Nanopipette-Based SERS Aptasensor for Subcellular Localization of

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A Nanopipette-based SERS Aptasensor for Subcellular Localization of Cancer Biomarker in Single Cells Sumaira Hanif, Hai-Ling Liu, Saud Asif Ahmed, Jin-Mei Yang, Yue Zhou, Jie Pang, Lina Ji, Xing-Hua Xia, and Kang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02147 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 22, 2017

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Analytical Chemistry

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A Nanopipette-based SERS Aptasensor for Subcellular

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Localization of Cancer Biomarker in Single Cells

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Sumaira Hanifa, Hai-Ling Liua, Saud Asif Ahmeda, Jin-Mei Yanga, Yue Zhoua, Jie Panga, Li-Na

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Jib*, Xing-Hua Xiaa and Kang Wanga*

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a: State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and

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Chemical Engineering, Nanjing University, Nanjing 210023, China.

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b: State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing

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University, Nanjing 210023, China;

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*Corresponding author: [email protected]; [email protected]

1 ACS Paragon Plus Environment


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Abstract: Single cell analysis is essential for understanding the heterogeneity, behaviors of

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cells, and diversity of target analyte in different subcellular regions. Nucleolin (NCL) is a

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multifunctional protein that is markedly overexpressed in most of the cancers cells. The variant

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expression levels of NCL in subcellular regions have marked influence on cancer proliferation

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and treatments. However, the specificity of available methods to identify the cancer biomarkers

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is limited because of the high level of subcellular matrix effect. Herein, we proposed a novel

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technique to increase both the molecular and spectral specificity of cancer diagnosis by using

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aptamers affinity based portable nanopipette with distinctive surface-enhanced Raman scattering

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(SERS) activities. The aptamers-functionalized gold-coated nanopipette was used to capture

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target, while p-mercaptobenzonitrile (MBN) and complementary DNA modified Ag

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nanoparticles (AgNPs) worked as Raman reporter to produce SERS signal. The SERS signal of

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Raman nanotag was lost upon NCL capturing via modified DNA aptamers on nanoprobe, which

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further helped to verify the specificity of nanoprobe. For proof of concept, NCL protein was

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specifically extracted from different cell lines by aptamers modified SERS active nanoprobe.

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The nanoprobes manifested specifically good affinity for NCL with a dissociation constant Kd of

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36 nM and provided a 1000-fold higher specificity against other competing proteins.

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Furthermore, the Raman reporter moiety has vibrational frequency in the spectroscopically silent

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region (1800-2300 cm-1) with a negligible matrix effect from cell analysis. The subcellular

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localization and spatial distribution of NCL were successfully achieved in various types of cells

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including MCF-7A, HeLa and MCF-10A cells. This type of probing technique for single cell

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analysis could lead to develop a new perspective in cancer diagnosis and treatment at cellular

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level.


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Keywords: Aptasensor, nanopipette, nucleolin, SERS signal on/off, single cells, spatial

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distribution

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Single cell analysis is essential for unveiling the heterogeneity, behaviors of cells, and diversity

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of target analyte localizations.1,2 The single cell heterogeneity dictates a multitude of functions

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that could be helpful to investigate the development of disease states (prognosis and proliferation)

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and get information of carcinogenic transformations.3-5 A number of different methods have been

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developed for single cells analysis, including capillary electrophoresis (CE),6,7 mass

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spectrometry,8, flow cytometry,9 and laser induced fluorescence (LIF)10,11 based assays. Besides

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these well-developed techniques, a huge space is available due to some unsolved problems in

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cancer diagnostics at cellular level.12-14

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Surface-enhanced Raman scattering (SERS) has emerged as an appealing potential

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detection tool for cell study due to its unprecedented optical properties.15 As well characterized

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SERS substrates, metal nanoparticles16-18 undergo indocile aggregation in cells19,20 and usually

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interferes with original SERS signal21. To overcome this issue, SERS substrates with fixed

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configuration have been used, such as carbon nanopipettes, optical fibers and SERS-active glass

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nanopipettes.

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biomarkers in the single living cells.23 However, to the best of our knowledge, biocompatible and

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portable aptamers based SERS nanoprobes have not been explored yet for subcellular

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localization of biological targets in single cells.

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Recently, SERS-active glass pipette has been successfully applied for probing

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As a multifunctional protein associated with numerous biomolecules (DNA, RNA, and

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other small molecules), nucleolin (NCL) plays an essential role in cell division.24 NCL is

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localized at various parts of the cells including nucleus, cytoplasm and cell membrane with the



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characteristic functionalities.25 More importantly, the altered expression levels of subcellular

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NCL are found to be closely related to various types of cancers,26 including colorectal cancer,27

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human breast and lung cancer cells.28 Usually, an immunofluorescence assay is used to examine

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the various localization of NCL in cells.29 However, immunofluorescence technique can hardly

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examine the spatial distribution of NCL at single cell level. Previously, a SERS-based analysis

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was reported via a DNA barcode-assay for NCL detection at the cells surface while its

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fabrication steps were complicated and it was not applicable for subcellular distribution.

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Therefore, it is highly desirable to develop an alternative and non-invasive method for detecting

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and mapping the expression of NCL at subcellular level.

