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In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets Ying Wang, Zhaohui Li, Thomas J. Weber, Dehong Hu, Chiann-Tso Lin, Jinghong Li, and Prof. Yuehe Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac400858g • Publication Date (Web): 11 Jun 2013 Downloaded from http://pubs.acs.org on June 16, 2013

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Analytical Chemistry

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In Situ Live Cell Sensing of Multiple Nucleotides Exploiting DNA/RNA Aptamers and Graphene Oxide Nanosheets 24

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Ying Wang†‡, Zhaohui Li‡ǂ, Thomas J. Weber‡, Dehong Hu‡, Chiann-Tso Lin‡, 28

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Jinghong Li†* and Yuehe Lin‡* 30

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† Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and 35

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Instrumentation, Tsinghua University, Beijing, China 100084 37

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‡ Pacific Northwest National Laboratory, Richland, Washington, USA 99352 38

ǂ College of Chemistry and Molecular Engineering, Zhengzhou University, 41

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Zhengzhou, China 450001 43

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Dr. Ying Wang and Dr. Zhaohui Li contributed equally to this work 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

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ACS Paragon Plus Environment


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1 2 3 4 6

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ABSTRACT: 7 8 9

Nucleotides, 10

for

example,

adenosine-5’-triphosphate

(ATP)

and

1

guanosine-5’-triphosphate (GTP), are primary energy resources for numerous 12 13

reactions in organisms including microtubule assembly, insulin secretion, ion channel 14 15

regulation and so on. In order to advance our understanding of the production and 16 17

consumption of nucleoside triphosphates, a versatile sensing platform for 18 19

simultaneous visualization of ATP, GTP, adenosine derivates and guanosine derivates 20 21

in living cells has been built up in the present work based on graphene oxide 2 23

nanosheets (GO-nS) and DNA/RNA aptamers. Taking advantage of the robust 24 25

fluorescence quenching ability, unique adsorption for single strand DNA/RNA probes, 26 27

and efficient intracellular transport capacity of GO-nS, selective and sensitive 28 29

visualization of multiple nucleoside triphosphates in living cells is successfully 30 31

realized with the designed aptamer/GO-nS sensing platform. Moreover, GO-nS 32 3

displays good biocompatibility to living cells and high protecting ability for 34 35

DNA/RNA probes from enzymatic cleavage. These results demonstrate that 36 37

aptamers/GO-nS based sensing platform is capable of selective, simultaneous, and in 38 39

situ detecting of multiple nucleotides, which hold a great potential for analyzing other 40 41

biomolecules in living cells. 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

2


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1 2 4

3

Introduction 6

5

Nucleotides, consisting of a nucleobase and a five-carbon sugar as well as one 8

7

phosphate group, are mainly considered as the basic building block units in nucleic 10

9

acids. However, some nucleotides also have many other important roles in 12

1

metabolism and in metabolic control. Among them, adenosine-5’-triphosphate (ATP) 14

13

and guanosine-5’-triphosphate (GTP) are found as typical energy molecules 16

15

regulating various biological processes.1,2 As the primary energy molecule in living 18

17

cells, ATP is generally called as "molecular unit of currency" for intracellular energy 20

19

transfer,3-5 which is highly necessary for some biochemical reactions such as muscle 2

21

contraction, bio-molecule synthesis and degradation, membrane transportation and 24

23

signal transduction, etc.6-9 Meanwhile, GTP plays important roles in protein synthesis 26

25

and holds great significance for signal transduction in living cells.10-13 Most 28

27

importantly, ATP and GTP could act coordinately to realize numerous reactions such 30

29

as microtubule assembly, insulin secretion, and ion channel regulation.14-17 Therefore, 32

31

the analysis especially in situ simultaneous visualization of ATP and GTP has great 34

3

importance to advance our understanding of their behavior, function and interaction 36

35

inside living cells.18-21 38

37

In the past decades, numerous attempts have been made to realize the detection 40

39

of either ATP or GTP. For example, biosensors based on fluorescent molecules like 42

41

acridine, polythiophene or imidazolium anthracene derivate have been used for ATP 4

detection.22-24 Luciferase (an ATP-consuming enzyme) and lymphoid ecto-adenylate 46

45

43

kinase have been combined to measure cellular ATP levels in some cases. In addition, 48

47

ATP aptamer-sensors making uses of fluorescent, electrochemical, and colorimetric 50

methods have been reported in previous studies.25,26 Similarly, synthesized fluorescent 52

51

49

dyes, such as water-soluble imidazolium anthracene derivative and benzimidazolium 54

53

with unique specificity, have been applied for GTP detection in buffer solutions as 56

well as biological fluids.19,21,27 However, most of the assays could only detect ATP or 58

57

5

GTP, respectively. Lack of methods is an obstacle to realize the simultaneous 60

59

detection of ATP and GTP inside living cells. Accordingly, a suitable in situ analyzing 3



