WO2007011660A2

WO2007011660A2 - Quantum dot probes

Info

Publication number: WO2007011660A2
Application number: PCT/US2006/027244
Authority: WO
Inventors: Jennifer L. West; Rebekah A. Drezek
Original assignee: William Marsh Rice University
Priority date: 2005-07-14
Filing date: 2006-07-14
Publication date: 2007-01-25
Also published as: WO2007011660A3

Abstract

A composition comprising a quantum dot, at least one metal nanoparticle, and at least one tether that is attached to the quantum dot and to the at least one metal nanoparticle. A method comprising providing at least one probe, introducing the at least one probe into a subject or a sample, and detecting the resulting luminescence of the quantum dot. A system comprising at least one probe and a detector capable of detecting luminescence from the quantum dot, wherein the detector is positioned in relation to the at least one probe such that luminescence can be detected.

Description

QUANTUM DOT PROBES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/699,108, filed July 14, 2005. STATEMENT OF GOVERNMENT INTEREST

This disclosure was developed at least in part using funding from the National Science Foundation, Award Number EEC-Ol 18007. The U.S. government may have certain rights in the invention.

BACKGROUND Field of Invention

The present disclosure, according to one embodiment, relates to nanoparticulate luminescent probes, and more particularly, to nanoparticulate luminescent probes having inherent signal amplification upon interaction with a targeted molecule

Background

Probes may be used to monitor molecular targets and pathways in vivo through optical imaging. Some probes may use organic fluorophores or dyes that are linked to a substrate in a variety of configurations. One configuration relies on fluorophores in close proximity to each other to auto-quench fluorescence. Upon separation of the fluorophores from the substrate, fluorescence may be unquenched.

Another approach is to monitor the loss of fluorescence resonance energy transfer (FRET) between donor/acceptor paired fluorophores. For example, FRET may be used to monitor protease activity in probes that link a fluorophore to a substrate through an enzymatically degradable peptide sequence. For imaging applications, probes consisting of a fluorescence emitter linked to a non- fluorescent absorber have been developed. These probes, however, lack general tunability of wavelength, requiring specific pairing between the donor and acceptor. In addition, these probes utilize organic fluorophores, which are often inherently unstable in aqueous environments and quickly photobleach, and may be destroyed under a variety of conditions (e.g. exposure to light, change of pH, or a change in temperature). This makes organic fluorophores a poor choice for fluorescent quantitation and long term in vivo studies.

SUMMARY

The present disclosure, according to one embodiment, relates to probes comprising: a quantum dot, at least one metal nanoparticle, and at least one tether that is attached to the quantum dot and to the at least one metal nanoparticle. Release of metal nanoparticles by disruption of the tether (e.g. through the action of a proteolytic enzyme on its target substrate within the tether) may restore radiative quantum dot luminescence. The quantum dot's luminescence may then be detected and quantified. An example of a method of the present invention is a method of quantifying quantum dot luminescence by providing at least one probe, introducing the at least one probe into a subject or a sample, and detecting the resulting luminescence of the quantum dot.

An example of a system of the present invention is a system comprising at least one probe and a detector capable of detecting luminescence from the quantum dot, wherein the detector is positioned in relation to the at least one probe such that luminescence can be detected.

Other and further features and advantages of the present invention will be readily apparent to those skilled in the art upon a reading of the description of the embodiments that follows. FIGURES

A more complete understanding of this disclosure may be acquired by referring to the following description taken in combination with the accompanying figures.

FIGURE 1 is a schematic depicting a probe, according to one embodiment of the present disclosure. FIGURE 2 is an illustration depicting activation of a probe, according to one embodiment of the present disclosure.

FIGURE 3 is a chart demonstrating the reduction in luminescence of a probe, according to one embodiment of the present disclosure.

FIGURE 4 is an emission scan of a probe, according to one embodiment of the present disclosure. FIGURE 5 is an activation plot of a probe, according to one embodiment of the present disclosure.

While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are described below in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as defined by the appended claims.

DESCRIPTION The present disclosure, according to one embodiment, relates to nanoparticulate luminescent probes, and more particularly to nanoparticulate luminescent probes having inherent signal amplification upon interaction with a targeted molecule. According to one embodiment, the present disclosure provides a probe that comprises a quantum dot (Qdot, QD), a metal nanoparticle, and a tether, in which the tether is attached to the QD and the metal nanoparticle.

