Polydopamine Nanospheres Nanocomplex for in Situ Molecular Sensing in Living Cells

Aptamer/Polydopamine Nanospheres Nanocomplex for in Situ Molecular Sensing in Living Cells

Weibing Qiang,Hongting Hu,Liang Sun,Hui Li,and Danke Xu*

State Key Laboratory of Analytical Chemistry for Life Science,School of Chemistry and Chemical Engineering,Nanjing University, 22Hankou Road,Nanjing,Jiangsu210093,China

*Supporting Information

semiquanti?cation.This design provides a strategy to

intracellular molecules analysis.For the advantages of

applications,such as gene and drug delivery,intracellular

onitoring and visualization of physical events and

molecules,the important method to understand cell biology,have a profound in?uence on advancement in biological science.Sensors with high spatiotemporal resolution and selective and quantitative signals for physical variables or biological molecules in living cells are a constant need in biological science.1,2For the high sensitivity and noninvasive-ness of?uorescent spectroscopic techniques,they o?er sensing for intracellular analysis and intracellular signaling.From the morphological analysis of anatomical structures to sensitive measurements of intracellular molecules,?uorescent bioimag-ing is widely used in the applications of biomedical sciences.3 As?uorescent bioimaging is a nondestructive,selective, sensitive,and real-time method without radioactivity,4it has some inherent advantages over those conventional imaging methods.Magnetic resonance imaging(MRI)is severely limited by nonspeci?c biodistribution and expensive con-struction,5and micro single-photon emission computed tomography/computed tomography(microSPECT/CT)is seriously limited by low spatial resolution.6The researchers have developed numbers of organic dyes and?uorescent proteins as powerful molecular probes for?uorescent biosensing.However,due to the poor photobleaching resistance,broad emission spectra,and largely shifted excitation bands of these molecular probes,there are some limitations for them to achieve reliable intracellular measurements.7Fur-thermore,the possible chemical interactions or steric hindrance with biomolecules of these molecular probes would cause biotoxicity or perturbation to the investigated systems.8 Currently,the“always on”strategy is mostly used in the

design of the detection probes,in which the probes are bound to the targets and then lead to an elevated signal with reference to surroundings.9?11For the absence of signal change during the targeting of these probes,to eliminate interference from excessive unbound probes,a time-consuming washing step is typically required in the in vitro utilization of“always on”probes,and during in vivo applications,the high background from constant signals in nontarget tissues often a?ects target speci?city and imaging contrast.As an alternative,some“signal on”activatable probes have been developed,12?14in which normally quenched signals are activated only after recognizing the targets,such as the molecular beacons(MBs).15,16 Some nanomaterials have be used as quenchers for developing“signal on”biosensors for the assay of biomolecules, such as nucleic acids and proteins.Typically,these nanoma-terials are capable of quenching the?uorescence from di?erent ?uorophores with various emission frequencies through nonresonant energy transfer or electron transfer.For their advantage as universal quenchers,?uorescence-quenching nanomaterials eliminate the selection issue of a?uorophore?

