Locked Nucleic Acid Based Beacons for SurfaceInteraction Studies and Biosensor Development

Karen Martinez, M.-Carmen Estevez, Yanrong Wu, Joseph A. Phillips, Colin D. Medley, andWeihong Tan*

Center for Research at the Bio/Nano Interface, Department of Chemistry and Department of Physiology andFunctional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute,University of Florida, Gainesville, Florida 32611

DNA sensors and microarrays permit fast, simple, andreal-time detection of nucleic acids through the design anduse of increasingly sensitive, selective, and robust mo-lecular probes. Specifically, molecular beacons (MBs)have been employed for this purpose; however, theirpotential in the development of solid-surface-based bio-sensors has not been fully realized. This is mainly aconsequence of the beacon’s poor stability because of thehairpin structure once immobilized onto a solid surface,commonly resulting in a low signal enhancement. Here,we report the design of a new MB that overcomes someof the limitations of MBs for surface immobilization.Essentially, this new design adds locked nucleic acidbases (LNAs) to the beacon structure, resulting in a LNAmolecular beacon (LMB) with robust stability after surfaceimmobilization. To test the efficacy of LMBs against thatof regular molecular beacons (RMBs), the properties ofselectivity, sensitivity, thermal stability, hybridizationkinetics, and robustness for the detection of target se-quences were compared and evaluated. A 25-fold en-hancement was achieved for the LMB on surface withdetection limits reaching the low nanomolar range. Inaddition, the LMB-based biosensor was shown to possessbetter stability, reproducibility, selectivity, and robustnesswhen compared to the RMB. Therefore, as an alternativeto conventional DNA and as a prospective tool for use inboth DNA microarrays and biosensors, these resultsdemonstrate the potential of the locked nucleic acid basesfor nucleic acid design for surface immobilization.

Over the past decade, DNA/RNA-based analytical methodshave been extremely important in molecular biology, diseasediagnosis, and gene expression studies. Biosensors, such as DNAmicroarrays and/or DNA chips, have gained popularity becausethey allow the measurement and detection of a high number ofsamples using a fast and simple setup. Moreover, biosensors allowfor real-time detection of nucleic acid molecules and geneexpression changes.1-6 However, these results can only be

achieved by efficient, reproducible and stable immobilization ofDNA probes onto specific surfaces.1-7 Most DNA biosensors arebased on the detection of DNA-DNA and DNA-RNA hybridswith the DNA probe immobilized on a transducer surface. Of themany varieties of DNA sensors, the most common are (i)electrochemical-based sensors which involve the immobilizationof the DNA probe onto an electrode surface such that targetmolecules are detected by changes in electron transfer8,9 and (ii)fluorescence-based sensors where DNA probe immobilization isusually performed on a glass surface such that target moleculesare detected upon changes in the fluorescence intensity whenhybrids are formed.3,10,11 Out of the many fluorescent-based DNAprobes, molecular beacons (MBs)12 are the most effective probesfor DNA biosensors as they have high sensitivity and can be usedfor detection without separation. In addition, because of theirhairpin structure, MBs have better selectivity than linear DNAprobes in sensing target nucleic acids. MBs are single-strandedoligonucleotide probes that contain a loop-and-stem structure.12

The loop portion of the beacon contains the sequence comple-mentary to a target (usually 15-30 bases), whereas the two stemsare complementary to each other (5-7 bases). A quencher iscovalently attached at the end of one stem, while a fluorophore iscovalently attached to the end of the other. Because the fluoro-phore and quencher are in close proximity, MBs do not fluoresceuntil hybridization with the target nucleic acid sequence. In thepresence of a target, the hairpin structure of MBs undergoesconformational changes that force the fluorophore and quenchermoieties to separate, thus restoring fluorescence intensity. MBprobes have been studied as DNA probes for DNA biosensorswith the aim of overcoming some drawbacks inherent in single-

* To whom correspondence should be addressed. E-mail: tan@chem.ufl.edu.Phone: 352-846-2410. Fax: 352-846-2410.

(1) Broude, N. E.; Woodward, K.; Cavallo, R.; Cantor, C. R.; Englert, D. NucleicAcids Res. 2001, 29, e91/1-e91/11.

(2) Culha, M.; Stokes, D. L.; Griffin, G. D.; Vo-Dinh, T. Biosens Bioelectron2004, 19, 1007–1012.