A QD is a semiconductor nanocrystal, whose radii are smaller than the bulk exciton Bohr radius, which constitutes a class of materials intermediate between molecular and bulk forms of matter. QDs are typically formed from inorganic, crystalline semiconductive materials and have unique photophysical, photochemical, and nonlinear optical properties arising from quantum size effects. QDs have therefore attracted a great deal of attention for their potential applicability in a variety of contexts. For example, QDs have been considered for use as detectable labels in biological applications, and as useful materials in the areas of photocatalysis, charge transfer devices, and analytical chemistry.

A QD may exhibit a number of unique optical properties due to quantum confinement effects. For example, QDs possess strong luminescence, photostability against bleaching and physical environments such as pH and temperature, and optical tunability, overcoming many of the shortcomings apparent with organic fluorophores. These properties make them ideal for optical imaging and have proven to be useful as in vitro and in vivo biological labels.

As noted above, in one embodiment, the present disclosure provides a probe that comprises a QD, a metal nanoparticle, and a tether, in which the tether is attached to the QD and the metal nanoparticle. As used herein, the term "attached" may include, but is not limited to, such attachments as covalent binding, adsorption, and physical immobilization. By way of explanation, and not of limitation, in such probes, the luminescence of the QD may be non-radiatively suppressed by the metal nanoparticle (e.g., "gold colloid" of Figure 1) when the QD and metal nanoparticle are attached by the tether (e.g., "peptide" of Figure 1). Release of metal nanoparticles by disruption of the tether (e.g., "peptide cleavage" illustrated in the middle of Figure 2) may restore radiative QD luminescence (e.g., "photoluminescence" illustrated in the middle of Figure 2) through non-radiative energy transfer to make a functional QD probe. Thus, the probe may be tuned by pairing different QDs with different tethers based on the desired result, or the desired application. For example, certain pairings of QDs and tethers may allow for the simultaneous imaging and quantification of numerous targets or activities in vivo. Accordingly, the probes may be used in conjunction with optical imaging techniques to monitor specific molecular targets and pathways. Such probes may be useful, among other things, as customizable agents for optical imaging, for example, for imaging in cancer detection and diagnosis. In addition, the probes of the present disclosure may be detected at high resolutions, among other things, because QDs have a small size and are luminescent. The small size and luminescence may be used, for example, to overcome the limited signal-to-background ratio problems often present in targeted imaging.

QDs may be formed from an inner core of one or more first semiconductor materials that optionally may be contained within an overcoating or "shell" of a second semiconductor material. A QD core surrounded by a semiconductor shell is referred to as a "core/shell" QD. The optional surrounding shell material will preferably have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the core substrate. Suitable semiconductor materials for the core and/or the optional shell include, but are not limited to, the following: materials comprised of a first element selected from Groups 2 and 12 of the Periodic Table of the Elements and a second element selected from Group 16 (e.g., ZnS, ZnSe, ZnTe, CDs, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like); materials comprised of a first element selected from Group 13 of the Periodic Table of the Elements and a second element selected from Group 15 (e.g., GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like); materials comprised of a Group 14 element (Ge, Si, and the like); materials such as PbS, PbSe and the like; and alloys and mixtures thereof. As used herein, all reference to the Periodic Table of the Elements and groups thereof is to the IUPAC system for numbering element groups, as set forth in the Handbook of Chemistry and Physics, 81st Edition (CRC Press, 2000). QDs may be made using techniques known in the art. See, e.g., U.S. Pat. Nos. 6,048,616; 5,990,479; 5,690,807; 5,505,928; and 5,262,357. QDs used in the present disclosure may absorb a wide spectrum of light, and may be physically tuned with emission bandwidths in various wavelengths. See, e.g., Badolato, et al., Science 208:1158-61 (2005). For example, the emission bandwidth may be in the visible spectrum (e.g., from about 3.5 to 7.5 μm), the visible-infrared spectrum (e.g., from about 0.1 to about 0.7 μm), or in the near-infrared spectrum (e.g., from about 0.7 to about 2.5 μm). QDs that emit energy in the visible range include, but are not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs. QDs that emit energy in the blue to near-ultraviolet range include, but are not limited to, ZnS and GaN. QDs that emit energy in the near-infrared range include, but are not limited to, InP, InAs, InSb, PbS, and PbSe. QDs with emission spectra in the near- infrared spectrum may be particularly suited for probes used in certain imaging applications. For example, such QDs may be suited for in vivo imaging, among other things, due to tissue's high optical transmissivity in the near-infrared spectrum.