1 2 3

assay for multiple bio-targets in living cells is highly desirable. 4 5

In recent years, graphene oxide (GO) has been emerging with several unique 6 7

properties including planar sheet structure, fluorescence quenching ability, easy 9

8

functionalization and good biocompatibility.28-32 For example, GO has been utilized 10 1

for the ultrasensitive detection of cyclin A2 with detection limit of 0.5 nM, 10-fold 13

12

better than that using single-walled carbon nanotubes.33 Meanwhile, polyethylene 14 15

glycol (PEG) modification is also under investigation to extend GO as an anti-cancer 17

16

therapeutic drug delivery agent.34 Based on the robust fluorescence quenching ability, 18 19

GO has been used in fluorescence resonance energy transfer (FRET) applications 20 21

when combined with either single-strand DNA probes or thrombin aptamers labeled 23

2

with fluorescence molecules.35,36,37 Besides, Fan et al. has reported a GO-based 24 25

multicolor fluorescent DNA nanoprobe that allows rapid, sensitive and selective 27

26

detection of multiple DNA targets in buffer solutions.38 Moreover, our previous efforts 28 29

to develop a proof of concept analyzing assay for small molecule detection in living 31

30

cells has been reported.30 This assay has employed modified GO-nS (graphene oxide 32 3

nanosheets) as a fluorescence quencher and cellular carrier for ATP detection in living 34 35

cells. The demonstrated dramatic delivery, protection, and sensing abilities should 36 37

facilitate the development of this technology to be a robust candidate for many other 38 39

small molecule detections inside living cells. 40 41

Herein, we further applied GO-based sensing technology for simultaneous cellular 42 43

imaging of adenosine derivates and guanosine derivates to address the issues 4 45

mentioned above. To validate the utility of this technology in our study, we employed 46 47

fluorescent dye labeled DNA/RNA aptamers and GO-nS to create ATP, GTP, 48 49

adenosine derivates and guanosine derivates sensing platform. Taking the advantages 50 51

of aptamers and GO-nS, ATP- and GTP-selective aptamer probes could be loaded 52 53

onto GO-nS and delivered through cell membrane successfully. Due to electron 54 5

acceptor effect of GO-nS, obvious fluorescent off/on switch and real-time target 56 57

detection in living cells was realized by aptamer/GO-nS sensing platform. In addition, 58 60

59

GO-nS shows excellent protection for DNA/RNA form enzymatic cleavage and good biocompatibility to living cells in the studies. The primary achievements indicated 4


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that this GO-nS based sensing system owned promising abilities for monitoring and 4 5

imaging of multiple nucleotides in living cells, which will enable it to be applied to 6 7

cellular simultaneous imaging studies of many other predicting biomarkers such as 8 9

mRNAs, and microRNAs. 10 1 12 13

■ EXPERIMENTAL SECTION 15

14 16

Materials. Fluorophore carboxy fluorescein (FAM)-labeled ATP aptamer (5′ 17 19

18

-FAM/AAC CTG GGG GAG TAT TGC GGA GGA AGGT-3 ′ ), cyanine-5 20 2

21

(Cy5)-labeled GTP aptamer (5′-Cy5/GGG ACG AAG UGG UUG GGU GUG AAA 23 24

ACG UCC C-3′) and AF546-labeled random DNA (5′-AF546/TCT AAA TCG CTA 25 27

26

TGG TCG C-3′) were synthesized by Integrated DNA Technologies, Inc. (San Diego, 28 29

CA). Agarose, 10 base-pair DNA ladder, 10×Tris/Borate/EDTA (TBE) buffer, gel 31

30

loading buffer (TrackIt™ cyan⁄yellow loading buffer), SYBR green nucleic acid gel 32 3

stain, DNase I along with the reaction and stop buffers, Trypanblue 0.4% were from 34 36

35

Invitrogen (Carlsbad, CA). Adenosine 5′-triphosphate (ATP) disodium salt hydrate, 37 38

guanosine 5′-triphosphate (GTP) disodium salt hydrate, cytidine 5′-triphosphate (CTP) 39 41

40

disodium salt, thymidine 5′-triphosphate (TTP) sodium salt and all other chemicals 43

42

including graphite, K2S2O8, P2O5, and H2SO4 were purchased from Sigma-Aldrich (St. 45

4

Louis, MI). All buffers and reagent solutions were prepared with water purified on a 47

46

Barnstead NANOPure UV Ultra Pure Water System (Boston, MA). Binding buffer for 49

48

ATP and GTP assay was 10 mM potassium phosphate with 200 mM KCl, 5 mM 51

50

MgCl2 and 0.1 mM EDTA. 53

52

Preparation and Characterization of GO-nS. Chemically synthesized GO 5

54

powder was produced by filtrated the production and dried then in vacuum overnight 57

