Core–Shell Nanorod Columnar Array Combined with Gold Nanoplate

Aug 30, 2016 - (27) 1 mL of AuNR solution was mixed into 5 mL of 0.1 M CTAB solution. ... Next, the gold seeds were settled for 2 h at room temperatur...
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Core-shell nanorod columnar array combined with gold nanoplatenanosphere assemblies enable powerful in-situ SERS detection of bacteria Li Qiu, WeiQiang Wang, AiWen Zhang, NanNan Zhang, Tibebe Lemma, Honghua Ge, J. Jussi Toppari, Vesa P. Hytönen, and Jin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b06674 • Publication Date (Web): 30 Aug 2016 Downloaded from http://pubs.acs.org on August 31, 2016

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Core-shell nanorod columnar array combined with gold nanoplate-nanosphere assemblies enable powerful in-situ SERS detection of bacteria

Li Qiu†a, WeiQiang Wang†b, AiWen Zhang†a, NanNan Zhangb, Tibebe Lemmac, HongHua Ge*b, J. Jussi Topparic, Vesa P. Hytönen*d, Jin Wang*a a.

Institute of Intelligent Machines, HeFei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui 230031,P. R. China b. Institute of Health Sciences and School of Life Science, AnHui University, Hefei, Anhui 230601, P.R. China c University of Jyväskylä, Department of Physics, Nanoscience Center, P.O. Box 35, FI-40014, University of Jyväskylä, Finland d BioMediTech, University of Tampere, FI-33520 Tampere, Finland and Fimlab Laboratories, FI-33520, Tampere, Finland

ABSTRACT Development of a label-free ultrasensitive nanosensor for detection of bacteria is presented. Sensitive assay for Gram-positive bacteria was achieved via electrostatic attraction-guided plasmonic bifacial superstructure/bacteria/columnar array assembled in one step. Dynamic optical hot-spots were formed in the hybridized nanoassembly under wet-dry critical state amplifying efficiently the weak vibrational modes of three representative food-borne Gram-positive bacteria, i.e., Staphylococcus xylosus, Listeria monocytogenes and Enterococcus faecium. These three bacteria with highly-analogous Raman spectra can be effectively differentiated through droplet wet-dry critical SERS approach combined with 3D PCA statistical analysis so that highly sensitive discrimination of bacterial species and samples containing mixtures of bacteria can be achieved.

KEYWORDS: superstructure, nanoarray, SERS, Gram-positive bacteria, 3D PCA

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1. INTRODUCTION As a powerful optical nanotechnology, surface enhanced Raman scattering (SERS) is being widely used for ultrasensitive detection of environmental contaminants, e.g., pesticides,1,2 heavy metals,3,4 toxins etc.5-7 Over the past years, SERS has also been proven as a highly efficient approach for sensing of bacterial pathogens.8-11 SERS-based bacterial assays have been developed utilizing two different strategies, i.e., discriminating bacterial species by using their intrinsic SERS spectra (direct detection),12-14 or employing Raman tags as bacterial indicators for recognition of bacteria (indirect detection).15,16 Unlike the single-step direct assay, the indirect assay of bacteria comprises of as several steps, i.e., bacterial antibody functionalization on the SERS-tagged plasmonic nanoparticle, recognition of bacteria via antibody-antigen interaction, and detection of bacteria by aid of the SERS probes. The advantage of the latter is the ultrasensitivity of the SERS tags compared to the inherently weak Raman signal of bacteria due to a much larger Raman scattering cross section of SERS probes. However, the disadvantage of the indirect assay is mainly the above mentioned more complicated assay. Especially the antibody functionalization of the plasmonic nanoparticles might bring extra complications. Also the availability of the suitable antibodies can be a limitation. Moreover, compromised antibody nanomaterial interface and the stability of plasmonic nanoparticles in bio-chemical reactions may further decrease the detection efficiency of bacteria. In contrast, increasing the efficiency of a SERS substrate to enhance the direct SERS signal and thus the discrimination sensitivity of bacteria, can promote the direct detection of bacteria to become feasible. Recently, assembling of plasmonic nanoparticles to form a long-ranged ordered superstructure with uniform optical hotspots has been widely utilized as a highly-sensitive SERS substrate.17,18

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Tian et al. employed ordered mesoporous silica structures as templates to prepare a highly ordered gold or silver superstructures with a controllable nanoparticle size and well-defined particle gaps; Moreover, the superstructure exhibited a high Raman enhancement of 109 as a SERS substrate, which originated from the uniform distribution of the hotspots.19 Very recently, Shi et al. have fabricated anisotropic ordered two-dimensional nanoparticle liquid crystalline superstructures (NLCS) from bipyramidal gold nanoparticles.20 Particularly, plasmonic superstructures, core-satellite type superstructures have proven to be excellent in terms of optical properties, e.g., superior SERS enhancement ascribed to an efficient generation of hot spots between the plasmonic core and its satellites,21-23 highly enhanced fluorescence imaging for controllable biological delivery24 and high plasmonic chirality.25 In the present work, we demonstrate a novel one-step assembling and SERS sensing strategy combining core-satellite superstructures and a columnar array. The hybridized super-substrate is fabricated under a wet-dry critical state via surface of intact bacteria, and it enables sensitive SERS recognition of bacteria based on the following advantages. Firstly, a columnar array of SH-polyethylene glycol-NH2 (SH-PEG-NH2) functionalized Au@Ag nanorods provides a large-scaled uniform highly-sensitive SERS substrate without destruction of bacteria. Secondly, SH-PEG-NH2-aided plasmonic superstructure bifacial assembly of triangular gold nanoplates gold nanospheres (TAuNPs-AuNSs) provides plenty of optical hot-spots for further SERS enhancement. Thirdly, negative-charged bacteria can be efficiently trapped within the TAuNPs-AuNSs superstructures on top of the columnar array of Au@Ag nanorods. Lastly, the wet-dry critical SERS state based on the hybridized nanoassembly can combine electromagnetic (EM) SERS effects and photothermal temperature gradient (PTG) SERS effects. Therefore, it

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provides highly dynamic optical hot-spots to maximize the SERS enhancement for sensitive direct detection of bacteria.

