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Measurement of Interferon Gamma Concentration Using an Electrochemical Immunosensor Yun Wang,a,∗,c Gerald H. Mazurek,b and Evangelyn C. Alociljaa,z a Department
of Biosystems & Agricultural Engineering, Michigan State University, East Lansing, Michigan 48824, USA b Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA Interferon gamma (IFN-γ) plays an important role in immune responses in a variety of infections and diseases. The measurement of IFN-γ concentration can facilitate disease diagnosis. In this study, measurement of IFN-γ concentration using a nanoparticle-based electrochemical immunosensor is reported. IFN-γ was captured by antibody-functionalized magnetic nanoparticles (MNPs) and labeled with gold nanoparticles (AuNPs) conjugated with a second anti-IFN-γ antibody and multiple cadmium sulfide nanoparticles (CdS-NPs), thus forming the MNP-IFN-γ-Au-CdS complex. After magnetic separation, the complex was resuspended in a dilute nitric acid solution, mixed with bismuth, and added to a screen-printed carbon electrode (SPCE) chip. The electrochemical signal from the CdS-NPs was measured using square wave anodic stripping voltammetry (SWASV). Maximum current at −0.87 V was linearly associated with IFN-γ concentration over the range of 0.01 to 1 IU/ml. Measurement of IFN-γ concentration with this immunosensor required about 1 hour. These characteristics suggest that, with optimization, this immunosensor may have clinical utility. © The Author(s) 2016. Published by ECS. This is an open access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives 4.0 License (CC BY-NC-ND, http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is not changed in any way and is properly cited. For permission for commercial reuse, please email:
[email protected]. [DOI: 10.1149/2.0271605jes] All rights reserved. Manuscript submitted August 24, 2015; revised manuscript received January 7, 2016. Published January 22, 2016.
Interferon gamma (IFN-γ) is a species-specific pleiotropic cytokine that participates in the regulation of a wide array of immune and inflammatory responses including activation, growth, and differentiation of T and B lymphocytes, macrophages, endothelial cells, and fibroblasts.1,2 It is a homodimer formed by antiparallel association of two glycosylated subunits of 143 amino acids.3 It is secreted primarily by Type 1 T helper (Th1) CD4+ lymphocytes in response to recognized antigens, and to a lesser extent by CD8+ lymphocytes, NK cells, B cells, and professional antigen-presenting cells.4–6 IFN-γ production is characteristic of Th1 differentiation and cellmediated immunity. Increased IFN-γ production is associated with a variety of infectious and autoimmune diseases including tuberculosis (TB),7 leprosy,8 Johne’s disease,9 berylliosis,10 rheumatoid arthritis,11 multiple sclerosis,12 graft-versus-host disease,13 systemic lupus erythematosus,14 and various malignancies.15,16 Enzyme-linked immunosorbent assays (ELISA) have been used to measure IFN-γ concentration since its discovery in 1965.17,18 Commercially available ELISAs for measuring human IFN-γ are being used to aid in the diagnosis of Mycobacterium tuberculosis infection.19 Although ELISAs are extremely useful in clinical medicine, their ability to precisely measure cytokine concentrations is limited, especially at the low concentrations typically encountered with diagnostic assays.20 Several investigators report the detection of IFN-γ using biosensors. Stigter, et al. used a surface plasmon resonance immunosensor to measure 250 to 1000 ng/ml IFN-γ in plasma.21 Tuleuova and Revzin employed a fluorescence resonance energy transfer (FRET)-based aptamer beacon to detect IFN-γ.22 Compared to other methods, electrochemical biosensors are attractive options for measuring IFN-γ due to their sensitivity, speed, and ability to convert a biological event directly to an electronic signal.23 Bart, et al. described an electrochemical impedance immunosensor prepared by immobilizing anti-IFN-γ antibodies on a self-assembled monolayer (SAM) of acetylcysteine deposited on polycrystalline gold.24 Dijksma, et al. also reported an electrochemical impedance immunosensor.25 Min, et al. developed an aptamer-based electrochemical impedance biosensor.26 Liu, et al. reported an electrochemical biosensor which could detect IFN-γ in the range of 0.06 to 10 nM (i.e., ∼1 to 169 ng/ml).27 ∗ Electrochemical Society Member. c Present address: U.S. Food and Drug Administration, Bedford Park, Illinois 60501, USA. z E-mail:
