Development of an Aptamer Beacon for Detection of Interferon-Gamma

Anal. Chem. 2010, 82, 1851–1857

Development of an Aptamer Beacon for Detection of Interferon-Gamma Nazgul Tuleuova,†,‡ Caroline N. Jones,† Jun Yan,† Erlan Ramanculov,‡ Yohei Yokobayashi,† and Alexander Revzin*,† Department of Biomedical Engineering, University of California, Davis, California, and National Center for Biotechnology, Astana, Kazakhstan Traditional antibody-based affinity sensing strategies employ multiple reagents and washing steps and are unsuitable for real-time detection of analyte binding. Aptamers, on the other hand, may be designed to monitor binding events directly, in real-time, without the need for secondary labels. The goal of the present study was to design an aptamer beacon for fluorescence resonance energy transfer (FRET)based detection of interferon-gamma (IFN-γ)san important inflammatory cytokine. Variants of DNA aptamer modified with biotin moieties and spacers were immobilized on avidin-coated surfaces and characterized by surface plasmon resonance (SPR). The SPR studies showed that immobilization of aptamer via the 3′ end resulted in the best binding IFN-γ (Kd ) 3.44 nM). This optimal aptamer variant was then used to construct a beacon by hybridizing fluorophore-labeled aptamer with an antisense oligonucleotide strand carrying a quencher. SPR studies revealed that IFN-γ binding with an aptamer beacon occurred within 15 min of analyte introductionssuggesting dynamic replacement of the quencher-complementary strand by IFN-γ molecules. To further highlight biosensing applications, aptamer beacon molecules were immobilized inside microfluidic channels and challenged with varying concentration of analyte. Fluorescence microscopy revealed low fluorescence in the absence of analyte and high fluorescence after introduction of IFN-γ. Importantly, unlike traditional antibody-based immunoassays, the signal was observed directly upon binding of analyte without the need for multiple washing steps. The surface immobilized aptamer beacon had a linear range from 5 to 100 nM and a lower limit of detection of 5 nM IFN-γ. In conclusion, we designed a FRET-based aptamer beacon for monitoring of an inflammatory cytokinesIFNγ. In the future, this biosensing strategy will be employed to monitor dynamics of cytokine production by the immune cells. Interferon-gamma (IFN-γ) is an important inflammatory cytokines secreted by immune cells in response to various patho* To whom correspondence should be addressed. Mailing address: Department of Biomedical Engineering University of California, Davis, 451 East Health Sciences Drive No. 2519, Davis, CA, 95616. E-mail: [email protected]. Phone: 530-752-2383. Fax: 530-754-5739. † University of California, Davis. ‡ National Center for Biotechnology. 10.1021/ac9025237  2010 American Chemical Society Published on Web 02/01/2010

gens.1 The levels of this protein provide diagnostic information about various infectious diseases and the ability of the body to mount an immune response. For example, in HIV infected patients, vigorous production of IFN-γ by T-helper (Th1) and cytotoxic T-lymphocytes correlates with low viremia and slow progression of the disease.2,3 Traditionally, secreted cytokines such as IFN-γ are detected using antibody-based sandwich immunoassays. While robust and well-established, these traditional strategies are time-consuming, require multiple washing steps, and provide no information about dynamics of cytokine production. Aptamer-based affinity sensing strategies are emerging as viable alternatives to antibody-based immunoassays.4 Aptamers are single-stranded DNA or RNA oligonucleotides selected in vitro to bind target analytes with high specificity and affinity.5 Because aptamers are short nucleic acid molecules they are more robust than antibodies so that aptamer-based biosensors can be regenerated and used multiple times. Even more importantly, the simplicity and robustness of aptamers makes them particularly amenable to chemical modification and inclusion of surface binding or sensing moieties.6-9 Several strategies of transforming aptamer-analyte interactions into electrochemical, mechanical, piezoelectric, or fluorescent signals have been reported.10-15 Among these methods, fluorescence-based signal transduction is quite powerful because such strategies as fluorescence resonance energy transfer (FRET) may be utilized to convert aptamers into (1) Boehm, U.; Klamp, T.; Groot, M.; Howard, J. C. Annu. Rev. Immunol. 1997, 15, 749–795. (2) Pantaleo, G.; Koup, R. A. Nat. Med. 2004, 10, 806–810. (3) Romagnani, S. Clin. Immunol. Immunopath. 1996, 80 (3), 225–235. (4) Jayasena, S. D. Clin. Chem. 1999, 45, 1628–1650. (5) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818–822. (6) Balamurugan, S.; Obubuafo, A.; Soper, S. A.; Spivak, D. A. Anal. Bioanal. Chem. 2008, 390, 1009–1021. (7) Kirby, R.; Cho, E. J.; Gehrke, B.; Bayer, T.; Park, Y. S.; Neikirk, D. P.; McDevitt, J. T.; Ellington, A. D. Anal. Chem. 2004, 76, 4066–4075. (8) Nutiu, R.; Li, Y. F. Methods 2005, 37, 16–25. (9) Luzi, E.; Minunni, M.; Tombelli, S.; Mascini, M. Trac-Trends Anal. Chem. 2003, 22, 810–818. (10) Lu, Y.; Zhu, N; Yu, P.; Mao, L. Anal. 2008, 133, 1256–1260. (11) Ikanovic, M.; Rudzinski, W. E.; Bruno, J. G.; Allman, A.; Carrillo, M. P.; Dwarakanath, S.; Bhahdigadi, S.; Rao, P.; Kiel, J. L.; Andrews, C. J. J. Fluorescence 2007, 17, 193–199. (12) Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. Anal. Chem. 2002, 74, 4488– 95. (13) Li, J. W. J.; Fang, X. H.; Tan, W. H. Biochem. Biophys. Res. Commun. 2002, 292, 31–40. (14) Yamamoto, R.; Baba, T.; Kumar, P. K. Genes Cells 2000, 5, 389–96. (15) Bang, G. S.; Cho, S.; Kim, B. G. Biosens. Bioelectr. 2005, 21, 863–70.