2. EXPERIMENTAL DETAILS 2.1.

Materials.

HAuCl4·4H2O

cetyltrimethylammonium

bromide

(99.9%), (CTAB)

NaBH4 (99%),

(99%),

Vitamin

AgNO3

C

(99.9%),

(99.9%), p-ATP

(para-aminothiophenol), sodium dodecyl sulfate (SDS) and trisodium citrate were purchased from Sigma-Aldrich. SH-PEG-NH2 (MW3400) was purchased from LaySan Bio company. Gram-positive Staphylococcus xylosus (S. xylosus, ZSL-3), Enterococcus faecium (E. faecium, yellow sea1006) and Listeria monocytogenes (L. monocytogenes, CVCC1598) were purchased from China General Microbiological Culture Collection Center (CGMCC). The deionized water that was used throughout the experiments was purified by using a Milli-Q system.

2.2. Synthesis of plasmonic nanoparticles Au@Ag core-shell nanorods: gold nanorods (AuNRs) were prepared via a reported seed-mediated approach.26 Firstly, gold seed solution was synthesized as follows: 600 µL 0.02 M ice-cold NaBH4 was added to solution prepared by mixing 10 mL 0.5 mM HAuCl4 and 10 mL 0.2 M CTAB. The gold seed solution was stirred efficiently for 2 min and allowed to settle at 25 ºC for 2h. Secondly, growth solution of AuNRs was prepared by mixing 100 mL of 0.2 M CTAB, 10 mL of 4 mM AgNO3, 100 mL of 1mM HAuCl4 solution and 1.4 mL of 0.08 M vitamin C at room temperature. Finally, the AuNRs were prepared by addition of 240 µL gold seed to the growth solution and letting the solution to settle at 27 ºC for overnight. The AuNRs were then centrifuged

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with 10000 rpm and redispersed in 20 mL of water. Au@Ag core-shell nanorods were synthesized according to our previous report.27 1 mL of AuNR solution was mixed into 5 mL of 0.1 M CTAB solution. Then, 0.75 mL 4 mM AgNO3 solution was added to the AuNRs colloidal solution, followed by addition of 0.1 mL 0.1 M vitamin C and 0.2 mL 0.1 M NaOH solution under gentle mixing. Finally, the formation of silver coating was observed from a colour change from yellow-reddish to green. Triangular gold nanoplates (TAuNPs): TAuNPs were synthesized via a three-step seed-mediated synthesis.28 0.5 mL of 10 mM sodium citrate and 0.5 mL of 10 mM HAuCl4 were diluted to 20 mL of water. 0.5 mL of 100 mM NaBH4 was quickly added to the mixture and stirred for 2 min to generate gold seeds. Then, the gold seeds were settled for 2h at room temperature. For the gold growth solution, the mixture containing 0.625 mL of 0.01M HAuCl4, 22.5 mL of 0.05 M CTAB, 0.125 mL of 0.1M NaOH, 0.125 mL of 0.1M vitamin C, and 0.1 mL of 0.01 M Potassium iodide, was stored in vial A. Subsequently, 2.25 mL of gold growth solution was transferred from the vial A to an empty vial B, and 250 µL of solution was again transferred from the vial B to an empty vial C. After that, 22.5 µL of gold seed solution was added to the vial A, and the solution in vial A was added to the vial B. Finally, the solution in vial B was transferred to vial C. The resulting gold growth solution was settled for overnight at 30°C. The as-prepared TAuNPs were centrifuged at 6000 rpm and redispersed in 20 mL of water. Nanospheres (AuNSs): The citrate-capped gold nanospheres with a size of 13 nm were prepared as follows: 50 mL of 2.5×10-4 M HAuCl4 was heated to boiling. Subsequently, 5 mL 1% sodium citrate solution was added to the boiling HAuCl4 solution. The color of the solution changed from purple to ruby red, indicating that the gold nanospheres with diameter of 13 nm were formed. The

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as-prepared AuNPs were centrifuged at 13000 rpm and redispersed in 20 mL of water.