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To detect and inhibit the proliferation of cancer cells, several efforts have been put to

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develop NCL binding molecules, such as aptamers (AS1411) and peptides.31-33 AS1411 is a 26-

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mer G-rich DNA Aptamer, which is highly stable against nucleases.34,35 Herein, we developed a

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simple SERS-based nanoprobe for subcellular localization of NCL in a single living cell via

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aptamers based affinity. SERS-active glass nanopipette was functionalized with AS1411 then

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hybridized with versatile Raman reporter and cDNA modified Ag nanotags for the recognization

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of NCL. The principle and procedure of aptamers affinity SERS assay are illustrated in Scheme 1.

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The thin layer of gold film coated on glass nanopipette with ~200 nm outer diameter was used as

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based substrate, while thiolated NCL aptamers were modified on the surface of Au-coated

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nanopipette. Meanwhile, Raman nanotags prepared by immobilizing thiolated complementary

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DNA (cDNA) of AS1411 and MBN as Raman reporter on 55±5 nm AgNPs were used for the

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detection. The developed SERS nanoprobe detection is relied on the SERS signal-off response of

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MBN on the exposure of target molecule due to release of Raman nanotags from hybridized

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SERS-active nanopipette. The bi-metallic nature of Au and Ag plasmon and MBN as Raman


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reporter with characteristic peak (2223 cm-1) in cell silent region provided a versatile detection

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platform for single cell analysis. For proof of concept, the SERS nanoprobe was precisely

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inserted into nucleus, cytoplasm and near cell surface in different living cell lines (MCF-7, MCF-

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10A and HeLa cells). The nanoprobe exhibits astonishing results for subcellular localized study

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and spatial distribution of biomarker in single cells. Thus, the burgeoning field of SERS active

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nanopipettes can be further explored by applying various aptamer-affinity approaches for single

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living cell study.

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EXPERIMENTAL SECTION

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Reagents and materials. Oligonucleotides used in the present study were purchased from

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Shanghai

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(China), and the sequences are given below:

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Nucleolin Aptamer (AS1411):

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5`-(CH2)6-S-S-(CH2)6-GGTGGTGGTGGTTGTGGTGGTGGTGG-3`

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Complementary ssDNA:

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3`-(CH2)6-S-S-(CH2)6-CCACCACCACCAACACCACCACCACC-5`

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4-mercaptobenzonitrile (MBN, 95%) was purchased from Nanjing Norris Pharm Technology Co.

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Ltd. Bovine serum albumin (BSA), horseradish peroxidase (HRP), β-Casein (β-Cas),

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cytochrome-c (Cyt-c), transferrin (Trf), TE buffer (100-X, pH 7.4) and nucleolin (NCL) from

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calf intestinal mucosa were all from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen

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tetrachloroaurate trihydrate (HAuCl4 3H2O, 99.9%) was purchased from Alfa-Aesar (Shanghai

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China). Silver nitrate (AgNO3), Triton-X 100, trisodium citrate, NaOH, NaH2PO4, Na2HPO4,

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glucose, potassium bicarbonate (KHCO3), NaOH, HCl (36%), NaCl, KCl, MgCl2, CaCl2,

Sangon

Biological

Engineering

Technology


&

Services

Co.,

Ltd.


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KH2PO4, K2HPO4, EDTA, glycerol and anhydrous ethanol of analytical grade were purchased

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from the Nanjing reagent company. All these reagents were used without further purification.

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Water was purified with a Milli-Q Advantage A10 (Millipore, Milford, MA, USA), and was used

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to prepare all solutions. Borosilicate glass capillaries with filament (0.58 mm I.D., 1.0 mm O.D.)

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were purchased from Sutter Instrument Co.

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Instrumentation. CO2-laser-based pipet puller (P-2000, Sutter Instrument Co.) was used to

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fabricate the glass nanopipettes. Scanning electron microscopic (SEM) characterization was

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carried out on a FE-SEM S-4800 system (Hitachi, Tokyo, Japan). Transmission electron

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microscopy (TEM) characterization was performed on a JEM-1011 system (JEOL, Tokyo,

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Japan). Raman and SERS experiments were conducted on a Renishaw InVia Reflex confocal

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microscope (Renishaw, UK) equipped with a high-resolution grating with 1800 grooves/cm,

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additional band-pass filter optics, and a CCD camera. All measurements were carried out using a

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He-Ne laser (λ = 633 nm; laser power at spot, 1 mW; excitation laser line: 1s integration time

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and 1 accumulation). The laser was focused onto the sample by using a ×50 objective lenses with

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(N.A. 0.75), providing a spatial resolution of 1 µm. Wavelength calibration was performed by

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measuring silicon wafers through a ×50 objective, assessing the first-order phonon band of

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silicon (Si) at 520 cm-1. The spectra were recorded using the Renishaw's WiRE (Windows-based

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Raman Environment) software and analyzed with Origin Pro 8.6 software. Each spectrum

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baseline was corrected except noise test. A three-dimensional manipulator equipped on an

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inverted microscope (Nikon Ti-E) was used to precisely insert extraction microprobes into single

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cells under investigation.

