- Email: [email protected]
Development of localized surface plasmon resonance biosensors for the detection of Brettanomyces bruxellensis in wine
Accepted Manuscript Title: Development of Localized Surface Plasmon Resonance biosensors for the detection of Brettanomyces bruxellensis in wine Author: Marisa Manzano Priya Vizzini Kun Jia Pierre-Michel Adam Rodica Elena Ionescu PII: DOI: Reference:
S0925-4005(15)30405-6 http://dx.doi.org/doi:10.1016/j.snb.2015.09.099 SNB 19084
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
23-4-2015 14-9-2015 18-9-2015
Please cite this article as: M. Manzano, P. Vizzini, K. Jia, P.-M. Adam, R.E. Ionescu, Development of Localized Surface Plasmon Resonance biosensors for the detection of Brettanomyces bruxellensis in wine, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.09.099 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development of Localized Surface Plasmon Resonance biosensors for the detection of
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Brettanomyces bruxellensis in wine
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Marisa Manzano*1, Priya Vizzini1, Kun Jia2,3, Pierre-Michel Adam2, Rodica Elena Ionescu2*
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Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy
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Laboratoire de Nanotechnologie et d’Instrumentation Optique, Institute Charles Delaunay,
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Universite’ de technologie de Troyes, UMR CNRS 6281, 12 Rue Marie-Curie CS 42060,
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10004 Troyes Cedex, France
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Technology of China, 610054 Chengdu, China (present address)
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School of Microelectronics and Solid-State Electronics, University of Electronic Science and
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*
corresponding authors:
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Marisa Manzano1,*, Department of Food Science, via Sondrio 2/A, 33100 Udine, Italy, tel:
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+390432558127, Fax: +390432558130 E-mail: [email protected]
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Rodica Elena Ionescu2*, Laboratoire de Nanotechnologie et d’Instrumentation Optique,
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Institute Charles Delaunay, Université de technologie de Troyes, UMR-CNRS 6281, 12 Rue
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Marie-Curie CS 42060, 10004 Troyes Cedex, France,
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tel: (33) 3 25 75 97 28; fax: (33) 3 25 71 84 56
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E-mail: [email protected]
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Abstract
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Incident light interacting with noble-metal nanoparticles with smaller sizes than the wavelength of
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the incident light induces Localised Surface Plasmon Resonance (LSPR). In this work a gold
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nanostructured surface was used for the immobilization of a 5' end Thiol modified DNA probe to
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develop a LSPR nanobiosensor for the detection of the spoiler wine yeast Brettanomyces
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bruxellensis. Gold was evaporated to obtain a gold thickness of 4 nm. DNA (2 µL) from the target
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microorganism and the negative control at various concentrations were used to test the specificity
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and sensitivity of the LSPR technique. Changes in the optical properties of the nanoparticles due to
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DNA-probe binding are reflected in the shift of LSPR extinction maximum (λ
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obtained using as target microorganism B. bruxellensis, and as negative control S. cerevisiae
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demonstrated the specificity of both the DNA-probe and the protocol. The LSPR spectrophotometry
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technique detects 0.1 ng/µL DNA target confirming the possibility to utilise this system for the
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detection of pathogen microorganisms present in low amount in food and beverage samples.
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Keywords: Localized Surface Plasmon Resonance (LSPR), Brettanomyces bruxellensis,
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genosensor, Scanning Electron Microscopy
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The results
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1. Introduction
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In the last decade the optical properties of noble metal nanoparticles that exhibit unique extinction
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spectra induced various authors to explore alternative strategies for the development of optical
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biosensors. The interaction between metals and biomolecules opened a wide field of applications
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for biosensors, from ecology, to medicine and food analysis. Moreover, optical properties of gold
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nanostructures, which show high chemical stability, allowed the utilization of Localized Surface
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Plasmon Resonance (LSPR) for the construction of sensitive biosensors [1].
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LSPR biosensors, analogously to SPR sensors, based on LSPR spectroscopy, transduce the small
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changes in the refractive index near a nanoscale noble metal surface into a measurable wavelength
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shift [2, 3]. LSPR is a collective oscillation of the conduction band electrons at the nanoparticles’
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surface that develops when incident electromagnetic radiation is of appropriate frequency. The
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properties of the particles (i.e. size, shape and dielectric function) influence the plasmonic
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oscillation that occurs at a specific resonance wavelength [4]. The possibility to change these
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parameters allows the application on various fields such as DNA detection. Biological molecules
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can be detected as their presence induces a modification of refractive index near the metal surface.
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All conditions and parameters have to be optimized to obtain repeatability and sensitivity of the
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LSPR biosensors. Metal nanoparticles size, shape, interparticle separation, and metal nanoparticle
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fabrication parameters (time, temperature, thickness of metal deposition layer, etc.) are important
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points for high specificity and sensitivity of LSPR biosensors. Thus the homogeneity of the
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nanoparticles has to be evaluated, mostly by using a Scanning Electron Microscope (SEM).
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As demonstrated by Spadavecchia et al. [5] using labelling strategies such as the utilization of gold
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nanorods (AuNRs) and gold nanostars (AuNSs) the LSPR biosensor reach a high sensitivity.
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Moreover, LSPR biosensors can be used for the high throughput monitoring both in proteomics and
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DNA research [6] requiring small volumes of analyte solutions [7, 10]. The common way to detect
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microorganisms present in samples using DNA as target is based on the utilization of one labeled
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DNA molecules, mostly by fluorescent tags.
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Today several methods are employed for the detection of biomolecules based either on methods
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such as ELISA assays, PCR assays and biosensors using specific hybridization steps [8].
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The aim of the present work is the development of LSPR nanobiosensors for the rapid and sensitive
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detection of Brettanomyces bruxellensis, a spoiler yeast worldwide well-known to produce
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unpleasant aromas in wine. As gold nanoparticles have been the system-of-choice in preparation of
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LSPR biosensors, a gold nanostructured surface was prepared and used for the immobilization of a
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thioled specific DNA probe used as receptor for the target DNA molecules extracted from wine
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yeasts.
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2. Materials and methods
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2.1 Materials and equipment
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The reagents used for the preparation of the buffer solutions listed below were acquired from
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Sigma–Aldrich (Switzerland). Three buffers: 1X PBS (10X, NaCl 1.5 M, Na2HPO4 anhydrous 81
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mM, NaH2PO4 anhydrous 19 mM); 1X SSPE (20X: NaCl 3M, NaH2PO4 anhydrous 230 mM,
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EDTA x 2H2O 25 mM and 1X TRIS-HCl (10X, Tris-HCl 0.5 M) were compared for their
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contribution over the biofunctionalization steps of the gold nanostructured surfaces. A complex
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made using the Thiol capture probe at 100 ng/µL and DNA from the target yeasts at 10 ng/µL were
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used to select the buffer for the hybridisations events.
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All LSPR measurements were obtained by using a home-built optical extinction setup [9].
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2.2
Preparation of gold nanoparticles on glass slides
and
their characterization through
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Scanning Electron Microscope (SEM) measurements
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Glass slides (Carol Roth & Co. KG, Germany) were cut at the size of about 25 × 8 mm2 with a
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diamond tip, washed in a solution of Decon 90 and deionized water (18.2 Ὼ) (2:8, v/v ratio) into an
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ultrasonic water bath at 50°C for 15 min according to [10]. 4 Page 4 of 27
Commercial TEM copper grids (200 mesh, diameter of 3.05 mm grids)(Strata TekTM Double
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Folding Grids, Ted Pella, Inc.) with double folding were fixed with scotch-tape onto the processed
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glass slides for a maximum of 3 double grids located on the same glass slide prior to gold
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evaporation, to obtain a pattern of 100 or 200 well per grid. Gold (4 nm) was evaporated by using a
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MEB 400 (PLASSYS, France) evaporator at the following conditions: 1.0 x 10-6 Torr at 40°C, and
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an evaporation rate of 0.08 nm/s. After evaporation, the TEM grids were removed and the gold
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covered glass slides were transferred in an oven (Naberthem, Germany) at 500 °C for 8 h to allow
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the formation of the gold nanopaticles (NPs) by thermal annealing before a washing step with an
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acetone-ethanol (1:1) mixture in an ultrasonic water bath at 30 °C for 30 min. To verify the gold
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surface of bare gold nanoparticles and their interspatial distance Scanning Electron Microscope
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analysis were made using a field emission scanning electron microscopy (Raith, SEM-FEG-eLine,
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France) at the following conditions: accelerating voltage of 10 kV, working distance of 8.8 mm,
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samples covered with an ultrathin layer (3 nm) of palladium by sputter-coating to suppress the
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charging effect.
