An electrochemical aptasensor for detection of bovine interferon gamma

Bruno P. Crulhas ab, Dustin Hadley a, Ying Liu ac, Dong-Sik Shin ad, Gulnaz Stybayeva eg, Meruyert Imanbekova ae, Ashley E. Hill f, Valber Pedrosa b and Alexander Revzin *ag
aDepartment of Biomedical Engineering, University of California, Davis, CA, USA. E-mail: revzin.alexander@mayo.edu
bDepartment of Chemistry and Biochemistry, Sao Paulo State University, Botucatu, SP, Brazil
cState Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, China
dDepartment of Chemical Engineering, Sookmyung Women's University, Republic of Korea
eNational Center for Biotechnology, Republic of Kazakhstan
fCalifornia Animal Health and Food Safety Laboratory, University of California, Davis, CA, USA
gDepartment of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA

Received 26th May 2017 , Accepted 10th July 2017

First published on 17th July 2017


Bovine tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) in which it is hard to identify the pathological symptoms. Release of bovine interferon gamma (BoIFN-γ) by T-cells provides an important diagnostic marker of MTB infections. In this work, we developed for the first time an electrochemical aptasensor for sensitive and specific determination of BoIFN-γ. A thiolated IFN-γ-binding aptamer was conjugated with methylene blue (MB) and immobilized on a gold electrode by self-assembly. Binding of IFN-γ to the electrode surface caused a conformational change in the aptamer, decreasing electron transfer efficiency. The redox current was quantified using square wave voltammetry (SWV) and was found to be specific for bovine IFN-γ with detection limit of 0.1 nM in pristine buffer and 0.9 nM in blood. The biosensor described here may, in the future, be used for on-site testing of bovine blood to help better identify and contain outbreaks of bovine TB.


Introduction

Bovine tuberculosis (TB) is a chronic infectious disease occurring in cattle, domestic small ruminants, humans and a wide range of wild mammals.1,2 TB is a complex and intractable disease caused by Mycobacterium bovis. It puts significant financial burden on several countries, including the United States, to assess and survey sites of infection and promote preventive measures.3,4

In cattle, only about ten percent of infected cows show non-specific clinical symptoms (e.g., weakness, anorexia, loss of body condition, cough, dyspnea, and lymph node enlargement)3 (Cousins 2001). Most infected animals remain unapparent carriers without developing clinical disease. However, this latent infection may develop into active TB if the organism has a weakened immune system or becomes immunocompromised.4

Pathological signs are difficult to detect in early stage of infection.5 As an intracellular pathogen, M. bovis replicates inside reticuloendothelial cells and has adapted to survive and thrive in the intra-macrophage environment using different strategies. These strategies include ways to subvert or incapacitate macrophage bactericidal activity and also block and neutralize various macrophage functions, affecting various immune factors, including IFN-γ.6,7

Interferon gamma (IFN-γ) is a cytokine produced primarily by activated T lymphocytes and natural killer cells in response to mitogen or antigen stimulation.8,9 At the early phase of TB infection, bovine IFN-γ (BoIFN-γ) levels released in response to specific antigen increase significantly.10,11 Therefore, BoIFN-γ produced by T-cells can be used as an indicator for early diagnosis of TB infection.12 In fact, commercial sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect BoIFN-γ for diagnosis of TB in cattle.13 However, ELISA assays are labor- and instrumentation-intensive, requiring a dedicated laboratory to conduct experiments.

In this paper, we describe the development of a different strategy for detecting BoIFN-γ. This strategy relies on aptamers, in our case DNA molecules, selected for binding to a target analyte of interest.14,15 When functionalized with redox reporters and immobilized on electrode surfaces, aptamers may be used for electrochemical sensing of proteins, often providing in sensitivity and specificity comparable to or better than that of antibody-based immunoassays. Furthermore, as shown in Fig. 1, aptasensors generate a signal directly upon binding of analyte without the need for washing, labeling and development steps associated with immunoassays. While a number of electrochemical aptasensors has been reported in the literature,14,16–19 an aptasensor for detection of BoIFN-γ has not been described to date. Herein we describe such an aptasensor and demonstrate it to be sensitive and specific for BoIFN-γ.


image file: c7ay01313b-f1.tif
Fig. 1 Principle of aptasensor operation. (A) DNA aptamer forming a hairpin on an electrode surface with redox reporter located close to the electrode surface. (B) Hairpin changes conformation upon binding with IFN-γ preventing electron transfer between redox reporter molecules and an electrode. (C) Square wave voltammetry technique measures differences in faradaic current with and without BoIFN-γ.