For attachment to certain tethers, the QD may comprise a functional group or attachment moiety. One example of such a QD that has a functional group or attachment moiety is a QD with a carboxylic acid terminated surface, such as those commercially available though, for example, Quantum Dot, Inc., Hayward, CA.

In general, any metal nanoparticle may be used in the probes of the present disclosure provided that the metal nanoparticle is capable of attachment to a tether and can quench the QD. Suitable metal nanoparticles may have any shape, for example, spherical, elliptical, hollow, and solid, and may have a diameter in the range of about 1 nm to about 1,000 nm (e.g., ~2 nm to ~50 nm or ~2 nm to -20 nm). In some examples, suitable metal nanoparticles may be formed from a biocompatible metal, for example, gold or silver. One example of a suitable metal nanoparticle is a gold nanoparticle (AuNP), such as a ~1.4 nm mono- maleimide functionalized AuNP commercially available from Nanoprobes, Yaphank, NY. Other suitable AuNPs include, but are not limited to, Au-AuS nanoshells, gold nanorods, and gold nanoshells. In general, the tether may be any molecule capable of binding a QD and a metal nanoparticle. Suitable tethers may have any length, provided the length does not exceed the energy transfer distance necessary for the metal nanoparticle to at least partially suppress the QD's luminescence. The distance at which energy transfer between two molecules is 50% efficient is known as the Fδrster radius (typically less than about 10 nm). The Fδrster radius is determined by a host of factors such as molecular dipole, quantum yield, refractive index, and spectral overlap, as described in J.R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer Academic/Plenum Publisher, New York (1999). Examples of molecules suitable for use as a tether include, but are not limited to, a molecule that is a substrate for a proteolytic enzyme, a peptide, a nucleic acid (e.g., DNA or RNA), a molecule having a hydrolyzable ester bond, and an N-isopropylacrylamide (NIP AAm) molecule. The particular tether chosen may depend on, among other things, the desired application and the desired target for the probe.

In one embodiment, in which the tether is a nucleic acid, the probe may be capable of detecting DNAses or RNAses. In such a probe, luminescence may be correlated with nucleic acid hybridization (e.g., as in a gene chip).

In another embodiment, in which the tether is a molecule having a hydrolyzable ester bond, the probe may operate as a pH sensor. Ester bonds are often pH sensitive. Accordingly, hydrolysis of an ester bond in a tether may result in luminescence of the QD, which may be correlated to pH.

In another embodiment, in which the tether is a NIPAAm molecule, the probe may operate as a temperature sensor. The conformation of NTPAAm may be dependent on temperature, and NIPAAm can be synthesized across a range of temperature sensitivities. Accordingly, when NIPAAm tethers are used, the probe may be capable of luminescence when the temperature is sufficient to disrupt the NIPAAm tether. In a related aspect, such probes may be used in conjunction with microelectromechanical devices (MEMS) with very broad sensitivity ranges.

In another embodiment, in which the tether is a substrate for a proteolytic enzyme, the probe may be capable of detecting protease activity. In such probes, the tether may comprise a proteolytically sensitive tether, for example, a peptide sequence that can be degraded by a protease, or a synthetic substrate for a proteolytic enzyme (Figure 2). Proteolytic activity critically impacts a wide range of biologic phenomena, including: development, cancer growth and metastasis, wound healing, cell migration, leukocyte extravasation, and degenerative diseases and conditions (e.g. arthritis). Accordingly, probes having tethers susceptible to proteolytic activity may be used to detect, quantify, and localize proteolytic activity to, among other things, better diagnose and treat precancers, cancers, and degenerative conditions, as well as to design new therapeutics to combat these ailments. In particular, the metastatic potential of a tumor could be assessed during imaging and diagnosis.