[email protected]
Nanomaterials are attractive for biosensor design because of their ability to enhance the attachment of sensing elements and increase the signal-to-noise ratio (S/N) compared to conventional materials.23,28–30 In this paper, we describe a nanoparticle-based electrochemical immunosensor for the measurement of IFN-γ concentration. As depicted in Figure 1, this biosensor relies on: 1) capturing IFN-γ with magnetic nanoparticles (MNPs) conjugated with anti-IFN-γ antibody; 2) labeling IFN-γ with gold nanoparticles (AuNPs) conjugated with a second anti-IFN-γ antibody and multiple cadmium sulfide nanoparticles (CdS-NPs); 3) magnetic separation of the MNP-IFN-γ-Au-CdS complex from unbound CdS-NPs; 4) measurement of bound Cd using a screen-printed carbon electrode (SPCE) chip and square wave anodic stripping voltammetry (SWASV). Experimental All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless stated otherwise. Deionized water from a Millipore Direct-Q system was used to prepare solutions including phosphate buffer (0.1 M, pH 7.4), two phosphate buffered saline (PBS) buffers (0.01 M, pH 7.4 and 0.1 M, pH 7.4), tris buffer (0.1 M, pH 7.6), tris buffer with 0.01% (w/v) casein,31 PBS buffer with 0.1% (w/v) bovine serum albumin (BSA), assay buffer32 (0.562 g Na2 HPO4 , 0.125 g NaH2 PO4 , 4.383 g NaCl and 0.5 g BSA in 500 ml water), dilute nitric acid (0.8 M), and acetate buffer (0.1 M, pH 4.5). Acetate buffer with 1 mg/l bismuth was prepared by adding 5 μl of stock bismuth solution (10,000 ppm in 3% HNO from Ricca Chemical Company, Arlington, TX) to 50 ml of acetate buffer.33 Preparation of nanoparticles.—Two monoclonal antibodies that recognize different regions of human IFN-γ, and three kinds of nanoparticles were used for this biosensor. Magnetic nanoparticles (MNPs), functionalized with an anti-human IFN-γ capture antibody (cAb), were used to recover IFN-γ from samples. Gold nanoparticles (AuNPs), functionalized with an anti-human IFN-γ detection antibody (dAb), were used to label the captured IFN-γ. CdS-NPs, conjugated to the functionalized AuNPs, were used to amplify the electrochemical signal. Nanoparticles were prepared in-house as described below, and characterized by transmission electron microscopy (TEM) and absorbance spectrophotometry. MNPs were coated with polyaniline (PANI) as previously described.31,34 PANI coated MNPs, 50 to 100 nm in size, were
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Journal of The Electrochemical Society, 163 (5) B140-B145 (2016)
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Figure 1. Schematic of the immunosensor for measurement of interferon gamma (IFN-γ) concentration. Target (i.e.,IFN-γ) is captured by magnetic nanoparticles (MNPs) conjugated with capture antibodies (MNP-cAb) and isolated from the remainder of the sample by magnetic separation. Then the target is labeled with gold nanoparticles (AuNPs) conjugated with detection antibodies and cadmium sulfide nanoparticles (labeling complex, dAb-AuNP-oligo-CdS-NP conjugates). The labeled target complexes (MNP-cAb-target-dAb-AuNP-CdS-NP) are isolated from the remainder of the sample and unbound label by magnetic separation. The labeled target complexes are resuspended in an acid that dissolves CdS-NPs. The solution is transferred onto a screen-printed carbon (SPCE) chip connected to a potentiostat for electrochemical measurement.