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2.3 DNA probes
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The specific DNA probe for B. bruxellensis (named Thiol-Brett-probe, or capture probe) (MWG,
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GmbH Ebersberg, Germany) [11, 12] modified at 5' end by the addition of a thiol group: 5'-
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ThiC6TGTTTGAGCGTCATTTCCTTCTCACTATTTAGTGGTTATGAGATTACACGAGG-3’ to
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allow the binding with the gold nanoparticles was used.
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A
Thiol-labelled
probe
(5′-
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CCTCGTGTAATCTCATAACCACTAAATAGTGAGAAGGAAATGACGCTCAAACA-3′
named
short
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sequence) without any chemical modifications was used as a positive control to verify the
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hybridization conditions. The Thiol-Brett-probe was used at 100 ng/µL, while the sequence
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complementary to the capture probe (short sequence) was standardized at 0.01, 0.1, 1 and 10 ng/µL
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prior to be used.
DNA
sequence
complementary
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the
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2.4 Biofunctionalization of gold nanoparticles with Thiol-Brett-DNA probe
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The immobilization of Thiol-Brett DNA probe (capture probe, at 100 ng/µL) on the surface of the
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gold nanoparticles (NPs) was performed through the thiol chemistry. For LSPR experiments, three
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buffer solutions (TRIS-HCl, PBS and SSPE, respectively) were investigated for their influences on
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DNA hybridization events with the target DNAs. Practically, 3 µL of the capture probe (Thiol-Brett
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DNA probe), previously denatured at 95°C for 5 min, were allowed to bind to the gold
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nanoparticles using 1X SSPE buffer at 4°C for 1, 2, and 6 h respectively.
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2.5 Microorganisms and DNA samples analysis
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Pure yeast strains cultures of Brettanomyces bruxellensis DSM70726 (Deutsche Sammlung von
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Microorganismen und Zellkulturen GmbM, Braunschweig, Germany) as positive sample, and
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Saccharomyces cerevisiae DSM70424 as negative sample, were used for testing the specificity and
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sensitivity of the biosensor. The yeasts were cultured in Malt Extract broth (Oxoid, Milan, Italy)
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and Malt Extract Agar (Oxoid) prior to be used for the experiments. The DNA of the reference
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strains was extracted and purified from 1 mL of overnight broth culture using the Wizards Genomic
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DNA Purification Kit (Promega, Milan, Italy) [11]. The purity and concentration of the DNA
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samples were evaluated by using a Varian Cary 100 UV-Vis spectrophotometer (Agilent
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Technologies). DNA samples were standardized using sterile ddwater.
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2.6 LSPR measurements and processing data
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The LSPR measured the extinction spectrum (absorption + scattering) of the nanoparticles obtained
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by recording the intensities of transmitted radiation for the different wavelengths of a white light
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source. The wavelength shift and optical density for bio-functionalization steps were compared.
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Changes in the optical properties of the nanoparticles due to DNA-probe binding are reflected in the
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shift of LSPR extinction maximum (λ
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The deposition of different DNA concentrations was 6 Page 6 of 27
controlled onto nanostructured supports thanks to the well-defined patterns of TEM grids.
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Specifically, four patterns were chosen for each grid located on the glass slide, and LSPR
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measurements were recorded on the same patterns after each bio-functionalization step. Thus, the
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resulted LSPR spectra were measured and compared before and after NPs biofunctionalization.
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SpectraSuite-Spectrometer Operating Software was used to acquire LSPR data and register the
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maximum spectra extinction. Furthermore, all the acquired data were processed with software
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Origin Pro 8.5.
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Pratically, LSPR experiments were carried out by using 2 µL of the DNA sequence complementary
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to the capture probe (positive control) at 0.01, 0.1, 1 and 10 ng/µL to optimize the sample-capture
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probe hybridization step. Finally, 2 µL of the DNA samples from two selected yeasts (B.
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bruxellensis DSM70726 and S. cerevisiae DSM70424) at 0.01, 0.1, 1 and 10 ng/µL were used to
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test the specificity and sensitivity of the LSPR technique. The short sequence (DNA sequence
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complementary to the Thiol-labelled probe) and DNA samples extracted from B. bruxellensis
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DSM70726 and S. cerevisiae DSM70424 were independently added to the biofunctionalized glass
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slide after denaturation at 95°C for 5 min, and allowed to hybridize to the capture probe for 2 h at
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room temperature.
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3. Results and Discussions
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3.1 SEM characterization of annealed gold nanoparticles
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By using Scanning Electron Microscopy (SEM) characterization, it was found that gold
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nanoparticles are homogeneous prepared at 500°C with an average size of 25 ± 3 nm by using 4 nm
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gold evaporation thicknesses, according to the authors previous work [10]. Au NPs sizes for LSPR
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biosensing can be obtained by using 4 nm evaporation thickness and annealing temperature of 500
167
°C, according to our previous work [10]. Thus, such gold structured substrates were chosen for the
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LSPR experiments (Figure 1).
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3.2 Optimization of the buffer for LSPR measurements
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The standard extinction spectrum of bare NPs measured from four patterns /TEM grid, among 3
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independent grids, had an average of 552.76 nm with an Optical Density (OD) of 0.2094.
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Generally, after each biofunctionalization step using the DNA-probe complex of Au NPs, the three
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resulted resonant wavelengths had a red shift compared to bare NP’s. Thus, the average wavelength
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of the Thiol-Brett-DNA probe in the presence of TRIS-HCl was 557.43 nm, of PBS was 566.33 nm
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and of SSPE was 574.45 nm, respectively (Table 1). Interestingly, the nanoparticles modified with
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Thiol-Brett-DNA probe suspended in 1X SSPE buffer showed the highest peak amplitude value of
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0.3545 OD. Based on these results, the SSPE buffer was selected for the subsequent LSPR
179
experiments. The LSPR extinction spectra measured for pure gold nanoparticles (Au NPs) and their
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biofunctionalized with Thiol-Brett-DNA probe suspended within TRIS-HCl, PBS and SSPE buffers
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are reported in Figure 2.
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3.3 Biofuncionalization of gold nanoparticles with Thiol-Brett-DNA probe
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The average value of resonant wavelength for bare AuNPs before biofunctionalization was 555.75
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nm whilst after the Thiol-Brett-DNA capture probe deposition onto AuNPs for 1h, 2h, and 6h,
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respectively, the average values of resonant wavelengths had proportional red-shifts of 560.73 nm
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566.50 nm and 569.23 nm respectively (Figure 3). In other words, the shift calculated from bare
188
NPs is of 4.98 nm, 10.75 nm and 13.48 nm respectively (Table 2). The maximum optical density
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(OD) values increased from 0.2928 OD for bare AuNPs to 0.3873 OD for gold nanoparticles
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exposed to Thiol-Brett-DNA probe for 6 h. Based on above LSPR data 1 h at 4°C was considered
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sufficient to obtain a well-defined red shift.
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3.4 Capability of the LSPR-biosensor to accurately and specifically detect B. bruxellensis.
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Figure 4 and Figure 5 depict LSPR extinction spectra for bare gold nanoparticles, for NPs
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functionalized with Thiol-Brett-DNA probe and for biofunctionalized particles after the addition of 8 Page 8 of 27
various DNA-contents from B. bruxellensis DSM70726 (Figure 4) and S. cerevisiae DSM70424
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(Figure 5). Values of maximum extinction and its corresponding resonant wavelength are
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summarized in Table 3 for B. bruxellensis and in Table 4 for S. cerevisiae, respectively. Thus, two
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sets of values of averaged extinction and resonant wavelength have been measured for gold
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nanoparticles (belonging to four TEM-grid patterns/set) modified with Thiol-Brett-DNA capture
201
probe such as: 0.18 /549.8 nm (Figure 4) and 0.22/568.62 nm (Figure 5). The differences in thiol-
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DNA-functionalization of particles are probably due to the slight variations of NPs size and shape
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distribution induced by the preparation and cleanness processes. However, this is not an issue for
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the development of sensitive LSPR-biosensors because the detection method is not based on the
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absolute value of these parameters but on their variation versus the target content.