A variety of peptide sequences susceptible to proteolytic cleavage are available. This allows wide applicability of such probes for applications ranging from imaging proteolytic activity of cells to assessing the metastatic potential of cancerous lesions, among other things, due to optical tunability and adjustable peptide sequences. In addition, the small size of the probes, among other things, may enable detection of single-cell precancers and single-cell cancers throughout a subject. In one example, a library of different wavelength emitting probes (each probe using spectrally distinct QD) may be formed, in which the probes have different enzyme specificities to allow for simultaneous imaging from many different proteases. In this way, the detection of multiple types of proteases in the same preparation may be possible, for example, by providing quantifiable data about several different proteases at once. In another example, the probes of the present invention may be crosslinked into a biomimetic scaffold to follow, for example, proteolytic activity of migrating cells over time.

Suitable peptide sequences may be synthesized using standard techniques, for example, Fmoc (9-flourenylmethloxycarbonyl) solid phase peptide synthesis. Once synthesized, the peptide sequence may be attached to a QD, for example, by covalently attaching the N-terminus of the peptide to the carboxylic acids on a QD surface under conditions specific for amine reactivity and allowing an amide to form. A thiol on the peptide then may be reacted with an AuNP to complete the attachment; and this attachment may be readily achieved, because the sulfur-gold bond is spontaneous under most conditions. Alternatively, Nanogold™ monomaleimide (Nanoprobes, Inc., Yaphank, NY) may be reacted with the peptide thiol, with the Nanogold™ serving as the AuNP. In some embodiments, the probes may be treated to, among other things, to increase biocompatibility. This may be useful in, for example, therapeutic applications. For example, the QDs or metal nanoparticles or both may be coated with a biocompatible moiety, for example, polyethylene glycol (PEG) (Figure 1).

In some embodiments, linkers may be used, between the QD and tether, or between the tether and a metal nanoparticle, or both, among other things, to maximize quenching and enzymatic accessibility. One example of a suitable linker is a PEG linker.

In some embodiments, the probes may be conjugated with one or more of an antibody, an antigen, and streptavidin.

According to another embodiment, a probe of the present disclosure may be used in therapeutic or diagnostic applications. For therapeutic applications, probes may be administered (e.g., by intravenous injection) to a subject (e.g., a human being or other mammal), and any luminescence from the probe may be detected. For diagnostic applications, probes may be applied to a sample (e.g., a biological sample) and any luminescence from the probe may be detected.

According to another embodiment, the present disclosure provides a system that comprises a probe of the present disclosure and a detector capable of detecting luminescence from a QD. To detect luminescence, the detector should be positioned in relation to the probe such that luminescence can be detected, and the detector should be adapted to detect luminescence. One example of a suitable detector is a fiuorimeter. In some embodiments, the system may further comprise a source of electromagnetic radiation, for example, an excitation monochromator. An excitation monochromator may be used to excide the probe at a specific wavelength to ensure the quality of emission detection.

To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. In no way should the following examples be read to limit or define the entire scope of the invention.

EXAMPLES

Quantum dot Synthesis: The QDs were core/shell structured CdSe/CdS synthesized as described in J.J. Li, et al., Am. Chern. Soc. 125:12567-75. Polyethylene glycol) (PEG₅ 750 Da) was used to increase the water-solubility and stability of the QDs. The resulting QDs are carboxylate-terminated with a peak emission at 620 nm. Peptide Synthesis: A collagenase-degradable specific peptide sequence (GGLGPAGGCG) was used. The GGLGPAGGCG degradable peptide sequence was synthesized using Fmoc solid phase peptide synthesis (Applied Biosystems, Inc., Foster City, CA). Cleavage from the polystyrene resin was effected with 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane. The cleaved peptide was precipitated in ether followed by dialysis against MiIIiQ water (Milli-Q Gradient, Millipore, Billerica, MA). The peptide was lyophilized and stored at -2O⁰C until use.

