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Plasmonic-Fluorescent and Magnetic-Fluorescent Composite Nanoparticle as Multifunctional Cellular Probe

Arindam Saha, SK Basiruddin, and Nikhil Ranjan Jana

Centre for Advanced Materials, Indian Association for the Cultivation of Science, Kolkata, India

1.1 Introduction

The interaction of fluorophore with metal has been a field of research since many decades. In 1970s, Drexhage found that fluorophore located near the metallic surfaces shows oscillations of the emissive lifetime depending on the distance from the metal surface [1, 2]. After this finding, many theoretical [3-9] and practical researches were directed in this field [10-16]. Now it is well known that when a metal surface interacts with an excited-state fluorophore, two phenomena can take place, either the fluorescence of the fluorophore is enhanced or it is quenched. When a metal stays very close to the fluorophore, typically within 20?nm, the electrons from the excited fluorophore transfer to the metal and hence result in fluorescence quenching. But when the metal surface stays 20-50?nm away from the fluorophore, the excited electrons of fluorophore interact differently with the metal electrons and it results in an increase in the rate of fluorescence decay as well as the rate of excitation. The effect is enhancement of fluorescence with decreased lifetime and increased photostability. This phenomenon is termed as metal-enhanced fluorescence (MEF) [14, 17, 18]. Most of this MEF has been observed for silver island film [17, 19-26], silver colloids [27-29], other silver nanostructures [30, 31], gold [32-35], iron oxide [36], tin [37], platinum [38], aluminium [39], and so on. Among different metals, nanosize silver and gold are studied more frequently as they have plasmonic properties in the visible region arising due to collective oscillations of surface electrons and that can significantly modulate the MEF properties.

Different options of a fluorophore under proper excitation are shown in Scheme 1.1. According to the aforementioned diagram, when a fluorophore is excited with an appropriate energy, it reaches to a higher electronic level and then undergoes decay in both radiative and non-radiative ways. Here G indicates the rate of radiative decay; Knr is the rate of non-radiative decay and rate of quenching collectively. When the fluorophore is within the distance of 20?nm from the metal surface, then Knr?>?G and as a result both quantum yield (QY) (Q0) and lifetime (t0) decrease. But when the distance between the metal surface and fluorophore is typically between 20 and 50?nm, G?>?Knr and hence Q0 increases but t0 decreases, which is indicated by MEF. Since the lifetime decreases, the fluorophore remains in the excited state for a shorter time period and their chance of non-radiative decay or other excited state reaction is decreased that results in increased photostability [18, 23, 40-42]. When the distance is larger than 50?nm, there is preferably no interaction between the fluorophore and metal surface.

Scheme 1.1 Different options of electron-hole recombination within a fluorophore. Here E and Em are energy of excitation for fluorophore and metal plasmon.

In fluorescence-based experiments (e.g., sensor application, cellular imaging, molecular probe design), metal-induced quenching is a critical drawback [43] and difficult to overcome completely. However, this quenching effect has been exploited as "Turn Off" detection of analyte in many cases, although this type of sensor design is neither specific nor accurate since many other factors can influence the quenching phenomenon. Alternative "Turn On" sensor design is of utmost importance where the presence of analyte is more selectively detected by the fluorescence enhancement effect [44]. In cellular imaging, the conventionally used molecular dyes often undergo photobleaching and hence long-term tracking of cellular activities is difficult. Although new dyes have been developed with reduced photobleaching properties, the quenching of the dye under complex cellular environment is unavoidable that often hinders the imaging. Various semiconductors and other fluorescent nanoparticles are emerging as promising alternatives, but toxicity issues question their application potential for in vivo imaging and long-time cellular tracking [45-48].

The most adverse effect of metal-induced quenching occurs in the preparation of multifunctional probe, which is one of the current research directions. Various multifunctional probes composed of metallic and fluorescent components have been developed and many are still under development stages. These probes can be used for multiple applications like sensing, detection, imaging, and separation [49-55]. However, synthesis of these probes is a major challenge since the metal-based plasmonic or magnetic component in the composite system quenches the fluorescence of the fluorophore component. In order to solve this problem, various methods have been developed via tuning the separation distance between fluorescent components and quencher components, and in some selected cases MEFs are also observed [56-64].

We are interested in the development of noble metal nanoparticle-based hybrid nanoprobes composed of plasmonic and fluorescent components so that they can be used for different cellular labeling applications. Although noble metal nanoparticles have tunable optical property due to surface plasmon, their strong quenching property creates a difficulty in making hybrid nanoprobes. Thus appropriate methods need to be developed for synthesis, keeping optimum separation distance between the plasmonic and fluorescent components. In addition the methods should be simple and cost effective with options for various functionalizations. We have developed different methods to prepare different plasmonic-fluorescent nanoprobes that are composed of gold/silver nanoparticles of different sizes/shapes as plasmonic components and fluorescein or CdSe/ZnS quantum dots (QDs) as fluorescent components [65-68]. In addition to plasmonic-fluorescent hybrid nanoparticle, we are also developing magnetic metal oxide-based magnetic-fluorescent hybrid nanoparticles that can be used for magnetic separation applications [67, 68]. In all these hybrid nanoprobes, the fluorescence of the fluorophore component is partially quenched with the final QY ranging between 7 and 20%. These hybrid nanoparticles can be used for both fluorescence and plasmon-based imaging probes. Here we will summarize different approaches for their synthesis, functionalization, and application potential.

1.2 Synthesis Design of Composite Nanoparticle

Our research goal is to develop synthetic approach for plasmonic-fluorescent, magnetic-fluorescent, magnetic-plasmonic, or a combination of all the three components and applications of all these composites in various biomedical fields, such as cellular imaging, cellular targeting and separation, protein detection, and study of carbohydrate-protein interaction. Here we will mainly focus on fluorescence-based composite materials. There are three general steps in the synthesis of these composite nanoparticles. First, high-quality hydrophobic nanoparticles, such as Au, Ag, CdSe/ZnS, and iron oxide; and hydrophilic nanoparticles, such as Au nanorod, Ag plate, Ag-coated Au nanorod have been synthesized by different reported methods [69-73]. Next, these as-synthesized nanoparticles are converted into polyacrylate-coated water-soluble nanoparticles when needed, using the reported methods [74-76]. Finally, composite materials have been synthesized using three different approaches shown in Scheme 1.2. Table 1.1 summarizes all types of composite nanoparticles prepared by these approaches along with their properties.

Scheme 1.2 Different synthesis approaches for multifunctional nanoparticle.

Table 1.1 Summary of different composite nanoparticles along with their properties and application potentials.