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Table 1. Sequences of IFN-γ Aptamer Oligonucleotides Used in the Experiments name 5′B 3′B 5′Bspacer 3′Bspacer FA Q

Figure 1. Schematic representation of an aptamer beacon for detection of IFN-γ. In duplex, fluorescence of an aptamer is effectively quenched by a FRET effect resulting from proximity of fluorophorelabeled aptamer to an acceptor-carrying complementary strand. Binding of IFN-γ disrupts the DNA duplex and results in a fluorescence signal.

real-time optical biosensors.8,16-18 The FRET-based aptamer beacons have been particularly popular.8,11,16,17,19,20 As shown in Figure 1 this sensing scheme involves formation of a duplex where a fluorophore-labeled aptamer is hybridized with an antisense oligonucleotide sequence carrying a quencher. The aptamer beacon shows no fluorescence in duplex; however, the addition of a target analyte results in displacement of the quencher-carrying strand, disruption of the FRET effect, and the appearance of the fluorescence signal. While a number of aptamer beacons been described in the literature,21,22 to the best of our knowledge, there have been no reports describing detection of IFN-γ using this sensing strategy. Given the importance of IFN-γ as a diagnostic immune response marker,1-3,23 we sought to design a novel aptamer-based immunosensor for the detection of this analyte. The IFN-γ-binding DNA aptamer previously described in the literature24,25 was biotinylated and immobilized on the surface via avidin-biotin interactions. Surface plasmon resonance (SPR) was used to investigate the effects of biotinylation, fluorophore attachment, and spacer incorporation on the ability of aptamer to bind IFN-γ. The 3′ end biotinylated aptamer without spacer was to found to have the highest affinity for IFN-γ (Kd ) 3.4 nM) and was used (16) Urata, H.; Nomura, K.; Wada, S.; Akagi, M. Biochem. Biophys. Res. Commun. 2007, 360, 459–463. (17) Nishihira, A.; Ozaki, H.; Wakabayashi, M.; Kuwahara, M.; Sawai, H. Nucleic Acids Symp. Ser. (Oxford) 2004, 135–6. (18) Babendure, J. R.; Adams, S. R.; Tsien, R. Y. J. Am. Chem. Soc. 2003, 125, 14716–7. (19) Tang, Z. W.; Mallikaratchy, P.; Yang, R. H.; Kim, Y. M.; Zhu, Z.; Wang, H.; Tan, W. H. J. Am. Chem. Soc. 2008, 130, 11268. (20) Li, W.; Yang, X. H.; Wang, K. M.; Tan, W. H.; Li, H. M.; Ma, C. B. Talanta 2008, 75, 770–774. (21) Hall, B.; Cater, S.; Ellington, A. D. Biotechnol. Bioeng. 2009, 103, 104901059. (22) Yang, C. J.; Jockusch, S.; Vicens, M.; Turro, N. J.; Tan, W. Proc. Nat. Acad. Sci. 2005, 102, 17278–17283. (23) Karlsson, A. C., J. N; Martin, S. R.; Younger, B. M.; Bredt, L.; Epling, R.; Ronquillo, A. V.; Deeks, S. C.; McCune, J. M.; Nixon, D. F.; Sinclair, a. E. J. Immunol. Methods 2003, 283, 141–153. (24) Lee, P. P.; Ramanathan, M.; Hunt, C. A.; Garovoy, M. R. Transplantation 1996, 62 (9), 1297–1301. (25) Balasubramanian, V.; Nguyen, L. T.; Balasubramanian, S. V.; Ramanathan, M. Mol. Pharmacol. 1998, 53, 926–32.