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Thus, the NPs biofunctionalization with the Thiol-Brett-DNA capture probe leads to increase of the
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extinction values and red shifts of the resonant wavelength (around +15nm) when comparing with
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LSPR spectra of bare AuNPs. Such results evidence the grafting of the DNA probe onto gold NP
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surface. For instance, the increase of the B. bruxellensis DNA-target content (up to 10 ng/mL) leads
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to an increase of the maximum extinction value and the red shift of the wavelength up to 18.4 nm.
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On the contrary, the maximum extinction value is kept almost constant whatever the used content of
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S. cerevisiae whilst, the red shift of the corresponding wavelength exhibited very low values (about
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1 nm). These results obtained using as target a DNA extracted from B. bruxellensis, and as negative
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control a DNA extracted from S. cerevisiae clearly demonstrated the specificity of both the probe
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and validated the step-by-step LSPR biosensor preparation protocol to detect 0.1 ng/ µL (means
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about 10 cells of yeast, considering that one yeast cell weight is around 10 pg).
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It should be noted that several washing steps with ddH2O were employed to avoid the false LSPR
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signals due to the presence of target DNA not-specific bound to the Thiol-Brett-DNA modified Au
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NPs annealed particles.
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It is worthwhile to notice that this steady red-shift of plasmon resonant wavelength as the increasing
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of DNA concentration was obtained using homogenous gold nanoparticles firmly embedded in
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glass substrate, thus the evolution of plasmonic wavelength versus DNA concentration is
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reproducible, meanwhile the optical responses of developed nanoparticles biosensors are
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independent of the external experimental conditions such as test temperature, which is
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advantageous over the classical biosensor platforms based on surface plasmon resonance.
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3.6 Dose –responses curves
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Figure 6 (a,b) shows the variation of the maximum extinction peak value and its corresponding
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wavelength versus B. bruxellensis and S. cerevisiae contents calculated from the data reported in
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Table 4 and Table 5.
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In the case of B. bruxellensis, the variation of the maximum peak extinction value and its
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corresponding wavelength shift continuously increases from 0 to 0.8 and from 0.95 nm to 18.4 nm
233
respectively, with the increase of DNA-target content. On the contrary, in the case of S. cerevisiae,
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the variation of the maximum peak extinction value is almost zero and its corresponding
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wavelength shift is kept lower than 1 nm. Thus, based on the two curves, a detection limit of 0.1
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ng/µL DNA was obtained using annealed active gold surfaces.
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The utilization of the LSPR technique for the detection of the DNA extracted from the wine spoiler
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yeast is free of fluorescencent markers, that elegantly reduce the number of required DNA probes
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(from two to one) commonly utilised in molecular biology.
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4. Conclusions
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An optimized protocol for LSPR-detection of B. bruxellensis onto gold nanostructured substrates by
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using a specific Thiol-Brett DNA probe is reported. Thus, the LSPR spectrophotometry technique
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confirmed that it is possible to detect 0.1 ng/µL Brett-DNA target. A clear discrimination between
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the specific hybridization with DNA from B. bruxellensis and the nonspecific binding with DNA
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from S. cerevisiae was successfully demonstrated using the genomic DNA extracted from pure 10 Page 10 of 27
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cultures and without the requirement of probe fluorescent labeling procedures. Due to the high
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sensitivity, since the LSPR nanobiosensors detect about 10 cells of yeast (considering that one yeast
249
cell weight is around 10 pg), it can also be used for the detection of traces contents of pathogen
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microorganisms in low amount in food and beverage samples.
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Acknowledgements
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The authors thank the University of Technology of Troyes for the Stratégique Program 2009–2012
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and the Region Champagne-Ardenne grants NANO’MAT for the electron microscopy
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characterization. Priya Vizzini thanks ERASMUS Programme for funding her stay at the Université
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de Technologie de Troyes between 28 of February to 31 of July, 2013. This work was performed in
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the context of the COST Action MP1302 Nanospectroscopy.
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simultaneous detection of the plasmon modes of gold nanostructure. Anal. Chem. 85 (2013) 3288-
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Table 1: Plasmonic properties (extinction and wavelength) were measured in 4 patterns of a
301
TEM/grid containing annealed gold nanoparticle modified with Thiol-Brett DNA probe (100
302
ng/ /µ µL) suspended in three independent buffers (TRIS-HCl, PBS and SSPE, respectively).
303
1
2
3
4
TRISHCl
556.27
557.05
557.05
559.37
557.43
0.25
0.22
0.24
0.24
0.24
PBS
565.56
567.11
564.79
567.88
566.33
0.29
0.29
0.29
0.28
0.29
574.07
575.61
574.84
573.30
574.45
0.34
0.36
0.35
0.36
Extinction
0.24
5.31
0.25
0.46
0.17
1.70
0.35
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SSPE
Wavelength
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Buffer
Relative Standard Deviation %
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Average wavelength extinction
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Resonant wavelength (nm), maximum extinction of the patterns:
14 Page 14 of 27
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Table 2. Plasmonic properties (extinction and wavelength) were measured in 4 patterns of a
305
TEM-grid containing annealed gold nanoparticles modified with Thiol-Brett-DNA probe (100
306
ng/µ µL) after incubation for 1, 2 and 6 h, respectively at +4°C.
307
6h
2
3
4
λ
560.15
562.47
560.15
560.73
OD
0.32
0.34
0.32
560.15 0.32
λ
567.88
567.11
566.24
564.79
566.50
OD
0.34
0.33
0.33
0.32
λ
568.66
570.2
570.2
567.88
OD
0.38
0.39
0.38
0.32
Relative Standard Deviation %
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1
Wavelength
Extinction
0.20
2.32
0.23
2.55
0.20
2.38
0.33
569.23
0.37
0.38
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Average wavelength extinction
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2h
Resonant wavelength(nm), maximum extinction (OD) of the patterns
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Time
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Table 3. Plasmonic properties (extinction and wavelength) measured in 4 patterns of a TEM-
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grid containing annealed gold nanoparticles modified with Thiol-Brett-DNA capture probe
311
(CP-100 ng/µ µL) and exposed to 4 different concentrations of DNA extracted from B.
312
bruxellensis (0.01, 0.1, 1 and 10 ng/µL, respectively). Hybridization between DNA probe and
313
Brett-DNA target was performed at room temperature for 2 h. measure
Resonant wavelength(nm), maximum extinction (OD) of the patterns: 2
3
4
λ
548.52
550.07
550.07
550.85
OD
0.18
0.18
0.18
0.18
λ
552.4
550.07
552.4
550.07
OD
0.18
0.18
0.18
DNA
λ
553.17
553.95
553.17
0,1
OD
0.19
0.19
DNA
λ
1
OD
563.24 0.25
DNA
λ
OD
10
549.87
an
551.23
0.18
0.18
553.17
553.36
M
Relative Standard Deviation %
Wavelength
Extinction
0.17
1.85
0.24
1.22
0.07
6.25
0.37
8.61
0.23
2.32
0.18
0.21
0.19
0.19
560.15
565.27
562.47
562.78
0,24
0.22
0.27
0.24
567.11
569.43
567.11
569.43
568.27
0.26
0.27
0.26
0.27
0.26
d
DNA 0,01
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CP
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1
Average wavelength extinction
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sample
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Table 4. Plasmonic properties (extinction and wavelength) measured in 4 patterns of a TEM-
315
grid containing annealed gold nanoparticles modified with Thiol-Brett-DNA capture probe
316
(CP-100 ng/µ µL) and exposed to 4 different concentrations of DNA extracted from S.
317
cerevisiae (0.01, 0.1, 1 and 10 ng/µL, respectively). Hybridization between DNA probe and
318
Saccharomyces-DNA target was performed at room temperature for 2 h.
