Signal-on Protein Detection via Dye Translocation between Aptamerand Quantum DotYeh-Hsing Lao,†,∥ Chun-Wei Chi,‡,∥,# Sarah M. Friedrich,§ Konan Peck,¶ Tza-Huei Wang,§,⊥

Kam W. Leong,*,† and Lin-Chi Chen*,‡

†Department of Biomedical Engineering, Columbia University, New York, New York 10027, United States‡Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei 10617, Taiwan§Department of Biomedical Engineering and ⊥Department of Mechanical Engineering, Johns Hopkins University, Baltimore,Maryland 21218, United States¶Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan

*S Supporting Information

ABSTRACT: A unique interaction between the cyanine dyeand negatively charged quantum dot is used to construct asignal-on biaptameric quantum dot (QD) Forster resonanceenergy transfer (FRET) beacon for protein detection anddistinct aptamer characterization. The beacon comprises a pairof aptamers, one intercalated with the cyanine dye (YOYO-3)and the other conjugated to a negatively charged, carboxyl-QD. When the target protein is present, structural folding andsandwich association of the two aptamers take place. As aconsequence, YOYO-3 is displaced from the folded aptamerand transferred to the unblocked QD surface to yield a targetconcentration-dependent FRET signal. As a proof-of-principle, we demonstrate the detection of thrombin ranging fromnanomolar to submicromolar concentrations and confirm the dye translocation using cylindrical illumination confocalspectroscopy (CICS). The proposed beacon provides a simple, rapid, signal-on FRET detection for protein as well as a potentialplatform for distinct aptamer screening.

KEYWORDS: quantum dot, aptamer, intercalating dye, FRET, dye translocation

■ INTRODUCTION

YOYO-3 (qu ino l in ium, 1 ,1 ′ - [1 ,3 -propaned iy lb i s -[(dimethyliminio)-3,1-propanediyl]]bis[4-[3-(3-methyl-2(3H)-benzoxazolylidene)-1-propenyl]]-tetraiodide) is one of thecyanine dyes that intercalates a DNA duplex backbone with acorresponding increase in its quantum yield.1 This property hasbeen exploited widely for nucleic acid detection.2,3 In additionto DNA, cyanine dyes (e.g., carbocyanine and indoleninecyanine families) are known to interact with nanomaterials suchas tin dioxide nanocrystallite,4 gold nanoparticle,5 and quantumdot (QD).6 In this study, we report a unique interactionbetween YOYO-3 and carboxyl-QD, which leads to a similarinteraction with DNA, changing YOYO-3’s quantum yield andthus enhancing its fluorescence response. If QD’s emission andYOYO-3’s excitation spectra overlap, the interaction induces aForster resonance energy transfer (FRET) between them.Hypothesizing that it can be used as a readout for sensingapplications, we have designed a beacon construct for proteindetection based on aptamer’s conformational change and thetranslocation of YOYO-3 between the aptamer and the QD.As a highly specific ligand, aptamer is an attractive antibody

alternative for diagnostics because of its small size, chemical andthermal stability, and easy modification and immobilization.7,8

Consequently, it has been applied in many formats ofimmunoassays.9−11 Moreover, a certain group of aptamerssuch as anti-thrombin, anti-PDGF (platelet-derived growthfactor), and anti-cocaine aptamers exhibit a target-inducedconformational change.12−15 This structural property inspiresanother dimension of specificity in target recognition: theconformational change as a subsidiary signal of the bindingevent. Herein, as illustrated in Scheme 1, we use this propertyto store YOYO-3 onto the backbone of the aptamer-recognizing thrombin exosite II (HTDQ2916) so that there isno YOYO-3 on the QD surface and no FRET emission in theabsence of thrombin. In the presence of thrombin, HTDQ29and the other aptamer on QD surface (HTQ37; the exosite Ibinding aptamer, HTQ3717) bind to the two distinct motifs ofthrombin and change their conformations. Consequently, thedye is expelled from the HTDQ29 backbone and translocatesonto the QD surface, which leads to a signal-on FRETresponse.

