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Plasmonic Approach to Enhanced Fluorescence for Applications in Biotechnology and the Life Sciences Wei Deng, and Ewa M Goldys Langmuir, Just Accepted Manuscript • DOI: 10.1021/la300332x • Publication Date (Web): 08 May 2012 Downloaded from http://pubs.acs.org on May 16, 2012

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PLASMONIC APPROACH TO ENHANCED FLUORESCENCE FOR APPLICATIONS IN BIOTECHNOLOGY AND THE LIFE SCIENCES

Wei Deng and Ewa M Goldys∗ ∗ MQ BioFocus Research Centre, Macquarie University, North Ryde 2113 NSW, Australia

Abstract. One of the most rapidly growing areas of physics and nanotechnology is concerned with plasmonic effects on the nanometre scale; these have applications in sensing and imaging technologies. Nanoplasmonic colloids such as Ag and Au have been attracting active interest and there has been a recent explosion in the use of these metallic nanostructures to favorably modify the spectral properties of fluorophores and to enhance the fluorescence emission intensity. In this review article, we summarize our work on a range of nanoplasmonics-assisted biological applications such as flow cytometry, immunoassays, cell imaging and bioassays where we use custom-designed plasmonic nanostructures (Ag and Au) to enhance fluorescence signatures. This fluorophore–metal effect offers unique advantages providing improved photostability and enhanced fluorescence signals. We discuss plasmonic enhancement of lanthanide fluorophores whose long and microsecond lifetimes offer the advantage of background-free fluorescence detection, but low photon cycling rates lead to poor brightness. We also show that plasmonic colloids are capable of enhancing the emission of fluorescent nanoparticles, including upconverting nanocrystals and lanthanide nanocomposites.

, To whom correspondence should be addressed. Telephone: +61-2-9850-8902. Fax:

∗

+61-2-9850-8115. E-mail: [email protected]

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1. Introduction Metal nanostructures have been receiving considerable attention due to their ability to guide and manipulate “light” at the nanoscale. They can interact with light in such a way that conduction electrons become collectively excited at resonance with the frequency of the incident radiation. Such collective excitations known as surface plasmons (SP) make metallic nanoparticles and nanostructures useful for many applications, including light guiding and manipulation at the nanoscale, biodetection at the single molecule level, enhanced optical transmission through subwavelength apertures, and high resolution optical imaging below the diffraction limit. For example, the sensitivity of SP bands to their immediate environment offers opportunities to detect adsorbed molecules (a particular protein or DNA) and environmental changes.1-3 In addition, Raman scattering, an exchange of energy between photons and molecular vibration, was enhanced by electromagnetic fields near the rough metal surface due to the presence of SP. This observation led to the now well established field of Surface Enhanced Raman Scattering (SERS).4 Among the various metals, Ag and Au colloids are of particular interest not only because they are comparatively inert but also because their SP absorption bands are in the visible and near-infrared spectral regions, which are most useful for practical applications.5-6 Numerous medical, biotechnology and biological research applications involve the detection of biomolecules with fluorescence.7 Fluorescent molecules are sensitive to the environment e.g. temperature, pH, polarity, oxidation state and the proximity of quenching groups which can alter quantum yield or wavelength of emitted light. The use of fluorescent markers is currently the most common labeling technique for immunoassays and cell imaging. A large range of fluorophores with distinctive spectral characteristics are available, providing many possibilities for multi-color biological labeling. Fluorescence allows in-situ studies in living cells and tissues including binding ACS Paragon Plus Environment

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of ligands such as a drug or hormone to a receptor, measurement of concentration of metabolites, study of structure, orientation, fluidity and distances between molecules. The technique is inherently sensitive so that single molecules have been been routinely detected in research laboratories. Yet in practical situations, where complex samples of biological origin must be used, such as in proteomics or clinical medicine the presence of interfering background makes fluorescence detection more difficult. Secondly, the extinction coefficient of common fluorophores is relatively low.8 Consequently, the detection of low concentrations of fluorophores (even with quantum yield close to unity) is limited by the small magnitude of the fluorescence signal. Another drawback of molecular fluorophores is their poor photostability. Following the excitation event, a fluorophore may become involved in certain unintended chemical reactions, especially with oxygen free radicals. This phenomenon, often referred to as photobleaching, causes the practically irreversible loss of the molecule fluorescence. Reducing the excitation power limits the photobleaching effects and thus delays the signal decay, however reduced excitation intensity obviously produces weaker fluorescence emission. Thus the need arises for the fluorescence signals to be amplified, and this is where plasmonics provides a novel approach referred to as Metal-Induced Fluorescence Enhancement (MIFE) which is the focus of our review. The MIFE effect increases fluorophore brightness and its photostability, and it also shows other desirable effects such as improved energy transfer.9-10 Due to these features, the MIFE technique has been widely employed in immunofluorescence, biosensors and bioimaging,11-12 see also excellent reviews.13-15 Plasmonic enhancement of fluorescence is a nanoscale phenomenon which is particularly pronounced in close proximity to metal nanostructures. This effect has been intensively studied including by Lakowicz and his collaborators,16 who proposed a widely utilised semi-empirical


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model. Briefly, in the proximity of metals the fluorophore radiative properties are modified, and a decrease in spontaneous emission lifetime is observed which is associated with concomitant increase in the radiative quantum efficiency. These are given by the following relationships.

Qm =

Γ + Γm Γ + Γm + k nr

/1/

τm =

1 Γ + Γm + knr

/2/

Here Qm is the quantum yield modified by the nanostructure, Γ is the radiative rate of the isolated fluorophore, knr is the non-radiative rate, and

is the additional rate induced by the nanostructure,

is the fluorophore lifetime modified by the nanostructure. The quantum yield is increased

because lower lifetime translates into faster cycling of the fluorophore. Electrodynamics explains this acceleration is due to increased density of photonic states in the proximity of metal nanoparticles; such increased density of photonics states is also present in other structures such as periodic dielectric microstructures known as photonic crystals, and also high Q resonant cavities (for example glass microbeads with whispering gallery modes). All such structures exhibit the fluorescence enhancement effect discussed here. The fluorophore emission intensity in the MIFE effect depends in a complex fashion on the distance between the metal and fluorophore. At very close distances, less than several nm, the fluorphore experiences a significant non-radiative decay component (Equation /1/ and / 2/), and consequently the emission is strongly quenched. At intermediate distances, around 5 nm for silver, the nonradiative decay process subsides somewhat and the enhancement effect begins to dominate, producing the overall fluorescence amplification peak, which, for spherical metal nanoparticles reaches a typical value in the order of 10. For longer distances, 10 nm and above, the amplification tapers off to eventually reach unity. The detailed modeling of the distance dependence of


