Aptamer-Based Electrochemical Biosensor For Interferon Gamma Detection
Aptamer-Based Electrochemical Biosensor For Interferon Gamma Detection
Aptamer-Based Electrochemical Biosensor For Interferon Gamma Detection
Department of Biomedical Engineering, University of California, Davis, California, and National Center for
Biotechnology, Astana, Kazakhstan
In this paper, we describe the development of an electro- expensive.4,5 While a number of strategies for miniaturization and
chemical DNA aptamer-based biosensor for detection of multiplexing of Ab-based cytokine immunoassays have been
interferon (IFN)-γ. A DNA hairpin containing IFN- proposed,6-10 these approaches are still hindered by the high cost
γ-binding aptamer was thiolated, conjugated with meth- and low stability of Abs as well as by the relative complexity of
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
ylene blue (MB) redox tag, and immobilized on a gold transducing antibody-antigen binding events.
electrode by self-assembly. Binding of IFN-γ caused the
Downloaded via UNIV ESTADUAL PAULISTA on December 24, 2020 at 05:49:14 (UTC).
10.1021/ac101409t 2010 American Chemical Society Analytical Chemistry, Vol. 82, No. 19, October 1, 2010 8131
Published on Web 09/03/2010
Figure 1. Schematic of aptamer-based electrochemical sensor for IFN-γ. (A) The aptamer hairpin was thiolated at the 5′ end allowing self-
assembly on gold electrodes. The redox label was attached at the 3′ end of the hairpin and was in close proximity to the electrode surface. (B)
Upon addition of IFN-γ aptamer, the hairpin changed confirmation and the redox label moved further away from the electrode, lowering the
electron-transfer efficiency. (C) The differences in faradaic current before and after addition of IFN-γ were quantified using square wave voltammetry
(SWV).
aptamer-analyte binding.18-20 Of particular note is the work of quence (IDT Technologies, San Diego, CA) was as follows: 5′-
Plaxco and colleagues who modified electrodes with redox tag- NH2-C6-GGGGTTGGTTGTGTTGGGTGTTGTGTCCAACCCC-
containing aptamers and demonstrated electrochemical detection C3-SH-3′. The aptamer was modified at the 5′-terminus with a
of a range of analytes.21-23 C6-disulfide [HO(CH2)6-S-S-(CH2)6-] linker and at the 3′-
The sensing strategy pursued in this study involved self- end with an amine group for redox probe (MB) conjugation.
assembly of thiolated aptamer hairpin molecules on gold electrode The aptamer was dissolved in 10 mM HEPES buffer (pH 7.4
surfaces. (See Figure 1.) The binding of IFN-γ molecules likely with 150 mM NaCl). This buffer was also employed in all IFN-γ
caused the hairpin to unfold, decreasing the efficiency of electron sensor experiments.
transfer from the redox label to the electrode. The change in redox Attachment of Redox Labels to Aptamers. The MB-tagged
current was quantified using square wave voltammetry (SWV) and aptamer was prepared using a procedure similar to that described
was found to depend on the concentration of IFN-γ. Overall, this by Plaxco and co-workers.23 Briefly, NHS-labeled MB was
electrochemical aptasensor was found to be sensitive and specific conjugated to the 3′-end of an amino-modified DNA aptamer
to IFN-γ, pointing to future applications in immunology, cancer through succinimide ester coupling. MB-NHS (1 µmol) was
research, and infectious disease monitoring. dissolved in 10 µL of DMF/50 µL of 0.5 M NaHCO3, then added
to 10 µL of 200 µM aptamer solution, stirred, and allowed to
react for 4 h at room temperature in the dark. After reaction,
MATERIALS AND METHODS
the sample was filtered using a centrifugal filter (Millipore,
Materials. The following reagents were used as received: 4-(2-
Amicon Ultra 3K 0.5 mL) in order to purify and concentrate
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium
MB-modified aptamers. The stock aptamer solution (20 µM)
bicarbonate (NaHCO3) (all reagent grade), 6-mercapto-1-hexanol
was stored at -20 °C for future use. Conjugation efficiency
(MCH), tris-(2-carboxyethyl) phosphine hydrochloride (TCEP),
was estimated by MALDI-MS analysis to be ∼30%. This
urea, bovine serum albumin (BSA) (98%) (all from Sigma-
efficiency is comparable to values reported previously.24
Aldrich, St. Louis, MO), IgG, anti-human IgG (from BD
Assembly of Aptamer Molecules on Gold Electrodes. The
Pharmingen), methylene blue (MB), carboxylic acid, succin-
IFN-γ sensor was fabricated using a gold working electrode (BAS,
imidyl ester (MB-NHS, Biosearch Technologies, INC, Novato,
L)1.6 mm). The electrodes were cleaned in “piranha” solution
CA), and cell culture medium RPMI 1640 (1×, with L-glutamine;
consisting of a 3:1 ratio of 30% w/v aqueous solutions of H2SO4
VWR). Medium was supplemented with fetal bovine serum
and H2O2 (Caution: this mixture reacts violently with organic
(FBS) and penicillin/streptomycin purchased from Invitrogen.
