Multifunctional nanoparticles, which incorporate diagnostic (quantum dots, magnetic, metallic, polymeric and silica nanoparticles) and/or therapeutic (magnetic and metallic nanoparticles) properties, are in the process of development. This review focuses on dye-doped silica nanoparticles and gold-based nanomaterials, which have been widely investigated by our group for bioanalytical purposes in molecular diagnostics and cancer therapy. A summary describes their main properties, as well as some recent relevant applications.
by Dr S. Bamrungsap, Dr Y-F. Huang, Dr M. Carmen Estevez and Dr W. Tan
Nanotechnology, which has received considerable attention in advanced biomedical science over the past decade, refers to the applied science involved with materials in the sub-100 nanometre range. With dimensions similar to biomacromolecules, nanoparticles can be engineered to have specific or multiple functions and can be used for investigating and pursuing an in-depth understanding of the mechanisms involved in biochemical processes. The unique characteristics of particles in the nanometre range, such as high surface-to-volume ratio or size-dependent optical and magnetic properties, are drastically different from those of their bulk materials and hold promise in the clinical field for disease diagnosis and therapeutics [1-4]. The successful transfer of nanotechnology into biological systems has been made feasible by combining the intrinsic properties of nanoparticles with immobilisation of specific ligands, such as proteins and oligonucleotides, on the surface for specific recognition. Thus, the development of multifunctional nanoparticles, which incorporate diagnostic (quantum dots, magnetic, metallic, polymeric and silica nanoparticles) and/or therapeutic (magnetic and metallic nanoparticles) properties, as well as specific targeting capability by surface modification with biomolecules, is a continuous topic of research [5-10]. In this review, we will mainly focus on a discussion of dye-doped silica nanoparticles and gold-based nanomaterials, which have been widely investigated by our group for bioanalytical purposes in molecular diagnostics and cancer therapy. A summary describing their main properties, as well as some recent relevant applications, is also given [Figure 1 and 2].
Dye-doped silica nanoparticles
The attractive properties of dye-doped silica nanoparticles have accelerated their use in bioanalysis. Specifically, their physically and chemically inert surface can protect trapped fluorescent dyes from the oxygenic environment, whereas the silanol surface allows for an easy and versatile chemical modification with various functional groups. There are two major approaches for the synthesis of dye-doped silica nanoparticles. One is reverse microemulsion, which is primarily used for the incorporation of hydrophilic inorganic dyes. In this water-in-oil (W/O) microemulsion system, water nanodroplets are formed in the bulk oil phase which acts as confined media or nanoreactors for discrete nanoparticle formation. Positively charged dye molecules, such as ruthenium complexes, can be successfully encapsulated in the negatively charged silica matrix through strong electrostatic interactions, resulting in the formation of highly uniform particles with diameters ranging from 15 to 200 nm. On the other hand, the Stöber method is based on the formation of particles by the hydrolysis of the silica precursor (e.g., tetraethylorthosilicate, TEOS) in ethanolic media containing water and ammonia as the basic catalyst. This method is highly efficient for the entrapment of more hydrophobic molecules and has been modified by adding different organosilanes with appropriate functional groups (i.e., amine-containing silane agents, such as 3-aminopropyltriethoxysilane, APTES), which can be linked with dye molecules, allowing the incorporation of covalently coupled molecules to the silica matrix. Thus, these particles make excellent multifunctional fluorescent probes for ultrasensitive detection in bioanalytical and biomedical areas because of 1) their extraordinary brightness, which is provided by the entrapment of hundreds, or even thousands, of dye molecules inside their matrix and 2) their extremely high stability which is conferred by the silica shell, thus avoiding photobleaching and
photodegradation of the dyes.
