Camera-Based Ratiometric Fluorescence Transduction of Nucleic

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Camera-Based Ratiometric Fluorescence Transduction of Nucleic Acid Hybridization With Reagentless Signal Amplification on a Paper-Based Platform Using Immobilized Quantum Dots as Donors M. Omair Noor, and Ulrich J. Krull Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Sep 2014 Downloaded from http://pubs.acs.org on September 26, 2014

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Analytical Chemistry

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Camera-Based Ratiometric Fluorescence Transduction of Nucleic

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Acid Hybridization With Reagentless Signal Amplification on a

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Paper-Based Platform Using Immobilized Quantum Dots as Donors

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M. Omair Noor and Ulrich J. Krull*

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Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto

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Mississauga, 3359 Mississauga Road, Mississauga ON, L5L 1C6, Canada.

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*Author to whom correspondence should be addressed: [email protected]

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ABSTRACT

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resource-limited settings and for point-of-care screening. Achievement of high sensitivity with

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precision and accuracy can be challenging when using paper substrates. Herein, we implement

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R-G-B color palette of a digital camera for quantitative ratiometric transduction of nucleic acid

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hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in

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fluorescence resonance energy transfer (FRET). A non-enzymatic means of signal enhancement

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for QD-FRET assays on paper-substrates is based on the use of dry paper substrates for data

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acquisition. This approach offered at least 10-fold higher assay sensitivity and at least 10-fold

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lower limit of detection as compared to hydrated paper substrates. The surface of paper was

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modified with imidazole groups to assemble a transduction interface that consisted of

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immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as

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donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close

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proximity to the surface of immobilized gQDs was responsible for a FRET sensitized emission

Paper-based diagnostic assays are gaining increasing popularity for their potential application in

ACS Paragon Plus Environment


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from the acceptor dye, which served as an analytical signal. A handheld UV lamp was used as an

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excitation source and ratiometric analysis using an iPad camera was possible by a relative

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intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels

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of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a

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sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an

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epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range

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spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by

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detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an

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important framework for the integration of QD-FRET methods with digital imaging for a

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ratiometric transduction of nucleic acid hybridization on a paper-based platform.

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INTRODUCTION

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There is a growing interest in the development of decentralized diagnostic assays that are low-

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cost, portable and that employ simple readout instrumentation to expand opportunities for use in

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remote and resource-limited settings. Such developments have potential to significantly reduce

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the cost of medical testing and to expedite global access to healthcare. In this regard, the use of

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paper as a solid support for the development of various biochemical assays has received renewed

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interest in bioanalytical community due to its low cost, flexibility, biocompatibility, ease of use,

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intrinsic filtering functionality, solution transport by capillary wicking action, ease of patterning

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and safe disposal by incineration to eradicate biohazards.1-2 Various formats for paper-based

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assays such as lateral flow devices, dipstick assays and microfluidic paper-based analytical

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devices (µPAD) have been reported.3-8 These formats have been implemented for the detection

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of targets such as ions9-11, nucleic acids12-13, protein markers14 and enzymatic activity15-16.


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From the standpoint of optical readout instrumentation, the use of smartphones and personal

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digital assistant (PDA) devices is attractive considering the ubiquity and technical capabilities of

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such technology.17-18 Smartphones and PDAs exhibit many attractive attributes that can

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potentially be applied in areas of environmental monitoring, telemedicine, point-of-care testing,

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and mobile diagnostics. These attributes include a capacity to store, process and transmit data;

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intrinsic color selectivity (red-green-blue color channels) for imaging; portability; ease of use;

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accessibility and relatively low cost.17-18 Examples of smartphone-based accessories with an

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integrated application for data processing include the detection of albumin in urine19,

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quantification of blood cholesterol levels20 and detection of biomarkers in saliva and sweat21.

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Zhu et al. reported the use of a smartphone camera as a detector for selective detection of E. coli

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O157:H7 using quantum dots (QDs) as labels with a detection limit of 5-10 cfu mL-1.22 Wang et

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al. reported selective detection of an ovarian cancer biomarker in urine using a smartphone

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camera as a detector with an integrated mobile application for readout of colorimetric ELISA

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results.23 Doeven et al. used R-G-B color selectivity of a digital camera in combination with

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electrochemical excitation to achieve a multiplexed detection of electrochemiluminescence

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intensity associated with three electrochemiluminophores.24 Petryayeva et al. reported the use of

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a smartphone camera as a detector for proteolytic assays using QD mediated fluorescence

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resonance energy transfer (FRET) on paper substrates16, and more recently solution-phase

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homogenous multiplexed detection of proteolytic activity using R-G-B imaging of multicolor

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QDs that served as donors in FRET.25 Wei et al. used aptamer functionalized plasmonic gold

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nanoparticles for a ratiometric detection of Hg2+ ions with ppb detection limits using a

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smartphone accessory with an integrated application for data processing.26 Song et al. recently

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reported visual detection of nucleic acid hybridization on paper substrates using magnetic beads



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of micrometer size as labels.27 Implementation of magnetic beads facilitated sample preparation,

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and the color readout associated with the magnetic beads made use of a digital camera. Despite

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the aforementioned progress, no work has appeared about the integration of a ratiometric

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detection method on a paper-based platform for solid-phase transduction of nucleic acid

