Nano Endoscopy with Plasmon-Enhanced Fluorescence for Sensitive

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Nano Endoscopy with Plasmon-enhanced Fluorescence for Sensitive Sensing inside Ultra Small Volume Samples Hang Yuan, Jie Liu, Yuexiang Lu, Zhe Wang, Guoyu Wei, Tianhao Wu, Gang Ye, Jing Chen, Sichun Zhang, and Xinrong Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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Nano Endoscopy with Plasmon-enhanced Fluorescence for Sensitive Sensing inside Ultra Small Volume Samples Hang Yuan,† Jie Liu, ‡ Yuexiang Lu,*,† Zhe Wang,† Guoyu Wei,† Tianhao Wu, ‡ Gang Ye, Jing Chen,*,† Sichun Zhang,‡ Xinrong Zhang‡ †

Institute of Nuclear and New Energy Technology, Collaborative Innovation Center of Advanced Nuclear Energy Technology, Tsinghua University, Beijing 100084, P. R. China ‡ Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, P. R. China. *Email: [email protected]; [email protected]. Fax no.: +86 10 62771740 ABSTRACT: Plasmon-enhanced fluorescence (PEF) generally requires the samples settled on a metal substrate and the effective enhancement distance is less than 100 nm, which limit its application in intracellular sensing. Herein, we report a nano endoscopy with PEF effect for sensing analytes inside the extremely small volume samples. The nano endoscopy was fabricated by assembling single nanoporous gold nanowire (PGNW) on the tip of a tungsten needle. It was accurately manipulated to insert into a micro droplet, and an effective sensing was realized at micrometre scale with submicrometer resolution. By taking lysozyme as a model sensing target, a 23-fold improvement of sensitivity was obtained, comparing with that of smooth gold nanowire (SGNW).These results indicated that the nano endoscopy can realize a high spatial resolution sensing, showing its potential application in intracellular sensing.

Plasmon-enhanced fluorescence (PEF) or metal-enhanced fluorescence (MEF) is an effective way to improve the fluorescence sensing performance.1-6 However, the realization of a substantial PEF effect generally requires metal substrate such as Ag, Au and Cu, with roughened surfaces or patterned nanostructures, and the effective enhancement distance is less than 100 nm, which has severely limited the practical application of PEF, especially in single cell sensing.7 Cells need to be transferred and live on the metal substrate, which may alter the behavior of the cells in their original environment. More importantly, as the diameter or height of a cell is about tens of micrometer, only fluorescent molecules near the cell membranes could be enhanced.8 The sensing sensitivity and imaging quality of the analytes inside the cell could not be improved, while the intracellular investigation is essential for better understanding the relationship between cellular functions, disease mechanisms and cell-drug interactions.9-12 Endocytosis of PEF nanoparticles (NPs) with good mobility may give a solution for intracellular biosensing.13-16 Unfortunately, moving a nanoparticle to a specific target zone inside a cell is still a big challenge.17-18 It is highly needed to develop new PEF strategy to bridge the gap between PEF and intracellular sensing application. A promising approach is single cell endoscopy, in which one-dimensional probes are accurately maneuvered and inserted into a live cell, allowing intracellular high spatial resolution optical studies. 19 Recently, nanowires and nanotubes with cylinder shape have been employed for fabricating single cell endoscopy as they showing less damage and affection to live cells comparing to large conical glass pipettes and optical fibers. 20-24 By combining nanowires with PEF effect into single

cell endoscopy, it is possible to realize intracellular sensing and imaging with both high resolution and high sensitivity.

Figure 1. The working principle of PEF based on different nanomaterials. (a) Film substrate. (b) Nanoparticles. (c) Nanowires. (d) The SEM image of a PGNW. The insert is the high magnification image. (e) SEM and (f) optical image of a PGNW assembled on the tip of the tungsten probe by the adhesion of epoxy.

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Scheme 1. Schematic illustration of the nanoporous nanowire modification and the interaction between the negative nanowire and the aptamer/ aptamer-lysozyme complex.