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sequence with modification 5′-biotin-GGG GTT GGT TGT GTT GGG TGT TGT GT-3′ 5′-GGG GTT GGT TGT GTT GGG TGT TGT GT-Biotin-3′ 5′-Biotin-C12-GGG GTT GGT TGT GTT GGG TGT TGT GT-3′ 5′-GGG GTT GGT TGT GTT GGG TGT TGT GT-C12-Biotin-3′ 5′-6-FAM- T GGG GTT GGT TGT GTT GGG TGT TGT GT-Biotin-3′ 5′- ACAACCAACCCCA-BHQ-1-3′

throughout the study. SPR experiments also revealed rapid binding of IFN-γ molecules with an aptamer/antisense duplex and suggested displacement of an antisense strand by the cytokine molecules. Disruption of the DNA duplex and formation of aptamer-IFN-γ complex was further confirmed with fluorescence assays involving soluble or surface-immobilized aptamer beacons. To highlight biosensing application of this approach, surface immobilization of aptamer beacons and detection of IFN-γ was demonstrated inside microfluidic devices. MATERIALS AND METHODS Chemicals and Materials. Glass slides (75 × 25 mm2) were obtained from VWR (West Chester, PA). 3-Aminopropyltrimethoxysilane was purchased from Gelest, Inc. (Morrisville, PA). Anhydrous toluene (99.9%), 2-hydroxy-2-methylpropiophenone (photoinitiator), bovine serum albumin (BSA), HEPES, KCl, EDTA, MgCl2, surfactant Tween20, and glutaraldehyde were obtained from Sigma-Aldrich (St. Louis, MO). Acetone was obtained from EMD Chemicals (Gibbstown, NJ), Neutravidin was purchased from Invitrogen (Carlsbad, CA). Recombinant human IFN-γ and Interleukin-2 were purchased from R&D systems (Minneapolis, MN) and Endogen (Woburn, MA), respectively. 96-Well plates, transparent optical flat bottom, black, were purchased from NUNC. 96-Well Reacti-bind neutroavidin covered plates, black, were obtained from Pierce. Cell culture medium RPMI 1640: 1X, with L-glutamine was purchased from VWR. The following buffers were used in this study: TKM buffer (50 mM TrisHCl, 10 mM KCl, 1 mM MgCl2, pH 8.6), HKE buffer (10 mM Hepes, 100 mM KCl, 1 mM EDTA, pH 7.4), HKMT washing buffer (10 mM Hepes, 100 mM KCl, 1 mM MgCl2, 0.05% Tween20, pH 7.4). IFN-γ aptamer sequences with 3′ and 5′ biotin modifications (3′B and 5′B) and sequences with biotin + spacer modifications (3′Bspacer and 5′Bspacer) were ordered from Bioneer (Alameda, CA). A 3′ biotinylated aptamer carrying carboxyfluorescein label (FA) and 3′BHQ-1-labeled complementary oligo (Q) were synthesized by IDT Technologies (San Diego, CA). Oligonucleotide sequences and modifications used in this study are listed in Table 1. Prior to their use, samples were heated at 95 °C for 3 min and then allowed to cool slowly to room temperature. Oligonucleotide samples were kept overnight at 4 °C until their use. Diluted solutions of oligos and recombinant protein for measurements were prepared in appropriate buffers.