56

at 25 °C. 0.2 mg/mL GO aqueous solution was prepared with pre-synthesized GO 60

59

58

powder and sonicated in water bath for 2 h followed by a strong sonification with power of 40 W (4 min/sonication × 5 sonications) in an ice bath. The ice bath was 5



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1 3

2

changed after each treatment to maintain sample temperature below 5 oC. The crude 4 5

GO was re-dissolved in 5 M NaOH and sonicated in water bath for 2 h, followed by 6 7

adding HCl and completely rinse with pure water to natural pH. The obtained GO 9

8

sample was then autoclaved at 80 oC for 60 min. The autoclaved solution was 10 1

centrifuged at 12,000 rpm for 10 min, and the supernatant was designated as GO-nS. 12 13

Characterization. AFM imaging was prepared by first treating a freshly cleaved 15

14

mica surface with 1 M MgCl2 for one minute, followed by addition of 10 μL of a 16 17

sample solution onto the mica surface. The mica substrate was tilted to allow the 18 19

droplet to spread on the surface. After adsorption for one minute, the mica surface was 20 21

washed twice with doubly distilled water, and dried with compressed air. The sample 2 23

was then scanned in tapping mode with a Nanoscope III, Digital Instrument atomic 24 25

force microscope (AFM). Transmission electron microscopy (TEM) images of GO-nS 26 27

and ATP aptamer-FAM/GO-nS were taken on a JEOL TEM 2010 microscope. Powder 28 29

X-ray diffraction (XRD) measurements were performed on Bruker D8-Advance X-ray 30 31

powder diffract meter using a graphite monochrometer with Cu Ka radiation (k= 32 3

1.5406 Å). Raman spectra were obtained using a confocal microprobe Raman system 34 35

Renishaw, RM200 (Gloucestershire, United Kingdom) using green (514 nm) laser 36 37

excitation. The 39

38

Fourier-transform

infrared

spectroscopy

(FT-IR)

spectrum

(800-4,000 cm-1) was measured using a Perkin Elmer Fourier transform infrared 40 41

spectroscopy spectrometer with pure KBr as the background. 42 43

Nucleotides detection with Aptamer/GO-nS Complex. 100 nM ATP 4 45

aptamer-FAM and/or GTP aptamer-Cy5 were incubated with different concentrations 46 47

of GO-nS aqueous solution in 10 mM potassium phosphate with 200 mM KCl, 5 mM 49

48

MgCl2 and 0.1 mM EDTA for 5 min at 25 oC to form the aptamer/GO-nS complex 51

50

39,40

52

. Herein, the 10 mM potassium phosphate with 200 mM KCl, 5 mM MgCl2 and

53

0.1 mM EDTA was reaction buffer for in vitro detection of nucleotides. The 54 5

aptamer/GO-nS were tested to determine the quenching ability of GO-nS. The in vitro 56 57

detection of GTP based on GTP aptamer-Cy5/GO-nS was carried out by adding 58 60

59

different concentrations of GTP into 100 nM GTP aptamer-Cy5/GO-nS in reaction buffer, incubation for 1 h at 25 oC and the mixture solution was scanned on a 6


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1 3

2

fluorometry41,42. Selective responding of different triphosphates was carried out by 4 5

incubation of 100 nM ATP aptamer-FAM/GTP aptamer-Cy5/GO-nS complex with 7

6

0.5 mM and 2 mM ATP, GTP, CTP and TTP in reaction buffer for 1 h at 25 oC by 8 9

recording the respective fluorescence intensity of FAM channel and Cy5 channel. In 10 1

addition, 0.5 mM and 2 mM ATP and GTP mixture solution was added into the 100 12 13

nM ATP aptamer-FAM/GTP aptamer-Cy5/GO-nS complex for the selectivity tests. 14 15

All of the in vitro detections were recorded on a Tecan Safire 2 microplate reader 16 17

(TECAN, Switzerland) directly without any other treatments and operations. 18 19

Electrophoresis Investigation. Agarose was prepared with 10 mM TBE buffer 20 21

containing 2 mM EDTA (TBE, pH 9.2) and heated to form 3.5% agarose-TBE 2 23

sol-gel. The prepared gel was cooled down to room temperature prior to 24 25

electrophoresis. 100 nM of FAM-ATP aptamer, free in solution and unbound to 26 27

GO-nS, was prepared in DNase I reaction buffer and incubated with 0.2 units/µL of 28 29

DNase I for 15, and 40 min at room temperature respectively. The digestion reaction 30 31

for ATP aptamer-FAM /GO-nS complex was carried out by adding 0.2 units/µL of 3

32

DNase I into 100 nM aptamer solution pre-mixture with 3 μg/mL GO-nS for 5 min, 34 35

and then incubated for 15 and 40 min, respectively. All samples were heated at 95 °C 36 37

for 5 min immediately followed by addition of loading buffer prior to gel 38 39

electrophoresis. Gel electrophoresis was performed in 10 mM TBE buffer at 100 V 40 41

for 1 h. After electrophoresis, the gel was stained by 5000-fold diluted SYBR green 42 43

nucleic acid gel stain in 10 mM TBE for 30 min. Then the gel was visualized via 4 45