recovered by filtration and washing with 20% methanol. PANI coated MNPs were functionalized with cAb as follows. PANI coated MNPs (2.5 mg in 150 μl of 0.1 M phosphate buffer) were mixed with cAb (0.1 mg of purified mouse anti-human IFN-γ #551221 from BD Pharmingen San Diego, CA in 100 μl) on a tube rotator for 5 min at 8 rpm. PBS buffer (25 μl of 0.1 M, pH 7.4) was added, and mixing was continued for an additional 55 minutes. Excess antibody was removed by magnetic separation. Exposed MNP surface not covered by cAb was blocked by incubation in 250 μl of tris buffer with 0.01% casein for 5 minutes and magnetic separation, which was repeated twice. cAbMNPs were resuspended in tris buffer with 0.01% casein and mixed on a tube rotator for 60 min at 8 rpm. After magnetic separation, cAb-MNPs were resuspended in 2.5 ml of 0.1 M phosphate buffer and stored at 4◦ C until used. AuNPs were synthesized from gold (III) chloride trihydrate under alkaline conditions with dextrin as described by Anderson, et al.35 AuNPs were functionalized with dAb (dAb-AuNP) by mixing 2 to 10 μg of purified mouse anti-human IFN-γ (BD Pharmingen #554549) with 1 ml of AuNPs for 30 minutes. The optimal amount of antibody to conjugate to the AuNPs was determined by the “antibody loading test” as described by Hill and Mirkin.32 The optimal amount of antibody was considered to be the maximum amount without preventing subsequent oligonucleotide binding. CdS-NPs were synthesized by mixing mercaptoacetic acid (2 μl) with 100 ml of 1 mM CdCl2 and adjusting the pH to 11 with 1 M NaOH. Oxygen-free nitrogen was bubbled through the solution for 24 hours with Na2 S (50 ml of 1.34 mM) added drop wise after the first half hour.33,36 A 3 -thiol-modified and 5 -amino-modified oligonucleotide with sequence 5 -AAA AAA AAA AAA AAA AAA AA-3 (Integrated DNA Technologies Inc., Coralville, IA) was used to attach CdS-NPs to functionalized AuNPs. The thiol group on the oligonucleotide was attached to AuNPs as described by Hill and Mirkin.32 Specifically, oligonucleotide (0.2 mM, 25 μl) was activated by the addition of 1,4-dithio-dl-threitol (DTT, 25 μl of 0.2 M) which cleaves oxidized
thiol groups. The activated oligonucleotide was purified using a Nap5 column and conjugated to 1 ml of dAb-AuNP with 3 h serial salt addition. The amine group on the oligonucleotide was then bound to the carboxylic group on the CdS-NPs as previously described.33 Specifically, the solution with oligonucleotide and conjugated dAbAuNPs was mixed with a solution containing 25 μl CdS-NPs, 5 mg of 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), 50 μl of 88 mg/ml N-hydroxysuccinimide (NHS) in dimethyl sulfoxide. The labeling conjugate (dAb-AuNP-oligo-CdS-NP) that formed was separated from unassociated CdS-NPs by centrifugation at 18,000 g at 4◦ C, washed with assay buffer followed by centrifugation (18,000 g, 4◦ C), resuspended and stored in assay buffer at 4◦ C until used. Measurement of IFN-γ concentration.—Lyophilized human IFN-γ from Cellestis Inc. (Valencia, CA) was reconstituted with deionized water to prepare a 10 IU/ml stock solution of IFN-γ. For comparison, 1 IU was considered equivalent to 40 pg of IFN-γ.7 Deionized water and serial dilutions of stock IFN-γ were used as samples to evaluate the biosensor. Interferon gamma-induced protein 10 (IP-10) was obtained from BD Pharmingen (#551130, San Diego, CA), diluted to a concentration of 100 pg/ml, and used to assess sensor specificity. IFN-γ concentration was measured by mixing 100 μl of sample, 100 μl PBS buffer, and 25 μl of MNP-cAb conjugate in a 2 ml sterile tube for 20 min on a tube rotator at 8 rpm. Surface blocking of the MNP-IFN-γ was done by adding 50 μl of PBS buffer with 0.1% BSA and mixing for 5 min. MNP-IFN-γ conjugates were separated by applying a magnetic field and then resuspended in 200 μl of assay buffer. Twenty five microliters of the labeling conjugate (dAbAuNP-oligo-CdS-NP) were added to the MNP-IFN-γ suspension, and mixed for 20 min on a tube rotator at 8 rpm. After magnetic separation, MNPs (with bound CdS-NPs when IFN-γ was present) were washed once with assay buffer and resuspended in nitric acid (10 μl of 0.8 M). The solution was incubated for 10 minutes at room temperature to release cadmium ions. After the 10 minute incubation,