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fluorescence enhancement is beyond the Lakowicz’s model and needs to be carried out by numerical simulations of Maxwell’s equations by using commercial software packages such a COMSOL or in-house code. Lakowicz’s model of fluorescence enhancement which focuses on fluorophore lifetime ignores an important plasmonic effect of concentration of electromagnetic field which must be added a posteriori. Metal nanostructures modify the incident electromagnetic field and increase field amplitude, especially close to sharp tips and edges as well as in tight junctions between metal nanostructures. The fluorophore emission intensity depends on the square of that local exciting field, as well as on the fluorophore cycling rate described by its lifetime (Equation /1/ and /2/). These two factors are referred to as “excitation enhancement” and “emission enhancement”. The overall fluorescence enhancement factor is a product of these two. Plasmonic enhancement of fluorescence can be described as the coupling of fluorophores (sources of radiation) to metal nanostructures which then act as antennas and relay the radiation outward. The nanostructure enhances the radiative scattering efficiency of a fluorophore which is related to the magnitude of its radiating dipole and this, in turn reflects the antenna size. Individual spherical metal nanoparticles of tens of nm size produce some increase of the radiating dipole which can be well described by the Mie theory calculations of the radiative scattering cross-section.17-18 However, the coupling of nanoparticles produces a much greater increase. It is important that one can engineer this dipole-dipole coupling, for example by using metal mirrors under the nanoparticle layer. In such engineered structures high values of fluorescence enhancement can be easily obtained. The investigations of fluorescence enhancement can be divided into two broad classes. Earlier investigations especially by Lakowicz, Geddes and their groups concentrated on metal


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nanostructures produced by chemical methods such as metal colloids, silver islands on glass substrates, and more recently on metal nanorods, more complex metal nanostructures such as core-shell, aggregates etc. These were usually accompanied by a demonstrated application, often to an important bio- or immunoassay. This is because, due to its sharp distance dependence, plasmonic enhancement of fluorescence is ideally suited to monitor biorecognition reactions. Other authors were concerned with the understanding of how the fluorescence enhancement effect is underpinned by deeper physical principles such as the coupling between individual nanoscale metal structures and the capacity of the structure to support large values of the radiating dipole. Such structures have been subsequently applied to the area of ultrasensitive bioassays.19-22 This work is currently being extended to periodic nanostructures produced by electron beam lithography where principles of rational design can be implemented more easily. Frequently, the emphasis was on the demonstration of a particular phenomenon or property in a largely empirical approach, and research was rarely driven by the specific demands of a particular biotechnology or life science application. While plasmonic fluorescence enhancement remains one of the selected physical phenomena with significant applications in biotechnology and life sciences, (alongside with SERS), it is not always closely compatible with real biological applications. Our group tried to address this compatibility based on a thorough understanding of the practical demands and limitations. The practical applications of MIFE are challenging for a number of fundamental reasons. For example it is well known that bright fluorophores are in high demand for cell imaging and also for whole-body imaging in clinical situations. Responding to this Lakowicz et al. in one of his early papers attempted to enhance fluorescence of indocyanine green,23 one of a few fluorescent dyes approved for clinical use as contrast agents. They used relatively small metal colloids which have limited radiative scattering, hence the net fluorescence enhancement was small, with a factor of several.


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However using larger colloids would not be ideal from an application point of view, as contrast agents must be able to be easily cleared from the body. This illustrates the nature of constraints the application may pose. Another type of constraint is the format of the metal enhancing substrate. Much of the fluorescence enhancement research, especially with refined electron beam lithography structures has been done in a planar format. However such planar structures are poorly suited to cell imaging, where events inside of the cell are of most interest. Only recently some authors have been able to develop significant applications in planar formats, for example in the studies of membrane receptors and imaging of target molecules in tissue samples.24-26 At the same time, some formats, very popular in biomolecular diagnostics, continue to be poorly compatible with plasmonics. Membranes are used as solid support for various biomolecular analyses, especially for the detection of specific proteins or peptides and nucleic acid sequences. Such membranes can be coated by metal nanoparticles owing to their charged surfaces. Taking advantage of the capacity of noble metal nanoparticles to bind proteins, Duchesne and Fernig demonstrated that such coated membranes are highly effective for the Western blot detection of proteins and peptides with small molecular weight (fewer than 10 kDa), where they capture the proteins during the electro-transfer, and thus prevent the protein loss.27 An improvement by a factor in the order of 10,000 was found compared with unmodified membranes. We verified whether this approach can also yield fluorescence enhancement, with moderate success so far, due to high extinction of metal nanoparticles which distribute in the membrane.28 In this article we present a brief overview of our work on selected biological applications of MIFE technology, such as flow cytometry and time-gated bioassays and bioimaging.29-32 Some of this work used silver and gold colloids deposited at planar surfaces, these are typical metal nanostructures widely used in this field. We also developed combinations of metals and inorganic


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materials in a core-shell geometry. As we know, silver colloids have some advantages such as narrow SP band and high scattering efficiency, while gold nanoparticles are more stable and easier to synthesise with good size uniformity. The choice of metal nanostructures and synthesis method was dictated by the demands of a particular application, and our review is organized with respect to these applications.

2. Technologies based on silver nanostructure-coated silica beads 2.1. Flow cytometry-based MIFE microsphere immunoassay29 Flow cytometry-based bead or microsphere assay is an important analytical technique commonly used in biotechnology. In this technique ensembles of antibody-coated fluorescent microspheres (beads) are mixed with a sample such as patient blood or some other complex, biological or environmental fluid. The coating on the microspheres is then allowed to react with target molecules or cells. Once the antibody-antigen specific reaction has occurred, the microspheres are analysed, one by one by using a flow cytometer, or they can also be analysed by other techniques. Such bead assays offer significant advantages over alternative planar microarrays including rapid assay times and readout, extremely small sample volumes, and compatibility with high dilution.33-34 They are also able to read multiple components of a complex mixture at once (multiplexing) by using several types of beads with different fluorophores or optically encoded microspheres with unique combinations of fluorescent dyes. However, this technology has not yet reached its full potential. One of the outstanding challenges is their limited detection sensitivity; while fluorescent microsphere signal is strong, the trace amount of analyte on the microsphere surface combined with rapid passage of the microspheres through the detection region makes sensitive measurements difficult. We were the first and only group to employ silver nanostructures on the microsphere surface with a view to amplify analyte fluorescence and improve the detection ACS Paragon Plus Environment