NucleoVision imaging system using UV irradiation. Images were captured using 46 47

GelExpert 2.0 software. 48 49

Cell Viability Assay of MCF-7 Cell Incubated with GO-nS. A human breast 50 51

cancer cell line MCF-7 (ATCC, HTB-22) was grown in the complete Eagle's 52 53

Minimum Essential Medium (completed EME medium, ATCC) with 0.01 mg/mL 54 5

bovine insulin and 10% fetal bovine serum (FBS). Cells were grown to confluence in 56 57

an incubator with humidified atmosphere of 5% CO2 and 95% air at 37 °C and kept in 58 60

59

a confluent state for 24 to 48 h before subculture. The cell culture methods were used for all of the cell studies in the present work. MCF-7 cells were trypsinized and 7



1 3

2

seeded at 5 × 104 cells/well into 12-well flat bottomed plate. After seeding for 24 h, 4 5

MCF-7 cells were treated with 1 - 9 µg/mL GO-nS for 12, 24 and 72 h, respectively. 6 7

Cell viability was determined using a standard trypan blue cell viability assay (0.05% 8 9

trypan blue staining for 5 min). 10 1

In Situ Live Cell Imaging of Multiple Nucleotides. For in situ investigations, 12 14

13

MCF-7 cells were incubated with ATP aptamer-FAM, GTP aptamer-Cy5, and 16

15

random DNA-Alex546N in the presence or absence of GO-nS at 37 °C for 6 h in 5% 18

17

CO2 atmosphere. To form the aptamer/GO-nS complex, aptamers labeled with 20

19

fluorophore were diluted to 5 µM in reaction buffer. 1 mg/mL GO-nS aqueous 2

21

solution was injected into the aptamer buffer to get a final concentration depending on 24

23

aptamer concentrations. Then the aptamer/GO-nS complex solution was diluted in 1 26

25

mL culture medium (completed EME medium, ATCC) to get the final concentration 28

27

of 25 nM, 50 nM, 100 nM and 200 nM, respectively for the cell imaging tests. After 30

29

incubation with aptamer/GO-nS complex, cells were washed with phosphate buffer 32

31

saline (PBS) completely and images were captured by confocal microscope on a Zeiss 34

3

LSM 710 NLO laser scanning confocal microscope with an upright Zeiss 36

35

Axioexaminer stand. The objective is W Plan-Apo 20X NA1.0 water dipping 38

37

objective. The max excitation wavelength and max emission wavelength for ATP 40

39

aptamer-FAM is 495 nm and 520 nm (show in green color), for GTP aptamer-Cy5 is 42

41

650 nm and 670 nm (show in red color), and for random DNA-Alex546N is 556 nm 4

43

and 573 nm (show in orange color). 45 46 48

47

■ RESULTS AND DISCUSSION 51

50

49

Configuration of DNA/RNA Aptamer/GO-nS Sensing Platform. Basic concept 53

52

of the proposed sensing platform is shown in Scheme 1. GO-nS and DNA/RNA 5

54

aptamers were employed to construct the aptamer/GO-nS sensing platform. Aptamers 57

56

are short, single-stranded oligonucleotides selected by an in vitro method known as 60

59

58

SELEX (systematic evolution of ligands by exponential enrichment).43 As we know, ATP aptamer has been demonstrated with expected performances from fundamental 8


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2

studies to therapeutic researches.44-46 However, no successful reports about GTP 4 5

aptamer untill a novel RNA aptamer with high specificity for GTP molecule reported 7

6

by Szostak and co-workers recently.47-50 This RNA aptamer is able to target GTP by 8 9

forming loop structure with Kds ranging from 9 nM to 8 µM. Based on the previous 10 1

studies, the multiple sensing platform is constructed through the assembly of 12 13

DNA/RNA aptamers on GO-nS. Large surface to volume ratio and the planar 14 15

structure of GO-nS provided a suitable substrate for multiple probes assembling. 16 17

Carboxyfluorescein (FAM) labeled ATP-aptamer and cyanine-5 (Cy5) modified 18 19

GTP-aptamer was introduced to generate multiple signals corresponding to different 21

20

targets. The assembly was induced by “π-π stacking” between aptamers and the 23

2

honeycomb lattice of GO.30,35,36,37 As shown in Scheme 1, binding of aptamers to 24 25

GO-nS guarantees the close proximity of dyes to graphene surface. The following 26 27

efficient long-range energy transfer from dye to GO-nS results in rapid and complete 28 29

quenching of fluorophores. In direct contrast, the conformation of aptamers could be 30 31

changed into a stable, internal loop structures after interaction with their targets. The 32 3

weak binding ability of loop-structured assembly of aptamer/target to GO-nS makes 34 35

fluorophores far away from the quencher surface, leading to fluorescence recovery of 36 37

