In Situ Detection and Imaging of Telomerase Activity in Cancer Cell

Article

In-Situ Detection and Imaging of Telomerase Activity in Cancer Cell Lines via Disassembly of Plasmonic Core-Satellites Nanostructured Probe Kan Wang, Li Shangguan, Yuanjian Liu, Ling Jiang, Fen Zhang, Yuanqing Wei, Yuanjian Zhang, Kang Wang, and Songqin Liu Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 31 May 2017 Downloaded from http://pubs.acs.org on May 31, 2017

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Analytical Chemistry

In-Situ Detection and Imaging of Telomerase Activity in Cancer Cell Lines via Disassembly of Plasmonic Core-Satellites Nanostructured Probe Kan Wang,† Li Shangguan,† Yuanjian Liu,† Ling Jiang,† Fen Zhang,† Yuanqing Wei,† Yuanjian Zhang, † Kang Wang, *‡ and Songqin Liu*† †

State Key Laboratory of Bioelectronics, Jiangsu Engineering Laboratory of Smart

Carbon-Rich Materials and Device, School of Chemistry and Chemical Engineering, Southeast University, Nanjing, 211189, People’s Republic of China ‡

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative

Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China

*

Corresponding author: Tel.: 86-25-52090613; Fax: 86-25-52091098. E-mail addresses:

[email protected] (S.Q. Liu); [email protected] (K. Wang)

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ACS Paragon Plus Environment


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ABSTRACT The label-free localized surface plasmon resonance (LSPR) detection technique has been identified as a powerful means for in-situ investigation of biological processes and localized chemical reactions at single particle level with high spatial and temporal resolution. Herein, a core-satellites assembled nanostructure of Au50@Au13 was designed for in-situ detection and intracellular imaging of telomerase activity by combining plasmonic resonance Rayleigh scattering spectroscopy with dark-field microscope (DFM). The Au50@Au13 was fabricated by using 50 nm gold nanoparticles (Au50) as core and 13 nm gold nanoparticles (Au13) as satellites, both of them were functionalized with single chain DNA and gathered proximity through the highly specific DNA hybridization with a nicked hairpin DNA (O1) containing a telomerase substrate (TS) primer as linker. In the presence of telomerase, the telomeric repeated sequence of (TTAGGG)n extended at the 3’-end of O1 would hybridized with its complementary sequences at 5’-ends. This led the telomerase extension product of O1 be folded to form a rigid hairpin structure. As a result, the Au50@Au13 was disassembled with the releasing of O1 and Au13-S from Au50-L, which dramatically decreased the plasmon coupling effect. The remarkable LSPR spectral shift was observed accompanied with a detectable color change from orange to green with the increase of telomerase activity at single particle level with a detection limit of 1.3×10-13 IU. The ability of Au50@Au13 for in-situ imaging intracellular telomerase activity, distinguishing cancer cells from normal cells, in-situ monitoring the variation of cellular telomerase activity after treated with drugs were 2


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also demonstrated.

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INTRODUCTION Recently, label-free localized surface plasmon resonance (LSPR) technique receives promoting interesting due to its wide applications in catalysis, phototherapy and biosensing.1-3 A LSPR signal comes from the coherent oscillation of electrons in the conduction band of the plasmonic nanoparticles, which depends on the morphology, sizes, composition, as well as surrounding dielectric environment of the nanoparticles.4 Au, Ag and Cu nanoparticles are ideal plasmonic materials owing to their low toxicity, good biocompatibility, photostability and much stronger scattering signal intensity than the fluorescence of dye or quantum dot.5-9 Moreover, the scattering light of an individual plasmonic nanoparticle can be readily observed under a dark-field microscope (DFM), providing a powerful means for the in-situ investigations of biological processes and localized chemical reactions with high spatial and temporal resolution.10-12 Nonetheless, the minor scattering spectral shift occurring in an individual nanoparticles limits their applications in biosensing.13 Several strategies,14-17 in particularly, controlled assembly of individual nanoparticles into dimers,18-20 Janus nanoparticles21 and core-satellites nanostructure22-24 have been developed to enhance the scattering spectral shift. The inter-particle coupling in close vicinity can largely enhance the local field and achieve the highly sensitive requirement for the analysis at single particle level. For example, Lee and co-workers utilized thiol-mediated adsorption and streptavidin-biotin binding to self-assemble core-satellite nanostructures with a sacrificial linking peptide, and demonstrated the use of scattered light from gold nanoparticle assembly and trypsin-mediated

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disassembly for detection of protease activity.22 Fan and co-workers designed a DNA-directed programmable assembly strategy to fabricate a halo-like Au nanostructure to indirectly monitor catalytic reactions at single-particle level.24 The prominent LSPR performances of the above nanoparticle assembled nanostructure greatly improve the detection sensitivity and signal-to-noise ratio, and pushed the feasibility for sensing in long-duration and real-time live cell studies.25-28 The first application of plasmonic rulers to in vivo monitor trajectories of single biomolecules in live cells was conducted by Alivisatos and co-workers.25 In their work, the plasmonic ruler comprised of peptide-linked gold nanoparticle satellites around a core particle was used as a probe to monitor caspase-3 activity in live cells for over 2 h, providing sufficient time to observe early-stage caspase-3 activation. Xu and co-workers proposed a strategy for sensitive detection of surviving mRNA based on resonance Rayleigh scattering of a single AuNP nanohalo probe that couples large gold nanoparticles with small AuNPs through the affinity interaction between streptavidin and biotin.28 In the present of mRNA, the small AuNPs were pushed away from the surface of the core nanoparticle and the interparticle distance increased notably, leading to a dramatic decrease in the plasmon coupling effect and a significant blue shift in the LSPR spectrum. All these works demonstrated the feasibility of core-satellites nanostructure assembly and disassembly for biosensing even in real-time cell studies. Human telomerase is a ribonucleoprotein reverse transcriptase composed of template RNA and protein.29 This holoenzyme catalyzes the addition of tandem 5