(3) Fang, X.; Liu, X.; Schuster, S.; Tan, W. J. Am. Chem. Soc. 1999, 121, 2921–2922.

(4) Preininger, C.; Chiarelli, P. Talanta 2001, 55, 973–980.(5) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000,

79, 975–981.(6) Steemers, F. J.; Ferguson, J. A.; Walt, D. R. Nat. Biotechnol. 2000, 18,

91–94.(7) Culha, M.; Stokes, D. L.; Griffin, G. D.; Vo-Dinh, T. J. Biomed. Opt. 2004,

9, 439–443.(8) Gibbs, J. M.; Park, S.-J.; Anderson, D. R.; Watson, K. J.; Mirkin, C. A.;

Nguyen, S. T. J. Am. Chem. Soc. 2005, 127, 1170–1178.(9) Fan, C.; Plaxco, K. W.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2003,

100, 9134–9137.(10) Fang, X.; Li, J. J.; Tan, W. H. Anal. Chem. 2000, 72, 3280–3285.(11) Ho, H.-A.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384–1387.(12) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303–308.

Anal. Chem. 2009, 81, 3448–3454

10.1021/ac8027239 CCC: $40.75 2009 American Chemical Society3448 Analytical Chemistry, Vol. 81, No. 9, May 1, 2009Published on Web 04/07/2009

stranded DNA probes3,13-15 and DNA array applications.3,4,6,7,16-19

Specifically, because of their inherent signal transduction mech-anism, MBs provide superior properties over single-stranded linearDNA probes as probes for surface biosensors.

In solution, MBs possess many attractive properties, such ashigh sensitivity, selectivity, and DNA detection that can beperformed in real-time, as the unbound probes do not need to beseparated out from the bound probes. However, the use of MBsfor surface hybridization has been limited, mainly becausefluorescence enhancement of the immobilized MBs decreasessignificantly when compared to that of MBs in solution. Thus,whereas MBs can typically achieve fluorescence enhancementsabove 25-fold in solution,3,14,15,17,18 these values can in some casesdrop to 2- to 5-fold once immobilized.12,20 This behavior is mainlya result of interactions between the MB and the surface whichpartially disrupt the loop and, consequently, destabilize stemstructure. This can cause inefficient quenching,18 which isreflected in high fluorescence background signal, thus affectingthe overall sensitivity of the MBs.14,15 Some published studies havereported ways of improving MB performance on the surface. Forexample, to have a more liquid-liquid environment, instead ofliquid-solid interface, polyacrylamide gel and agarose gel havebeen used for MB immobilization.2,17 In other reports, physico-chemical parameters, such as pH and ionic strength, as well asthe distance of the MB to the surface, have been investigated,although with limited improvement.18 Despite these advances,further development of new DNA probes with a higher stabilityon the surface, which, at the same time, allow a simple design,low background signal, and high hybridization efficiency, is stillneeded to recreate the performance and sensitivity of MBs insolution. In addition, improving the surface performance of MBswill be easily transferable to biotechnologies based on immobiliza-tion of nucleic acid probes on surfaces such as DNA arrays andprotein arrays based on aptamers.

In this study, we have optimized the design of molecularbeacons by incorporating locked nucleic acids (LNAs) to createa highly stable MB and therefore improve the performance ofthese probes on a solid surface. LNA is a nucleic acid containinga bicyclic furanose unit locked in an RNA mimicking sugarconformation.21 The methylene bridge that connects the 2′-oxygenand the 4′-carbon of the ribose ring confers higher structurerigidity to the LNA base pair, thus preventing potential interactionswith the surface and other molecules that might be present inthe solution. Furthermore, LNAs contain a structure resemblingRNA, which allows a superior affinity and specificity toward the

target strand when compared to that of DNA.22 Other attractiveproperties of LNA bases are high resistance against degradationand thermostability, which is reflected in low background signal,and efficient target hybridization.23,24 These properties typicallyallow LNA bases to increase the sensitivity and selectivity of anymolecular probes, thus allowing the development of LNA-basedmicroarrays for miRNA profiling.25-28 In spite of these advantages,the hybridization kinetics of the fully modified LNA MB is slowerwhen compared to that of the DNA MBs. This apparent flaw,which can be attributed to the slow stem dehybridization rate,24

is discussed in detail below; however, it can be noted here thatthe hybridization kinetics of LNA MBs can be improved substan-tially by decreasing the percent of LNA bases in the beacon’sdesign.24 Therefore, considering the advantages that molecularbeacons can offer over conventional DNA probes, we have studiedthe performance of beacons incorporating LNA bases in thestructure after immobilization onto a glass surface in terms ofstability, background signal, thermodynamics, kinetics, selectivity,and sensitivity. Specifically, we have synthesized a MB with acombination of DNA and LNA bases, and it has been comparedwith MB containing only regular DNA bases. This newly designedLMB can potentially be used for a more sensitive DNA arraydetection and other biotechnological applications.