2.3. Fabrication of plasmonic TAuNPs-AuNSs bifacial superstructures and the columnar array of Au@Ag nanorods. 2 mL of 0.1 mg/mL SH-PEG-NH2 added to 1mL TAuNP colloids can give core-satellite TAuNP-NSs superstructured nanoparticles with high-density NSs. 50 µL of 0.1 mg/mL SH-PEG-NH2 added to 1mL TAuNP colloids can give the edge-assembled TAuNP-NSs superstructured nanoparticles. 500 µL of 0.1 mg/mL SH-PEG-NH2 added to 1mL TAuNP colloids can give core-satellite TAuNP-NSs superstructured nanoparticles with low density NSs. Hence, 1 mL of TAuNPs colloidal solution was mixed with 2 mL of 0.1 mg/mL SH-PEG-NH2 under stirring for 2 h. Then, 0.5 mL 10% sodium dodecyl sulfate (SDS) solution was added to the SH-PEG-NH2 functionalized TAuNPs solution. Subsequently, 1 mL Au nanospheres (5 nm or 13 nm in size) were added to the mixed solution to yield TAuNP-AuNSs bifacial superstructures. A silicon chip was cleaned by ultrasonication in acetone, ethanol and MilliQ water for 1 h in each. Subsequently, the silicon chip was immersed in piranha solution (H2SO4:H2O2 = 3:1) at 80˚C for 1 h and rinsed by MilliQ water. The clean silicon chip was let to dry at ambient condition. 1 mL of as-prepared bimetallic core-shell nanorods were centrifuged at 8000 rpm for 10 min to remove the excess CTAB surfactants and the pelleted nanorods were redispersed in 30 µL MilliQ water. Then, 5 µL of nanorods colloidal solution was dropped on the silicon substrate. The samples were kept in Petri dish with cover at 25˚C and the humidity ca. 80% for 24 h. The columnar array of nanorods is formed on a chip during this incubation.

2.4. Bacterial culturing. Lyophilized powders containing Staphylococcus xylosus (S. xylosus,

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ZSL-3), or Enterococcus faecium (E. faecium, yellow sea1006) were resuspended to 5 mL LB nutrient and cultivated for 14 h in a shaking incubator (200 rpm), respectively. Listeria monocytogenes (L. monocytogenes, CVCC1598) was dissolved into BHR nutrient and cultivated for 14 h in the shaking incubator (200 rpm). Bacterial suspension was plated on an LB agar plate using a sterile plastic inoculating loop to carry out plate streaking and the plate was settled in a constant temperature incubator and cultivated for 14 h to obtain single colonies. The single colonies were collected using the sterile plastic inoculating loop and dissolved into 1 mL of aquae sterilisata. 100 µL of bacterial suspension was coated on LB agar base and incubated in constant temperature incubator at 37°C. Subsequently, the bacteria were dispersed in Milli-Q water and centrifuged gently for 10 min at 3000 rpm to protect the cell membrane. Finally, the supernatant liquid was discarded and the bacterial cells were redispersed in Milli-Q water. The purified bacteria were diluted to a concentration of 5×102 cfu/mL. The density of the bacteria cells was determined by counting the number of colonies grown on the Petri dish after 12 h of cultivation.

2.5. One-step assembling and wet-dry critical state SERS measurement of bacteria, and data analysis. 10 µL of aqueous bacterial suspension was drop-casted on a columnar array of nanorods. Subsequently, 5 µL of as-prepared bifacial superstructured TAuNP-AuNSs colloidal solution was drop-casted on the sample. The SERS spectra collection could be started when shrinking of the liquid droplet was recorded. Surface enhanced Raman spectra were collected through 100 × objective lens with excitation laser power of 8.5 mW laser power and 10 s integration time. The Raman spectra were continually collected (one spectrum per minute) during the change from the wet state to a dry state.

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All data analysis was performed by using the Unscrambler® X software (version 10.3, CAMO Software; Oslo, Norway) including Unscrambler Service Pack 2014. Linear baseline correction with baseline offset, and standard normal variate (SNV) were used prior to chemometric analysis. Subsequently, mean centered principal component analysis (PCA) was performed in the 400 cm-1 to 1800 cm-1 range.

2.6. Characterization. Scanning electron microscope (SEM) images were acquired with a Quanta200FEG. UV-Vis-NIR absorption spectra were recorded by using a Solidspec-3700 spectrophotometer. The transmission electron microscopy (TEM) images were obtained with a JEOL JEM2010 instrument operated at 100kV. SERS experiment were performed via Renishaw Invia Reflex Raman spectrometer equipped with a 280 mW semiconductor laser emitting at a 785 nm line.

3. Results and Discussion 3.1. Analysis of TAuNP-AuNSs superstructure and a columnar array of nanorods. Recently, Au nanoplates have been utilized as highly active SERS substrates for chemical or biosensing.29,30 The previous investigation demonstrated that the maximum SERS intensity increase with the edge of the triangle, reaching a maximum enhancement factor of 1013.31 Hence, we choose the triangular gold nanoplates with the size of ca. 120 nm in order to achieve the optimized SERS ability and maximum assembling AuNSs. For the TAuNPs-AuNSs superstructures, we prepared citrate-stabilized gold nanospheres (AuNSs) as described above, and triangular gold nanoplates (TAuNPs) with high quality using a

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finely-tuned seed-mediated approach. Figure 1. shows that TAuNPs with the clear and sharp triangular tips can be obtained via the present synthesis protocol. Furthermore, as seen from the UV-Vis-NIR spectrum of TAuNPs (Figure. S1B in the Supporting Information), the absence of the localized surface plasmon resonance (LSPR) of AuNSs at 520 nm indicates that high purity of the TAuNPs sample without excess AuNSs can be obtained, and thus effectively utilized for assembling of the superstructures. For the production of the TAuNP-AuNSs superstructures, we selected SH-PEG-NH2 polymer molecules on the basis of the following reasons: (I) strong bonding of Au-S can retain large amounts of amine group; (II) strong electrostatic attractive interaction between the SH-PEG-NH2 functionalized TAuNP and citrate-stabilized AuNSs; (III) flexible polymer chain can yield narrow-gapped junction between TAuNP and AuNSs. Hence, this facile electrostatic assembly strategy enables anchoring of AuNSs to the TAuNP to yield TAuNP-AuNSs plasmonic superstructures (see Figure 1).