Received: March 7, 2016Accepted: April 21, 2016Published: April 21, 2016

Research Article

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© 2016 American Chemical Society 12048 DOI: 10.1021/acsami.6b02871ACS Appl. Mater. Interfaces 2016, 8, 12048−12055

To date, the vast majority of QD-aptamer beacons rely onDNA strand-displacement for target protein detection,18−24

which may require laborious optimization;23 otherwise,undesired strand interactions (e.g., when the interaction withinthe beacon is stronger than the beacon−protein interaction)would reduce the sensitivity. In contrast, this method provides asimple and rapid approach for protein sensing. Using thrombinas a model protein, we demonstrate the operating principle ofthis beacon design. We also propose that this design may beuseful for the screening of two distinct aptamers binding to thesame target protein, a feature that no other aptameric-QDbeacons can offer.18−28

■ EXPERIMENTAL SECTIONAptamers. Two reported anti-thrombin DNA aptamers, HTQ3717

(CCCGG TTGGT GTGGT TGGAT TGATC GTAGG TACAA CC;an aptamer derived from a G-quadruplex aptamer HTQ1529) andHTDQ2916 (AGTCC GTGGT AGGGC AGGTT GGGGT GACT),were synthesized and investigated in this study. HTQ37’s parentalaptamer HTQ15 (the underlined part of HTQ37 sequence) and itscomplementary sequence were synthesized as well in this study.HTQ37 was modified with an amino C6 linker at the 5′-end forcoupling with carboxyl QD. Our previous work and another publishedwork have already shown that the modification with an amino C6linker does not significantly influence the affinity of anti-thrombinaptamers.30,31 All of the oligonucleotides were obtained from eitherPurigo Biotech, Inc. (Taipei, Taiwan) or Integrated DNATechnologies (Coralville, IA). The lengths of aptamer probes wereconfirmed by either urea-polyacrylamide gel electrophoresis (PAGE)or electrospray ionization−mass spectrometry (ESI−MS).Preparation of QD-HTQ37 Conjugate. We slightly modified the

protocol for QD-aptamer conjugation from our previous work.25

Through EDC/sulfo-NHS chemistry, QD was conjugated withaptamer probe. First, 0.5 nmol carboxyl-QD (Qdot 565 ITK;Invitrogen) was activated by an EDC/sulfo-NHS mixture in PBSbuffer (10 mM phosphate, pH 7.4, 137 mM NaCl and 3 mM KCl) for15 min. Subsequently, 5 nmol amine-modified HTQ37 was added tothe activated mixture and incubated for 2 h. QD-aptamer reporter waspurified with a 50 kDa molecular weight cutoff centrifugal filter(Amicon Ultra-0.5, Milipore Corp., Billeria, MA). Prior to use, QD-HTQ37 reporters were quantitated by UV−vis and stored at 4 °C inthe dark.

Determination of the Conjugation Efficiency and the Ratioof Free to Conjugated HTQ37s. The conjugation efficiency andfree to conjugated HTQ37 ratio were determined by gel electro-phoresis. TOTO-3 (Invitrogen) was used to stain free and conjugatedHTQ37s because the excitation (peak at 642 nm) and emission (peakat 660 nm) spectra of TOTO-3 do not overlap with those of QD565.To calculate the conjugation efficiency, unpurified QD-HTQ3 sampleswere directly stained with 500 nM of TOTO-3 and subsequentlyloaded on a 0.5% agarose-TBE gel. The gel was run and then visualizedby Typhoon 9410 (GE Healthcare) with a 633 nm excitation laser anda 670 bandpass filter for TOTO-3 signal detection. The conjugationefficiency was determined using ImageJ with eq 1:

=F

FConjugation efficiency (%)

conjugatedHTQ37

totalHTQ37signals (1)

where F represents the fluorescent intensity at 670 nm. The ratio offree to conjugated HTQ37s was determined with a similar fashion.The QD/HTQ37 conjugation mixtures (5 μL) after the first throughthe fifth purification were sampled and stained with TOTO-3 (500nM). Free and conjugated HTQ37 was also visualized on the 0.5%agarose-TBE gel and detected by Typhoon 9410. The ratio wastherefore determined by the signal of free HTQ37 divided by that ofconjugated HTQ37.