Received:August11,2015

Accepted:November10,2015

quencher pair in conventional“signal on”biosensors.So far, gold nanoparticles(AuNPs)17or nanorods,18carbon nanoma-terials,such as carbon nanotubes(CNTs),19carbon nano-particles(CNPs),20and graphene,21MoS2nanosheets,22WS2 nanosheets,23carbon nitride nanosheets,24copper oxide nanobelts,25metal?organic framework(MOF),26and polyani-line nano?bers27have been proved to be?uorescence-quenching nanomaterials.However,in the consideration of low cytotoxicity,only few of those?uorescence-quenching nanomaterials could be used for intracellular sensors,such as AuNPs,gold nanorods,and carbon nanotubes or gra-phene.28?31In the intracellular biosensors,these nanomaterials act as not only a universal?uorescence quencher but also a nanocarrier for the recognition probes to improve the delivery. In the previous studies,the polydopamine nanospheres (PDANS)have been employed as a nanoquencher to develop the sensing platforms for the assay of biomolecules,32?34and some reports have demonstrated that polydopamine has excellent biocompatibility and biodegradability.35Here,a “signal on”?uorescence biosensing nanocomplex was devel-oped,based on the polydopamine nanospheres and the aptamers.Aptamers are single-strand DNA/RNA oligonucleo-tides with high speci?city and a?nity against many given targets,ranging from small inorganic and organic molecules to macromolecules and even cells.36?38For the advantages of high a?nity and speci?city,low immunogenicity,as well as facile synthesis and modi?cation,39,40aptamers promise to be the next-generation recognition molecules in molecular diagnosis. In our study,we employed the carboxy?uorescein(FAM)-labeled DNA aptamer and PDANS to create a biosensor for the adenosine triphosphate(ATP)sensing in living cells.ATP is the primary energy molecule in living cells,and it is generally called as the“molecular unit of currency”for intracellular energy transfer.41,42It is highly necessary for some biochemical reactions such as muscle contraction,membrane transportation, biomolecule synthesis and degradation,and signal transduction, etc.43,44In the absence of the target,the aptamer/PDANS nanocomplex was initially in the“o?”state due to the e?cient ?uorescence quenching of FAM adjacent to the surface of PDANS.After the nanocomplex was incubated with sample containing the target or transported into cells,due to the binding of the aptamer by ATP,the nanocomplex would change into the“on”state as a result of the dissociation of the FAM from the surface of PDANS,thus providing greatly enhanced?uorescence emission intensity.The primary achieve-ments indicated that this PDANS-based biosensor owned promising abilities for sensing of ATP in living cells,which will enable it to be applied in cellular imaging of the predicting

biomarkers such as mRNAs and microRNAs.

■MATERIALS AND METHODS

Materials and Reagents.The96microwell plates were obtained from Coring incorporated(New York,U.S.A.). Dopamine hydrochloride,oligomycin,ATP,cytidine triphos-phate(CTP),guanosine triphosphate(GTP),and uridine triphosphate(UTP)were obtained from Sangon Biotech Co., Ltd.(Shanghai,China).DNase I was obtained from Sigma-Aldrich(St.Louis,MO,U.S.A.).1×PBS,complete Dulbecco’s modi?ed Eagle’s medium(DMEM)(with10%fetal bovine serum,100U/mL penicillin,and0.1mg/mL streptomycin), trypsin?EDTA,HeLa cells(human cervical carcinoma),and the MTT cell proliferation and cytotoxicity detection kit were obtained from KeyGEN Biotech Co.,Ltd.(Nanjing,China).MCF-7cells(human breast cancer)were obtained from Hunan University.Glass bottom cell culture dishes(Φ15mm)were purchased from Nest Biotechnology Co.,Ltd.(Wuxi,China). Other reagents from commercial suppliers were analytical grade and used without further puri?cation.Ultrapure water(electric resistance>18.25MΩ)obtained through a Millipore Milli-Q water puri?cation system(Billerica,MA,U.S.A.)was used throughout the experiments.1×PBS(with200mM KCl,5 mM MgCl2,and0.1mM EDTA)was used as the binding bu?er for ATP assay.FAM-labeled ATP aptamer P1(5′-FAM-ACCTGGGGGAGTATTGCGGAGGAAGGT-3′)and FAM-labeled random ssDNA P2(5′-FAM-AAAAAAGCTTGTGTT-CGTTGGAAAAAA-3′)were synthesized and puri?ed by Sangon.

Apparatus.Scanning electron microscopy(SEM)images were recorded using an S-4800scanning electron microscope (Hitachi,Japan).The absorption and?uorescence emission were measured on a Synergy H1multimode reader(BioTek, U.S.A.).A confocal scanning laser microscope(CSLM)(TCS SP5,Leica,Germany)was used to take cell images.The laser excitation for FAM was488nm,the bright-?eld image was recorded simultaneously by transmission PMT,and the ?uorescence detection band was set to505?575nm for FAM. Preparation of the P1/PDANS Nanocomplex.PDANS was synthesized according to our previous report with some modi?cations.32Brie?y,50mg of dopamine hydrochloride was added to a mixture of100mL Tris bu?er(10mM)and20mL of ethyl alcohol with stirring.After stirring for72h,the polydopamine nanospheres PDANS was obtained.The suspension was centrifuged and washed/resuspended with water several times.The precipitate was dried for the following experiments.After the synthesis of PDANS,enough PDANS was introduced into the binding bu?er containing100nM P1, and the mixture was incubated at room temperature for10min to form the P1/PDANS nanocomplex.Then the obtained P1/ PDANS nanocomplex was stored at4°C before further usage. The P2/PDANS nanocomplex was also prepared as the above descriptions.

