膀胱癌相关miRNA灵敏检测的框架核酸线性放大平台构建

doi:10.3969/j.issn.1674-8115.2025.03.001

上海交通大学学报（医学版） ›› 2025, Vol. 45 ›› Issue (3): 253-260.doi: 10.3969/j.issn.1674-8115.2025.03.001

• 论著 · 基础研究 • 下一篇

膀胱癌相关miRNA灵敏检测的框架核酸线性放大平台构建

毛晨宙¹(), 张瑞赟², 陈海戈²(), 尹芳菲¹(), 左小磊¹()

^1.上海交通大学医学院附属仁济医院分子医学研究院，上海 200127
^2.上海交通大学医学院附属仁济医院泌尿科，上海 200127

收稿日期:2024-10-24 接受日期:2025-01-21 出版日期:2025-03-28 发布日期:2025-03-28
通讯作者: 左小磊，教授，博士；电子信箱：zuoxiaolei@sjtu.edu.cn
尹芳菲，助理教授，博士；电子信箱：yinfangfei@sjtu.edu.cn
陈海戈，教授，学士；电子信箱：chenhage@renji.com。
作者简介:毛晨宙（2002—），女，博士生；电子信箱：mcz1588_dpz@sjtu.edu.cn。
基金资助:
国家重点研发计划(2021YFF1200300);国家自然科学基金(22025404);上海市自然科学基金(19JC1410300)

Framework nucleic acid-based linear amplification platform for sensitive detection of bladder cancer-related miRNAs

MAO Chenzhou¹(), ZHANG Ruiyun², CHEN Haige²(), YIN Fangfei¹(), ZUO Xiaolei¹()

^1.Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Department of Urology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China

Received:2024-10-24 Accepted:2025-01-21 Online:2025-03-28 Published:2025-03-28
Contact: ZUO Xiaolei, E-mail: zuoxiaolei@sjtu.edu.cn
YIN Fangfei, E-mail: yinfangfei@sjtu.edu.cn
CHEN Haige, E-mail: chenhage@renji.com.
Supported by:
National Key R&D Program of China(2021YFF1200300);National Natural Science Foundation of China(22025404);Natural Science Foundation of Shanghai(19JC1410300)

摘要/Abstract

摘要：

目的·构建框架核酸线性放大平台，用于膀胱癌相关微RNA（microRNA，miRNA）灵敏定量检测，实现膀胱癌的早期筛查和精准分型诊断。方法·结合等离子荧光增强芯片与四面体框架核酸高性能探针，以miRNA为靶标，构建框架核酸线性信号放大平台，实现多靶标精准、高通量定量分析。首先利用原子力显微镜（atomic force microscope，AFM）验证四面体结构的有效合成。通过聚丙烯酰胺凝胶电泳（polyacrylamide gel electrophoresis，PAGE）和全内反射荧光显微镜（total internal reflection fluorescent microscope，TIRFM）验证报告单元的信号线性放大能力。比较传感界面基底性能，选取具有信号放大的金岛芯片。通过界面特异度实验，验证检测体系的特异度。选取5种膀胱癌相关miRNA，构建靶标标准曲线，用于定量检测。结果·AFM验证了四面体单体及二聚体结构的有效合成。PAGE和TIRFM表征验证了1~6价荧光报告单元的荧光信号线性放大。为进一步实现信号放大，比较了等离子金岛芯片和传统的玻璃芯片，结果表明金岛芯片具有等离子效应，可显著增强近红外荧光，相较于玻璃芯片信号放大最高可达13.6倍。特异度验证实验显示，该体系信噪比为7~10，特异度良好。基于体系的高特异度，结合框架核酸界面良好的界面调控能力与线性放大，最终实现了靶标双色并行检测，各靶标工作范围为100 fmol/L~10 nmol/L（R²≥0.991），检出限低至100 fmol/L。结论·该平台的构建为生物标志物的高灵敏度定量分析开辟了新的途径。此外，所开发的框架核酸检测平台在膀胱癌及其他重大疾病的临床诊断和预后方面具有很大的潜力。通过早期检测和精准分型，医师可以为患者制定更加个性化的治疗方案，提高治疗效果，减少不必要的治疗和随之而来的不良反应。液体活检技术不仅为膀胱癌的早期筛查提供了新的可能性，也为其他类型癌症的研究和临床应用提供了借鉴。