FAM or Cy5. Referring to previous studies about drug delivery and tumor diagnostics 38 39

using graphene derivates, we proposed that GO-nS could be suitable for visualization 41

40

of multiple nucleotides based on the fluorescent off/on switch mechanism.30,34,37 42 43

Hence, cell imaging tests were carried out on MCF-7 cells (human breast cancer cell) 4 45

incubated with aptamer/GO-nS complex consisting of ATP aptamer-FAM, GTP 46 47

aptamer-Cy5 and GO-nS. The fluorescenct signal was captured by a confocal 48 49

microscope. As a result, the pictures would show bright fluorescent signals 50 51

corresponding to FAM and Cy5 tagged on DNA/RNA aptamers releasing from the 52 53

complex after interaction with cellular adenine derivates (including ATP, AMP and 54 5

adenine) and guanosine derivates, since both of ATP- and GTP- selective aptamers 56 57

were lack of distinguishing adenine and guanosine derivates. 58 59 60

9



1 2 3

Characterization of GO-nS. GO-nS were demonstrated with smaller size and 4 5

narrow size distribution relative to GO (Figure S1). To illustrate other features of 6 7

GO-nS, characterizations of XRD (Figure S2), Raman (Figure S3) and FT-IR spectra 8 9

(Figure S4) were carried out and detailed in supporting information. TEM images of 10 1

GO (Figure S5), GO-nS and aptamer/GO-nS (Figure S6) were illustrated to show 12 13

their morphology. Detail discussion about GO-nS characterizations were illustrated in 15

14

the Supporting Information51-55. 16 17

In Vitro Detection of Multiple Nucleotides. Fluorescence quenching ability of 18 19

GO-nS was evaluated prior to in vitro detection of ATP and GTP (Figure 1). 20 21

Single-aptamer/GO-nS complex was formed by mixing 100 nM ATP aptamer-FAM 2 23

or 100 nM GTP aptamer-Cy5 with 0.5 - 5 µg/mL GO-nS for 5 min in reaction buffer. 24 25

Quenching ability of GO-nS to ATP aptamer-FAM (Figure 1A) and GTP 26 27

aptamer-Cy5 (Figure 1B) was evaluated, respectively. Fluorescence intensity from 28 29

FAM or Cy5 decreased sharply as increasing concentration of GO-nS due to FRET 30 31

between fluorophore and GO-nS. Meanwhile, multi-aptamer/GO-nS complex 32 3

including ATP aptamer-FAM and GTP aptamer-Cy5 simultaneously was test to 34 35

determine the optimized GO-nS concentration for dual detection of ATP and GTP. As 36 37

shown in Figure 1C, fluorescence intensity derived from ATP aptamer-FAM and GTP 38 39

aptamer-Cy5 is mostly being quenched when concentration of GO-nS reaching 3 41

40

μg/mL. However, it is not stable until the concentration of GO-nS is up to 5 μg/mL. 43

42

Consequently, 5 μg/mL GO-nS was considered optimal for dual probing of ATP and 45

4

GTP simultaneously while 3 μg/mL GO-nS was taken as the optimized amount for 46 47

individual ATP- or GTP- aptamer. 49

48

ATP detection has been performed in our previous study.30 Herein we are mainly 50 51

focusing on the dissociation of GTP aptamer-Cy5 from aptamer/GO-nS complex and 52 53

the resulted fluorescence recovery (Figure 2). As shown in Figure 2A, after 5 min 54 5

incubation of GTP aptamer-Cy5 with GO-nS, a nearly 100 % fluorescence quenching 56 57

with very fast kinetics was observed for GTP aptamer-Cy5/GO-nS complexes. Being 58 60

59

incubated with 10 µM - 2 mM of GTP for 60 min, GTP aptamer-Cy5 fluorescence was recovered linearly over the range of GTP concentrations (inset of Figure 2A), 10


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suggesting that GTP aptamer-Cy5 was specifically liberated from the GO-nS surface. 4 5

Tracing the cause, the weak binding ability of loop-structured assembly of 6 7

aptamer/target to GO-nS makes fluorophores far away from the quencher surface and 8 9

leading to fluorescence recovery of Cy5. Figure S7 illustrates specific dissociation of 10 1

GTP aptamer only in the presence of GTP, but not ATP, CTP nor TTP. The large 12 13

planar surface of GO-nS raise the possibility for adsorption of multiple aptamers 14 15

whose dissociation could then be specifically monitored using the appropriate Ex/Em 16 17

wavelengths for FAM and Cy5 dyes (FAM Ex495/Em520; Cy5 Ex650/Em670). As shown in 18 19