MATERIALS AND METHODSProbe Preparation. The MBs, cDNA, and mismatch targets

were synthesized and are listed in Table 1. All the DNA and LNAreagents for the synthesis of the beacons and targets werepurchased from Glen Research. The probes and DNA comple-mentary targets were synthesized with an ABI3400 DNA/RNA(13) Li, J.; Tan, W. H.; Wang, K. M.; Xiao, D.; Yang, X. H.; He, X. X.; Tang, Z. Q.

Anal. Sci. 2001, 17, 1149–1153.(14) Liu, X. J.; Farmerie, W.; Schuster, S.; Tan, W. H. Anal. Biochem. 2000,

283, 56–63. Du, H.; Strohsahl, C. M.; Camera, J.; Miller, B. L.; Krauss, T. D.J. Am. Chem. Soc. 2005, 127, 7932–7940.

(15) Liu, X. J.; Tan, W. H. Anal. Chem. 1999, 71, 5054–5059.(16) Lou, H. J.; Tan, W. H. Instrum. Sci. Technol. 2002, 30, 465–476.(17) Wang, H.; Li, J.; Liu, H. P.; Liu, Q. J.; Mei, Q.; Wang, Y. J.; Zhu, J. J.; He,

N. Y.; Lu, Z. H. Nucleic Acids Res. 2002, 30 e61/1-e61/9.(18) Yao, G.; Tan, W. H. Anal. Biochem. 2004, 331, 216–223.(19) Epstein, J. R.; Leung, A. P. K.; Lee, K. H.; Walt, D. R. Biosens. Bioelectron.

2003, 18, 541–546.(20) Bonnet, G.; Tyagi, S.; Libchaber, A.; Kramer, F. R. Proc. Natl. Acad. Sci.

U.S.A. 1999, 96, 6171–6176.(21) Koshkin, A. A.; Nielsen, P.; Meldgaard, M.; Rajwanshi, V. K.; Singh, S. K.;

Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252–14253.

(22) Kaur, H.; Babu, R.; Maiti, S. Chem. Biochem. 2007, 107, 4672–4697.(23) Wang, L.; Yang, C. Y. J.; Medley, C. D.; Benner, S. A.; Tan, W. H. J. Am.

Chem. Soc. 2005, 127, 15664–15665.(24) Yang, C. J.; Wang, L.; Wu, Y.; Kim, Y.; Medley, C. D.; Lin, H.; Tan, W.

Nucleic Acids Res. 2007, 35, 4030–4041. Wu, Y.; Yang, C. J.; Moroz,L. L.; Weihong, T. Anal. Chem. 2008, 80, 3025–3028.

(25) Castoldi, M.; Benes, V.; Hentze, M. W.; Muckenthaler, M. U. Methods 2007,43, 146–152.

(26) Castoldi, M.; Schmidt, S.; Benes, V. RNA 2006, 12, 913–920.(27) Fang, S.; Lee, H. J.; Wark, A. W.; Corn, R. M. J. Am. Chem. Soc. 2006,

128, 14044–14046.(28) Neely, L. A.; Patel, S.; Garver, J.; Gallo, M.; Hackett, M.; McLaughlin, S.;

Nadel, M.; Harris, J.; Gullans, S.; Rooke, J. Nature (London) 2006, 3, 41–46.

Table 1. Molecular Beacons and Target Sequences

a Red letters represent LNA bases. b Blue letter represents themismatch base.