关键词: 膀胱癌, 框架核酸, 生物传感, 荧光芯片

Abstract:

Objective ·To construct a framework nucleic acid-based linear amplification platform for the sensitive and quantitative detection of bladder cancer-related microRNAs (miRNAs), facilitating early screening and accurate diagnosis of bladder cancer. Methods ·This study combined a plasma fluorescence-enhanced chip with high-performance tetrahedral framework nucleic acid (tFNA) probes, targeting miRNAs as biomarkers, to construct a framework nucleic acid-based linear signal amplification platform for precise and high-throughput quantitative analysis of multiple targets. First, atomic force microscope (AFM) was used to verify the efficient synthesis of tFNA. The signal linear amplification capability of the reporter unit was verified by polyacrylamide gel electrophoresis (PAGE) and total internal reflection fluorescent microscope (TIRFM). The performance of the sensing interface substrates was compared, and the golden island chip with signal amplification was selected. The specificity of the detection system was verified by an interface specificity experiment. Five bladder cancer-related miRNAs were selected to construct standard curves for quantitative detection. Results ·The efficient synthesis of tetrahedral monomer and dimer structures was verified by AFM. PAGE and TIRFM characterization verified the linear amplification of fluorescence signals from 1 to 6 valence fluorescence reporter units. In order to achieve further signal amplification, the plasma island chip and the traditional glass chip were compared. The results showed that the gold island chip exhibited a plasmonic effect, which significantly enhanced the near-infrared (NIR) fluorescence, with a signal amplification of up to 13.6 times compared to the glass chip. The specificity verification experiment showed that the signal-to-noise ratio of the system ranged from 7 to 10, demonstrating high specificity. Based on the high specificity of the system, along with the good interface regulation ability and linear amplification of the framework nucleic acid-based interface, dual-color parallel detection of the targets was finally realized. The working range was 100 fmol/L‒10 nmol/L (R²≥0.991), and the detection limit was as low as 100 fmol/L. Conclusion ·The establishment of this platform opens new avenues for highly sensitive quantitative analysis of biomarkers. Furthermore, the developed framework nucleic acid-based detection platform holds great potential for clinical diagnosis and prognosis of bladder cancer and other major diseases. Through early detection and precise subtype diagnosis, doctors can formulate more personalized treatment plans for patients, improving treatment efficacy and reducing unnecessary treatment plans and associated side effects. Therefore, this liquid biopsy technology not only provides new possibilities for early screening of bladder cancer but also serves as reference for research and clinical applications in other types of cancer.

Key words: bladder cancer, framework nucleic acid, biosensing, fluorescence chip

中图分类号:

O652.7

毛晨宙, 张瑞赟, 陈海戈, 尹芳菲, 左小磊. 膀胱癌相关miRNA灵敏检测的框架核酸线性放大平台构建[J]. 上海交通大学学报（医学版）, 2025, 45(3): 253-260.

MAO Chenzhou, ZHANG Ruiyun, CHEN Haige, YIN Fangfei, ZUO Xiaolei. Framework nucleic acid-based linear amplification platform for sensitive detection of bladder cancer-related miRNAs[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2025, 45(3): 253-260.

