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.
Analytical Chemistry, Vol. 82, No. 5, March 1, 2010
1851
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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Analytical Chemistry, Vol. 82, No. 5, March 1, 2010
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.
Analytical Chemistry, Vol. 82, No. 5, March 1, 2010
1853
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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Analytical Chemistry, Vol. 82, No. 5, March 1, 2010
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
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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.
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