基于双色荧光传感器的癌细胞成像及microRNA定量检测

doi:10.7503/cjcu20200026

摘要/Abstract

摘要：

癌细胞中microRNA(miRNA)的灵敏成像对于疾病的诊断治疗具有重要意义, 其中miRNA-21通常在多种癌细胞中异常表达. 本文将DNA功能化的金纳米颗粒与发射波长分离的荧光染料FAM和Cy5.5修饰的DNA通过含有光控基团PC-linker的DNA4作为桥梁进行自组装, 构建了纳米传感器GDC. 将302 nm紫外光作为启动开关, 用其照射该体系时, Cy5.5修饰的DNA3被释放, 其荧光强度可作为内参比信号, 用于标定进入细胞的组装体含量; 细胞中miRNA-21作为催化分子, 与外加燃料Fuel DNA共同作用下可实现催化放大, FAM修饰的DNA2被释放且被猝灭的荧光信号得以恢复, 并作为检测信号. 通过2种荧光信号强度(FL)的检测及FL_FAM/FL_Cy5.5比值的计算, 达到定量分析细胞中miRNA含量的目的. 该体系可扣除因细胞内组装体含量不同造成的背景信号误差, 不仅能显著提高检测准确度, 还因存在催化循环而大大降低了检出限, 比传统方法至少降低了3个数量级. 该传感器的检出限为23.1 pmol/L, 通过定量计算得出HeLa细胞中miRNA的含量为0.0236 nmol/L.

关键词: 金纳米颗粒, 催化放大, MicroRNA, 荧光成像, 定量分析

Abstract:

MicroRNA(miRNA) plays an important role in cellular function and gene expression with abnormal expression level or varieties. Given this, miRNA imaging in cancer cells was regarded as the effective approach for disease diagnosis and treatment. Herein, we built a sensitive nanosensor with gold nanoparticles(GNP) and two type of dye-modified DNA, FAM-DNA2 and Cy5.5-DNA3. DNA4 containing photocleavable linker(PC-linker), was used as the bridge for self-assembly of nanosensor. 302 nm UV light was used as the startup switch. Cy5.5-DNA3 was released from assembly with the irradiation of UV light, the fluorescence intensity was used as internal reference signal to standardize the quantity of nanoparticles into cells. MiRNA-21 that working as catalytic molecule can induce the assembly of nanosensors, resulting to release FAM-DNA2 in the presence of Fuel DNA, at the same time, the recovery fluorescence of FAM was used as detection signal. By combination of the fluorescence intensity of FAM and Cy5.5(FL_FAM/FL_Cy5.5), we can calculate the quantity of miRNA and nanosensors in cancer cells, which avoided the existence of background signal caused by the different contents of intracellular nanoparticles. Importantly, this nanosensor is of great stability, biocompatibility, and can be used for cellular fluorescence imaging. In addition, the limit of detection(LOD) was about three orders of magnitude lower than traditional non-catalytic method and calculated to be 23.1 pmol/L. The quantity of miRNA-21 in Hela cells was calculated to be 0.0236 nmol/L.

Key words: Gold nanoparticles, Catalytic amplification, MicroRNA, Fluorescence imaging, Quantitative analysis

中图分类号:

O657

TrendMD:

白翠婷, 岳仁叶, 罗列高, 马楠. 基于双色荧光传感器的癌细胞成像及microRNA定量检测. 高等学校化学学报, 2020, 41(6): 1252.

BAI Cuiting, YUE Renye, LUO Liegao, MA Nan. Quantitative Analysis of MicroRNA Content by Fluorescence Imaging in Cancer Cells Using Dual-color Fluorescence Nanosensor ^†. Chem. J. Chinese Universities, 2020, 41(6): 1252.

图/表 10

Table 1 DNA sequences*

Name	DNA Sequence(5'→3')
DNA1(thiolated DNA)	SH-AAAAAAAAAATCTCACTAACTTACGG
DNA2(FAM)	FAM-CCCTATAGCTTATCAGACT
DNA3(Cy5.5)	Cy5.5-GATGTTGACTCGAGAC
DNA4(PC Linker DNA)	GTCTCGAG//TCAACATCAGTCTGATAAGCTATAGGGCCGTAAGTTAGTGAGA
Fuel DNA(F)	CTAACTTACGGCCCTATAGCTTATCAGACT
Catalyst DNA(C')	TAGCTTATCAGACTGATGTTGA
MiRNA-21(C)	UAGCUUAUCAGACUGAUGUUGA
FAM-DNA1	SH-AAAAAAAAAATCTCACTAACTTACGG-FAM
FAM-DNA4	GTCTCGAGTCAACATCAGTCTGATAAGCTATAGGGCCGTAAGTTAGTGAGA-FAM
1-Mismatch[C'(mis-1)]	TAGCTTATCAGACTGATCTTGA
2-Mismatches[C'(mis-2)]	TAGCTTATCAGTCTGATCTTGA
3-Mismatches[C'(mis-3)]	TAGCTAATCAGTCTGATCTTGA

