Organic Cyanide Decorated SERS Active Nanopipettes for

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Organic cyanide decorated SERS active nanopipettes for quantitative detection of hemeproteins and Fe3+ in single cells Sumaira Hanif, Hai-Ling Liu, Ming Chen, Pir Muhammad, Yue Zhou, Jiao Cao, Saud Asif Ahmed, Jing-Juan Xu, Xing-Hua Xia, Hong-Yuan Chen, and Kang Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04689 • Publication Date (Web): 23 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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Analytical Chemistry

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Organic cyanide decorated SERS active nanopipettes for quantitative

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detection of hemeproteins and Fe3+ in single cells

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Sumaira Hanif, Hailing Liu, Ming Chen, Pir Muhammad, Yue Zhou, Jiao Cao, Saud Asif Ahmed,

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Jingjuan Xu, Xinghua Xia, Hongyuan Chen, and Kang Wang*

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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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*Corresponding author: [email protected]

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Tel: +86-25-89687436

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1 ACS Paragon Plus Environment


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Abstract: It is challenging to develop a robust nanoprobe for real-time operational and

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accurate detection of heavy metals in single cells. Fe-CN coordination chemistry has

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been well studied to determine the structural characteristics of hemeproteins by different

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techniques. However, the frequently used cyanide ligands are inorganic molecules that

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release cyanide anion under particular conditions and cause cyanide poisoning. In the

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present study, organic cyanide (4-mercaptobenzonitrile, MBN) was utilized for the first

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time in developing a facile nanoprobe based on surface-enhanced Raman scattering

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(SERS) for quantitative detection of hemeproteins (oxy-Hb) and trivalent iron (Fe3+) ions.

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The nanoprobe prepared by coating the glass capillary tip (100 nm) with a thin gold film,

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which enables highly localized study in living cell system. The cyanide stretching

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vibration in MBN was highly sensitive and selective to Fe3+ and oxy-Hb with excellent

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binding affinity (Kd 0.4 pM and 0.1 nM, respectively). The high sensitivity of the

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nanoprobe to analyte (Fe3+) was attributed to the two adsorption conformations (–SH and

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–CN) of MBN to the gold surface. Therefore, MBN showed an exceptional dual-

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peak (2126 and 2225 cm-1) behavior. Furthermore, the special Raman peaks of cyanide in

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2100-2300 cm-1 (silent region of SERS spectra) are distinguishable from other

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biomolecules characteristic peaks. The selective detection of Fe3+ in both free and

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protein-bound states in aqueous solution is achieved with 0.1 pM and 0.08 µM levels of

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detection limits, respectively. Furthermore, practical applicability of fabricated

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nanoprobe was validated by detection of free Fe3+ in pretreated living HeLa cells by

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direct insertion of a SERS active nanoprobe. Regarding the appropriate precision, good

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reproducibility (relative standard deviation, RSD 7.2-7.6%), and recyclability (retain

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good Raman intensity even after three renewing cycles) of the method, the developed


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sensing strategy on a nanopipette has potential benefits for label-free, qualitative and

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quantitative recognition of heavy metal ions within nanoliter volumes.

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Keywords: Surface-enhanced Raman Scattering, iron-cyanide chemistry, nanopipette,

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hemeproteins, single cell

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Nanoprobes assimilation for metal ions is still following an ascendant slope in these days.

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These probes have potential applications in a variety of fields such as health care, food

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contamination, environmental safety, and security.1 Indeed many metal ions play a key role as

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reactive centers of enzymes in all living organisms, though in normal conditions the

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concentration of these ions are usually low (nanomolar levels)2 whereas in certain pathological

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conditions the concentration might raise to several orders of magnitude higher.3 Amongst the

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different metal ions in the living body, iron in a trivalent state is one of the essential elements for

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all organisms which plays an important role in cellular metabolism and enzyme catalysis.4 The

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fluctuations of Fe3+ both in the deficiency (hypoferremia) and iron overload (hyperferremia) are

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associated with many diseases that lead to dysfunction of organs, certain cancers5 and may cause

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anemia.6 Usually iron inside body is found associated with proteins (hemeprotein) or other

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biological molecules, such as DNA, carbohydrates, and phospholipids.7

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Mammalian cells uphold steady level of metabolically active iron (non-protein bound iron)

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through the regulation of iron uptake and storage in the form of hemeproteins.8 The elevation of

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free iron represents a potential liability to cells because of the participation of iron in the

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metabolism of reactive oxygen species.9 However, metabolically active intracellular free iron is

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present in the form of a cytosolic labile iron pool, classically referred to as the chelatable iron

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pool, not easy to access by simple detection tools.10 Thus to detect the elevated free iron levels in

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cells is highly important to understand the process of ferroptosis. Although numerous techniques

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including fluorescence,11 colorimetric,12 spectroscopic13 and electrochemical analysis14 have

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been established to monitor the free iron at intracellular levels, they are hard to distinguish the

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different forms of iron (Fe2+ to Fe3+). Moreover, these techniques are also insensitive to capture

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the bound iron (metalloproteins) both in vitro and in vivo analysis. Previously, organic ligand4 ACS Paragon Plus Environment

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metal ion complex has been used as an artificial receptor for bio-molecules recognition.15 Such

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an organic-metal chelating approach implies the possibility of detecting metal containing

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biomolecules (metalloproteins) in real world. Hence, it is highly worthy to develop an

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appropriate biosensor that can be used for free metal ion (iron) detection within living system

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also for bio-molecular recognition.