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hybridization using a digital camera as a detector. The ratiometric detection method as compared

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to absolute intensity measurements is less susceptible to signal fluctuations imposed by

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variations in sample dilution, detector response, excitation source intensity and ambient light

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conditions.28-29 It should be noted that the variation in ambient light conditions has been reported

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to be a significant challenge for colorimetric detection methods using smartphone and digital

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devices.19-21, 30

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QDs can be integrated as donors for a FRET based ratiometric assay development.31 QDs exhibit

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a number of unique optoelectronic properties that make them excellent donors for FRET based

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transduction.32 In this work, we implement digital imaging for quantitative transduction of

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nucleic acid hybridization on a paper-based platform using immobilized QDs as donors in FRET,

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providing a ratiometric detection platform of low cost and that has potential for field portable

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and remote diagnostic applications. As shown in Figure 1a, the surface of paper was modified

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with imidazole groups to immobilize QD-probe oligonucleotide conjugates that were assembled

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in solution. The green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor.

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Association of Cy3 dye in close proximity to the QD surface as a result of a hybridization event

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introduced FRET sensitized emission from the acceptor dye, which served as an analytical

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signal. The photoluminescence (PL) intensities of gQDs and Cy3 were associated with the green

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(G) and red (R) imaging channels of an iPad mini camera after R-G-B splitting of the acquired


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colored digital images (Figure 1b). Hybridization assays were demonstrated in direct (Figure 1a

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(i)) and sandwich formats (Figure 1a (ii)), where the latter format served as a label-free target

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detection approach. We also report for the first time a reagentless and non-enzymatic means of

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signal enhancement on paper substrates for QD-FRET assays that is based on imaging the paper

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substrates in dry format. Comparative studies of the hybridization assays in a sandwich format

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were also done using an epifluorescence microscope that was equipped with a laser excitation

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source and diode array spectrometer as a detector. The selectivity of nucleic acid hybridization

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assay is demonstrated by single nucleotide polymorphism (SNP) detection.

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Figure 1. (a) Design of paper-based solid-phase QD-FRET nucleic acid hybridization assay using green-emitting QDs (gQDs, 525 nm PL maximum) as donors with Cy3 as an acceptor. Paper-substrates that were modified with imidazole groups were used to immobilize QD-probe conjugates, and hybridization assays in either (i) a direct format or (ii) a sandwich format provided the proximity for FRET sensitized emission from the acceptor dye upon illumination of the paper substrates with a handheld UV lamp (excitation source). (b) Paper substrates were imaged using an iPad mini, where increase in Cy3 PL and corresponding decrease in gQD PL with increasing target concentration were associated with R and G channels, respectively.



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EXPERIMENTAL SECTION

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used in these experiments can be found in the Supporting Information (SI).

A detailed description of reagents, experimental procedures, data analysis and instrumentation

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The oligonucleotide sequences used in the hybridization assays are listed in Table 1. The SMN1

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sequence is a genetic marker for spinal muscular atrophy disorder that codes for survival of

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motor neuron (SMN) protein.33

124 Table 1. Probe, targets and reporter oligonucleotide sequences used in the hybridization assays. Name Sequence SMN1 probe

DTPA-5′-ATT TTG TCT GAA ACC CTG T-3′ Direct Assay Format

SMN1 Cy3 FC P TGT SMN1 Cy3 FC D TGT SMN1 Cy3 1 BPM D TGT SMN1 Cy3 NC TGT

Cy3-3′-TAA AAC AGA CTT TGG GAC A-5′ 3′-TAA AAC AGA CTT TGG GAC A-5′-Cy3 3′-TAA AAC ACA CTT TGG GAC A-5′-Cy3 Cy3-3′-TGT CCC AAA GTC TGT TTT A-5′ Sandwich Assay Format

SMN1 FC TGT

3′-TAA AAC AGA CTT TGG GAC ATT CCT TTT ATT TCC T-5′

SMN1 1 BPM TGT SMN1 Cy3 Rep

3′-TAA AAC ACA CTT TGG GAC ATT CCT TTT ATT TCC T-5′

SMN1 NC TGT

3′-GAA TGA AGG TAC TAA AGA AAT TGA-5′

Cy3-5′-AA GGA AAA TAA AGG A-3′

TGT = target, FC = fully-complementary, 1 BPM = 1 base pair mismatch, Rep = reporter, NC = noncomplementary, DTPA = dithiol phosphoramidite, P = proximal, D = distal. The mismatched base in SMN1 1 BPM TGT and SMN1 Cy3 1 BPM D TGT is bolded and underlined. Note: The sequences have been aligned to represent complementary nucleotides that undergo hybridization.

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Preparation of QD-probe Oligonucleotide Conjugates

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were made water soluble by a ligand exchange reaction with glutathione (GSH) and

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subsequently modified with SMN1 probes (see SI for details).

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Fabrication of Paper Zones, Immobilization of QD-Probe Conjugates and Hybridization Assays

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Whatman cellulose chromatography paper substrates (Grade 1) were patterned using a Xerox

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ColorQube 8570DN solid ink printer. The details can be found in SI. Each paper device was 25

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mm by 60 mm in dimensions and contained 32 circular zones of 3 mm diameter, arranged in a 4

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by 8 array. The paper zones were chemically modified with aldehyde groups and then imidazole

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groups for the immobilization of QD-probe conjugates (see SI for details). Each imidazole

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modified paper zone was spotted with a 3 µL aliquot of QD-probe conjugates solution at ca. 300

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nM and allowed to incubate at room temperature for 1 hour. The paper was then washed with 50

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mM borate buffer (BB, pH 9.25).