319 Average wavelength extinction
2
3
4
λ
570.75
568.72
567.88
567.13
OD
0.25
0.25
0.25
0.25
λ
570
567.88
568.66
571.75
OD
0.25
0.25
0.24
0.25
0.25
DNA
λ
568.34
567.88
567.88
570.98
568.77
0,1
OD
0.24
0.25
0.25
0.27
0.25
DNA
λ
568.66
569.43
570.11
570.12
569.58
1
OD
0.25
0.25
0.25
0.25
DNA 10
568.62
Wavelength
Extinction
0.27
1.67
0.29
1.80
0.26
4.51
0.12
0.85
0.31
3.79
0.25
569.57
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DNA 0,01
Relative Standard Deviation %
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Resonant wavelength(nm), maximum extinction (OD) of the patterns: 1
CP
320
measure
us
sample
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λ
571.75
567.54
568.66
569.00
569.23
OD
0.26
0.24
0.25
0.25
0.25
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320
Figure captions:
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Figure 1. Scanning Electron Microscope (SEM) images) of bare gold nanoparticles after 8 h at
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500°C.
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Figure 2. LSPR spectra of bare gold nanoparticle and gold NPs modified with the Thioled-
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DNA probe (100 ng/ /µ µL) in the presence of the three buffers SSPE, PBS and Tris-HCl
326
respectively.
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Figure 3. LSPR spectra of gold nanoparticles modified with Thiol-Brett DNA probe (100
329
ng/µ µL) after 1, 2 and 6 h respectively at +4 °C.
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Figure 4. LSPR spectra of bare gold nanoparticles, modified Au NPs (with Thiol-Brett DNA
332
probe), and after addition of B. bruxellensis DNA (target microorganism) at 0.01, 0.1, 1 and
333
10 ng/µL. The hybridisation between the gold surface linked DNA Thiol probe and the yeast
334
DNAs was conducted at room temperature for 2 h.
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Figure 5. LSPR spectra of bare gold nanoparticles, modified Au NPs (with Thiol-Brett DNA
337
probe), and after addition of S. cerevisiae DNA at 0.01, 0.1, 1 and 10 ng/µL.
338
The hybridisation between the gold surface linked DNA Thiol probe and the yeast DNAs was
339
conducted at room temperature for 2 h.
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Figure 6. Dose-response curves showing the variation of the maximum extinction peak value
342
versus the B. Bruxellensis different contents (a) and the variation of the corresponding red
343
shift of the wavelength versus the B. Bruxellensis different contents (b).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Graphical Abstract
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Highlights
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1) LSPR genosensors were designed for the rapid and sensitive detection of B. bruxellesis
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2) Substrates based on annealed gold nanoparticles were modified with oligonucleotides
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3) LSPR technique shows high specificity for B. bruxellesis detection (0.1ng/mL)
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Marisa Manzano: Master degree in Natural Sciences at the University of Padua. 1984-1989 independent researcher at the University of Udine, Department of Food Science. 1990 Researcher, and from 2005 Associate Professor at University of Udine. member of various official Committees: Academic, Collegiate of Doctorate Research, Didactic Committee, State Exam Commission; and member of the "Quality System in the laboratory of Microbiology" (UNICHIM) Commettee. Teaching activity: Molecular Biology Techniques, Selection and Use of Enological Yeasts, Biotechnology of Microorganisms, Genetic of Microorganisms. Author of the Patent - detection of Listeria monocytogenes by PCR, 1996, C12Q. Speaker in various conferences: Nanotechnology & Biotechnoogy workshop, Ireland, 2014; European Biotechnology Congress, Slovakja, 2013; La diagnostica di laboratorio. Italy 2012; European Biotechnology Week, Spain, 2012; iit (Istituto Italiano Tecnologie), Italy, 2012; Optical Biosensors and Nanobiophotonics Congress, Israel, 2011; 25 Page 25 of 27
Eurobiotechnology Congress, Turkey 2011; Erasmus Workshop, Spain, 2009; NATO Conference, Italy, 2008. Author of 123 papers, 10 book chapters. Supervisor of 60 thesis; 3 PhD thesis. Topics of Interest: development of DNA probes for the detection of pathogen and spoiler microorganisms using biosensors; optimization of molecular methods (PCR, RT-PCR, DNA array, DGGE, TTGE, PFGE, RFLP, SSCP) for the detection and identification of pathogens in food and beverage and for the differentiation of microorganisms isolated from food and beverage.
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Pierre-Michel Adam has obtained his PhD in Physics in 1995 (Université de Bourgogne), and is at present Full Professor at the Université de technologie de Troyes (ICD-LNIO). Title of his PhD is “Photon Scanning Tunneling Microscope (PSTM) with a polychromatic incoherent light source. Near-field investigation of test samples and surface plasmons.” In january 1995, P.M .Adam has joined the Université de technologie de Troyes as an Assistant Professor and has been appointed Full Professor in February 2013. His fields of research are near-field microscopy and spectroscopy, surface plasmons, surface enhanced Raman scattering. He is currently in charge of a research group “Nanospectroscopy” at the ICD-LNIO. He is vice-president of a European network COST action MP1302 “Nanospectroscopy”. He is editor in chief of the International review “Nanospectroscopy”.
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Elena Rodica Ionescu earned one Ph.D. from the Ben-Gurion University of Negev, Israel in Biotechnological Engineering in 2004 and a second Ph.D. in Chemistry, from the University of Bucharest, in 2007. She has performed several post-doctoral positions in France in the field of electrochemical biosensors. In 2008, Dr. Ionescu was a researcher fellow at National Nanotechnology Laboratory, Lecce (Italy) working on impedimetric cell-chips based interdigitated microlectrodes. Since 2009, Dr. Ionescu is an Associate Professor at the Université de Technologie de Troyes, France. From April 1st, 2014, Dr. Ionescu was invited to conduct research in a POC-NRF project at the Nanyang Technological University, Singapore under an international CREATE-NTU-HUJ-BGU program. Her current research activities include acoustic, electrochemical and optical biosensors, functionalization of surfaces, AFM nanopipette applications, synthesis of nanoparticle, and toxicity of nanoparticles to living cells.
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Dr. Kun JIA received his B.S. and M.S. degree in Chemistry from University of Electronic Science and Technology of China (UESTC) in 2007 and 2010, respectively. He obtained his Ph.D. degree from Laboratory of Nanotechnology and Instrumentation Optics (LNIO) at University of Technology of Troyes (UTT) in France under the supervision of Prof. Elena Rodica IONESCU at the end of 2013. In March 2014, Dr. JIA joined the School of Microelectronics and Solid-States Electronics of UESTC as an associate professor in Chemistry. His current research interests lie in the field of plasmonic controlled polymer fluorescence, optical (bio)chemical sensors on flexible substrate, and synthesizing of functional polymer/quantum dots nanocomposites.
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Priya Vizzini was born in India in 1985. She obtained bachelor’s degree in Biomedical Laboratory Technician at the University of Udine (Italy) in 2011. She won an Erasmus grant to spend 5 months at Troyes University of technology (France) working on biological applications of QCM and Localized Surface Plasmon Resonance under the supervision of the Prof. Rodica Elena Ionescu. She got the master’s degree in Plant and Animal Biotechnology in 2014 at the University of Udine (Italy). At present she collaborates as postgraduate in the Department of Food Science working on the development of molecular biology methods for the detection of pathogens in food.