图/表 6

表1 合成四面体所需DNA链

Tab 1 DNA sequence for DTN

DNA	Sequence (5′→3′)
A37-1	CCCTGTACTGGCTAGGAATTCACGTTTTAATCTGGGCTTTGGGTTAAGAAACTCCCCG
A37-2	CGCTGGAGGCGCATCACCGTTTGCGTATGTGTTCTGTGCGGCCTGCCGTCCCGTGTGGG
B37-1	CGGTGATGCGCCTCCAGCGCGGGGAGTTTCTTAACCCTTTCCGACTTACAAGAGCCGG
B37-2	GCGAGACTCAGGTGGTGCCTTTGGCATTCGACCAGGAGATATCGCGTTCAGCTATGCCC
C37-1	CCCATGAGAATAATACCGCCGATTTACGTCAGTCCGGTTTCCCACACGGGACGGCAGGC
C37-2	CGCACAGAACACATACGCTTTGGGCATAGCTGAACGCGATATCTCCTGGTCGAATGCC
D37-1	GCCCAGATTAAAACGTGAATTCCTAGCCAGTACAGGGTTTCCGGACTGACGTAAATCGG
D37-2	CGGTATTATTCTCATGGGTTTGGCACCACCTGAGTCTCGCCCGGCTCTTGTAAGTCGG

表2 miRNA靶标序列

Tab 2 miRNA sequence for DTN

miRNA	Sequence (5′→3′)
miRNA-10a-5p	UACCCUGUAGAUCCGAAUUUGUG
miRNA-182-5p	UUUGGCAAUGGUAGAACUCACACU
miRNA-1-3p	UGGAAUGUAAAGAAGUAUGUAU
miRNA-30a-5p	UGUAAACAUCCUCGACUGGAAG
miRNA-100-5p	AACCCGUAGAUCCGAACUUGUG

表3 特异度验证所需序列

Tab 3 DNA sequence for specificity

DNA	Sequence (5′→3′)
Signal-10a-Cy5	Cy5-CACAAATTCGGATCTACTAA
Signal-182-Cy5	Cy5-AGTGTGAGTTCTACCATTGA
Signal-1-Cy5	Cy5-ATACATACTTCTTTACATGA
Signal-30a-Cy5	Cy5-CTTCCAGTCGAGGATGTTGA
Signal-100-Cy5	Cy5-CACAAGTTCGGATCTACTGA

图1 框架核酸线性信号放大平台设计示意图Note： A. Diagram of high-throughput target detection. B. Signal amplification via signal group labeling. C. Workflow diagram for target detection.

Fig 1 Schematic illustration of the design of a high-throughput multi-target detection platform

图2 框架核酸线性信号放大平台台性能对比Note： A. Signal reporter based on DNA tetrahedral dimers. B. Polyacrylamide gel electrophoresis characterization of 1‒6 valence fluorescent signal reporter. Up—Gelred channel; Bottom—Cy5 channel. C. Fluorescence characterization of 1‒6 valence fluorescent signal reporter. D. Single-molecule fluorescence characterization of 1‒6 valence fluorescent signal reporter. E. Signal output comparison between gold substrate and glass substrate for two types of miRNA.

Fig 2 Performance comparison of the high-throughput multi-target joint detection platform

图3 框架核酸线性信号放大平台性能验证Note： A. Photograph of the high-throughput multi-target detection platform (left); schematic of high-throughput target detection and AFM characterization (right). B. Standard curve for miRNA-100-5p. C. Specificity validation of the detection system. D. Parallel detection standard curves for the high-throughput multi-target detection platform (scale bar: 250 μm).