In Vitro Detection of ATP by the P1/PDANS Nano-complex.In a typical assay,di?erent concentrations of ATP (ranging from0to2mM)or some other molecules were added into100nM P1/PDANS nanocomplex in the binding bu?er and the mixture was incubated at37°C for1h.After incubation,the?uorescence of the resulting solution was measured.The?uorescence emission spectra were recorded from508to650nm at an excitation wavelength of480nm,and the?uorescence intensity at523nm was used for quantitative analysis.

Cytotoxicity Assays and Live Cell Imaging with the P1/PDANS Nanocomplex.HeLa cells and MCF-7cells were grown in complete DMEM medium(with10%fetal bovine serum,100U/mL penicillin,and0.1mg/mL streptomycin)at 37°C in a humidi?ed atmosphere containing5%CO2.The MTT assay was used for the assessment of the cytotoxic e?ects from PDANS.HeLa cells were seeded into a96-well cell-culture plate at104cell/well and then incubated for24h at37°C under5%CO2.After incubating the cells with various concentrations of PDANS for12h,the standard MTT assay was carried out to determine the cell viabilities relative to the control untreated cells.In the cell imaging experiments,cells were seeded in glass bottom cell culture dishes and grown for 24h.When the cells were about90%con?uent,500μL of fresh cell growth medium supplemented with the P1/PDANS

nanocomplex,the P2/PDANS nanocomplex,the PDANS only,or P1was added in the dishes,respectively.After an incubation of 12h,1×PBS was employed to wash the cells three times.The ?uorescence imaging of the cells was observed with a confocal scanning laser microscope,and three-dimensional images were taken by scanning the samples every 2μm along the z -axis across a de ?ned section.

■

RESULTS AND DISCUSSION

Principle of the P1/PDANS Nanocomplex for Molec-ular Sensing.As shown in Scheme 1,the aptamer and PDANS were employed to construct the aptamer/PDANS nanocomplex.As it has been proven that single-strand DNA sequences (ssDNA)could be assembled on the polydopamine surface with strong a ?nity,45the nanocomplex is constructed through the assembly of FAM-labeled ATP aptamer (P1)on https://www.360docs.net/doc/c01380712.html,rge surface-to-volume ratio of PDANS makes it a suitable substrate for probes assembling.The assembly was caused by “π?πstacking ”interactions between the nucleobases of the aptamer and the aromatic groups of PDANS.45As shown in Scheme 1,the binding of the aptamer on PDANS guarantees the close proximity of ?uorophores to the surface of PDANS.The following rapid and complete quench of ?uorophores was led by the e ?cient energy transfer from ?uorophores to PDANS.In direct contrast,after interaction with ATP,the conformation of the aptamer would change into a stable,hairpin structure.46The weak binding ability of the hairpin-structured aptamer/target complex to PDANS makes ?uo-rophores far away from the quencher surface,resulting in the ?uorescence recovery of FAM.Referring to previous studies about the PDANS-based biosensors and the excellent biocompatibility of the polydopamine,we proposed that the P1/PDANS nanocomplex could be suitable for sensing of ATP based on the ?uorescent “signal on ”strategy.Hence,cell imaging studies were carried out on HeLa cells,which were incubated with the P1/PDANS nanocomplex.The ?uorescent signal was observed by a confocal microscopy.As a result,bright ?uorescent signals of FAM would be shown in the pictures corresponding to the aptamer releasing from the nanocomplex after interaction with cellular adenine derivatives (including ATP,ADP,AMP,and adenine)rather than only

ATP,because of the lack of distinguishing ability of ATP-selective aptamer to adenine derivatives.

