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Contents lists available at SciVerse ScienceDirect

Analytica Chimica Acta

j ourna l ho me page: www.elsev ier .com/ locate /aca

ptamer sensor for cocaine using minor groove binder based energy transfer

inwen Zhou, Amanda V. Ellis, Hilton Kobus, Nicolas H. Voelcker ∗

chool of Chemical and Physical Sciences, Flinders University, Adelaide, SA 5001, Australia

r t i c l e i n f o

rticle history:eceived 26 September 2011eceived in revised form0 December 2011ccepted 3 January 2012

a b s t r a c t

We report on an optical aptamer sensor for cocaine detection. The cocaine sensitive fluorescein isothio-cyanate (FITC)-labeled aptamer underwent a conformational change from a partial single-stranded DNAwith a short hairpin to a double-stranded T-junction in the presence of the target. The DNA minor groovebinder Hoechst 33342 selectively bound to the double-stranded T-junction, bringing the dye within theFörster radius of FITC, and therefore initiating minor groove binder based energy transfer (MBET), and

vailable online xxx

eywords:ptamerinor groove binder based energy transfer

ocaineolydimethylsiloxane

reporting on the presence of cocaine. The sensor showed a detection limit of 0.2 �M. The sensor was alsoimplemented on a carboxy-functionalized polydimethylsiloxane (PDMS) surface by covalently immobi-lizing DNA aptamers. The ability of surface-bound cocaine detection is crucial for the development ofmicrofluidic sensors.

© 2012 Elsevier B.V. All rights reserved.

. Introduction

Cocaine, or benzoylmethylecgonine, is a common illicit drugncountered by law enforcement, border protection and forensiccience authorities [1,2]. Current field tests for cocaine are eitherresumptive and require confirmatory analysis in the laboratoryr necessitate high levels of training for effective operation [3].hemical sensors for cocaine which are simple to operate andtill offer high sensitivity and specificity are therefore required.

range of chemical sensors has been developed in recent yearsnvolving fluorescence [4–12], colorimetric [13–21], chemilumi-escence [22–24], electrochemical [17,25–37], surface-enhancedaman scattering [38–40], surface plasmon resonance [31,41] andurface acoustic wave [42,43] based transducers. Fluorescence- andolorimetric-based sensors are particularly desirable due to theimple detection procedures involved [16].

Stojanovic et al. [4] were the first to report the use ofptamers for the detection of cocaine in fluorescence-based sen-ors. Aptamers are nucleic acid based receptors that are obtainedhrough a combinatorial selection process known as systematicvolution of ligands by exponential enrichment (SELEX) [44,45].hese molecules have significant advantages over antibodies, such

Please cite this article in press as: J. Zhou, et al., Aptamer sensor for cocain(2012), doi:10.1016/j.aca.2012.01.011

s convenient synthesis and chemical modification, high affinityven to small molecular targets and resistance to biodegrada-ion [14,22,26,46]. Among fluorescence-based aptamer sensors,

∗ Corresponding author. Tel.: +61 8 8201 5338; fax: +61 8 8201 2905.E-mail addresses: nico.voelcker@unisa.edu.au, nico.voelcker@flinders.edu.au

N.H. Voelcker).

003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2012.01.011

fluorescence resonance energy transfer (FRET)-based aptamer sen-sors are particularly attractive for cocaine detection because ofthe inherent sensitivity of FRET to detect conformation-associatedchange in donor/acceptor dye separation [8]. In the presence ofcocaine, FRET between fluorescein and dabcyl dye both attached tothe aptamer was stimulated by the formation of a ligand-inducedbinding pocket based on terminal stem-closure [4,5]. Quantum dot(QD)-stimulated FRET with organic dyes on DNA aptamers wasrecently reported for cocaine detection [7,47]. Furthermore, Cy5labeled cocaine aptamers were hybridized with complementaryDNA attached to gold nanoparticles. In the presence of cocaine, theaptamer strands were released resulting in deactivation of FRETbetween the gold nanoparticles and Cy5, and recovery of Cy5 fluo-rescence [8].

It is generally acknowledged that microfluidic devices are par-ticularly useful for the implementation of lab-on-a-chip sensorsdue to reduced sample and reagent consumption, shorter analysistimes and increased levels of automation [48]. Hilton et al. [11] firstused a polydimethylsiloxane (PDMS)/glass based microchamberpacked with aptamer-functionalized microbeads as a FRET-basedsensor for cocaine detection.