Detection of Protein Analytes. Before detection of proteinanalytes, 40 base-paired poly-TA duplex, HTDQ29 or HTQ37 (200nM) was stained by 500 nM YOYO-3 dyes for 1 h. The QD-HTQ37reporter (15 nM) and human α-thrombin (Haematologic Technolo-gies Inc., Essex Junction, VT) were incubated with YOYO3-stainedaptamers for an additional 1 h. The fluorescent changes were measuredby SpectraMax Gemini EM (with 365 nm excitation); the signal wasnormalized and presented as a relative signal compared to the signalintensity at the emission peak of QD (565 nm). In addition, toevaluate the specificity of this system, the confirmation of nonspecificbinding was carried out using 500 nM of BSA (Sigma-Aldrich) asanalyte, and the thrombin detection was also tested in the presence of0.1% and 1% of 0.22 μm-filtered human serum (Sigma-Aldrich). Eachmeasurement was performed in triplicate to determine the standarderrors of this sensing format.

Dye Translocation Measurement on the CICS Platform viaFree Solution Hydrodynamic Separations. Free solution hydro-dynamic separations were performed in a fused silica microcapillary(Polymicro, Molex) with a nominal inner diameter of 2 μm and alength of 48 cm. A 1 cm section of the polyimide coating was burnedaway from the capillary to create a viewing window of low backgroundfluorescence approximately 40 cm from the capillary inlet. The elutionbuffer used for all separations was PBS with 0.5% polyvinylpyrrolidone(PVP).

For the samples used on the CICS platform, HTDQ29 or HTQ37(1.67 μM) was first stained with TOTO-3 for 30 min at RT (dye-to-DNA molar ratio = 1:1) and subsequently incubated with 500 nMthrombin and 15 nM QD-HTQ37 conjugate for an additional 30 min.To perform a separation, the capillary was first filled with the elutionbuffer. A short sample plug was loaded into the capillary for 10 s at 200psi. The sample was driven down the length of the capillary bypressure driven flow of the elution buffer at 400 psi. The capillary wasflushed for at least 20 min at 450 psi with elution buffer before a newsample was injected for analysis.

The separated fluorescent species were detected using the CICSplatform described elsewhere.32 The center of the capillary viewingwindow was aligned within the 1 × 7 μm2 CICS observation volume. AHe−Ne laser with a power of 3 mW served as the excitation source,and fluorescence was detected with an avalanche photodiode. Photoncounts were collected in 0.1 ms bins using a custom LabVIEWprogram. The raw fluorescence data were corrected for chromaticinefficiency and rebinned in 1-s intervals for plotting.

■ RESULTS AND DISCUSSIONInteractions of YOYO-3 on QD and Dye Translocation.

In this work, we report a new protein sensing approach based

Scheme 1. Beacon Comprises a QD565-Aptamer Conjugateand a Nonconjugated Aptamer Stained with YOYO-3. TheRecognition of Thrombin Target Causes Structural Foldingand Sandwich Association of Two Aptamers. YOYO-3 IsThen Transferred from the Nonconjugated Aptamer to theQD’s Unblocked Surface To Yield a FRET Signal

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on the conformational change of aptamers induced by targetrecognition, facilitated by a protein that has two distinctaptamer-binding epitopes. In this proof-of-concept study,human thrombin binds to HTQ37 which is immobilized onthe surface of a QD as well as HTDQ29 in solution and stainedwith YOYO-3. When a HTQ37-captured thrombin moleculeinteracts with HTDQ29 in solution, the conformational changeof the latter expels the YOYO-3 and deposits the dye on theQD surface (Scheme 1). YOYO-3 interacts with the carboxyl-QD surface to yield a significant FRET output (Figure S1).Therefore, this can be used as a readout. To maximize thiseffect, different concentration ratios of QD to YOYO-3 werefirst tested. We incubated YOYO-3 in various concentrations(from 100 to 500 nM) with a fixed amount of unconjugatedand unblocked QD (15, 20, or 25 nM) for 1 h. The FRETresponse (F619/F565) was proportional to the concentration ofYOYO-3 but inversely proportional to the concentration of QD(Figure 1). Theoretically, YOYO-3 shows very low quantum

yield in the absence of double-stranded DNA (<0.01);33 yet, wehave observed that YOYO-3 interacted with the unblockedcarboxyl-QD surface and responded to energy transfer from theQD. Presumably, it is due to the electrostatic interaction.Unblocked QD surface provides additional negative charges toattract the dimeric cyanine dye, although this interaction shouldbe different from the known DNA/YOYO-3 interaction.Similar interactions have been observed on QD withthiacyanine6 and QD with Ru complex (Ru(bpy)2(dppz)