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Preparation of Raman nanotags


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Synthesis of silver nanoparticles. Silver nanoparticles (AgNPs) with a diameter of 55±5 nm

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were prepared according to a method by Lee and Meisel.36 In brief, AgNO3 (36 mg) was

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dissolved in 200 mL water and brought to boil under continuous stirring. Then, 4 mL of 1% (w/v)

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trisodium citrate was added. The mixture was boiled with stirring for about 1 h and then cooled

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down to room temperature naturally. The solution was stored at 4 °C in refrigerator for further

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use.

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Immobilization of cDNA and Raman reporter on AgNPs. The cDNA of NCL aptamers and

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Raman reporter was immobilized on AgNPs by using following procedure.26 10 µL of 100 µM

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cDNA of aptamer(3’-thiol) were added to freshly prepared AgNPs (1 mL), and incubated at 4 °C

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for 6 h. The obtained solution was centrifuged for 10 min at 10,000 rpm to remove the excess

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reagents, the precipitate was washed and centrifuged repeatedly for three times. The resulting

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cDNA-Ag nanoprobe was dispersed into 1 mL phosphate buffer solution (PBS, 0.01 M, pH 7.4).

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To modify Raman reporter, 1mL cDNA-AgNPs were incubated with 1 µL of MBN (1 mM

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dissolved in 0.2 M NaOH) for 1 h at room temperature under constant stirring. Then cDNA-

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AgNPs were washed with 0.01 M PBS (pH 7.4) three times by centrifugation and re-suspended

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in 1 mL PBS (pH 7.4) , stored at 4 °C for further use.

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Fabrication of SERS nanoprobe

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Fabrication of glass nanopipette. Borosilicate glass without filament (0.58 mm I.D., 1.0 mm

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O.D., Sutter Instrument Co.) was fabricated by a CO2-laser-based pipette puller (P-2000, Sutter

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Instrument Co.).

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Preparation of Au coated nanoprobe. For the preparation of Au-coated nanopipette, a gold

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layer was coated onto the surface of glass nanopipette by a previously reported chemo-deposition

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method.37 Briefly, the nanopipettes (200 nm O.D.) were immersed in a solution containing 12

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mM HAuCl4, 0.5 M KHCO3 and 25 mM glucose for 3-4 h at 45 °C until a clear gold layer

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formed on the surface of each nanopipette. After washing with water and anhydrous ethanol 2-3

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min for each, the Au-coated nanoprobes dried in air at room temperature.

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Immobilization of aptamers on Au-nanoprobe. The aptamers modification on nanoprobe was

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performed by immersing in 50 µL of 100 µM 5’-thiol aptamer in 0.01 M PBS and incubated at

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4 °C in dark for 5 h. Then, excess reagent was removed by washing with 0.01 M PBS (pH 7.4)

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for 2 min. The resulting aptamer-modified nanoprobes were stored in refrigerator at 4 °C for

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further use.

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NCL detection assay in solution

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Hybridization of Raman nanotag on SERS nanoprobe. Au nanoprobes modified with

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aptamers were immersed in 50 µL of Raman nanotag solution and incubated at room temperature

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for 1 h. Then non-hybridized Raman nanotags were removed by washing with PBS (pH 7.4) for

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2 min and air dried before Raman measurement.

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Hybridized SERS nanoprobe for NCL detection. NCL was dissolved in protein binding buffer

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(PBB) to at a concentration of 0.1 µM. PBB was prepared by adding 140 mM NaCl, 5 mM KCl,

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1 mM MgCl2 and 1 mM CaCl2 in 1× TE Buffer (pH 7). The as prepared SERS nanoprobe was


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immersed in NCL solution for 5 min at room temperature then washed with PBB and air dried

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before Raman analysis.

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Selectivity test. The selectivity of the SERS-nanoprobe was investigated against different

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proteins including BSA, Trf, HRP, Cyt-c and β-casein. The SERS nanoprobes were incubated in

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1 nM NCL or 1 µM interferants in PBB (pH 7) for 5 min each. Then nanoprobes were washed

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with PBB (pH 7) for 2 min, dried at room temperature and detected by the Raman.

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Cellular analysis

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Cell culture. NCL-positive MCF-7, normal MCF-10A, HeLa cells were purchased from the Cell

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Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai). The cells were

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cultured in DMEM (Gibco) media containing 10% fetal bovine serum (Gibco), 100 mg/mL

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streptomycin and 100 U/mL penicillin at 37 °C for 48 h under 5% CO2. Finally, cell culture

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media were removed and the cells lingered on the cell culture dishes were washed with 1× PBS

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buffer for three times then kept in 1× PBS buffer for further use.

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SERS nanoprobe for single cells analysis. With the help of an inverted microscope (Nikon Ti-

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E equipped with a three-dimensional manipulator), the nanoprobe was inserted into single cells

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anchored on the bottom of a petri dish. The nanoprobe was precisely localized at different

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regions (nucleus, cytoplasm and cell surface) of single cells under microscopy and kept still for 5

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min for each measurement. Meanwhile, the cells were kept in 1× PBS at room temperature for all

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measurements. After NCL extraction from each region of cells, the nanoprobes were washed

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with 1× PBS for 2 min to remove unwanted matrix from the nanoprobe, then dried at room



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temperature and measured for SERS signals. For spatial distribution study, the nanoprobe was

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kept horizontal when it approached the nucleus of single cells by passing the cytoplasmic region.