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S0925-4005(15)30405-6 http://dx.doi.org/doi:10.1016/j.snb.2015.09.099 SNB 19084
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
23-4-2015 14-9-2015 18-9-2015
Please cite this article as: M. Manzano, P. Vizzini, K. Jia, P.-M. Adam, R.E. Ionescu, Development of Localized Surface Plasmon Resonance biosensors for the detection of Brettanomyces bruxellensis in wine, Sensors and Actuators B: Chemical (2015), http://dx.doi.org/10.1016/j.snb.2015.09.099 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Development of Localized Surface Plasmon Resonance biosensors for the detection of
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Brettanomyces bruxellensis in wine
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Marisa Manzano*1, Priya Vizzini1, Kun Jia2,3, Pierre-Michel Adam2, Rodica Elena Ionescu2*
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Department of Food Science, University of Udine, via Sondrio 2/A, 33100 Udine, Italy
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2
Laboratoire de Nanotechnologie et d’Instrumentation Optique, Institute Charles Delaunay,
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Universite’ de technologie de Troyes, UMR CNRS 6281, 12 Rue Marie-Curie CS 42060,
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10004 Troyes Cedex, France
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3
12
Technology of China, 610054 Chengdu, China (present address)
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School of Microelectronics and Solid-State Electronics, University of Electronic Science and
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*
corresponding authors:
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Marisa Manzano1,*, Department of Food Science, via Sondrio 2/A, 33100 Udine, Italy, tel:
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+390432558127, Fax: +390432558130 E-mail: [email protected]
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Rodica Elena Ionescu2*, Laboratoire de Nanotechnologie et d’Instrumentation Optique,
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Institute Charles Delaunay, Université de technologie de Troyes, UMR-CNRS 6281, 12 Rue
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Marie-Curie CS 42060, 10004 Troyes Cedex, France,
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tel: (33) 3 25 75 97 28; fax: (33) 3 25 71 84 56
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E-mail: [email protected]
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Abstract
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Incident light interacting with noble-metal nanoparticles with smaller sizes than the wavelength of
25
the incident light induces Localised Surface Plasmon Resonance (LSPR). In this work a gold
26
nanostructured surface was used for the immobilization of a 5' end Thiol modified DNA probe to
27
develop a LSPR nanobiosensor for the detection of the spoiler wine yeast Brettanomyces
28
bruxellensis. Gold was evaporated to obtain a gold thickness of 4 nm. DNA (2 µL) from the target
29
microorganism and the negative control at various concentrations were used to test the specificity
30
and sensitivity of the LSPR technique. Changes in the optical properties of the nanoparticles due to
31
DNA-probe binding are reflected in the shift of LSPR extinction maximum (λ
32
obtained using as target microorganism B. bruxellensis, and as negative control S. cerevisiae
33
demonstrated the specificity of both the DNA-probe and the protocol. The LSPR spectrophotometry
34
technique detects 0.1 ng/µL DNA target confirming the possibility to utilise this system for the
35
detection of pathogen microorganisms present in low amount in food and beverage samples.
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Keywords: Localized Surface Plasmon Resonance (LSPR), Brettanomyces bruxellensis,
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genosensor, Scanning Electron Microscopy
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The results
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max).
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1. Introduction
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In the last decade the optical properties of noble metal nanoparticles that exhibit unique extinction
42
spectra induced various authors to explore alternative strategies for the development of optical
43
biosensors. The interaction between metals and biomolecules opened a wide field of applications
44
for biosensors, from ecology, to medicine and food analysis. Moreover, optical properties of gold
45
nanostructures, which show high chemical stability, allowed the utilization of Localized Surface
46
Plasmon Resonance (LSPR) for the construction of sensitive biosensors [1].
47
LSPR biosensors, analogously to SPR sensors, based on LSPR spectroscopy, transduce the small
48
changes in the refractive index near a nanoscale noble metal surface into a measurable wavelength
49
shift [2, 3]. LSPR is a collective oscillation of the conduction band electrons at the nanoparticles’
50
surface that develops when incident electromagnetic radiation is of appropriate frequency. The
51
properties of the particles (i.e. size, shape and dielectric function) influence the plasmonic
52
oscillation that occurs at a specific resonance wavelength [4]. The possibility to change these
53
parameters allows the application on various fields such as DNA detection. Biological molecules
54
can be detected as their presence induces a modification of refractive index near the metal surface.
55
All conditions and parameters have to be optimized to obtain repeatability and sensitivity of the
56
LSPR biosensors. Metal nanoparticles size, shape, interparticle separation, and metal nanoparticle
57
fabrication parameters (time, temperature, thickness of metal deposition layer, etc.) are important
58
points for high specificity and sensitivity of LSPR biosensors. Thus the homogeneity of the
59
nanoparticles has to be evaluated, mostly by using a Scanning Electron Microscope (SEM).
60
As demonstrated by Spadavecchia et al. [5] using labelling strategies such as the utilization of gold
61
nanorods (AuNRs) and gold nanostars (AuNSs) the LSPR biosensor reach a high sensitivity.
62
Moreover, LSPR biosensors can be used for the high throughput monitoring both in proteomics and
63
DNA research [6] requiring small volumes of analyte solutions [7, 10]. The common way to detect
64
microorganisms present in samples using DNA as target is based on the utilization of one labeled
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DNA molecules, mostly by fluorescent tags.
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Today several methods are employed for the detection of biomolecules based either on methods
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such as ELISA assays, PCR assays and biosensors using specific hybridization steps [8].
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The aim of the present work is the development of LSPR nanobiosensors for the rapid and sensitive
69
detection of Brettanomyces bruxellensis, a spoiler yeast worldwide well-known to produce
70
unpleasant aromas in wine. As gold nanoparticles have been the system-of-choice in preparation of
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LSPR biosensors, a gold nanostructured surface was prepared and used for the immobilization of a
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thioled specific DNA probe used as receptor for the target DNA molecules extracted from wine
73
yeasts.
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2. Materials and methods
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2.1 Materials and equipment
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The reagents used for the preparation of the buffer solutions listed below were acquired from
79
Sigma–Aldrich (Switzerland). Three buffers: 1X PBS (10X, NaCl 1.5 M, Na2HPO4 anhydrous 81
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mM, NaH2PO4 anhydrous 19 mM); 1X SSPE (20X: NaCl 3M, NaH2PO4 anhydrous 230 mM,
81
EDTA x 2H2O 25 mM and 1X TRIS-HCl (10X, Tris-HCl 0.5 M) were compared for their
82
contribution over the biofunctionalization steps of the gold nanostructured surfaces. A complex
83
made using the Thiol capture probe at 100 ng/µL and DNA from the target yeasts at 10 ng/µL were
84
used to select the buffer for the hybridisations events.
85
All LSPR measurements were obtained by using a home-built optical extinction setup [9].
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2.2
Preparation of gold nanoparticles on glass slides
and
their characterization through
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Scanning Electron Microscope (SEM) measurements
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Glass slides (Carol Roth & Co. KG, Germany) were cut at the size of about 25 × 8 mm2 with a
90
diamond tip, washed in a solution of Decon 90 and deionized water (18.2 Ὼ) (2:8, v/v ratio) into an
91
ultrasonic water bath at 50°C for 15 min according to [10]. 4 Page 4 of 27
Commercial TEM copper grids (200 mesh, diameter of 3.05 mm grids)(Strata TekTM Double
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Folding Grids, Ted Pella, Inc.) with double folding were fixed with scotch-tape onto the processed
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glass slides for a maximum of 3 double grids located on the same glass slide prior to gold
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evaporation, to obtain a pattern of 100 or 200 well per grid. Gold (4 nm) was evaporated by using a
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MEB 400 (PLASSYS, France) evaporator at the following conditions: 1.0 x 10-6 Torr at 40°C, and
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an evaporation rate of 0.08 nm/s. After evaporation, the TEM grids were removed and the gold
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covered glass slides were transferred in an oven (Naberthem, Germany) at 500 °C for 8 h to allow
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the formation of the gold nanopaticles (NPs) by thermal annealing before a washing step with an
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acetone-ethanol (1:1) mixture in an ultrasonic water bath at 30 °C for 30 min. To verify the gold
101
surface of bare gold nanoparticles and their interspatial distance Scanning Electron Microscope
102
analysis were made using a field emission scanning electron microscopy (Raith, SEM-FEG-eLine,
103
France) at the following conditions: accelerating voltage of 10 kV, working distance of 8.8 mm,
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samples covered with an ultrathin layer (3 nm) of palladium by sputter-coating to suppress the
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charging effect.
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2.3 DNA probes
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The specific DNA probe for B. bruxellensis (named Thiol-Brett-probe, or capture probe) (MWG,
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GmbH Ebersberg, Germany) [11, 12] modified at 5' end by the addition of a thiol group: 5'-
110
ThiC6TGTTTGAGCGTCATTTCCTTCTCACTATTTAGTGGTTATGAGATTACACGAGG-3’ to
111
allow the binding with the gold nanoparticles was used.
112
A
Thiol-labelled
probe
(5′-
113
CCTCGTGTAATCTCATAACCACTAAATAGTGAGAAGGAAATGACGCTCAAACA-3′
named
short
114
sequence) without any chemical modifications was used as a positive control to verify the
115
hybridization conditions. The Thiol-Brett-probe was used at 100 ng/µL, while the sequence
116
complementary to the capture probe (short sequence) was standardized at 0.01, 0.1, 1 and 10 ng/µL
117
prior to be used.