Ternary alloyed green-emitting CdSeS/ZnS core/shell QDs (gQDs) with PL maximum at 525 nm

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Hybridization assays were conducted in two formats, direct assay and sandwich assay (see

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Figure S2 in SI). In the case of the direct assay, oligonucleotide targets (SMN1 Cy3 FC TGT and

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SMN1 Cy3 1 BPM TGT) were labeled with Cy3 fluorophore, while in case of sandwich assay,

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unlabeled oligonucleotide targets (SMN1 FC TGT and SMN1 1 BPM TGT) were used in

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combination with a Cy3 labeled reporter (SMN1 Cy3 Rep) that were sequentially introduced.

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The details of the hybridization assays can be found in SI.

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PL Spectra and Digital Images Acquisition

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microscope (Nikon, Mississauga, ON). The excitation source was a 25 mW diode laser with an

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output of 402 nm (Radius 402, Coherent Inc., Santa Clara, CA). The detector was a diode array

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spectrometer (QE65000, Ocean Optics Inc., Dunedin, FL). See SI for further details. Digital

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color images from paper substrates were acquired using an iPad mini (Apple, Cupertino, CA,

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USA). For collecting digital images, paper substrates were illuminated at a distance ca. 10 cm

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using a handheld ultraviolet (UV) lamp (UVGL-58, LW/SW, 6W The Science Company,

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Denver, CO, USA) that was operated at the long wavelength (365 nm) setting (see Figure S1 for

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the schematic). Data collection from the paper substrates was done in both dry and hydrated

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formats. The paper substrates were dried using a vacuum desiccator. The FRET ratios from the

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PL spectra and the R/G ratios from the digital images were calculated using Equations S1 and

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S2, respectively (see SI).

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PL spectra from paper substrates were acquired using a Nikon Eclipse L150 epifluorescence

RESULTS AND DISCUSSION

The FRET pair The FRET pair used in this work was gQD/Cy3 (donor/acceptor). This FRET pair has been

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previously characterized in our earlier studies using the Förster formalism, and the Förster

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distance for this FRET pair was determined to be 6.6 nm.34

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Solution-Phase Quantitative Hybridization Assays

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In previous studies, we have demonstrated that the interfacial chemistry for solid-phase QD-

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FRET nucleic acid hybridization assays on a paper-based platform can be assembled using pre-


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formed QD-probe conjugates.34-35 These QD-probe conjugates were immobilized on paper-

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substrates that were modified with imidazole groups. This approach was in contrast to other

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studies that have also reported solid-phase QD-FRET nucleic acid hybridization assays on

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various solid supports (e.g. glass beads36, optical fibers37-40, microtitre plates41 and glass surface

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of hybrid polydimethylsiloxane/glass microfluidic channels42-43) where the immobilization of

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QDs on a solid support and subsequent conjugation of oligonucleotide probes to the surface of

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immobilized QDs was done sequentially in two separate steps. In our previous studies using pre-

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formed QD-probe conjugates, the QDs were incubated with 20 times molar excess of SMN1

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probes during the preparation of QD-probe conjugates.34-35 Solution-phase quantitative

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hybridization assays that involved titration of a constant aliquot of QD-probe conjugates with

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increasing stoichiometry of acceptors (SMN1 Cy3 FC P TGTs; see Figure S2 for schematics of P

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and D labeled TGTs) and absorption spectroscopy showed that on average between 6 to 9 SMN1

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probes were conjugated to the surface of QDs with such a preparation of QD-probe conjugates.34

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It should be noted that the self-assembly of SMN1 probes to the surface of QDs in our previous

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studies and as well as in this study was done using the same coupling chemistry (see SI for

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details).

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We hypothesized that the analytical performance of the paper-based solid-phase QD-FRET

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nucleic acid hybridization assay could be improved by conjugation of a greater number of probes

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to the surface of QDs, which would provide for a greater number of acceptors to interact with a

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QD donor upon hybridization. In this work, two approaches were concurrently explored in order

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to increase the loading density of SMN1 probes to the surface of QDs. One approach was

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incubating the QDs with a greater number of oligonucleotide probes (40 times molar excess)



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during the self-assembly step. The other approach was salt aging of QD-probes conjugates. Salt

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aging has been previously reported to increase the loading density of oligonucleotide probes to

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the surface of gold nanoparticles.44 Salt aging allows for a greater number of probes to assemble

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to the surface of a nanoparticle by screening the electrostatic repulsion associated with the

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negative charge of the DNA backbone. Solution-phase hybridization experiments with SMN1 FC

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Cy3 P TGTs demonstrating the effects of various preparations of QD-probe conjugates are

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shown in Figure 2a. Quantitative assessment of the analytical signal was done by assigning a

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FRET ratio to each background corrected and normalized PL spectrum (see Equation 1 in SI).