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sensitivity of the flow cytometry readout. Here we use silver as a metal whose plasmon band is narrower, and scattering efficiency is much higher than gold, both of which promote fluorescence enhancement. In our method, a seed Au colloid is first covalently bound to (3-aminopropyl)-trimethoxysilane (APTMS) modified silica microspheres (400 nm and 5 µm in diameter) (see Figure 1a), followed by silver shell overcoating (see Figure 1b). To test the ability of such nanostructured surfaces to enhance fluorescence we used a model immunoassay, rabbit IgG-IgG rabbit antibody, labeled with AlexaFluor 430, on the glass slide (see Figure 1c). The enhancement factor was determined as the ratio of the corrected fluorescence intensity on the silvered surface of silica beads to one on the surface of unmodified silica beads based on the fluorescence spectra. We firstly measured the fluorescence intensities from the wells on our glass slides with deposited Ag-coated and uncoated silica beads but without immunoassay as control samples. Then we measured the samples of supernatants of AlexaFluor 430-labeled IgG antibody collected from each well after immunoassay binding and before washing, followed by comparing their fluorescence intensity with the original AlexaFluor430-labeled protein solution under the same experimental conditions. Subsequently, we estimated how much protein was attached to the silica beads with and without the Ag layer, respectively (binding efficiency). Then we divided the observed fluorescence intensity from the wells for each sample by its corresponding binding efficiency and thus obtained the corrected fluorescence intensity. This value was equivalent to fluorescence intensity for the uniform coverage on each glass slide for the different samples. Finally, we compared the corrected fluorescence intensities from the model immunoassay on silica beads with Ag enhancement and on unmodified silica beads. The average enhancement factors for 400 nm silica beads with 3 min Ag enhancing


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time was 8.5. In the case of large 5 µm silver-coated silica beads with silver enhancing of 3 min, the enhancement factor was about 10.1. In addition, laser scanning microscopy at 405 nm excitation wavelength was also used to show the enhancement of fluorescence emission for the samples with the Ag layer. When the silver enhancing time was increased up to 3 min, the fluorescence signal observed from the silvered area was much greater than that from the pure silica beads surface, demonstrating fluorescence enhancement (see Figure 2). Finally, we demonstrated the flow cytometry readout of this immunoassay and we have shown that it was enhanced, by examining the fluorescence-intensity histograms of silica beads with and without Ag nanostructures. Figure 3 shows flow cytometry scanning results for 5.0 µm diameter fluorescence beads. This figure shows that Ag surface modification obviously affects side scatter (SSC). This result is consistent with the highly non-uniform surface coverage, where denser Ag nanostructure on the silica beads surface resulted in stronger 90o angle scattering. The flow cytometry results show that the Ag deposition for 3 min produced enhancing fluorescence intensity, with the enhancement factor of 3.7, compared to silica beads with the same immunoassay but without Ag nanostructures. Since our work was published, only one more publication appeared where plasmonic effects have been used in conjunction with flow cytometry,35 however these authors utilized enhanced plasmonic scattering of metal nanorods and not enhanced fluorescence.

2.2. Metal-enhanced bioassay based on lanthanide labeled streptavidin (SA)-biotin binding system30 Lanthanide chelates are attracting increasing interest for fluorescence labeling as a result of expanding applications in a wide variety of bioanalytical assays, in diagnostics, sensing and in


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imaging, especially with time-gating that offers exceptionally high background rejection.36-40 However such long lifetimes lead to limited fluorophore brightness. To achieve the brighter luminescence signal, we employed silver nanostructures as MIFE substrates to demonstrate their effects

on

a

tetradentate

ß-diketone

ligand,

4,4’-bis(1”,1”,1”,2”,2”,3”,3”-heptafluoro-4”,6”-hexanedion-6”-yl)chlorosulfo-o–terphenyl (BHHCT), which forms a very stable chelate with Eu3+ with desirable luminescence properties. In this work, Ag nanostructure-coated silica beads were deposited on glass slides and used as the substrates. Figure 4 demonstrates the luminescence enhancement and lifetime decrease of BHHCT-Eu3+ by using these beads in an IgG assay. We checked that Ag nanostructures did not change the spectral shape of the emission. Indeed, in the samples with and without the Ag nanostructures as shown in Figure 4a, we were able to observe a very similar spectrum, with the emission maximum at around 615 nm, typical of Eu3+. The Ag nanostructures clearly enhance luminescence of BHHCT-Eu3+, with enhancement factor of 11.3. What’s more, the measured decay curves of samples indicate a clearly decreased luminescence lifetime observed in the presence of the silver nanostructures near BHHCT-Eu3+, from 380 µs without Ag enhancement to 192 µs with 3 min Ag enhancement (Figure 4b). We should add here that we were not the first to observe the MIFE effect with lanthanide chelates and the pioneering work in this area was led by Lakowicz’s group.41-43 However ours is the first effort to use MIFE on Ag nanostructure-coated silica beads for immunoassays and take advantage of reduced reaction times which such beads provide, especially important at low analyte concentrations. Our work has thus improved the compatibility of MIFE with the demands of ultrasensitive bioassays.

3. Improved time-gated cell imaging based on MIFE30 ACS Paragon Plus Environment

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Bioimaging is a major application area for fluorescence as fluorescence imaging is one of the foundations of the current cell biology. An ongoing demand exists for fluorophores with ever increasing brightness and unique properties. The application of metal enhancement to traditional fluorophores such as fluorescent dyes or genetically targeted fluorescent proteins and their applications in bioimaging are challenging due to limited enhancement conferred by conjugation of metal nanoparticles. These conditions can be somewhat relaxed for fluorophores in a nanoparticle format. Our recent work focused on lanthanide fluorophores which are suitable for time-gated imaging with suppressed autofluorescence background, in particular on BHHCT-Eu3 +. In order to demonstrate beneficial effects of silver nanostructure modification of BHHCT-Eu3+ for imaging, we used a model microorganism Giardia lamblia where BHHCT-Eu3+-labeled streptavidin (SA) conjugate was attached to the cell surface. The organisms were placed on a glass slide covered with silver nanostructure-coated silica beads. From the corresponding fluorescence intensity histograms, we found the mean image signal is enhanced by a factor of 2. The fluorescence intensity distribution is much broader in comparison with one without Ag nanostructures

(Figure

5a).

The

representative

cell

images

with

and

without

Ag

nanostructure-coated silica beads shown in Figure 5b. The observed lower enhancement factor obtained in the cell imaging is attributed to following reasons. Firstly, it is known that the fluorescence enhancement is most pronounced only when the fluorophore is localized at an optimal distance close to the surface of metal nanostructures that is in the range of 5-30 nm.44-45 In our experiment on the biological cells the distances of fluorophore molecules to metal nanostructures cover a much wider range. The excitation enhancement is thus averaged over the cell thickness, resulting in the lower average enhancement. In addition, emission enhancement plays an important role for very thin samples. However, the strength of the emission enhancement decreases as the


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distance from the metal nanostructures increases.46 Therefore, the observed signal enhancement in cell imaging is mainly caused by the increase of the fluorophore excitation rate. We emphasized earlier limited compatibility of MIFE with bioimaging and highlighted new work concerned with imaging of membrance receptors and targeted biomolecules in tissue.24-26 Our work shows, for the first time that MIFE is also able to assist with imaging of intact cells. In particular, using MIFE in conjunction with lanthanide makes it possible to suppress autofluorescence background. We are not aware of any other relevant reports.

