide-Based Fluorescent Aptasensor for the Turn-on Detection of CCRF-CEM
A convenient, low-cost, and highly sensitive fluorescent aptasensor for detection of leukemia has been developed based on graphene oxide-aptamer complex (GO-apt). Graphene oxide (GO) can absorb carboxyfluorescein-labeled Sgc8 aptamer (FAM-apt) by π-π stacking and quench the fluorescence through fluorescence resonance energy transfer (FRET). In the absence of Sgc8 target cell CCRF-CEM, the fluorescence is almost all quenched. Conversely, when the CCRF-CEM cells are added, the quenched fluorescence can be recovered rapidly and significantly. Therefore, based on the change of fluorescence signals, we can detect the number of CCRF-CEM cells in a wide range from 1 × 102 to 1 × 107 cells/mL with a limit of detection (LOD) of 10 cells/mL. Therefore, this strategy of graphene oxide-based fluorescent aptasensor may be promising for the detection of cancer.
KeywordsAptamer Graphene oxide Leukemia CCRF-CEM
Atomic force microscopy
FAM-labeled Sgc8 aptamer
Fluorescence resonance energy transfer
Graphene oxide-aptamer complex
Leukemia is an aggressive and common malignant hematologic disease, which is a threat to the survival of human beings and health, especially for children and adolescents [1, 2]. It affects not only the body’s normal hematopoietic cells but also the bone marrow, as well as the immune system [3, 4, 5]. Therefore, the early diagnosis of leukemia for the treatment and the improvement of the quality of life of patients is essential. At present, the commonly used method for detecting leukemia is taking peripheral blood cells and bone marrow, after that many kinds of analysis , including cell morphology, cytochemistry [7, 8, 9], immunophenotype [10, 11], immunohistochemical [12, 13], and aptamer-based flow cytometry [14, 15], have been carried out. These methods can detect leukemia cells, but they still have many shortcomings such as high cost, low sensitivity, and being complicated. Therefore, it is very urgent to find a low-cost, highly sensitive, and simple method for detecting leukemia.
Aptamers, which are short single-stranded DNA (ssDNA) or RNA, were screened by in vitro screening of systematic evolution of ligands by exponential enrichment (SELEX) [16, 17]. Based on the special tertiary structures, aptamers have robust binding affinity and high specificity with targets, including small organic molecules, proteins, and even cells [18, 19, 20]. Moreover, aptamers also have the characteristics of being easily synthesized and modified so that they are widely used as cancer detection probes . Functionalized nanomaterials based on aptamers for detection of cancer are also hotspots in recent years [22, 23], such as quantum dots and silica nanoparticles .
Graphene oxide (GO), as a novel two-dimensional planar carbon nanomaterials, has received substantial attention owing to its unique properties including good aqueous solubility , large specific surface area, and excellent fluorescence quenching ability [26, 27]. Based on these properties, GO is considered to be an excellent energy receptor in fluorescence resonance energy transfer (FRET), which makes GO have a broad application prospect in fluorescence aptasensor . Moreover, GO can bind to aptamers by π-π stacking interactions, but not with double-stranded DNA or aptamer-target complexes [19, 29, 30]. Hence, the graphene-based aptamer sensor can improve the stability of the aptamer compared to the free aptamer probe .
At present, a great deal of researches reported that the strategy of graphene oxide-based fluorescent aptasensor for detection target is feasible [21, 32]. Nevertheless, few studies have been carried out using a GO-based aptasensor for leukemia cells, so far. Here, we designed a new strategy for the signal ‘turn-on’ detection of leukemia cells based on GO and carboxyfluorescein-labeled Sgc8 aptamer (FAM-apt). GO and aptamer were used as a fluorescence quencher and target agent, respectively. In the absence of leukemia cells, GO can interact with FAM-apt and quenched almost all the fluorescence, and the detection signal turned off. However, when the target cells are present, the aptamers actively target cells and fall off from GO, resulting in fluorescence recovery in the detection system, and the detection signal turned on. Therefore, the target cell concentration can be measured correspondingly according to the change in fluorescence intensity.
The FFAM-apt with a sequence of 5′-FAM-AT CTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3′ was synthesized by the Sangon Biotech Co., Ltd. (Shanghai, China). In this work, self-regulating Tris-HCl buffer was employed, including 20 mM Tris-HCl (pH 7.4), 5 mM MgCl2, and 100 mM NaCl. The aptamers used in this experiment were dissolved by Tris-HCl buffer. Graphene oxide powder was purchased from the Xianfeng Nano Materials Tech Co., Ltd. (Nanjing, China). All solutions were prepared with ultrapure water of 18 MΩ purified from a Milli-Q purification system (Millipore, Bedford, MA, USA).
CCRF-CEM (human acute leukemic lymphoblast cell lines), Ramos (human Burkitt’s lymphoma cell lines), 293T (human embryonic kidney cell lines), and H22 (murine hepatocellular carcinoma cell lines) cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured at 5% carbon dioxide and 37 °C, and the medium of 1640 contains 10% fetal bovine serum (FBS; HyClone) and 100 U/mL penicillin-streptomycin (Gibco, Grand Island, NY, USA).
All fluorescence spectra and fluorescence intensity were measured and recorded by an F-7000 fluorescence spectrophotometer (Hitachi Company, Tokyo, Japan). A 700-μL quartz cuvette was used to hold the sample solution. Owing to the characteristic peak wavelengths of carboxylfluorescein (FAM), the luminescence intensity was monitored by exciting the sample at 490 nm and measuring the emission at 518 nm.
