by Dr David E. Benson
Why is there such interest in using cadmium-containing semiconductors in biotechnological applications? There are three major advantages that small, synthetic particles of semiconductors (also termed “quantum dots”) posses relative to organic or metallo-organic fluorophores: size and material tunable emission wavelengths, high quantum yields and high resistance to photo-degradation [1]. The semiconductor where the majority of these concepts have been developed is based on nanoparticles of cadmium selenide, where the diameters range from 25 to 60 Å and the emission colours range from blue to red. Other less toxic semiconductor materials, indium phosphide [2] and manganese doped zinc selenide [3], can be used as well; however, the synthetic chemistry and photophysics are less developed. Semiconductor nanoparticles with high quantum yields are typically coated with a shell of insulating material (coating or shell), such as zinc sulphide, and are termed core-shell materials. Molecules that coordinate the surface cations are also used in synthesis and keep these nanoparticles in solution (capping groups). Water-soluble semiconductor nanoparticles are commercially available [3-5] with a range of capping groups and polymers [Figure 1], which are the first portion of these nanoparticles encountered by a biological system. For developing a biotechnological application of semiconductor nanoparticles, the nanoparticle core, coating and capping ligand/polymer need to be carefully considered for the particular application.
The attributes of semiconductor nanoparticles have led to use in fluorescence microscopy of living cells and deep-tissue imaging of mammals. While intravenous injection of semiconductor nanoparticles is used for deep tissue imaging of the mammalian vascular system, nanoparticle uptake into living cells is being investigated [6]. Uptake of semiconductor and metallic nanoparticles seem to readily occur, depending on the cell line and nanoparticle constitution. Ready uptake of nanoparticles by cells has also presented concerns about the toxicity of semiconductor nanoparticles with respect to deep tissue imaging, but also inhalation and skin exposure in the laboratory. Control of nanoparticle uptake into cells and specific cells is under investigation, but initial results suggest capping groups present on the nanoparticle exterior control cell uptake. Once inside the cell, semiconductor nanoparticles can be localised to vacuoles, localised to the cytoskeleton, or distributed through the cytoplasm. More work has been performed on nanoparticle capping groups that facilitate release from vacuoles. Clearly the role of capping ligands/polymers in cellular uptake and localisation remain critical to using semiconductor nanoparticles in fluorescence microscopy and deep tissue imaging. Once the distribution of fluorescence from semiconductor nanoparticles in living cells and mammals is relatively uniform [7], one can envision using nanoparticle emission intensity to report the concentrations of certain nucleic acids, proteins or small molecules.
Molecular detection by semiconductor nanoparticles in living cells or cell cultures is a long-term goal driving the biotechnological development of these fluorophores. These aspirations can be divided into macromolecule detection (proteins and nucleic acids) and small molecule detection. Macromolecule detection has been primarily reported due to large spatial distributions of macromolecular concentrations. Large spartial distribution allows fluorescence microscopy with semiconductor nanoparticle fluorophores to be used in a qualitative method, despite a uniform background emission from non-detecting nanoparticles. Macromolecule detecting functions are provided by covalent/coordinate attachment of antibodies, peptides or oligonucleotides to semiconductor nanoparticles. One of two detection methods are used for these biosensors; nanoparticle localisation or enzymatic release of nanoparticle quenchers. Enzymatic release strategies bring an important dynamic to fluorescent probe development, whether a single event or a real-time measurement needs to be performed. The localisation strategy allows for real-time monitoring, while the enzymatic method only observes a one-time event when the nanoparticle has been covalently modified (an irreversible response). Sometimes irreversible
responses are ideal, such as for low concentrations of enzymes where the irreversible response increases the method sensitivity. Despite enzymatic signal amplification, the photostability of semiconductor nanoparticles suggests real-time monitoring methods will more effectively out compete organic and metallo-organic fluorophores.
Detection of low-molecular weight molecules in living cells also requires real-time microscopic detection. The goal of these studies is not the detection of the average concentration of a molecule (e.g. glucose) in a cell. Average molecule concentrations can more easily be addressed by a variety of bioanalytical techniques that can even involve cell lysis. The advantage provided by fluorescence microscopy in small molecule detection is the ability to image concentration fluxes as a function of a stimulus. Such a desire fueled development of calcium ion-sensitive organic fluorophores: to image calcium ion fluxes involved in neural signalling. In this era of “-omics,” these studies have been termed fluxomics. There are biosensors based on fluorescent proteins that have been used to image glucose, phosphate and glutamate fluxes in cells [8]. Using semiconductor nanoparticle-based biosensors would provide longer event time monitoring and use in cells with high background fluorescence (e.g. flavins) or absorbance (e.g. red blood cells). The fluorescent protein-based sensors, however, demonstrate the need for a biosensor that only has the analyte alter the molecular weight of the biosensor during detection (termed reagentless or unimolecular). The unimolecular criterion allows for reversible analyte detection and real-time monitoring.