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90 μl of acetate buffer with bismuth was added and 100 μl of the suspension was transferred to a screen-printed carbon electrode (SPCE) chip (Gwent Inc. England) which has a carbon working electrode and a silver/silver chloride (Ag/AgCl) counter and reference electrode. A potentiostat/galvanostat (263A, Princeton Applied Research, MA) connected to a computer with PowerSuite software (Princeton Applied Research, MA) was used for electrochemical measurements. Measurements were performed at room temperature by applying −1.2 V to the chip for a 10 min deposition, and then conducting a SWASV from −1.2 V to 0.0 V as described previously.33,37 Each sample was measured three times and the average maximum current at −0.87 V was used as the signal from this immunosensor. At least three samples of each concentration of IFN-γ were tested, and the average and standard deviation of the maximum current at −0.87 V for each concentration of IFN-γ was determined. S/N was calculated by dividing the average signal for each concentration by the average signal from deionized water. Results and Discussion Characterization of nanoparticles.—The PANI coating on MNPs facilitates the attachment of the cAb through electrostatic interaction between the negatively charged Fc fragment of the antibody and the positively charged PANI. A TEM image of PANI-coated MNPs is presented in Figure 2a. TEM images of AuNPs and CdS-NPs are shown in Figures 2b and 2c, respectively. AuNPs are spherical with an average diameter of about 16 nm. The CdS-NPs appear close to spherical and have an average diameter of about 5 nm. AuNPs showed an absorption peak at 520 nm (data not shown), which is consistent with previous studies.38,39 The absorption spectrum of CdSNPs (Figure 3, black line) shows absorption in the range of 220 nm to 500 nm with a well-defined peak at 228 nm. The absorption peak of CdS-NPs is blue-shifted compared to the absorption peak of bulk CdS (not shown) at 512 nm due to the smaller particle size of the NPs.40 The “antibody loading test” demonstrated that 10 μg of dAb was the optimal amount of the antibody to add to the 1 ml AuNPs. With smaller amounts of antibody, uncoated or poorly coated AuNPs aggregate rapidly after adding salt, and the color of the colloid changes to blue. With excess antibody, AuNPs are fully coated with antibodies and do not aggregate with the addition of salt, and the solution remains red in color. However, with an optimal amount of antibody, AuNPs form small aggregates slowly when salt is added and the color of the solution shifts toward blue to become purple.41 Three tubes of AuNPs conjugated with different concentrations of dAb (0 μg/ml, 2 μg/ml, and 10 μg/ml) are shown in Figure 4 after adding NaCl. The tube with no antibody and the tube with 2 μg/ml antibody showed extensive aggregation and turned blue. The tube with 10 μg/ml antibody turned purple and formed some aggregates slowly, confirming that this was the optimal amount. Figure 3 shows ultraviolet and visible light (UV–Vis) absorption spectra of the solution containing CdS-NPs (black line) alone and of the second solution containing the labeling conjugate (dAb-AuNPoligo-CdS-NP, red line). Free CdS-NPs were separated from the labeling complex by centrifugation as mentioned in Experimental section. Therefore, the only CdS-NPs measured in the second solution were those conjugated to AuNPs. Peak absorbance for the CdS-NPs is at 228 nm. The spectrum of the labeling conjugate, has peaks at 228 nm, 260 nm, and 280 nm. The peak at 228 nm is consistent with that expected for CdS-NPs; the peak at 260 nm is characteristic of oligonucleotides,42 and the peak at 280 nm is characteristic of proteins43 (e.g., antibody in the conjugate). The presence of these peaks verified that CdS-NPs, oligos and antibodies were attached to the AuNPs. The absorption peak of AuNPs is absent in the scanned wavelength range from 220 nm to 748 nm, which may be attributed to the changes occurring to the AuNP surfaces due to the conjugation and the resultant shift of the absorption band out of the scanned range.44 Biosensor sensitivity and specificity.—We observed a peak in current at −0.87 V in the sensorgram for samples containing IFN-γ, as