Fig 3 Performance of the high-throughput multi-target detection platform

参考文献 34

1	VAN HOOGSTRATEN L M C, VRIELING A, VAN DER HEIJDEN A G, et al. Global trends in the epidemiology of bladder cancer: challenges for public health and clinical practice[J]. Nat Rev Clin Oncol, 2023, 20(5): 287-304.
2	BRAY F, LAVERSANNE M, SUNG H, et al. Global cancer statistics 2022: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2024, 74(3): 229-263.
3	JASSIM A, RAHRMANN E P, SIMONS B D, et al. Cancers make their own luck: theories of cancer origins[J]. Nat Rev Cancer, 2023, 23(10): 710-724.
4	YUAN S P, ALMAGRO J, FUCHS E. Beyond genetics: driving cancer with the tumour microenvironment behind the wheel[J]. Nat Rev Cancer, 2024, 24(4): 274-286.
5	TRAN L, XIAO J F, AGARWAL N, et al. Advances in bladder cancer biology and therapy[J]. Nat Rev Cancer, 2021, 21(2): 104-121.
6	WAHIDA A, BUSCHHORN L, FRÖHLING S, et al. The coming decade in precision oncology: six riddles[J]. Nat Rev Cancer, 2023, 23(1): 43-54.
7	KON E, AD-EL N, HAZAN-HALEVY I, et al. Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects[J]. Nat Rev Clin Oncol, 2023, 20(11): 739-754.
8	HIAM-GALVEZ K J, ALLEN B M, SPITZER M H. Systemic immunity in cancer[J]. Nat Rev Cancer, 2021, 21(6): 345-359.
9	LU Z H, CHEN Y, LIU D, et al. The landscape of cancer research and cancer care in China[J]. Nat Med, 2023, 29(12): 3022-3032.
10	DYRSKJØT L, HANSEL D E, EFSTATHIOU J A, et al. Bladder cancer[J]. Nat Rev Dis Primers, 2023, 9(1): 58.
11	LENIS A T, LEC P M, CHAMIE K, et al. Bladder cancer: a review[J]. JAMA, 2020, 324(19): 1980-1991.
12	SANLI O, DOBRUCH J, KNOWLES M A, et al. Bladder cancer[J]. Nat Rev Dis Primers, 2017, 3: 17022.
13	LOPEZ-BELTRAN A, COOKSON M S, GUERCIO B J, et al. Advances in diagnosis and treatment of bladder cancer[J]. BMJ, 2024, 384: e076743.
14	MATULEWICZ R S, DELANCEY J O, MEEKS J J. Cystoscopy[J]. JAMA, 2017, 317(11): 1187.
15	MAAS M, TODENHÖFER T, BLACK P C. Urine biomarkers in bladder cancer: current status and future perspectives[J]. Nat Rev Urol, 2023, 20: 597-614.
16	ROSE K M, HUELSTER H L, MEEKS J J, et al. Circulating and urinary tumour DNA in urothelial carcinoma: upper tract, lower tract and metastatic disease[J]. Nat Rev Urol, 2023, 20: 406-419.
17	DONG H F, LEI J P, DING L, et al. MicroRNA: function, detection, and bioanalysis[J]. Chem Rev, 2013, 113(8): 6207-6233.
18	LIU Z L, WANG H, LIU J, et al. MicroRNA-21 (miR-21) expression promotes growth, metastasis, and chemo- or radioresistance in non-small cell lung cancer cells by targeting PTEN[J]. Mol Cell Biochem, 2013, 372(1/2): 35-45.
19	CAUSA F, ALIBERTI A, CUSANO A M, et al. Supramolecular spectrally encoded microgels with double strand probes for absolute and direct miRNA fluorescence detection at high sensitivity[J]. J Am Chem Soc, 2015, 137(5): 1758-1761.
20	GE Z L, LIN M H, WANG P, et al. Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor[J]. Anal Chem, 2014, 86(4): 2124-2130.
21	YIN F F, LIU H Q, LI Q, et al. Trace microRNA quantification by means of plasmon-enhanced hybridization chain reaction[J]. Anal Chem, 2016, 88(9): 4600-4604.
22	DUAN R X, ZUO X L, WANG S T, et al. Lab in a tube: ultrasensitive detection of microRNAs at the single-cell level and in breast cancer patients using quadratic isothermal amplification[J]. J Am Chem Soc, 2013, 135(12): 4604-4607.
23	LIN M H, WANG J J, ZHOU G B, et al. Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection[J]. Angew Chem Int Ed, 2015, 54(7): 2151-2155.
24	SONG P, SHEN J W, YE D K, et al. Programming bulk enzyme heterojunctions for biosensor development with tetrahedral DNA framework[J]. Nat Commun, 2020, 11(1): 838.
25	LI F Q, MAO X H, LI F, et al. Ultrafast DNA sensors with DNA framework-bridged hybridization reactions[J]. J Am Chem Soc, 2020, 142(22): 9975-9981.
26	SQUIRES T M, MESSINGER R J, MANALIS S R. Making it stick: convection, reaction and diffusion in surface-based biosensors[J]. Nat Biotechnol, 2008, 26(4): 417-426.
27	LIU Q, GE Z L, MAO X H, et al. Valency-controlled framework nucleic acid signal amplifiers[J]. Angew Chem Int Ed, 2018, 57(24): 7131-7135.
28	YIN F F, ZHAO H P, LU S S, et al. DNA-framework-based multidimensional molecular classifiers for cancer diagnosis[J]. Nat Nanotechnol, 2023, 18(6): 677-686.
29	ZAIDI N, SIDDIQUI Z, SANKHWAR S N, et al. Urinary microRNA-10a levels in diagnosis and prognosis of urinary bladder cancer[J]. J Cancer Res Ther, 2023, 19(5): 1324-1329.
30	GU C H, ZHAO K Y, ZHOU N C, et al. UBAC2 promotes bladder cancer proliferation through BCRC-3/miRNA-182-5p/p27 axis[J]. Cell Death Dis, 2020, 11(9): 733.
31	YAMASAKI T, YOSHINO H, ENOKIDA H, et al. Novel molecular targets regulated by tumor suppressors microRNA-1 and microRNA-133a in bladder cancer[J]. Int J Oncol, 2012, 40(6): 1821-1830.
32	ZHANG C, MA X, DU J, et al. MicroRNA-30a as a prognostic factor in urothelial carcinoma of bladder inhibits cellular malignancy by antagonising Notch1[J]. BJU Int, 2016, 118(4): 578-589.
33	XU C L, ZENG Q S, XU W D, et al. miRNA-100 inhibits human bladder urothelial carcinogenesis by directly targeting mTOR[J]. Mol Cancer Ther, 2013, 12(2): 207-219.
34	BLANCA A, SANCHEZ-GONZALEZ A, REQUENA M J, et al. Expression of miR-100 and miR-138 as prognostic biomarkers in non-muscle-invasive bladder cancer[J]. APMIS, 2019, 127(8): 545-553.