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Surface-enhanced Raman scattering (SERS) provides a highly sensitive, cost-effective, and

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expeditious rapid diagnostic tool for medical and clinical analysis. SERS exhibits several

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significant advantages, including ultrahigh sensitivity, less susceptible to sample environment,

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rapid readout speed and the possibility for on-site or field detection.16-18 Even though SERS was

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extensively used for in vitro detection of metal ions as As3+, Hg2+, Pb2+, Cu2+ and Cd2+,19 it is

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still challenging to obtain relevant SERS data within living cells. A SERS active nanopipette can

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detect minimal amount of analytes in nanoliter volumes, and the action of the pipette can be

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precisely controlled by commercially available micro-manipulators.20 Although some SERS-

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enabled nanopipettes have been used for in situ analysis, the lacking of appropriate label for

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specific targets obstructs the selectivity and provides inadequate cellular information.21,22 To

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work out the intracellular processes on nanoscale, carbon nanotube with plasmonic effect has

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been used, which carry its own limitations.23-25 Previously, organic cyanide derivatives (nitriles)

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have been successfully implied as excellent Raman reporters in the Raman imaging of cells due

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to their peculiar features.26 Recently, Kim et al27 used cyanide derivative as reporter ligand for

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the detection of Cr3+, Fe3+, Fe2+, Ni2+ and Mn2+ by monitoring the shift of the -C≡N stretch on

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complexing via SERS. An obvious disadvantage of such inorganic cyanide derivatives

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(KCN/HCN) under physiological analysis is that the uncertain liberation of high-toxic free -C≡N

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moiety may cause cyanide poisoning.28



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In the present study, we developed a SERS-based nanoprobe for iron detection in free (Fe3+)

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and bound states (hemeproteins) with chemically modified MBN on a gold coated nanopipette.

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Due to the versatile interaction mode of MBN to the gold surface, two characteristic peaks of -

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C≡N (at 2126 and 2225 cm-1) within the SERS silent region were observed. The variation in

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SERS intensity ratio of these two peaks was likely to be sensitive for the formation of Fe-C≡N

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complex on iron exposure. This dual-peak behavior of organic cyanide moiety is quite different

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from the single-peak behavior of typical cyanide derived ligands, which enables MBN to

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quantify iron content. As a proof-of-concept, the fabricated SERS nanoprobe was successfully

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applied to analyze the intracellular iron concentration. To the best of our knowledge, there is no

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method reported yet which could detect free and bound state iron simultaneously inside the

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biological system. Thus, we manifest that the MBN modified SERS-active nanopipette can be

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used for biorecognition and living cell detection.

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EXPERIMENTAL SECTION

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Reagents and materials. 4-mercaptobenzonitrile (MBN, 95%) was purchased from Nanjing

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Norris Pharm Technology Co., Ltd (Nanjing, China). Oxidized hemoglobin (oxy-Hb), sodium

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dithionite, Hemin B, myoglobin (Mb), cytochrome-c (Cyt-c), reduced horse-reddish peroxidase

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(HRP) and Apo-transferrin (Apo-Trf) were all from Sigma-Aldrich (St. Louis, MO, USA).

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Hydrogentetrachloroaurate (III) trihydrate (HAuCl4 3H2O 99.9%) was purchased from Alfa-

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Aesar (Shanghai China). Glucose, potassium bicarbonate (KHCO3), NaOH, ZnSO4, HCl (36%),

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NaCl, KCl, MgCl2, ZnCl2, CuCl2, Ni(CH₃CO₂)₂.2H₂O, FeCl3, Pb(NO3)2, ZnCl2, Mn

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(CH₃COO)₂.2H2O, MgCl2, CaCl2, NaH2PO4, Na2HPO4, ethylenediaminetetraacetic acid (EDTA),

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acetonitrile (≥ 99.00%) and anhydrous ethanol were of analytical grade and purchased from the


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Nanjing reagent company (Nanjing, China). All these reagents were used without further

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purification. Other chemicals were of analytical grade or higher. Water was purified with a Milli-

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Q Advantage A10 (Millipore, Milford, MA, USA), and was used to prepare all solutions.

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Borosilicate glass capillaries with filament (0.58 mm I.D., 1.0 mm O.D.) were purchased from

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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 pipettes. Scanning electron microscopic (SEM) characterization was carried

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out on a FE-SEM S-4800 system (Hitachi, Tokyo, Japan). Raman and SERS experiments were

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conducted on a Renishaw InVia Reflex confocal microscope (Renishaw, UK) equipped with a

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high-resolution grating with 1800 grooves/cm, additional band-pass filter optics, and a CCD

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camera. All measurements were carried out using a He-Ne laser (633 nm: laser power: 1 mW).

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The laser was focused onto the sample by using a ×50 objective lenses with (N.A. 0.75), and the

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diameter of the laser spot was 1 µm. Wavelength calibration was performed by measuring silicon

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wafers through a ×50 objective, assessing the first-order phonon band of Si at 520 cm-1. The

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spectra were recorded using the Renishaw WiRE (Windows-based Raman Environment)

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software and analyzed with Origin Pro 8.6 software. Each spot was detected from 5 to 6 min

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with different locations. Each spectrum baseline was corrected except noise test. A three-

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dimensional manipulator provided by a local company was equipped on an inverted microscope

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(Nikon Ti-E), and was used to precisely insert extraction microprobes into single cells under

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investigation.

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Fabrication of glass nanopipettes. Borosilicate glass with filament (0.58 mm I.D., 1.0 mm O.D.,

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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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Fabrication of SERS nanoprobe. For the preparation of Au-coated nanopipette probes, 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.29 Briefly, the nanopipettes (100 nm I.D. and 200 nm O.D.) were immersed in a solution

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containing 12 mM HAuCl4, 0.5 M KHCO3 and 25 mM glucose for 3-4 h at 45 °C until a clear

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gold layer formed on the surface of each nanopipette. After washed with water and anhydrous

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ethanol for 3 min, the Au-coated nanoprobes were dried in air at room temperature. The Au film

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thickness was estimated to be 20-30 nm thus the total O.D of nanoprobe was about 250 nm.

