Multiplexed Tracking of Protease Activity Using a Single Color of

Article pubs.acs.org/ac

Multiplexed Tracking of Protease Activity Using a Single Color of Quantum Dot Vector and a Time-Gated Förster Resonance Energy Transfer Relay W. Russ Algar,*,†,§,# Anthony P. Malanoski,† Kimihiro Susumu,‡,⊥ Michael H. Stewart,‡ Niko Hildebrandt,∥ and Igor L. Medintz*,† †

Center for Bio/Molecular Science and Engineering, Code 6900, and ‡Optical Sciences Division, Code 5611, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States § College of Science, George Mason University, Fairfax, Virginia 22030, United States ∥ Institut d’Electronique Fondamentale, Université Paris-Sud, 91405 Orsay Cedex, France ⊥ Sotera Defense Solutions, Annapolis Junction, Maryland 20701, United States S Supporting Information *

ABSTRACT: Semiconductor quantum dots (QDs) are attractive probes for optical sensing and imaging due to their unique photophysical attributes and nanoscale size. In particular, the development of assays and biosensors based on QDs and Förster resonance energy transfer (FRET) continues to be a prominent focus of research. Here, we demonstrate the application of QDs as simultaneous donors and acceptors in a time-gated FRET relay for the multiplexed detection of protease activity. In contrast to the current state-of-the-art, which uses multiple colors of QDs, multiplexing was achieved using only a single color of QD. The other constituents of the FRET relay, a luminescent terbium complex and fluorescent dye, were assembled to QDs via peptides that were selected as substrates for the model proteases trypsin and chymotrypsin. Loss of prompt FRET between the QD and dye signaled the activity of chymotrypsin; loss of time-gated FRET between the terbium and QD signaled the activity of trypsin. We applied the FRET relay in a series of quantitative, real-time kinetic assays of increasing biochemical complexity, including multiplexed sensing, measuring inhibition in a multiplexed format, and tracking the proteolytic activation of an inactive pro-protease to its active form in a coupled, multienzyme system. These capabilities were derived from a ratiometric analysis of the two FRET pathways in the relay and permitted extraction of initial reaction rates, enzyme specificity constants, and apparent inhibition constants. This work adds to the growing body of research on multifunctional nanoparticles and introduces multiplexed sensing as a novel capability for a single nanoparticle vector. Furthermore, the ability to track both enzymes within a coupled biological system using one vector represents a significant advancement for nanoparticle-based biosensing. Prospective applications in biochemical research, applied diagnostics, and drug discovery are discussed. Fö rster resonance energy transfer (FRET) for “on/off” signaling,12−15 this task is considerably more complex due to the integration of multiple colors/populations of QDs with multiple acceptors/donors. Challenges include adequately resolving a greater number of PL contributions, reliably deconvolving the broad and asymmetric PL of non-QD donors/acceptors, accommodating variations in FRET efficiency between different FRET pairs, and optimizing the amount of each FRET pair present on the basis of relative brightness or biological activity. A possible solution to these challenges is to forego the current state-of-the-art where N unique colors of QDs are used to detect N analytes of interest

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here is fervent interest in harnessing the unique optical properties of semiconductor quantum dots (QDs) for biological imaging and bioanalysis.1 The bright, narrow, and size-tunable photoluminescence (PL), nanoscale size, and high surface area-to-volume ratio of QDs have all been shown to be highly advantageous for cellular and in vivo imaging,2,3 energytransfer-based biosensing,4 and drug delivery.5 Within all of these contexts, the striking ability of QDs to facilitate multiplexing has been highly touted. This capacity arises not only from the PL properties of QDs, but also from their broad absorption spectra and common synthetic methodology for producing different colors (i.e., size-tuning for a given semiconductor material). Multiplexed immunoassays and multicolor imaging have been widely demonstrated and tend to be straightforward since the PL from well-resolved colors of QDs is directly measured.6−11 Although multiplexing has also been demonstrated in biosensing applications that utilize © 2012 American Chemical Society