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Figure 1. TEM images of assembled TAuNP-AuNSs. A: bifacial TAuNP-AuNSs superstructure with the high density distribution of AuNSs (13 nm size); B: bifacial TAuNP-AuNSs superstructure with the low density distribution of AuNSs (13 nm size); C: edge-coated TAuNP-AuNSs superstructures of AuNSs (13 nm size); D: bifacial TAuNP-AuNSs superstructures with the high density distribution of AuNSs (5 nm size)

Compared to triangular plane {111} of the TAuNP, the edge facet is relatively high-enengy crystal facet.32 The different facet activity may lead to faster ligand exchangement to start from higher-energy side crystal facet,33 which can allow guided assembly of TAuNPs-AuNSs, i.e., transformation from edge-coated TAuNP-AuNSs superstructure to bifacial TAuNP-AuNSs superstructure.

A closer observation of the transmission electron microscope (TEM) images (shown in Figure 1) shows that the AuNSs are randomly distributed on the surface of a TAuNP. Moreover, the immobilization takes place on the both sides of the nanoplates, indicated by the dark and light dots. It should be pointed out that the plasmonic bifacial superstructure includes multiple optical hot-spot areas, which are ascribed to TAuNP-AuNSs contacts and aggregates of AuNSs-AuNSs. Additionally, yielding efficiency of TAuNP-AuNSs superstructure is quite high (see Figure S1A in the Supporting Information). The UV-Vis-NIR spectrum of the TAuNP-AuNSs superstructures (Figure S1B in the Supporting Information) shows that a very strong extinction peak at the 1153 nm, corresponding to redshift of the dipolar LSPR band of the plain TAuNP at the 1045 nm. This indicates plasmonic coupling between the TAuNP and AuNSs. The plasmonic properties of TAuNP-AuNSs superstructures can be tuned by size and quantity of the AuNSs satellites. Herein, two sizes of AuNSs, i.e., 5 nm and 13 nm, were selected to explore the SERS enhancement ability. As observed in TEM analysis (Figure 1D), numerous small AuNSs 10

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with size of 5 nm can be assembled on the edge/triangular face of TAuNP to form a quasi-continuous spiky shell. In contrast, the AuNSs with size of 13 nm can be adhered on the surface of TAuNP as a discrete state, which is shown in Figure 1A-C. In addition, the number of AuNSs assembled on the TAuNP can be adjusted by the amount of SH-PEG-NH2. Accompanied with the increasing number of SH-PEG-NH2, the assembled pattern can be transformed from an edge-assembly to a core-satellite bifacial pattern (see Figure 1). Different plasmonic superstructures were used for the SERS enhancement measurements. As shown in Figure 2, fingerprint peaks of p-ATP located at 1079 cm-1, 1191 cm-1, 1475 cm-1, 1577 cm-1 of p-ATP could be clearly observed if p-ATP is covalently interacted with the TAuNP-AuNSs superstructures by aid of Au-S bonding. The transformation from p-ATP to DMAB can be supported by the oxidative formation of azo species from aniline group presence in Au/TiO2.34 Additionally, p-aminobenzonic acid can react to form azo molecules on the SERS-active Ag surface.35 As reported by previous investigation, local heating effect from plasmonic nanointerface in the SERS measurement, can effectively induce the surface reaction or change the surface process.35

Hence,

chemical

transformation

from

para-aminothiophenol

(p-ATP)

to

4,4’-dimercaptoazobenzene (DMAB), could happen and be proven according to SERS observations and surface mass spectroscopic measurement.36 The DMAB can be produced by the oxidation of two adsorbed p-ATP. As confirmed by experimental work and theoretical simulations,37 ag modes of DMAB at 1140 cm-1, 1388 cm-1, and 1438 cm-1, are obviously different from 1075 cm-1, 1594 cm-1, which can be ascribed the fact that DMAB has ca. 100-folds larger Raman scattering cross-section in contrast with p-ATP.37 Herein, the presence of other very strong fingerprint bands, i.e., 1144 cm-1, 1393 cm-1 and 1437

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cm-1, which can be assigned as C-N and N=N stretching vibrations, respectively, are suggesting that large amounts of p-ATP are efficiently transformed to 4,4’-dimercaptoazobenzene (DMAB). In contrast, if p-ATP is absorbed on plain TAuNP, intensities of the vibrational bands from p-ATP are comparable to those of DMAB, implying that the efficiency of p-ATP changing to DMAB is very low. In the case of AuNSs, if p-ATP is applied on AuNSs, intensities of the vibrational bands from p-ATP are slightly stronger than those of DMAB, which indicate that p-ATP can react to form DMAB on the surface of AuNSs. It is reasonable to believe that coupling of isotropic nanoparticles upon aggregation tends to be more easily performed, leading to enormous SERS enhancement of p-ATP as Raman reporter. However, previous investigations suggested that nanoplates assembled into more complicated configurations in aggregated form as compared to silver nanospheres, e.g., face-to-face, edge-to-edge or edge-to face, which also lead to large Raman enhancement.38 Herein, we can observe enhancement of AuNSs is only slightly stronger than TAuNP and obviously weaker than TAuNP-NSs superstrucutre if p-ATP molecules are interacted with them. In addition, intensity of the vibrational bands of p-ATP is the lowest on the TAuNPs without AuNSs decoration, suggesting that the plasmonic coupling from optical hotspots between TAuNP and assembled AuNSs can significantly improve the SERS enhancing ability.

