Streptavidin was purchased from Promega Corporation (Madison, WI, USA). Biotin-QD525 was synthesized by Beijing Zhongke Wu Yuan Biotechnology Co., Ltd. (Beijing, China) and the biotin-Sgc8 aptamer was synthesized by Shanghai Sangon Biotechnology (Shanghai, China). The sequence was: biotin-T₁₀, 5′-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3′; and biotin-labeled random DNA library (biotin-Lib) as control biotin-T₁₀, 5′-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3′. Sterile phosphate-buffered saline (PBS; 10 mM, pH, 7.4) was used as the buffer. All solutions were prepared with Milli-Q water.

All cells were provided by the National Center for International Research of Biological Targeting Diagnosis and Therapy, Guangxi Medical University (Nanning, Guangxi, China). CEM and Ramos cells were used as the positive and negative cells, respectively, for the QDs-bsb-Sgc8 complex. 293T cells were used to assess the toxicity of the complex. All of the cells were cultured in 1640 medium, which was supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (both from Gibco Co., Grand Island, NY, USA). All of the cell lines were cultured at 37°C, in a humid environment with 5% CO₂. Nude mice were provided by the Guangxi Laboratory Animal Center (Guangxi, China), and were raised and cared for following the Federation of European Laboratory Animal Science Association guidelines. All protocols were approved by the Animal Ethics Committee of Guangxi Medical University.

Biotin-QD525 was decorated by streptavidin, and then conjugated with the biotin-modified Sgc8 aptamer. Firstly, streptavidin and biotin-QDs were mingled at a ratio of 10:1 and incubated for 40 min at 37°C to obtain streptavidin-QDs. Secondly, QDs-bsb-apt was synthesized followed by addition of the biotin-modified Sgc8 aptamers and incubated sequentially at a ratio of 40:1. Then, the mixture was co-cultured for 40 min at 37°C. Finally, unconjugated biotin-aptamers were eliminated, and conjugated QDs-bsb-aptamer complexes were retrieved by centrifugal filtration at 14,000 rpm for 25 min. Following washing in PBS (10 mM; pH, 7.4), an ultrasonic processor (Scientz-IID; Ningbo Scientz Biotechnology Co., Ltd., China) was used to disperse the aggregated conjugates by sonication (25 kHz; amplitude, 15 µm; sonication time, 3 min) and stored at 4°C.

The cultured cells were collected and centrifuged for 8 min at 1,200 rpm at 4°C, and washed two or three times in cold PBS. To determine the cell density, a counting plate was used. Then, 3–4×10⁵ cells were resuspended in 200 µl PBS. Cell samples were incubated with the QDs-bsb-apt complexes in cell culture medium or binding buffer (200 µl) containing FBS [10% (vol/vol)] on ice for 50 min, followed by washing using washing buffer and suspension in serum (200 µl). The detection sensitivity of this complex was determined by a flow cytometer (Epics XL; Beckman Coulter, USA). EXPO32 ADC analysis software was applied to analyze the data.

CEM and Ramos cells were cultured in a 6-well cell culture plate at a density of 3–4×10⁵ cells/ml. After incubation for 24 h, the cells were then placed on ice for 30 min to avoid non-specific binding. The cells were washed with cold PBS three times, and then fixed with 4% polyoxymethylene (Sigma-Aldrich). After incubation with the complexes (10 nM) in binding buffer (1 mM CaCl₂, 1 mM MgCl₂, 1 mg/ml bovine serum albumin and 10% FBS) on ice for 50 min, the cells were then washed three times. Next, the cells were mounted onto microscope slides using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Life Co., USA) to perform imaging analysis via a fluorescence microscope (DS-Ri1; Nikon Corporation, Tokyo, Japan).

Evaluation of cell toxicity of the QDs-bsb-apt was carried out by the methylthiazol tetrazolium (MTT) assay. The process involved in the MTT assay is briefly described as follows. Firstly, 293T cells (2×10⁴ in 100 µl) were seeded into 96-well plates and cultured overnight in an incubator. Then, the cells were treated with the complexes (10, 20, 30, 50 and 100 nM) or PBS as a control group for 24 or 48 h. After that, 10 µl of MTT (5 mg/ml) was added to each well and the cells were incubated for a further 3 h at room temperature in a lucifugal place. Before adding the ethanol/dimethyl sulfoxide (DMSO) into the well, the medium in the well was discarded. After 2 min, the absorbance was tested at λ=570 nm with an ELISA microplate reader (Thermo Scientific, USA).