Here, we demonstrate an optical cocaine sensor where acocaine-sensitive aptamer labeled with FITC changes conformationfrom a partial single-stranded oligonucleotide with a short hair-pin to a double-stranded T-junction, thereby trapping cocaine. Thedouble-strand specific DNA minor groove binder Hoechst 33342

e using minor groove binder based energy transfer, Anal. Chim. Acta

binds to the double-stranded T-junction allowing the dye uponexcitation at 360 nm to instigate energy transfer to FITC. The resultof this effect which we term minor groove binder based energytransfer (MBET) is green fluorescence at 520 nm.

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Scheme 1. Schematic illustration of MBET aptamer se

We also demonstrate the MBET cocaine sensor on a PDMS sur-ace for ready integration into microfluidic chips. We fabricate

carboxy-functionalized PDMS surface by simply curing a mix-ure of undecylenic acid (UDA) and PDMS prepolymer on a goldoated glass slide, which had been pretreated with a hydrophilicelf-assembled monolayer (SAM) of 3-mercaptopropionic acidScheme S-1). FITC labeled 5′-amino-terminal single-stranded DNAptamers are covalently attached to the UDA modified PDMS sur-ace via amide linkages (Scheme 1).

. Experimental

.1. Materials and reagents

PDMS Sylgard 184 was purchased from Dow Corning Corpora-ion (USA) as a two-component kit, including pre-polymer (basegent) and cross-linker (curing agent). Gold slides were purchasedrom Platypus Technologies (USA). Lucifer Yellow CH dipotassiumalt, Hoechst 33342 were purchased from Invitrogen (USA). Cocaineas obtained from Forensic Science, Adelaide, South Australia,ustralia. All other chemicals were purchased from Sigma–Aldrich

USA). The aptamer sequence used here was based on a literatureequence [5,7] and was adapted to suit our application. 5′-amino-spacer18)2 AGACAAGGAAAATCCTTCAATGAAGTGGGTCTC-FITC-3′

as purchased from GeneWorks Pty Ltd (Australia). The buffersere prepared as follows: PBS (pH 4.8): NaH2PO4 (30 mM),

0 mM PBS (pH 7.4), NaCl (137 mM), KCl (54 mM), Na2HPO410 mM), KH2PO4 (2 mM) and tris(hydroxymethyl)aminomethaneTris) buffer (pH 8.4): Tris (25 mM), NaCl (100 mM), MgCl2 (1 mM).or pH adjustment of the buffers, NaOH (0.1 M) and HCl (0.1 M)ere used.

.2. PDMS sample preparation

The UDA modified PDMS surface was prepared according to ourrevious work [49]. Briefly, the PDMS (10:1 weight ratio of base anduring agents) and 2 wt% UDA were thoroughly mixed and degassedo remove air bubbles and then poured onto the hydrophilicold slide which was pre-coated with a 3-mercaptopropionic acidonolayer. The sample was left on the bench under ambient con-

ition for 24 h and then cured at 80 ◦C for 2 h. Following this,he sample was immersed in MilliQ water for 4 h so that theured UDA modified PDMS can be easily peeled off from the gold

Please cite this article in press as: J. Zhou, et al., Aptamer sensor for cocain(2012), doi:10.1016/j.aca.2012.01.011

ubstrate. The UDA modified PDMS was finally rinsed sequen-ially with MilliQ water and ethanol and dried under a stream ofitrogen (Scheme S-1). Native PDMS without UDA was used as aontrol.

for cocaine detection on UDA modified PDMS surface.

2.3. Surface characterization

2.3.1. X-ray photoelectron spectroscopy (XPS)XPS analysis of PDMS samples was performed on an AXIS

HSi spectrometer (Kratos Analytical Ltd, GB), equipped with amonochromatized Al K� source. The pressure during analysis wastypically maintained at 5 × 10−9 kPa. High resolution spectra werecollected at a pass energy of 40 eV. Binding energies were calibratedagainst the aliphatic hydrocarbon peak at 285.0 eV.

2.3.2. Streaming zeta-potential analysisZeta potential data were obtained using a ZetaCAD instrument

equipped with an RS232 C bi-directional interface as well as a pro-grammable in/out board for automation of the measurements withthe aid of a Keithley 2400 high accuracy multimeter. The approachpublished by Karkhaneh et al. [50] was adopted where potassiumchloride (1 mM) was used as a background electrolyte in all exper-iments. Potassium hydroxide (0.1 M) and hydrochloric acid (0.1 M)were used for pH adjustment. The tested PDMS substrates wereimmersed in the electrolyte solution overnight for equilibrationprior to testing. The measurements were repeated three times atpH 4 to pH 12 at room temperature, and the results were averaged.