DNA
sequence
complementary
to
the
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2.4 Biofunctionalization of gold nanoparticles with Thiol-Brett-DNA probe
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The immobilization of Thiol-Brett DNA probe (capture probe, at 100 ng/µL) on the surface of the
121
gold nanoparticles (NPs) was performed through the thiol chemistry. For LSPR experiments, three
122
buffer solutions (TRIS-HCl, PBS and SSPE, respectively) were investigated for their influences on
123
DNA hybridization events with the target DNAs. Practically, 3 µL of the capture probe (Thiol-Brett
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DNA probe), previously denatured at 95°C for 5 min, were allowed to bind to the gold
125
nanoparticles using 1X SSPE buffer at 4°C for 1, 2, and 6 h respectively.
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2.5 Microorganisms and DNA samples analysis
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Pure yeast strains cultures of Brettanomyces bruxellensis DSM70726 (Deutsche Sammlung von
129
Microorganismen und Zellkulturen GmbM, Braunschweig, Germany) as positive sample, and
130
Saccharomyces cerevisiae DSM70424 as negative sample, were used for testing the specificity and
131
sensitivity of the biosensor. The yeasts were cultured in Malt Extract broth (Oxoid, Milan, Italy)
132
and Malt Extract Agar (Oxoid) prior to be used for the experiments. The DNA of the reference
133
strains was extracted and purified from 1 mL of overnight broth culture using the Wizards Genomic
134
DNA Purification Kit (Promega, Milan, Italy) [11]. The purity and concentration of the DNA
135
samples were evaluated by using a Varian Cary 100 UV-Vis spectrophotometer (Agilent
136
Technologies). DNA samples were standardized using sterile ddwater.
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2.6 LSPR measurements and processing data
139
The LSPR measured the extinction spectrum (absorption + scattering) of the nanoparticles obtained
140
by recording the intensities of transmitted radiation for the different wavelengths of a white light
141
source. The wavelength shift and optical density for bio-functionalization steps were compared.
142
Changes in the optical properties of the nanoparticles due to DNA-probe binding are reflected in the
143
shift of LSPR extinction maximum (λ
max).
The deposition of different DNA concentrations was 6 Page 6 of 27
controlled onto nanostructured supports thanks to the well-defined patterns of TEM grids.
145
Specifically, four patterns were chosen for each grid located on the glass slide, and LSPR
146
measurements were recorded on the same patterns after each bio-functionalization step. Thus, the
147
resulted LSPR spectra were measured and compared before and after NPs biofunctionalization.
148
SpectraSuite-Spectrometer Operating Software was used to acquire LSPR data and register the
149
maximum spectra extinction. Furthermore, all the acquired data were processed with software
150
Origin Pro 8.5.
151
Pratically, LSPR experiments were carried out by using 2 µL of the DNA sequence complementary
152
to the capture probe (positive control) at 0.01, 0.1, 1 and 10 ng/µL to optimize the sample-capture
153
probe hybridization step. Finally, 2 µL of the DNA samples from two selected yeasts (B.
154
bruxellensis DSM70726 and S. cerevisiae DSM70424) at 0.01, 0.1, 1 and 10 ng/µL were used to
155
test the specificity and sensitivity of the LSPR technique. The short sequence (DNA sequence
156
complementary to the Thiol-labelled probe) and DNA samples extracted from B. bruxellensis
157
DSM70726 and S. cerevisiae DSM70424 were independently added to the biofunctionalized glass
158
slide after denaturation at 95°C for 5 min, and allowed to hybridize to the capture probe for 2 h at
159
room temperature.
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3. Results and Discussions
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3.1 SEM characterization of annealed gold nanoparticles
163
By using Scanning Electron Microscopy (SEM) characterization, it was found that gold
164
nanoparticles are homogeneous prepared at 500°C with an average size of 25 ± 3 nm by using 4 nm
165
gold evaporation thicknesses, according to the authors previous work [10]. Au NPs sizes for LSPR
166
biosensing can be obtained by using 4 nm evaporation thickness and annealing temperature of 500
167
°C, according to our previous work [10]. Thus, such gold structured substrates were chosen for the
168
LSPR experiments (Figure 1).
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3.2 Optimization of the buffer for LSPR measurements
171
The standard extinction spectrum of bare NPs measured from four patterns /TEM grid, among 3
172
independent grids, had an average of 552.76 nm with an Optical Density (OD) of 0.2094.
173
Generally, after each biofunctionalization step using the DNA-probe complex of Au NPs, the three
174
resulted resonant wavelengths had a red shift compared to bare NP’s. Thus, the average wavelength
175
of the Thiol-Brett-DNA probe in the presence of TRIS-HCl was 557.43 nm, of PBS was 566.33 nm
176
and of SSPE was 574.45 nm, respectively (Table 1). Interestingly, the nanoparticles modified with
177
Thiol-Brett-DNA probe suspended in 1X SSPE buffer showed the highest peak amplitude value of
178
0.3545 OD. Based on these results, the SSPE buffer was selected for the subsequent LSPR
179
experiments. The LSPR extinction spectra measured for pure gold nanoparticles (Au NPs) and their
180
biofunctionalized with Thiol-Brett-DNA probe suspended within TRIS-HCl, PBS and SSPE buffers
181
are reported in Figure 2.
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3.3 Biofuncionalization of gold nanoparticles with Thiol-Brett-DNA probe
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The average value of resonant wavelength for bare AuNPs before biofunctionalization was 555.75
185
nm whilst after the Thiol-Brett-DNA capture probe deposition onto AuNPs for 1h, 2h, and 6h,
186
respectively, the average values of resonant wavelengths had proportional red-shifts of 560.73 nm
187
566.50 nm and 569.23 nm respectively (Figure 3). In other words, the shift calculated from bare
188
NPs is of 4.98 nm, 10.75 nm and 13.48 nm respectively (Table 2). The maximum optical density
189
(OD) values increased from 0.2928 OD for bare AuNPs to 0.3873 OD for gold nanoparticles
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exposed to Thiol-Brett-DNA probe for 6 h. Based on above LSPR data 1 h at 4°C was considered
191
sufficient to obtain a well-defined red shift.
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3.4 Capability of the LSPR-biosensor to accurately and specifically detect B. bruxellensis.
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Figure 4 and Figure 5 depict LSPR extinction spectra for bare gold nanoparticles, for NPs
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functionalized with Thiol-Brett-DNA probe and for biofunctionalized particles after the addition of 8 Page 8 of 27
various DNA-contents from B. bruxellensis DSM70726 (Figure 4) and S. cerevisiae DSM70424
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(Figure 5). Values of maximum extinction and its corresponding resonant wavelength are
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summarized in Table 3 for B. bruxellensis and in Table 4 for S. cerevisiae, respectively. Thus, two
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sets of values of averaged extinction and resonant wavelength have been measured for gold
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nanoparticles (belonging to four TEM-grid patterns/set) modified with Thiol-Brett-DNA capture
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probe such as: 0.18 /549.8 nm (Figure 4) and 0.22/568.62 nm (Figure 5). The differences in thiol-
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DNA-functionalization of particles are probably due to the slight variations of NPs size and shape
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distribution induced by the preparation and cleanness processes. However, this is not an issue for
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the development of sensitive LSPR-biosensors because the detection method is not based on the
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absolute value of these parameters but on their variation versus the target content.
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Thus, the NPs biofunctionalization with the Thiol-Brett-DNA capture probe leads to increase of the
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extinction values and red shifts of the resonant wavelength (around +15nm) when comparing with
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LSPR spectra of bare AuNPs. Such results evidence the grafting of the DNA probe onto gold NP
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surface. For instance, the increase of the B. bruxellensis DNA-target content (up to 10 ng/mL) leads
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to an increase of the maximum extinction value and the red shift of the wavelength up to 18.4 nm.
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On the contrary, the maximum extinction value is kept almost constant whatever the used content of
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S. cerevisiae whilst, the red shift of the corresponding wavelength exhibited very low values (about
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1 nm). These results obtained using as target a DNA extracted from B. bruxellensis, and as negative
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control a DNA extracted from S. cerevisiae clearly demonstrated the specificity of both the probe
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and validated the step-by-step LSPR biosensor preparation protocol to detect 0.1 ng/ µL (means
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about 10 cells of yeast, considering that one yeast cell weight is around 10 pg).
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It should be noted that several washing steps with ddH2O were employed to avoid the false LSPR
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signals due to the presence of target DNA not-specific bound to the Thiol-Brett-DNA modified Au
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NPs annealed particles.