Conjugation Reaction: QDs (2 nmol) in deionized water were activated with EDC and sulfo-NHS (Pierce, Rockford, IL) to form an active ester leaving group. The N-terminus of the synthesized peptide was then covalently linked to the QDs at the active ester site to form an amide. Activation of the C-terminus of the peptide was prevented by reacting residual EDC with β-mercaptoethanol prior to peptide addition. During the coupling reaction, peptide was added in a 30-fold molar excess to ensure sufficient coupling onto the QD. The reaction was allowed to proceed overnight in the dark at room temperature. The solution was then dialyzed with 5,000 MWCO cellulose ester membrane (Spectrum Laboratories, Houston, TX) to remove any unreacted peptide or byproducts. Following dialysis, the solution was split into two aliquots. One aliquot was reacted with gold nanoparticles while the other aliquot served as control. The control underwent identical steps as the conjugate except it was reacted with equal volumetric amounts of deionized water rather than gold nanoparticles. The quantum dot-peptide conjugate was concentrated using a 50,000 MWCO Vivaspin Ultrafiltration concentrator (Vivascience AG, Hannover, Germany) and centrifuged at 2,000 x g for 20 min. The purified QD-peptide conjugate was resuspended to 400 μL of deionized water.

Mono-maleimide functionalized AuNps (1.4 nm; Nanoprobes, Yaphank, NY) were covalently linked to the sulfhydryl group on the cysteine residue of the QD-peptide conjugate at a ratio of 6:1 AuNP:QD. A centrifuge filter (Vivaspin 6 MWCO 50,000) was then used to remove unbound AuNPs and the probe was resuspended in sterile HEPES-buffered saline (HBS: 135 niM NaCl, 5 mM KCl, 1 mM MgSO₄, 1.8 mM CaCl₂, 10 mM HEPES, pH 7.4). Luminescence measurements were made on the control and conjugate to compare quenching of the quantum dots by the gold nanoparticles. Activation of Probe: Following initial luminescence measurements, collagenase Type XI (Sigma-Aldrich, St. Louis, MO) was added to the probes at a final concentration of 0.2 mg/mL. Control samples (QD probe without collagenase) were monitored simultaneously. Extinction measurements were made of varying concentrations of collagenase in HBS to examine effects on turbidity which may affect luminescence measurements. Studies demonstrated minimal effects on turbidity at wavelengths > 450nm for concentrations of 0.2 mg/mL and lower. Fluorescence measurements of collagenase (0.2 mg/mL) in HBS (n=3) were used to subtract autofluorescence from collagenase in all probe samples containing collagenase. QD probes with collagenase were incubated at room temperature. Photoluminescence was measured to observe unquenching of QDs in HBS over time. Studies were limited to less than 48 hours due to the progressive loss of collagenase bioactivity in solution.

Spectroscopy Measurements: All measurements were performed at room temperature with a 500 μL stoppered quartz cuvette to prevent evaporation (Starna Cells Inc., Atascadero, CA) on a Horiba Jobin Yvon SPEX FL3-22 Fluorimeter (Edison, NJ) with dual excitation and emission monochromators. Time-integrated photoluminescence was measured before and after conjugation to the AuNP to observe quenching of QD probes. Photoluminescence measurements were also taken over time to observe proteolytic activation of the probe. Baseline values were taken for all measurements of QD probes in deionized water and HBS. QDs were excited at 360 nm, and emission scans measured from 400-700 nm. Bandpass slits and integration time were set to 3 nm/3 nm and 100 ms, respectively on the fluorimeter. All values were normalized over time to a rhodamine 6G standard to avoid any artifacts that could arise from possible lamp fluctuations. Extinction measurements from 200-800 nm were also acquired for each sample on a Varian Gary 50 Bio spectrophotometer (Walnut Creek, CA).

Results: Results of quantum dot quenching by AuNPs are shown in Figure 3. All measurements reported consist of a sample size n=3. For a 6:1 ratio of AuNP:QD, QD- peptide-AuNP luminescence indicates a 71% quenching of signal compared to unreacted QD- peptide. In addition, initial measurements at t = 0 hours indicate no statistical difference between probe without collagenase and probe with collagenase after subtraction of collagenase autofluorescence. Luminescence of each sample was measured at the following time points: 0, 18, 38, and 47 hours. Figure 4 illustrates the spectral profile of increased QD luminescence over time. At collagenase concentrations of 0.2 mg/niL, there is minimal collagenase autofluorescence past 550 nm. A rise over time in luminescence of the probe with collagenase is observed in Figure 5. At 47 hours, an average luminescence rise of 52% is observed. The control group containing inactivated probe (QD probe without collagenase) in buffer solution indicated statistically insignificant changes in luminescence during the same time intervals.