Figure 2. TEM images of: (a) polyaniline-coated magnetic nanoparticles (MNPs) (b) gold nanoparticles (AuNPs), and (c) cadmium sulfide nanoparticles (CdS-NPs).
shown in Figure 5a. This indicates that CdS-NPs were present. For the deionized water sample, no peak in current at −0.87 V was observed and the average maximum current at −0.87 V was 4.63 μA. Table I shows the average maximum current at −0.87 V and standard deviations for samples with IFN-γ concentrations ranging from 0 to 10 IU/ml. Figure 5b shows the relationship between maximum current at −0.87 V and various IFN-γ concentrations. The relationship between IFN-γ and signal was linear over a range of concentration from 0.01 to 1 IU/ml (Figure 5c), with a R2 of 0.97. The signal at 10 IU/ml was lower than predicted by our linear model. This “hook effect”, as seen with other immunological tests, is attributed to incomplete target capture due to excess target or inadequate time to reach equilibrium.45 Longer binding times or more antibody conjugated nanoparticles could extend the linear range of measurement. The signals from all concentrations were significantly greater than
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Journal of The Electrochemical Society, 163 (5) B140-B145 (2016)
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Figure 3. Ultraviolet–visible (UV–Vis) absorption spectra of a solution containing cadmium sulfide nanoparticles (CdS-NPs, black line) alone and of a second solution containing labeling complex (dAb-AuNP-oligo-CdS-NP conjugates, red line).
the signal from deionized water (all P values < 0.01). S/N ranged from 2.55 for 0.01 IU/ml to 4.19 for 1 IU/ml, as shown in Table I. The limit of detection was at or below 0.01 IU/ml (0.4 pg/ml), which is at least 5 times lower than the limit quoted for the ELISA used to detect M. tuberculosis infections.46 Measurement of IFN-γ with the immunosensor took about 1 hour, less than half the time required for similar measurements with ELISA. By using MNPs for efficient target separation, only simple magnet manipulation is needed for this immunosensor as well. Figure 6 shows differences in maximum current at −0.87 V for deionized water, and solutions containing 0.1 IU/ml (4 pg/ml) of IFNγ, or 100 pg/ml of IP-10. It demonstrates the potential for sensitive measurement of IFN-γ concentrations with a high degree of specificity. Despite having a concentration 25 times higher, the signal from IP-10 solution is less than the signal from the solution containing IFN-γ, and identical to the signal from deionized water. Our preliminary assessment suggests that the described electrochemical immunosensor may overcome some of the limitations affecting current methods for measuring IFN-γ with ELISA, such as improved detection limit and less time and cost requirement. Clinical application of the immunosensor may facilitate improvements in detecting a variety of diseases such as TB. For TB diagnosis,
Figure 4. Results of “antibody loading test” for determining optimal amount of detection antibody (dAb) for the preparation of labeling complex (dAbAuNP-oligo-CdS-NP conjugates). Each tube with gold nanoparticles (AuNPs) was conjugated with different amounts of dAb: 0, 2, or 10 μg/ml (from left to right). After adding salt (100 μl of 2 M NaCl), the two left tubes turned blue with aggregates. The tube on the right with 10 μg/ml turned purple with fewer aggregates.