Received: October 4, 2012 Accepted: October 17, 2012 Published: November 5, 2012 10136

dx.doi.org/10.1021/ac3028068 | Anal. Chem. 2012, 84, 10136−10146

Analytical Chemistry

Article

Figure 1. (A) Principle of the time-gated Tb → QD → A647 FRET relay. Optical excitation of the conjugates yields excited-state Tb and an excitedstate QD (*), and FRET2 is observed on a prompt time scale (emission 90% and ∼80% of their putative completion. Gel electrophoresis confirmed digestion of both substrates (without QDs), but there were indications of a small indigestible fraction of SubTRP(Tb). Furthermore, when assembled to the QDs, a small fraction of the bound peptides may be poorly accessible. Regardless, incomplete digestion has no analytical impact since the rate of digestion is important here; the end point is of minimal, if any, value. From the progress curves, we derived initial reaction rates, vo, and specificity constants, kcat/Km, for TRP and ChT activity (Supporting Information, Table S3, p S34). In the context of biosensing, vo is useful since it reflects changes in overall activity with changes in the protease concentration. In contrast, kcat/Km is an effective second-order rate constant for enzyme activity.39,40 For ideal Michaelis−Menten (MM) kinetics, kcat/ Km is independent of both the enzyme and substrate concentrations, and we found this to be approximately true herein: (kcat/Km)TRP was ca. 5 s−1 mM−1 across the TRP, multiplexed, and activation assays; (kcat/Km)ChT was ca. 6 s−1 mM−1 in both ChT and multiplexed assays. Commercial trypsin assay kits (i.e., substrates different from ours) have (kcat/Km)TRP values in the range of 0.4−150 s−1 mM−1, and a fluorescent protein construct has been reported to have (kcat/Km)TRP ∼200 s−1 mM−1.41 The values we report for our configuration are without optimization of the peptide substrates and are already competitive with those of commercial kits. Superior values of kcat/Km may be achievable through refinement of the peptide sequences; the QD is not anticipated to be a hindrance in light of recent reports of enhanced hydrolytic enzyme activity at nanoparticle interfaces.42,43 However, the 33−50% relative standard deviations in our measured kcat/Km values between individual assays (Table S3) may potentially reflect digestion kinetics that do not fully adhere to the classic MM model due 10143



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downstream protease,41 cannot currently detect the upstream protease using the same construct and must rather rely on two biosensors. Having demonstrated proof-of-concept with pro-ChT and TRP, we expect that our novel time-gated QD-FRET relay configuration will be applicable to sensing the activation and coupled activity of other more clinically relevant enzymes. For example, the matrix metalloproteinases (MMPs) are extracellular proteases that are synthesized as inactive pro-enzymes and have both increased expression and activation in a broad range of human cancers.45 MMPs can be activated by plasmin (a serine protease, like TRP and ChT, that is activated upstream in a cascade) or, alternatively, by other upstream MMPs.46 Given the relevance of the MMPs to cancer growth, invasion, metastasis, and angiogenesis,45 a single nanoparticle vector that can monitor the coupled activity of an MMP and its activator is a potentially powerful tool. Another clinically relevant system is the proteolytic cascade associated with the activation of the intracellular caspase enzymes. These enzymes have important roles in apoptosis and inflammation,47 and their misregulation has been implicated in cancer and neurodegenerative diseases.48 The time-gated QD-FRET relay is a promising candidate for visualizing and quantitating coupled MMP or caspase activity, which may help elucidate important aspects of the above pathologies and aid in developing new diagnostics or therapies.