2+).28

However, the underlying mechanism is unclear, and it warrantsfurther investigation.We subsequently confirmed that the dye translocation would

occur during the target recognition of anti-thrombin aptamers,which is directly related to the DNA structural selectivity ofYOYO-3. If the dye did not show affinity difference towarddifferent aptamer conformations, YOYO-3 would not bereleased from the folded HTD29 aptamer. To investigatethis, we used HTQ37’s parental aptamer HTQ15 as a control,which has no intrabase parings and folds into a pure G-quadruplex (GQ) structure with the assistance of potassiumions.12,13 Here, the duplex and GQ structure of HTQ15 wereformed through a temperature-gradient annealing in thepresence of its complementary sequence and in the presenceof potassium ion, respectively. As shown in Figure 2, YOYO-3bound to the DNA duplex with high nanomolar affinity. Thedissociation constant against the duplex structure is 280.6 nM,five-fold higher than that against a GQ structure. This indicatesthat YOYO-3 dissociates from the aptamer during the duplex-

GQ transition. Both HTDQ2934 and HTQ15 family13 havebeen reported to fold into GQ-like structures when they bindto thrombin, so this result justifies the basic principle of ourapproach: YOYO-3 can be stored on the HTDQ29 backboneand be released when the aptamer interacts with thrombin andundergoes a conformational change.

QD-HTQ37 Preparation. To maximize the sensitivity ofthis system, in addition to HTDQ29, another anti-thrombinaptamer HTQ37 was conjugated onto QD surface, whichhelped to localize the YOYO-3 near QD surface. For theHTQ37 conjugation, sulfo-NHS was used to enhance theefficiency, and the ratio between sulfo-NHS and EDC wasoptimized. The QD-HTQ37 conjugates obtained underdifferent ratios of sulfo-NHS to EDC were visualized through0.5% agarose gel with TOTO-3 staining. The conjugationefficiency of QD and HTQ37 was determined to be 11.7 ±1.99% at the sulfo-NHS/EDC ratio of 0.1, which could beimproved to 26.8 ± 0.88% as the ratio of sulfo-NHS to EDCwas increased to 0.5 (Figure 3A). When the ratio was greaterthan 0.5, no significant enhancement in QD-HTQ conjugationwas observed. Under this optimal condition, the mean aptamer-to-QD ratio was determined to be 2.68, which is close to thevalue obtained by analyzing the unconjugated aptamer in thereaction mixture (3.20 ± 0.80). In addition, after the QD wasdecorated with HTQ37s, we could observe a mobility change ofQD under gel electrophoresis analysis (Figure 3B) although thezeta potential of QD was not significantly changed (Figure 3C).This may be because HTQ37 itself is also negatively charged.We thus expect that the interaction between negatively chargedQD and YOYO-3 would remain after the aptamer conjugation.On the other hand, if there was free HTQ37 in solution, it

may interfere with the capture of YOYO-3 on the QD surfaceand reduce the detection sensitivity. To minimize this potentialinterference, we used a molecular weight-cutoff column topurify the QD-HTQ37 conjugates and to remove free HTQ37from the conjugation mixture. For complete removal of freeHTQ37, we carried out five rounds of purification, and theproducts were sampled and analyzed after each purification. Asshown in Figure 3, panel D, the free-to-conjugated HTQ37ratio was reduced from 2.16 ± 0.08 to 0.65 ± 0.15 after fiverounds of purification.

Protein Detection. For protein detection, HTDQ29 (200nM) was first stained with 500 nM YOYO-3. The secondarystructure of HTDQ29 comprises only five base-pairs (bp)

Figure 1. Relative dye emission signal (excitation at 365 nm) as afunction of dye concentration with respect to a given amount ofQD565 in PBS after 1 h of incubation. Inset: the FRET spectra in thecase of 15 nM QD. Data are represented as mean ± SEM (n = 3).