Figure 2B, multi-aptamer/GO-nS complex was formed by mixing of 100 nM ATP 20 21

aptamer-FAM and 100 nM GTP aptamer-Cy5 with 5 µg/mL GO-nS for 5 min in 2 23

reaction buffer. ATP aptamer-FAM/GTP aptamer-Cy5/GO-nS complex was then 24 25

incubated with 0.5 mM and 2 mM ATP, GTP, CTP or TTP, respectively. Selective 26 27

release of ATP aptamer or GTP aptamer due to the high specific interaction between 28 29

aptamer and the target was observed by recording the fluorescence change. However, 30 31

for CTP and TTP, no obvious change was obtained. This observation successfully 32 3

facilitates the following ATP/GTP detections inside living cells. 34 35 36 37

Cleavage Protection and Cell viability Assay of GO-nS. As we know, most 38 39

biological probes, such as messenger RNA and molecular beacons, are easily to be 40 41

degraded by cellular enzymes or digested by cellular nucleases, which seriously 42 43

limited their further applications in living cell studies. Therefore, delivery of aptamer 4 45

probes into cells while protecting the fluorescent aptamers from enzymatic cleavage is 46 47

significant to facilitate biological application of aptamers. To date, only a few 48 49

nanomaterials (such as carbon nanotubes, silica nanoparticles, and gold nanoparticles) 51

50

have demonstrated with protection capabilities during molecular transport.43,44,46 52 53

Agarose gel electrophoresis was used to demonstrate GO-nS dependent protection of 54 5

aptamer from enzymatic cleavage. In order to separate aptamer and GO-nS, 57

56

electrophoresis was carried out right after heating of the samples at 95 ˚C for 5 min 58 60

59

(Figure 3A). DNase I, which can nonspecifically cleave single- and double-stranded DNA, was employed to simulate enzymatic cleavage functions in living cells. 11



1 2 3

Incubation of ATP aptamer-FAM with DNase I (0.2 units/µL) for 15 or 40 min 4 5

resulted in cleavage and loss of detection, relative to ATP aptamer-FAM control 6 7

without any DNase I shown as lane 2 in Figure 3A. In contrast, the ATP 8 9

aptamer-FAM/GO-nS complex is hard to be cleaved by DNase I after 15 or 40 min 10 1

incubation, suggesting that the GO-nS provides strong protecting ability to aptamers 12 13

against enzymatic cleavage. 14 15

To realize the in situ target monitoring in living cells, aptamer/GO-nS complex 16 17

are expected to be with good biocompatibility and low toxicity. Consequently, we 18 19

investigated whether GO-nS reduced cell viability in MCF-7 cells as an initial test 20 21

case (Figure 3B). MCF-7 cells were incubated with 1-9 µg/mL of GO-nS for 24 - 72 2 23

h and cell was determined under trypan blue assay, which measures plasma 24 25

membrane integrity as an index of cell viability. GO-nS showed negligible effects on 27

26

cell viability at concentrations of ≤ 7 µg/mL (Figure 3B). Certain toxicity could be 28 29

observed at the highest concentration (9 µg/mL) by 72 h. The results demonstrated 30 31

that GO-nS exhibited negligible effects on the growth in MCF-7 cells with 32 3

concentration lower than 7 µg/mL while the post-culture time was less than 72 h. 34 35

Cells treated with large amount of GO-nS for longer culture time resulted in low cell 36 37

proliferations because of mild cytotoxicity. 38 39 40 41

In situ Live Cell Imaging of Multiple Nucleotides. We have demonstrated that 42 43

multiple aptamers could be adsorbed onto GO-nS while retaining good specificity for 4 45

their respective triphosphates to dissociate from the aptamer/GO-nS complex. To 46 47

further test whether this platform could be used for intracellular simultaneous imaging 48 49

studies, MCF-7 cells were employed to be incubated with multi-aptamer/GO-nS 50 51

consisting of ATP aptamer-FAM, GTP aptamer-Cy5, and random DNA-Alex546N 52 53

for 6 hours. Random DNA-Alex546N was designed as a reference probe to evaluate 54 5

the specificity of this platform in living cells (Figure 4). MCF-7 cells incubated with 56 57

ATP aptamer-FAM/GTP aptamer-Cy5/random DNA-Alex546N without assistance of 58 60

59

GO-nS was taken as control to prove the transport ability of GO-nS. As shown in Figure 4, fluorescence signal derived from ATP aptamer-FAM and GTP aptamer-Cy5 12


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was clearly observed (Figure 4 a, b) while little/no fluorescent signal from the random 4 5

DNA aptamer could be observed (Figure 4c). Meanwhile, fluorescence could not be 6 7

observed without GO-nS (Figure 4 f-j). In addition, merged images showed both 8 9

co-localization and discrete sub-cellular localization profiles associated with the ATP10 1

and 12

GTP-aptamers.