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Figure 2. A: Raman spectra of p-ATP on (A) bifacial TAuNP-AuNSs superstructures with the high density distribution of AuNSs (13 nm size); (B) bifacial TAuNP-AuNSs superstructures with the low density distribution of AuNSs (13 nm size); (C) TAuNP-AuNSs superstructure with edge-coated AuNSs (13 nm size); (D) bifacial TAuNP-AuNSs superstructure with the high density distribution of AuNSs (5 nm size); (E) AuNSs with 13 nm; (F) TAuNP

As far as the three superstructures are concerned, the SERS enhancing ability of TAuNP-AuNSs with 5 nm AuNSs is dramatically weaker than that of TAuNP-AuNSs with 13 nm AuNSs, possibly due to the lower scattering cross section of 5 nm AuNSs. Additionally, the SERS enhancing ability is dependent on the number of the assembled AuNSs due to the numbers of optical hotspots. We also controlled the amount of SH-PEG-NH2 on the surface of TAuNP to obtain edge-assembled superstructure and core-satellite bifacial superstructure. As compared to edge-assembled TAuNP-AuNSs superstructure, the bifacial superstructure exhibited higher SERS enhancement and efficiently transforming efficiency from p-ATP to DMAB, which is also visible in Figure 2. Therefore, TAuNP-AuNSs bifacial superstructure with 13 nm size AuNSs could be an excellent candidate for the in-situ assembling and detection of bacteria using direct SERS detection mode. The effect of nanorod length, i.e., aspect raio, on the SERS performance has been detailed investigated by a previous report.39 As for silver nanorods, aspect ratio 3.5 nanorods with 5x107 enhancement factor is higher than aspect ratio 10 nanorods with 1.3x107 enhancement factor. In the case of gold nanorods, aspect ratio 1.7 nanorods with 7.1x106 enhancement factor and aspect ratio 1.7 nanorods with 9x106 enhancement factor are dramatically higher than aspect ratio 16 nanorods with 1.2x104 enhancement factor. Hence, the gold nanorods with aspect ratio between 1.5~5 can be appropriately selected as good SERS substrates. Columnar array of the Au@Ag nanorods, in which the long axes of the NRs are oriented vertically, 13

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can be fabricated via a controllable slow evaporation. The formation of a columnar nanorod array should be highly dependent on the concentration of the anisotropic nanoparticles. In the present case, increasing the concentration of the nanorods can lead to an alteration of array, i.e., from a horizontally assembled nanorod array to a vertically orientated one. We propose that the nanorods constantly precipitated from the droplet can be supported by the neighboring nanorods under the high coverage limit around the “coffee ring”. As a result, under a high surface coverage of the nanorods on a chip, they can stand up with their long axis vertical to the surface, which can be detected from a small dot-like distribution on the SEM images (shown in Figure 3). However, it should be mentioned that the distribution of the dots can be dependent on the composition of the nanorods. Herein, we prepared two different Au@Ag NRs with a thin and a thick Ag shell. As far as Au@Ag NRs with the thin Ag shell are concerned, as shown in Figure 3A, the columnar array standing on the chip is highly ordered assembly with a quasi-vertical pattern. In the case of the Au@Ag NRs with the thick Ag shell, the columnar array standing on chip can be changed from a quasi-vertical pattern to a completely vertical way, which is shown in Figure. 3B. As reported in a previous investigation, the silver shell in the transverse direction was thicker than that in the longitudinal direction. Because CTAB capping preferentially bound to their {110} or {100} end faces compared to {111} side faces, silver formation could give dumb-bell or boat-like silver shell,40, 41 leading to density difference between the two ends and sides of the nanorods. As far as the Au nanorods are concerned, they can be assembled in a lean standing orientation. If the silver shell is formed on the gold nanorods, it can be expected that the transformation from a lean standing orientation to complete perpendicular standing pattern could be obtained. Hence, by aid of the

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gravity, a complete columnar array appearing as dot-like distribution could be yielded.

Figure 3 A: SEM image of Au@Ag nanorods with a thin Ag shell forming a quasi-vertical nanoarray B: SEM image of a columnar nanoarray of dots-like distributed Au@Ag nanorods with a thick Ag shell columnar nanoarray. Inset picture means expansion of SEM image.

As revealed by the very recent study on a 2D bipyramid plasmonic nanoparticle liquid crystalline superstructures with four distinct packing orientation orders, the SERS enhancement factor of the vertical alignment is 77-fold greater than that of the horizontal alignment and 19 fold greater than that obtained with circular arrangement.20 Therefore, it can be expected that a similar fully vertical columnar array of core-shell nanorods will yield the highest SERS enhancement for the sensitive detection of bacteria.

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3.2. SERS analysis of Gram positive bacteria via plasmonic bifacial TAuNP-AuNSs superstructures on nanorod columnar array. SH-PEG-NH2 functionalized nanorods yield positively charged nanointeface due to the very strong Au-S bonding, leading to high amounts of accessible amine groups. It is well known that the surface of Gram positive bacteria has high negative charge due to peptidoglycan of the cell wall, resulting in electrostatic attractive interactions between bacteria and a SH-PEG-NH2 functionalized nanorod columnar array. As mentioned above, columnar array of plasmonic nanorods can be fabricated by a controllable evaporation to form a coffee-ring on a chip so as to efficiently accumulate bacteria along the coffee-ring line in specific area by aid of SH-PEG-NH2 functionalization.