3449Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

synthesizer (Applied Biosystems, CA). Blackhole quencher 2(BHQ2) core pore glass (CPG) was used for all MB synthesis;Cy3 phosphoramidite was used to label the probes with Cy3, andSpacer phosphoramidite 18, which incorporates PEG as linker intothe beacons. A biotin phosphoramidite was introduced for labelingat the 5′ terminus of the MBs. MBs that contained LNA baseswere synthesized using LNA phosphoramidites. LMBs wereprepared by alternating LNA and DNA bases in both, the stemand the loop (every other base). This design was chosen to takeadvantage of LNA bases without compromising the hybridizationrate. In addition, targets were prepared in such a way that thecomplementary sequence was complementary not only to thetarget but also to the stem as well, to ensure that the beaconsopen in the presence of the targets. Deprotection of the probeswas performed using overnight incubation with ammoniumhydroxide at room temperature. The solution resulting fromdeprotection was precipitated in cold ethanol. Subsequently, theprecipitates were dissolved in 0.5 mL of 0.1 M triethylammoniumacetate (TEAA, pH 7.0) for further purification with reverse phasehigh-pressure liquid chromatography (RP-HPLC). RP-HPLC wasperformed on a ProStar HPLC Station (Varian, CA) equipped witha fluorescent and a photodiode array detector using a C-18 reversephase column (Econosil, C18, 5 µM, 250 × 4.6 mm) from Alltech(Deerfield, IL). The product collected from the HPLC was vacuum-dried and then added with TEAA for a second round of HPLC.The final product was collected, vacuum-dried, and dissolved in200 µL of acetic acid (80%) for 20 min, followed by 200 µL of coldethanol and then vacuum-dried. Quantification of the probes andtargets was performed using a UV-vis Spectrometer (Cary Bio-300, Varian, CA).

Immobilization of the MBs on the Glass Surface. Themicroscope slides and coverslips used in the experiments wereobtained from Fisher (optical borosilicate glass with a size of 18× 18 mm and 0.13 to 0.17 mm thickness). The surfaces werecleaned with a 3:1 ratio of conc. H2SO4 to H2O2 (30%) to removeorganic impurities. Subsequently, the treated microscope slideswere washed thoroughly with deionized water and dried withcompressed nitrogen. Strips of double-sided tape (3M) wereplaced 3 mm apart on a microscope slide, and a cover glasswas placed on top. Channels were filled by capillary action.Solution exchange was performed by simultaneously pipettingsolution at one end and withdrawing fluid from the other endwith P8 filter paper (Fisher).29 All the steps were performedat room temperature, where both RMB and LMB form a stablehairpin structure in the absence of target (in solution, themelting temperatures of the beacons are ∼56 °C for RMB and>85 °C for LMB24). The channels were washed twice with 10mM phosphate buffer (PBS) at pH 7.4 before use. Next, 5 µLof 1 mg ·mL-1 of avidin was incubated in the channels for 5min. The excess of avidin was removed with PBS washes (20µL, three times). The biotinylated MB (2.5 µM in PBS buffer)was incubated with avidin-treated channels for 10 min, and theexcess of MB was washed away with washing steps of PBSbuffer. A solution of the cDNA (cDNA, 10 µM prepared in 20mM Tris, 50 mM of NaCl, and 5 mM of MgCl2, pH 7.5) wasincubated with the immobilized MB and the fluorescence wasmonitored at different times depending on the experiment. An

end-point fluorescence was determined after 10 min of incuba-tion. Several images were collected at every step to obtain anaverage fluorescence, and the procedure was repeated andmeasured several times. The sensitivity of the MB was testedby measuring the fluorescence enhancement at different targetconcentrations (from 1 nM to 100 µM).

Stability of the MBs at Different Temperatures. Thestability of the MBs at different temperatures was studied byincubating the glass slide on a surface block dry bath (BarnsteadThermolyne type 17600 Dri-Bath) for the temperatures rangingfrom 20 to 50 °C. The samples tested at 4 °C were kept in therefrigerator prior to use to achieve the desired temperature. Toprevent any light damage to the probes, aluminum foil was usedto cover them. All the steps were performed under the conditionspreviously described. Images of the glass slide were taken at everystep as detailed earlier.

Stability of the MBs in Complex Matrixes. The stability ofthe system in complex matrixes was studied by performing theexperiments in cell lysate and serum media. Cell lysate solutionwas prepared following commercial specifications from CellSignaling Technology, Inc. (Danvers, MA). Cell lysate was keptat -20 °C until analysis. Fetal Bovine Serum (FBS, heat-inactivated, GIBCO) was obtained from Invitrogen (Carlsbad, CA).For this set of experiments, buffer, cell lysate, and FBS wereincubated for 10 min with the immobilized MBs. In addition, targetsolutions were prepared in buffer, cell lysate or FBS for a finalconcentration of 10 µM and subsequently incubated with theimmobilized beacon. Then, fluorescence measurements wereperformed following the same immobilization conditions previ-ously described.