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MBN functionalization on SERS nanoprobe. The Au-coated nanoprobes were immersed in a

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solution of MBN in acetonitrile at room temperature for 30 min, and then rinsed with acetonitrile

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to remove unmodified reagents. The MBN-functionalized Au-coated nanoprobes were stored at

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room temperature for further use.

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In vitro free iron (Fe3+) detection by SERS nanoprobe. The MBN modified nanoprobes were

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separately immersed in different concentrations of ferric chloride aqueous solution (1 fM to 1

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µM) for 15 min at room temperature. After capturing the analyte, nanoprobes were washed with

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water for 2 min to remove unbound analyte. These SERS nanoprobes were dried and Raman

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spectra were collected.

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In vitro hemoglobin (oxy-Hb) detection by SERS nanoprobe. The MBN modified nanoprobes

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were separately immersed in different concentrations of oxidized hemoglobin (oxy-Hb: 0.1 nM

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to 100 mM) in 10 mM sodium phosphate buffer (pH 7.4) for 30 min at room temperature. After

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capturing the analyte, nanoprobes were washed with 10 mM sodium phosphate buffer (pH 7.4)

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for 2 min to remove unbound analyte, dried and Raman spectra were collected.

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Selectivity test. The selectivity of the substrate for labile iron (Fe3+) was checked against a

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variety of interfering agents including Fe2+, Cu2+, Ni2+, Pb2+, Zn2+, Mn2+, Ca2+, Mg2+, Na+ and K+.

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The concentrations of interfering agents were kept at 1 mM against the Fe3+ standard

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concentration (1 µM) i.e. 1000-fold lowered as compared to these interference constituents in

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solution. For each constituent, SERS nanoprobe was immersed in for 15 min at room

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temperature. After washed with water for 2 min, the SERS substrate was dried and analyzed

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under Raman spectrograph. Meanwhile, control experiment, MBN nanoprobe without trapped

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Fe3+ was conducted. Results are represented as mean of at least three independent experiments.

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The selectivity of the nanoprobe for bound iron (oxy-Hb) was tested against the

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following interfering hemeproteins including Mb, Cyt-c, red-Hb, HRP and Hemin B in 10 mM

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sodium phosphate buffer (pH 7.4) and the other steps were the same except incubation time of 30

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min. For preparation of red-Hb, 9.763 mg of sodium dithionite in 3.0 mL of stock solution

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containing 1 mg oxy-Hb to get maximum reduced protein. The concentrations of interfering

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agents were kept at 1 M and for target analyte (oxy-Hb) 1 mM. Meanwhile, control experiments,

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named as positive control MBN nanoprobe without trapped oxy-Hb and negative control Apo-

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Trf were also conducted. Results are represented as mean of at least three independent

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experiments.



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Reusability of SERS nanoprobe. The reusability of SERS nanoprobe was evaluated via treating

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with strong multidentate ligand, EDTA. The nanoprobe was repeatedly treated by Fe3+ (1 mM,15

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min, washed in water for 2 min), and EDTA (10 mM, 30 min, washed in water for 2 min) at

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room temperature. Raman spectra were recorded after each step.

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Reproducibility of SERS nanoprobe. The SERS signal reproducibility were evaluated for

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different concentrations (1 µM and 100 nM) of Fe3+ and SERS scan were collected from 10

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random spots on the surface of nano-tip. In the meantime, a control experiment was also

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evaluated without trapped Fe3+ at identical conditions. The corresponding SERS intensity ratio at

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2126 cm-1 and 2225 cm-1 was chosen to further evaluate the reproducibility. Meanwhile, control

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experiment, MBN nanoprobe without trapped Fe3+ was conducted. Results are represented as

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mean of at least three independent experiments.

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Stability of SERS nanoprobe. Stability of the nanoprobes was investigated through monitoring

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the Raman signal intensity ratio of MBN for identical concentration up to ten weeks. Briefly, the

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MBN nanoprobe was immersed in aqueous solution of Fe3+ (1 mM) for 15 min at room

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temperature, then washed with water for 2 min, dried and detected by Raman spectrograph. All

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SERS nanoprobes were stored at room temperature for further use. In the meantime, control

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experiment of MBN nanoprobe was also conducted without addition of Fe3+ at identical

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conditions. Results are represented as mean of at least three independent experiments.

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Intracellular iron (Fe3+) detection in HeLa cell


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Culture of HeLa cells. HeLa cells were from the Cell Bank of Type Culture Collection of

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Chinese Academy of Sciences (Shanghai). The cells were cultured in Dulbecco’s modified eagle

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medium (DMEM, Gibco) containing 10% fetal bovine serum, 100 mg/mL streptomycin and 100

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U/mL penicillin (37 °C in 5% CO2 for 48 h). HeLa cells were seeded in culture medium for 2 to

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3 days at 37 °C and 5% CO2. After the cell culture medium was removed, the cells lingering on

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the cell culture dishes were washed with 1× phosphate-buffered saline (PBS) solution for 2 min

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and then the attached cells were kept in 1× PBS buffer for further use.

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Fe3+ treatment to HeLa cells. Manipulation of the intracellular labile iron (Fe3+) content was

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carried out by iron supplementation or deprivation treatments30. HeLa cells seeded on glass-

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bottom dishes were loaded with different concentration of iron (1, 50 and 100 µM) in culturing

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buffer (1× PBS, pH 7.4). After 15 min of incubation at room temperature, the cells were washed

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twice and kept in buffer 1× PBS buffer till used for analysis.

