Article pubs.acs.org/ac
Proteolytic Assays on Quantum-Dot-Modified Paper Substrates Using Simple Optical Readout Platforms Eleonora Petryayeva and W. Russ Algar* Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada S Supporting Information *
ABSTRACT: Paper-based assays are a promising diagnostic format for point-of-care applications, field deployment, and other low-resource settings. To date, the majority of efforts to integrate nanomaterials with paper-based assays have utilized gold nanoparticles. Here, we show that semiconductor quantum dots (QDs), in combination with Förster resonance energy transfer (FRET), are also suitable nanomaterials for developing paper-based assays. Paper fibers were chemically modified with thiol ligands to immobilize CdSeS/ZnS QDs, the QDs were self-assembled with dye-labeled peptides to generate efficient FRET, and steady-state and fluorescence lifetime imaging microscopy (FLIM) were used for characterization. Peptides were selected as substrates for three different proteases and a series of kinetic assays for proteolytic activity was carried out, including multiplexed assays and pro-enzyme activation assays. Quantitative results were obtained within 5−60 min at levels as low as 1−2 nM of protease. These assays were possible using simple optical readout platforms that did not negate the low cost, ease of use, and overall accessibility advantages of paper-based assays. A violet light-emitting diode (LED) excitation source and color imaging with either a digital camera, consumer webcam, or smartphone camera were sufficient for analysis on the basis of a red/green color intensity ratio. At most, a universal serial bus (USB) connection to a computer was required and the instrumentation cost orders of magnitude less than that typically utilized for in vitro bioanalyses with QDs. This work demonstrates that QDs are valuable probes for developing a new generation of paper-based diagnostics.
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to seed silver enhancement.21 The latter has been the basis of highly sensitive “scanometric” assay formats, requiring only benchtop scanners for readout.6,22,23 Few paper-based assays, however, have been developed around other nanomaterials, including luminescent semiconductor quantum dots (QDs), which offer excellent brightness, size-tunable photoluminescence (PL), and superior resistance to photobleaching. QDs have emerged as outstanding probes for both cellular and in vivo imaging and, most pertinently, in vitro assays.24−28 To date, however, QD-based in vitro assays have largely relied on spectroscopic equipment that, although available in research laboratories, is not suitable for field work or point-of-care settings. In addition to lateral flow assays with QD-labeled antibodies on nitrocellulose membranes,29−31 some researchers have begun to explore the possibility of using QDs as part of paper-based assays to ameliorate these limitations. Noor et al. recently reported a quantitative DNA hybridization assay using spots of QDs immobilized on a paper substrate;32 however, readout was done using a sophisticated fluorescence microscope equipped with a laser excitation source. While the assay chemistry was elegant, this readout system largely negated the advantages of the paper substrate. Yuan et al. deposited mixed
iagnostic assays are a critical frontline for healthcare and should be sensitive, selective, and rapid in their response to target analytes. Improved benchmarks for these figures of merit continue to be reported across a myriad of different analyses, frequently with the aid of nanomaterials and nanotechnology.1−7 Unfortunately, many of these improvements require sophisticated instrumentation for readout. While such methods are potential healthcare solutions in some settings, it has been recognized that simple approaches are essential for developing diagnostics that are suitable for pointof-care applications, field deployment, or other low-resource settings.8−10 In this context, other important criteria for assays include low cost, robustness, portable supporting instrumentation, few (if any) external reagents, and ease of use. Over the past decade, paper has become a preferred solid support for developing assays that can meet these additional criteria.9,11,12 Paper is inexpensive and ubiquitous, robust, easily patterned and chemically modified, biocompatible, and has intrinsic wicking action that can drive fluid flow.9,13 Dipstick assays, lateral flow assays, and microfluidic paper-based analytical devices (μPADs) have been prominent developments in this area.8,14−19 Many researchers have sought to integrate nanomaterials with paper-based assay formats. The vast majority of these developments have utilized gold nanoparticles (AuNPs),20 which support visual detection with their strong red color, pronounced color change upon aggregation,1 and their ability © 2013 American Chemical Society
Received: July 2, 2013 Accepted: August 12, 2013 Published: August 27, 2013 8817
dx.doi.org/10.1021/ac4020066 | Anal. Chem. 2013, 85, 8817−8825
Analytical Chemistry
Article
Figure 1. (A) Design of a paper-based assay to monitor protease activity via FRET with QD donors. Acceptor dye-labeled (A555) peptide substrates containing a cleavage site for trypsin (TRP), chymotrypsin (ChT), and enterokinase (EK) were assembled on immobilized QDs. Protease activity was measured through changes in the intensity and color of PL from the spots of immobilized QDs and peptides. (B) Synthetic steps used for modification of cellulose paper fibers with bidentate thiol surface ligands for the immobilization of QDs. (C) Color images of paper-immobilized QDs and A555-labeled QD-Sub(TRP) peptide conjugates under white light and their PL under UV illumination.