Figure 4. Schematic illustration of the one-step assembling and detection of bacteria via plasmonic bifacial TAuNP/AuNSs superstructures on a Au@Ag nanorod columnar array

As schematically shown in Figure 4, the Au@Ag NRs array – TAuNP@AuNSs superstructure hybridized plasmonic nanoassemblies can be obtained by aid of the negative charged intact bacteria. The columnar array of SH-PEG-NH2 functionalized Au@Ag NRs array can provide a positively charged nanointerface for immobilization of the negatively charged bacteria, while the retaining large amounts of positive charged amine groups of TAuNP-AuNSs superstructures can again lead to strong electrostatic attractive interaction between the TAuNP-AuNSs and the surface of bacteria. 16

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TEM analysis revealed an uneven distribution of AuNSs on the TAuNP (Figure 5), which results in a triangular plane of the TAuNP with positive charge binding with bacteria in a parallel pattern. Additionally, the distribution of AuNSs tends to be concentrated on the edges and corners of TAuNP, which causes the cross-overlapping of TAuNPs due to electrostatic attractive interaction. Generally, the SERS enhancement mechanism can be divided into electromagnetic and chemical enhancement; however, temperature gradient inherently created by the electron-photon coupling and heat dissipation, can also remarkably improve SERS performance by aid of introducing fluidic motion and thermal diffusion.42 Recently,dynamic surface enhanced Raman scattering (D-SERS) technology as a novel detection approach, has been proven for highly-sensitively amplifying the signal of analytes in contrast with traditional SERS approach.43,44 Also, D-SERS approach has been demonstrated two significant benefits compared to tranditional SERS method, i.e., removal of instrumental and normal Raman interference in SERS spectroscopy of adsorbate population on SERS-active particle.45 Very recently, a comprehensive investigation on formation of three-dimensional and time-ordered SERS hotspot matrix during the evaporation of a droplet of citrate-Ag sol on a silicon wafer has been reported.46 Detailed experimental results of dark-field optical microscopy, in-situ UV-Vis spectrum and in-situ SR-SAXS revealed that 3D geometry of the Ag particles with minimal polydispersity of particle size and maximal uniformality of interparticle distance can be formed in wet-dry critical state, which is different from the dry state. Moreover, their investigation results proves that 3D geometry can certainly produce a large number of hotspots in 3D space for improving SERS enhancement. At the final stage of the evaporation, when the droplet is dried, the hotspots disappear due to formation of very big clusters, leading to decreasing of Raman

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enhancement.47 As shown in the schematic graph in Figure 4, we performed the in-situ assembling and SERS detection of bacteria on wet-dry critical state based on the bio-nanointerface due to generation of efficient optical hotspots and photothermal temperature gradient effect for additional SERS enhancement.

Figure 5. A: SEM image of L. monocytogenes bacteria adhered to a SH-PEG-NH2 functionalized nanorod columnar array; B: SEM image of L. monocytogenes bacteria adhered to SH-PEG-NH2 functionalized bifacial TAuNP-AuNSs superstructures on a nanorod columnar array; C:SEM image of S. xylosus bacteria adhered to a SH-PEG-NH2 functionalized nanorod columnar array; D: SEM image of S. xylosus bacteria adhered to SH-PEG-NH2 functionalized bifacial TAuNP-AuNSs superstructures on a nanorod columnar array; E: SEM image of E. faecium bacteria adhered to a SH-PEG-NH2 functionalized nanorod columnar array; F: SEM image of E. faecium bacteria adhered to SH-PEG-NH2 functionalized bifacial TAuNP-AuNSs superstructure on nanorod columnar array

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Herein, a unique nanoassembly of plasmonic columnar array and the bifacial discrete superstructures was developed for maximizing SERS performance via wet-dry critical SERS approach. Heat can be generated from the multiple electrical hotspots in the nanoassembly and propagated along the surface of nanorods columnar array due to high thermal conductivity of gold (318 W m-1 K-1). Under the state of liquid droplet, bacteria drift towards the coffee-ring region, i.e., columnar nanorods array, by aid of the photothermal effect and electrostatic attractive interaction, and enriched in the large quantities on hotspots of plasmonic columnar array. Moreover, bacterial cells can promote the bifacial plasmonic TAuNP-AuNSs superstructures via electrostatic attractive interaction to yield new additional hotspots for further amplifying SERS effects. On the other hand, the brownian motion principle can also give reasonable explanation for the SERS enhancement from the wet-dry critical state. At the initial evaporation phase, collision amongst nanoparticles is quite weak due to a wide dispersion of bacteria in the liquid droplet, leading to a weak Raman signal. Moreover, accompanied with the shrinking of the liquid droplet transforming from liquid to solid state, the Raman signal from the bacteria is remarkably enhanced. At the stage of the shrinking of the droplet and temperature increment from the laser irradiation, collisions amongst bifacial superstructured nanoparticles, which are accumulated around the bacteria via electrostatic attractive interaction, causes dramatic increase in energy transfer, leading to a rapid generation of dynamic optical hotspots visible in the SERS measurements. After gradual drying at the evaporation phase, dynamic assembling of bacteria and superstructures on the nanorods columnar array will be terminated. Simultaneously, renovation and increasing of optical hotspots will be stopped, accompanied with solidified surface on the bio-nanointerface, resulting in a decrease of Raman signal. In this state, the Raman signal from the bacteria should be