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Then the nanoprobe was washed with 1× PBS for 2 min after extraction, dried and measured for

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SERS signals. All the experiments were repeated in triplicates to confirm the results. Meanwhile,

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blank as negative control experiment was also conducted before each analysis.

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RESULTS AND DISCUSSION

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Characterization of the SERS nanoprobe and Raman nanotags. In this study, a smooth and

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uniform gold layer on was coated the surface of glass nanopipettes in order to generate

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plasmonic effect. The combination of bimetallic nanostructures (i.e. Au film and AgNPs) to

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generate an enhanced plasmon has gained more interest in SERS based detection materials

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because of its unique plasmon coupling effect and other physiochemical properties.38 With an

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outer diameter of ~250 nm and a taper angle of 33˚, the Au coated glass nanopipette is sharp

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enough to penetrate into the nucleus of a living cell. The SEM images of SERS nanoprobe before

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and after its hybridization with Raman nanotag are shown in Figure 1A and 1B. For the

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synthesis of Raman nanotags, AgNPs with an average diameter of 55±5 nm were prepared

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(Figure S1A). The AgNPs were modified with MBN as Raman reporter and thiolated aptamers

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complementary DNA as binding receptors for Raman nanotags. TEM images in Figure S1B and

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S1C showed that the AgNPs retained its shape and dispersity before and after the modification

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with MBN and cDNA. The localized surface plasmon resonance (LSPR) characterization of

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MBN and cDNA modified AgNPs showed no apparent shifts in the LSPR spectrum, suggesting

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that the modified AgNPs were well dispersed (Figure S2).


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The optical response of hybridized SERS nanoprobe against NCL is also presented by the

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SEM images in Figure S3. The SERS response of aptamers modified Au-coated nanoprobe is

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shown in Figure S4, which represents many characteristic peaks in DNA fingerprint region while

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no peaks at cell silent region (highlighted in grey). The detailed characteristic peaks with

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assigned vibrations are listed in Table S1. The sharp peak at 2223 cm-1 is assigned to

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characteristic vibration of cyanide ν(-C≡N) functional group of MBN, and is selected for further

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experimental characterizations. The MBN is a robust Raman reporter molecule for cell based

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study due to its simple structure and sharp Raman peak at cell silent region (~2223 cm-1).39,40

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The SERS response of Raman nanotags before and after modification is given in Figure S5,

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which indicates a sharp and clear peak at 2223 cm-1 while the other cDNA characteristic peak

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was overcome by MBN peaks in DNA fingerprint region.

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The incubation time and concentration of thiolated aptamers and cDNA were optimized

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before analysis. The 5’ labeled DNA sequences (MB labeled aptamers and alkyne labeled cDNA)

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were used to optimize DNA concentrations on the surface of Au coated glass nanopipette and

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AgNPs, respectively. The SERS response at peak 1623 cm-1 of MB labeled aptamers modified

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nanoprobe is shown in Figure S6A with other characteristic peaks given in Table S1. The

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optimized concentration and incubation time for aptamers modification on Au nanoprobe were

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100 µM and 5 h, respectively (Figures S6B). Similarly, the alkyne tagged cDNA gave a

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characteristic peak at 2010 cm-1 that was taken as a reference peak for optimization (Figure S7A).

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The optimized concentration and incubation time for cDNA modification on AgNPs were 100

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µM and 6h, respectively (Figures S7B). Meanwhile, the previously optimized concentration and

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the maximum incubation time of MBN modification on metal surface were 1 mM and 1 h to get

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a maximum saturation point.39 It is important to note that a consistent increase in SERS



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intensities is observed with gradually increasing incubation time for the DNA sequences

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modifications even for lower concentrations (10 and 50 µM for aptamers and cDNA). However,

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higher concentrations have been chosen in order to get maximum Raman signal of modified

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DNAs on the plasmonic surfaces of Au nanoprobe and Ag nanotag.

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NCL detection. The aptamer modified SERS nanoprobe was incubated with Raman nanotags to

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form hybridization complex between complementary DNA sequences. This interaction between

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Au surfaces of nanoprobe with MBN-attached Raman nanotags resulted in the amplification of

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SERS signal at peak position of 2223 cm-1. The amplified signal within the hot spots of Au

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nanoprobe and Raman nanotags was specifically lost if target protein NCL was present in the

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solution as shown in Figure 1C. The binding tendency of modified aptamers towards the target

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NCL was much higher as compared to complementary sequence hybridization.41 As shown in

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Figure S8, the complete SERS spectra including DNA and protein fingerprint regions manifest

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the alterations in characteristic peaks region of DNA after capturing protein. The reaction time of

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SERS nanoprobe for NCL detection was also optimized to be 5 min (Figure S9). Similar

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response of modified aptamers towards NCL was also observed in previous reports,42 indicating

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that the binding efficiency of SERS nanoprobe modified aptamers for the target molecule can be

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utilized for single cell analysis. Furthermore, SERS mapping image in Figure S10 indicates the

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even distribution of Raman nanotags after hybridization on SERS nanoprobe. Therefore the

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fabricated nanoprobe has the ability to provide quantitative information about the target analyte

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(NCL) within complex biological samples.

