Quantitative Analysis of MicroRNA Content by Fluorescence Imaging in Cancer Cells Using Dual-color Fluorescence Nanosensor †

doi:10.7503/cjcu20200026

Abstract

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

CLC Number:

O657

TrendMD:

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

Figures/Tables 10

References 39

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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

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

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

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