Fluorescent silica NPs have been used for the development of an ultrasensitive DNA
hybridisation assay [11]. The sandwich assay design was based on the immobilsation of two different DNA capture sequences, one onto a glass surface and the other one attached to dye-doped silica nanoparticles. The unlabelled target DNA sequence was complementary to both capture sequences. After specific DNA hybridisation onto the solid surface, and with the subsequently added NPs, an enhanced fluorescent signal could then be monitored and quantified. Because of the effective surface modification, only minimal nonspecific binding of the NPs onto the glass surface with no aggregation of nanoparticles was observed. Furthermore, the NPs have proved to be effective as luminescent probes in commercial microarray systems, such as Affymetrix GeneChips [12]. In an Affymetrix GeneChips system, multiple probes homologous to different regions of target RNA are designed and immobilised on the arrays. The biotinylated cRNA is then hybridised to the GeneChips. After hybridisation, the arrays are washed and stained with Ruby dye-doped nanoparticles [Figure 1a]. Thus, by their simplified staining procedure, higher photobleaching threshold, and enhanced fluorescent signal allowing a concentration detection limit of 50 fM, nanoparticles have been found to be superior to the traditional protein Streptavidin-Phycoerythin
(SA-PE) approach.
These probes have also been used for the detection of whole living entities, such as bacteria and cancer cells. For instance, a simple, rapid, and sensitive fluorescence-based immunoassay has been developed using bioconjugated silica nanoparticles for the detection of single E. coli O157:H7 [13]. Antibodies against single E. coli O157:H7 were conjugated to dye-doped silica nanoparticles to form nanoparticle-antibody complexes which could specifically bind to the antigens expressed on the E. coli O157:H7 surface. The high signal intensity, together with the presence of a high number of antigens on the target bacteria surface which are available for specific recognition, provided an extremely strong fluorescent signal allowing single bacterial cell detection within 20 minutes. Moreover, accurate enumeration of 1-400 bacterial cells in 1 g of spiked ground beef sample was demonstrated with the methodology developed. Furthermore, the ability to encapsulate not only one type of dye, but also a combination of fluorophores, all of which undergo efficient energy transfer, resulted in different nanoparticles with tunable final emission using a single excitation source, which opens the door to multiplexing analysis. Thus, by using three different NPs encapsulating different ratios of three different dyes,
simultaneous analyses of three different bacteria were conducted by immobilising specific and
distinctive antibodies to each one [14] [Figure 1b].
On the other hand, in the area of early diagnostics, there is a continuing need for effective and sensitive methodologies which allow accurate detection of disease in the early stages. In this context, ultrasensitive detection of low concentrations of abnormal cells is required. A recently developed and straightforward approach to this problem combined dye-doped silica nanoparticles and magnetic core-silica shell nanoparticles for the collection and detection of cancer cells [15]. Both types of NPs were conjugated to specific aptamers, which were selected for the specific detection of leukaemia cancer cells. This protocol involved the selective extraction and separation of the target cancer cells using the aptamer-conjugated magnetic nanoparticles (AMNPs) under the application of an external magnetic field. Then, the corresponding aptamer-conjugated fluorescent nanoparticles (AFNPs) were used for cellular detection. As a consequence of the high brightness of the silica NPs, an extremely low detection limit of approximately 250 cells was achieved, with a wide dynamic range covering more than two orders of magnitude using pure samples. The assay was fast (less than 30 minutes) and easy, compared with other methodologies, such as PCR-based assays or immunophenotyping. Since it worked in a similar manner in more complex matrices, such as serum and whole blood, the effectiveness of the initial extraction step was firmly established. Moreover, a multiple successive extraction of three different types of cancer cells has been further performed by using three different dye-doped nanoparticles [16] [Figure 1c]. Overall, these findings clearly demonstrate the high potential of nanoparticles for ultrasensitive detection of bacterial pathogens and cells in the clinical, environmental and food fields.