Figure 2. Comparison between the fluorescence intensity of aYOYO3-stained duplex (formed by HTQ15 and its antisensesequence) and that of a YOYO-3-stained quadruplex (obtained byannealing of HTQ15 in Tris-HCl buffer with KCl) DNA structures.Data are fitted by the total binding assay model, and the dissociationconstant of YOYO-3 against HTQ15 duplex is determined to be 280.6nM. Data are represented as mean ± standard error of the mean(SEM; n = 3).

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(predicted using IDT OligoAnalyzer 3.1), so we used a bp-to-dye ratio of 2. We assumed that in this condition there wouldbe minimal excess free dye after staining because the minimumbinding site for YOYO family is 3.2 ± 0.6 bp per dye,35 which isclose to the ratio used in this study. As shown in Figure 4, panelA, before target recognition, the QD and the YOYO-3 are apartfrom each other, and this results in no FRET response (FREToff). When thrombin is present, HTDQ29 and HTQ37associate with thrombin together by binding to differentepitopes. As a result, a sandwich complex of QD-HTQ37/thrombin/HTDQ29 is formed, and both aptamers becomefolded, which then induces the dye translocation and turns onthe beacon (FRET on). Figure 4, panel B shows the stability aswell as the responsive rate of this beacon system. Whenthrombin was introduced, the dye translocation-triggeredFRET signal reached the saturation at once and was maintainedfor at least 1 h. Less than 10% signal decay was observed during1 h of continuous measurement. Compared with conventionalimmunoassays, this beacon design is advantageous for on-siteand rapid diagnosis.Protein detection using our QD-HTQ37/HTDQ29 FRET

system is shown in Figure 5, panel A. The FRET signal indeedincreases with thrombin concentration, a clear signal-on feature.Our biaptameric QD beacon was able to detect thrombinranging from 10−500 nM. This satisfies the need to monitorthrombin generation at the clotting initiation phase forthrombosis diagnostics.36,37 The previous work demonstratingQD-Ru(bpy)2(dppz)

2+ interaction was also designed forthrombin detection.28 However, in that design, the aptamerwas used to compete with Ru(bpy)2(dppz)

2+ from QD surfaceto trigger dye (Ru(bpy)2(dppz)

2+) translocation. Comparedwith that study, our design enhances the LOD of dyetranslocation sensing approach. On the other hand, testingbovine serum albumin (BSA, 500 nM) as nontarget analyte, we

observed that the target signal at the same concentration wassignificantly higher than that of BSA (Figure 5A and Figure S2),but BSA also contributed to a notable signal, which may becaused by a false-negative response. To further evaluate the

Figure 3. (A) Comparison of QD-aptamer conjugation efficiency in different ratios of sulfo-NHS to EDC. (B) A gel image and (C) zeta potentials ofunconjugated QD and QD-HTQ37 conjugates. (D) Investigation on postconjugation purification. Conjugation efficiency and free/conjugatedHTQ37 ratio were determined by ImageJ. Data are represented as mean ± SEM (n = 3).

Figure 4. (A) FRET spectra of the beacon system, which comprisesYOYO-3-bearing HTDQ29 and QD-HTQ37 reporter, before andafter incubation with 500 nM of thrombin. (B) FRET signal stability ofthe beacon system. Relative signal (%) is obtained by normalizing theFRET florescence at each time point to that at t = 0. Data arerepresented as mean ± SEM (n = 3).

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source of the false-negative response, we first tested our designwith thrombin-spiked human serum. We could detect thrombinfrom the protein complexes, but the detection encounteredinterference with a high level of serum proteins (>1%), whichresulted in a significant signal decrease (Figure S3). In addition,we used a 40 bp double-stranded polyTA to investigate anotherpossible false-negative response to thrombin, which wasgenerated from this nonfunctional, dummy DNA. As shownin Figure 5, panel B, the false-negative response to thrombinwas not significantly higher than the background signal; we cantherefore exclude the possibility that the response is from thenonspecific interaction between thrombin and any types ofDNA, and we conclude that the specificity of this system isprimarily based on the selectivity of YOYO-3-brearing aptamer.Although the false-positive response restricts this dye trans-location approach to a detection scenario only with a lowconcentration of background proteins, compared with otherQD-aptamer beacons, the system presented in this work stilloffers comparable performance (Table S1) with a rapid readoutand a long-term stability for protein sensing in pure buffercondition. More importantly, this strategy does not rely onreporter strand displacement, so it can be more easily adaptedfor other target-sensing applications.18−28