Formation of the P1/PDANS Nanocomplex.Prior to the formation of the nanocomplex,the PDANS was prepared through a facile and low-cost method.We prepared monodisperse polydopamine nanospheres PDANS with a diameter of approximately 62.7nm (Figure S1,Supporting Information ).The SEM data indicates that PDANS falls within a size range that favors cellular uptake by mammalian cells.47As PDANS shows broad-band absorbance in the UV ?vis spectrum (Figure S2,Supporting Information ),it would quench the ?uorescence of the ?uorophores with di ?erent emission wavelengths due to Fo r ster resonance energy transfer (FRET).32The P1/PDANS nanocomplex was formed by mixing 100nM P1with PDANS for 10min in the binding bu ?er.The quenching ability of PDANS toward P1was evaluated (Figure 1).Fluorescence intensity of FAM decreased

sharply during the increase of PDANS concentration due to energy transfer between the ?uorophore and PDANS.When the concentration of PDANS added reached 0.15mg/mL,the ?uorescence of P1was nearly completely quenched,and the quenching e ?ciency was 95.05%.Consequently,0.15mg/mL PDANS was considered optimal for the construction of the P1/PDANS nanocomplex with 100nM P1.

In Vitro Detection of ATP.The P1/PDANS nanocomplex was used for the in vitro detection of ATP prior to sensing in living cells.Due to the energy transfer from FAM to PDANS,the ?uorescence of FAM was quenched,and the nanocomplex was initially in the “o ?”state.On being incubated with ATP for 1h,the ?uorescence of the nanocomplex was recovered (Figure 2A).Tracing the cause,the weak a ?nity of the hairpin-structured aptamer/target complex to PDANS makes the ?uorophore far away from the surface of PDANS and leads to ?uorescence recovery of FAM.The ?uorescence intensity was found to be linear with the concentration of ATP in the range of 0.01?2mM,and the limit of detection (LOD)was calculated to be 0.01mM (Figure 2A).The linear range of the nanocomplex met the need for the sensing of ATP in living cells,as the concentration of ATP in living cells is typically 1?10mM.48

Scheme 1.Schematic Illustration of Aptamer/PDANS Nanocomplex for the Molecular Sensing in Living

Cells

Figure 1.Quenching e ?ciency of 100nM P1upon the introduction of di ?erent concentrations of PDANS.Inset:?uorescence emission spectra of 100nM P1with di ?erent concentrations of PDANS (0,0.05,0.10,0.15,0.20,and 0.25mg/mL).

Furthermore,the speci ?city of the nanocomplex for the detection of ATP was tested (Figure 2B).The target ATP led to the enhancement of the ?uorescence of the P1/PDANS nanocomplex,while after the incubation of P1/PDANS with the three analogues CTP,GTP,and UTP for 1h,the ?uorescence intensity of FAM was almost the same with the blank,and the nanocomplex was still in the “o ?”state.This indicated that the P1/PDANS nanocomplex was selective for the assay of ATP,and this result successfully facilitates the following ATP sensing in living cells.Meanwhile,the nanocomplex constructed by the random DNA P2and PDANS was also used for the speci ?city study.When the analyte was not presented,P2was adsorbed on the surface of PDANS for the interaction between ssDNA and PDANS.As FAM was quenched through FRET,the P2/PDANS nano-complex was in the “o ?”state,while the addition of 2mM ATP did not lead to the recovery of the ?uorescence,and the P2/PDANS nanocomplex was still in the “o ?”state.The reason was that P2could not react with ATP to change the conformation;the ?uorescence was kept quenched as P2adsorbed on the surface of PDANS.The above results indicated that only ATP could lead to the ?uorescence recovery of P1/PDANS,and none but P1/PDANS,the nanocomplex constructed by the aptamer and PDANS could be used for the sensing of ATP.

