PMID- 25058642
OWN - NLM
STAT- MEDLINE
DCOM- 20150402
LR  - 20211021
IS  - 1750-2799 (Electronic)
IS  - 1750-2799 (Linking)
VI  - 9
IP  - 8
DP  - 2014 Aug
TI  - In situ simultaneous monitoring of ATP and GTP using a graphene oxide 
      nanosheet-based sensing platform in living cells.
PG  - 1944-55
LID - 10.1038/nprot.2014.126 [doi]
AB  - Here we present a detailed protocol for in situ multiple fluorescence monitoring 
      of adenosine-5'-triphosphate (ATP) and guanosine-5'-triphosphate (GTP) in MCF-7 
      breast cancer cells by using graphene oxide nanosheet (GO-nS) and DNA/RNA 
      aptamers. FAM-labeled ATP aptamer and Cy5-modified GTP aptamer are used to 
      construct the multiple aptamer/GO-nS sensing platform through 'pi-pi stacking' 
      between aptamers and GO-nS. Binding of aptamers to GO-nS guarantees the 
      fluorescence resonance energy transfer between fluorophores and GO-nS, resulting 
      in 'fluorescence off'. When the aptamer/GO-nS are transported inside the cells 
      via endocytosis, the conformation of the aptamers will change on interaction with 
      cellular ATP and GTP. On the basis of the fluorescence 'off/on' switching, 
      simultaneous sensing and imaging of ATP and GTP in vitro and in situ have been 
      realized through fluorescence and confocal microscopy techniques. In this 
      protocol, we describe the synthesis of GO and GO-nS, preparation of aptamer/GO-nS 
      platform, in vitro detection of ATP and GTP, and how to use this platform to 
      realize intracellular ATP and GTP imaging in cultured MCF-7 cells. The 
      preparation of GO-nS is anticipated to take 7-14 d, and assays involving 
      microscopy imaging and MCF-7 cells culturing can be performed in 2-3 d.
FAU - Wang, Ying
AU  - Wang Y
AD  - Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and 
      Instrumentation, Tsinghua University, Beijing, China.
FAU - Tang, Longhua
AU  - Tang L
AD  - Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and 
      Instrumentation, Tsinghua University, Beijing, China.
FAU - Li, Zhaohui
AU  - Li Z
AD  - Pacific Northwest National Laboratory, Richland, Washington, USA.
FAU - Lin, Yuehe
AU  - Lin Y
AD  - 1] Pacific Northwest National Laboratory, Richland, Washington, USA. [2] School 
      of Mechanical and Materials Engineering, Washington State University, Pullman, 
      Washington, USA.
FAU - Li, Jinghong
AU  - Li J
AD  - Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and 
      Instrumentation, Tsinghua University, Beijing, China.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20140724
PL  - England
TA  - Nat Protoc
JT  - Nature protocols
JID - 101284307
RN  - 7782-42-5 (Graphite)
RN  - 86-01-1 (Guanosine Triphosphate)
RN  - 8L70Q75FXE (Adenosine Triphosphate)
SB  - IM
MH  - Adenosine Triphosphate/*analysis
MH  - Graphite
MH  - Guanosine Triphosphate/*analysis
MH  - MCF-7 Cells
MH  - Microscopy, Fluorescence/*methods
MH  - Nanostructures
MH  - Nanotechnology/*methods
EDAT- 2014/07/25 06:00
MHDA- 2015/04/04 06:00
CRDT- 2014/07/25 06:00
PHST- 2014/07/25 06:00 [entrez]
PHST- 2014/07/25 06:00 [pubmed]
PHST- 2015/04/04 06:00 [medline]
AID - nprot.2014.126 [pii]
AID - 10.1038/nprot.2014.126 [doi]
PST - ppublish
SO  - Nat Protoc. 2014 Aug;9(8):1944-55. doi: 10.1038/nprot.2014.126. Epub 2014 Jul 24.