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The PL spectra were normalized to the gQD emission PL maximum. When GSH-QDs were

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incubated with 20 times molar excess of SMN1 probes in the absence of the salt aging step, the

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hybridization assay yielded a FRET ratio response of 0.44. This corresponded to an average

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assembly ca. 9 SMN1 probes to the surface of QDs. Increasing the stoichiometric ratio of SMN1

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probes to GSH-QDs from 20:1 to 40:1 in the absence of the salt aging step yielded a FRET ratio

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response of 1.0, which corresponded to an average assembly of ca. 17 SMN1 probes to the

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surface of QDs. However, when 40:1 preparation of QD-probe conjugates was subjected to the

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salt aging step, the FRET ratio response increased to 6.1. This corresponded to an average

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assembly of ca. 40 SMN1 probes to the surface of QDs. It should be noted that the

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aforementioned hybridization assays were conducted under the condition of probe saturation,

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i.e., if the stoichiometry of probe to QD ratio was 20:1, then 20 targets were incubated with QD-

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probe conjugates. An estimation of the number of SMN1 probes that were conjugated to the

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surface of QDs was done using solution-phase quantitative hybridization assays with SMN1 FC

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Cy3 P TGTs shown in Figure 2b. This calibration curve was generated using a 40:1 preparation

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of QD-probe conjugates that were subjected to the salt aging step and purified from any excess


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unconjugated probes by purification of QD-probe conjugates via a precipitation step (see SI for

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details). Figure 2c shows solution-phase quantitative hybridization experiments with SMN1 FC

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Cy3 D TGT. It can be seen that the implementation of a target sequence that was labeled at the D

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end showed significantly reduced assay sensitivity (ca. 100 fold) as compared to a target

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sequence that was labeled at the P end. This suggests that the oligonucleotide probes are oriented

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away from the surface of a QD instead of adsorbing to the surface of a QD. In all of the solution-

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phase experiments, the presence of SMN1 Cy3 NC TGT showed no significant signal above the

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background as reported previously.34-35 The 40:1 preparation of QD-probe conjugates that was

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subjected to the salt aging step were subsequently used for the development of paper-based solid-

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phase QD-FRET nucleic acid hybridization assays.

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Figure 2. (a) FRET ratio responses for solution-phase hybridization reactions of SMN1 Cy3 FC P TGTs with various preparations of QD-probe conjugates under the condition of probe saturation. 20 X and 40 X correspond to the relative stoichiometry of SMN1 probe to QD. Solution-phase quantitative hybridization assays with 40 X (salt aging) preparation of QD-probe conjugates with increasing stoichiometry of (b) SMN1 Cy3 FC P TGTs and (c) SMN1 Cy3 FC D TGTs. (Insets) PL spectra corresponding to the data points. Note the difference in the sensitivity response of (b) and (c) by considering that the ordinate scale in (b) and (c) is different.



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Solid-Phase Hybridization Assays on Paper Substrates

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improved sensitivity, reusability, amelioration of non-specific interactions and ease of assay

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assembly (cf. issues of maintaining colloidal stability with solution-phase assays).28,

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results for the solid-phase nucleic acid hybridization assays on paper substrates are shown in

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Figure 3. Initial experiments were conducted in a direct assay format using SMN1 Cy3 FC D

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TGT. Figure 3a shows the response of the assay to increasing concentration of SMN1 Cy3 FC D

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TGT, where the data acquisition from the hydrated paper substrates was done using an

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epifluorescence microscope (laser excitation source and diode array spectrometer as a detector).

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The limit of detection (LOD) of the assay from hydrated paper substrates was 300 fmol and

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corresponded to a FRET ratio signal that was three standard deviations above the average

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background FRET ratio (i.e., FRET ratio in the absence of target DNA). The upper limit of

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dynamic range of the assay was 45 pmol. This corresponds to a dynamic range of greater than

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two orders of magnitude. Figure 3b shows the response of the same assay from the paper

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substrates that were dried inside a desiccator prior to data collection. The LOD of SMN1 Cy3

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FC D TGT in case of the dry paper was 47 fmol and showed almost an order of magnitude lower

270

LOD as compared to hydrated paper substrates. The upper limit of dynamic range of the assay

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with dry paper substrates was the same as in the case of hydrated paper substrates (45 pmol).

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This corresponds to a dynamic range of three orders of magnitude. The dynamic range of three

273

orders of magnitude that is reported herein is two orders of magnitude larger than previous solid-

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phase QD-FRET nucleic acid hybridization assays that have been reported by our group using

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various solid supports such as optical fibers38, microfluidic channels42, microtitre plates41 and

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hydrated paper substrates34-35. This improvement in the dynamic range can be attributed to a

Solid-phase QD-FRET assays are advantageous from the standpoint of containment of QDs,


31

The

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greater number of oligonucleotide probes that were assembled on the surface of QDs by the salt

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aging step, which subsequently allowed for a greater number of acceptors to interact with a

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single QD donor, and the improvement in the LOD offered by the dry paper substrates. Previous

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solid-phase QD-FRET (gQD/Cy3 FRET pair) nucleic acid hybridization assays that were

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reported by our group using hydrated paper substrates for selective detection of SMN1 Cy3 FC P

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TGTs offered a LOD of 150 fmol.34 The LOD of 47 fmol that is reported in this work using the

283

same FRET pair is 3-fold lower than the previously reported assay despite 100-fold lower assay

284

sensitivity (see Figure 2) offered by SMN1 Cy3 FC D TGT (this work) as compared to SMN1

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Cy3 FC P TGT (previous work34). Hybridization assays conducted with non-complementary

286

target (SMN1 Cy3 NC TGT) showed excellent resistance to non-specific adsorption of

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oligonucleotides and showed no significant response above the background FRET ratio (see

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Figure S3 in SI for details).