Recognition Mechanism. We have demonstrated that thebiaptameric QD FRET beacon featuring target-induced dyetranslocation can be used for protein detection. To confirm themechanism behind this detection approach, we used CICScoupled to free solution hydrodynamic separation (FSHS) toverify whether thrombin led to a dye translocation. The CICSplatform creates a highly uniform observation volume thatspans the cross-section of a microchannel for enhanced masssensitivity of fluid scanning. This technology has been used toanalyze circulating DNA integrity32 and to determine the DNAloading level of polyplex.38 Moreover, integration of thismodified confocal spectroscopy technique with pressure-drivenflow through a long microcapillary can achieve highly sensitivefree solution hydrodynamic separation and characterization ofDNA molecules based on their length and conformation.39

Herein, we used this integrated separation and spectroscopyplatform to separate the dye-QD complexes from freeHTDQ29 and free dye. HTDQ29 was first stained withTOTO-3, followed by incubation with thrombin and QD-HTQ37. HTDQ29- or QD-bound TOTO-3 was detected usinga 633 nm excitation laser and a 670/40 nm band-pass filter. In

the presence of thrombin and QD-HTQ37, TOTO-3 wasreleased from HTDQ29, which resulted in a significant TOTO-3 signal decrease at the free HTDQ29 peak (Figure 6), and it

translocated onto QD-HTQ37 (Figure 6, inset). In the absenceof thrombin, this phenomenon was not observed (Figure S4).These results confirmed the working principle of this protein-sensing approach.We propose two possible recognition mechanisms to explain

how our beacon works. (i) Thrombin is first captured by QD-HTQ37 and then HTDQ29/YOYO-3 docks on the otherexosite of thrombin. In this case, the dye is directly transferredfrom the folded HTDQ29 to the vacant QD surface. (ii) Therecognition sequence is reversed and based on two-step dyetranslocation. The dye is transferred from the folded HTDQ29to unfolded HTQ37 first, and then the subsequent folding ofHTQ37 pushes the dye to the vacant QD surface. It has beenreported that HTDQ29 holds subnanomolar affinity againstthrombin exosite II, but the affinity of exosite I-binding aptamer(HTQ family) is nearly ten-times weaker.16 As a result, wespeculate that mechanism ii is likely predominant (Scheme 2).The dye is transferred from the folded HTDQ29 to unfoldedHTQ37 on the QD surface, which causes the first stage of dyetranslocation. Second stage of dye translocation occurs when

Figure 5. (A) Detection of thrombin in PBS using the signal-on QD-apt FRET beacon composed of QD-HTQ37 and HTDQ29:YOYO-3. (B) Theinvestigation of false negative response to thrombin using a dummy DNA (poly TA; 40bp). Samples were incubated for 1 h at room temperatureprior to detection. Data are represented as mean ± SEM (n = 3). Two-tailed Student t test was used for p-value calculation. The significant level isrepresented as ∗ (p < 0.05), ∗∗∗ (p < 0.001), or n.s. (no significance).

Figure 6. Dye translocation measured by CICS. HTDQ29 (1.67 μM)was first stained with 1.67 μM of TOTO-3 and then incubated withthrombin (500 nM) and QD-HTQ37 (15 nM) or without boththrombin and QD-HTQ37 for 30 min at RT. The sample wasseparated, and the TOTO-3 signal was measured on the CICSplatform. Background subtraction was performed on each data set. Thebackground was calculated as the average fluorescent intensity of thefirst 5 s of minute 15 and was subtracted from the total fluorescenceintensity for the full time trace.

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HTQ37 interacts with thrombin, and the dye is subsequentlylocalized on the carboxyl-QD surface.Potential Use for Bivalent Aptamer Screening. To

date, only a few protein targets have more than two distinctaptamers for different binding site recognition.16,40 Mostscreening techniques do not allow for bi- or multiple-valentaptamer identification.7 To take advantage of the workingprinciple of the proposed beacon system, we also explored thepossibility of using this beacon for aptamer screening inaddition to protein sensing. We tested both an “identical” anti-thrombin pair (YOYO-3-bearing HTQ37 with QD-HTQ37conjugate) and a “distinct” pair (HTD29+QD-HTQ37) ofaptamers to study on their protein detection performance anddye translocation. Although the identical pair was able to detectthrombin, sensitivity was reduced by 36% (p < 0.001; Figure 7).

