核酸适配体功能化近红外量子点结合流式细胞术快速检测白血病细胞

doi:10.7503/cjcu20140527

高等学校化学学报 ›› 2014, Vol. 35 ›› Issue (10): 2093.doi: 10.7503/cjcu20140527

核酸适配体功能化近红外量子点结合流式细胞术快速检测白血病细胞

汤进录^1,^2,⁴, 石慧^1,^2,⁴, 何晓晓^1,^3,⁴(), 王柯敏^1,^2,^3,⁴(), 李朵^1,^3,⁴, 颜律安^1,^2,⁴, 雷艳丽^1,^3,⁴, 刘剑波^1,^2,⁴

1. 湖南大学化学生物传感与计量学国家重点实验室长沙 410082
2. 湖南大学化学生物传感与计量学国家重点实验室, 化学化工学院
3. 湖南大学化学生物传感与计量学国家重点实验室, 生物学院
4. 生物纳米与分子工程湖南省重点实验室, 长沙 410082

收稿日期:2014-06-11 出版日期:2014-10-10 发布日期:2014-09-30
作者简介:联系人简介: 王柯敏, 男, 博士, 教授, 博士生导师, 主要从事纳米尺度和分子水平上的生物分析化学研究. E-mail: kmwang@hnu.edu.cn. 何晓晓, 女, 博士, 教授, 博士生导师, 主要从事生物分析化学和生物纳米技术研究. E-mail: xiaoxiaohe@hnu.edu.cn
基金资助:
国家自然科学基金(批准号: 21190044, 21221003, 21205033, 21322509, 21305038)和中央高校基本科研业务费资助

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

TANG Jinlu^1,^2,⁴, SHI Hui^1,^2,⁴, HE Xiaoxiao^1,^3,^4,^*(), WANG Kemin^1,^2,^3,^4,^*(), LI Duo^1,^3,⁴, YAN Lüan^1,^2,⁴, LEI Yanli^1,^3,⁴, LIU Jianbo^1,^2,⁴

1. State Key Laboratory of Chemo/Biosensing and Chemometrics
2. College of Chemistry and Chemical Engineering
3. Institute of Biology
4. Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University,Changsha 410082, China

Received:2014-06-11 Online:2014-10-10 Published:2014-09-30
Contact: HE Xiaoxiao,WANG Kemin E-mail:xiaoxiaohe@hnu.edu.cn;kmwang@hnu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(Nos.21190044, 21221003, 21205033, 21322509, 21305038) and the Fundamental Research Funds for the Central Universities, China

摘要/Abstract

摘要：

利用作为肿瘤细胞识别分子的核酸适配体(Aptamer)的高特异性和高亲和力以及作为信号报告单元的近红外量子点(QDs)的高荧光发射强度和低生物背景干扰特性, 构建了一种基于Aptamer功能化近红外QDs的新型纳米荧光探针, 并进一步结合流式细胞术在单细胞荧光分析方面的高通量、简便和快速等优势, 建立了一种检测白血病细胞的新方法. 以基于Cell-SELEX(Cell-based systematic evolution of ligands by exponential enrichment)技术针对CCRF-CEM人急性白血病细胞筛选的特异性Aptamer Sgc8c为模型, 构建了Sgc8c-QDs探针, 其仅需与细胞样品培育30 min即可实现对缓冲液和血清中靶细胞的简单、快速和高特异性检测. 与传统荧光染料标记技术相比, 该方法不仅大大提高了分析灵敏度, 还显示出对血清等复杂生物样品的高适用性和优良检测性能. 鉴于Cell-SELEX技术在其它白血病细胞Aptamer筛选领域的应用潜力, 该方法有望作为一种通用技术在白血病诊断及预后监测等方面发挥重要作用.

关键词: 核酸适配体, 量子点, 流式细胞术, 白血病, 癌细胞检测

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

中图分类号:

O657.3

TrendMD:

汤进录, 石慧, 何晓晓, 王柯敏, 李朵, 颜律安, 雷艳丽, 刘剑波. 核酸适配体功能化近红外量子点结合流式细胞术快速检测白血病细胞. 高等学校化学学报, 2014, 35(10): 2093.

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^†. Chem. J. Chinese Universities, 2014, 35(10): 2093.

图/表 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)

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核酸适配体功能化近红外量子点结合流式细胞术快速检测白血病细胞

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

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核酸适配体功能化近红外量子点结合流式细胞术快速检测白血病细胞

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

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