Materials and methods

Materials

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium bicarbonate (NaHCO3), magnesium chloride (MgCl2), potassium chloride (KCl), sodium chloride (NaCl), 6-mercapto-1-hexanol (MCH), tris-(2-carboxyethyl)phosphine hydrochloride (TCEP), bovine serum albumin (BSA) (98%) and lectin from Phytolacca americana (pokeweed) and nucleopore track-etch membrane (13 mm, 0.2 μM) were purchased from Sigma-Aldrich (St. Louis, MO). Methylene blue (MB), carboxylic acid, succinimidyl ester were purchased from Biosearch Technologies, Inc. (Novato, CA). Cell culture medium RPMI 1640 (1×, with L-glutamine; VWR) was supplemented with fetal bovine serum (FBS) and penicillin/streptomycin purchased from Invitrogen. Recombinant bovine IFN-γ was purchased from R&D systems (Minneapolis, MN). The aptamer for BoIFN-γ was discovered for us by Base Pair Biotechnologies (Houston, TX). The 32-mer BoIFN-γ-binding aptamer sequence (IDT Technologies, San Diego, CA) was as follows: 5′-NH2-C6-TT GCC CAA CGT GGT GTT TAT CAC CTT GGA TTC-C3-SH-3′. Aptamer was modified at 3′-terminus with C3-disulfide linker for binding to gold electrode and at 5′-end with an amine group for redox probe (MB) conjugation. 10 mM of HEPES buffer was used to dissolve aptamer (pH 7.4 with 150 mM NaCl) and was employed in all BoIFN-γ sensor experiments.

Conjugating redox labels to aptamers

MB-tagged aptamer was prepared using a similar procedure described by Plaxco and coworkers.20 Briefly, NHS-labeled MB was chemically conjugated to 5′-end of an amino-modified DNA aptamer through succinimide ester coupling. MB-NHS (1 μmol) was dissolved in 10 μL of DMF/50 μL of 0.5 M NaHCO3 and 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, the sample was filtered using desalting column – a centrifugal filter (Millipore, Amicon Ultra 3K 0.5 mL) – in order to purify and concentrate MB-modified aptamers. The stock aptamer solution (50 μM) was stored at −20 °C.

Assembly of aptamers onto electrodes

Electrodes were prepared by sputtering 5 nm Cr adhesion layer followed by 100 nm layer of Au onto silicon wafers (LGA Thin Films, Santa Clara, CA). Prior to electrode modification, aptamer stock solution (50 mM) was diluted in HEPES buffer to achieve the desired aptamer concentration of 0.5 μM. For aptamer immobilization, gold electrodes were kept in a solution of thiolated aptamer for 18 h in the dark at 4 °C. Following incubation, the electrodes were rinsed with 70% ethanol and DI water. The electrodes were then immersed in an aqueous solution of 3 mM 6-mercapto-1-hexanol (MCH) for 1 h to displace nonspecifically adsorbed aptamer molecules and to passivate the electrode surface. Lastly, the electrodes were washed again with DI water and were ready to use immediately for electrochemical measurements or to be stored in buffer solution at 4 °C.21,22

Electrochemical characterization of aptasensors and detection of BoIFN-γ

Electrochemical measurements were made using a CHI 840C Electrochemical Workstation (CHInstruments, Austin, TX) with a three electrode system consisting of Ag/AgCl (3 M KCl) as reference electrode, Pt wire as counter electrode, and a gold working electrode. Electrochemical experiments were performed by using square wave voltammetry (SWV) with a 40 mV amplitude signal at a frequency of 60 Hz, over the range from −500 to 0.00 mV versus Ag/AgCl references.

Electrodes were modified with aptamer molecules as described above and were placed into a custom-made Teflon electrochemical cell. The sensor was allowed to equilibrate, as determined by the stable faradaic current. For control experiments (without BoIFN-γ), aptamer-modified working electrodes were tested either in HEPES buffer, RPMI with 10% of bovine serum or whole blood by making SWV measurements every 15 min for 2 h. When constructing calibration curves, aptasensors were challenged with several concentrations of bovine recombinant IFN-γ (ranging from 1 ng mL−1 to 230 ng mL−1). Before each measurement the sensor was allowed to react with analyte for 15 min.