materials and must be handled with extreme care), then thor-
Recombinant human IFN-γ was purchased from R&D systems
oughly washed with DI H2O and ethanol, and dried under
(Minneapolis, MN). The 34-mer IFN-γ-binding aptamer se-
nitrogen. Prior to modification of the electrodes, aptamer stock
(18) Li, D., Song, S., Fan, C. Acc. Chem. Res. 2010, 43, 631-641. solution (0.02 mM) was reduced in 10 mM TCEP for 1 h to
(19) Lu, Y.; Li, X. C.; Zhang, L. M.; Yu, P.; Su, L.; Mao, L. Q. Anal. Chem. 2008, cleave disulfide bonds. This solution was then diluted in HEPES
80, 1883–1890.
buffer to achieve the desired aptamer concentration (from 0.5
(20) Radi, A. E.; Acero Sanchez, J. L.; Baldrich, E.; O’Sullivan, C. K. J. Am. Chem.
Soc. 2006, 128, 117–24. to 8 µM). For aptamer immobilization, the gold electrodes were
(21) Ricci, F.; Bonham, A. J.; Mason, A. C.; Reich, N. O.; Plaxco, K. W. Anal. kept in a solution of thiolated aptamer for 16 h in the dark at
Chem. 2009, 81, 1608–1614.
(22) Lubin, A. A.; Plaxco, K. W. Acc. Chem. Res. 2010, 43, 496–505. (24) Kang, D.; Zuo, X.; Yang, R.; Xia, F.; Plaxco, K. W.; White, R. J. Anal. Chem.
(23) Xiao, Y.; Lai, R. Y.; Plaxco, K. W. Nat. Protoc. 2007, 2, 2875–2880. 2009, 81, 9109–9113.
5′ end of DNA sequence. The choice of a redox tag was based on conformational changes in the hairpin structure of the aptamer,
previous reports in the literature describing MB-conjugated the surface density of probe molecules was hypothesized to be
nucleic acid molecules as offering better shelf life and performance related to sensitivity of the aptasensor. The surface density of
in serum compared to other redox molecules (e.g., ferrocene).24,27 MB-aptamer surface density was calculated on the basis of the
As shown in Figure 1A, in the absence of the analyte, hairpin intensity of the reduction peaks collected during SWV experi-
molecules were closed and MB redox tags were positioned next ments. Such an approach is frequently used to determine grafting
to the electrode surface. Binding of IFN-γ molecule likely caused density of DNA strands.33,34 In our calculations, we assumed that
hairpins to open, moving MB redox tags away from the electrode one MB molecule was conjugated to one aptamer, a reasonable
surface and decreasing electron transfer/current at the electrode. assumption for MB-NHS to NH2-aptamer conjugation chem-
(See Figure 1B.) SWV was used to monitor changes in the istry. Another point to note is that MB labeling efficiency was
intensity of the reduction current of MB as shown in Figure 1C. ∼30%, so that the surface density values reported below refer
The peak current intensity dropped as a function of increasing only to MB-modified aptamer molecules.
IFN-γ concentration. For determining aptamer surface density, the gold electrodes
The choice of frequency for SWV analysis has been noted as were incubated with MB-aptamer solution of either 0.5, 2.0 or
an important parameter in defining sensitivity of the aptasensor.28 8.0 µM and were subsequently passivated with MCH. The amount
We characterized behavior of aptamer-containing electrodes at of total charge passed during the redox reaction was calculated
frequencies ranging from 3 to 1000 Hz in the presence of 10 nM by integrating the current with respect to time. The relationship
IFN-γ (concentration) and determined that the signal change was between the total charge passed (Q, in coulombs) and the surface
greatest for frequencies ranging from 60 to 100 Hz. (See Figure density of the MB moiety (Γ) is described as Q ) nFAΓ, where
1 in Supporting Information.) Therefore, all subsequent SWV n is the number of electrons transferred per MB moiety () 2), A
experiments were performed at a 60 Hz sampling frequency. is the electrode surface area, and F is the Faraday constant. The
Figure 3A shows a typical SWV voltammogram with MB reduction MB-aptamer surface densities were estimated to be (4.17 ± 0.59)
peak appearing at -0.25 V (vs Ag/AgCl). Upon increasing the × 1012 molecules/cm2, (8.53 ± 0.56) × 1012 molecules/cm2, and
concentration of IFN-γ in the vicinity of the biosensor, the peak (6.57 ± 0.55) × 1013 molecules/cm2 for 0.5, 2.0, and 8.0 µM
current decreased, suggesting unfolding of the hairpin molecules. aptamer solution concentrations. Our surface density is in
As shown in Figure 3A, the loss in signal due to binding of IFN-γ general agreement with other reports employing MB-tagged