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Extraction of iron (Fe3+) from HeLa cells. An inverted microscope (Nikon Ti-E equipped with

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a three-dimensional manipulator) was used to precisely insert SERS nanoprobe into single cells

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under investigation by optimized extraction time periods. A control experiment for comparative

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study was also performed under similar conditions except iron treatment to the cell as a blank.

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RESULTS AND DISCUSSION

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SERS response of MBN modified nanoprobe. The SERS active nanopipettes were fabricated

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by smearing a thin layer of gold on the pulled glass nanopipette of 100 nm orifice. Figures 1A

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and B display the typical SEM images of the gold coated nanopipette. The outer surface of the



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nanopipette was evenly coated with an ultrathin Au film at the proximal end to keep the shape.

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The fabricated Au coated nanoprobe and optical images are shown in Figures S1A and B in

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supporting information. The outer diameter (O.D.) of nanopipette after Au coating was about 250

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nm and cone angle of 33˚ (details shown in Figure S2 in supporting information). The plasmonic

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response of fabricated nanopipette, imputed via MBN modification was shown in Scheme 1A.

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The optimized MBN concentration (10 mM) and incubation time (30 min) were used for the

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modification on SERS active nanopipette (Figures S3 and S4 in supporting information). The

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SERS enhancement factor for MBN on gold nanopipette was estimated to be ∼1.5 × 104 (details

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are mentioned in supporting information under Figure S5). The acquired SERS spectrum from

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MBN modified nanopipette is shown in Figure 1C. The Raman spectrum with detailed

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characteristic vibrational bands for MBN on SERS active nanopipette is given in Table S1 in

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supporting information. Remarkably, the SERS spectrum of MBN exhibits two distinct and sharp

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peaks at 2126 and 2225 cm-1 for cyanide υ(-C≡N) vibrations. This dual-peak behavior of MBN is

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associated with -C≡N stretching originating from direct interaction of -C≡N and -SH groups

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within the same molecule to Au surface, which is comparable to the previous studies (Table S2

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in supporting information).30 In order to further characterize the ratio of the two adsorption sites

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of MBN molecules on the gold surface (Figure S6), the SERS intensity ratio Ir = I1/(I1+I2) of

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MBN was used (I1 and I2 refer to the peak intensity at 2225 and 2126 cm-1, respectively). The

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nanoprobe formation under optimized conditions requires an extremely short time period of 30

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min. That makes it superior to the other typical SERS substrate fabrication assays, which

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required hours or even overnight procedures.31

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Mechanism of dual peak behavior of MBN. We observed dual SERS characteristic peaks of

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cyanide at 2126 and 2225 cm-1 for MBN modified gold surface of nanoprobe due to the distinct

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interaction mode (Figure 1D). MBN has numerous anchoring sites to interact with the gold

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surface; (i) sulfur/thiolate (-SH), (ii) nitrogen of the nitrile group and (iii) benzene ring π-electron

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system. Based on these sites, two possible binding conformations are likely to be observed;

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conformation-I is the most probable anchoring position of the MBN to gold surface via thiol (-

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SH) group. Conformation-II reveals the involvement of strong electron withdrawing group (-

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C≡N) at para-position to give a bent conformation of MBN. There are two primary factors for

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the distinctive conformations of MBN. Firstly, the strong electron-withdrawing (-C≡N) present

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at peculiarly effective para-position destabilizes the C-S bond of Au adsorbed MBN to provide

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the additional interaction sites.31 Secondly, because of the native feature of -C≡N to coordinate

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with transitional metal ions, the interaction between cyanide and gold surface leads to an unusual

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but quite stable bent conformation.32 Therefore, exceptional dual-peak behavior of -C≡N with

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two peaks at 2225 and 2126 cm-1 originated from structures differentiation, which was also

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verified by previous reports.33 The bent conformation of adsorbed MBN molecule provides the

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significant SERS response to trace analyte through direct interaction with -C≡N group. Therefore

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unlike the typical cyanide derived SERS reporter ligands, where single characteristic peak

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(∼2222 cm-1) appear, current organic cyanide with dual-peak response can precisely elaborate the

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chemical mechanism of cyanide coordination with the analyte.

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Detection of target molecules (Fe3+/oxy-Hb). Generally, cyanide -C≡N is one of the few

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ligands which interact with many metal ions with different affinity.34 Scheme 1B shows the

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binding mechanism of immobilized MBN on nanoprobe and its interaction with highly



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electropositive ion (Fe3+) metal ion as target. Upon binding, the MBN reactive site (-C≡N) shows

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a conformation change in the structure of a bent position and the change of corresponding SERS

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spectrum is shown in Figure 2A. This conformational change is the result of a strong carbon

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directed bond of Fe-CN chelation effect. Due to the release of bent moiety of cyanide, the SERS

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intensity increased at 2225 cm-1 and decreased at 2126 cm-1, which resulted in an increase in Ir

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on the exposure of Fe3+ (Figure S7 in supporting information). Figure 2B showed that cyanide-

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Fe3+ chelating affinity was maintained for the ferric ion enclosed in hemeproteins (oxy-Hb). The

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optimized capturing time of 15 min for Fe3+ and 30 min for oxy-Hb was used for detection

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(shown in Figures S8 and S9 in supporting information). The fluctuating response of SERS

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intensities to analytes (Fe3+/oxy-Hb) with respect to the time intervals verified the phenomena

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that the expansion in SERS spectra was not random but closely related to the iron-cyanide

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complex formation by MBN. The combination of the cyanide group to iron in hemoglobin with

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high affinity has been attributed to similarity in structural stereochemistry of oxygen, carbon

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monoxide and cyanide.35 The presence of hemoglobin on the MBN modified nanoprobe was

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further verified by collecting the whole SERS spectra (200-3200 cm-1) including fingerprint

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region of hemoglobin. The major characteristic peak regions at 1500-1650 and 1300-1400 cm-1

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were assigned to porphyrin and pyrrole ring stretching, respectively. Meanwhile, region 600-

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1200 cm-1 gave pyrrole breathing for symmetric pyrrole deformation mode.36 Fe-C≡N has its

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prolonged recognition history to access the ferric heme iron without hindrance from the heme

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model compounds and with very stable association equilibrium constant (10-5 M).37 Hence, the

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present iron chelating strategy can be considered to have the ability of detecting Fe3+ in both

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ionic and protein bounded states.