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telomeric DNA repeats (TTAGGG)n on the telomeric ends of chromosomal DNA to protect telomeres from erosion during cell division.30 As it reported, telomerase activity is reactivated and over-expressed in over 85% of cancer cells and the length of telomeres is maintained, which makes cancer cells divide indefinitely.31 While in normal human cells, telomerase activity is inhibited and telomeres are shortened after each replication cycle, which leads to cell senescence and death.32 The differential expression between cancer cells and normal cells makes telomerase a valuable tumor biomarker and a potential therapeutic target.33 Therefore, sensitive detection and in-situ monitoring of telomerase activity and its inhibition is significant to tumor diagnostic, therapy, and monitoring.34 Since the discovery of telomerase in 1985 by Greider and Blackburn, various analytical methods have been developed to detect telomerase activity, 35 including the polymerase chain reaction PCR-based telomerase repeat amplification protocol (TRAP) assay,36 chemiluminescence,37 colorimetry38 and electrochemistry.39 However, these works failed to in-situ detect and acquire spatiotemporal variation of telomerase activity in living cells. Recently, Ju and co-workers designed a nicked molecular beacon-functionalized probe for in-situ fluorescent imaging and detection of intracellular

telomerase

activity.40

Gu

and

co-workers

reported

a

gold

nanoparticle-based molecular beacon for the visualization of intracellular telomerase activity in living cells and the precise drug delivery to cancer cells.41 These pioneering work evidently demonstrate that fluorescent imaging is a powerful technique for determining and visualizing telomerase activity at cell level with high sensitivity and 6


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low interference. However, the inherent low signal intensities, photobleaching and complex blinking phenomena limitations of dyes or quantum dot hinder the ability for the long-duration live cell studies. In this context, developing sensitive methods for in-situ monitoring intracellular telomerase activity using non-fluorescent imaging techniques is still a great challenge. In this work, we have designed a telomerase-triggered nicked hairpin DNA and successfully prepared a core-satellites assembled nanostructure of Au50@Au13 via DNA hybridization. The core-satellites assembled Au50@Au13 was disassembled by substitutional hybridization of the telomerase-triggered nicked hairpin DNA elongation. The dramatic decrease of the plasmon coupling effect of Au50@Au13 produced a telomerase activity related LSPR spectral shift accompanied with a detectable color change from orange to green. Thus, a novel LSPR sensing for in-situ detection and intracellular imaging of telomerase activity at single particle level was first developed by using core-satellites nanostructured probe. EXPERIMENTAL SECTION In-situ Assembly of Core−Satellites Nanostructure of Au50@Au13 on a Glass Slide. AuNPs with diameter of 13 nm (Au13) and 50 nm (Au50) were synthesized by trisodium citrate reduction method and seed-growth method.42,43 The concentration of the as-prepared Au13 and Au50 solution was calculated to be 11 nM and 0.1 nM with the extinction coefficient of 2.7×108 M-1cm-1 and 1.5×1010 M-1cm-1.44,45 The obtained DNA-modified AuNPs were prepared and denoted as Au13-S, Au50-L and Au50-L-O1, respectively.46 The fluorescein-modified DNA (L-DNA-FAM and O1-FAM) were 7



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bounded to AuNPs to measure the amount of S-DNA, L-DNA and O1 assembled on each particle.47 For in-situ construction of Au50@Au13 on ITO slides, Au50-L-O1 solution was dropped onto the amino-functionalized ITO slides. To eliminate the possible influence of amino-groups on further assembly of Au50-L-O1 with Au13-S, the ITO slide was subsequently immersed in sulfo-SMCC in PBS to neutralize the charge on the ITO surface.48 Details and the further experimental measurements can be found in the Supporting Information “Materials and Methods”. RESULTS AND DISCUSSION Synthesis and Properties of Core-Satellites Nanostructure of Au50@Au13. The construction of core-satellites nanostructure of Au50@Au13 was illustrated in Scheme 1. The highly specific DNA hybridization event brings the core and satellites in close proximity to fabricate core-satellites nanostructured probe. Gold nanoparticles with average diameter of 50 (Au50) and 13 nm (Au13), both modified with two short thiolated single-stranded DNA (L-DNA and S-DNA), respectively, were acted as core and satellites. L-DNA and S-DNA were two complementary sequence of a nicked hairpin DNA (O1), where L-DNA was designed to hybridize with the stem part of the 5’ terminal region of O1, while S-DNA opened the hairpin sequence of O1 according to the competitive hybridization with the inner chain. The hybridization of L-DNA and S-DNA with O1 led to assembly of core-satellites structures along with the conformation changes of O1 from a nicked hairpin state to an extended state. The length of hybridized oligonucleotides (18 bp) on O1 controlled the distance between Au50 and Au13, and the narrow gap (

In Situ Detection and Imaging of Telomerase Activity in Cancer Cell

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