Characterization of IFN-γ Binding to an Immobilized Aptamer. SPR experiments were performed on a four-channel BIAcore T3000 instrument (Uppsala, Sweden) using streptavidin (SA) sensor chips obtained from BIAcore. Biotinylated aptamer was diluted in HKE buffer to 1 µM and injected into SPR instrument at a flow rate of 20 µL/min. All SPR experiments were performed at 20 °C in filtered degassed HKE buffer. One channel of SA sensor chip was designated as reference and was blocked with biotin (without aptamer) to prevent other binding events from happening. IFN-γ solutions ranging in concentration from 12 to 120 nM were prepared HKE buffer and injected into the SPR instrument at flow rate of 20 µL/min. Binding of IFN-γ to the immobilized aptamer was followed in real-time to determine the time required for reaching equilibrium. In a typical experiment, the contact time of 180 s was sufficient to reach saturation of the binding signal and 360 s was allotted for dissociation. Washing in between binding steps was performed using HKE buffer. Four aptamer variants were tested to determine aptamer immobilization/modification strategy leading to highest binding affinity of IFN-γ. The variants were: 3′ biotinylated aptamer with or without spacer and 5′ biotinylated aptamer with or without spacer. Kd values were obtained using affinity in solution fitting model on BIAvaluation 4.0 software. From the SPR experiments described above, 3′ biotinylated aptamer without spacer was found to have the lowest Kd and was used for construction of the DNA duplex-based aptamer beacon. Hybridization of antisense strand to aptamer and its displacement with IFN-γ molecules were investigated by SPR. In these experiments, aptamer-containing surfaces were primed by injecting 5 µL of 20 mM NaOH at the flow rate of 30 µL/ min, followed by washing with HKE running buffer for 1200 s. Hybridization was initiated by injecting 45 µL of 2 µM complementary oligonucleotide at 30 µL/min followed by a flow of HKE running buffer at 30 µL/min for 300 s. Equilibrium constant for aptamer-antisense hybridization was determined as described in the previous paragraph. In SPR experiments investigating analyte binding to the molecular beacon duplex, 100 µL of 2 µM quenching oligonucleotide was injected and flowed at 30 µL/min for 300 s, followed by injection of 100 µL of 100 nM IFN-γ. Measuring Fluorescence Signal Due to Aptamer-IFN-γ Binding. In addition to characterizing cytokine-aptamer interactions with SPR, fluorescence spectroscopy and microscopy were used to detect changes in fluorescence signal due to analyte binding. The function of surface immobilized aptamer beacon was tested using known concentration dissolved in either buffer or cell culture medium supplemented with 10% serum. Biotinylated and fluorophore-labeled aptamer was immobilized in neutravidincoated 96-well plates by incubation of 50 nM aptamer solution for 2 h at room temperature. After washing three times with TKMT washing buffer, quenching-labeled antisense oligo was added at a concentration 500 nM and incubated overnight to allow for hybridization to occur. After washing with TKMT, IFN-γ concentrations ranging from 1 to 200 nM were prepared either in TKMT buffer or RPMI medium supplemented with 10% serum and were added into the wells of the microplate. Change in fluorescence intensity was measured with a Safire2 microplate reader (Tecan) at 483 nm excitation and 525 nm emission. The fluorescence signal

was normalized to the background fluorescence of the solution without any input molecules and presented as fold fluorescence increase. Several oligos of varying lengths and nucleotide sequence were tested in terms of quenching efficiency and displacement by IFN-γ in order to identify a suitable candidate (see Table 1 for the sequence of the complementary strand). Detecting IFN-γ in Aptamer-Modified Microfluidic Devices. To demonstrate a proof-of-concept microdevice for detection of IFN-γ, aptamer beacon molecules were immobilized inside avidin-coated poly(dimethyl siloxane) (PDMS) microchannels. Prior to avidin coating, glass slides were cleaned using “piranha” solution as described by us previously26 and stored in the oven at 200 °C prior to use. Immediately before silanization, a glass slide was exposed to oxygen plasma for 5 min at 300 W (YES3, Yield Engineering Systems, Livermore, CA) and then placed for 10 min in a 2% v/v solution of aminopropyl-triethoxysilane in acetone. After silanization, the slides were rinsed with DI water, dried under nitrogen, cured at 100 °C for 1 h, and incubated in a 2% v/v aqueous solution of glutaraldehyde for 1 h. PDMS microfluidic devices were fabricated using standard SU-8 processing and soft lithography protocols. The design of the microfluidic devices used in these experiments has been described in our previous publications.27,28 Briefly, the microfluidic device contained two flow chambers with width-length-height dimensions of 3 × 10 × 0.1 mm and a network of independently addressed auxiliary channels. The auxiliary channels were used to apply negative pressure (vacuum suction) to the PDMS mold and to reversibly secure it on top of a glass substrate. This strategy allowed to seal a fluid conduit on top of the glass slide without compromising the aminosilane layer. The inlet/outlet holes were punched with a blunt 16 gauge needle. A 5 mL syringe was connected to silicone tubing (1/32 in. i.d., Fisher), which was attached to the outlet of the flow chamber with a metal insert cut from a 20 gauge needle. A blunt, shortened 20 gauge needle carrying a plastic hub was inserted in the inlet. A pressure-driven flow in the microdevice was created by withdrawing the syringe positioned at the outlet with a precision syringe pump (Harvard Apparatus, Boston, MA). Aminosilane- and glutaraldehyde-modified glass slides were outfitted with PDMS microchannels and incubated with 1 mg/ mL neutravidin solution in 1× PBS. Biotinylated and fluoresceinlabeled aptamer was then injected in the microfluidic channels at concentration of 10 µM and incubated for 2 h at room temperature. After washing with TKMT buffer, quencher-labeled antisense oligonucleotide was injected into channels at concentration of 50 µM and hybridized with aptamer overnight at room temperature. This step resulted in immobilization of an aptamer-fluorescein/ antisense-quencher duplex on the surface of the microfluidic channels. During cytokine detection experiments, IFN-γ was injected into the microfluidic device at concentrations ranging from 1 to 100 nM in TKM buffer. The change in fluorescence due to cytokineaptamer beacon interactions was monitored using a Zeiss 200 M (26) Jones, C. N.; Lee, J. Y.; Zhu, J.; Stybayeva, G.; Ramanculov, E.; Zern, M. A.; Revzin, A. Anal. Chem. 2008, 80, 6351–7. (27) Zhu, H.; Macal, M.; Jones, C. N.; George, M. D.; Dandekar, S.; Revzin, A. Anal. Chim. Acta 2008, 608, 186–96. (28) Zhu, H.; Stybayeva, G.; Macal, M.; Ramanculov, E.; George, M. D.; Dandekarb, S.; Revzin, a. A.; Lab Chip 2008, 8, 2197–2205.