2.3.3. Fluorescence labelingThe presence of carboxylic acid groups on the UDA mod-

ified PDMS surface was confirmed by fluorescence labelingusing the Lucifer Yellow CH dipotassium salt dye. 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) and Lucifer Yellow CHdipotassium salt dye were dissolved in MilliQ water (0.4 M EDACand 1 mg mL−1 dye). Native PDMS and UDA modified PDMS sam-ples were immersed into the EDAC/dye solution for 4 h at roomtemperature after which the samples were removed and rinsedsequentially with MilliQ water and ethanol and finally dried undera stream of nitrogen gas. The samples were then imaged under afluorescence microscope (Leitz Laborlux fluorescence microscope).

2.3.4. Other analysesIn addition, water contact angle (WCA) measurements and

Fourier transform infrared-attenuated total reflection (FTIR-ATR)spectroscopy analysis were also performed on native PDMS andthe UDA modified PDMS surfaces [49].

2.4. MBET aptamer sensor for cocaine detection in solution

To optimize the temperature protocol for cocaine detection

e using minor groove binder based energy transfer, Anal. Chim. Acta

using the FITC-labeled cocaine aptamer, three parallel experi-ments were performed. (a) A solution containing aptamer (0.1 �M)and cocaine (0.1 �M) in Tris buffer (pH 8.4) was maintainedat room temperature for 20 min; (b) A solution containing

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Fig. 1. Fluorescence emission spectra upon excitation at 360 nm recorded for solu-tions of aptamer/cocaine/Hoechst 33342 after different incubation protocols: (a) asolution containing aptamer (0.1 �M) and cocaine (0.1 �M) in Tris buffer (pH 8.4)was then maintained at room temperature for 20 min; (b) a solution containingaptamer (0.1 �M) and cocaine (0.1 �M) in Tris buffer (pH 8.4) was heated at 80 ◦Cfor 10 min, and then cooled in fridge to 4 ◦C for 10 min; (c) a solution containingaptamer (0.1 �M) and cocaine (0.1 �M) in Tris buffer (pH 8.4) was heated at 80 ◦Cfor 10 min, and then cooled to room temperature for 10 min. Hoechst 33342 (0.1 �M)

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ptamer (0.1 �M) and cocaine (0.1 �M) in Tris buffer (pH 8.4)as heated at 80 ◦C for 10 min, and then cooled to 4 ◦C in the

ridge for 10 min; (c) A solution containing aptamer (0.1 �M)nd cocaine (0.1 �M) in Tris buffer (pH 8.4) was heated at 80 ◦Cor 10 min, and then cooled to room temperature for 10 min.oechst 33342 (0.1 �M) was then added into the above threeptamer/cocaine solutions and reacted for 30 min. Fluorescencemission spectra from aptamer/cocaine/Hoechst solutions wereollected at room temperature on a Cary-Eclipse fluorescence spec-rometer from Varian (Australia). The fluorescence spectrometeras set to an excitation wavelength of 360 nm and emissionas monitored at 520 nm. In addition, fluorescence of solutions

f 100 nM aptamer, Hoechst 33342, cocaine, aptamer/cocaine,ptamer/Hoechst, cocaine/Hoechst solutions was also measured.

To investigate the selectivity of MBET aptamer sensor forocaine in solution, two cocaine metabolites, benzoyl ecgonine0.1 �M) and ecgonine methyl ester (0.1 �M), were used as ana-ogues to replace cocaine using temperature protocol (c).