307


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308

Validation of nanoprobe for iron sensing. In addition, for quantitative information a good

309

correlation was observed between SERS intensity ratio Ir and iron (Fe3+) ion concentrations in a

310

range from 10-6 to 10-15 M. As evident in Figure 2C, clear variations exist for different iron

311

concentrations with respect to the control SERS spectrum at two vibrational modes at 2126 and

312

2225 cm−1. This intensity ratio was increased with increasing Fe3+ exposure, owing to a decrease

313

in peak intensity at 2126 cm-1 and a successive increase at 2225 cm-1. At the higher

314

concentrations (>1 µM) of Fe3+, the Ir increased up to 1 with disappearance of peak at 2126 cm-1

315

(data is not shown). In the meantime, SERS intensity response of MBN nanoprobe was also

316

observed against different oxy-Hb concentrations (10-1 to 10-8 M). As shown in Figure 2D, the

317

SERS intensity at 2126 cm-1 consistently decreased with increasing oxy-Hb concentration. The

318

SERS intensity ratio (Ir) increased dynamically at higher concentration (>1 µM) because

319

globular protein interfere destabilizes the interaction between -C≡N and gold surface.

320

The plot fittings of SERS intensity ratio Ir as function of varying concentrations of Fe3+

321

and oxy-Hb have provided good correlations (r2 = 0.999 for Fe3+ and 0.999 for oxy-Hb) as

322

shown in Figures 2E and 2F, respectively. The dissociation constants (Kd) calculated by

323

concentration-response curve fittings for free Fe3+ and oxy-Hb were 0.4 pM and 0.1 nM,

324

respectively, (detailed procedure is shown in supporting information). Such a high binding

325

affinity for iron indicates a strong chelation by more than one -C≡N group for single Fe3+ on

326

MBN modified nanopipette. A good affinity of small MBN for a macromolecule as oxy-Hb

327

(molecular weight 16 kDa) showed the evidence that 2 or more possible heme sites (within

328

single Hb) interact with -C≡N on the nanopipette. Further elaboration of Hb binding behavior on

329

the nanoprobe has been shown in Figure S10 in supporting information, where a negligible

330

affinity for Apo-protein while maximum interaction for hemin was observed. These SERS



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Page 16 of 34

331

responses of MBN are in good agreement with the previous study of -C≡N interaction to

332

hemoprotein with detailed mechanism.38 A good linear trend was observed for labile Fe3+ and

333

oxy-Hb (r2 = 0.998

334

respectively. The experimentally calculated limit of detection (LOD) by using standard deviation

335

formula (S/N > 3) were 0.1 pM for in vitro Fe3+ and 0.08 µM for oxy-Hb, comparable to the

336

previous reports.39, 40

for Fe3+ and 0.997 for oxy-Hb) as shown in of Figures 2E and 2F,

337 338

Selectivity of the nanoprobe. The selectivity of the nanoprobe was investigated against various

339

metal ions including Fe2+, Zn2+, Mn2+, Ca2+, Mg2+, Na+ and K+ in aqueous solution. The

340

concentration of interfering metal ions was kept 1000-fold higher than the target metal ion. As

341

shown in Figure 3A, clear Raman shifts (20-65 cm-1) in both peaks around 2126 and 2225 cm-1

342

of -C≡N were observed, verifying the direct involvement of cyanide of MBN in metal ions

343

chelation.27 Figure 3B showed that all these metal ions yielded less decrease in SERS intensity

344

and smaller Ir value compared with the Fe3+ (ratio was measured considering the shifted

345

positions). It is worth to note that the nanoprobe has peculiar feature to differentiate the different

346

ionic states of iron in Fe2+ and Fe3+ with high precision. Figure 4A shows the selectivity of

347

nanoprobe for oxy-Hb against other Fe3+ containing proteins (Mb and Cyt-c) and Fe2+ containing

348

proteins (red-Hb and HRP). The concentration of oxy-Hb was 0.1 mM while the other proteins

349

were kept at 1 mM. In Figure 4B, the SERS intensity ratios Ir shows the exceptional behavior of

350

nanoprobe for red-Hb, representing a totally different interaction mechanism to the Fe2+

351

containing Hb. Such an inverse behavior can be attributed to the strong electrostatic coupling of

352

the terminal amino acids in red-Hb with cyanide group. Hemoglobin is well known to has

353

charged amino acids crowding around the heme pocket, which facilitated the combination of


Page 17 of 34

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354

heme with O2 and CO2. Cyanide group is also reported having strong interaction with these

355

amino acids.

356

towards the gold surface (as shown in type II moiety in Fig. 1D), which resulted in a much

357

higher peak intensity at 2126 cm-1. The other Fe2+ containing protein (HRP) and protein without

358

Fe (Apo-Trf) provided no significant SERS response. Thus an iron (Fe3+ and Fe2+) differentiation

359

phenomenon of nanoprobe was retained even for metalloproteins. Additionally, the appearance

360

of characteristic protein peaks between 600 and 1400 cm-1 further revealed the presence of

361

protein on nanoprobe. The varying response of probe to the Fe3+ containing proteins (Mb and

362

Cyt-c) can be attributed to the dissimilar Fe3+ content in hemeproteins as; Hb has 4 hemes, while

363

Mb and Cyt-c have single heme, individually. Besides, the different protein residual part

364

(charged amino acid) also induced the electrostatic effect which hindered the accessibility of Fe3+

365

by MBN.42 Thus a general binding affinity trend of -C≡N with hemeproteins will follow the

366

order of hemoglobin> myoglobin > cytochrome.