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epifluorescence microscope (Carl Zeiss MicroImaging, Inc. Thornwood, NY) equipped with xioCam MRm (CCD monochrome, 1300 pixels × 1030 pixels). Objectives, camera, and fluorescence filters were computer controlled through a PCI interface. Image acquisition and fluorescence analysis were carried out using AxioVision software (Carl Zeiss MicroImaging, Inc. Thornwood, NY). RESULTS AND DISCUSSION The goal of this study was to develop an aptamer beacon for FRET-based detection of IFN-γ. Several key parameters pertaining to orientation of the immobilized aptamer and the design of aptamer-antisense duplex were characterized by SPR as well as fluorescence spectroscopy and microscopy. As a proof-of-concept biosensor demonstration, aptamer beacon molecules were immobilized inside microfluidic channels and were shown to produce a fluorescence signal in response to different concentrations of IFN-γ. Characterization of IFN-γ Binding to Surface Immobilized Aptamer. Avidin-biotin interactions have been used widely for surface binding of functional biomolecules, including aptamers;7,29 therefore, this immobilization scheme was chosen for our study. While the nucleic acid sequence of aptamer specific for IFN-γ has been reported in the literature,24 the position of the sensing nucleotides on the DNA strand was not known. Given that chemical modification may negatively impact the affinity of aptamer for the analyte,6,30 we investigated the effects of placing biotin at the 3′ vs 5′ end of the aptamer. In addition, insertion of PEG spacer between the aptamer and biotin was explored as a means of making nucleotides more accessible to the target analyte. The aptamer variants including 5′ biotin, 3′ biotin, 5′ biotin w/spacer, and 3′ biotin w/spacer (see Table 1) were synthesized and immobilized on avidin-coated sensor chips. SPR was used to test the ability of aptamer variants to bind IFN-γ molecules. A representative experiment is shown in Figure 2 where an SPR sensor chip containing four aptamer variants described above was challenged with 100 nM IFN-γ. As seen from this sensogram, the highest level of cytokine binding was observed on a 3′ biotinylated aptamer without spacer. By repeating binding experiments for IFN-γ concentrations ranging from 1 to 100 nM, equilibrium binding constants Kd for aptamer variants were determined using simple affinity fitting model with BIAcore software 4.0. As seen from Table 2, the Kd values ranged from 28 nM in the case of lowest affinity aptamer biotinylated at the 5′ to 3 nM for the highest affinity aptamer biotinylated at the 3′. These results show that attachment of biotin at the 5′ end hinders analyte binding and may mean that the nucleotides responsible for recognition of IFN-γ are located at the 5′ end of the aptamer. It is not entirely clear at this time why incorporation of a spacer at the 3′ end increases Kd but may suggest that inclusion of PEG-based spacers hinders interaction with cytokine molecules due to hydration of PEG. Testing other space chemistries will help address this question in the future. Overall, the IFN-γ aptamer immobilized via 3′ end without the spacer was found to have the best affinity constant (Kd ) 3 nM) and therefore was chosen as the basis for the molecular beacon described (29) Collett, J. R.; Cho, E. J.; Ellington, A. D. Methods 2005, 37, 4–15. (30) Walter, J. G.; Kokpinar, O.; Friehs, K.; Stahl, F.; Scheper, T. Anal. Chem. 2008, 80, 7372–7378.

