Skip to main content
SpringerLink
Log in

Multiplexed determination of intracellular messenger RNA by using a graphene oxide nanoprobe modified with target-recognizing fluorescent oligonucleotides

  • Short Communication
  • Published:
Microchimica Acta Aims and scope Submit manuscript

Abstract

A multiplexed graphene oxide (GO) fluorescent nanoprobe is described for quantification and imaging of messenger RNAs (mRNAs) in living cells. The recognizing oligonucleotides (with sequences complementary to those of target mRNAs) were labeled with different fluorescent dyes. If adsorbed on GO, the fluorescence of the recognizing oligonucleotides is quenched. After having penetrated living cells, the oligonucleotides bind to target mRNAs and dissociate from GO. This leads to the recovery of fluorescence. Using different fluorescent dyes, various intracellular mRNAs can be simultaneously imaged and quantified by a high content analysis within a short period of time. Actin mRNA acts as the internal control. This GO-based nanoprobe allows mRNA mimics to be determined within an analytical range from 1 to 400 nM and a detection limit as low as 0.26 nM. Up to 3 intracellular mRNAs (C-myc, TK1, and actin) can be detected simultaneously in a single living cell. Hence, this nanoprobe enables specific distinction of intracellular mRNA expression levels in cancerous and normal cells. It can be potentially applied as a tool for detection of cancer progression and diagnosis.

A multiplexed graphene oxide (GO)-based fluorescent nanoprobe is described for quantification and imaging of intracellular messenger RNAs. After penetrating living cells, the recovered fluorescence of the dissociated recognizing oligonucleotides can be analyzed , and this allows for simultaneous detection of up to 3 intracellular messenger RNAs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Schwarzenbach H, Hoon DS, Pantel K (2011) Cell-free nucleic acids as biomarkers in cancer patients. Nat Rev Cancer 11(6):426–437

    Article  CAS  Google Scholar 

  2. Morris S, Vachani A, Pass HI, Rom WN, Ryden K, Weiss GJ, Hogarth DK, Runger G, Richards D, Shelton T, Mallery DW (2018) Whole blood FPR1 mRNA expression predicts both non-small cell and small cell lung cancer. Int J Cancer 142(11):2355–2362

    Article  CAS  Google Scholar 

  3. VanGuilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44(5):619–626

    Article  CAS  Google Scholar 

  4. Chen JJ (2007) Key aspects of analyzing microarray gene-expression data. Pharmacogenomics 8(5):473–482

    Article  CAS  Google Scholar 

  5. Li W (2011) Application of volcano plots in analyses of mRNA differential expressions with microarrays. J Bioinf Comput Biol 10(06):1231003

    Article  Google Scholar 

  6. Lyubimova A, Itzkovitz S, Junker JP, Fan ZP, Wu X, van Oudenaarden A (2013) Single-molecule mRNA detection and counting in mammalian tissue. Nat Protoc 8(9):1743–1758

    Article  Google Scholar 

  7. Chen KH, Boettiger AN, Moffitt JR, Wang S, Zhuang X (2015) RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348(6233):aaa6690

    Article  Google Scholar 

  8. King BR (2018) Visualization of arenavirus RNA species in individual cells by single-molecule fluorescence in situ hybridization (smFISH) suggests a model of cyclical infection and clearance during persistence. J Virol 92(12):e02241–e02217

    Article  CAS  Google Scholar 

  9. Qiu L, Wu C, You M, Han D, Chen T, Zhu G, Jiang J, Yu R, Tan W (2013) A targeted, self-delivered, and photocontrolled molecular beacon for mRNA detection in living cells. J Am Chem Soc 135(35):12952–12955

    Article  CAS  Google Scholar 

  10. Giraldo-Vela JP, Kang W, McNaughton RL, Zhang X, Wile BM, Tsourkas A, Bao G, Espinosa HD (2015) Single-cell detection of mRNA expression using nanofountain-probe electroporated molecular beacons. Small 11(20):2386–2391

    Article  CAS  Google Scholar 

  11. Lee K, Rouillard JM, Kim BG, Gulari E, Kim J (2009) Conjugated polymers combined with a molecular beacon for label-free and self-signal-amplifying DNA microarrays. Adv Funct Mater 19(20):3317–3325

    Article  CAS  Google Scholar 

  12. Adinolfi B, Pellegrino M, Giannetti A, Tombelli S, Trono C, Sotgiu G, Varchi G, Ballestri M, Posati T, Carpi S, Nieri P, Baldini F (2017) Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells. Biosens Bioelectron 88:15–24

    Article  CAS  Google Scholar 

  13. Prigodich AE, Randeria PS, Briley WE, Kim NJ, Daniel WL, Giljohann DA, Mirkin CA (2012) Multiplexed nanoflares: mRNA detection in live cells. Anal Chem 84(4):2062–2066

    Article  CAS  Google Scholar 

  14. Li N, Chang C, Pan W, Tang B (2012) A multicolor nanoprobe for detection and imaging of tumor-related mrnas in living cells. Angew Chem Int Ed 51(30):7426–7430

    Article  CAS  Google Scholar 

  15. Zheng D, Seferos DS, Giljohann DA, Patel PC, Mirkin CA (2011) Aptamer nano-flares for molecular detection in living cells. Nano Lett 9(9):3258–3261

    Article  Google Scholar 

  16. Yang Y, Huang J, Yang X, Quan K, Wang H, Ying L, Xie N, Ou M, Wang K (2015) FRET nanoflares for intracellular mRNA detection: avoiding false positive signals and minimizing effects of system fluctuations. J Am Chem Soc 137(26):8340–8343

