Increased understanding of cellular and viral functions and regulating mechanisms is anticipated to lead to novel approaches for therapy and novel therapeutic agents. There is accordingly much interest in cellular and viral nano-tools as a means of investigating aspects of cellular and viral function and mechanisms. Dahm et al., (Journal of Biochemistry 111:279-290, 2004) describe the use of ultra-stable zeolite particles as a tool for in-cell chemistry. In particular Dahm et al., describe the use of labeled Y zeolite particles which were absorbed into THP-1 cells by phagocytosis and concluded that these zeolites could be used as effective carriers for delivery of low molecular weight molecules into cells. The zeolite particles are made ultra-stable by de-aluminating the natural structure.
Natural zeolites are minerals that are the result of a very low-grade metamorphism, typically found in the cavities or vesicles of volcanic rock. They are framework aluminosilicate consisting of interlocking tetrahedrons of SiO4 and AlO4, wherein the corner-sharing SiO4 and AlO4 tetrahedra of the crystalline aluminosilicate give rise to one-dimensional channels arranged in a hexagonal structure. Each aluminum entity in their framework contributes a negative charge that is compensated by an exchange of cations such as sodium, calcium and the like that reside in the large vacant spaces and cages in the structure (see Breck in Zeolite Molecular Sieves, 752, Wiley, New York, 1974; and Baerlocher et al., in Atlas of Zeolite Framework Types, 19, Elsevier, Amsterdam, 5th edition, 2001). The stoichiometry is (K)9[Al9Si27O72].nH2O, where n is 21 in fully hydrated materials and 16 at about 22% relative humidity. Out of 9 potassium cations per unit cell, 3.6 can be exchanged by other monovalent cations, or an equivalent amount of divalent or trivalent cations.
Many forms of Zeolite are known, each form exhibiting different geometrical and chemical characteristics. For example, Zeolite L exhibits one-dimensional channels running through the whole crystal, with an opening of 0.71 nm, a largest free diameter of 1.26 nm and a unit cell length of 0.75 nm (see FIG. 1a). The centre-to centre distance between two channels is 1.84 nm. As an example a crystal with a diameter of 550 nm consists of about 80,000 parallel channels. Zeolite L channels can be filled with suitable organic guest molecules, although only guests that can pass through the opening are able to enter the channels. Due to the channel entrances, the chemical and physical properties of the base and coat of the cylindrical crystals are different. Exemplary guest molecules include certain dyes possessing desired emission properties (see Calzaferri et al., Angew Chem Int Ed 42:3732, 2003). Zeolite L crystals can also be prepared in different sizes (diameter and length). For example the length of a zeolite L crystal can be from 30 nm to several thousand nanometers (see Ruiz et al., Monatshefte Fur Chemie 136:77, 2005). Pure zeolite L crystals with lengths between 30 and 7000 nm have been synthesized previously (Megelski and Calzaferri, Adv Funct Mater 11:277, 2001).
Dye-loaded zeolites are described in European Patent No 1 335 879 (University Bern). This patent teaches the loading of a dye into interior channels within a zeolite L crystal. The entrances to the channels are terminated or blocked by closure molecule, especially by a so-called stopcock moiety. The teachings of this patent document are directed toward to the construction of a luminescent optical device, an optical sensor device, a light-emitting device and a photonic energy harvesting device.
It is known that the channel entrances of a zeolite L crystal can be blocked or closed by a closure molecule. Such closure molecule may be chemically modified to carry for example an amino group (see Huber et al., Angew Chem Int Ed 43:6738, 2004) or a carboxylate group (H. Li, Z. Popovic, L. De Cola, G. Calzaferri, Micr Mes Mat, 95:112, 2006). In this process preferably a stop-cock molecule, i.e., a molecule having a head group a and a tail, comprising for example a hydrophilic anchor group and a spacer, partially enters a channel of the zeolite L crystal. The anchor group, such as methoxysilane, and the spacer enter the channel allowing the anchor to attach to the zeolite L crystal. The bulky head group, e.g. a fluorenylmethylcarbamate group, remains outside the channel entrance due to size restriction imposed by the channel dimensions. Depending on the reactivity of the label, attachment to the crystal can either be reversible or irreversible. The head group is then chemically detached to e.g. result in an amino functional group at the end of a channel.
Much of the work on zeolites to date has been on the use of the zeolite crystals in photonic applications (as reported in the University of Bern patent). The inventors of the present application have, however, realized that zeolites are used in diagnostic assays for both in-vivo and in-vitro diagnostics. The inventors have realized that it is possible to attach an affinity binding agent e.g. via the stopcock moiety to for a conjugate. Such conjugate comprising an affinity binding agent under appropriate conditions will bind to another biological moiety or chemical moiety. The dye loaded in the channels or at the outside of the zeolites allows the simple detection of the conjugate bound to the biological moiety or the chemical moiety.
USPTO Backgrounds
USPTO Backgrounds