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Figure 2. SPR analysis of IFN-γ binding as a function of aptamer modification. Vertical arrows represent a washing step where buffer is introduced. (A) Sensograms of four aptamer variants differing in the placement biotin (B) and inclusion of spacer in addition to biotin (BS). These sensograms compare response four aptamer variants binding 100 nM of IFN-γ. Multiple concentrations of IFN-γ were tested for each aptamer variant to determine Kd values listed in Table 2. (B) SPR sensograms comparing aptamer carrying a fluorophore (F) at the 5′ end and biotin at the 3′ end to an aptamer without a fluorophore and biotin 3′ end. These variants were challenged with different concentrations of IFN-γ to determine Kd values. Table 2. Dissociation for Different Aptamer Variants Investigated in This Studya aptamer

IFN-γ KD (nM)

5′B 5′B 3′B 3′BS F

28.1 ± 1.09 14.9 ± 1.3 3.44 ± 0.2 6.56 ± 0.8 5.73 ± 1.1

a The abbreviations are as follows: aptamer modified with biotin (B), biotin and spacer (BS), or fluorophore (F).

in the subsequent sections of this paper. Importantly, Kd values of the aptamer-IFN binding were comparable to concentrations of cytokine secreted by cells in vitro or observed in

blood,2,27 pointing to the potential use of aptamer-based sensor for monitoring physiological levels of IFN-γ. Design and Characterization of the Aptamer-Antisense Duplex. The aptamer beacon designs may be broadly categorized into monochromophoric and bichromophoric approaches.8 The first strategy is suitable in the case where analyte binding causes significant structural reorganization of the aptamer leading to a change in the spectroscopic properties of the fluorophore. Fluorescence spectra of 6FAM labeled IFN-γ aptamer were not much different before and after IFN-γ binding (data not shown). This result suggests that the binding of IFN-γ did not cause the change to the aptamer and/or fluorescent properties of the fluorophore. Therefore, we chose to pursue a bichromophoric strategy involving fluorophore-labeled aptamer and quencherlabeled complementary (antisense) oligonucleotide strand. As shown diagrammatically in Figure 1, the molecular beacon was comprised of two DNA oligonucleotides: an aptamer modified with a fluorophore at the 5′ end and an antisense strand labeled with quencher at the 3′ end. The quencher-carrying strand was a 12mer sequence complementary to the 5′ region of the aptamer. In the absence of the target, the two DNA molecules assembled into the duplex structure where fluorophore and quencher were in close proximitysallowing for the FRET effect. The displacement of the quencher-carrying oligo strand with the IFN-γ was hypothesized to disrupt the quenching of the fluorophore leading to a fluorescence signal. In order for disruption of the DNA duplex by the target analyte to occur rapidly, the affinities of aptamer-antisense and aptamer-IFN-γ needed to be similar. SPR experiments were first carried out to determine the Kd value for an aptamer modified with fluorescein at the 5′ and biotin at the 3′ end. The affinity constant for IFN-γ binding to this aptamer construct was determined to be 5.73 ± 1.1 nM, suggesting that fluorophore attachment did not appreciably impact the binding IFN-γ (see Table 2 for comparison of Kd values for different aptamer modifications). SPR was also used to determine the equilibrium binding constant for the hybridization of the quencher-antisense strand and a fluorophore-aptamer construct (SPR sensograms not shown). The Kd value for this interaction was determined to be 1.09 ± 0.4 nM. The similarity of Kd values for aptamer-IFN-γ and aptamer-antisense interactions suggested that displacing the antisense strand in DNA duplex by the cytokine molecules was indeed possible. Specificity is one of the most important characteristics of a biosensor. SPR experiments were used to demonstrate that our aptamer was responding specifically to IFN-γ. Figure 3 shows a representative experiment where two SPR channels containing aptamer are challenged with 100 nM concentration of IFN-γ and IL-2. As seen from the data, the binding signal was observed only in the channel containing the correct analytessuggesting specificity of the aptamer. Further proof of aptamer beacon specificity in serum is presented in the next section. In order to verify competitive binding of IFN-γ, we performed additional SPR and fluorescence spectroscopy experiments. A representative SPR sensogram is shown in Figure 4. In this experiment, one channel was coated with aptamer while the other channel contained aptamer-antisense duplex. Importantly, no appreciable dissociation of the DNA duplex was observed after

Figure 3. Specificity of aptamer-IFN-γ interaction: SPR sensogram showing binding curves of aptamer-modified surfaces challenged with 100 nM IFN-γ vs 100 nM IL-2. No signal is observed for IL-2 binding. Arrows represent a washing step.