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It is worthwhile to notice that this steady red-shift of plasmon resonant wavelength as the increasing
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of DNA concentration was obtained using homogenous gold nanoparticles firmly embedded in
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glass substrate, thus the evolution of plasmonic wavelength versus DNA concentration is
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reproducible, meanwhile the optical responses of developed nanoparticles biosensors are
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independent of the external experimental conditions such as test temperature, which is
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advantageous over the classical biosensor platforms based on surface plasmon resonance.
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3.6 Dose –responses curves
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Figure 6 (a,b) shows the variation of the maximum extinction peak value and its corresponding
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wavelength versus B. bruxellensis and S. cerevisiae contents calculated from the data reported in
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Table 4 and Table 5.
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In the case of B. bruxellensis, the variation of the maximum peak extinction value and its
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corresponding wavelength shift continuously increases from 0 to 0.8 and from 0.95 nm to 18.4 nm
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respectively, with the increase of DNA-target content. On the contrary, in the case of S. cerevisiae,
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the variation of the maximum peak extinction value is almost zero and its corresponding
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wavelength shift is kept lower than 1 nm. Thus, based on the two curves, a detection limit of 0.1
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ng/µL DNA was obtained using annealed active gold surfaces.
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The utilization of the LSPR technique for the detection of the DNA extracted from the wine spoiler
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yeast is free of fluorescencent markers, that elegantly reduce the number of required DNA probes
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(from two to one) commonly utilised in molecular biology.
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4. Conclusions
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An optimized protocol for LSPR-detection of B. bruxellensis onto gold nanostructured substrates by
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using a specific Thiol-Brett DNA probe is reported. Thus, the LSPR spectrophotometry technique
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confirmed that it is possible to detect 0.1 ng/µL Brett-DNA target. A clear discrimination between
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the specific hybridization with DNA from B. bruxellensis and the nonspecific binding with DNA
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from S. cerevisiae was successfully demonstrated using the genomic DNA extracted from pure 10 Page 10 of 27
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cultures and without the requirement of probe fluorescent labeling procedures. Due to the high
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sensitivity, since the LSPR nanobiosensors detect about 10 cells of yeast (considering that one yeast
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cell weight is around 10 pg), it can also be used for the detection of traces contents of pathogen
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microorganisms in low amount in food and beverage samples.
251
Acknowledgements
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The authors thank the University of Technology of Troyes for the Stratégique Program 2009–2012
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and the Region Champagne-Ardenne grants NANO’MAT for the electron microscopy
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characterization. Priya Vizzini thanks ERASMUS Programme for funding her stay at the Université
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de Technologie de Troyes between 28 of February to 31 of July, 2013. This work was performed in
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the context of the COST Action MP1302 Nanospectroscopy.
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REFERENCES
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Nano Today 4 (2009) 244-251.
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[2] A.J. Haes, R.P. Van Duyne, A higly sensitive and selective surface-enhanced nanobiosensor.
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Mat. Res. Soc. Symp. Proc. 723 (2002) O3.1.
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[3] L.S. Yung, C.T. Campbell, T.M. Chinowsky, M.N. Mar, S.S. Yee, Quantitative interpretation of
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the response of surface plasmon resonance sensors to adsorbed films. Langmuir 14 (1998) 5636-
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5648.
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[4] C. Tabor, R. Murali, M. Mahmoud, M.A. El-Sayed, On the use of plasmonic nanoparticle pairs
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as a plasmon ruler: the dependence of the near-field dipole plasmon coupling on nanoparticle size
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and shape. J. Phys. Chem. A 113 (2009) 1946-1953.
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[5] J. Spadavecchia, A. Barras, J. Lyskawa, P. Woisel, W. Laure, C.-M. Pradier, R. Boukherroub, S.
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Szunerits, Approach forplasmonic based DNA sensing: amplification of the wavelenght shift and
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simultaneous detection of the plasmon modes of gold nanostructure. Anal. Chem. 85 (2013) 3288-
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3296.
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[6] W.K. Jung and K.M. Byun, Fabrication of nanoscale Plasmonic Structures and their
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applications, Biomed Eng Lett 1(2011) 153-162.
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[7] I. Abdulhaim, M. Zourob, A. Lakhtakia, Overview of optical biosensing techniques, in: R.S.
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Marks, D.C. Cullen, I. Karube, C.R. Lowe, H.H. Weetall (Eds), Handbook of Biosesors and
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Biochips, J. Wiley & Sons Ltd, West Sussex, England, 2007, pp. 413-446.
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[8] Y. Hong, Y.-M. Huh, D.S. Yoon, J. Yang, Nanobiosensors based on localized Surface Plasmon
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Resonance for biomarker detection. J. Nanomat. (2012) 1-13.
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[9] K. Jia , J.L. Bijeon, P.M. Adam, R.E. Ionescu, Sensitive Localized Surface Plasmon Resonance
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multiplexing Protocols, Anal. Chem. 84 (2012) 8020-8027.
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[10] K. Jia, J.L. Bijeon, P.M. Adam, R.E. Ionescu, A facile and cost-effective TEM grid approach
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to design gold nano-structured substrates for high throughput plasmonic sensitive detection of
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[11] F. Cecchini, M. Manzano, Y. Mandaby, E. Perelman and R.S. Marks, Chemiluminescent DNA
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[12] F. Cecchini, L. Iacumin, M. Fontanot, P. Comuzzo, G. Comi, M. Manzano, Dot Blot and PCR
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for Brettanomyces bruxellensis detection in red wine. Food Control, 34 (2013) 40-46.
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Table 1: Plasmonic properties (extinction and wavelength) were measured in 4 patterns of a
301
TEM/grid containing annealed gold nanoparticle modified with Thiol-Brett DNA probe (100
302
ng/ /µ µL) suspended in three independent buffers (TRIS-HCl, PBS and SSPE, respectively).
303
1
2
3
4
TRISHCl
556.27
557.05
557.05
559.37
557.43
0.25
0.22
0.24
0.24
0.24
PBS
565.56
567.11
564.79
567.88
566.33
0.29
0.29
0.29
0.28
0.29
574.07
575.61
574.84
573.30
574.45
0.34
0.36
0.35
0.36
Extinction
0.24
5.31
0.25
0.46
0.17
1.70
0.35
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an
SSPE
Wavelength
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Buffer
Relative Standard Deviation %
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Average wavelength extinction
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Resonant wavelength (nm), maximum extinction of the patterns:
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Table 2. Plasmonic properties (extinction and wavelength) were measured in 4 patterns of a
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TEM-grid containing annealed gold nanoparticles modified with Thiol-Brett-DNA probe (100
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ng/µ µL) after incubation for 1, 2 and 6 h, respectively at +4°C.
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6h
2
3
4
λ
560.15
562.47
560.15
560.73
OD
0.32
0.34
0.32
560.15 0.32
λ
567.88
567.11
566.24
564.79
566.50
OD
0.34
0.33
0.33
0.32
λ
568.66
570.2
570.2
567.88
OD
0.38
0.39
0.38
0.32
Relative Standard Deviation %
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1
Wavelength
Extinction
0.20
2.32
0.23
2.55
0.20
2.38
0.33
569.23
0.37
0.38
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Average wavelength extinction
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2h
Resonant wavelength(nm), maximum extinction (OD) of the patterns
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Time
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Table 3. Plasmonic properties (extinction and wavelength) measured in 4 patterns of a TEM-
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grid containing annealed gold nanoparticles modified with Thiol-Brett-DNA capture probe
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(CP-100 ng/µ µL) and exposed to 4 different concentrations of DNA extracted from B.