.5. MBET aptamer sensor for cocaine detection on PDMS surface

The protocol for detecting cocaine using the UDA modifiedDMS can be divided into three steps. Step 1: the preparationf aptamer-linked PDMS surface. UDA modified PDMS surfacesere first exposed to EDAC (0.4 M) in PBS buffer (pH 4.8) for

0 min (followed by washing with PBS buffer (pH 4.8)), and thenptamer (10 �M) in PBS buffer (pH 7.4) for 2 h (followed by wash-ng with PBS buffer (pH 7.4)). Step 2: the bonding between theptamer and cocaine. The aptamer-linked PDMS surfaces from step

were immersed into cocaine solutions with different concentra-ions (0 �M, 1 �M, 2 �M, 3 �M, 4 �M, 5 �M, 10 �M prepared in PBSuffer (pH 7.4)), then kept at 80 ◦C for 10 min, and finally cooledt room temperature for 10 min (followed by washing with PBSuffer (pH 7.4)). Step 3: the activation and readout of the MBETystem. Cocaine/aptamer-linked PDMS surfaces were exposed tooechst 33342 (20 �M) in Tris buffer (pH 8.4) for 30 min, and then

insed with the Tris buffer (pH 8.4) and dried under a stream ofitrogen gas. The dried PDMS surfaces were then imaged undern IX81 inverted fluorescence microscope from Olympus (Japan)sing a custom filter (excitation at 360–370 nm and emission at10–550 nm). All images were taken with a 1 s exposure and fluo-escence levels were quantified using Image J software.

In addition, to test the stability of MBET aptamer sensor, therepared aptamer-linked PDMS according to step 1 was stored at◦C for 4 days before being used for cocaine detection. After keep-

ng the previous stored aptamer-linked PDMS at room temperatureor 10 min, it was exposed to cocaine (3 �M) and Hoechst 3334220 �M) according to steps 2 and 3. The fluorescence image washen obtained and quantified as described above.

. Results and discussion

.1. MBET aptamer sensor for cocaine detection in solution

En route to developing an aptamer sensor for cocaine detec-ion on the PDMS surface, we first tested the performance ofhe MBET based detection in solution. In our system (as shownn Scheme S-2), a partial single-stranded aptamer with a shortairpin labeled with FITC forms a characteristic double-stranded-junction structure in the presence of cocaine, resulting in theormation of an aptamer/cocaine complex [5,7]. Hoechst 33342

Please cite this article in press as: J. Zhou, et al., Aptamer sensor for cocain(2012), doi:10.1016/j.aca.2012.01.011

electively binds to the double-stranded T-junction, bringing itithin the Förster radius of the FITC dye on the aptamer. Upon

xcitation at 360 nm, MBET between the Hoechst 33342 donornd the FITC acceptor results in fluorescence emission at 520 nm.

was then added into the above three aptamer/cocaine solutions and incubated for30 min.

Three parallel experiments were performed to optimize the incuba-tion conditions for aptamer-based cocaine detection: (a) a solutioncontaining aptamer and cocaine was maintained at room temper-ature for 20 min; (b) a solution containing aptamer and cocainewas heated at 80 ◦C for 10 min and then cooled to 4 ◦C in thefridge for 10 min; (c) a solution containing aptamer and cocainewas heated to 80 ◦C for 10 min, and then maintained at roomtemperature for a further 10 min. Hoechst 33342 dye was thenadded to the three solutions. Fluorescence emission spectra overthe range of 400 nm to 600 nm upon excitation at 360 nm werecollected from the three aptamer/cocaine/Hoechst 33342 solu-tions, as shown in Fig. 1. A fluorescein emission peak at 520 nmwas observed in all cases. However, it is evident that the fluo-rescence intensity is affected by different treatment conditions.The (b and c) aptamer/cocaine/Hoechst 33342 solutions, havingundergone heating and cooling steps, showed a higher intensityfluorescein emission than (a) without the heating and cooling.For the (c) aptamer/cocaine/Hoechst 33342 solutions, after slowcooling to room temperature, showed a higher intensity than (b)aptamer/cocaine/Hoechst 33342 solutions cooled in the fridge. Weconclude from these results that the heating and slow coolingassisted in aptamer re-conformation and binding to cocaine in thehairpin loop. Fluorescence spectra of solutions of aptamer, cocaine,Hoechst 33342, aptamer/cocaine, aptamer/Hoechst 33342 andcocaine/Hoechst 33342 only after heating and slow cooling stepsat 360 nm excitation were also acquired (Fig. S-1). No fluoresceinemission was observed except for the solution of aptamer/Hoechst33342, where weak MBET fluorescence indicates a certain level ofMBET was initiated in the absence of cocaine due to the particu-lar aptamer conformation [5]. Nevertheless, the fluorescence levelwas more than four times lower than in the presence of cocaine at0.1 �M concentration.