40 min in a running buffer. Injection of 100 nM IFN-γ resulted in rapid appearance of binding signals of comparable magnitude in both channels (see Figure 4). The response time (time to 90% of signal) for IFN-γ binding was 15 min for both channels. These data were suggestive of displacement of an antisense strand and binding of IFN-γ to the aptamer; however, there remained a possibility that IFN-γ attached to the DNA duplex without dislodging the quencher oligo strand. Fluorescence spectroscopy and microscopy experiments described in the following section were conducted to exclude this scenario. Quantifying Fluorescence Response of Surface Immobilized Aptamer Beacon. To conclusively demonstrate displacement of the quenching oligo strand by the cytokine molecules, the biotinylated aptamer-antisense duplex was immobilized in avidin-coated 96-well plates. IFN-γ was then added into the plates at concentrations of 1, 5, 10, 50, and 100 nM and the fluorescence intensity was measured after 10 min of incubation. This time was chosen based on the SPR studies of the dynamics of IFN-γ to the aptamer-antisense complex described in the previous section. The aptamer beacon response was quantified using a microplate reader. As shown in Figure 5, the fluorescence signal change of our aptamer beacon was linear from 5 to 100 nM IFN-γ and the lowest detected concentration of analyte was 5 nM. The detection limit of our aptamer sensor is sufficient to monitor physiological levels of the cytokine secreted by the immune cells.2,27 Our laboratory is interested in placing immunosensors at the site of the cells in order to characterize dynamics of cytokine release;28,31 therefore, we sought to characterize response of the aptamer beacon in cell culture media. In these experiments, IFN-γ was dissolved in RPMI (media commonly used for culturing immune cells) supplemented with 10% fetal bovine serum or in HEPES buffer (pH 7.4) were compared. As shown in Figure 5, aptamer beacons remained functional in the cell culture media and showed concentration dependent changes in fluorescence signal. This result is very important as it demonstrates that the aptamer beacon remains responsive in a sample where concentration of extraneous proteins exceeds IFN-γ concentration by (31) Zhu, H.; Stybayeva, G. S.; Silangcruz, J.; Yan, J.; Ramanculov, E.; Dandekar, S.; George, M. D.; Revzin, A. Anal. Chem. 2009, 81, 8150–8156.


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Figure 4. SPR sensogram demonstrating IFN-γ binding to an aptamer beacon. Aptamer molecules were immobilized in two channels of an SPR instrument. A quenching strand was injected into channel 1 forming an aptamer-quencher duplex. Channel 2 contained only aptamer and was used as a reference. In the next step, we injected 100 nM of IFN-γ into both channels and observed comparable binding signals in both channels. This suggested disruption of a DNA duplex and displacement of the quenching strand with IFN-γ.

Figure 5. Fluorescence signal from an aptamer beacon challenged with varying concentrations of IFN-γ. Analyte was dissolved in either HEPES buffer (pH 7.4) or RPMI1640 media supplemented with 10% serum. Aptamer beacon responses to varying concentrations of IFN-γ were measured using a microplate reader. The measured fluorescence intensities were normalized by the values obtained in the absence of analyte molecules. Data are averages of three independent experiments (n ) 3).

100-10 000 fold. An ∼20 to 30% loss of signal for aptamer beacons operating in serum-containing media may be attributed to low level nonspecific binding of serum components. Despite this, our results are highly encouraging as they demonstrate reproducible and robust responses of an aptamer beacon in a physiological medium and point to immediate applicability of this sensing strategy for real sample analysis. Biosensing applications frequently require integration of the recognition molecules into microdevices to enable analysis of small sample volumes. In this paper, we demonstrate modification of the microfluidic device with aptamer beacon molecules and in situ detection of IFN-γ binding. Because PDMS is solvated easily by organic solvent, we chose to first modify the glass slides with aminosilane and glutaraldehydesmaking these substrates protein 1856