Cleavage Protection and Cell Viability Assay of PDANS.To our knowledge,most nucleic acid probes,such as molecular beacons,are easily digested by cellular nucleases or degraded by cellular enzymes,which seriously limits their further applications in the studies of biomolecules and physical events in living cells.Therefore,the challenge in the biological application of aptamers in living cells is the delivery of aptamer probes into cells while protecting them from enzymatic cleavage.However,only a few nanomaterials (such as gold nanoparticles,carbon nanotubes,graphene,and silica nano-particles)29,49have been proved with protection capabilities during molecular transport.Here,DNase I was employed to simulate enzymatic cleavage functions in living cells,which would nonspeci ?cally cleave single-and double-stranded DNA.If PDANS could not protect the aptamer from enzymatic cleavage,P1would be cleaved by DNase I that would cause the release of FAM from the surface of PDANS and the ?uorescence recovery.However,the result showed that the introduction of DNase I did not lead to the ?uorescence enhancement of P1/PDANS (Figure 2B),suggesting that PDANS has the protection capability to ssDNA against enzymatic cleavage,which has also been proved by a recent work.34Then,the aptamer P1would target ATP after the delivery of the P1/PDANS nanocomplex into the cells.

To realize the in situ target sensing in living cells,it is expected that the P1/PDANS nanocomplex is with good biocompatibility and low toxicity.Consequently,the MTT assay,the standard cell viability assay,was employed for the evaluation of the cytotoxicities of PDANS on HeLa cells.After the HeLa cells were incubated with PDANS concentrations up to 0.45mg/mL for 12h,higher than 90%cell survival rate was observed (Figure S3,Supporting Information ).Throughout the present study for cell imaging,the concentration of PDANS used was less than 0.15mg/mL,which would ensure high viability of all the tested cells.

In Situ Live Cell Imaging of ATP.It has been demonstrated that the aptamer could be assembled on the surface of PDANS while retaining good speci ?city for ATP to dissociate from the aptamer/PDANS nanocomplex.To further test the employment of this nanocomplex for intracellular imaging study,HeLa cells were used to be incubated with P1/PDANS for 12h.The nanocomplex consisting of random DNA P2(P2/PDANS)was employed as a reference probe for the evaluation of the speci ?city of the P1/PDANS nanocomplex in living cells,and HeLa cells incubated with ATP aptamer P1without PDANS were chosen as control to prove the transport ability of PDANS.As shown in Figure 3,a signi ?cant FAM ?uorescence image was obtained from the cells cultured with P1/PDANS (Figure 3C),indicating successful intracellular aptamer delivery and ATP sensing in HeLa cells,while almost no ?uorescence signal was observed from HeLa cells incubated either with or without P1as well as with P2/PDANS or PDANS only (Figure 3,parts A,B,D,and E).

To con ?rm the internalization of P1/PDANS in HeLa cells,Z -scanning confocal imaging was further performed (Figure S4,Supporting Information ).After the incubation of HeLa cells with P1/PDANS for 12h,the images were taken by scanning the samples every 2μm along the z -axis across a de ?ned section.Bright FAM ?uorescence was clearly present throughout the whole cells,suggesting the e ?cient delivery of this nanocomplex to the cytosol.

Furthermore,HeLa cells were incubated with P1/PDANS at concentrations of 25,50,and 100nM,and after a 12

h

Figure 2.(A)Fluorescence emission spectra of 100nM P1/PDANS nanocomplex in the presence of di ?erent concentrations of ATP (0,0.01,0.1,0.5,1,1.5,and 2mM).Inset:calibration curve for ATP detection.FI =2351.4c +630.1(R 2=0.9978).(B)Fluorescence intensity of 100nM P1/PDANS nanocomplex with blank (a),2mM ATP (b),2mM CTP (c),2mM GTP (d),2mM UTP (e),and 20U DNase I (h)and ?uorescence intensity of 100nM P2/PDANS nanocomplex without (f)and with 2mM ATP (g).

incubation,images were captured by a confocal microscope (Figure S5,Supporting Information ).Fluorescence intensity corresponding to cellular ATP increased with increasing nanocomplex concentration.This result demonstrated that this nanocomplex successfully realize the in situ sensing of ATP in HeLa cells.

To further con ?rm the ?uorescence signal resulting from the endogenously produced ATP of the HeLa cells,an assay for in situ ATP semiquanti ?cation was designed,and the results are shown in Figure 4.Before culture with P1/PDANS,the HeLa cells were treated with 10μM oligomycin (a well-known inhibitor of ATP 50)or with 5mM Ca 2+(a commonly used ATP inducer 51)for 30min.As shown in Figure 4,the FAM ?uorescence decreased dramatically upon treatment with oligomycin (Figure 4A),while a signi ?cant enhancement was observed when the cells were preincubated with Ca 2+(Figure 4C).These results demonstrate that the P1/PDANS nano-complex could achieve reliable intracellular measurement of ATP in HeLa cells.