An evaluation of the selectivity of the MBET aptamer sensorwas performed by monitoring the fluorescence emission peak at520 nm, after exposing the sensors to benzoyl ecgonine, ecgoninemethyl ester and cocaine, respectivity. As shown in Fig. 2, two times

e using minor groove binder based energy transfer, Anal. Chim. Acta

higher fluorescence intensity at the peak of 520 nm was observedin the presence of cocaine, compared to in the presence of ben-zoyl ecgonine or ecgonine methyl ester. This result proved that

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Fig. 2. Fluorescence emission spectra upon excitation at 360 nm recordedfor solutions of (a) aptamer (0.1 �M)/Hoechst 33342 (0.1 �M), (b) aptamer(0.1 �M)/benzoyl ecgonine (0.1 �M)/Hoechst 33342 (0.1 �M), (c) aptamer(0.1 �M)/ecgonine methyl ester (0.1 �M)/Hoechst 33342 (0.1 �M), and (d) aptamer(0.1 �M)/cocaine (0.1 �M)/Hoechst 33342 (0.1 �M) after the same incubationph3

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rotocol: the above solutions without Hoechst 33342 in Tris buffer (pH 8.4) wereeated at 80 ◦C for 10 min, and then cooled to room temperature for 10 min. Hoechst3342 (0.1 �M) was then added into the solutions and incubated for 30 min.

he MBET aptamer sensor was selective for cocaine. Nevertheless,he fluorescence level in the presence of cocaine metabolites waslightly higher than without any analytes.

Having proven the principle of the MBET-based detection andhe selectivity of the MBET sensor for cocaine in solution, the nexttep was to implement the sensor on the PDMS surface.

.2. MBET aptamer sensor for cocaine detection on anptamer-modified PDMS surface

We first prepared a PDMS surface functionalized with carboxyliccid groups to allow covalent attachment of aptamers. A templatingrocedure previously developed in our group [49] was used here,nd has been described briefly in Section 2.

XPS was used to investigate the change in the PDMS surfacehemistry after UDA modification. An additional C 1 s high-esolution peak at 290.0 eV corresponding to O C O groupsppeared in the spectrum for UDA modified PDMS (Fig. S-2),

Please cite this article in press as: J. Zhou, et al., Aptamer sensor for cocain(2012), doi:10.1016/j.aca.2012.01.011

howing that carboxylic acid groups are available on the sur-ace. In addition, the presence of carboxyl moieties on the UDA

odified PDMS surface could also be detected by FTIR-ATR spec-roscopy (Fig. 3). Two characteristic carboxyl peaks were present at

Fig. 3. FTIR-ATR spectra of (a) native PDMS and (b) UDA-modified PDMS.

Fig. 4. Streaming zeta potential measurements for native PDMS and UDA modifiedPDMS at pH 4, 6, 8, 10 and 12; (n = 3).

1715 cm−1 and 1730 cm−1, corresponding to hydrogen bonded car-boxylic groups and free carboxylic acid, respectively, while thesepeaks were absent on FTIR-ATR spectrum of native PDMS between1600 and 1800 cm−1.

The change of PDMS surface chemistry after UDA modifica-tion was also verified by the comparison of the zeta potentialon native PDMS and UDA modified PDMS over the pH rangefrom 4 to 12 (Fig. 4). For UDA modified PDMS, the zeta poten-tial was −14.1 ± 3.0 mV at pH 4, compared to −24.3 ± 1.7 mV fornative PDMS. This difference was attributed to the presence ofprotonated carboxylic acid moieties from the UDA on the sur-face. As the pH increased, the zeta potential on the UDA modifiedPDMS decreased more rapidly than for native PDMS. At pH ≥ 8,more negative zeta potentials were measured on the UDA mod-ified PDMS (−43.4 ± 4.0 mV at pH 8, −51.0 ± 0.8 mV at pH 10 and−55.9 ± 1.8 mV at pH 12) compared to native PDMS (−30.0 ± 2.0 mVat pH 8, −33.2 ± 1.7 mV at pH 10 and −38.3 ± 2.0 mV at pH 12), dueto the deprotonated carboxylate functionalities present on the sur-face at higher pH. The slow decrease of zeta potential on nativePDMS with the increase of pH might result from the deprotonationof the surface Si OH groups on the native PDMS surface at highpH and/or physisorbed cations within the Stern layer [51]. Finally,the surface wettability after UDA modification was also improvedshowing a difference in WCA between native PDMS (109.8 ± 0.4◦)and the UDA modified PDMS surfaces (91.4 ± 2.0◦) [49] (as shownin Fig. S-3).