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Binding affinity of SERS nanoprobe. The binding affinity of SERS nanoprobe in vitro was

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determined by using increasing concentrations of NCL (0.5 to 500 nM). As shown in Figure 2A,

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the SERS signal of MBN gradually decreased with the increasing concentration of NCL. Figure

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2B showed that the decrease in SERS intensity at peak 2223 cm-1 became almost static when

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NCL concentration was above 100 nM, suggesting the NCL capturing by SERS nanoprobe might

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reach to saturation. The plot of SERS intensity at 2223 cm-1 against varying concentration of

265

NCL showed a significant binding affinity with an apparent Kd of 36 nM (the detailed is shown

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in supporting information). The experimentally calculated limit of detection by using standard

267

deviation formula (S/N>3) was 0.8 nM for in vitro NCL with a linear range of 1-50 nM (inset of

268

Figure 2B), which was comparable to the previous report.42 Such a good binding affinity ensures

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the formation of a stable complex between aptamers and NCL with significant detection limit to

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release the hybridized Raman nanotags into the solution.43

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Selectivity of the SERS nanoprobe. The selectivity of SERS nanoprobe was checked against a

273

variety of interfering proteins such as BSA, HRP, β-Cas, Cyt-C and Trf. As shown in Figure 3,

274

the SERS nanoprobe exhibited excellent specificity against the interfering proteins because no

275

SERS signal variation was observed for them as compared to blank. Even though the

276

concentration of interfering proteins was 1000-fold higher than that of NCL (especially for β-Cas

277

being phosphoprotein similar to NCL), the interfering proteins showed no or minimal SERS

278

response. Thus, the SERS nanoprobes are highly selective for the target molecule due to

279

aptamers based affinity.

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Reproducibility of SERS nanoprobe. The SERS signal reproducibility of the SERS nanoprobe

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was determined in terms of the coefficient of variation for hybridized nanoprobes before and

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after NCL capturing. The SERS responses of 10 different Au nanoprobes were measured for

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peak position at 2223 cm-1 before and after NCL detection (Figure S11). The relative standard

285

deviation (RSD) value of 8.6% manifested the good reproducibility of fabricated SERS-based

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detection approach on Au coated glass nanopipette to work in sample with nanoliter volume.

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Subcellular localization and spatial distribution of NCL in single cells. To understand the

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subcellular detection and spatial distribution of NCL, SERS nanoprobe was precisely inserted

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within single cancer cells (MCF-7 and HeLa) and normal (MCF-10A) cells. For the subcellular

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localizations of NCL, different SERS nanoprobes were individually inserted in perpendicular

292

way to the various cellular compartments as in nucleus, cytoplasm and kept near inner cell

293

membrane for 5 min at each region as shown in Figure 4A. The SERS spectra in Figure S12

294

depicted that the overexpressed NCL levels in cancer cells led to a tremendous decrease in SERS

295

signal of MBN at peak 2223 cm-1 in all regions including cytoplasm and cell surface. Meanwhile,

296

no significant decrease in SERS signal was observed in cytoplasmic and cell surface regions of

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normal MCF-10A cells. Interestingly, due to the occurrence of SERS signal of MBN within

298

silent region of cell, negligible cell matrix effect and no SERS spectral overlapping were

299

observed as compared to blank. To further investigate the semi-quantitative analysis, percentage

300

loss in SERS spectra intensity was observed for NCL localization in different cell lines (Figure

301

4B). For instance, blank nanoprobes has 100% SERS signal and after NCL capturing the

302

intensity dropped to 11.2%, 12.5% and 12% in nucleus, cytoplasm and cell surface of MCF-7

303

cancer cell, respectively. For HeLa cells, the SERS intensity dropped to 11.8%, 34.6% and 47%


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304

for nucleus, cytoplasm and cell surface regions, respectively. Conversely, for normal cell (MCF-

305

10A) the drop of SERS intensity was not consistent in all regions. The SERS intensity of MCF-

306

10A dropped to 12.2% in nucleus, which is the native synthetic region for NCL in all normal

307

cells. The relatively higher percentage loss of SERS signal in cytoplasmic and cell surface

308

regions of cancer cells also verified the overexpressed level of NCL in these regions.

309

Furthermore, the expression levels of NCL in cancer and normal cells were also detected in

310

cytoplasmic and nuclear extracts of these cell lines (Figure S13). Figure S14 showed a low

311

degree of relative SERS intensity variations among different cellular regions, indicating that

312

NCL was homogenously distributed in cellular extracts and excessive free NCL molecules were

313

available for capturing. All these results for the subcellular detection of NCL and variation

314

within different cancer lines are in well agreement with previously published reports.44 Besides,

315

the spatial distribution of NCL on single nanoprobe was also investigated by inserting the Au

316

nanoprobe horizontally passing by the cytoplasm towards the nucleus of cell. After extraction the

317

SERS spectra were collected by starting from nucleus (N) inserted region towards the cell

318

surface (S) region of the nanoprobes with the total available surface area of 7-8 µm for Raman

319

measurements, and the schematic presentation is given in Figure S15. SERS spectral response in

320

Figure 5A showed a uniform distribution of SERS signal of MBN at peak position 2223 cm-1

321

before extraction of any NCL from the cells. The corresponding SERS spectra in Figure 5B, 5C

322

and 5D showed that the NCL was not evenly distributed in MCF-7 MCF-10A and HeLa cells.