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bruxellensis (0.01, 0.1, 1 and 10 ng/µL, respectively). Hybridization between DNA probe and
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Brett-DNA target was performed at room temperature for 2 h. measure
Resonant wavelength(nm), maximum extinction (OD) of the patterns: 2
3
4
λ
548.52
550.07
550.07
550.85
OD
0.18
0.18
0.18
0.18
λ
552.4
550.07
552.4
550.07
OD
0.18
0.18
0.18
DNA
λ
553.17
553.95
553.17
0,1
OD
0.19
0.19
DNA
λ
1
OD
563.24 0.25
DNA
λ
OD
10
549.87
an
551.23
0.18
0.18
553.17
553.36
M
Relative Standard Deviation %
Wavelength
Extinction
0.17
1.85
0.24
1.22
0.07
6.25
0.37
8.61
0.23
2.32
0.18
0.21
0.19
0.19
560.15
565.27
562.47
562.78
0,24
0.22
0.27
0.24
567.11
569.43
567.11
569.43
568.27
0.26
0.27
0.26
0.27
0.26
d
DNA 0,01
Ac ce pt e
CP
us
1
Average wavelength extinction
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sample
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Table 4. Plasmonic properties (extinction and wavelength) measured in 4 patterns of a TEM-
315
grid containing annealed gold nanoparticles modified with Thiol-Brett-DNA capture probe
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(CP-100 ng/µ µL) and exposed to 4 different concentrations of DNA extracted from S.
317
cerevisiae (0.01, 0.1, 1 and 10 ng/µL, respectively). Hybridization between DNA probe and
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Saccharomyces-DNA target was performed at room temperature for 2 h.
319 Average wavelength extinction
2
3
4
λ
570.75
568.72
567.88
567.13
OD
0.25
0.25
0.25
0.25
λ
570
567.88
568.66
571.75
OD
0.25
0.25
0.24
0.25
0.25
DNA
λ
568.34
567.88
567.88
570.98
568.77
0,1
OD
0.24
0.25
0.25
0.27
0.25
DNA
λ
568.66
569.43
570.11
570.12
569.58
1
OD
0.25
0.25
0.25
0.25
DNA 10
568.62
Wavelength
Extinction
0.27
1.67
0.29
1.80
0.26
4.51
0.12
0.85
0.31
3.79
0.25
569.57
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DNA 0,01
Relative Standard Deviation %
cr
Resonant wavelength(nm), maximum extinction (OD) of the patterns: 1
CP
320
measure
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λ
571.75
567.54
568.66
569.00
569.23
OD
0.26
0.24
0.25
0.25
0.25
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320
Figure captions:
321
Figure 1. Scanning Electron Microscope (SEM) images) of bare gold nanoparticles after 8 h at
322
500°C.
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Figure 2. LSPR spectra of bare gold nanoparticle and gold NPs modified with the Thioled-
325
DNA probe (100 ng/ /µ µL) in the presence of the three buffers SSPE, PBS and Tris-HCl
326
respectively.
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Figure 3. LSPR spectra of gold nanoparticles modified with Thiol-Brett DNA probe (100
329
ng/µ µL) after 1, 2 and 6 h respectively at +4 °C.
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Figure 4. LSPR spectra of bare gold nanoparticles, modified Au NPs (with Thiol-Brett DNA
332
probe), and after addition of B. bruxellensis DNA (target microorganism) at 0.01, 0.1, 1 and
333
10 ng/µL. The hybridisation between the gold surface linked DNA Thiol probe and the yeast
334
DNAs was conducted at room temperature for 2 h.
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Figure 5. LSPR spectra of bare gold nanoparticles, modified Au NPs (with Thiol-Brett DNA
337
probe), and after addition of S. cerevisiae DNA at 0.01, 0.1, 1 and 10 ng/µL.
338
The hybridisation between the gold surface linked DNA Thiol probe and the yeast DNAs was
339
conducted at room temperature for 2 h.
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Figure 6. Dose-response curves showing the variation of the maximum extinction peak value
342
versus the B. Bruxellensis different contents (a) and the variation of the corresponding red
343
shift of the wavelength versus the B. Bruxellensis different contents (b).
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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6a
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Graphical Abstract
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Highlights
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1) LSPR genosensors were designed for the rapid and sensitive detection of B. bruxellesis
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2) Substrates based on annealed gold nanoparticles were modified with oligonucleotides
372
3) LSPR technique shows high specificity for B. bruxellesis detection (0.1ng/mL)
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Marisa Manzano: Master degree in Natural Sciences at the University of Padua. 1984-1989 independent researcher at the University of Udine, Department of Food Science. 1990 Researcher, and from 2005 Associate Professor at University of Udine. member of various official Committees: Academic, Collegiate of Doctorate Research, Didactic Committee, State Exam Commission; and member of the "Quality System in the laboratory of Microbiology" (UNICHIM) Commettee. Teaching activity: Molecular Biology Techniques, Selection and Use of Enological Yeasts, Biotechnology of Microorganisms, Genetic of Microorganisms. Author of the Patent - detection of Listeria monocytogenes by PCR, 1996, C12Q. Speaker in various conferences: Nanotechnology & Biotechnoogy workshop, Ireland, 2014; European Biotechnology Congress, Slovakja, 2013; La diagnostica di laboratorio. Italy 2012; European Biotechnology Week, Spain, 2012; iit (Istituto Italiano Tecnologie), Italy, 2012; Optical Biosensors and Nanobiophotonics Congress, Israel, 2011; 25 Page 25 of 27
Eurobiotechnology Congress, Turkey 2011; Erasmus Workshop, Spain, 2009; NATO Conference, Italy, 2008. Author of 123 papers, 10 book chapters. Supervisor of 60 thesis; 3 PhD thesis. Topics of Interest: development of DNA probes for the detection of pathogen and spoiler microorganisms using biosensors; optimization of molecular methods (PCR, RT-PCR, DNA array, DGGE, TTGE, PFGE, RFLP, SSCP) for the detection and identification of pathogens in food and beverage and for the differentiation of microorganisms isolated from food and beverage.
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Pierre-Michel Adam has obtained his PhD in Physics in 1995 (Université de Bourgogne), and is at present Full Professor at the Université de technologie de Troyes (ICD-LNIO). Title of his PhD is “Photon Scanning Tunneling Microscope (PSTM) with a polychromatic incoherent light source. Near-field investigation of test samples and surface plasmons.” In january 1995, P.M .Adam has joined the Université de technologie de Troyes as an Assistant Professor and has been appointed Full Professor in February 2013. His fields of research are near-field microscopy and spectroscopy, surface plasmons, surface enhanced Raman scattering. He is currently in charge of a research group “Nanospectroscopy” at the ICD-LNIO. He is vice-president of a European network COST action MP1302 “Nanospectroscopy”. He is editor in chief of the International review “Nanospectroscopy”.
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Elena Rodica Ionescu earned one Ph.D. from the Ben-Gurion University of Negev, Israel in Biotechnological Engineering in 2004 and a second Ph.D. in Chemistry, from the University of Bucharest, in 2007. She has performed several post-doctoral positions in France in the field of electrochemical biosensors. In 2008, Dr. Ionescu was a researcher fellow at National Nanotechnology Laboratory, Lecce (Italy) working on impedimetric cell-chips based interdigitated microlectrodes. Since 2009, Dr. Ionescu is an Associate Professor at the Université de Technologie de Troyes, France. From April 1st, 2014, Dr. Ionescu was invited to conduct research in a POC-NRF project at the Nanyang Technological University, Singapore under an international CREATE-NTU-HUJ-BGU program. Her current research activities include acoustic, electrochemical and optical biosensors, functionalization of surfaces, AFM nanopipette applications, synthesis of nanoparticle, and toxicity of nanoparticles to living cells.
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Dr. Kun JIA received his B.S. and M.S. degree in Chemistry from University of Electronic Science and Technology of China (UESTC) in 2007 and 2010, respectively. He obtained his Ph.D. degree from Laboratory of Nanotechnology and Instrumentation Optics (LNIO) at University of Technology of Troyes (UTT) in France under the supervision of Prof. Elena Rodica IONESCU at the end of 2013. In March 2014, Dr. JIA joined the School of Microelectronics and Solid-States Electronics of UESTC as an associate professor in Chemistry. His current research interests lie in the field of plasmonic controlled polymer fluorescence, optical (bio)chemical sensors on flexible substrate, and synthesizing of functional polymer/quantum dots nanocomposites.
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Priya Vizzini was born in India in 1985. She obtained bachelor’s degree in Biomedical Laboratory Technician at the University of Udine (Italy) in 2011. She won an Erasmus grant to spend 5 months at Troyes University of technology (France) working on biological applications of QCM and Localized Surface Plasmon Resonance under the supervision of the Prof. Rodica Elena Ionescu. She got the master’s degree in Plant and Animal Biotechnology in 2014 at the University of Udine (Italy). At present she collaborates as postgraduate in the Department of Food Science working on the development of molecular biology methods for the detection of pathogens in food.
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