Gold-based nanoparticles
Such fascinating features as ease of synthesis and surface functionalisation with thiol-containing molecules, non-cytotoxicity, high biocompatibility, as well as broad-based optical properties, make gold-based nanoparticles still another attractive nanomaterial and one of the most studied in the bioanalytical field. Gold nanoparticles with tunable size (~0.8 - 60 nm in diameter) and narrow size distribution (± 10% in deviation) can be synthesised by physical methods, including photochemistry, sonochemistry and radiolysis, and by chemical methods, including reduction of HAuCl4, microemulsion and seeding growth. Owing to the quantum size effect, gold nanoparticles possess strong surface plasmon resonance (SPR) bands in the visible wavelength range. Their SPR frequencies are also strongly influenced by the interparticle distance. When the gold nanoparticles in solution are in close proximity with others, their overall change in surface plasmon is translated into absorption spectra shifts resulting in a change in sample colour. In past years, this characteristic has been exploited to develop various techniques for selective colourimetric detection of ions, genes, proteins and saccharides. It has also been recently applied to the direct detection of cancer cells [17] by conjugating specific aptamers onto the surface of the gold nanoparticles. Once the gold nanoparticle-aptamer complexes selectively bind and assemble around the target cells, the aggregation of gold nanoparticles causes a red shift of absorption spectra, resulting in a noticeable colour change in solution [Figure 2a]. The results can be observed with the naked eye without sophisticated and expensive instrumentation and can also be quantified using conventional UV-visible spectrometers. The assay performed well in complex matrices, such as serum samples, and showed excellent sensitivity and selectivity for the target cells in the complex system, with low nonspecific binding in control cells.
By changing the shape of gold nanoparticles from spheres to elongated rods, another SPR band at a longer wavelength arises from the plasmon oscillation of electrons along the longitudinal axis. This longitudinal SPR band can be shifted into the near-infrared region (NIR) by an increase in the nanorod aspect ratio, which is the ratio of length to diameter. It has been demonstrated that multimetallic nanocomposites, such as gold-silver nanorods, also possess sharper and stronger longitudinal SPR bands, as well as higher molar absorptivity, than spherical gold nanoparticles or gold nanorods. Thus, gold-silver nanorods are considered attractive candidates for photothermal therapy as a consequence of their excellent absorption in the near infrared (NIR) range and their high efficiency in converting light energy to local heat. Recently, we reported the use of aptamer-conjugated gold-silver nanorods for specific target cell recognition and photothermal therapy [18]. Highly specific aptamers selected against leukaemia cancer cells were conjugated on the nanorod surface through thiol linkage. The aptamer-nanorod conjugates exhibited excellent specific recognition of both suspension- and adherent-targeted cells. Our nanorods exhibited high molar absorptivity by utilising only 8.5×104 W/m2 laser exposure to induce 93% cell death as compared to gold nanoshells or gold nanorods which required 1×105 ~ 1×1010 W/m2 of laser irradiation. Moreover, results demonstrated that about 50% of target (CEM) cells were severely damaged after laser exposure, while 87% of control (NB-4) cells still remained intact in suspension cell mixture [Figure 2b].
Conclusions
Nanobiotechnology has become an attractive and promising research area with potential application in many diversified fields, and it has played a particularly important role in biomedicine. Nanoparticles have emerged as promising nanoplatforms for efficient diagnostics and therapeutics by merging the characteristic properties they possess at the nanometre scale with the feasible immobilisation of specific ligands on the surface. Therefore, they have become ideal candidates for molecularly sensitive detection, highly efficient contrast agents for molecular imaging, as well as carriers for targeted drug and gene delivery, and therapeutical reagents for targeted photothermal therapy. Nonetheless, a better fundamental understanding of the behaviour of nanomaterials in biological systems needs to be addressed, as well as the engineering of novel nanoparticles, which can overcome the drawbacks related to currently developed nanomaterials, including nonspecific binding, aggregation, toxicity and biodistribution.
Acknowledgements
This work was supported by NIH NCI grant and by State of Florida Center for NanoBiosensors. S. B. acknowledges financial support from The Royal Thai Government, Thailand. M.-C.E acknowledges financial support from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya, Spain.
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The authors
Suwussa Bamrungsap, Yu-Fen Huang, M.- Carmen Estevez and Weihong Tan
Center for Research at the Bio/Nano Interface,
Department of Chemistry and Physiology and Functional Genomics,
Shands Cancer Center and UF Genetics Institute,
University of Florida,
Gainesville,
Florida, 32611-7200,
USA