323

Furthermore, the spatial distribution of NCL in various cell lines was also monitored via SERS

324

mapping technique as shown in Figure S16. Therefore, we can extract molecular information

325

about heterogeneously overexpressed NCL levels among different types of cancer cell lines (as

326

HeLa and MCF-7 are used in current study) which is highly important in cancer diagnosis.45



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Page 16 of 31

327

Hence, the SERS nanoprobes have the tendency to distinguish the subcellular NCL levels and

328

also can be implied for study of spatial distribution of any cancer biomarker with high precision

329

in individual cells. Moreover, our proposed approach with some alteration (cDNA modified

330

nanopipette and hybridization with aptamers attached nanoparticles) could be further useful to

331

develop an extended drug delivery setup or cancer therapy platform for localized region in

332

cancer cells. Such a simple, specific and sensitive probing strategy of cancer cells will also be

333

helpful for accessing biomolecular information at cellular level by merging it to in-situ SERS

334

detection platform.

335 336

Conclusion

337

In conclusion, we proposed a novel design of the SERS nanoprobe for cancer biomarker

338

detection at a subcellular level. The fabricated SERS nanoprobe has provided several beneficial

339

properties. First, the Raman reporter labeled AgNPs can aggregate on Au nanotip, and provide

340

significantly enhanced and reproducible SERS signal, which is selectively lost in the presence of

341

target. Second, the characteristic peak of MBN at 2223 cm-1 in the Raman silent region (cell and

342

other biomolecules) enables to detect the distribution of biomarker in single cells and reflect the

343

physiological and pathological states of the cancer cells. Third, the recognition nanoprobe

344

contains aptamers that can be easily replaced by the other types of aptamers to develop diverse

345

ranges of detecting platforms for different biological targets. Lastly, the design of fabricated

346

detection nanoprobe is highly flexible to be converted into the cancer treatment therapy setup

347

with a little variation, as our future perspective. Particularly, the successful detection of NCL at

348

subcellular level along the spatial distribution for the first time may provide a potential approach


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349

for detection of other proteins at low expression level in a single living cell and opens a new

350

horizon for cancer cell research.

351



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Page 18 of 31

352

ASSOCIATED CONTENT

353

Supporting Information

354

The Supporting Information is available free of charge on the ACS Publications website.

355

Synthesis routes, additional optimization, additional characterization and additional validation

356

are provided in the supporting information.

357 358

AUTHOR INFORMATION

359

* Corresponding Author: [email protected], [email protected]

360

Tel.: +86-25-89687436

361 362

ACKNOWLEDGEMENTS

363

This work was supported by the grants from the National Natural Science Foundation of China

364

(21575059 and 21627806) and the National Science Fund for Creative Research Groups

365

(21121091).


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REFERENCES

367

1 Schubert, C. Nature 2011, 480, 133-137.

368

2 Altschuler, S. J.; Wu, L. F. Cell 2010, 141, 559-563.

369

3 Chen, X.; Love, J. C.; Navin, N. E.; Pachter, L.; Stubbington, M. J.; Svensson, V.; Sweedler,

370

J. V.; Teichmann, S. A. Nat. Biotechnol. 2016, 34, 1111-1118.

371

4 Rubakhin, S. S.; Romanova, E. V.; Nemes, P.; Sweedler, J. V. Nat. Methods 2011, 8, 20-29.

372

5 Huang, B.; Wu, H.; Bhaya, D.; Grossman, A.; Granier, S.; Kobilka, B. K.; Zare, R. N.

373

Science 2007, 315, 81-84.

374

6 Xue, Q.; Yeung, E. S. Anal. Chem. 1994, 66, 1175-1178.

375

7 Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202.

376

8 Rubakhin, S. S.; Sweedler, J. V. Nat. Protoc. 2007, 2, 1987-1997.

377

9 Irish, J. M.; Hovland, R.; Krutzik, P. O.; Perez, O. D.; Bruserud, Ø.; Gjertsen, B. T.; Nolan,

378

G. P. Cell 2004, 118, 217-228.

379

10 Cai, L.; Friedman, N.; Xie, X. S. Nature 2006, 440, 358-362.

380

11 Yu, J.; Xiao, J.; Ren, X.; Lao, K.; Xie, X. S. Science 2006, 311, 1600-1603.

381

12 König, K. J. Microsc. 2000, 200, 83-104.

382

13 Zipfel, W. R.; Williams, R. M.; Christie, R.; Nikitin, A. Y.; Hyman, B. T.; Webb, W. W.

383

Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7075-7080.

384

14 Widengren, J.; Rigler, R. Bioimaging 1996, 4, 149-157.

385

15 Lee, K.; Drachev, V. P.; Irudayaraj, J., ACS Nano 2011, 5, 2109-2117.

386

16 Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K. Nano Lett. 2006, 6, 2225-

387

2231.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

388

17 Kneipp, K.; Haka, A. S.; Kneipp, H.; Badizadegan, K.; Yoshizawa, N.; Boone, C.; Shafer-

389

Peltier, K. E.; Motz, J. T.; Dasari, R. R.; Feld, M. S. Appl. Spectrosc. 2002, 56, 150-154.