    Article  CAS  Google Scholar 

  17. Zu F, Yan F, Bai Z, Xu J, Wang Y, Huang Y, Zhou X (2017) The quenching of the fluorescence of carbon dots: a review on mechanisms and applications. Microchim Acta 184(7):1899–1914

    Article  CAS  Google Scholar 

  18. Lu CH, Yang HH, Zhu CL, Chen X, Chen GN (2009) A graphene platform for sensing biomolecules. Angew Chem Int Ed 48(26):4785–4787

    Article  CAS  Google Scholar 

  19. Ueno Y, Furukawa K, Matsuo K, Inoue S, Hayashi K, Hibino H (2015) On-chip graphene oxide aptananoprobe for multiple protein detection. Anal Chim Acta 866:1–9

    Article  CAS  Google Scholar 

  20. He Y, Lin Y, Tang H, Pang D (2012) A graphene oxide-based fluorescent aptananoprobe for the turn-on detection of epithelial tumor marker mucin 1. Nanoscale 4(6):2054–2059

    Article  CAS  Google Scholar 

  21. Ryoo SR, Lee J, Yeo J, Na HK, Kim YK, Jang H, Lee JH, Han SW, Lee Y, Kim VN, Min DH (2013) Quantitative and multiplexed microRNA sensing in living cells based on peptide nucleic acid and nano graphene oxide (PANGO). ACS Nano 7(7):5882–5891

    Article  CAS  Google Scholar 

  22. Mangalath S, Abraham S, Joseph J (2017) pH-responsive fluorescence enhancement in graphene oxide–naphthalimide nanoconjugates: a fluorescence turn-on nanoprobe for acetylcholine. Chemistry 23(47):11404–11409

    Article  CAS  Google Scholar 

  23. Ikeguchi M, Hirooka Y (2004) Expression of c-myc mRNA in hepatocellular carcinomas, noncancerous livers, and normal livers. Pathobiology 71(5):281–286

    Article  CAS  Google Scholar 

  24. Broët P, Romain S, Daver A, Ricolleau G, Quillien V, Rallet A, Asselain B, Martin PM, Spyratos F (2001) Thymidine kinase as a proliferative marker: clinical relevance in 1,692 primary breast cancer patients. J Clin Oncol 19(11):2778–2787

    Article  Google Scholar 

  25. Chen CC, Chang TW, Chen FM, Hou MF, Hung SY, Chong IW, Lee SC, Zhou TH, Lin SR (2006) Combination of multiple mRNA markers (PTTG1, Survivin, UbcH10 and TK1) in the diagnosis of Taiwanese patients with breast cancer by membrane array. Oncology 70(6):438–446

    Article  CAS  Google Scholar 

  26. Cui L, Chen Z, Zhu Z, Lin X, Chen X, Yang CJ (2013) Stabilization of ssRNA on graphene oxide surface: an effective way to design highly robust RNA probes. Anal Chem 85(4):2269–2275

    Article  CAS  Google Scholar 

  27. Tang Z, Wu H, Cort JR, Buchko GW, Zhang Y, Shao Y, Aksay IA, Liu J, Lin Y (2010) Constraint of DNA on functionalized graphene improves its biostability and specificity. Small 6(11):1205–1209

    Article  CAS  Google Scholar 

  28. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M (2008) Circulating microRNAs as stable blood-based markers for cancer detection. PNAS 105(30):10513–10518

    Article  CAS  Google Scholar 

  29. Li M, Zeringer E, Barta T, Schageman J, Cheng A, Vlassov AV (2014) Analysis of the RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Philos Trans R Soc B 369(1652):20130502

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful for financial support from the National Natural Science Foundation of China (81502589, 81802104), the Natural Science Foundation of Guangdong Province, China (2014A030310481, 2018A030310229), the Medical Science and Technology Research Foundation of Guangdong Province, China (A2017448), the Science and Technology Project of Shenzhen (CXZZ20130515092016300, JCYJ20160422142707177, JCYJ20170306093259065), the Department of Education of Guangdong Province (2017KQNCX069), and the Department of Education, Guangdong Government under the Top-tier University Development Scheme for Research and Control of Infectious Diseases.

Author information

Authors and Affiliations

  1. Department of Pharmacy, Shantou University Medical College, No. 22 Xinling Road, Shantou, 515041, China

    Hongyan Jiang, Wei Li & Kai Ling

  2. Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University, Shenzhen, 518035, China

    Hongyan Jiang

  3. Translational Medicine Collaborative Innovation Center, The Second Clinical Medical College (Shenzhen People’s Hospital), Jinan University, Shenzhen, 518020, China

    Fu-Rong Li & Kai Ling

  4. Department of Pharmacy, Affiliated Hospital of Qingdao University, Shandong, 266003, China

    Xiaodong Lu

Authors
  1. Hongyan Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fu-Rong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Wei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaodong Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kai Ling
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kai Ling.

Ethics declarations

The author(s) declare that they have no competing interests.

Electronic supplementary material

ESM 1

(DOC 5592 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jiang, H., Li, FR., Li, W. et al. Multiplexed determination of intracellular messenger RNA by using a graphene oxide nanoprobe modified with target-recognizing fluorescent oligonucleotides. Microchim Acta 185, 552 (2018). https://doi.org/10.1007/s00604-018-3090-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s00604-018-3090-1

Keywords

Search

Navigation