Aptamer-functionalized Near-infrared Quantum Dots Combined with Flow Cytometry for Rapid Detection of Leukemia Cells†

doi:10.7503/cjcu20140527

Abstract

Abstract:

As a haematological malignancy, leukemia poses a great threat to human health and life. Its early and rapid diagnosis is crucial for the improvement of the cure and survival rate of patients. In this paper, a novel leukemia cell assay strategy has been proposed based on aptamer-functionalized quantum dots(QDs) combined with flow cytometry. In this strategy, a biotin-labeled cancer-specific aptamer was adopted as the recognition molecule, and avidin-modified QDs with near-infrared fluorescence emission were utilized as the signal generator. Through the “biotin-avidin” interaction, QDs could be functionalized with aptamers to construct a novel aptamer-QDs fluorescent nano-probe. This probe could specifically bind to target cell surface via the interaction between aptamers and receptors on cell membrane, thus indicating the presence of the target after analysis with a flow cytometer. As proof of concept, the detection of human acute lymphoblastic leukemia CCRF-CEM cells was performed using the specific aptamer, Sgc8c, as a demonstration. Results showed that the modification with Sgc8c did not markedly influence the fluorescence emission and size of QDs. With a simple incubation with cell samples for just 30 min, this Sgc8c-QDs nano-probe could successfully achieve the highly selective detection of CCRF-CEM cancer cells both in buffer and in serum. By comparison with the traditional fluorescent dye labeling method, this Sgc8c-QDs-based strategy exhibited a substantial enhancement in analysis sensitivity for CCRF-CEM cells in buffer, which realized about 4.3 folds signal-to-background ratio of the FAM-labeled Sgc8c(FAM-Sgc8c) strategy. In particular, when used for serum sample analysis, the Sgc8c-QDs nano-probe still reserved a perfect applicability and displayed a relatively high signal-to-background ratio of about 9, while FAM-Sgc8c nearly lost the detection efficiency at the same concentration. It has been clearly verified that this aptamer-QDs strategy is facile, fast, washing-free, specific and sensitive, which might hold a great potential as a versatile technique for diagnosis and prognosis applications in cancer researches.

Key words: Aptamer, Quantum dots, Flow cytometry, Leukemia, Cancer cell detection

CLC Number:

O657.3

TrendMD:

TANG Jinlu, SHI Hui, HE Xiaoxiao, WANG Kemin, LI Duo, YAN Lüan, LEI Yanli, LIU Jianbo. Aptamer-functionalized Near-infrared Quantum Dots Combined with Flow Cytometry for Rapid Detection of Leukemia Cells^†[J]. Chem. J. Chinese Universities, 2014, 35(10): 2093.

Figures/Tables 8

Table 1 DNA sequences used in experiments

DNA probe	Sequence
Biotin-Sgc8c	5'-Biotin-T₁₀-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'
Biotin-Lib	5'-Biotin-T₁₀-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'
FAM-Sgc8c	5'-FAM-ATCTAACTGCTGCGCCGCCGGGAAAATACTGTACGGTTAGA-3'
FAM-Lib	5'-FAM-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'

Fig.1 Schematic representation of the principle for leukemia cell detection using aptamer-functionalized near-infrared QDs combined with flow cytometry

Fig.2 UV absorption spectra of aptamer solutions before(a) and after conjugation with Avidin-QDs(b)

Fig.3 Electrophoresis analysis of aptamer solutions before(a) and after conjugation with Avidin-QDs(b)

Fig.4 Fluorescence spectra(A) and size distribution(B) of Avidin-QDs(a) and Sgc8c-QDs(b)

Fig.5 Flow cytometry analysis of CCRF-CEM cells using different concentrations of Lib-QDs(A) or Sgc8c-QDs(B) and the corresponding signal-to-background ratio(C) Concentration of nucleicacid-QDs/(nmol·L-1): a. 0.1; b. 0.25; c. 0.5.