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decreased down to a stable value, which is as shown from Figure S2 in the Supporting Information. Therefore, with one-step assembling and Raman signal collection, it could be possible to discriminate fine differences amongst highly-analogous SERS spectra of bacteria. Herein, we selected three typical food-borne Gram positive bacteria, i.e., Enterococcus faecium (E. faecium), Listeria monocytogenes (L. monocytogenes) and Staphylococcus xylosus (S. xylosus) to be tested in this assay. The bacteria were chosen due to their very silimar SERS characteristics. The strongest vibrational band located at 730 cm-1 is common for the bacteria studied here. It can be assigned to a glycosidic ring stretching vibration of adenine of the nucleic acids (shown in Figure 6 and Table S1 in the Supporting Information). Another strong vibrational band at 1326 cm-1, which could be attributable to NH2 stretching vibration of cytosine and uracil of the nucleic acids, is also common for the three bacteria. So, it is really difficult to discriminate the three bacterial species only based on the information provided by the two vibrational bands.

Figure 6. SERS spectra of S. xylosus (A); L. monocytogenes (B); and E. faecium (C) obtained by bifacial TAuNP-AuNSs superstructure on nanorod columnar array

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Herein, multifold enhancements from the plasmonic superstructures and the nanoarray were utilized, providing possibility for amplifying weak vibrations of all the three bacteria. For example, the E. faecium has no vibrational band at 753 cm-1, while both of the other two bacteria have very small vibrational band at 753 cm-1, corresponding to ring stretching of thymine of the nucleic acids. Also, in contrast with E. faecium and L. monocytogenes, S. xylosus has relatively obvious vibrational band between 1600 cm-1 and 1700 cm-1, ascribed to C=O stretch of amide I of a protein. As far as SERS spectra of the three Gram positive bacteria are concerned, the SERS spectrum of E. faecium is relatively simple, and SERS spectrum of S. xylosus is more complicated. The profiles of the weak bands at 800~1100 cm-1, corresponding to the C-O vibrations of the lipid layer of the cell wall and membrane of the three bacteria studied here, are different, implying that by using the bifacial plasmonic superstructures on a nanoarray, it is possible to efficiently amplify Raman signals and discriminate bacteria even with highly analogous SERS spectra. Moreover, SERS spectra of mixtures of these bacteria were also studied on the hybridized plasmonic superstructures / nanoarray substrate. As observed from Figure S3 in the Supporting Information, the SERS spectrum of mixture of S. xylosus and E. faecium bacteria is obviously simpler than that of the S. xylosus bacteria alone, implying that it is possible to discriminate the mixtures from the monospecies samples. The SERS spectrum of mixture of S. xylosus and L. monocytogenes bacteria (refer to Figure S4 in the Supporting Information), is more complicated than that of L. monocytogenes due to observations of the weak vibrational bands, which indicates that differentiating the mixture from a pure L. monocytogenes sample could be possible to obtain via the plasmonic superstructures / nanoarray. Similarly, as seen from Figure S5 in the Supporting Information, the difference of the SERS spectra between mixed species and monospecies, i.e., E.

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faecium and L. monocytogenes, is reflected from different profiles of the bands at 900~1100 cm-1 and 1400~1500 cm-1. We plotted the SERS peak intensity at ca. I730 against the three bacterial concentration (Fig. S6-8), respectively, so as to obtain limit of Detection (LOD) via the present assay protocol. As for each concentration, mean and standard deviation of the peak intensities can be obtained on the basis of the collected 6 spectra from two different substrates. The LOD of the SERS detection can be determined by the lowest concentration at which a robust bacterial SERS spectrum can be obtained. As shown from Fig. S6-8, the lowest concentration of Staphylococcus xylosus, Listeria monocytogenes and Enterococcus faecium bacteria that gave detectable signal (LOD) is 50 CFU/mL, 100 CFU/mL and 100 CFU/mL, respectively. The resuability of the assays can be performed by repeatable wet-dry experiments of the identical assembled TAuNPs-AuNSs/bacteria/nanorods array. We found that after two round wet-dry state transformation, the SERS intensity of S. xylosus bacteria under critical wet-dry state drops ca. 50 percentage (as shown from Fig. S9). However, the SERS intensity of bacteria at dry state are quite stable. It should be pointed out that the SERS spectra of S.xylosus bacteria are almost identical after several round wet-dry experiments (shown in Fig. S9), indicating that the present assay can be repeatedly used for bacteria recognition with sustainable performance.

3.3. Discriminating monospecies and mixed-species of the three Gram-positive bacteria via 3D-PCA chemometric analysis based on the SERS spectra. As far as Gram-positive bacteria with similar cell structure is concerned, it is difficult to completely discriminate the bacteria and their mixtures by aid of visual classification of their SERS spectra. Principal component analysis

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converts a set of observations into a set of values of variables called principal components. The first principal component (variable) explains the highest amount of variance in the data (in this case, in the measured spectra). In three-dimensional (3D) PCA analysis, selected PCA variables are plotted in 3D scatter plot. Representation of the principal analysis scores using 3D plot enables more sensitive discrimination in between various sample groups as compared to 2D plot. In addition, use of 3D plot instead of a matrix of 2D plots saves space in the visualization of the PCA analysis results. Herein, chemometrics from PCA statistical analysis combined with the nanoplasmonic SERS technology can be expected to confine the same species to a specific area to differentiate these similar Gram positive bacteria. All the PCA analysis can be carried out based on the clustered SERS spectra of the bacterial monospecies and mixed species (shown from Figure S10-S16 in the Supporting Information). The 3D score plot (shown from Figure 7A) of the top three principle components (PCs), i.e., PC1-PC2-PC3, shows that the clusters of S. xylosus can be easily separated from those of L. monocytogenes and E. faecium, suggesting that S. xylosus has unique chemical features as compared to L. monocytogenes and E. faecium bacteria. Additionally, although the clusters of L. monocytogenes are close to those of E. faecium in both PC1-PC2-PC3 3D score plots and PC1-PC2-PC4 3D score plots (see Figure 7A-B), which can be ascribed to a very high similarity between the two species, they can still be distinguished, indicating that the present detection protocol based on plasmonic superstructures / nanoarray is quite powerful for identification of bacteria.