However, there is few report on this kind of single cell endoscopy, as achieving effective and reproducible PEF on single nanowire is really challenging.25 At the same time, due to the small scale and delicate nature of single-cell analysis, there are numerous design challenges for intracellular probes, such as probe size, shape, maneuverability and surface chemistry.19 In our previous work, we have found that single nanoporous gold nanowire (PGNW) is an effective onedimension platform for plasmon-enhanced fluorescence.26 Here, we report a nano endoscopy, in which single PGNW with PEF effect and micrometers length was assembled on the tip of a tungsten needle. The fabricated nano endoscopy could be accurately maneuvered and inserted into the sample. Then the nano endoscopy could serve as a PEF substrate inside the sample and could be moved to different area for sensing (Figure 1). The virtue of such a nano endoscopy is that it could realize sensing the analytes in situ, instead of transfer the samples to metal substrate. Also it could be utilized for highsensitivity sensing inside the sample with micrometers depth. For evaluation the application in intracellular sensing, the sensing performance and maneuverability of the nano endoscopy were evaluated by chosen lysozyme as sensing target. The nano endoscopy were fabricated by using porous gold nanowires with diameter of about 250 nm and length of about 15 µm (Figure 1d-f). A ‘turn-on’ aptasensor was designed to investigate the fluorescence sensing performance on single PGNWs (Scheme 1). First, negatively charged carboxyl groups were introduced to the surface of the PGNW by a layer of mercaptopropionic acid (MPA) via Au-S bond. Then, the positively charged poly(allylamine hydrochloride) (PAH) and negatively charged poly(sodium-p-styrenesulfonate) (PSS) were assembled via layer-by-layer self-assembly to obtain a negatively charged surface and serve as spacer layers for adjusting the distance between the nanowire and fluorescence dye. As lysozyme (isoelectric point 11.0~11.4) is positively charged in the buffer (pH=7.5), it could be adsorbed on the nanowire surface. When the Cy5-labbled aptamer interact with the lysozyme via specific binding, the Cy5 dye could be introduced to the surface of the PGNWs. As shown in Figure 2a, a strong fluorescence signal was observed on the nanowire in the presence of lysozyme. However, there was nearly no detectable fluorescence signal without lysozyme, since the

negatively charged aptamer could not be adsorbed on the surface of PGNWs alone because of the electrostatic repulsion. It was also found that among five proteins with different charges, only lysozyme could cause intense fluorescence response, since the specific binding between lysozyme and its aptamer is nessary for obtaining strong fluorescence signals (Figure 2b).

Figure 2. (a) The fluorescence spectra of PGNW adsorbing 1.5 µM Cy5 labeled aptamer and aptamer-lysozyme complex. (b) The response of PGNW to solution containing 1.5 µM different proteins. The fluorescence response was defined as (I-I0)/I0, in which I and I0 are the fluorescent intensity (at 656 nm) on the nanowires in the presence and absence of the target protein.

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The strong fluorescence intensity of Cy5 was benefited from the PEF effect of PGNWs. As the PEF effect is sensitive to the interval between the nanomaterial and the fluorophore, the number of PAH/PSS layers was also adjusted.27-28 The maximum fluorescence intensity was obtained when only one pair of PAH/PSS layer was used (Figure S1). If more PAH/PSS layers were used, the PEF effect became weaker gradually as the distance increased. When there was no PAH/PSS layer, Cy5 was too close to the nanowire and the fluorescence quenching effect of the metal nanomaterial appeared.

demonstrated that PGNW was an excellent platform for the development of nano endoscopy.

Figure 4. Photographs of the PGNW on the tip of the tungsten inserting to (a-c) and extracting from (d-f) a micro droplet of solution. (g) Scheme of the nano endoscopy can be manipulated to be partly immersed into a micro droplet. The fluorescence (h) and bright field (i) images of the PGNW after sensing. Inset is the fluorescence intensity distribution along the nanowire.