molecules was proportional to the cytokine concentration. This hairpin aptamers.31,32 Increasing aptamer solution concentration
loss in signal could be replotted as the change in reduction current above 8.0 µM did not result in higher surface density of the probe,
(∆I) before and after addition of IFN-γ. Figure 3B shows a suggesting steric hindrance or electrostatic repulsion of negatively
calibration curve of signal change vs cytokine concentration and charged DNA molecules at higher concentrations. Because
demonstrates that our aptasensor had a limit of detection of 0.06 binding of analyte results in unfolding of aptamer probes and
nM with linear range extending to 10 nM. The detection limit of decrease in redox current, the sensor response was reported in
our aptasensor is in line with concentration of IFN-γ in serum29 terms of signal loss or suppression. Signal suppression was
or in the proximity of activated immune cells.30 calculated by determining the difference in faradaic current before
Effects of Aptamer Surface Density on Biosensor Perfor- and after addition of IFN-γ. For a given cytokine concentration,
mance. An important factor affecting sensitivity and performance biosensors with larger signal suppression were deemed to be more
of an aptasensor is the packing density of aptamer molecules on sensitive. When challenged with 20 nM of IFN-γ, electrodes with
the surface.31,32 Given that the binding of IFN-γ likely induces
(31) Ricci, F.; Lai, R. Y.; Heeger, A. J.; Plaxco, K. W.; Sumner, J. J. Langmuir
(27) Ferapontova, E. E.; Gothelf, K. V. Langmuir 2009, 25, 4279–4283. 2007, 23, 6827–6834.
(28) White, R. J.; Plaxco, K. W. Anal. Chem. 2010, 82, 73–76. (32) White, R. J.; Phares, N.; Lubin, A. A.; Xiao, Y.; Plaxco, K. W. Langmuir
(29) de Metz, J.; Sprangers, F.; Endert, E.; Ackermans, M. T.; ten Berge, I. J. M.; 2008, 24, 10513–10518.
Sauerwein, H. P.; Romijn, J. A. J. Appl. Physiol. 1999, 86, 517–522. (33) Reeves, J. H.; Song, S.; Bowden, E. F. Anal. Chem. 1993, 65, 683–688.
(30) Zhu, H.; Stybayeva, G.; Macal, M.; Ramanculov, E.; George, M. D.; (34) Yang, W. R.; Ozsoz, M.; Hibbert, D. B.; Gooding, J. J. Electroanalysis 2002,
Dandekar, S.; Revzin, A. Lab Chip 2008, 8, 2197–2205. 14, 1299–1302.
ACKNOWLEDGMENT
Figure 4. Specificity of aptasensor response to IFN-γ. (A) SWV
measurements of aptasensor response to 100 nM concentration of We thank Prof. Yohei Yokobayshi in the Department of
nonspecific proteins (IgG, anti-IgG, BSA) as well as to 3.6 nM (60 Biomedical Engineering at UC Davis for helpful discussions and
ng/mL) of IFN-γ. These results show that biosensor did not respond comments. We also thank Dr. Eugene Kamarchik, Dr. Dianlu
to nonspecific protein but did respond to IFN-γ. This electrode had Jiang, and Dr. Weitao Jia for their help and advice. This work
medium packing density of aptamer molecules. (B) Aptasensor
was supported financially by an NSF EFRI award and by an NIH
response in HEPES buffer, RPMI media, and RPMI supplemented
with 10% serum. Varying concentrations of IFN-γ were spiked into grant (REB006519Z) awarded to A.R.
serum-containing media as highlighted by the dashed box. (C) Close-
SUPPORTING INFORMATION AVAILABLE
up view of the aptasensor response to IFN-γ spiked into serum-
Additional information as noted in text. This material is
containing media. These data show that, while changes in current
were dampened in the presence of serum, sensor response to varying available free of charge via the Internet at http://pubs.acs.org.
IFN-γ concentration was still discernible. This electrode had the low
packing density of aptamer molecules.
Received for review May 29, 2010. Accepted August 25,
2010.
modifications in the geometry or length of the aptamer molecules
may lead to future development of more sensitive IFN-γ aptasensors. AC101409T
Compared to other electrochemical biosensors for IFN-γ (36) Min, K.; Cho, M.; Han, S.-Y.; Shim, Y.-B.; Ku, J.; Ban, C. Biosens. Bioelectron.
reported in the literature, our aptsensor has similar sensitivity to 2008, 23, 1819–1824.
the biosensor described by Min et al. who used impedance (37) Dijksma, M.; Kamp, B.; Hoogvliet, J. C.; Van Bennekom, W. P. Anal. Chem.
2001, 73, 901–907.
spectroscopy to detect picomolar levels of IFN-γ binding to (38) Yan, J.; Sun, Y. H.; Zhu, H.; Marcu, L.; Revzin, A. Biosens. Bioelectron. 2009,
aptamer-modified electrodes.36 In another study, van Bennekom 24, 2604–2610.