Figure 5. Electrochemical measurements: (a) typical sensorgrams from square wave anodic stripping voltammetry (SWASV) for solutions containing 0 (black dots) or 0.1 IU/ml IFN-γ (red dots) with a peak in current at −0.87 V from cadmium; (b) mean and standard deviations in maximum current at −0.87 V for solutions containing 0 to 10 IU/ml IFNγ; and (c) relationship between maximum current at −0.87 V and IFN-γ concentration.
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Table I. Immunosensor signals from solutions containing 0 to 10 IU/ml IFN-γ. Concentration (IU/ml)
0
0.01
0.1
1
10
Average Maximum Current (μA) Standard Deviation (μA) Signal to noise ratio (S/N) P value
4.63 2.10 N/A N/A
11.81 2.41 2.55 8.87E-06
16.87 2.19 3.64 1.33E-07
19.42 3.96 4.19 1.06E-06
17.06 3.15 3.68 2.30E-07
Note. P value was calculated for comparison 0 IU/ml with other concentrations by a one-tailed paired t test. The critical value, P = 0.01.
0.35 IU/ml (14 pg/ml) cutoff is approved by the FDA for ELISA tests to separate positive and negative responses to combined Mtb antigens.7 For individual patients, IFN-γ level can vary from a few pg/ml or less to hundreds of pg/ml.7 Therefore, the lower detection limit of this immunosensor (0.4 pg/ml) may enhance the accuracy of diagnosis. For clinical samples, a serial dilution of the samples can be used for analysis in order to meet the linear range of the immunosensor. Additional research will evaluate this immunosensor for the measurement of IFN-γ in plasma samples and compare with IGRA results when IFN-γ concentrations are measured by ELISA and this immunosensor.
Acknowledgment This study was funded by Michigan State University (MSU) Foundation through the Strategic Partnership grant (SPG), Michigan Initiative for Innovation and Entrepreneurship (MIIE), and Targeted Support Grants for Technology Development (TSGTD). References in this manuscript to any specific commercial products, process, service, manufacturer, or company does not constitute its endorsement or recommendation by the U.S. Government or the Centers for Disease Control and Prevention (CDC). The findings and conclusions are those of the authors and do not necessarily represent the views of the U.S. Government or the CDC. References
Conclusions We describe the development of an electrochemical immunosensor for measuring IFN-γ concentrations based on nanoparticle conjugates for target separation, target labeling, and signal enhancement. We demonstrated the feasibility of the immunosensor by measuring the amount of IFN-γ in samples with known concentrations. We observed a linear relationship between the electrochemical signal (i.e., maximum current at −0.87 V) and IFN-γ concentration from 0.01 IU/ml to 1 IU/ml. Specificity was high and measurement of IFN-γ concentrations took about 1 hour. These characteristics suggest that, with optimization, this immunosensor may have clinical utility.
1. 2. 3. 4. 5. 6. 7.
8.
9. 10. 11. 12. 13.
14. 15. 16.
17. 18. 19. 20.
21. 22. 23. 24. 25. 26.
Figure 6. Assessment of immunosensor specificity. The maximum current at −0.87 V from a solution with 100 pg/ml of IP-10 was the same as the signal from the water sample (3 vs 3 μA), and smaller than the signal from a solution with 0.004 ng/ml (0.1 IU/ml) of IFN-γ (3 vs 10 μA).
27. 28. 29.
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