For measurements using peripheral blood mononuclear cells (PBMCs), blood samples were collected from healthy cattle by venipuncture into tubes containing EDTA. PBMCs were isolated using Lymphoprep™ (Axis-Shield, Dundee, Scotland). Briefly, blood was diluted with equal volume of 0.9% NaCl, after which 6 mL of diluted blood was dispensed over 3 mL Lymphoprep™ and centrifuged at 800 × g for 20 minutes at 20 °C. After centrifugation, mononuclear cells were removed using a Pasteur pipette and cultured in 75 cm2 tissue flasks in RPMI 1640 supplemented with 10% FBS, 100 U mL−1 penicillin/streptomycin, L-glutamine, under a 5% CO2/95% air humidified atmosphere at 37 °C.

Electrochemical measurements with bovine PBMCs were made using the same procedures as described above with minor modifications. A Teflon electrochemical cell was placed into tissue culture incubator (TS1500, Techne, Burlington, NJ) operating at 5% CO2/95% air humidified atmosphere at 37 °C. Prior to electrochemical measurements of PBMC secretions, the electrochemical signal was allowed to stabilize for 1 h. Subsequently, PBMCs were stimulated to commence secretion of cytokines, including IFN-γ, by incubation with mitogen (10 μg mL−1 pokeweed).

Institutional animal use and care review was not required because no animal experimentation was involved. Blood specimens were obtained with permission through a statewide regulatory program. Blood specimens came from farms that sent blood samples to the blood analysis facility at UC Davis.

Results and discussion

Characterization and optimization of aptamer-modified electrodes

Electrodes were functionalized with aptamers containing redox molecules and characterized by voltammetry. Square wave voltammetry (SWV) has emerged as the electrochemical technique of choice sensitive detection of redox events on an electrode surface.7,21 SWV was carried out in HEPES buffer serving as electrolyte solution. Based on our previous experience with DNA aptamers of similar length we chose to use 60 Hz frequency for SWV measurements.22Fig. 2A shows typical electrochemical responses of surfaces, with a reduction peak at −0.25 V (vs. Ag/AgCl) indicative of MB being present on the surface. The current was normalized and converted to signal suppression by following a simple formula – (initial peak current − final peak current)/initial peak current. The results in Fig. 2B show that signal suppression values stabilized at 10% after 30 min and remained stable for at least 2 h.
image file: c7ay01313b-f2.tif
Fig. 2 Aptasensor stability in the absence of analyte. (A) SWV curves in HEPES buffer only ((a–i) represents measurements made every 15 min). (B) Peak current from SWV curves plotted as signal suppression – ((initial current − final current)/initial current) × 100.

Detection of BoIFN-γ on aptamer-modified electrodes

Following experiments described above, we sought to detect recombinant BoIFN-γ using our aptasensor. The DNA molecules were designed to form a hairpin with double stranded DNA region obstructing the aptamer region. Presumptive mode of action for this electrochemical beacon is shown in Fig. 1, whereby binding to BoIFN-γ causes conformational switch of the hairpin and is associated with lower electron transfer (current).

Responsiveness of an aptasensor to target analyte was characterized by spiking different concentrations of BoIFN-γ into HEPES buffer and measuring electrode responses by SWV. Fig. 3A shows SWV responses for concentrations ranging from 1 ng mL−1 to 230 ng mL−1. Upon converting peak current into signal suppression, we constructed a calibration curve as shown in Fig. 3B and determined the limit of detection to be 0.1 nM. Furthermore, as highlighted by data in Fig. 3C, the aptasensor responded specifically to bovine and not human IFN-γ.


image file: c7ay01313b-f3.tif
Fig. 3 Characterizing aptasensor sensitivity and specificity to BoIFN-γ. (A) SWV responses of sensing electrodes to different concentrations of BoIFN-γ ((a–q) represents concentrations ranging from 1 ng mL−1 to 230 ng mL−1). (B) Calibration curve constructed by spiking BoIFN-γ into HEPES buffer. (C) Responses of aptasensors challenged with 100 ng mL−1 bovine IFN-γ vs. 500 ng mL−1 of human IFN-γ.