289 290

It is interesting to note the difference in the assay sensitivities with dry and hydrated paper

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substrates. The assay sensitivities with hydrated and dry paper substrates were 0.0131(±0.0003)

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pmol-1 and 0.1436(±0.0044) pmol-1, respectively. This corresponds to ca. 11-fold higher assay

293

sensitivity with dry paper substrates as compared to hydrated paper substrates. The origin of this

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enhancement is hypothesized to be an increase in the “effective” density of QD donors upon

295

drying of paper substrates. Cellulose fibers in a paper matrix are known to undergo contraction

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upon dehydration.45 It is anticipated that the collapse of cellulose fibers upon dehydration

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provides for an increase in the number of energy transfer pathways that originate from multiple

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donor-multiple acceptor interactions introduced by nearest-neighbor proximity as compared to

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hydrated paper substrates. Such multiple donor-multiple acceptor interactions have been



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previously reported to serve as a source of signal enhancement for a FRET-based transduction

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using QD-dye FRET pair assemblies that were immobilized as a monolayer on the surface of

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glass substrates43 and within a 3-dimensional hydrated paper matrices when compared with

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solution-phase QD-dye FRET pair centrosymmetric constructs34-35. However, the signal

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enhancement of QD-dye FRET pair assemblies associated with drying of paper substrates has

305

not been previously reported. Control experiments that involved immobilization of QD-probe

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conjugates that were hybridized to SMN1 Cy3 FC D TGT on glass coverslips, a substrate that

307

cannot undergo contraction upon drying, showed no difference in the FRET ratio response in the

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hydrated and dry formats (see Figure S4 in SI). This suggests that the enhancement of FRET

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signal associated with drying of paper substrates is not governed by a change in the interaction

310

distance between Cy3 dye and gQD interface, or by solvation effects. Interestingly, exposure of

311

the paper substrates to different levels of relative humidity (21%, 43% and 56%) after they had

312

been dried in a desiccator showed no dependency on the assay sensitivity (see Figure S5 in SI). It

313

is anticipated that higher levels of relative humidity can potentially contribute to a variation in

314

the assay sensitivity, however a control of the degree of dryness of the paper substrates can be

315

achieved by means of a vacuum desiccator followed by imaging of the paper substrates

316

immediately, as was done in this work.

317 318

Comparing the solution-phase assay response of SMN1 Cy3 FC D TGT shown in Figure 2c with

319

the solid-phase assay response with hydrated and dry paper substrates shown in Figure 3, the

320

hydrated and dry paper substrates respectively provided 6.5-fold and 57-fold higher FRET ratio

321

signal as compared to solution-phase assay response. These results are consistent with our earlier


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studies that have shown that solid-phase QD-FRET assays offer improved assay sensitivity as

323

compared to solution-phase QD-FRET assays.28

324

325 326 327 328 329 330 331 332 333 334 335 336 337 338 339

Figure 3. Solid-phase direct hybridization assays with SMN1 Cy3 FC D TGT on paper substrates in the (a) hydrated and (b) dry formats. (a) (i) FRET ratio responses and the corresponding (ii) PL spectra response of the hydrated paper with increasing concentration of SMN1 Cy3 FC D TGT. (b) (i) FRET ratio responses and the corresponding (ii) PL spectra response of the dry paper with increasing concentration of SMN1 Cy3 FC D TGT. (Inset) Response of the assay at fmol quantities of target DNA. Note the difference in the sensitivity response of (a) and (b) by considering that the ordinate scale in (a) and (b) is different. Each error bar represents one standard deviation of n = 4 replicates.

Ratiometric Detection with Digital Imaging and Sandwich Assay Optical transduction that is based on ratiometric detection requires concurrent detection at two

340

wavelength bands. For a FRET-based transduction, one wavelength band is associated with a

341

donor PL and another wavelength band is associated with an acceptor PL. In this work,

342

concurrent detection of two detection channels using a digital camera was achieved by red-

343

green-blue (R-G-B) splitting of the colored digital images. PL intensities of gQDs and Cy3 were



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344

associated with the G and R channels respectively. Color imaging in a digital camera is done by

345

means of a Bayer mosaic color filter array, where each photosensitive element (pixel) is covered

346

with either a R, G or B filter to interrogate the long, middle and short wavelength visible lights,

347

respectively.46 A demosaicing algorithm is then used to assemble a composite color image using

348

the information from each colored pixel.47 The digital images that were acquired using an iPad

349

mini in this work were split into corresponding R-G-B color channels using ImageJ software.