Figure 3. (a) The fluorescence response of lysozyme at 100 nM and 1500 nM on PGNWs and SGNWs. (b) The fluorescence response of lysozyme at different concentration and the fitting lines of PGNWs (orange) and SGNWs (blue). The inserts are the SEM images of the nanowires. Subsequently, the analysis performance of this nano endoscopy was investigated. As shown in Figure 3a, an obvious turn on fluorescence was observed at a lysozyme concentration as low as 100 nM. However, no effective response signal was detected on smooth gold nanowire (SGNW) at the same condition. At a higher protein concentration (1500 nM), the PGNWs showed a response signal as high as 8.78, while it was only 0.38 on SGNWs which is much lower than that on PGNWs. The fluorescence response signal and the protein concentration showed a good linear relationship (Figure 3b) in the range of 0~1.5 µM which can be expressed as y= 0.0064x+0.0023 (R2=0.9828) for PGNWs. The slope of linear fitting curve for PGNWs is 23fold higher than that of SGNWs (y=0.0003x+0.0116, R2=0.9173). The limit of detection (LOD) for PGNWs was calculated to be 68.5 nM, which is much lower than that on the SGNWs (445.6 nM). According to our previous study, the PGNWs could enhance the Cy5 fluorescence better than SGNWs and show lower background emission.26 These results

To prove that the nano endoscopy could be utilized in fluorescence sensing inside a small volume sample, the nano endoscopy was manipulated with the assistance of a threedimensional (3D) micromanipulator under an optical microscope. The nano endoscopy was approached to a droplet of lysozyme solution (5 µL, 1.5 µM) and partly immersed into it for capturing the analytes (Figure 4a-c). Subsequently, the nanowire was withdrawn from the sample solution and treated with 1.5 µM aptamer solution (Figure 4e-f). It was found that only the front end of PGNW, which was immersed into the sample solution, emitted fluorescence, while the back end showed no signal (Figure 4g-i). The fluorescence intensity distribution along the nanowire clearly showed that about 4 µm of the probe exhibited high fluorescence signal, indicating that the probe could get the informantion of the sample in the depth of micrometres with a submicrometre resolution (the insert of Figure 4i). These results indicated that the nanowire based probe could be precisely manipulated to realize high spatial resolution fluorescence microanalysis, which could be hardly done by using two-dimensional films or zerodimensional nanoparticles. In conclusion, we have developed a nano endoscopy approach for PEF based on single PGNW, which not only allow the probed substance to be on a generic substrate but also could realize a high spatial resolution sensing inside the sample. This single PGNW based nano endoscopy has several advantages that make it a good candidate for single cell PEF endoscopy. First, the gold nanowire has cylinder shape, small diameter and good biocompatibility, which could minimized the damage in live cell monitoring. Second, the PEF effect

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could provide much improved sensitivity. Third, the probe is a general sensing platform, as the surface of gold could be easily modified with Au-S bind or DNA interaction. By changing the sensing system, many targets could be analyzed. Although further studies will be needed, single nanoporous gold nanowire has shown their potential to act as a general platform for single cell PEF endoscopy. The application of this endoscopy in biosensing of important biomarkers inside single live cell is undergoing.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experiments details of the preparation of nanowires, the fabrication of nano endoscopy, quantitative analysis of lysozyme, and SEM image of SGMW. (PDF)

AUTHOR INFORMATION

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Corresponding Author

G.; Gogotsi, Y. Nanomedicine 2014, 9, 153-168.

*[email protected] *[email protected]

(20) Vitol, A. E.; Orynbayeva, Z.; Bouchard, J. M.; AzizkhanClifford, J.; Friedman, G.; Gogotsi, Y. ACS Nano 2009, 3, 3529-3536. (21) Singhal, R.; Orynbayeva, Z.; Sundaram, R. V. K.; Niu, J. J.;

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT We acknowledge the National Natural Science Foundation of China (Grant No. 21390413, No. 51425403 and No. 21405090), the Ministry of Science and Technology of China (2012YQ12006003) and Program for Changjiang Scholars and Innovative Research Team in University (IRT13026).

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