While embodiments of this disclosure have been depicted, described, and are defined by reference to embodiments of the disclosure, such references do not imply a limitation on the disclosure, and no such limitation is to be inferred. The subject matter disclosed is capable of considerable modification, alteration, and equivalents in form and function, as will occur to those ordinarily skilled in the pertinent art and having the benefit of this disclosure.

The depicted and described embodiments of this disclosure are examples only, and are not exhaustive of the scope of the disclosure.

Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part; by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising: a quantum dot; at least one metal nanoparticle; and a least one tether that is attached to the quantum dot and to the at least one metal nanoparticle.

2. The composition of claim 1, wherein the metal nanoparticle comprises a nanoshell or a nanorod.

3. The composition of claim 1 , wherein the metal nanoparticle is a gold nanoparticle.

4. The composition of claim 1, wherein the quantum dot has an emission bandwidth in a range chosen from the visible spectrum, the visible-infrared spectrum, the near-infrared spectrum, and the infrared spectrum.

5. The composition of claim 1, wherein the quantum dot comprises a functional group or attachment moiety.

6. The composition of claim 1, wherein the tether comprises at least one molecule chosen from a molecule that is a substrate for a proteolytic enzyme, a peptide, a nucleic acid, a DNA molecule, an RNA molecule, a molecule having a hydrolyzable ester bond, and an N- isopropylacrylamide molecule.

7. The composition of claim 1, further comprising a polyethylene glycol molecule, wherein the polyethylene glycol molecule is attached to one or more of the quantum dot, the metal nanoparticle, and the tether.

8. The composition of claim 1, further comprising at least one conjugate chosen from an antibody, an antigen, and streptavidin.

9. The composition of claim 1, wherein the ratio of the metal nanoparticles to the quantum dot is about six to about one.

10. The composition of claim 1, wherein the at least one metal nanoparticle has a diameter in the range of about 1 nm to about 1,000 nm.

11. The composition of claim 1, further comprising a biocompatible moiety.

12. A method comprising: providing at least one probe comprising: a quantum dot; at least one metal nanoparticle; and at least one tether that is attached to the quantum dot and to the at least one metal nanoparticle; introducing the at least one probe into a subject; and detecting luminescence from the at least one probe in the subject.

13. The method of claim 112, wherein the tether comprises at least one molecule chosen from a molecule that is a substrate for a proteolytic en2yme, a peptide, a nucleic acid, a DNA molecule, an RNA molecule, a molecule having a hydrolyzable ester bond, and an N- isopropylacrylamide molecule.

14. The method of claim 12, wherein the at least one probe is attached to a biomimetic scaffold.

15. A method comprising: providing at least one probe comprising: a quantum dot; at least one metal nanoparticle; and at least one tether that is attached to the quantum dot and to the at least one metal nanoparticle; introducing the at least one probe into a sample; and detecting luminescence from the at least one probe in the sample.

16. The method of claim 15, wherein the tether comprises at least one molecule chosen from a molecule that is a substrate for a proteolytic enzyme, a peptide, a nucleic acid, a DΝA molecule, an RΝA molecule, a molecule having a hydrolyzable ester bond, and an N- isopropylacrylamide molecule.

17. The method of claim 15, wherein the at least one probe is attached to a biomimetic scaffold.

18. A system comprising: at least one probe comprising: a quantum dot; at least one metal nanoparticle; and at least one tether that is attached to the quantum dot and to the at least one ^■ metal nanoparticle; and a detector capable of detecting luminescence from the quantum dot, wherein the detector is positioned in relation to the at least one probe such that luminescence can be detected.

19. The system of claim 18, wherein the tether comprises at least one molecule chosen from a molecule that is a substrate for a proteolytic enzyme, a peptide, a nucleic acid, a DNA molecule, an RNA molecule, a molecule having a hydrolyzable ester bond, and an N- isopropylacrylamide molecule.

20. The system of claim 18, wherein the detector is a fiuorimeter.

21. The system of claim 18 further comprising a source of electromagnetic radiation.

22. The system of claim 18 further comprising an excitation monochromator.