films of QDs and glucose oxidase or tyrosinase on paper substrates for detecting glucose and phenol, respectively.33 The byproducts of the enzyme catalyzed reactions quenched the QD PL. While this quenching could be visualized with an ultraviolet (UV) lamp, it was not quantitatively analyzed. Here, we show that QDs can be used to develop paper-based assays with quantitative readout methods that are relatively lowcost and suitable for point-of-care and field diagnostic applications. Expensive and electrical power-intensive laser and lamp sources are replaced with a violet light-emitting diode (LED; $0.50) as the excitation source, while sophisticated instruments are replaced with three different detection systems that require no more than a universal serial bus (USB) connection to a computer. These detection systems are much less expensive than those commonly used in research laboratories (e.g., fluorimeters, plate readers, microscopes) and progressively scale-down in cost from a miniature fiberoptic spectrometer ($2000) to an educational-grade digital microscopy camera ($250) to a consumer webcam ($40). We further show that a smartphone camera can be substituted for the webcam and the LED powered from batteriesa format amenable to the concept of telemedicine.34 The diagnostic utility of QD-modified paper substrates with each of the
foregoing detection systems is demonstrated through a series of proteolytic assays based on Förster resonance energy transfer (FRET). We build on previous work demonstrating detection of proteolytic activity in solution-phase assays with QD-peptide conjugates2,35−37 and adapt this format for paper-based assays. As shown in Figure 1A, QDs are immobilized on paper and conjugated with fluorescent dye-labeled peptide substrates for protease targets of interest, where the dye is chosen to be a FRET acceptor for the QDs. We use Alexa Fluor 555 (A555) as an acceptor for alloyed CdSeS/ZnS QD donors with green PL emission. In the absence of proteolytic activity, the dye remains in close proximity to the QD such that FRET is “on” and very efficient, resulting in the observation of yellow/orange emission from the A555. Proteolysis cleaves the peptide allowing the dye to diffuse away from the QD, turning FRET “off” with recovery of green emission from the QD. These fluorescence color changes, which are visible by eye, can be tracked with digital color imaging for quantitative analysis. We apply these readout modes in various model assays of pancreatic protease activity, including multiplexed assays and pro-enzyme activation assays. Activity-based assays (cf. protease concentration) are crucial for evaluation of protease function.38 Our primary motivation was to demonstrate that QDs can be combined with low-cost and 8818
dx.doi.org/10.1021/ac4020066 | Anal. Chem. 2013, 85, 8817−8825
Analytical Chemistry
Article
Table 1. Peptide Substrate Sequences
a
amino acid sequence (written N-terminal to C-terminal)a
abbreviation
HHHHHH SPPPPPPS GQGEGEGNSGR↓GGSGNG C(A555) HHHHHH SPPPPPPS GQGEGEGNSAAY↓ASGNG C(A555) HHHHHH SPPPPPPS GQGEGGNSDDDDK↓SGNG C(A555)
Sub(TRP) Sub(ChT) Sub(EK)
Protease recognition sites are indicated in bold and the cut position is indicated by the downward arrow.
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RESULTS AND DISCUSSION Assay Design and Characterization. Figure 1B summarizes the chemistry used to modify cellulose paper for the immobilization of QDs. Water-soluble QDs coated with DHLA or GSH ligands readily immobilized on the modified paper substrates (