Detection of BoIFN-γ in serum and whole blood

Upon optimization in buffer, we evaluated the stability and specificity of the aptasensor in complex environment such as serum-containing media and blood. As shown in Fig. 4A, changing from HEPES buffer to RPMI caused MB peak potential to shift towards more negatives values, while addition of 10% serum to RPMI caused a decrease in current. Despite this signal loss, it was possible to detect changes in redox current due to varying concentrations of BoIFN-γ as shown in Fig. 4B.
image file: c7ay01313b-f4.tif
Fig. 4 Aptasensor responses to BoIFN-γ in complex media. (A) Aptasensor response in HEPES buffer, RPMI media and RPMI with 10% serum. (B) Response of aptasensor to BoIFN-γ spiked into RPMI with 10% serum. Calibration curve of current vs. BoIFN-γ concentration ((a–g) represents: 5, 10, 30, 50, 100, 150 and 200 ng mL−1 of BoIFN-γ). (C) Signal suppression due to BoIFN-γ spiked into (image file: c7ay01313b-u1.tif) HEPES buffer, (image file: c7ay01313b-u2.tif) RPMI/serum and (image file: c7ay01313b-u3.tif) bovine whole blood. Experiments were done in triplicates.

To evaluate possibility of using this platform for clinical diagnostics, we tested our aptasensor in whole bovine blood solution with several concentrations of spiked recombinant BoIFN-γ and signal suppression was similar to RPMI with serum (Fig. 4C). The limits of detection of IFN-γ aptasensors operating in HEPES buffer, RPMI with 10% serum and bovine blood were 2.2 ng mL−1 (0.1 nM, R2 = 0.99), 15.7 ng mL−1 (0.9 nM, R2 = 0.94) and 14 ng mL−1 (0.8 nM, R2 = 0.95) respectively.

Monitoring BoIFN-γ released by bovine PBMCs

Next we evaluated suitability of our biosensor for detecting BoIFN-γ release from bovine PBMCs. In these experiments, 200[thin space (1/6-em)]000 cells were placed into an electrochemical cell containing sensing electrodes as shown in Scheme 1. To ensure that electrodes did not become fouled due to cell attachment, a nanoporous filter was placed on top of the electrode surface prior to introduction of PBMCs.
image file: c7ay01313b-s1.tif
Scheme 1 Detection of IFN-γ release from cells. Aptasensor is covered with nanoporous filter which prevents direct contact between cells and the electrode. Cytokines produced upon stimulation of cells diffuse through the filter and bind to the electrode surface underneath.

Subsequently, PBMCs in the e-cell were stimulated with mitogen (pokeweed) to induce cytokine production and were placed into custom made incubator operating under physiological conditions.

The results of this experiment, shown in Fig. 5, highlight the fact that mitogen stimulated PBMCs, which contain 40 to 50% of T-cells, released IFN-γ. Importantly, measurements of IFN-γ release were done dynamically and did not require labeling or washing steps that are typical of immunoassays. In the absence of stimulant, electrochemical signal remained relatively stable with signal suppression not exceeding 20% over the course of a 3 h experiment. In the presence of cell stimulant, signal suppression reached a maximum of ∼50% – a value that corresponded to 50 ng mL−1 based on the calibration curve in Fig. 4C.


image file: c7ay01313b-f5.tif
Fig. 5 Detecting BoIFN-γ release from leukocytes ((image file: c7ay01313b-u4.tif) unstimulated; (image file: c7ay01313b-u5.tif) stimulated cells).

Conclusions

In this paper, we developed a new electrochemical aptasensor for detecting BoIFN-γ. The aptasensor had high specificity for the target analyte and remained functional in complex samples such as serum-containing media or whole blood. The limit of detection was 0.1 nM in pristine buffer and ∼0.9 nM in serum containing media or blood. This aptasensor is not as sensitive as commercial ELISA for BoIFN-γ; however, sensitivity may be increased further in the future by enhancements to the electrode surfaces, redox reporter or structure of DNA probe. The aptasensor described here makes the assay for BoIFN-γ release significantly simpler and obviates the need for specialized laboratory. Therefore, it is conceivable that our aptasensor may, in the future, be used for on-site screening of bovine TB and may help break with current practice of shipping bovine blood samples for testing at centralized facilities.

Acknowledgements

This work was supported the grant from California Dairy Research Foundation (CDRF). Additional funding was provided by the Ministry of Education and Science of the Republic of Kazakhstan (grant no. 4132/GF4) and National Council for Scientific and Technological Development (CNPQ-Brazil).

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