Scheme 1 Schematic illustration of dual-color GDC sensor for quantitative analysis of microRNA (A) Photo-activated and miRNA-catalyzed disassemble of DNA2, DNA3 with GNP through two-step DNA strand displacement reactions(SDR); (B) DNA sequences for GDC and chemical structure of the photocleavage group(PC-linker).

Fig.1 Absorbtion spectra of GNP(a) and GNP-DNA1(b)(A), low-magnification TEM image of GNP-DNA1(B) and dynamic light scattering measurements(C)

Fig.2 Characterization of photocleavage DNA4 with UV irradiation(302 nm, 6 W) (A) Native PAGE of DNA4 with different UV irradiation time; (B) fraction of photocleaved products with different UV irradiation time; (C) agarose Gel Electrophoresis characterization of GNP assembly with DNA1, DNA4, DNA2 and DNA3, and the catalytic disassembly of GDC in the present or absence of UV irradiation, catalyst DNA(C') and Fuel DNA. Line 1: GNP-DNA1; line 2: GNP-DNA1+DNA4; lane 3: GNP-DNA1+DNA4+DNA2; lane 4: GNP-DNA1+DNA4+DNA2+DNA3(GDC); lane 5: GDC+1×F+1×C'; lane 6: GDC+hv; lane 7: GDC+ hv+1×F; lane 8: GDC+ hv+1×C'; lane 9: GDC+ hv+1×F+1×C'; lane 10: GDC+hv+1×F+0.1×C'; lane 11: GDC+ hv+0.1×C'; lane 12: GNP-DNA1+DNA4+DNA2+1×F+0.1×C'.

Fig.3 Agarose Gel electrophoresis characterization of disassembled products(A) and photoluminescence spectra of disassembled FAM-DNA2(B) with different UV irradiation time

Fig.4 Agarose gel electrophoresis characterization of disassembled products with different C'/DNA4 molar ratios(A) and photoluminescence spectra of released FAM-DNA2 with different C'/DNA4 molar ratios(B), photoluminescence spectra of GNP-DNA sensor for C' detection with or without UV irradiation(C) and calibration curve for quantitative analysis of miRNA(D) (A) a. GDC+1×F+1×C'(NO UV); b. GDC+1×F+1×C'(UV 5 min); c. GDC+1×F+0.5×C'(UV 5 min); d. GDC+1×F+0.2×C'(UV 5 min); e. GDC+1×F+0.1×C'(UV 5 min); f. GDC+1×F+0.02×C'(UV 5 min); g. GDC+1×F+0.01×C'(UV 5 min); h. GDC+1×F+0.004×C'(UV 5 min); i. GDC+1×F+0×C'(UV 5 min).

Fig.5 Specificity evaluation of GDC sensor (A) Agarose gel electrophoresis of catalytic DNA C' and three mutation sequences[C'(mis-1), C'(mis-2) and C'(mis-3)] containing one, two, three mismatches respectively. The sample without catalyst DNA is used as the control. All samples are irradiated with UV light in the presence of Fuel DNA; (B) corresponding photoluminescence spectra of the above-mentioned samples.

Fig.6 Photocontrolled catalytic imaging of FAM and Cy5.5 fluorescence dyes in living HeLa cells with different UV irradiation time

Table 2 FL intensity of FAM and Cy 5.5

10³ C'/L molar ratio	FL intensity/a.u.		10³ C'/L molar ratio	FL intensity/a.u.
10³ C'/L molar ratio	FAM	Cy5.5	10³ C'/L molar ratio	FAM	Cy5.5
0	1298	20635	15	26138	23284
4	7351	24341	20	33258	22946
10	16737	23975

Fig.7 Standard curve of FLFAM, 520 nm/FLCy 5.5, 714 nm vs. C'/L molar ratios

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