reactivesand then to place PDMS channels on top. The PDMS fluidic conduits were effectively sealed on glass by using negative pressuresan approach first described by Schiff et al. and employed by us in previous studies.28,31,32 The fluidic channels with protein-reactive glass surfaces were treated with neutravidin and then incubated with fluorophore-labeled and biotinylated aptamer molecules. Figure 6A shows a microfluidic device with two channels where the lower channel contains aptamer-fluorophore while the upper channel has been quenched by introduction of the quencher-carrying antisense strand. This image, as well as corresponding fluorescence intensity measurements seen in Figure 6B, demonstrates that injection of a quenching strand into the fluidic channel decreased the fluorescence by ∼10-fold. This is suggestive of DNA duplex formation and effective FRET quenching in the microdevice. Importantly, injection of 10 nM IFN-γ into the channel and subsequent displacement of the quenching strand caused reappearance of the fluorescence signal (Figure 6C). The signal observed in the microfluidic channel was also a function of analyte concentration so that introduction of 100 nM resulted in higher fluorescence compared to 10 nM of IFN-γ (Figure 6D; see also Figure 6E). The results shown in Figure 6 are highly significant as they demonstrate integration of aptamer beacon molecules into a microdevices and in situ detection of IFN-γ. Our study describes an aptamer beacon for detection of IFNγsan important clinical indicator of immune function. In contrast to traditional approaches employing antibodies and sandwich immunoassays which require multiple washing steps and serve as an end-point measurement, the biosensor described here emits fluorescence signal directly upon binding of the cytokine molecules. This surface immobilized aptamer beacon provides a simple, one-step immunoassay and may therefore be used for dynamic monitoring of cytokine release. The detection limit of our aptamer beacon (nanomolar range) is not as low as in the work by Min et al. who used impedance spectroscopy to detect (32) Schaff, U. Y.; Xing, M. M.; Lin, K. K.; Pan, N.; Jeon, N. L.; Simon, S. I. Lab Chip 2007, 7, 448–56.

Figure 6. Immobilization of aptamer beacons in microfluidic devices. (A) Image showing two fluidic channels where the bottom channel contains only aptamer-fluorophore while the upper channel contains aptamer-fluorophore-quencher duplex. (B) Fluorescence microscopy characterization of quenching observed in part A. An ∼10 fold quenching was observed. (C-E) Response of the fluidic channels to low (C) and high (D) concentrations of IFN-γ, and the corresponding fluorescence intensity measurement (E).

pM level IFN-γ binding to aptamer interactions.33 This discrepancy is likely due to the fact that ac impedance measures nonequilibrium molecular binding events at the concentrations far below the Kd. For example, the van Bennekom group working with antibody-containing surfaces reported detection of attamolar levels of IFN-γ using impedance34 and nanomolar levels with SPR.35 CONCLUSIONS This paper describes development of an aptamer beacon for FRET-based detection of IFN-γ. SPR was used to establish equilibrium binding constants (Kd) for different aptamer variants in order to determine how aptamer modification with biotin and fluorophore molecules affected analyte binding. These studies revealed that biotinylation of aptamer at the 3′ end resulted in the lowest Kd of 3.44 nM. Fluorescence spectroscopy and microscopy were used to demonstrate that attachment of IFN-γ to an aptamer beacon duplex resulted in fluorescence (33) Min, K.; Cho, M.; Han, S.-Y.; Shim, Y.-B.; Ku, J.; Ban, C. Biosens. Bioelectr. 2008, 23, 1819–1824. (34) Dijksma, M.; Kamp, B.; Hoogvliet, J. C.; Van Bennekom, W. P. Anal. Chem. 2001, 73, 901–907. (35) Stigter, E. C. A.; de Jong, G. J.; van Bennekom, W. P. Biosens. Bioelectr. 2005, 21, 474–482.

signal that changed in analyte concentration dependent fashion. The appearance of fluorescence signal suggested displacement of the quenching strand and disruption of the FRET effect, thus validating function of the aptamer sensor. To highlight possibility of sensor miniaturization, aptamer beacon molecules were immobilized inside microfluidic channels and were shown to be responsive in situ to different IFN-γ concentrations. The possibility for direct and dynamic sensing of cytokine binding provided by this aptamer beacon will be leveraged in the future for detecting cell-secreted cytokines in real-time. ACKNOWLEDGMENT We thank Prof. Laura Marcu and Dr. Yinghua Sun in the Department of Biomedical Engineering at UC Davis for use of fluorescence microscope. NT was supported by a fellowship from the National Center for Biotechnology, Republic of Kazakhstan. These studies were supported by an NSF EFRI grant awarded to AR.

Received for review November 4, 2009. Accepted January 20, 2010. AC9025237


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Development of an Aptamer Beacon for Detection of Interferon-Gamma

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