Fluorescence Imaging. Fluorescence imaging was performedwith a confocal microscope setup consisting of an Olympus IX-81inverted microscope with an Olympus Fluoview 500 confocalscanning system and a green HeNe laser (543 nm), with aphotomultiplier tube (PMT) for detection. The images were takenwith a 10×/0.30 NA objective. Cy3-labeled MBs were excited at543 nm and collected at 560 nm with a long pass filter. The datacollected from the confocal microscope were analyzed using theFluoview analysis software.

To make sure the images were obtained from the brightestpoint of the channel, the microscope was focused onto the surfaceof the microslide, followed by a zeta section scan. The zeta scanallowed us to capture the brightest image at a specific zetaposition. Subsequently, all the images from avidin, immobilizedMB, and target addition were taken at the same position for thatparticular channel. This position may vary for each channel;therefore, the zeta position was calculated with each immobilization.

The data collected from the images were analyzed, and thefluorescence enhancement was calculated using the followingequation:

where SMB open is the signal of the beacon in the open state formor hybridized with the target, SMB close is the signal of the MBin the closed state form or unhybridized, and Bavidin is thebackground fluorescence intensity corresponding to the im-mobilized avidin in buffer solution. An average of the fluores-(29) Howard, J.; Hunt, A. J.; Baek, S. Methods Cell Biol. 1993, 39, 137–147.

Fluorescence Enhancement ) (SMB open - Bavidin)/

(SMB close - Bavidin)

3450 Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

cence was determined by taking several images from the samechannel.

Kinetics experiments were performed using confocal micros-copy by taking images of the hybridization with the target every3 s for 10 min. Each image was analyzed by taking the averagefluorescence intensity at the determined zeta position.

RESULTS AND DISCUSSIONMolecular Beacon Design and Surface Immobilization. In

previous reports, fully modified LNA beacons have shown highersensitivity but relatively slower hybridization rates than theircounterpart DNA.23,24 However, by decreasing the percentage ofLNA bases in the stem and the loop, LMBs can retain the affinityand the stability of the LNA bases without compromising thekinetics and hybridization rate of the assay. Thus, LMBs withalternating LNA and DNA bases in both the stem and the loop(every other base) were utilized for this study. In addition, it iswell-known that LNA bases have a strong affinity with comple-mentary LNA bases.22 Therefore, targets were prepared such thatthe cDNA sequence was not only complementary to the loop ofthe MBs, but to the stem as well (also known as shared stemtarget).

In this study, MBs were immobilized on a glass surface forsurface hybridization kinetics studies and for biosensor develop-ment. To achieve this, the avidin-biotin interaction was selected.The biotin molecule was attached at the 5′ end of the MB followedby the poly(ethylene glycol) (PEG) linker (see Scheme 1). PEGwas chosen as a linker to increase the distance of the MBs to theglass surface. In addition, PEG possesses great solubility andhydrophilicity and can be easily incorporated into DNA sequencesusing standard phosphoramidite chemistry. Furthermore, PEGhas no charge, which results in limited electrostatic interactionsand minimizes the precipitation or aggregation of the probes at

the glass surface.30 Finally, since PEG adds flexibility to anotherwise rigid oligonucleotide structure, PEG linker facilitates amore efficient immobilization onto the solid surface and furtherhybridization with target molecules.

The length of the PEG is a key factor in our MB design, andcare was taken to ensure that the linker was neither too shortnor too long, either of which can affect hybridization efficiency.Therefore, sets of LMBs and RMBs with different distancesbetween the biotin group and the sequence were initially designedby incorporating 0, 6, and 12 units of PEG. The sequences of theMBs are summarized in Table 1.