[1]	谢莎莎, 吕叶辉, 林涧. 四面体框架核酸在医学领域的应用与研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(3): 380-384.
[2]	杨晨凯, 李威, 曹向乾, 何磊, 李圣洲, 沈兵. 基于纳米技术的膀胱癌治疗方法研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(12): 1562-1568.
[3]	戚炀炀, 熊鹰. Galectin-9阳性肿瘤相关巨噬细胞在肌层浸润性膀胱癌中的表型、功能及临床治疗意义[J]. 上海交通大学学报（医学版）, 2022, 42(12): 1666-1676.
[4]	王晓寒, 叶云林, 肖康华, 赵志鑫, 吴静雅. 敲减免疫调节蛋白B7-H3基因对膀胱癌细胞恶性增殖、侵袭和干细胞样特性的影响[J]. 上海交通大学学报(医学版), 2021, 41(11): 1454-1460.
[5]	周坤, 陈虞梅, 刘建军, 黄钢. 膀胱癌根治性切除术前利尿延迟¹⁸氟-氟代脱氧葡萄糖正电子发射计算机断层显像的预后价值[J]. 上海交通大学学报(医学版), 2021, 41(10): 1336-1343.
[6]	伍科1，姚智显 1，郑重1*，程蕾蕾 2，刘志宏 1. 顺铂通过杀伤粒样髓系抑制细胞促进膀胱癌小鼠心力衰竭[J]. 上海交通大学学报（医学版）, 2019, 39(9): 1011-.
[7]	黄盛松, 朱子奇, 孙莉, 等. 膀胱癌组织JARID1B表达及其慢病毒表达载体的构建及功能研究[J]. , 2010, 30(6): 693-.

膀胱癌相关miRNA灵敏检测的框架核酸线性放大平台构建

Framework nucleic acid-based linear amplification platform for sensitive detection of bladder cancer-related miRNAs

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 34

相关文章 7

编辑推荐

Metrics