Photoluminescent-based assays are highly desirable in sensing applications as a result of their excellent contrast, high specificity, and fast response times. The workhorses of these systems are photoluminescent dyes that are sensitive to changes in their environment. Typically these dyes have been employed in a variety of sensing platforms, including free in solution, thin films, membranes, and optical fibers. However, many of these platforms can affect the responsiveness of these dyes, e.g., their ability to respond to environmental conditions or analytes. In view of these drawbacks, Burns et al. seeks to provide a sensing system that can operate in a variety of environments.
Burns et al. discloses silica-based sensors comprising a reference dye and a sensor dye. The emission spectrum of the reference dye is substantially insensitive to environmental conditions and/or analytes, whereas the emission spectrum of the sensor dye has a known response to a given external condition and/or analyte. Thus, the reference dye serves as an internal standard to which the response of the sensor dye can be compared, much the same way that the residual solvent peak in proton NMR serves as an internal standard to which the proton resonances of an analyte are compared.
The silica-based sensors can have various structural configurations, but typically comprises a silica core surrounded by one or more outer shells. The outermost shell typically contains the sensor dye to allow interaction with the external environment, whereas the reference dye is typically included in the silica core or an intermediate shell to prevent the reference dye from contacting the environment.
The periphery of the silica can be tailored to allow targeted interactions to take place. For example, the silica can be functionalized with amino groups thereby imparting a positive charge to the periphery of the silica, which promotes adhesion of the amino-functionalized silica to cell surfaces. Alternatively, the silica surface can be functionalized with hydrophobic groups, which allows the silica to intercalate within the lipid bilayer of a cell wall, for example.
The sensor dyes can be sensitive/responsive to a wide range of external stimuli, such as hydrophobicity/hydrophilicity, presence or absence of metal ions, changes in pH, or identification of small molecules such as oxygen.
One specific disclosed potential application is in vivo or in vitro determination of the redox state of a cell. It is well-known that the internal cell environment is typically a highly regulated reducing environment. Any deviation from this reducing state could indicate the presence of a disease, such as cancer, or an injury, such as stroke.
Sensing environmental conditions and/or analytes is typically performed by (1) comparing the emission intensity ratios of the reference and sensor dyes, (2) comparing spectral shifts of the reference and sensor dyes (i.e., the difference between lambda maxes), and/or (3) comparing the excited state lifetimes of the reference and sensor dyes. Of course, the emissions of these dyes in various environments must be previously known via the construction of a calibration curve in order to be able to identify a certain condition or analyte in an unknown solution/medium.
The sole example in Burns et al. demonstrates the determination of the pH of an unknown sample. A silica-based sensor is first synthesized by covalently attaching a tetramethylrhodamine reference dye to a silica core, followed by layering over this core a shell comprised of a fluorescein sensor dye covalently bonded to silica. A calibration curve is then determined for this sensor by measuring the ratio of the reference and sensor dye emission intensities over a pH range of 5-9. Thus, when this sensor is added to a solution having an unknown pH, the ratio of emission intensities of the reference and sensor dyes can be measured and compared with the calibration curve, thereby determining the pH of the solution.
Burns et al. only contains product claims. Claim 1 is very broad and minimally requires (1) a reference dye and a sensor dye that are chemically different, (2) at least one of these dyes is covalently attached to a silica core, (3) the reference dye has a relatively constant emission in different environments, and (4) the sensor dye exhibits different emission in different environments or in the presence of varying concentrations of analytes.
As filed, the application originally also contained method claims that were restricted out and ultimately canceled in response to the restriction requirement. Two methods were claimed: (I) a method of determining an analyte in an unknown environment, and (II) a method of introducing a silica-based particle into an organism. Perhaps these method claims will appear in a divisional application at some point in the future, though no related applications are currently listed in PAIR.