Evaluation of systemic toxicity was carried out by analysis of the histologic evidence from the heart, liver, kidney, spleen and lung. Six-week-old nude mice were administered an intravenous injection of QDs-bsb-apt (15 mg/kg) or PBS in the control experiments through the tail vein. Then, changes in the clinical behavior of the treated mice were monitored every day. Mice were sacrificed after being treated for 20 days, for histological examination. Tissue samples of the organs, which were preserved in 10% formaldehyde solution, were dehydrated, following a routine strategy, and then embedded in paraffin. The paraffin sections were deparaffinized and immersed in distilled water, following a routine strategy. Then, hematoxylin and eosin staining was carried out in the following steps: i) coloration with hematoxylin solution for 5–15 min; ii) separation of color with acid or ammonia solution for a few seconds, respectively; iii) washing with running water for half an hour; iv) dehydration in 70% and 90% alcohol for 10 min sequentially; v) eosin staining for 2–3 min; and vi) dehydration, clearing and mounting with neutral gums. The negative control group was implemented using the same steps as described above. Hematoxylin and eosin solutions were purchased from Beijing Solarbio Co., China.

Student's t-test was applied to make comparisons and one-way analysis of variance (ANOVA) was applied to compare three means or more. Data are shown as means ± SD. All statistical analyses were two-tailed. P≤0.05 was considered to indicate a statistically significant result. Statistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

Recently, QDs and aptamers have been combined and are widely used for cell imaging and biomedical application. In most cases, QD-aptamer conjugates are connected directly for cell recognition, such as biotin-QDs conjugated with a streptavidin-aptamer (29). Using such an approach, the aptamers in recognizing target cells may be baffled by steric hindrance caused by QDs, and the limited amplification may cause insensitive examination in detection processing. To avoid these issues, a strategy of linking biotin-QDs and the biotin-aptamer was proposed in which biotin-QDs were coupled to streptavidin, and the streptavidin-appended QDs were then bound to the biotin-aptamer to allow the target cancer cells to be detected by fluorescence microscopy and flow cytometry (Fig. 1). CEM cells, a common leukemia cancer cell line, were treated as target cells. Sgc8, an aptamer of CEM cells, that has high affinity and specificity, was selected by cell-SELEX. This method may provide an efficient approach for tumor cell detection.

Various characteristics of the prepared QDs-bsb-apt complex were analyzed via the following methods. Transmission electron microscopy (TEM; H-7650; Hitachi, Ltd., Tokyo, Japan) was employed to image the dissemination and the size. It was observed, via the image, that the QDs-bsb-apt complexes were well disseminated and the size was increased (Fig. 2A). The hydrodynamic diameter of the QDs-bsb-apt complexes was tested by dynamic light scattering. It was 18.92±3.05 nm (Fig. 2C). The fluorescence emission peak of the QDs with or without the aptamer was 525 nm and the absorbance peak of the aptamers with or without QDs was 260 nm (Fig. 2D). Electrophoretic mobility shift assay was performed using 3% agarose gel electrophoresis. As shown in Fig. 2B, the band in lane 2 indicates the QDs-bsb-apt complex. Its size is >500 bp. The band in lane 1 represents the aptamer alone. Its size is ~50 bp).

At first, feasibility analysis of the QDs-bsb-apt strategy was carried out using fluorescence microscopy. Human Burkitt's lymphoma Romas cells were used as negative control and the random DNA library (lib) was used as a control sequence. In the fluorescence images, CEM cells exerted a distinctly stronger fluorescence intensity on their surface with the presence of QDs-bsb-Sgc8 than that of FITC-Sgc8, while no fluorescence signal was observed for the random library (Fig. 3A). When CCRF-CEM cells were replaced by Ramos cells, no obvious fluorescence signal was observed (Fig. 3B). This result indicated that due to the the specific properties of the Sgc8 aptamer, QDs-bsb-Sgc8 complexes were able to be applied in the detection of the CEM cells with high sensitivity.

Flow cytometric analysis confirmed that the positive CEM cells shown a stronger fluorescent signal property in serum than the QDs-bsb-Lib, Ramos cells and the FITC-Sgc8 (Fig. 4). CEM cells (71.6%) could be detected by employing the QDs-bsb-Sgc8 complex, while 57.5% of cells were detected by FITC-Sgc8. The detection efficiency of QDs-bsb-Sgc8 was 1.2-fold higher than the traditional organic dye modified aptamer FITC (Fig. 4).

MTT assay demonstrated that various concentrations of the QDs-bsb-apt complexes were non-toxic to normal cells such as 293T when co-incubated with the QDs-bsb-apt complexes (Fig. 5A). Additionally, after injecting the QDs-bsb-apt complexes into nude mice, hematoxylin and eosin staining was conducted. The staining results demonstrated that the complexes were non-toxic to the entire organism (Fig. 5B).