Furthermore,

MCF-7

cells

were

incubated

with

the

13

multi-aptamer/GO-nS complex (ATP aptamer-FAM/GTP aptamer-Cy5/random 14 15

DNA-Alex546N/GO-nS) at concentrations ranging from 50 nM to 200 nM and 16 17

images were captured by confocal microscope at 6 h post-incubations (Figure 5). 18 19

Fluorescence intensities corresponding to cellular ATP (green color, Figure 5 a, e, i, 20 21

m) and GTP (red color, Figure 5 b, f, j, n) increased with increasing complex 2 23

concentration. 24

In

contrast,

fluorescent

signal

corresponding

to

random

25

DNA-Alex546N remained below detection as shown in Figure 5 c, g, k, and o. These 26 27

results demonstrated that this sensing platform could deliver multiple aptamer probes 28 29

into living cells and successfully realize the in situ visualization of ATP and GTP 30 31

simultaneously. 32 3 34 36

35

■ CONCLUSIONS 38

37

In summary, an advanced in situ sensing platform for multiple nucleotides 40

39

detection has been fabricated with DNA/RNA aptamers and GO-nS. GO-nS exhibited 42

41

strong loading ability to multiple DNA/RNA aptamer probes while remaining their 4

43

biological functions as expected. Meanwhile, GO-nS displayed good biocompatibility 46

45

to living cells, efficient intracellular transport capacity, and high protecting ability for 48

47

DNA/RNA probes from enzymatic cleavage. Moreover, the GTP-RNA aptamer 50

49

utilized in this work would be the foremost demonstration of this RNA aptamer in 52

51

practical investigation. The positive results achieved here might become initial guide 54

53

for the potential applications of this GTP RNA aptamer in biochemical studies. In 56

5

conclusion, this platform showed great advantages and hold great potential to be 58

57

applied in many other cellular studies such as multiple imaging of DNA, protein, and 60

59

RNA, etc. The apparent biocompatibility, efficient intracellular transport capacity and 13



1 2 3

ability of GO-nS to protect DNA/RNA against cleavage might find important 4 5

applications in drug delivery and gene therapy associated with predicting biomarkers 6 7

such as single-nucleotide polymorphisms and microRNAs. 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

14


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1 2 3

■ AUTHOR INFORMATION 6

5

4

Corresponding Author 8

7

* E-mail: [email protected] (Jinghong Li); [email protected] (Yuehe Lin) 9 10 12

1

■ ACKNOWLEDGMENT 13 15

14

This work was financially supported by National Basic Research Program of 17

16

China (No. 2011CB935704), the National Natural Science Foundation of China (No. 19

18

21235004, No. 21128005) and Tsinghua University Initiative Scientific Research 21

20

Program. This work was supported by a laboratory-directed research and development 23

2

program at Pacific Northwest National Laboratory (PNNL). Part of the research 25

24

described in this paper was performed using EMSL. PNNL is operated for DOE by 27

26

Battelle under Contract DE-AC05-76RL01830. The authors are very grateful to Dr. 29

28

Alan Scott Lea (PNNL), Prof. Li Yu (Tsinghua University), Prof. Dongsheng Liu 31

30

(Tsinghua University), Mr. Yunpeng Huang (Tsinghua University) for professional 3

32

advices. 34 35 37

36

■ Supporting Information 38 39

Additional information about synthesis of chemically prepared graphene oxide (GO) 40 41

and GO-nS, characterizations of GO and GO-nS and in vivo selective responding of 42 43

GTP as noted in text. This material is available free of charge via the Internet at 4 45

http://pubs.acs.org. 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

15



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1 2 4

3

Legends 5 6 8

7

Scheme 1. Schematic illustration of multiple sensing platform. Binding of ATP 9 10

aptamer-FAM and GTP aptamer-Cy5 to GO-nS led to fluorescence off due to FRET 1 13

12

effect between fluorophores and GO-nS. After incorporating the analytes (ATP or 14 16

15

GTP), loop-structured assemblys of aptamer-ATP and aptamer-GTP were released 17 18

from GO-nS and resulted in fluorescence on. In situ simultanous probing of ATP and 19 21

20

GTP in living cells was realized consequently by using this fluorescence off/on switch 2 24

23

concept. 25 26 27 29

28

Figure 1. Normalized fluorescence intensity of 100 nM ATP aptamer-FAM (A), 100 30 32

31

nM GTP aptamer-Cy5 (B), and the mixture of 100 nM ATP aptamer-FAM and 100 34

3

nM GTP aptamer-Cy5 (C) versus the concentration of GO-nS from 0.5 μg/mL to 5 35 37

36

μg/mL in reaction buffer. Error bars were obtained from three parallel experiments. 38 40

39

Excitation wavelength for FAM: 470 nm; Cy5: 650 nm. 41 42 43 45

4

Figure 2. (A) Fluorescence emission spectra of 100 nM GTP aptamer-Cy5 quenched 46 48