b 4. MIFE for enhanced upconversion (UC) luminescence Unlike conventional fluorescent reporters, up-converting phosphors (UCP) transfer low energy infrared (IR) radiation to high-energy visible light by multi-photon absorption and subsequent emission of dopant-dependant phosphorescence.47 They absorb two or more pump photons via intermediate long-lived energy states followed by the emission of the output radiation at a shorter wavelength than the pump wavelength, which is referred to as upconversion. Different rare earth ions can produce upconversion luminescence at various emission wavelengths characteristic to the lanthanide ion with a single near-infrared excitation wavelength. UCP containing only one type of rare earth ion as the dopant, such as Er3+ or Tm3+, are capable of anti-Stokes photoluminescence, but the upconversion processes can be more efficient when Yb3+ is also present.48 The most efficient upconversion materials known to date are NaYF4: Er,Yb and NaYF4: Tm,Yb.48 These nanoparticles produce red and green emission typical of Er ions when excited at 980 nm. They can be synthesized with quantum yield as high as 0.3, which makes these nanoparticles brighter than the best current two-photon dyes used in background-free multiphoton imaging. We used MIFE to enhance the upconversion fluorescence and/or bring about the enhancement of resonance energy transfer, responsible for delivery of excitation energy from sensitizer Yb to Er.31 Three more publications ACS Paragon Plus Environment

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have appeared since our work was published, one was a parallel work from our collaborators and two more recent ones.49-51 Below we provide more details about our approach.

4.1. Silver-nanostructure enhanced UC nanoparticles31 NaYF4: Er,Yb UC nanoparticles produced by an established method52 were first made water-soluble by deposition of a thin silica coating (Figure 6a). This silica coating additionally ensured nanoscale separation between nanoparticles and silver nanostructures, which is required to prevent metal quenching. Further, these nanoparticles were conjugated to silver nanostructures deposited on the surface of dielectric silica beads. The conjugation was achieved by using a simple bioassay, where SA-labeled UC nanoparticles were bound to the biotinylated anti-mouse IgG antibody. This assay was carried out on silica beads with and without Ag nanostructures under the same experimental conditions. The Ag nanostructures on silica beads had a broad absorption band around 400 nm. We first assessed the effect of silver nanostructures on UC luminescence amplification using SA-biotin binding system. The glass slide was coated with Ag-silica beads and biotinylated IgG antibody molecules; SA-UC nanoparticle conjugates were then bound to silica beads via the avidin-biotin recognition mechanism. The impact of the nearby Ag nanostructures on luminescence was measured. We checked the effect of these nanostructures on the spectral shape of the UC emission. In the samples with and without Ag nanostructures as shown in Figure 6b, we were able to observe very similar spectral signatures, with the emission bands typical of Er3+ around 525 nm, 545 nm and 660 nm. Ag nanostructures clearly enhanced UC luminescence with enhancement factors of ~4.4 and ~3.5 for green and red emission, respectively, compared to the control sample without Ag nanostructures. These values were obtained by dividing the integrated luminescence intensities for


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samples with Ag nanostructures after background subtraction (integrated luminescence intensities around 525 nm, 545 nm and 660 nm wavelengths only, from Ag nanostructures without the bioassay) by those without Ag nanostructures. The intensity values were also corrected for the differences in the number of nanoparticles bound in each sample, determined from the Er fluorescence of the top solution remaining after the completion of bioassays.

In order to obtain a more complete characterization of the effect of Ag nanostructures on UC luminescence we also carried out the lifetime measurements. As discussed in the literature, MIFE is characterized by an increase in the quantum yield and a decrease in the lifetime of a fluorophore located in the proximity of a metallic nanostructure.53 The measured decay curves of UC luminescence from samples with and without Ag nanostructures indicate a clearly decreased lifetime observed in the presence of the silver nanostructures near these nanoparticles (see Figure 7), which was reduced from 211 µs to 127 µs for green emission and from 654 µs to 276 µs for red emission, respectively. Such reduced lifetime has two major implications, higher photostability and a higher number of emitted photons per unit time under the same excitation conditions. Both these properties make it possible to use shorter exposure time or obtain higher signal to noise for comparable exposure times, thus increasing sensitivity and detectivity in luminescence measurements.

4.2. Gold shell-coated UC nanoparticles31 This study mainly focused on another metal nanostructure, gold, which is also widely used in nanoplasmonics.54-56 The gold nanoshells had a strong absorbance in the green region of the visible spectrum and they did not inhibit emission from Er3+ centered around 525 and 545 nm (Figure 8a). This is in direct contrast to the case of quantum dots, where the fluorescence emission from the quantum dots is quenched by the gold shell in the core-shell architecture.57 The synthesis of the gold


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shell-coated nanoparticles used a facile one-pot technique and the prepared NaYF4: Er,Yb nanoparticles were coated with ~5-8 nm thick gold shells (Figure 8b).58 We first evaluated the effect of gold nanoshell on UC luminescence intensity of NaYF4: Yb, Er. As shown in Figure 9, in samples with gold nanoshells both green and red emissions were increased, by a factor of ~9.1 times and ~6.7 times, respectively. Investigations on metal surface enhanced luminescence of nanocrystals indicate that the degree of fluorescence enhancement is correlated with the spectral overlap between the absorption or emission band of the phosphors and the surface plasmon band of metals.59 Therefore this larger enhancement (~9.1-fold) for green emission could, in principle be attributed to better spectral overlap of the gold plasmon band between 500-550 nm with the green luminescence, which can cause improved plasmonic coupling. However further investigations of another key MIFE factor in this system, the lifetime, do not confirm this explanation. Figure 10 shows that the red emission in gold shell-coated samples had a longer lifetime (~1190 µs) than in uncoated ones (~ 812 µs), while the lifetimes of the green emission in these two samples are similar (~882 µs). These lifetime values can be interpreted when we consider the effect of surface recombination which is known to affect the green/red upconversion in a major way.60-61 This surface recombination may be different in gold-shell coated and uncoated nanoparticles. Its effect is superimposed and may dominate over the plasmonic effects. The plasmonic effect of the gold shell coating is expected to reduce the lifetime in both green and red band, while the same coating may protect nanoparticles from surface recombination, producing the opposite effect of extending the lifetimes. Thus we interpret the similarity of lifetimes in Au-coated nanoparticles and uncoated nanoparticles is due to surface recombination counterbalancing the plasmonic effects.