to the heterogeneous character of the QD interface, as has been suggested recently for a similar system with QDs and dyelabeled peptide substrates.42 Furthermore, the range of QD and protease concentrations used in this work transitioned from a regime with a >10-fold excess of QD per enzyme to one where there was a small excess of enzyme, albeit that the bulk equivalent concentration of substrate was always in excess over the enzyme concentration. Deviations from ideal behavior (i.e., kcat/Km varies) could be observed over this range of stoichiometry even if MM kinetics were to be a suitable description from first principles (e.g., insufficient excess of substrate for the MM assumptions). Another consideration is that, in multiplexed experiments, some of the variation in kcat/Km may have been due to crossreactivity. For example, this appeared to be the case in activation assays (Supporting Information, Table S4, p S35) where, at lower concentrations of TRP, the increase in (kcat/ Km)TRP with increasing pro-ChT is attributed to the low specificity of background ChT activity in the samples. In contrast, at higher concentrations of TRP, faster activation of pro-ChT and subsequent hydrolysis of TRP by ChT are presumed to have caused the decrease in (kcat/Km)TRP with increases in pro-ChT. Substrate cross-reactivity may be practically eliminated by further optimization of peptide sequences; however, the potential for digestion of one enzyme by another is largely intrinsic to the biological system under study. Regardless of these considerations and any departure from MM kinetics, the overall agreement of the average kcat/Km values between assays confirmed the fidelity of quantitative, multiplexed detection using the time-gated FRET relay. Pro-Enzyme Activation and Coupled Activity. Most proteases are initially biosynthesized in vivo as inactive proenzymes (or zymogens). ChT is a well-known example: prochymotrypsin (pro-ChT), also known as chymotrypsinogen A, is activated to chymotrypsin via the activity of trypsin, which hydrolyzes the Arg15−Ile16 amide bond of pro-ChT.44 The proChT/TRP pair was selected as a model pro-enzyme/enzyme system due to the extensive characterization44 and wide availability of these proteins. The progress curves for SubChT(A647) digestion were expected to be a convolution of nested hydrolysis reactions: (1) the action of TRP on pro-ChT and (2) the subsequent action of chymotrypsin on SubChT(A647). Despite background chymotrypsin-like activity in our pro-ChT sample, unambiguous evidence for the activation of pro-ChT to ChT by TRP was observed (see Figure 5). The progress curves for the digestion of SubChT(A647) by TRP + pro-ChT had an inflection that was clearly distinct from digestion with only pro-ChT, ChT, or TRP + ChT. This inflection is indicative of the transition from inactive pro-protease to active protease. Thus, even without controls for the baseline activity of pro-ChT, this feature enables at least qualitative observation of activation. End point assays, however, would not be able to distinguish mixed TRP + ChT activity from the activation of pro-ChT by TRP. Activation would be even more evident in kinetic assays with near zero background activity from the pro-ChT. Here, the large background activity is likely to be a limitation of the assupplied in vitro materials; enzyme activity is more tightly controlled in real biological systems. Another important advantage of this configuration is that the proteolytic activity of both the upstream activating protease and the downstream activated protease can be tracked. In contrast, fluorescent protein-based sensors, while capable of visualizing an activated

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CONCLUSIONS We have demonstrated that a single color of QD, in combination with a time-gated FRET relay, can be used for multiplexed protease sensing. The development of single QD vectors with multiple capabilities is a very active area of research and includes theranostic probes49 and multimodal imaging probes that combine QDs with noble metals or magnetic ironbased materials,50 paramagnetic lanthanides,51 or radioisotopes.52 Our work now adds multiplexed sensing based on a single detection modality (i.e., PL) to this growing multifunctional nanotoolbox. The time-gated FRET relay is expected to be widely applicable since all assays were done using a commercially available fluorescence plate reader. We demonstrated quantitative application of the time-gated FRET relay in a series of kinetic protease assays with increasing biochemical complexity: sensing the activity of TRP, ChT, or TRP + ChT, evaluating inhibition in a multiplexed format, and tracking the activation of pro-ChT to ChT. Real-time substrate/product concentrations, initial reaction rates, enzyme−substrate specificity constants (kcat/Km), and apparent inhibition constants (Ki) were measured through a robust, empirical calibration of prompt and time-gated ratiometric PL signals. The ability to track coupled proteolytic activity and evaluate inhibition in a multiplexed format is very promising for future biosensing applications that target the regulation of protease activity through pro-enzyme activation and/or endogenous inhibitors. It is anticipated that this new method can be extended to the study of clinically relevant protease systems and provide quantitative sensing and visualization of activity or be incorporated into drug discovery assays.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental methods (materials, labeling and assay protocols, measurement and analysis methods), additional results and discussion (spectral overlap, enzyme−substrate 10144



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specificity, PL spectra, calibration parameter values, signal modality, raw PL data from assays, MM analysis and data tables, complexation between Tb and A647), and inverse configuration data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (W.R.A.); [email protected]. mil (I.L.M.). Present Address #

Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the U.S. Naval Research Laboratory (NRL) and NRL Nanoscience Institute (NSI), Office of Naval Research (ONR), Defense Threat Reduction Agency-Joint Science and Technology Office (DTRA-JSTO) (MIPR no. B112582M), and the European Commission (FP7 project NANOGNOSTICS). WRA is grateful to the Natural Sciences and Engineering Research Council (NSERC) of Canada for support through a postdoctoral fellowship.

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Multiplexed Tracking of Protease Activity Using a Single Color of

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