41

Thus we postulated that the presence of red-Hb forced the cyanide bending

367 368

Reversibility of the nanoprobe. To the best of author knowledge, the SERS nanoprobes for

369

Fe3+ detection with renewable and recyclable properties have not been reported yet. To check the

370

reusability, EDTA was used as multidentate ligand to release the captured Fe3+ from MBN as

371

shown in Figure 5A. The resulted Raman spectra are presented in Figure 5B. The SERS

372

intensities at 2126 cm−1 were recovered on EDTA treatment after each regenerating cycle,

373

indicating the good recyclability of SERS nanoprobe. Quantitative characterization of the

374

reusability was done by measuring the SERS intensity ratios after each response-regeneration

375

cycles (Figure S11 in supporting information). After three cycles, the SERS intensity ratio

376

recovered to its initial (blank) state.



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Page 18 of 34

377 378

Stability and reproducibility of the nanoprobe. Variation of the SERS signal by an increment

379

of Fe3+ concentration on MBN nanoprobe with multiple scans near the tip was observed in term

380

of RSD (7.2 to 7.6%). Signal repeatability for Fe3+ detection at different concentrations at single

381

run experiment is shown in Figure S12 in supporting information. The signal was read from 10

382

random points on the SERS nanoprobe substrate, thus the MBN modified nanopipette generates

383

a uniform and reproducible hot spot with reference values.

384

The SERS spectra of the nanoprobes were checked from a period of freshly prepared to

385

ten weeks aged at room temperature. Meanwhile, the SERS spectra of the nanoprobes after Fe3+

386

exposure were also tested. The MBN on the nanopipette maintained its SERS activity even after

387

ten weeks period ageing, with stable binding affinity for iron (Figure S13 in supporting

388

information). Even though a small shift in peak at 2225 cm-1 after 4 weeks was observed that

389

might be the possible thiol bond displacement due to prolong storage time of modified gold

390

substrate.43 Owing to chemical stability of the Au film, the MBN on Au surface can be

391

environmentally benign to the surrounding atmospheric factors. As a result, the developed

392

nanoprobe convincingly demonstrates the feasibility in long-time stability with reproducible

393

SERS intensity response.

394 395

Detection of intracellular Fe3+ in cell. The good performance and special shape of the

396

nanoprobe make it possible to serve as a promising candidate for identification of Fe3+ in single

397

cell analysis. To get the actual efficacy of the developed nanoprobe, HeLa cells were used as the

398

model system. Monitoring the intracellular Fe3+ in target cells were attained by direct insertion of

399

MBN modified nanopipette into adherent living cell. Briefly, HeLa cells were treated with Fe3+


Page 19 of 34

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400

for 15 min in culture medium then washed with PBS (pH 7.4). Subsequently MBN modified

401

SERS nanopipette was directly inserted into HeLa cell using a 3-D manipulator to extract Fe3+

402

from the cell (Figure 6A). The dynamics of the Raman signal upon the staying time of the

403

nanoprobe inside the cell was found reach equilibrium within 15 min (Figure S14 in supporting

404

information). The penetration depth of nanoprobe in cell was 2-3 µm with the biggest diameter

405

of the nanoprobe that entered in the cell is about 1.4 µm. Therefore, the laser spot of the confocal

406

Raman can be easily recorded on the nanoprobe. The data shown in Figure 6B represent the

407

averaged Raman spectra of at least three different measurements, conducted on multiple cells

408

with SERS nanoprobes. The appearance of multiple peaks between 1000 to 1650 cm-1 indicated

409

the adsorption of proteins (1600 cm-1 for amide-I; 1300, 1250 and 1240 cm-1 for amide-III), and

410

possibly lipid (1500-1650 cm-1)19 on the nanoprobe. Moreover, decreasing SERS intensity at

411

peak ~1570 cm-1 as compared blank also indicating the presence of other iron conjugated

412

biomolecules on nanoprobe. The extraction process occurs primarily in the first several minutes

413

after the insertion of nanoprobe to the cell. These results ensure that the MBN modified SERS

414

nanopipette can be applied in the single cell analysis with high metal matrix effect in SERS

415

fingerprint region, where the characteristic active vibrations (2126 and 2225 cm-1) of MBN

416

undergo significant variations for Fe3+ treated cell. It is also important to emphasize that

417

nanoprobe insertion does not cause fatal damage to the cell.

418 419

CONCLUSION

420

In conclusion, we developed an exceptional plasmonic sensing assay on nanopipettes of 100 nm

421

tip size coated with a thin Au film with enhanced SERS-activity. MBN, an organic cyanide

422

ligand for SERS based Fe3+ detection, showed very high selectivity and reproducibility that can



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Page 20 of 34

423

be used in detecting Fe3+ in free and bounded states. The SERS intensity ratio of the two specific

424

peaks from MBN showed strong relevance to the concentration of Fe3+, which made the

425

nanoprobe a quantitative tool in Fe3+ analysis. The adaptability of nanopipette on microcontroller

426

and as the support for SERS substrate made the MBN modified nanoprobe suitable for single cell

427

study. All of these merits of MBN modified SERS-enabled nanopipettes open a new insight to

428

prepare more effective metal ion nanoprobes for the detection of heavy metal ions.


Page 21 of 34

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429


ASSOCIATED CONTENT

430

Supporting Information

431

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

432

Synthesis routes, additional optimization, additional characterization and additional validation

433

are provided in the supporting information.