One of the major reasons why LMBs can be used for effectivesurface studies and biosensor development is their low back-ground signal which is a result of the rigid structure of LNA basesin the MB. We evaluated the initial background fluorescence ofthe beacons in the absence of cDNA, as well as the fluorescencechange after target addition. The results are summarized in Table2. For the RMB, the background fluorescence slightly increasedwhen 6 units of PEG were incorporated in the sequence. However,for the RMB with 12 PEG, the resulting background was ∼5-foldhigher than MBs containing zero and 6 PEG linker units. On theother hand, for the LMB, the background intensity remained thesame and stable, regardless of the length of the linker. Mostimportantly, the background intensity of LMB was found to belower than that of RMB (around 2.5 times). Moreover, the signalwas comparable to the one obtained from the system background(avidin-coated surface, ∼160 ± 10). These data support previousreports, which concluded that destabilization of regular DNA-based MB occurs after immobilization, resulting in incompletequenching which is reflected in high background signal.3,18 Thisis one of the major limitations of using MBs for surface im-mobilization. Nevertheless, the addition of LNA bases into thebeacon for hybridization studies brings stability and minimizessurface-glass interactions. Also, LNA bases in the stem keep thefluorophore/quencher moieties in close contact, which is reflectedin the low background signal.

On the other hand, higher fluorescence change of the RMBafter target addition was obtained when a PEG linker wasincorporated between the biotin and the sequence, reaching amaximum signal after the addition of 6 PEG units (∼ 8-foldenhancement signal). This result proves that PEG increases thedistance to the surface and facilitates the accessibility of themolecular beacon for an efficient hybridization with the target.Similarly, LMB with 6 PEG units produced the highest fluores-

(30) Kidambi, S.; Chan, C.; Lee, I. J. Am. Chem. Soc. 2004, 126, 4697–4703.

Scheme 1. Schematic Representation of theMolecular Beacon onto a Glass Surfacea

a Images in the closed and open state posses a value of 500and 2500 counts, respectively, at the fixed conditions used tomeasure the fluorescence.

Table 2. Effect of Different PEG Units on theBackground Intensity and the Overall FluorescenceChange after Target Addition

probes background intensity (a.u.) fluorescence enhancement

RMB0 PEG 510 ± 14 2.8 ± 0.46 PEG 608 ± 113 7.9 ± 0.612 PEG 2540 ± 220 1.2 ± 0.1

LMB0 PEG 200 ± 14 2.8 ± 0.66 PEG 258 ± 6 25 ± 512 PEG 239 ± 28 6.1 ± 2

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cence enhancement upon target addition (up to 25-fold signalenhancement) compared to 2.8 and 6.1 for 0 and 12 PEG units,respectively. Thus, considering that similar background intensitieswere observed for LMB with different PEG units, we can concludethat signal enhancement, in this case, results from a more efficienthybridization with the target when the 6-PEG linker is used. Thismay, therefore, also represent the optimum probe-to-surfacedistance. The addition of more PEG units (12 units) results in alonger and perhaps a more flexible linker. However, the extendedlength and higher bulkiness of the linker could, at the same time,hinder the accessibility of the target hampering an efficienthybridization, resulting in overall lower signal enhancement.Considering these results, the probes with 6 PEG units wereselected for further experiments.

Thermal Stability of the Beacons. To prove the improvedstability and robustness of the sensor using LNA bases, theinfluence of incubation temperature was investigated. Figure 1shows the background intensities of the beacons before targetaddition and the signal enhancements of the MBs after additionof the target sequence at 4, 20, 30, 40, and 50 °C. As can beobserved in the graph, the highest signal enhancement for RMBswas recorded at 20 °C, with a 9.4- fold enhancement. Subsequenttemperature increases caused a decrease in the signal enhance-ment of the RMBs to 3.4-fold, as a result of a significant andgradual increase in the overall background signal at highertemperatures. These results suggest thermal destabilization of thebeacon structure, thus compromising the stability of the probe-target complex. In addition, this increase in temperature is likelyto unzip the bases of the stem and thereby decrease the rigidityof the RMB, forcing the beacon to melt and form into fluorescentrandom coils.20 The random coil forces the fluorophore andquencher to separate, which causes an increase of backgroundintensity. In contrast, LMBs showed better stability, as can beobserved from the steady background throughout the entire arrayof temperatures studied. This resulted in a stable signal enhance-ment in the whole range of temperatures, which, in all cases, was∼22, but also clearly higher than the results for RMB, asdemonstrated in the previous experiments. This improved robust-ness is a result of the intrinsic stability of LNA bases, bringingrigidity to the probe, which is necessary to maintain the beaconin a closed state form, even at higher temperatures. These resultsalso confirm previous data obtained regarding the higher melting

temperature of the beacon containing the LNA bases.24 Thus, bythe addition of the LNA bases in the beacon design, we havedemonstrated that MBs with higher stability and reproducibilitycan be obtained, thus increasing the versatility of the sensor forextended applications.

