Anal Bioanal Chem (2003) 376 : 284286 DOI 10.

1007/s00216-002-1734-8

TRENDS

Dale M. Willard

Nanoparticles in bioanalytics

Published online: 15 February 2003 Springer-Verlag 2003

Inorganic compounds with nanometer dimensions display properties unlike the bulk material due to electronic confinement effects. The result is a new class of compounds called nanoparticles. Since the 1970s, primarily physicists and material scientists investigated nanoparticles to identify and exploit their unique properties. Applications originally centred on electronic and optical devices, however, research is now proving that some of the most exciting applications will be in bioanalytics. This brief review outlines recent advances using nanoparticles in bioanalytics with emphasis on small metal colloids and semiconductor quantum dots. Nanoparticles are primarily being introduced into bioanalytics as an improved alternative to conventional fluorescent labels or label-free sensing elements, such as replacements for organic dyes or metallic thin layers used in Surface Plasmon Resonance (SPR). Like metallic thin films, Au and Ag nanoparticles display a Plasmon Resonance that is sensitive to refractive index changes in the surrounding environment. However, unlike metallic thin films that display a polarization-dependent infrared Plasmon Resonance, because of confinement effects, nanoparticles display a Localized Surface Plasmon Resonance (LSPR) displaying an absorption peak in the visible spectrum and excitable with non-polarized light. In addition, these nanoparticles produce intense Surface Enhanced Raman Spectra (SERS) effects. Semiconductor nanoparticles, commonly called quantum dots, have dimensions smaller than the Bohr radius of an electron in the bulk material. This confines the electron/hole pairs such that behaviour mimics a particle in a box. The most popular quantum dots have large fluorescence quantum yields, resistance to photobleaching, good chemical stability, and Gaussian emission profiles (~30 nm FWHM). One can tune the properties of nanoparticles by controlling the

size, shape, and preparation procedures. For example, the emission from CdSe quantum dots can be adjusted nearly continuously from 450650 nm. Nanoparticle sizes, typically on the order of 10 nm, are comparable to biological proteins. Nanoparticles can be suspended in solution for in-vivo studies or aggregated in certain conditions, thus providing a simple separation tool. Figure 1 is an example of the brilliant images that are possible when biological samples are labelled with CdSe quantum dots. Several recent reviews discuss the properties of nanoparticles in greater detail [1, 2, 3]. To date, the stability and solubility in aqueous solutions is the largest limiting factor that keeps nanoparticles from being pervasive in bioanalytical laboratories. To be biologically relevant, nanoparticles need to have surface

D. M. Willard () Institute for Physical Chemistry, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany e-mail: dale.willard@ipc.uni-tuebingen.de

Fig. 1 Microtubules in 3T3 cells labelled with Qdot 605 Streptavidin Conjugate after the cells were incubated with monoclonal anti-tubulin antibodies and biotinylated goat anti-mouse IgG. Nuclei were counter-stained with blue dye Hoechst. Image was captured with a Nikon E-800 fluorescent microscope equipped with a CCD camera. Image courtesy of Quantum Dot Corporation, Hayward, CA, USA. www.qdots.com

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Fig. 2 A family of quantum dots emits a spectrum of colours when excited with a single light source. Image copyrighted: Felice Frankel, Envisioning Science, the Design and Craft of the Science Image

functionality amenable to biological derivatisation, solubility and long term stability in a range of buffered saline solutions and pHs, and limited non-specific binding. Many recent studies have addressed these issues. The Alivasatos group developed CdSe quantum dots coated with siloxane and poly(ethylene glycol) (PEG) layers [4, 5]. These quantum dots were derivatised with DNA that was then used to capture the quantum dots on DNA labelled surfaces. Mattoussi and coworkers developed a novel electrostatic coupling of quantum dots and proteins [6, 7, 8]. Others have developed new thiolyting groups to stabilise the surface chemistry [9, 10]. Another study found that Ag nanoparticles can be stabilised by adding a thin layer of Au without disrupting the underlying Ag LSPR property [11]. Two papers developed different quantum dot derivatisation procedures that suppress non-specific binding and targets them to specific sites within biological samples. The first study demonstrated fluorescence in-situ hybridization experiments [9]. Quantum dots were derivatised
Fig. 3 Graphic depicting how quantum dots can be inserted into polystyrene beads to create distinct spectral barcodes. Image courtesy of Quantum Dot Corporation, Hayward, CA, USA. www.qdots.com