350

Ratiometric analysis was done by dividing the mean PL intensity of R channel by the mean PL

351

intensity of G channel for a given spot (see Equation S2 in SI). Ratiometric analysis for

352

quantification, such as by using the FRET ratio or R/G ratio, is advantageous as the relative

353

donor and acceptor PL intensities self calibrate each other to provide a means to account for

354

signal variations that are embedded in absolute PL measurements (e.g. variation in excitation

355

intensity and detector sensitivity response).29 However, ratiometric detection does not take into

356

account automatic image contrast adjustments, such as exposure time, illumination intensity,

357

lighting conditions and white balance of an image, that are made by the iPad Camera software

358

during image acquisition.46 Variations in the automatic image contrast adjustments can

359

potentially compromise the reproducibility of digitization of the color intensity response

360

(numerical value of R/G ratio). To provide some control of these adjustments, paper substrates in

361

this work were imaged in the dark to alleviate automatic image contrast adjustment caused by

362

variations in the ambient light conditions. Moreover, control spots consisting of just immobilized

363

QD-probe conjugates were imaged in parallel with the spots subjected to the hybridization

364

reactions. These spots served as the brightest spots in the field of view and provided a means to

365

control automatic image contrast adjustments made by the software.

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367

Figure 4a shows colored digital PL images and associated pseudo-colored PL images in the R

368

and G channels upon R-G-B splitting for the response of the assay with increasing concentration

369

of SMN1 FC TGT in a sandwich assay format. As compared to the direct assay format that was

370

earlier presented, the sandwich assay format is advantageous as it avoids reliance on direct

371

labeling of targets, and hence serves as label-free target detection approach. The paper substrates

372

were dried in a vacuum desiccator prior to data collection as the dry format offered higher

373

analytical sensitivity. It can be seen from Figure 4a that with increasing concentration of SMN1

374

FC TGT, the PL color response of spots changed from green to yellow, where the increase in

375

color intensity of a spot in the R channel and a concurrent decrease in the color intensity of the

376

same spot in the G channel were commensurate with increasing target concentration. Multiple

377

PL images of the same paper substrate that were acquired using the same device and two

378

different devices showed no difference in the R/G ratio response as a function of target

379

concentration (see Figures S6a and S6b in SI). Additionally, iPad imaging offered reproducible

380

sensitivity over the detection window (see Figure S6c). Figure 4b shows the normalized spectral

381

response of the assay with increasing concentration of SMN1 FC TGT that was acquired using

382

an epifluorescence microscope (laser excitation source and spectrometer as a detector). The data

383

shows an increase in the FRET sensitized Cy3 emission with increasing concentration of SMN1

384

FC TGT. Note that the spectra in Figure 4b are normalized to the gQD emission maximum and

385

hence show no decrease in the gQD emission with increasing target concentration. Figures 4c

386

and 4d offer a comparison of the ratiometric response of the sandwich assay from the

387

epifluorescence microscope (FRET ratio) and R-G-B digital imaging (R/G ratio). The

388

relationship between the R/G ratio and the FRET ratio was modeled as a power function (see

389

Figure S7 in SI). It can be seen that the two readout platforms exhibited similar response curves



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390

as a function of target concentration. The upper limit of dynamic range of the sandwich assay for

391

both the microscope data and R-G-B imaging data was 45 pmol, which is consistent with the

392

direct hybridization assay results presented earlier. The difference between the two readout

393

platforms was observed at low target concentrations as shown in Figure 4d. The data acquired

394

from the epifluorescence microscope provided almost an order of magnitude lower LOD (30

395

fmol) as compared to the data acquired from R-G-B imaging (LOD 450 fmol). However, digital

396

imaging using an iPad mini in combination with a handheld UV lamp as an excitation source

397

provided an optical readout platform that was orders of magnitude lower in cost as compared to

398

an epifluorescence microscope (laser excitation source and diode array spectrometer as a

399

detector). The LOD of 30 fmol reported herein for the sandwich assay format is competitive with

400

other FRET based nucleic acid diagnostic assays, such as the strand displacement based nucleic

401

acid hybridization assay that was recently reported by Scida et al. using origami PADs (LOD ca.

402

30 fmol)48 and solid-phase QD-FRET nucleic acid hybridization assays that were reported within

403

microfluidic channels (LOD ca. 23 fmol)42. In case of the hydrated paper substrates, the R-G-B

404

digital imaging provided a LOD of 7.5 pmol, which is at least an order of magnitude higher than

405

the LOD achieved using dry paper substrates (see Figure S8 in SI). These results are consistent

406

with the data seen for direct hybridization, where the dry paper substrates offered lower LOD as

407

compared to hydrated paper substrates. Hybridization assays in a sandwich format also showed

408

excellence resistance to non-specific adsorption of oligonucleotides (see Figure S9 in SI).

409 410


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411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429


Figure 4. (a) Colored digital image and pseudo-colored PL images of gQDs (G channel) and Cy3 (R channel) upon R-G-B splitting of the colored digital image for the hybridization of (i) 0 pmol (ii) 0.94 pmol (iii) 1.9 pmol (iv) 3.8 pmol (v) 7.5 pmol (vi) 15 pmol (vii) 30 pmol and (viii) 45 pmol of SMN1 FC TGT in a sandwich assay format. The white dashed circles indicate the location of the spots that are not otherwise clearly visible. (b) Normalized PL spectra for the hybridization of (i) 0 pmol (ii) 0.057 pmol (iii) 0.12 pmol (iv) 0.23 pmol (v) 0.47 pmol (vi) 0.94 pmol (vii) 1.9 pmol (viii) 3.8 pmol (ix) 7.5 pmol (x) 15 pmol (xi) 30 pmol and (xii) 45 pmol of SMN1 FC TGT in a sandwich assay format. (c) Calibration curves showing the R/G ratio response (black) and the FRET ratio response (red) of the sandwich assay with increasing amounts of SMN1 FC TGT. (d) R/G ratio and FRET ratio responses of the assay at fmol quantities of SMN1 FC TGT. Each error bar represents one standard deviation of n = 4 replicates.