434 435

AUTHOR INFORMATION

436

* Corresponding Author: [email protected]

437

Tel.:+86-25-89687436

438

ACKNOWLEDGEMENTS

439

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

440

(21327902, 21575059, and 21275071), the National Science Fund for Creative Research Groups

441

(21121091), and State Key Laboratory of Analytical Chemistry for Life Science

442

(5431ZZXM1502).



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443 444

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Esposito, B. P.; Breuer, W.; Sirankapracha, P.; Pootrakul, P.; Hershko, C.; Cabantchik, Z. I. Blood 2003, 102, 2670-2677.

10 Breuer, W.; Ronson, A.; Slotki, I. N.; Abramov, A.; Hershko, C.; Cabantchik, Z. I. Blood 2000, 95, 2975-2982.

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11 Wang, S.; Meng, X.; Zhu, M. Tetrahedron Lett. 2011, 52, 2840-2843.

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12 Yin, W.; Cui, H.; Yang, Z.; Li, C.; She, M.; Yin, B.; Li, J.; Zhao, G.; Shi, Z. Sens. Act. B.

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13 Lunvongsa, S.; Oshima, M.; Motomizu, S. Talanta 2006, 68, 969-973.

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15 Ali, M.; Nasir, S.; Nguyen, Q. H.; Sahoo, J. K.; Tahir, M. N.; Tremel, W.; Ensinger, W. J. Am. Chem. Soc. 2011, 133, 17307-17314.

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16 Qian, X. M.; Nie, S. M. Chem. Soc. Rev. 2008, 37, 912-920.

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17 Porter, M. D.; Lipert, R. J.; Siperko, L. M.; Wang, G.; Narayanan, R. Chem. Soc. Rev. 2008,

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18 Lane, L. A.; Qian, X.; Nie, S. Chem. Rev. 2015, 115, 10489-10529.

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19 Tu, A.T. Raman, Spectroscopy in Biology: Principles and Applications, John Wiley and

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20 Vitol, E. A.; Orynbayeva, Z.; Friedman, G.; Gogotsi, Y. J. Raman. Spect. 2012, 43, 817–827.

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21 Vitol, E. A.; Orynbayeva, Z.; Bouchard, M. J.; Azizkhan-Clifford, J.; Friedman, G.; Gogotsi,

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Y. ACS Nano 2009, 3, 3529–3536. 22 Masson, J. F.; Breault-Turcot, J.; Faid, R.; Poirier-Richard, H. P.; Yockell-Lelievre, H.; Lussier, F.; Spatz, J. P. Anal. Chem. 2014, 86, 8998-9005. 23 Li, Y.; Zhou, W.; Wang, H.; Xie, L.; Liang, Y.; Wei, F.; Idrobo, J. C.; Pennycook, S. J.; Dai, H. Nat. Nanotechnol. 2012, 7, 394-400.

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24 Niu, J. J.; Schrlau, M. G.; Friedman, G.; Gogotsi, Y. Small 2011, 7, 540-545.

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25 Lussier, F.; Brule, T.; Vishwakarma, M.; Das, T.; Spatz, J. P.; Masson, J. F. Nano Lett. 2016,

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27 Kim, K.; Lee, J. W.; Shin, K. S. Analyst 2013, 138, 2988-2994.

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28 Gracia, R.; Shepherd, G. Pharmacotherapy 2004, 24, 1358-1365.

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



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31 Holze, R. J. Sol. State. Electrochem. 2013, 17, 1869-1879.

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32 Aragay, G.; Pons, J.; Merkoci, A. Chem. Rev. 2011, 111, 3433-3458.

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33 Jacobs, M. B.; Jagodzinski, P. W.; Jones, T. E.; Eberhart, M. E. J. Phy. Chem. C, 2011, 115,

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38 Pinakoulaki, E.; Vamvouka, M.; Varotsis, C. Inorg. Chem. 2004, 43, 4907-4910.

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39 Yoshikawa, S.; O'Keeffe, D. H.; Caughey, W. S. J. Biol. Chem. 1985, 260, 3518-3528.

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40 Guo, X.; Li, M.; Hou, T.; Wu, H.; Wen, Y.; Yang, H. Sens and Act. B. Chem. 2016, 224, 16-

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509

Figure Captions

510

Scheme 1. A) Schematic of nanoprobe surface modified with MBN and its interaction with Fe3+

511

for detection. B) Suggested mechanism for Fe3+ detection process by Au-MBN nanoprobe.

512 513

Figure 1. A) SEM image of nanoprobe after Au-film fabrication kept the conical shape of

514

nanoprobe. B) SEM image of outer surface of nanoprobe near tip after au-film fabrication. C)

515

Raman spectrum of MBN powder and MBN-modified nanoprobe at 10 mm concentration for 30

516

min incubation at room temperature. D) Schematic representation of two possible conformations

517

of MBN on Au-coated nanoprobe depending on interaction sites.

518 519

Figure 2. A) SERS intensity response of MBN modified nanoprobe after iron (Fe3+) of 1 mM

520

concentration exposure. B) SERS spectrum of MBN modified nanoprobe (black) and captured

521

oxy-Hb by nanoprobe (red) at 1 mM concentration for 30 min incubation at room temperature. C)

522

SERS intensity responses of MBN modified nanoprobe to various concentrations of Fe3+ (from

523

top to bottom 1 fM to 1 µM) in water incubated for 15 min. (D) SERS intensity responses of

524

MBN modified nanoprobe to various concentrations of oxy-Hb (from top to bottom 0.1 nM to

525

100 mM) in phosphate buffer (pH 7.4) incubated for 30min. E) Plots of SERS intensity ratio Ir

526

versus log of Fe3+ concentration (from 1 µM to 1 fM), linear fitting at r2 = 0.998 for Fe3+. F)

527

Plots of SERS intensity ratio Ir versus log of oxy-Hb concentration (from 0.1 nM to 100 mM),

528

linear fitting at r2 0.997 for oxy-Hb. Error bars were estimated from three replicate measurements.