with a hydroxyl-disulfide organic cap to reduce non-specific binding and further derivatised with oligonucleotides that targeted them to chromosomes in human sperm cells. A more recent study found that quantum dots derivatised with PEG and polypeptides can be used for in-vivo studies [12]. The PEG reduced non-specific binding and the peptides directed the quantum dots to the lungs of mice. Since quantum dots were first demonstrated as biological probes [13, 14], they have been promoted for multilabel experiments. Different sets of nanoparticles can be simultaneously excited with a single laser source, detected individually by their unique emission/scattering, and biologically derivatised with similar procedures (see Fig. 2). Recently, Mattoussi and co-workers demonstrated a dual-quantum dot assay [6]. Other papers utilized quantum dots in Energy-Transfer studies [15, 16]. Mirkin and co-workers demonstrated a dual-Au nanoparticle assay with detection via scattering [17]. In addition, two papers found Au nanoparticles to be very efficient fluorescence quenchers and can be effectively utilized in Molecular Beacon or Molecular Beacon-like assays [18, 19]. Nanoparticles hold particular promise as the next generation barcodes for multiplexing experiments (see Fig. 3). Genomic and proteomic research demands greater information from single experiments. Conventional experiments utilize multiple organic fluorophores to barcode different analytes in a single experiment, but positive identification is difficult because of the cross-talk signal between fluorophores. Han et al. encapsulated CdSe quantum dots in polystyrene beads with varying concentrations and sizes [20]. The narrow Gaussian emission profile of quantum dots reduces cross-talk. Therefore, eight bead variations were clearly identifiable with many more possible. Another study took advantage of the large SERS effect next to Au nanoparticles plated with Ag [21]. Nanoparticles labelled with dyes produced a clear fingerprint revealed by the Raman Spectra. Another study prepared nanometer-micrometer length metal rods coded with strips of differing composition that could be identified optically via reflectivity [22]. Yet another study found that

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spherical Ag nanoparticles can be converted to triangular prisms with simple UV irradiation, adding shape as a distinguishing feature of mixed nanoparticles in solution [23]. Several studies demonstrated Au [24, 25] or Ag [26, 27] functionalised colloids/nanoparticles can be utilised as an alternative to SPR. The assays are label-free with surface binding events inducing a shift in the LSPR. As opposed to conventional SPR, changes in the LSPR can be monitored with simple UVvisible spectrometers and the technology is easily transferred to high density array platforms. However to date, nanoparticles based assays can not match the limit of detection obtained by conventional SPR. Functionalised nanoparticles dispersed in solution can be aggregated by adding a cross-linking reagent. The LSPR is very sensitive to aggregation and can be monitored with UVvisible spectrometry. In addition, aggregates can be separated from other solvated components by centrifugation. Three papers utilized these advantages to develop assays for lectin [28], anti-Protein A antibodies [29], and metal ions [30]. Another study extended these advantages by labelling the nanoparticles with oligonucleotide-recognition elements [31]. This permitted multianalyte assays since separated aggregates could then be identified by their oligonucleotide-elements with either thermal de-hybridisation or chip-based detection. Three final, but no less noteworthy articles can be viewed as unique research. Hamad-Schifferli et al. [32] remotely heated Au functionalised nanoparticles by a radio frequency magnetic field. The local heating of the nanoparticles induced DNA de-hybridisation that could then be re-hybridised when the field was removed, in effect producing electronically controlled nano-machinery. Park et al. [33] developed a very sensitive DNA assay detected with simple electrical conductance measurements. They developed methods to grow Ag wires between micro-gates containing Au nanoparticles. The Au nanoparticles were functionalised with oligonucleotides that permitted binding to the gate region of the chip only when target analytes are present; 500 fmol L1 concentrations were detected. The authors propose that the assay is easily amenable to micro-array formats. Finally, Gearhert et al. [34] used Au nanoparticles as a template for the study of DNA curvature conformations. Utilizing SERS effects, they measured oligonucleotides breathing vibrations to identify the presence of straight, bent, or kinked DNA. Clearly, nanoparticles have a promising future in bioanalytics. Their utilisation will be driven by the need for smaller detection platforms with lower limits of detection. Once nanoparticles become commercially available and their solubility and stability issues addressed, we can expect great things from these little packages.

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