Single Nucleotide Polymorphism Discrimination Mismatch discrimination at the single base pair level, also known as single nucleotide

430

polymorphism (SNP) detection, is important for many nucleic acid diagnostic applications that

431

involve detection of genetic mutations and identification of gene copy numbers. In this work, the

432

stringency conditions for SNP discrimination were optimized by using a combination of ionic

433

strength and formamide concentration as shown in Figure 5a. Hybridization assays conducted in

434

50 mM borate buffered saline (BBS buffer, 100 mM NaCl, pH 9.25) provided a negligible SNP



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Page 20 of 33

435

contrast ratio of 1.07 to1 between SMN1 Cy3 FC D and SMN1 Cy3 1 BPM D TGTs (direct

436

assays), respectively. Washing the paper substrates for 10 minutes with BB buffer (no NaCl

437

added) containing increasing concentration (% v/v) of formamide provided improved SNP

438

contrast ratio. As can be seen from Figure 5a(i) and the corresponding R-G-B images in Figure

439

5a(ii), the highest SNP contrast ratio of 149:1 (from PL spectra) was observed at 12% (v/v)

440

formamide concentration. Under these optimized conditions, SNP discrimination was also

441

possible in a sandwich assay format as can be seen from the R/G ratio plot and the corresponding

442

R-G-B images shown in Figure 5b(i) and Figure 5b(ii), respectively. The R/G ratios associated

443

with SMN1 FC TGT and SMN1 1 BPM TGT after washing the paper substrates with 12% (v/v)

444

formamide were 0.614(±0.018) and 0.010(±0.004), respectively. This corresponds to a SNP

445

contrast ratio of 60 to 1 (from R-G-B images) between SMN1 FC and SMN1 1 BPM TGTs,

446

respectively. It should be noted that the use of a hydrogen bond disrupter such as formamide to

447

lower the melt temperature of a DNA duplex for SNP discrimination is advantageous as this

448

method avoids reliance on the use of external heaters and allows SNP discrimination at room

449

temperature. Additionally, temperature variation is known to affect the optical properties of QDs

450

and molecular fluorophores.49

451


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452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471


Figure 5. (a) Optimization of SNP contrast ratio using direct hybridization assays with SMN1 Cy3 FC D TGT and SMN1 Cy3 1 BPM D TGT. (i) SNP contrast ratio acquired from the PL spectra (epifluorescence microscope) as a function of increasing (% v/v) formamide concentration in BB buffer. The highest contrast ratio is observed at 12 % (v/v) formamide concentration. (ii) Pseudo-colored PL images of gQDs (G channel) and Cy3 (R channel) after RG-B splitting for the hybridization of FC and 1 BPM TGTs in BBS buffer and after exposure to 12 % (v/v) formamide in BB buffer for 10 minutes. (b) SNP discrimination in a sandwich assay format with SMN1 FC TGT and SMN1 1 BPM TGT. (i) R/G ratio response and (ii) pseudocolored PL images of gQDs and Cy3 after exposure to 12 % (v/v) formamide in BB buffer for 10 min. For reference, pseudo-colored PL images of just immobilized QDs in the G and R channels are also shown. The red arrow indicates SNP discrimination. The white dashed circles indicate the location of the spots that are not otherwise clearly visible. Each error bar represents one standard deviation of n = 4 replicates. Abbreviation: formamide = F.

Detection in Complex Matrices The performance of the paper-based solid-phase QD-FRET nucleic acid hybridization assay in a

472

sandwich format was also challenged in the presence of three potentially interfering matrices.

473

The three matrices that were investigated were salmon sperm (SS) DNA (2000 base pair

474

fragment), bovine serum albumin (BSA) and goat serum (GS) that were present at a

475

concentration of 0.8 mg mL-1, 40 mg mL-1 and 85% (v/v) respectively. These matrices were

476

spiked with the target DNA (SMN1 FC TGT or SMN1 NC TGT) at a concentration of 20.7 µg

477

mL-1 (2.0 µM or 6 pmol). The results for the hybridization assays conducted in the absence (just



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Page 22 of 33

478

BBS buffer) and presence of the complex matrices are given in Figure 6. As can be seen from the

479

R-G-B images in Figure 6a and the corresponding R/G ratio plots in Figure 6b, the hybridization

480

assays conducted with SMN1 FC TGT in the presence of complex matrices yielded the same

481

R/G ratio response (within experimental precision) as in the absence of the potentially interfering

482

matrices. In the presence of SMN1 NC TGT, no significant response (R/G ratio) was observed in

483

the presence and absence of complex matrices. These results suggest that the selective chemistry

484

was not occluded in the presence of large background of the complex matrices and the presence

485

of these matrices did not compromise the selectivity of the assay.