5. Technologies based on silver/core-silica/shell nanocomposites


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5.1 MIFE bioassay based on strepavidin (SA)-biotin binding system32 Metal enhancement not only increases the fluorescence signal but it also greatly moderates the problem of fluorescence saturation, unavoidable in long lifetime fluorophores. By using silver/core-silica/shell (Ag@SiO2) nanocomposites we demonstrated high enhancement factors of lanthanide chelates integrated into silica nanoparticles with silver cores as well as their applications. Such Ag@SiO2 nanoparticles have been previously utilized by other authors, most notably by Geddes as well as Cheng.62-63. In this work we have chosen a new Eu chelate, BHHCT-Eu-DPBT, whose absorption is red-shifted compared to previously used BHHCT-Eu3+. In order to control the fluorescence enhancement of BHHCT-Eu-DPBT doped into Ag@SiO2 nanocomposites, we designed our chemistry to modify both the silver-fluorophore distance and the Ag-core size. The Ag core size is controlled by the growth of citrate-reduced silver colloids using metal deposition. The thickness of the first silica shell is varied by using different amounts of tetraethyl orthosilicate (TEOS) which is able to produce silica sols on the surface of silver nanoparticles. Once the silver core has been covered with

the

first

shell,

3-aminopropyl(triethoxy)silane

(APS)-linked

BHHCT-Eu-DPBT

(APS-BHHCT-Eu-DPBT) is then covalently linked and embedded in second, < 3 nm thick exterior silica shell (see Figure 11a). In order to evaluate the effects of metal core on BHHCT-Eu-DPBT in this Ag@SiO2 system, the corresponding “control” fluorophore-doped hollow silica nanoshells were also prepared by using the etching process where the metallic silver is oxidized to Ag(CN)2- by cyanide in the presence of air (see Figure 11b). It is worth emphasizing that at high excitation intensities BHHCT-Eu-DPBT-doped Ag@SiO2 nanocomposites showed significantly increased fluorescence enhancement factors, owing to the fact that the control samples without Ag cores showed distinctive fluorescence saturation, while in the composites the onset of saturation was not observed within the examined intensity range. This is


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because fluorescence saturation occurs when the rate of absorption becomes comparable to the decay rate. As the decay rate in the samples with silver core is higher, the onset of saturation takes place at higher powers. Figure 12 shows a comparison of fluorescence intensity as a function of laser power, obtained after 10 millisecond light exposure. It increases rapidly and linearly in the nanoparticles with metal core, while in the control samples the increase is much slower. The sample with 81 nm core diameter and 25 nm shell thickness had the highest fluorescence enhancement factor of 146. Our results show that presence of silver nanoparticle alleviates the fluorescence saturation problem typical of lanthanide fluorophores.

In order to demonstrate the utility of these nanocomposites in biological applications, we carried out a fluorescent bioassay using streptavidin-biotin binding. To this aim the nanocomposites with 12 nm SiO2 shell and with (or without) 52 nm Ag core were conjugated with biotynilated IgG antibody molecules. These were presented to streptavidin-coated glass slides where they were bound due to the avidin-biotin recognition mechanism (Figure 13a). The enhancement factors were obtained by comparing the fluorescence intensity of bioassays with and without the Ag core at the same experimental conditions. The enhancement factor observed at high excitation intensity of 32.3 mW/cm2 was ~120 times (Figure 13b). The detection limit, calculated with the concentration corresponding to three standard deviations of background signal, is 17.6 pg ml-1. Additionally, various concentrations of biotinylated IgG antibody-conjugated nanocomposites were measured when concentration of streptavidin on the glass slide was fixed as 50µg ml-1. The corresponding calibration curve is shown in Figure 13c.

5.2. Single lanthanide nanoparticle imaging facilitated by MIFE32


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BHHCT-Eu-DPBT-doped Ag@SiO2 nanocomposites were exceptionally bright and could be observed as single particles, both in conventional and time-gated imaging. This is documented in Figure 14 which shows the fluorescence images of nanocomposites with 52 nm Ag core and 25 nm SiO2 shell excited at 405 nm (Figure 14a) and the time-gated image of the same sample (Figure 14b). Both images were taken in dry conditions on a ruled TEM grid, to allow comparison with a TEM image of the same region (Figure 14c and 14d). The nanoparticles in all images are labeled for individual identification. The magnified image shown in Figure 14d confirms that single nanoparticles have been recorded both in conventional and time-gated fluorescence images.

6. Conclusions and further outlook In this paper we reviewed our recent work on colloidal metal nanostructures for enhanced-fluorescence applications in biotechnology and the life sciences. These studies mainly focused on silver and gold nanostructures that offer enhanced spectral properties of the nearby fluorophores, such as increased quantum yields (enhanced fluorescence intensities) and enhanced fluorophore photostability (reduced lifetimes). We first demonstrated the use of silver nanostructures on the silica beads surface as an amplification mechanism for a flow cytometry bead immunoassay. The Ag nanostructure-coated silica beads after 3 min silver enhancing time produced strong enhancement factors (8.5-fold and 10.1-fold) of emission intensity of Alexa 430 fluorophore excited at 430 nm for 400 nm and 5 µm beads, respectively. This enhancement was mediated by plasmon resonance in clustered silver nanoparticles. Both 400 nm and 5 µm silica beads were compatible with a flow cytometry readout at 488 nm, although lower enhancement factors of 3.0 and 3.7 were obtained, which were consistent with less favorable overlap of the plasmon resonance with 488 nm excitation wavelength used in the flow cytometry experiment. We then have shown that such Ag nanostructures were able to produce brighter luminescence images of cells labeled by BHHCT-Eu3+, ACS Paragon Plus Environment

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suggesting that such substrates are applicable for background-free time-gated luminescence bioimaging. We also investigated the feasibility of using both silver nanostructures and gold nanoshells for UC luminescence enhancement. These two types of noble metal nanomaterials produced clear luminescence enhancement (~4.4-fold for green emission and ~3.5-fold for red emission in the case of silver nanostructures; ~9.1-fold and ~6.7-fold in the case of gold nanoshells). These results prove that UC emission can be enhanced by noble metals such as silver and gold, which promise to yield a broader range of applications of metal–UC nanoparticle composites in bioassays, bioimaging and energy conversion that require ultrahigh sensitivity and low background. Finally, we reported an ultrabright time-gatable nanocomposite by doping the Eu chelate, BHHCT-Eu-DPBT into Ag@SiO2 core-shell nanostructure. We showed feasibility of bioassays with such nanocomposites and quantitatively interpreted the characterisation results. These nanocomposites did not show obvious fluorescence saturation in our study, and thus they can be used at high laser powers, where they offer greatly amplified fluorescence compared with nanocomposites without plasmonic effects. These results indicate the high potential of the MIFE technique as a generic platform for highly sensitive immunoassay development as well as for any general bioaffinity reactions on surfaces and in suspension. Although the MIFE technique allows scientists to readily address the issues of assay sensitivity and photostability, several challenges still remain. One major challenge is to determine the specific influence of a nanostructure on the fluorescence emission, as the detected signal results from a product of excitation and emission processes where we can tune each of the resonances separately. In our opinion the concept of dipole dipole coupling has not yet been fully exploited; and our group is pursuing this direction. Here the absence of analytical plasmonic models has been particularly limiting as accurate numerical simulations are time-consuming. Another main challenge in the


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synthesis of MIFE substrates is adequate control of their physical properties such as obtaining uniform particle size distributions, identity of shapes, morphology, chemical composition and crystal structure. Here, we see a significant promise in the application of microsphere assisted metal deposition and in the use of nanoimprint lithography. They combine low cost and large areas with good periodicity which enables structure optimization by numerical simulations. We believe that the successful combining of membranes with plasmonic enhancement would be a very significant development due to their significance in lateral flow assays, and this would lead to rapid pathogen detection in medicine and in the food industry and lead to new consumer products.