Two methods have been reported for unimolecular biosensors of small molecules that use semiconductor nanoparticles [Figure 2]. Energy transfer (photonic coupling) from cadmium selenide nanoparticles to a Cy3 fluorophore has been reported for unimolecular maltose biosensing [9]. This method could be thought of as maltose binding to the protein, maltose binding protein, causing a conformation change [Figure 2, top right] in the protein and that conformation change altering the nanoparticle-Cy3 distance. The spectra from this system show no increase in the nanoparticle emission, but only a change in the Cy3 emission. This points to a biosensing mechanism where maltose-driven conformation change alters the association of the pendant Cy3 with the protein surface/interior [Figure 2, bottom right]. Therefore, the energy transfer strategy relies on the fluorescence of an organic fluorophore whose photo stability relies on the organic fluorophore. Furthermore, for each emission wavelength the energy transfer method will need to develop a new nanoparticle/organic fluorophore pair. Electron transfer provides a solution to unimolecular biosensing with semiconductor nanoparticles that uses the nanoparticle emission intensity. A maltose-dependent change in the distance between a redox-active metal complex and the surface of a cadmium selenide nanoparticle was driven by conformation change [Figure 2, top left] in maltose binding protein [10, 11]. Differences in distance-dependent sensitivity between energy and electron transfer, where electron transfer has a sharper response function, is the primary reason for nanoparticle emission response with electron transfer. The biosensors mentioned above that use energy transfer between fluorescent proteins [8], like the nanoparticle-Cy3 system [Figure 2, bottom right], do not operate in a distance dependent fashion but change the relative orientation of one fluorophore to the other. Such an orientation-dependent energy transfer strategy would be difficult with a semiconductor nanoparticle due to the spherical emission, as opposed to planar emission, from organic fluorophores. Since electron transfer provides a unimolecular nanoparticle emission response, the size-dependent, material-dependent tunable emission wavelengths and photostability of semiconductor nanoparticles can be utilised in biosensor development.
There are two issues with the electron transfer method for semiconductor nanoparticle-based biosensing: the ability to use binding pocket solvation and generating a ratiometric emission wavelength response. Ratiometric wavelength responses will require new nanomaterials to be developed. One could envision using nanorods for this biosensing, but specific protein attachment to the rod terminus might be required. Alternatively, nanoparticles that have emission coming from both the core and the coating (type-II semiconductor nanoparticles) could provide the needed ratiometric emission wavelength response. With respect to binding-pocket solvation as a biosensing method, it was recently shown that using a redox-active metal complex with ammine ligands allows for active site hydration (Figure 2, bottom left) to be reflected by nanoparticle emission [12]. This work generated fatty acid biosensors from rat intestinal fatty acid binding protein and demonstrated an additional, orthogonal axis for developing semiconductor nanoparticle-based biosensors.
Taken together, semiconductor nanoparticle-based biosensors will soon be used in live-cell imaging of small molecule fluxes. Once the toxicity issues surrounding semiconductor nanoparticles are better understood, deep tissue imaging of mammals could be used to measure molecular fluxes in the vascular system. These biotechnological innovations will enable long-term imaging of molecular fluxes, so that linkages between disease and molecular fluxes can be better understood. However, nanoparticle components and biosensing mechanisms need to be considered together in order to develop these biosensors.
REFERENCES
1. Alivisatos AP et al. Ann. Rev. Biomed. Eng 2005;7:55-76.
2. Xie R et al. Colloidal InP. J Amer Chem Soc 2007; 129:15432-15433.
3. NN-Labs LLC, www.nn-labs.com
4.Invitrogen, www.invitrogen.com/site/us/en/home/brands/Product-Brand/Qdot.html
5. Evident Technologies, www.evidenttech.com
6. Smith AM et al. Adv Drug Delivery Rev 2008;60:1226-1240.
7. Yezhelyev MV et al. J Amer Chem Soc 2008;130:9006-9012.
8. Wiechert W et al. Curr Opin Plant Biol 2007;10:323-330.
9. Medintz IL et al. Adv Mater 2005;7:2450-2455.
10. Sandros et al. . J Amer Chem Soc 2005;127:12198-12199.
11. Sandros MG et al. . Analyst 2006;131:229-235.
12. Aryal BP & Benson DE. J Amer Chem Soc 2006;128:15986-15987.
THE AUTHOR
Dr David E. Benson
Associate Professor
Department of Chemistry & Biochemistry
Calvin College
1726 Knollcrest Circle SE
Grand Rapids, MI 49546, USA
email: dbenson@calvin.edu