Hybridization Kinetics of the MBs. The kinetics propertiesof the LMBs are relatively different from those of the RMBs.Depending on the percentage of LNA bases, hybridization ef-ficiency in these MBs can be greatly affected, usually by slowingdown the hybridization rate with the target DNA. To study andcompare the hybridization kinetics of both molecular beacons withthe target onto the glass surface, the fluorescence enhancementwas monitored in the confocal microscope every 3 s for 10 minafter addition of the target. As shown in Figure 2, the fluorescencesignal of RMB reached hybridization equilibrium within 3 min,whereas LMBs slowly increased the fluorescence signal over time.This behavior, already observed in previous work, can result fromthe presence of LNA-LNA base-pair in the stem of the LMB.24

In this study, LMBs possessed three LNA-LNA pairs in the stem(50%), which provide great stability and affinity to the beacon.This base-pairing percentage made stem opening difficult andunfavorable, slowing down the hybridization rate with the target,which is consistent with, and supports, our results. To know howLMB and RMB hybridization rates vary, we calculated the initialrate for hybridization of the beacons from the linear fraction ofthe hybridization kinetics (inset Figure 2). In this manner, therate of each beacon was determined by the slope of the lineargraph of the fluorescence intensity versus time. The slope of theRMB is ∼7 times higher than that of the LMB, quantitativelyconfirming that RMB possesses faster hybridization rate. Interest-ingly, this hybridization rate can be drastically accelerated withthe replacement of some LNA bases in the stem of the LMB byDNA bases (i.e., decreasing the percentage of LNA in the stem);on the other hand, this reduction of LNA bases can also result indecreasing the stability of LMB.24 Notwithstanding these results,we believe that the hybridization rate exhibited by the LMBcontaining 50% LNA and DNA, despite being slower than RMB,is still very reasonable for nucleic acid-based biosensors since itcan completely open the beacon in just a few minutes. Thus, theperformance of the biosensor is minimally compromised, or evenless, considering the improvement achieved in terms of signalenhancement and stability when LNA are incorporated in theprobe.

Sensitivity of the Immobilized MBs. In DNA arrays andbiosensors, it is important to determine the minimum concentra-tion of target molecules that can provide a measurable signal.Thus, in the following experiment, we investigated the sensitivityof the immobilized MBs by testing different concentrations oftarget, ranging from 1 nM up to 100 µM. The same concentrationof MBs was used to immobilize on the surface (initial concentra-tion of 2.5 µM). However, it should be noted that only the initialconcentration of MBs used for immobilization was known. Asexpected, the signal enhancements of both the LMB and the RMBwere observed to be proportional to the target concentration, andthe saturation point for each beacon was 5 µM (see Figure 3)with detection limits (DL) for RMB and LMB of 25 nM and 7.5nM, respectively. Evidently, the high background intensity of theRMBs (around 2.5 times higher) limits the amount of target that

Figure 1. Temperature effect on the stability of the MBs immobilizedonto the surface. Black straight line: fluorescence enhancement inLMB; black dashed line: background intensity of LMB; gray straightline: fluorescence enhancement in RMB; gray dashed line: back-ground intensity of RMB.

3452 Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

is detectable. On the other hand, the lower background intensityof the LMBs allows the detection of the target, even at lowconcentrations. In addition, it has been documented that LNAbases possess higher affinity22 for DNA bases than DNA itself,which could also explain the lower detection limit of the LMBs.

Biosensor selectivity is critical, particularly in DNA-baseddiagnostic devices. Thus, the selectivity of MBs immobilized ontothe glass surface was also investigated, and for this purpose, asequence with a single base mismatch was synthesized andevaluated. The mismatch was positioned in the loop and comple-mentary to a DNA base (see Table 1), not a LNA base, in the twobeacons. The signal enhancement was obtained for the perfectmatch and mismatch targets to compare the detection capabilitiesof RMB and LMB. Similar to what was observed in solution,23

LMB showed a slightly superior selectivity to that of the RMB(see Figure S1 in Supporting Information), proving that thispattern was also maintained when the probe is immobilized on aglass surface. The better discriminatory capability of the LMBcan be directly attributed to the high affinity of the LNA basestoward DNA target molecules.31