In addition, the

benefits of these MIFE substrates need to be weighed with any potential cost to the environment and public health. Despite these challenges we believe the MIFE technique can be further developed and optimized to fully realize its promising prospects in medicine and the life sciences.

Acknowledgements

We acknowledge contributions from D. Jin, K.Drozdowicz-Tomsia, L. Sudheendra, J. Zhao, J. Fu, I. Kennedy, J. Yuan.

Supporting Information Available None


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References (1) Maier, S. Plasmonics: Fundamentals and Applications. Springer ((New York): 2007. (2) Brongersma, M.; Kik, P. Surface Plasmon Nanophotonics. Springer Series in Optical Sciences: 2007, 131. (3) Noginov, M. A. et al. The effect of gain and absorption on surface plasmons in metal nanoparticles, Appl. Phys. B 2007, 86, 455–460. (4) Fleischmann, M.; Hendra, P.J.; McQuillan, A.J. Raman spectra of pyridine adsorbed at a silver electrode, Chem. Phys. Lett. 1974, 26, 163-166. (5) Daniel, M-C; Astruc, D. Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology, Chem. Rev. 2004, 104, 293–346. (6) Tao, A.; Sinsermsuksakul, P.; Yang, P. Tunable plasmonic lattices of silver nanocrystals, Nature Nanotechnology 2007, 2, 435 – 440. (7) Goldys, E. Fluorescence applications in biotechnology and life sciences, Wiley-Blackwell: 2009. (8) Seydack, M. Nanoparticle labels in immunosensing using optical detection methods, Biosens. Bioelectron. 2005, 20, 2454–2469. (9) Sokolov, K.; Chumanov, G.; Cotton, T. Enhancement of molecular fluorescence near the surface of colloidal metal films, Anal. Chem. 1998, 70, 3898-3905. (10) Geddes, C.; Parfenov, A.; Roll, D.; Fang, J.; Lakowicz, J. Electrochemical and laser deposition of silver for use in metal-enhanced fluorescence, Langmuir 2003, 19, 6236-6241. (11) Stewart, M.; Anderton, C.; Thompson, L.; Maria, J.; Gray, S.; Rogers, J.; Nuzzo, R. Nanostructured plasmonic sensors, Chem. Rev. 2008, 108, 494–521. (12) Zhang, J.; Fu, Yi.; Liang, D.; Zhao, R.; Lakowicz, J. Reduced blinking and long-lasting ACS Paragon Plus Environment

Page 22 of 44

Page 23 of 44

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

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fluorescence of single fluorophores coupling to silver nanoparticles ， Langmuir 2008, 24, 12452-12457. (13) Lakowicz, J. Radiative decay engineering: biophysical and biomedical applications, Anal. Biochem. 2001, 298, 1–24. (14) Lakowicz, J.R. Plasmonics in biology and plasmon-controlled fluorescence, Plasmonics 2006, 1, 5-33. (15) Aslan, K.; Gryczynski, I.; Malicka, J.; Matveeva, E.; Lakowicz, J.; Geddes, C. Metal-enhanced fluorescence: an emerging tool in biotechnology, Curr. Opin. Biotechnol. 2005, 16, 55–62. (16) Lakowicz, J.; Shen, Y.; D’Auria, S.; Malicka, J.; Fang, J.; Gryczynski, Z.; Gryczynski, I. Radiative decay engineering 2. effects of silver island films on fluorescence intensity, lifetimes, and resonance energy transfer, Anal. Biochem. 2002, 301, 261-277. (17) Kreibig, U.; Vollmer, M. Optical properties of Metal Clusters, Springer-Verlag (Berlin): 1995. (18) Mie, G.; Beiträge zur Optik trüber Medien, speziell Kolloidaler Metallösungen, Ann. Phys.

1908, 330, 377-445. (19) Shtoyko, T.; Matveeva, E.; Chang, I.; Gryczynski, Z.; Goldys, E.; Gryczynski, I. Enhanced fluorescent immunoassays on silver fractal-like structures, Anal.Chem. 2008, 80, 1962-1966. (20) Barnett, A.; Matveeva. E.; Gryczynski, I.; Gryczynski, Z.; Goldys, E. Coupled plasmon effects for the enhancement of fluorescent immunoassays, Physica B: Condensed Matter 2007, 394, 297-300. (21) Matveeva, E.; Gryczynski, I.; Barnett, A.; Goldys, E.; Akopova, I.; Borejdo, J.; Gryczynski, Z. Enhanced fluorescent immunoassays on metal mirrors coated with metal particles, J. Biophys

2007, 519A-519A Suppl.S (22) Goldys, E.; Drozdowicz-Tomsia, K.; Xie, F.; Shtoyko, T.; Matveeva, E.; Gryczynski, I.;


Langmuir

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

Gryczynski, Z. Fluorescence amplification by electrochemically deposited silver nanowires with fractal architecture J. Am Chem Soc 2007, 129, 12117-12122. (23) Geddes, C.; Cao H.; Gryczynski, I.; Gryczynski, Z.; Fang, J.; Lakowicz, J. Metal-enhanced fluorescence (MEF) due to silver colloids on a planar surface: potential applications of indocyanine green to in vivo imaging, J. Phys. Chem. A 2003, 107, 3443–3449. (24) Wang, S.; Ota, S.; Guo, B.; Ryu, J.; Rhodes, C.; Xiong, Y.; Kalim, S.; Zeng, L.; Chen, Y.; Teitell, M.; Zhang, X. Subcellular resolution mapping of endogenous cytokine secretion by nano-plasmonic resonator sensor array, Nano Lett 2011, 11, 3431 -3434. (25) Zhang, J.; Fu, Y.; Xu, X.; Lakowicz, J. Target molecule imaging in tissue specimens by fluorescent metal nanoprobes, J. Biomed. Optics, 2011, 16, 116004. (26) Zhang, J.; Fu, Y.; li, G.; Zhao, R.; Lakowicz, J. Direct observation of chemokine receptors 5 on T0 lymphocyte cell surface s using fluorescent nanoprobes 2: approximation of CCR5 populations, Biochemical and Biohysical Research Communications 2011, 407, 63-67. (27) Duchesne, L.; Fernig, D. Silver and gold nanoparticle–coated membranes for femtomole detection of small proteins by dot and Western blot, Anal Biochem 2007, 362, 287-289. (28) Xie, F.; Shtoyko, T.; Drozdowicz-Tomsia, K.; Goldys, E. Silver and gold nanoparticle coated membranes applied to protein dot blots, J. Nanopart Res 2011, 13, 613-624. (29) Deng, W.; Drozdowicz-Tomsia, K.; Jin, D.; Goldys, E. Enhanced flow cytometry-based bead immunoassays using metal nanostructures, Anal. Chem. 2009, 81, 7248–7255. (30) Deng, W.; Jin, D.; Drozdowicz-Tomsia, K.; Yuan, J.; Goldys, E. Europium chelate (BHHCT-Eu3+) and its metal nanostructure enhanced luminescence applied to bioassays and time-gated bioimaging, Langmuir, 2010, 26, 10036–10043. (31) Deng, W.; Sudheendra, L.; Zhao, J.; Fu, J.; Jin, D.; Kennedy, I.; Goldys, E. Upconversion in