529 530

Figure 3. A) SERS response of MBN modified nanoprobe for free Fe3+ in water against different

531

metal ions: interferants concentrations are 1 mM for each and Fe3+ (as target) with concentration



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

532

1 µM for identical capturing conditions. B) Bar graph of plotted for SERS intensity ratio Ir

533

versus different metal ions including blank (no metal ion) under similar conditions as for graph-

534

A. Error bars were estimated from three replicate measurements.

535 536

Figure 4. A) SERS response of MBN modified nanoprobe for oxy-Hb in phosphate buffer (pH

537

7.4) against different hemeproteins: concentrations of interferants are 1 M and oxy-Hb (1 mM as

538

target) under identical capturing conditions. (B) Bar graph of plotted for SERS intensity ratio Ir

539

versus different hemeproteins including blank (no protein) under similar conditions as for graph-

540

A. Error bars were estimated from three replicate measurements.

541 542

Figure 5. A) Schematic illustration of regeneration of SERS nanoprobe through the

543

supplementation of EDTA (10 mM for 30 min). (B) SERS spectra of MBN modified nanoprobe

544

is recycling for three times in the detection of 1 mM Fe3+.

545 546

Figure 6. A) Micrographs of the MBN modified nanoprobe inserted into HeLa cell. The scale

547

bars are 5 µm. (B) SERS intensity response of MBN nanoprobes after extraction of labile iron

548

(Fe3+) treated different cell for different iron concentrations (1, 50 and 100 µM) with respect to

549

blank cell (without iron).

550 551 552 553 554


Page 26 of 34

Page 27 of 34

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555

556

A 557 558 559

B

560 561 562

563

Scheme 1

564

565

566

567

568

569

570

571

572



573

574

575 576 577 578 579 580 581

582

C

MBN Powder MBN SERS

5000 counts

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

400

800

1200

1600

2000

2400

Raman Shift (cm-1)

Figure 1

583

584 585 586 587 588 589 590 591 592 593


Page 28 of 34

Page 29 of 34

594 595 596

598

2225 cm-1

2126 cm-1

2000

2100

2200

2300

2400

Au-MBN Au-MBN-oxy-Hb

400

-1

800

601

2100

D

0M 1 fM 10 fM 100 fM 1 pM 10 pM 100 pM 1 nM 10 nM 100 nM 1 µM

5000 counts

2000

2200

2300

2000

2400

2100

0.95

2200

2300

2400

Raman Shift (cm )

F 1.00

Fe3+ Dose Response fitting Linear fitting

oxy-Hb Dose Response fitting Linear fitting

0.95

0.90

0.90

0.85

0.85

Ιr

603

2800

-1

-1

E 1.00

2400

0M 1nM 10 nM 100 nM 1 µM 10 µM 100 µM 1 mM 10 mM 100 mM

5000 counts

Raman Shift (cm )

602

2000

SERS intensity

600

1600

Raman Shift (cm )

SERS intensity

C

1200

-1

Raman Shift (cm )

599

604

5000 counts

SERS intensity

597

B

Au-MBN Au-MBN-Fe3+

5000 counts

SERS intensity

A

Ιr

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


0.80 0.75

0.80 0.75

3+

0.70

605

606

Sample Blank without Fe -15 -14 -13 -12 -11 -10 -9 3+

Log[Fe

-8

-7

-6

0.70 -5

-8

(M)]

-7

-6

-5

-4

-3

Log[Hb (M)]

Figure 2

607

608

609

610


-2

-1

0


611

612

613

A

614

B1.0

5000 cps

3+

Fe = 1 µM Other metal ions= 1 mM

0.8

615 nk + la 2 B

Ιr

0.6

2+

617

618

Figure 3

619

620

621

622

623

624

625

626

627 30 ACS Paragon Plus Environment

3+

Fe

1+

2+

1+

K

N a

2+

2+

2+

M g Fe

a C

-1

Raman Shift (cm )

2+

+

0.0

2400

2+

2300

2+

2200

u N i M n Pb

2100

la nk Zn 2

2000

0.2

B

616

0.4

C

Fe n 2+ 1+ M u C Na 1+ 2+ K Mg 2+ 2+ a C Zn 2i+ 2+ N b 3+ P e F

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 34

Page 31 of 34

628

629

631

B 1.2

5000 cps

1.0 0.8

Oxy-Hb Red-Hb

0.6

Mb Cyt c HRP APO-Trf

632

oxy-Hb= 1 mM Other interfering proteins= 1 M

Ιr

630

A SERS intensity

0.4 0.2

Blank

634

2400

2800

Figure 4

635

636

637

638

639

640

641

642

643


M b Re dHb O xy -H b

1200 1600 2000 -1 Raman Shift (cm )

Cy tc

633

800

HR P Ap oTr f

0.0

400

Bl an k

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



645

646

B

5000 counts

647

648

EDTA treatment

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 32 of 34

3+

2nd Fe

EDTA treatement 3+

1st Fe

649

Blank 2000

2100

2200

2300 -1

Raman Shift (cm )

650

651

Figure 5

652

653

654

655

656

657

658

659

660


2400

Page 33 of 34

662

663

664

B

Control (0M Fe3+) 1 µM Fe3+ 50 µM Fe3+ 100 µM Fe3+

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


5000 counts

665 400

800

1200

1600

2000 -1

Raman Shift (cm )

666

667

Figure 6

668

669

670

671

672

673

674

675

676


2400

2800


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

677

678

TOC graphic:

679 680 681 682


Page 34 of 34

Organic Cyanide Decorated SERS Active Nanopipettes for

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