MB Sensitivity in the Presence of Complex Matrixes.Another important aspect in the study of RMBs and LMBs to beused in surface immobilized biosensors is the ability of probes todetect a target in complex matrixes. This is especially importantin the early detection of various genetic diseases where rapid andsensitive detection is needed. Ideally, the MB signal should only

light up from the detection of target molecules. However, this isnot always the case since degradation of the MB can occurbecause of DNA binding proteins and nucleases that are foundinside the cells, resulting in false positive signals. Moreover, theseproteins can hinder the ability of the MB to bind with the targets,thereby decreasing overall signal enhancement. Therefore, it isimperative to evaluate the performance of the molecular beacon-based biosensor in complex matrixes. Two different media, fetalbovine serum (FBS) and cell lysate, were selected, and the resultsare summarized in Figure 4. These media were selected becausethey contain many proteins, amino acids, sugars, and lipids that

(31) Mouritzen, P.; Nielsen, A. T.; Pfundheller, H. M.; Choleva, Y.; Kongsbak,L.; Moller, S. Expert Rev. Mol. Diagn. 2003, 3, 27–38.

Figure 2. Hybridization kinetics of RMB and LMB immobilized onto the surface after target addition (10 µM). Right figure shows the initial rateof the immobilized beacons. The curves correspond to the average of three independent experiments. The Y axis represents fluorescence(arbitrary units), and the values have been divided by 100 to clarify the graphics.

Figure 3. Comparison of the influence of target concentration on hybridization of RMBs and LMBs. Each point corresponds to the average ofthree measurements. The detection limit (DL) was calculated from the expression DL ) meanblank + 3SDblank (graphic on the right).

Figure 4. Normalized fluorescence enhancement of the immobilizedRMB and LMB after treatment of the target with fetal bovine serum(FBS), cell lysate, and PBS buffer solution.

3453Analytical Chemistry, Vol. 81, No. 9, May 1, 2009

can potentially interfere with hybridization efficiency and/ordegrade the MB. As expected, the data revealed a decrease inthe signal enhancement of the RMB in the presence of FBS, aswell as cell lysate. The background intensity of the RMBs didnot change significantly (data not shown), which indicates thatboth the FBS and cell lysate solutions impede the ability of RMBsto bind with the target. Also, it is possible that degradation of theloop sequence has taken place since the DNA bases in the loopare more exposed and, therefore, more prone to degradation thanthe DNA bases in the stem. This effect is not as significant inLMBs since the signal enhancement appears to be unaffected aftertreatment with FBS and cell lysate. The degradation of the loopis less likely because of the inherent resistance of the LNA basesto degradation typically caused by nucleases. Nonetheless, en-dogenous materials can still block hybridization with the target,which, to some degree, explains the reduction of the signalenhancement observed for the LMB. Overall, these results showthat LMBs on solid surface retain the ability to detect the targetin complex matrixes.

CONCLUSIONIn this paper, we have developed a surface-immobilized MB,

which possesses superior stability for surface hybridization stud-ies. The incorporation of LNA bases in the beacon design allowsus to take combined advantage of the sensitivity of the MBs andthe high binding affinity and stability of the LNAs bases.Moreover, the incorporation of a PEG linker with optimum lengthhas considerably improved the target hybridization. Overall, theintroduction of these modifications in the MB sequence it hasmade possible to achieve a signal-to-background of 25-fold,

considerably better than the previously best reported values ofonly 5.5-fold with regular MBs immobilized probes. The biosensordeveloped with these MBs was highly stable and robust, as wellas selective and sensitive, resulting in a promising design for usein a wide variety of biological and biotechnology applications.Future investigation will address the immobilization of multipleLMBs on solid surfaces, targeting different gene sequences forrobust biosensor assay development.

SAFETY CONSIDERATIONSPiranha solution is a dangerous and highly corrosive substance.

It must be handled extremely carefully under the hood and usingprotective equipment. Fresh solutions should be prepared rightbefore use, and should not be stored in a closed container.

ACKNOWLEDGMENTThis work was supported by NIH NCI and NIGMS grants and

by the State of Florida Center for Nano-Biosensors. M.-C.E.acknowledges financial support from the Departament d’Univer-sitats, Recerca i Societat de la Informació de la Generalitat deCatalunya, Spain.

SUPPORTING INFORMATION AVAILABLEFurther details are given in Figure S1. This material is available

free of charge via the Internet at http://pubs.acs.org.

Received for review December 22, 2008. Accepted March11, 2009.

AC8027239

3454 Analytical Chemistry, Vol. 81, No. 9, May 1, 2009