Fig.6 Flow cytometry analysis of CCRF-CEM cells in buffer using Sgc8c-QDs(A), FAM-Sgc8c(B, C) and the corresponding signal-to-background ratio(D)

Fig.7 Flow cytometry analysis of CCRF-CEM cells in serum using Sgc8c-QDs(A), FAM-Sgc8c(B, C) and the corresponding signal-to-background ratio(D)

References 21

[1]	Nelson B. P., Treaba D., Goolsby C., Williams S., Dewald G., Gordon L., Peterson L. C., Leuk. Lymphom., 2006, 47(7), 1352—1359
[2]	Canellos G.P., Lister T. A., Young B. D., The Lymphomas, Saunders Elsevier, Philadelphia, 2006, 490—502
[3]	Bennett J., Catovsky D., Daniel M. T., Flandrin G., Galton D., Gralnick H., Sultan C., Br. J. Haematol., 1981, 47(4), 553—561
[4]	Onciu M., Clin. Haematol., 2009, 23(4), 655—675
[5]	Wang C., Li M., Liu Y. P., Huang J. F., International Journal of Laboratory Medicine,2009, 30(6), 538—539
	(王超, 李明, 刘跃平, 黄君富. 国际检验医学杂志, 2009, 30(6), 538—539)
[6]	Belov L., de la Vega O., dos Remedios C. G., Mulligan S. P., Christopherson R. I., Cancer Res., 2001, 61(11), 4483—4489
[1]	Wang Y., Hammes F., De Roy K., Verstraete W., Boon N., Trends Biotechnol., 2010, 28(8), 416—424
[8]	Al-Mawali A., Gillis D., Lewis I., Am. J. Clin. Pathol., 2009, 131(1), 16—26
[9]	Hermann T., Patel D. J., Science,2000, 287(5454), 820—825
[10]	Mayer G., Angew. Chem. Int. Ed., 2009, 48(15), 2672—1289
[11]	Song Y., Zhu Z., An Y., Zhang W., Zhang H., Liu D., Yu C., Duan W., Yang C. J., Anal. Chem., 2013, 85(8), 4141—4149
[12]	Xiong X., Liu H., Zhao Z., Altman M. B., Lopez-Colon D., Yang C. J., Chang L. J., Liu C., Tan W., Angew. Chem. Int. Ed., 2013, 125(5), 1512—1519
[13]	Guo Q. P., Yang X. H., Wang K. M., Meng X. X., Li J., Zhong Z. H., Chem. J. Chinese Universities,2010, 31(2), 274—278
	(郭秋平, 羊小海, 王柯敏, 孟祥贤, 李军, 仲志鸿. 高等学校化学学报, 2010, 31(2), 274—278)
[14]	Leutwyler W. K., Bürgi S. L., Burgl H., Science,1996, 271(5251), 933—937
[15]	Wu X., Liu H., Liu J., Haley K. N., Treadway J. A., Larson J. P., Ge N., Peale F., Bruchez M. P., Nat. Biotechnol., 2003, 21(1), 41—46
[16]	Resch-Genger U., Grabolle M., Cavaliere-Jaricot S., Nitschke R., Nann T., Nat. Methods,2008, 5(9), 763—775
[17]	Li Z. H., Wang K. M., Tan W. H., Li J., Fu Z. Y., Wang Y. L., Liu J. B., Yang X. H., Chin. Sci. Bull., 2005, 5(13), 1318—1322
	(李朝辉, 王柯敏, 谭蔚泓, 李军, 付志英, 王益林, 刘剑波, 羊小海. 科学通报, 2005, 5(13), 1318—1322)
[18]	Zhang L. P., Hu B., Wang J. H., Chem. J. Chinese Universities,2011, 32(3), 688—693
	(张立佩, 胡博, 王建华. 高等学校化学学报, 2011, 32(3), 688—693)
[19]	Cao M. R., Hou J., Zhang Q., Bai F., Bai G., Chem. J. Chinese Universities,2012, 33(3), 437—441
	(曹美荣, 侯洁, 张奇, 白芳, 白钢. 高等学校化学学报, 2012, 33(3), 437—441)
[20]	Shangguan D., Li Y., Tang Z., Cao Z. C., Chen H. W., Mallikaratchy P., Sefah K., Yang C. J., Tan W., Proc. Natl. Acad. Sci. USA,2006, 103(32), 11838—11843
[21]	Gu Y. P.,Cui R., Zhang Z. L., Xie Z. X., Pang D. W., J. Am. Chem. Soc., 2012, 134(1), 79—82

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