Page 24 of 44

Page 25 of 44

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

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NaYF4: Yb, Er nanoparticles amplified by metal nanostructures, Nanotechnology 2011, 22, 325604. (32) Deng, W.; Jin, D.; Drozdowicz-Tomsia, K.; Yuan, J.; Wu, J.; Goldys, E. Ultrabright Eu–doped plasmonic Ag@SiO2 nanostructures: time-gated bioprobes with single particle sensitivity and negligible background, Adv. Mater 2011, 23, 4649-4654. (33) Salas, V. M.; Edwards, B. S.; Sklar, L. A. Advances in multiple analyte profiling, Adv Clin Chem

2008, 45, 47-74. (34) Battersby, B.; Trau, M. Optically encoded particles and their applications in multiplexed biomedical assays, Aust. J Chem. 2007, 40, 343-353. (35) Crow, M.; Marinakos, S.; Cook, J.; Chilkoti, A.; Wax, A. Plasmonic flow cytometry by immunolabelled nanorods, Cytometry A 2011, 79A, 57-65. (36) Yuan, J.; Matsumoto, K. A new tetradentate β-diketonate-europium chelate that can be covalently bound to proteins for time-resolved fluoroimmunoassay, Anal. Chem. 1998, 70, 596-601. (37) Bornhop, D.; Hubbard, D.; Houlne, M.; Adair, C.; Kiefer, G.; Pence, B.; Morgan, D. Fluorescent tissue site-selective lanthanide chelate, Tb-PCTMB for enhanced imaging of cancer，Anal. Chem.

1999, 71, 2607-2615. (38) Petoud, S.; Cohen, S.; Bünzli, J.; Raymond, K. Stable lanthanide luminescence agents highly emissive in aqueous solution: Multidentate 2-hydroxyisophthalamide complexes of Sm3+, Eu3+, Tb3+, Dy3+, J. Am. Chem. Soc. 2003, 125, 13324-13325. (39) Weibel, N.; Charbonnière, L.; Guardigli, M.; Roda, A.; Ziessel, R. Engineering of highly luminescent lanthanide tags suitable for protein labeling and time-resolved luminescence imaging, J. Am. Chem. Soc. 2004, 126, 4888-4896.


Langmuir

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

(40) Karvinen, J.; Laitala, V.; Makinen, L.; Mulari, O.; Tamminen, J.; Hermonen, J.; Hurskainen, P.; Hemmila, I. Fluorescence quenching-based assays for hydrolyzing enzymes. Application of time-resolved fluorometry in assays for caspase, helicase, and phosphatase, Anal. Chem. 2004, 76, 1429-1436. (41) Wu, M.; Lakowicz, J.; Geddes, C. Enhanced lanthanide luminescence using silver nanostructures: opportunities for a new class of probes with exceptional spectral characteristics, J. Fluoresc 2005, 15, 53-59. (42) Zhang, J.; Fu, Y.; Lakowicz, J. Luminescent silica core/silver shell encapsulated with Eu(III) Complex, J. Phys. Chem. C 2009, 113, 19404–19410. (43) Zhang, J.; Gryczynski, I.; Gryczynski, Z.; Lakowicz, J. Dye-labeled silver nanoshell−bright particle, J. Phys. Chem. B 2006, 110, 8986-8991. (44) Zhang, J.; Malicka, J.; Gryczynski, I.; Leonenko, Z.; Lakowicz, J. Surface-enhanced fluorescence of fluorescein-labeled oligonucleotides capped on silver nanoparticles, J. Phys. Chem. B 2005, 109, 7643-7648. (45) Mertens, H.; Koenderink, A.; Polman, A. Plasmon-enhanced luminescence near noble-metal nanospheres: comparison of exact theory and an improved Gersten and Nitzan model, Phys. Rev. B 2007, 76, 115123: 1-12. (46) Moal, E.; Fort, E.; Lévêque-Fort, S.; Cordelières, F.; Fontaine-Aupart, M.; Ricolleau, C. Enhanced fluorescence cell imaging with metal-coated slides, J. Biophys. 2007, 92, 2150-2161. (47) Corstjens, P.L.A.M.; Li, S.; Zuiderwijk, M.; Kardos, K.; Abrams, W.R.; Niedbala, R.S.; Tanke, H.J. Infrared up-converting phosphors for bioassays, IEE Proc.-Nanobiotechnol. 2005, 152, 65-72. (48) Auzel, F. Upconversion and anti-stokes processes with f and d ions in solids, Chem. Rev. 2004,


Page 26 of 44

Page 27 of 44

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

Langmuir

104, 139-173. (49) Sudheendra, L.; Ortalan, V.; Dey, S.; Browning, N.; Kennedy, I. Plasmonic enhanced emissions from cubic NaYF4:Yb:Er/Tm nanophosphors, Chem Mater, 2011, 23, 2987-2993. (50) Priyam, A.; Idris, N.; Zhang, Y. Gold nanoshell coated NaYF4 nanoparticles for simultaneously enhanced upconversion fluorescence and darkfield imaging, J. Mater. Chem. 2012, 22, 960-965. (51) Xing, H.; Bu, W.; Zhang, S.; Zheng, X.; Li, M.; Chen, F.; He, Q.; Zhou, L.; Peng, W.; Hua, Y.; Shi, J. Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging, Biomaterials 2012, 33, 1079-1089. (52) Boyer, J.; Cuccia, L.; Capobianco, J. Synthesis of colloidal upconverting NaYF4: Er3+/Yb3+ and Tm3+/Yb3+ monodisperse nanocrystals, Nano Lett. 2007, 7, 847-852. (53) Li. K.; Stockman, M.; Bergman, D. Enhanced second harmonic generation in a self-similar chain of metal nanospheres, Phys. Rev. B 2005, 72, 153401-5. (54) Lee, S.; Sasaki, D.; Perroud, T.; Yoo, D.; Patel, K.; Lee , L. Biologically functional cationic phospholipid−gold nanoplasmonic carriers of RNA, J. Am. Chem. Soc. 2009, 131, 14066–14074. (55) Choi,Y.; Kang, T.; Lee, L. Plasmon resonance energy transfer (PRET)-based molecular imaging of cytochrome c in living cells, Nano Lett. 2009, 9, 85–90. (56) Dahlin, A.; Jonsson, M. Höök, F. Specific self-assembly of single lipid vesicles in nanoplasmonic apertures in gold, Adv Mater 2008, 20, 1436–1442. (57) Zhang, J.; Badugu, R.; Lakowicz, J. Fluorescence quenching of CdTe nanocrystals by bound gold nanoparticles in aqueous solution, Plasmonics 2008, 3, 3-11. (58) Sudheendra, L.; Han, J.; Kennedy, I. Forster resonance energy transfer measurement from a gold coated upconverting nanophosphor, Proc. of SPIE. 2010, 7576, 75760Y-5.