To demonstrate the reactivity of carboxylic acid groups onthe UDA modified PDMS, Lucifer Yellow CH was coupled tothe carboxylic acid groups via EDAC carbodiimide coupling.Fig. 5(a) depicts the reaction scheme. The fluorescence microscopyimages clearly demonstrate rather homogenous yellow fluores-cence across the UDA modified PDMS surface (Fig. 5(b) right),indicating the dye has successfully attached to the carboxy-functionalized PDMS surface. Meanwhile, no fluorescence wasdetected on the native PDMS surface (Fig. 5(b) left) after incubationwith EDAC and Lucifer Yellow CH.

The UDA modified PDMS surface was reacted with 5′-amino-terminal cocaine aptamer using carbodiimide coupling. The thusfabricated aptamer sensor was then used for cocaine detectionunder the optimized treatment conditions (heating at 80 ◦C for10 min, and then cooling to room temperature for another 10 min),followed by incubation with Hoechst 33342 dye (Scheme 1).Using a custom filter in a fluorescence microscope, the aptamer-

e using minor groove binder based energy transfer, Anal. Chim. Acta

linked PDMS surface was excited at 360–370 nm and fluorescenceemission from 510 nm to 550 nm was collected. As shown inFig. 6, cocaine concentrations ranging from 0 �M to 10 �M werereadily detected using the aptamer sensor. Fluorescence intensity

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ig. 5. (a) Reaction scheme of Lucifer Yellow CH labeling on a UDA modified PDMSia EDAC coupling and (b) fluorescence images of native PDMS (left) and Luciferellow CH labeled UDA modified PDMS (right).

ncreased rapidly with increasing cocaine concentration from 0o 5 �M, and then remained almost constant when the cocaineoncentrations increased further from 5 to 10 �M. For compari-on, the same process was repeated for aptamers incubated on aative PDMS surface, reacted with aptamer under identical reac-ion conditions as for the UDA modified PDMS, exposed to cocaineolution and then Hoechst 33342. The results showed negligibleuorescence emission over the entire concentration range from

to 10 �M. The fluorescence intensity (I) on the UDA modifiedDMS surfaces correlated linearly with the logarithm of cocaine

Please cite this article in press as: J. Zhou, et al., Aptamer sensor for cocain(2012), doi:10.1016/j.aca.2012.01.011

oncentration over the range of 1 to 5 �M (Fig. 6 insert). Theinear regression equation was: I = 4.1139 Log[cocaine] + 5.1608,nd the correlation coefficient was 0.9882. The detection limit for

ig. 6. MBET response using a 510–550 nm bandpass filter for the aptamer-basedensor to cocaine at varying concentrations on native and UDA modified PDMS sur-aces after functionalization with aptamer and incubation with cocaine and Hoechst3342. The insert shows a plot of fluorescence intensity for the aptamer-basedensor against the logarithm of cocaine concentrations.

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PRESS Acta xxx (2012) xxx– xxx 5

cocaine was 0.20 �M (S N−1 = 3), which is better than many existingaptamer sensors for cocaine detection [6,16,25,38,47]. After storageat 4 ◦C for 4 days, the aptamer-linked PDMS was used for cocainedetection to test the stability of the MBET aptamer sensor. Com-pared to a freshly prepared aptamer-linked PDMS, a small decreaseof fluorescence intensity of 9% ± 3.1% was observed on the surfaceafter exposing to cocaine and Hoechst 33342, showing that thesensor can be stored for several days before use.

4. Conclusion

In conclusion, we have demonstrated a MBET aptamer sensorwhich has potential application for cocaine detection. The exposureof the aptamer sensor to solutions containing cocaine resulted in aconspicuous change of aptamer conformation from a partial single-stranded DNA with a short hairpin to a double-stranded T-junction,facilitating MBET between the FITC labeled 3′ end of the aptamerand Hoechst 33342 bound to the double-stranded T-junction. Wealso prepared a PDMS surface functionalized with carboxylic acidgroups and immobilized 5′ amino-terminated aptamers via amidelinkages. This allowed us to implement an aptamer sensor forcocaine on a PDMS surface, implying the possibility of incorpo-rating this sensor into a microfluidic device. The aptamer sensoron PDMS surface had a detection limit of 0.2 �M. Moreover, thesensor showed stable after storage at 4 ◦C for 4 days.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.aca.2012.01.011.

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