All the atomic force microscopy (AFM) imaging was taken by a SPI3800N microscope (Seiko Instruments Industry Co., Tokyo, Japan).
Zeta potential of the GO, FAM-apt, and graphene oxide-aptamer complex (GO-apt) was determined by a nanoparticle size, zeta potential, and absolute molecular weight analyzer (Zetasizer Nano ZS, Malvern, UK).
UV-visible absorbance spectra of GO, FAM-apt, and GO-apt were recorded on NanoDrop 2000 (Thermo, USA).
Preparation of GO-apt Fluorescent Aptasensor
The graphene oxide powder was dissolved and scattered in Milli-Q purified water and then dispersed by ultrasonic to obtain a homogeneous black solution with the concentration of 1 mg/mL. Diluting the stock solution by 20 mM Tris-HCl buffer, we obtained the concentration of 20 nM FAM-apt. And after that, 1 μL FAM-apt (10 μM) and 10 μL GO solution (1 mg/mL) as prepared were mixed and then diluted with Tris-HCl buffer to 500 μL.
CCRF-CEM and Ramos cells were cultured for 12 h in six-well plates (5 × 105 cells per well). Cells were washed two times with cold phosphate-buffered saline (PBS) and incubated with GO-apt solution at 4 °C in the dark for 30 min. Then, cells were washed three times and fixed for 20 min with 4% polyoxymethylene. Cells were washed again with PBS and stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Co., USA) for 5 min in the dark. Finally, cells were washed three times with PBS and examined by fluorescence microscopy (Nikon DS-Ri1; Japan).
Detections of CCRF-CEM Cells
CCRF-CEM cells were collected by centrifugation and suspended in 1 mL of PBS. The different concentrations of CCRF-CEM cells (0 to 1.0 × 107/mL) were incubated with a GO-apt fluorescent aptasensor at 4 °C in the dark for 30 min. After incubation, the CCRF-CEM cells were detected by fluorescence spectroscopy in the wavelength range of 560–500 nm. The limit of detection (LOD) is estimated based on the 3σ / S calculation, where σ is the standard deviation for the GO-apt solution (n = 10) and S is the slope of the linear equation .
To investigate the specificity of GO-based fluorescent aptasensor, we tested the system with several different cells, including Ramos cells, H22 cells, and 293T cells. Each of the 100-μL reaction systems included 1 × 106 cells.
Each experiment was repeated three times. The data was processed by the software SigmaPlot 12.5, and statistical analyses were performed using GraphPad Prism 6.02 (GraphPad Software, San Diego, CA, USA). The threshold of significance in all analyses was P < 0.0001.
Results and Discussion
Principle of GO-apt Fluorescent Aptasensor for Detection of CCRF-CEM
Fluorescence Quenching and Recovery
Characterizations of GO-apt Fluorescent Aptasensor
Fluorescence Microscopy of Cells
Optimization of Experimental Conditions for Detection of CCRF-CEM
In order to make the fluorescent aptasensor more sensitive to the detection of CCRF-CEM, the reaction system used to optimize the GO concentration becomes indispensable. Figure 5c, which clearly illustrates our strategy, shows the effect of different concentrations of GO on the fluorescence intensity of FAM-apt in the absence (Fig. 5c, curve a) and in the presence (Fig. 5c, curve b) of CCRF-CEM. As we have seen from Fig. 5c, upon the addition of GO, the fluorescence signal background is significantly reduced. Figure 5d shows the restored fluorescence of the FAM-apt by 1 × 106 CEM cells as a function of GO concentration. From Fig. 5d, we can find that when the GO concentration is 20 μg/mL, the ratio of F/F0 (where F0 and F are the fluorescence intensities of FAM at 518 nm in the absence and presence of CCRF-CEM, respectively) gets the highest value, which is 13.0354. Therefore, 20 μg/mL was considered to be the optimal GO concentration.
CCRF-CEM Detection with GO-apt Fluorescent Aptasensor
Comparison of analytical properties for CCRF-CEM cytosensors
Detection limit (/mL)
3.30 × 103–2.69 × 103
1.00 × 103–1.00 × 105
Quartz crystal microbalance
8.00 × 103–1.00 × 105
Electrochemical impedance spectroscopy
1.00 × 103–1.00 × 107
7.50 × 103–6.25 × 105
4.00 × 102–5 × 106
1.00 × 102–1 × 107
Specificity of GO-apt Fluorescent Aptasensor
We have developed a convenient, low-cost, and highly sensitive fluorescent aptasensor for detection of CCRF-CEM cells. This strategy cleverly uses the non-covalent bond interaction by the π-π stacking between graphene and single-stranded DNA and the superior performance of graphene-quenching fluorescence. Compared with the aptamer, the binding of the CEM-aptamer complex to GO is weak, so the fluorescence quenched by the graphene can be gradually restored. Under optimized conditions, the limit of detection is regarded as less than 100 cells. Therefore, based on its excellent performance, the fluorescent aptasensor has a broad prospect in tumor cell detection.
YXZ, JT, and ZQL designed the experiments. ZHZ, RZ, and PPH performed the experiments. JS, HS, NY, and YH analyzed the data. SFZ, JT, and ZQL wrote the manuscript. All authors read and approved the final manuscript.
The present study was supported by the Key Project of National Natural Scientific Foundation of China (No. 81430055), the Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China (No. IRT_15R13), and the National Natural Scientific Foundation of China (Nos. 81372362 and 81360335).
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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