Moreover, the CICS result confirmed that there was only subtletranslocation happening between YOYO-3 stained HTQ37 andQD-HTQ37 conjugate (Figure S5). As a result, the avidityeffect afforded by two distinct aptamers would show bettersensitivity. Owing to the signal difference between twosynergistic and competitive aptamers, we believe this systemcan be useful for post-SELEX aptamer screening to assesswhether two aptamer candidates recognize the same epitope ofthe target protein, which cannot be done by other aptamericQD beacons.

■ CONCLUSIONSA unique interaction between cyanine dye and carboxyl-QDanalogous to that of cyanine-DNA interaction is used to design

a beacon for protein sensing. We report a rapid and stablesignal-on QD biaptameric beacon composed of a YOYO-3-bearing HTDQ29 and a QD-HTQ37 conjugate for thrombindetection. When thrombin is sandwiched between HTDQ29and HTQ37, the conformational change of HTDQ29 releasesYOYO-3 and transfers the dye to the HTQ37 or QD surface togenerate a FRET signal. Two characteristics have beenvalidated to support the rationale of this beacon design. First,YOYO-3 selectively binds to duplex DNA rather than a GQstructure to allow for the dye translocation as confirmed byCICS spectroscopy. Second, an unblocked carboxyl-QDspontaneously associates with the translocated YOYO-3 toyield a stable FRET signal. This sensing format does notrequire a reporter strand optimization, rendering it readilyadaptable to other protein detection. As a proof-of-concept, thisbeacon can instantaneously detect submicromolar to nano-molar thrombin. The signal is highly stable, with less than a10% FRET signal decay over 1 h of measurement. In addition,this dye translocation approach may allow a post-SELEXscreening for two distinct aptamers.

■ ASSOCIATED CONTENT*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.6b02871.

Fluorescence spectra, CICS detections for dye trans-location measurement (PDF)

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: kam.leong@columbia.edu.*E-mail: chenlinchi@ntu.edu.tw.Present Address#Department of Biomedical Engineering, City College, TheCity University of New York, New York, New York 10031,United States.Author Contributions∥These authors contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors gratefully thank Dr. Shu-Chuan Jao (BiophysicsCore Facility, Academia Sinica) and Dr. Chaihoon Quek andDr. Bo Peng (Columbia University) for the instrumentalsupport and the input for manuscript preparation. Y.-H.L.acknowledges fellowship support from the Ministry of

Scheme 2. Possible Mechanism for the Target-Induced Dye Translocation: Thrombin Is First Captured by HTDQ29, andYOYO-3 Is Released Due to the Conformational Change of HTDQ29. The Dye Is Accordingly Transferred from the FoldedHTDQ29 to Unfolded HTQ37, and Then the Subsequent Folding of HTQ37 Pushes the Dye to the Negatively-ChargedCarboxyl-QD Surface

Figure 7. Emission signals at 619 nm for the beacons with a distinctaptamer pair (QD-HTQ37+YOYO-3/HTDQ29) or an identical pair(QD-HTQ37+YOYO-3/HTDQ37) recognition. Data are representedas mean ± SEM (n = 3). Two-tailed Student t test was used for p-valuecalculation. The significant level is represented as ∗∗∗ (p < 0.001).

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Education, Taiwan. This work is supported by MOST, Taiwan(NSC 98-2221-E-002-099-MY3), National Taiwan University(103R7820) (L.-C.C.), the Department of Defenses(W81XWH-12-1-0261 to K.W.L.), and the Grant-In-Aid ofResearch Program from Sigma Xi Research Society(G20131015280632 to Y.-H.L.).

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ACS Applied Materials & Interfaces Research Article

DOI: 10.1021/acsami.6b02871ACS Appl. Mater. Interfaces 2016, 8, 12048−12055

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strategy to generate aptamer pairs that bind to distinct sites on proteintargets. Anal. Chem. 2012, 84, 5365−5371.

ACS Applied Materials & Interfaces Research Article

DOI: 10.1021/acsami.6b02871ACS Appl. Mater. Interfaces 2016, 8, 12048−12055

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