Page 20 of 31

390

18 Nabiev, I. R.; Morjani, H.; Manfait, M. J. Eur. Biophys. 1991, 19, 311-316.

391

19 Schatz, G. C. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6885-6892.

392

20 Moskovits, M. J. Raman Spectrosc. 2005, 36, 485-496.

393

21 Kickhoefer, V. A.; Han, M.; Raval-Fernandes, S.; Poderycki, M. J.; Moniz, R. J.; Vaccari, D.;

394 395 396 397 398 399 400 401 402 403 404 405 406

Silvestry, M.; Stewart, P. L.; Kelly, K. A.; Rome, L. H., ACS Nano 2008, 3, 27-36. 22 Vitol, E. A.; Orynbayeva, Z.; Friedman, G.; Gogotsi, Y., J. Raman Spectrosc. 2012, 43, 817827. 23 Liu, J.; Yin, D.; Wang, S.; Chen, H. Y.; Liu, Z. Angew. Chem., Int. Ed. 2016, 55, 1321513218 24 Qiu, W.; Zhou, F.; Zhang, Q.; Sun, X.; Shi, X.; Liang, Y.; Wang, X.; Yue, L. APMIS 2013, 121, 919-925. 25 Fähling, M.; Steege, A.; Perlewitz, A.; Nafz, B.; Mrowka, R.; Persson, P. B.; Thiele, B. J. Biochim. Biophys. Acta 2005, 1731, 32-40. 26 Pichiorri, F.; Palmieri, D.; De Luca, L.; Consiglio, J.; You, J.; Rocci, A.; Talabere, T.; Piovan, C.; Lagana, A.; Cascione, L. J. Exp. Med. 2013, 210, 951-968. 27 Shen, N.; Yan, F.; Pang, J.; Wu, L.-C.; Al-Kali, A.; Litzow, M. R.; Liu, S. Oncotarget 2014, 5, 5494-5509.

407

28 Huang, Y.; Cai, D.; Chen, P. Anal. Chem. 2011, 83, 4393-4406.

408

29 Walling, M. A.; Shepard, J. R. Chem. Soc. Rev. 2011, 40, 4049-4076.

409

30 Li, Y.; Qi, X.; Lei, C.; Yue, Q.; Zhang, S. Chem. Commun. 2014, 50, 9907-9909.


Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

410 411


31 Bates, P. J.; Laber, D. A.; Miller, D. M.; Thomas, S. D.; Trent, J. O. Exp. Mol. Pathol. 2009, 86, 151-164.

412

32 Koutsioumpa, M.; Papadimitriou, E. Recent Pat. Anticancer Drug Discov. 2014, 9, 137-152.

413

33 Krust, B.; El Khoury, D.; Nondier, I.; Soundaramourty, C.; Hovanessian, A. G. BMC Cancer

414

2011, 11, 333.

415

34 Gao, H.; Yang, Z.; Zhang, S.; Pang, Z.; Liu, Q.; Jiang, X. Acta Biomater. 2014, 10, 858-867.

416

35 Lee, K. Y.; Kang, H.; Ryu, S. H.; Lee, D. S.; Lee, J. H.; Kim, S. J. Biomed. Biotechnol. 2010,

417

168306.

418

36 Lee, P.; Meisel, D. J. Phy. Chem. 1982, 86, 3391-3395.

419

37 Zhou, F.; Wang, M.; Yuan, L.; Cheng, Z.; Wu, Z.; Chen, H. Analyst 2012, 137, 1779-1784.

420

38 Muhammad, P.; Tu, X.; Liu, J., Wang, Y., Zhen, L. ACS Appl. Mater. Interfaces 2017, 9,

421 422 423 424 425 426 427

12082-12091. 39 Hanif, S.; Liu, H.; Chen, M.; Muhammad, P.; Zhou, Y.; Cao, J.; Ahmed, S. A.; Xu, J.; Xia, X.; Chen, H.; Wang, K., Anal. Chem. 2017, 89, 2522-2530. 40 Kennedy, D. C.; Tay, L. L.; Lyn, R. K; Rouleau, Y.; Hulse, J.; Pezacki, J. P., ACS Nano 2009, 3, 2329-2339. 41 Borghei, Y. S.; Hosseini, M.; Dadmehr, M.; Hosseinkhani, S.; Ganjali, M. R.; Sheikhnejad, R. Anal. Chim. Acta 2016, 904, 92-97.

428

42 Li, Y. R.; Liu, Q.; Hong, Z.; Wang, H. F., Anal. Chem. 2015, 87, 12183-12189.

429

43 Noaparast, Z.; Hosseinimehr, S. J.; Piramoon, M.; Abedi, S. M., J. Drug Targeting 2015, 23,

430 431 432

497-505. 44 Malik, M. T.; O'Toole, M. G.; Casson, L. K.; Thomas, S. D.; Bardi, G. T.; Reyes-Reyes, E. M,; Bates, P., Oncotarget 2015, 6, 22270-22281.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

433 434

45 Tulchin, N.; Chambon, M.; Juan, G.; Dikman, S.; Strauchen, J.; Ornstein, L.; Monteiro, A. N., Am. J. Pathol. 2010, 176, 1203-1214.


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435

FIGURE CAPTIONS

436

Scheme 1. Schematic illustration of aptamers affinity based SERS nanoprobe for probing NCL

437

in single cells.