47

with 3 μg/mL GO-nS (red bottom line) and fluorescence recovery by addition of GTP 49 50

with concentration ranging from 0.01 - 2 mM (from bottom to top) in reaction buffer 51 53

52

for 1 h at 25 oC. Inset: Linear relationship between (F-F0)/F0 (relative fluorescence 54 56

5

intensity, where F0 and F are the fluorescence intensity without and with the presence 57 58

of GTP) and GTP concentration, error bars were obtained from three parallel 59 60

experiments. (B) Selective responding to ATP and GTP based on aptamer/GO-nS 21



1 2 4

3

sensing platform by recording the respective fluorescence channel (green color 5 6

presents the FAM channel and red color presents the Cy5 channel). Normalized 7 9

8

fluorescence intensity of the mixture of 100 nM ATP aptamer-FAM and 100 nM GTP 10 12

1

aptamer-Cy5 in reaction buffer is shown in column (ATP aptamer and GTP aptamer). 13 14

After injection of GO-nS, fluorescence was quenched shown as column (with 5 15 17

16

µg/mL GO-nS). By incubation of ATP aptamer-FAM/GTP aptamer-Cy5/GO-nS with 18 19

0.5 and/or 2 mM ATP, GTP, CTP or TTP for 1 h at 25 oC respectively (shown in the 21

20 2

corresponding columns), fluorescence recovery was obtained. Excitation wavelength 23 25

24

for FAM: 470 nm; for Cy5: 650 nm. 26 27 28 30

29

Figure 3. (A) Agarose gel electrophoresis image for enzymatic cleavage protection 31 32

assay: lane 1, DNA size ladder for 100 bp; lane 2, aptamer-FAM; lane 3, 3 35

34

aptamer-FAM reacted with DNase I for 15 min; lane 4, aptamer-FAM reacted with 36 38

37

DNase I for 40 minutes; lane 5, aptamer-FAM/GO-nS; lane 6, aptamer-FAM/GO-nS 39 40

incubated with DNase I for 15 min; lane 7, aptamer-FAM/GO-nS incubated with 41 43

42

DNase I for 40 min. Aptamer concentration is 100 nM, GO-nS is 3 μg/mL, and DNase 4 46

45

I is 0.2 units/µL. Excitation wavelength for SYBR Green I: 494 nm. (B) Cell viability 47 48

determined using a trypan blue assay after treatment of MCF-7 cells with 1 - 9 µg/mL 49 51

50

GO-nS for 12 h (red), 24 h (green) or 72 h (blue). Values represent the mean ± se, n=3. 52 54

53

*Significantly different from control, p < 0.05. 5 56 57

60

59

58

Figure 4. Confocal images of in situ visualization for ATP and GTP. Images represent

22


Page 22 of 30

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1 2 4

3

MCF-7 cells incubated in completed EME medium for 6 h with 100 nM ATP 5 6

aptamer-FAM/GO-nS (panel a), 100 nM GTP aptamer-Cy5/GO-nS (panel b), 100 nM 7 9

8

random DNA-Alex546N/GO-nS (panel c) and the merged fluorescent panels for 10 12

1

confocal images (panel d). Merged confocal images with bright field image (panel e). 13 14

Corresponding images for MCF-7 cells incubated with ATP aptamer-FAM (panel f), 15 17

16

GTP aptamer-Cy5 (panel g), random DNA-Alex546N (panel h) alone without 18 20

19

assistance of GO-nS, the merged fluorescent panels for confocal images (panel i) and 21 2

merged confocal images with bright field image (panel j) are illustrated. Images were 23 25

24

captured by confocal microscope after extensive washing of cells with PBS. Scale bar: 26 28

27

50 µm. 29 30 31 3

32

Figure 5. In situ cell imaging of MCF-7 cells with 25 nM (a-c), 50 nM (e-g), 100 nM 34 36

35

(i-k) and 200 nM (m-o) ATP aptamer-FAM/GTP aptamer-Cy5/Random DNA37 38

Alex546N/GO-nS for 6 h in completed EME medium. Images of cells were captured 39 41

40

using the respective Ex/Em wavelengths for each fluorophore and equal exposure 42 4

43

times followed by completely rinsing with PBS. Merged images are illustrated in 45 46

panels d, h, i, and p. Scale bar: 10 µm. 47 48 49 50 51 52 53 54 5 56 57 58 59 60

23



1 2 4

3

Scheme and Figures 6

5

Scheme 1 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

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Figure 1 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

25



1 3

2

Figure 2 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

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Figure 3 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

27



1 3

2

Figure 4 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

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Figure 5 4 5 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

29



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For TOC Only 6 7 8 9 10 1 12 13 14 15 16 17 18 19 20 21 2 23 24 25 26 27 28 29 30 31 32 3 34 35 36 37 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60

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RNA

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