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Fig. 7 Monospecies 3D PC1-PC2-PC3 PCA score plot of E. faecium, L. monocytogenes and S. xylosus bacteria (A); monospecies 3D PC1-PC2-PC4 PCA score plot of E. faecium, L. monocytogenes and S. xylosus bacteria (B)

Besides discriminating monospecies of the three Gram positive bacteria, differentiation among the mixtures of the three positive bacteria was also performed. Comparison between Figure 8A and 7A, reveals that the top three PCs of the mixtures represent 79% of the total variance (the former) compared to 69% (the latter), indicating that more variance and exists in the mixed species in contrast to monospecies. Compared to the results from PC1-PC2-PC3 score plots, the three mixed species in PC1-PC2-PC4 score plots clusters the bacteria more closely together. However, separation amongst these mixed species can still be obtained. Both Figure 8A and 8B suggest that mixed bacterial species can be identified via SERS collected under wet-dry critical state based on plasmonic superstructures / nanoarray combined with 3D PCA analysis.

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Fig. 8 Mixture species 3D PC1-PC2-PC3 PCA score plot of E. faecium, L. monocytogenes and S. xylosus bacteria with the ratio 1:1 (A); Mixture species 3D PC1-PC2-PC4 PCA score plot of E. faecium, L. monocytogenes and S. xylosus bacteria with the ratio 1:1 (B)

In addition, on the basis of the excellent performance in differentiating monospecies and mixed species, determining ratio of different bacteria in mixed species has been explored here. Two bacterial species, i.e., S. xylosus and L. monocytogenes, were chosen for this test. As observed from 3D PC1-PC2-PC3 score plot in Figure 9A, mixed species of S. xylosus and L. monocytogenes with the ratio of 1:1 are clustered quite close to the pure S. xylosus leading to a slight overlapping. However, discriminating the mixed species with the ratio of 1:1 from the pure S. xylosus sample is possible. Moreover, clusters of the mixed species with the ratio of 1:1 are away from the pure L. monocytogenes sample. Figure 9A indicates that clusters of mixed species with the ratio of 1:4 can be discriminated from the monospecies and mixed species with the ratio of 1:1, suggesting the ratio of the two bacteria could be measured with this method. As far as the PC1-PC2-PC4 score plots are concerned, all of the monospecies and mixed species with the different ratio were found to be well separated.

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Fig. 9 3D PC1-PC2-PC3 PCA score plot of of L. monocytogenes and S. xylosus bacteria with the ratio 1:1 and 1:4 plotted with monospecies of L. monocytogenes and S. xylosus (A); Mixture species 3D PC1-PC2-PC4 PCA score plot of L. monocytogenes and S. xylosus bacteria with the ratio 1:1 and 1:4 and monospecies of L. monocytogenes and S. xylosus (B)

To sum up, the monospecies and mixed species of bacteria can be identified with SERS when the highly-sensitive SERS substrate of bifacial superstructures / nanorods columnar array under wet-dry critical state combined with 3D PCA analysis is used. Moreover, the ratio of different bacteria can also be effectively determined by aid of the chemometric analysis on the basis of the clustered SERS spectra.

4. CONCLUSION Plasmonic bifacial TAuNP-AuNSs superstructures and a columnar array of nanorods can be dynamically assembled by aid of negative-charged bacteria by utilizing electrostatic attraction. One-step assembling and detection of bacteria can be achieved by temporal collection of SERS spectra during a wet-to-dry transformation. Electromagnetic enhancement effects combined with photothermal temperature gradient effects allow an ultrasensitive SERS assay of bacteria on the

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basis of numerous optical hotspots existing in the dynamic nanoassembly formed under wet-dry critical state. We demonstrated that the three representative food-borne Gram positive bacteria as well as their mixtures and even ratios in them, can be well discriminated by aid of this novel detection protocol combined with a 3D PCA chemometric analysis.

ASSOCIATED CONTENT

Supporting Information The SEM images, UV-Vis-NIR spectrum, SERS spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION †

These authors contributed equally to this work

Corresponding Author Corresponding author. Emails: [email protected]; co-corresponding authors. Emails: [email protected]; [email protected]

ACKNOWLEDGEMENTS This work is supported by MOST China-Finland International Cooperation Project (Grant No

2014DFG42290) and Tekes – the Finnish Funding Agency for Innovation (project #1185/31/2013 and 1191/31/2013), the National Natural Science Foundation of China (Grant No. 31270770, 21077106, 31400641,). BioNavis, Fimlab Laboratories, PBL Brewing Laboratory (VTT) and Orion Diagnostica are

gratefully acknowledged. Biocenter Finland is acknowledged for infrastructure support. Dr Meng Fu and 27

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JieHong Mao from Merieux NutriSciences-Sino Analytica (QingDao and NingBo) company are gratefully

acknowledged for discussion.

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Chem. 2010, 82, 2766−2772.

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