486 487 488 489 490 491 492 493 494 495 496 497 498 499 500

Figure 6. Hybridization experiments conducted in a sandwich assay format in the presence of complex background matrices. (a) Pseudo-colored PL images of gQDs (G channel) and Cy3 (R channel) and (b) corresponding R/G ratios for the exposure of SMN1 FC TGT and SMN1 NC TGT that was dissolved either in BBS, SS DNA, BSA or GS. For reference, pseudo-colored PL images of just immobilized QDs in the G and R channels are also shown. The amount of target DNA that was spotted was 6 pmol. The white dashed circles indicate the location of the spots that are not otherwise clearly visible. Each error bar represents one standard deviation of n = 4 replicates.

CONCLUSION Detection using portable devices such as smartphones and PDAs for the development of low cost

501

paper-based assays holds great promise for point-of-care screening and diagnosis. This work

502

investigated the use of R-G-B color selectivity of a digital camera for a ratiometric transduction


Page 23 of 33

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503

of nucleic acid hybridization assay on a paper-based platform using immobilized QDs as donors

504

in FRET. A hybridization event was transduced by a simultaneous “turn-on” signal from the

505

FRET sensitized emission from Cy3 acceptor dye and a “turn-off” signal from the loss in the

506

intensity of gQD donor emission with increasing target concentration. The PL intensities of Cy3

507

and gQD were respectively associated with the red and green imaging channels of a digital

508

camera for a ratiometric transduction of nucleic acid hybridization. Hybridization assays were

509

also demonstrated in a sandwich format, which avoided the need to directly label the target

510

sequences. The signal enhancement associated with the QD-FRET transduction that was offered

511

by the dried paper substrates provided at least one order of magnitude higher assay sensitivity

512

and at least one order of magnitude lower LOD as compared to the hydrated paper substrates.

513

This enhancement facilitated detection of sub-picomole (LOD 450 fmol) quantities of

514

oligonucleotide targets using the digital camera imaging, while the epifluorescence microscope

515

detection platform offered a LOD of 30 fmol. The stringency conditions for SNP discrimination

516

were optimized by using a combination of ionic strength and formamide concentration and

517

allowed SNP discrimination at a contrast ratio of 60 to 1 using a digital camera. The

518

hybridization assays were also functional in the presence of large background of non-

519

complementary matrices and showed excellent resistance to non-specific adsorption of

520

oligonucleotides. This work further extends the advantages of a paper-based substrate for the

521

development of QD-FRET assays by providing a simple and reagentless means of signal

522

enhancement, where the advantageous ratiometric transduction is facilitated by R-G-B color

523

selectivity of a digital camera.

524 525 526 527



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528 529 530

ACKNOWLEDGEMENTS

531

Canada (NSERC) for financial support of this research. M.O.N is also grateful to the Ontario

532

Ministry of Training, Colleges and Universities (MTCU) for provision of an Ontario Graduate

533

Scholarship (OGS). The authors also thank Syeda Natasha Hasan for her assistance with

534

preliminary experiments.

Page 24 of 33

The authors gratefully acknowledge the Natural Sciences and Engineering Research Council of

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ASSOCIATED CONTENT Supporting Information Available Detailed experimental procedures, equations used in the data analysis, description of instrumentation and additional results and discussion. This information is available free of charge via the Internet at http://pubs.acs.org/.

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TOC 389x180mm (300 x 300 DPI)



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Figure 1 374x178mm (300 x 300 DPI)


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Figure 2 292x93mm (300 x 300 DPI)



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Figure 3 221x172mm (300 x 300 DPI)


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Figure 4 345x188mm (300 x 300 DPI)



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Figure 5. (a) Optimization of SNP contrast ratio using direct hybridization assays with SMN1 Cy3 FC D TGT and SMN1 Cy3 1 BPM D TGT. (i) SNP contrast ratio acquired from the PL spectra (epifluorescence microscope) as a function of increasing (% v/v) formamide concentration in BB buffer. The highest contrast ratio is observed at 12 % (v/v) formamide concentration. (ii) Pseudo-colored PL images of gQDs (G channel) and Cy3 (R channel) after R-G-B splitting for the hybridization of FC and 1 BPM TGTs in BBS buffer and after exposure to 12 % (v/v) formamide in BB buffer for 10 minutes. (b) SNP discrimination in a sandwich assay format with SMN1 FC TGT and SMN1 1 BPM TGT. (i) R/G ratio response and (ii) pseudo-colored PL images of gQDs and Cy3 after exposure to 12 % (v/v) formamide in BB buffer for 10 min. For reference, pseudocolored PL images of just immobilized QDs in the G and R channels are also shown. The red arrow indicates SNP discrimination. The white dashed circles indicate the location of the spots that are not otherwise clearly visible. Each error bar represents one standard deviation of n = 4 replicates. Abbreviation: formamide = F. 314x196mm (300 x 300 DPI)


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Figure 6. Hybridization experiments conducted in a sandwich assay format in the presence of complex background matrices. (a) Pseudo-colored PL images of gQDs (G channel) and Cy3 (R channel) and (b) corresponding R/G ratios for the exposure of SMN1 FC TGT and SMN1 NC TGT that was dissolved in BBS, SS DNA, BSA and GS. For reference, pseudo-colored PL images of just immobilized QDs in the G and R channels are also shown for reference. The amount of targets that was spotted was 6 pmol. The white dashed circles indicate the location of the spots that are not otherwise clearly visible. Each error bar represents one standard deviation of n = 4 replicates. 236x168mm (300 x 300 DPI)


Camera-Based Ratiometric Fluorescence Transduction of Nucleic

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