As the above results have demonstrated,the P1/PDANS nanocomplex could deliver the aptamer probe into HeLa cells and successfully achieve the in situ sensing of ATP.As ATP is

the primary energy molecule in all living cells,we further test whether this nanocomplex could be used for intracellular sensing of ATP in other cells.MCF-7cells were used to be incubated with P1,P1/PDANS,and P2/PDANS for 12h,respectively.As shown in Figure 5,?uorescence signal corresponding to FAM tagged on P1in the nanocomplex was clearly observed (Figure 5B),while little/no ?uorescence signal from P2/PDANS could be observed (Figure 5

C).

Figure 3.Fluorescence images,bright-?eld images,and the merge of ?uorescence and bright-?eld images of HeLa cells after incubation without (A)or with 100nM P1(B),100nM P1/PDANS nanocomplex (C),100nM P2/PDANS nanocomplex (D),and 0.15mg/mL PDANS (E)for 12h at 37°C.Scale bar:75μ

m.

Figure 4.Fluorescence images,bright-?eld images,and the merge of ?uorescence and bright-?eld images of HeLa cells treated with 10μM oligomycin (A),medium (B),or 5mM Ca 2+(C)followed by incubation with 100nM P1/PDANS nanocomplex for 12h at 37°C.Scale bar:75μ

m.

Figure 5.Fluorescence images,bright-?eld images,and the merge of ?uorescence and bright-?eld images of MCF-7cells after incubation with 100nM P1(A),100nM P1/PDANS nanocomplex (B),and 100nM P2/PDANS nanocomplex (C)for 12h at 37°C.Scale bar:75μm.

Meanwhile,?uorescence of FAM could not be observed from P1without PDANS(Figure5A).This indicated that the P1/ PDANS nanocomplex could also be used for the intracellular sensing of ATP in MCF-7cells.All these results demonstrate that the P1/PDANS nanocomplex can a?ord an excellent intracellular biosensor for high-contrast?uorescence imaging of

biomolecules in living cells.

■CONCLUSION

In summary,a nanocomplex has been developed based on the ?uorescence aptamer and the polydopamine nanospheres for in situ sensitive and selective assay of biomolecules.In vitro assays demonstrated that the P1/PDANS nanocomplex was a robust, sensitive,and selective biosensor for quantitative detection of ATP,and the confocal?uorescence microscopy experiments with HeLa cells and MCF-7cells further suggested that the P1/ PDANS nanocomplex was e?ciently delivered into living cells and worked as an in situ“signal on”biosensor for speci?c,high-contrast imaging of target molecules.Excellent biocompatibility of PDANS along with noncovalent binding between oligonucleotides and PDANS has proved that PDANS was an e?cient cargo and an excellent protector for cellular delivery of nucleic acids and possibly peptides or proteins in due course. What’s more,superquenching ability suggests PDANS as a universal sensing platform appropriate for various?uorescent probes.In conclusion,these advantages of polydopamine make it an excellent candidate for the employment in many biological ?elds,such as the assay of DNA and protein,the delivery of gene and drug,and intracellular tracking,as well as in vivo

monitoring,etc.

■ASSOCIATED CONTENT

*Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.anal-chem.5b03075.

SEM image and UV?vis spectrum of PDANS,cell

viability of HeLa treated with PDANS,and?uorescence

microscopy and bright-?eld images of HeLa cells

incubated with P1/PDANS(PDF)

■AUTHOR INFORMATION

Corresponding Author

*Phone/Fax:+00862583595835.E-mail:xudanke@https://www.360docs.net/doc/c01380712.html,. Notes

The authors declare no competing?nancial interest.■ACKNOWLEDGMENTS

We acknowledge the?nancial support of the National Basic Research Program of China(973Program,2011CB911003), the National Natural Science Foundation of China(Grant Nos. 21227009,21175066,21328504,and21475060),the Funda-mental Research Funds for the Central Universities (20620140439),and the National Science Funds for Creative

Research Groups(21121091).

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