438 439

Figure 1. SEM images of aptamers modified Au-nanoprobe before (A) and after hybridization

440

(B) with Raman nanotag. (C) SERS response of hybridized nanoprobe before and after nucleolin

441

(NCL) detection. SERS nanoprobe was incubated in 0.1 µM NCL solution for 5 min at room

442

temperature.

443 444

Figure 2. (A) Concentration-dependent SERS spectra of hybridized nanoprobe for NCL in PBB

445

(pH 7). (B) Plots of SERS intensity at 2223 cm-1 versus different concentrations of NCL. Binding

446

isotherm data was calculated with Hill fitting (R2 = 0.995). Inset graph indicates the linear fitting

447

for different NCL concentrations from 1 to 50 nM with value of R2 = 0.985. Error bars were

448

estimated from three repeated measurements.

449 450

Figure 3. Selectivity of SERS nanoprobe in the presence of different interferants. Bar chart

451

plotted for SERS intensity at 2223 cm-1 versus different proteins and blank (having no protein)

452

under similar conditions. Sample: 100 nM of target NCL protein and 100 µM of other competing

453

proteins dissolved in PBB (pH 7) while blank contain only buffer. Error bars were estimated

454

from three repeated measurements.

455 456

Figure 4. Optical images show the NCL extraction from (A) nucleus, (B) cytoplasm and (C) cell

457

surface of individual cell. The scale bar is 5 µm. (D) SERS intensities at 2223 cm-1 for each



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

458

nanoprobe inserted into different cell lines (MCF-7, MCF-10A and HeLa) within different

459

regions of cells (nucleus, cytoplasm and cell surface).

Page 24 of 31

460 461

Figure 5. SERS spectra for spatial distribution of NCL probing in different cell lines. (A) Blank

462

SERS nanoprobe (B) MCF-7 cells (C) MCF-10A cells and (D) HeLa cells. Raman analysis was

463

performed for 7-8 µm region of SERS nanoprobe with a roughly estimated step distance of 1 µm

464

for each measurement, while N and S referred as nucleus and surface regions of the cells,

465

respectively.

466


Page 25 of 31

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467 468 469 470 471 472 473 474 475 476

Scheme 1

477

Scheme 1

478 479 480 481 482 483 484 485 486 487 488 489



490 491 492 493 494 495 496 497 498 499

C

Aptamer modified SERS nanoprobe

1000 counts

Raman nanotags hybridized SERS nanoprobe Hybridized SERS nanoprobe after NCL detection

SERS intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 31

500 2000

2100

2200

2300

501

Raman Shift (cm )

502

Figure 1

-1

503 504 505 506 507 508 509 510 511 512


2400

Page 27 of 31

513

515

0 nM 0.5 nM 1 nM 10 nM 25 nM 50 nM 100 nM 200 nM 300 nM 400 nM 500 nM

516 517 518

2000

2100

2200

2300

2400

520

6000 4000

Raman Shift (cm )

7695 6840 5985 5130 4275 3420 0

2000

10 20 30 40 50 Conentration of NCL (nM)

Hill Fitting

0 0

-1

519

Sample Blank without NCL

8000

SERS intensity at 2223 cm-1

A

514

B10000 1000 counts

SERS intensity at 2223 cm-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

200

300

400

Concentration of NCL (nM)

Figure 2

521 522 523 524 525 526 527 528 529 530 531 532 533 534 535


500


536 537 538 539 540 541 542

10000 SERS intensity at 2223 cm-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

8000 6000 4000 2000 0

NCL Blank BSA HRP β-Cas Cyt-C Trf

543 544

Figure 3

545 546 547 548 549 550 551 552 553 554 555 556 557 558


Page 28 of 31

Page 29 of 31

559 560 561 562 563 564 565 566 567 568

D12000 SERS intensity at 2223 cm-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nucleus Cytoplasm Cell Surface

10000 8000 6000 4000 2000 0

569 570

Blank

MCF-10A

MCF-7

Figure 4

571 572 573 574 575 576 577 578 579 580 29 ACS Paragon Plus Environment

HeLa


581

8000 6000

584

4000 2000

585

5000

B

SERS Intensity

10000

A

583

4000 3000 2000 1000 0

0

S

2000

2100

2200

2300

on / it i

N 2000

2400

2100

2200

2300

Po s

N

587

Po si tio n/ µm

586

µm

S

2400

Raman Shift (cm-1)

Raman Shift (cm-1)

10000

C

8000 6000

590

4000 2000

591

10000

D

SERS Intensity

589

8000 6000 4000 2000 0

0

S

n/ µ t io N

593 2000

594

2100

2200

2300

N 2000

2400

2100

2300

Raman Shift (cm-1)

Raman Shift (cm-1)

595 596

2200

Figure 5

597 598 599 600 601


2400

Po si tio n/

m

µm

S

592

SERS Intensity

588

SERS Intensity

582

Po si

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 31

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Table of Content

602

603 604

An aptamer modified gold nanopipette was decorated by Raman reporter-covered AgNPs to

605

form a signal-off nanoprobe. The subcellular localization and spatial distribution of NCL were

606

probed in various types of cells


Nanopipette-Based SERS Aptasensor for Subcellular Localization of

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