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(59) Chen, Y.; Munechika, I.; Plante, J.; Munro, A.; Skrabalak, S.; Xia, Y.; Ginger, D. Excitation enhancement of CdSe quantum dots by single metal nanoparticles, Appl. Phys. Lett 2008, 93, 053106. (60) Lü, Q.; Guo, F.; Sun, L.; Li, A.; Zhao, L. Surface modification of ZrO2: Er3+ nanoparticles to attenuate aggregation and enhance upconversion fluorescence, J. Phys. Chem. C 2008, 112, 2836-2844. (61) Wang, F.; Wang, J.; Liu, X. Direct evidence of a surface quenching effect on size-dependent luminescence of upconversion nanoparticles, Angew. Chem. Int. Ed. 2010, 122, 7618 –7622. (62) Aslan, K.; Wu, M.; Lakowicz, J.; Geddes, C. Fluorescent core-shell Ag@SiO2 nanocomposites for metal-enhanced fluorescence and single nanoparticle sensing platforms, J. Am Chem Soc

2007, 129, 1524-1525. (63) Cheng,D.; Xu,Q. Separation distance dependent fluorescence enhancement of fluorescein isothiocyanate by silver nanoparticles, Chem. Commun. 2007, 248–250.


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Figure 1. TEM images of Au-coated (a) and Ag nanostructure-coated silica beads; (c) schematic illustration of the immunoassay on a glass slide, reproduced from Ref [15]. 101x94mm (300 x 300 DPI)


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Figure 2. Laser scanning microscopy images of samples with 3 min Ag enhancement (a) and without Ag enhancement (b), reproduced from Ref [29]. 101x35mm (300 x 300 DPI)


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Figure 3. Flow cytometry results for 5 µm silica beads: AlexaFluor 430-antibody on the silica beads without Ag enhancing (a) and with 3 min Ag enhancing (b). Left, forward scattering (FSC) versus side scattering (SSC); Right, fluorescence histograms, reproduced from Ref [15]. 101x89mm (300 x 300 DPI)


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Figure 4. Luminescence intensities of BHHCT-Eu3+ with and without Ag nanostructures, reproduced from Ref [30]. 266x203mm (300 x 300 DPI)


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Figure 5. Representative fluorescence lifetime decay curves of BHHCT-Eu3+ labeled assay with and without Ag nanostructures, reproduced from Ref. [30] 266x203mm (300 x 300 DPI)


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Figure 6. (a) Signal intensity histograms of stained Giardia lamblia cells with and without Ag nanostructures, (b) representative images of Giardia cysts stained by BHHCT-Eu3+ on the silica beads without (upper panel) and with Ag nanostructures (lower panel), reproduced from Ref [16]. 101x38mm (300 x 300 DPI)


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Figure 7. TEM image of SiO2-coated NaYF4: Er,Yb (a) and the UC luminescence spectra observed from NaYF4: Er,Yb nanoparticles with and without Ag nanostructures (b), reproduced from Ref [17].

101x44mm (300 x 300 DPI)


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Figure 8. Representative UC luminescence decay times of green (a) and red (b) emissions from NaYF4: Er,Yb nanoparticles with and without Ag nanostructures, reproduced from Ref [17].

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Figure 9. Absorption spectra of Au coated and uncoated nanoparticles (a) and TEM image of gold shellcoated NaYF4: Er,Yb nanocrystals, reproduced from Ref [17]. 101x40mm (300 x 300 DPI)


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Figure 10. The UC luminescence spectra observed from NaYF4: Er,Yb nanoparticles with and without gold nanoshells, reproduced from Ref [17]. 101x76mm (300 x 300 DPI)


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Figure 11. Representative UC luminescence decay times of green (a) and red (b) emissions from NaYF4: Er,Yb nanopaticles with and without gold nanoshells, reproduced from Ref [17]. 101x39mm (300 x 300 DPI)


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Figure 12. TEM images of BHHCT-Eu-DPBT-doped Ag@SiO2 nanocomposites (a) and BHHCT-Eu-DPBT-doped SiO2 nanoshell without Ag core (b). 101x76mm (300 x 300 DPI)


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Figure 13. Luminescence of the nanocomposites at high excitation intensities, relative to the luminescence intensity observed at low, 1 mW/cm2 excitation. a) 33 nm core 25 nm shell and the control sample. b) 81 nm core 25 nm shell and the control sample. c) samples with fixed core of 52 nm, diamonds - 25 nm shell thickness, circle - 12 nm shell thickness, triangles - 57 nm shell thickness, stars – control sample, for 12 nm shell thickness, reproduced from Ref [18]. 50x81mm (300 x 300 DPI)


Langmuir

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Figure 14. (a) Bioassay using Ag@SiO2 nanocomposites as labels, (b) luminescence spectra of nanocomposite-labeled antibody after bioassay . Solid line - BHHCT-Eu-DPBT-doped Ag@SiO2 with 12 nm silica shell and 52 nm Ag core, dotted line - BHHCT-Eu-DPBT-doped hollow nanocomposites with 12 nm silica shell and without 52 nm Ag core. (c) The calibration curve of biotinylated IgG antibody-conjugated nanocomposites when concentration of streptavidin was 50 µg ml-1, reproduced from Ref [18]. 101x73mm (300 x 300 DPI)


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Langmuir

Figure 15. Images of single BHHCT-Eu-DPBT-doped Ag@SiO2 nanocomposites with 25 nm silica shell and 52 nm Ag core. a) conventional fluorescence image excited at 365 nm b) time-gated fluorescence image excited at 365 nm, c) TEM image at the same magnification as the fluorescence images d) enlarged image of spots in the central rectangle confirming single nanoparticles. Fluorescence images were taken at × 60 magnification in a time-gated fluorescence microscope at 10 s exposure time. Capital letters identify individual nanoparticles, reproduced from Ref [18]. 101x28mm (300 x 300 DPI)


Langmuir

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TOC 47x29mm (300 x 300 DPI)


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Plasmonic Approach to Enhanced Fluorescence ... - ACS Publications

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