Electrochemical Aptasensor of Cardiac Troponin I for the Early

Sep 9, 2015 - Cardiac troponin I (cTnI) is well-known as a promising biomarker for the early diagnosis of acute myocardial infarction (AMI). In this w...
3 downloads 0 Views 1MB Size
Download PDF
Article pubs.acs.org/ac

Electrochemical Aptasensor of Cardiac Troponin I for the Early Diagnosis of Acute Myocardial Infarction Hunho Jo,†,§ Hyunwoo Gu,‡,§ Weejeong Jeon,† Hyungjun Youn,† Jin Her,† Seong-Kyeong Kim,† Jeongbong Lee,‡ Jae Ho Shin,*,‡ and Changill Ban*,† †

Department of Chemistry, Pohang University of Science and Technology, 77, Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 790-784, South Korea ‡ Department of Chemistry, Kwangwoon University, Wolgye-Dong, Nowon-Gu, Seoul 139-701, South Korea S Supporting Information *

ABSTRACT: Cardiac troponin I (cTnI) is well-known as a promising biomarker for the early diagnosis of acute myocardial infarction (AMI). In this work, single-stranded DNA aptamers against cTnI were identified by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) method. The aptamer candidates exhibited a high selectivity and sensitivity toward both cTnI and the cardiac Troponin complex. The binding affinities of each aptamer were evaluated based on their dissociation constants (Kd) by surface plasma resonance. The Tro4 aptamer that had the highest binding capacity to cTnI showed a very low Kd value (270 pM) compared with that of a cTnI antibody (20.8 nM). Furthermore, we designed a new electrochemical aptasensor based on square wave voltammetry using ferrocene-modified silica nanoparticles. The developed aptasensor demonstrated an excellent analytical performance for cTnI with a wide linear range of 1−10 000 pM in a buffer and a detection limit of 1.0 pM (24 pg/mL; S/N = 3), which was noticeably lower than the cutoff values (70−400 pg/mL). The specificity of the aptamers was also examined using nontarget proteins, demonstrating that the proposed sensor responded to only cTnI. In addition, cTnI was successfully detected in a human serum albumin solution. On the basis of the calibration curve that was constructed, the concentrations of cTnI in a solution supplemented with human serum were effectively measured. The calculated values correlated well with the actual concentrations of cTnI. It is anticipated that the highly sensitive and selective aptasensor for cTnI could be readily applicable for the accurate diagnosis of AMI.

A

immunoassay (RIA), which are both based on a selective antigen−antibody interaction.7−12 However, these antibodybased methods have several limitations such as the low stability at high temperatures, high cost for the production of antibodies, and difficulty of the chemical modification of the antibodies for biological detection. Therefore, aptamers, peptide molecules, or oligonucleic acids which can bind to specific targets have been developed as alternatives to antibodies for both therapeutic and diagnostic applications as they offer many advantages to overcome the limitations of antibodies.13−17 Such aptamerbased diagnostic tools are considered to be a new generation methodology in various fields of study. A highly sensitive detection system is an indispensable element to achieve accurate diagnosis of target diseases. Among various detection techniques, the electrochemical method has been known as one of the most common techniques because it is simple, direct, rapid, and sensitive and has been broadly

cute myocardial infarction (AMI) is one of the leading causes of death in the world, and the age- and sex-adjusted incidence rates of AMI are 200 cases per person year.1 The rapid and accurate diagnosis and prognosis of AMI has received much attention by many research groups and companies. Although various biomarkers such as the isoform of creatine kinase, lactate dehydrogenase, and myoglobin (Mb) have been identified for the diagnosis of AMI, these biomarkers showed low specificity.2−4 On the other hand, cardiac troponin I (cTnI) and the troponin complex composed of cTnI, cardiac troponin T (cTnT), and cardiac troponin C (cTnC) in the cardiac muscle tissue have been shown to be promising biomarkers for AMI, which are released into the blood circulation right after AMI.5,6 They showed a high specificity and sensitivity toward AMI, suggesting that the detection and monitoring of cTnI and troponin complex is a valuable approach for the early diagnosis of AMI. Numerous studies have been carried out to develop more sensitive and specific diagnostic methods for cTnI. In the clinical setting, the cTnI levels are commonly monitored by enzyme-linked immunosorbent assay (ELISA) and radio© XXXX American Chemical Society

Received: June 19, 2015 Accepted: September 9, 2015

A

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry utilized to quantify target proteins.18−22 In particular, electrochemical analysis based on ferrocene (Fc)-modified nanoparticles has been actively utilized because Fc is a good mediator for electron transfer reaction.23−25 Fc-modified silica nanoparticles (Fc-SiNPs) have special benefits such as high stability under harsh condition, easy modification, and high electroactivity.26 Electrochemical detection using Fc-SiNPs provides great signal amplification via electron transfer, leading to the successful quantification of target molecules. Herein, we have designed an aptamer-based detection platform for cTnI using Fc-SiNPs. First, single-stranded DNA (ssDNA) aptamers against cTnI were selected by the Systematic Evolution of Ligands by Exponential enrichment (SELEX) method,27,28 and their binding affinities were measured by surface plasmon resonance (SPR). For the electrochemical analysis of cTnI, Fc-SiNPs were synthesized and characterized via various techniques such as zeta potential measurements, transmission electron microscopy (TEM) imaging, and cyclic voltammetry (CV). This novel detection system offers great sensitivity for signals and enables accurate measurements for target molecules. Using these nanoparticles, a highly sensitive and selective label-free quantification of cTnI was successfully carried out. The proposed biosensor is rapid and accurate and can be applied to detect cTnI in human serum for the diagnosis of AMI.

binding buffer) was added to the magnetic beads. The mixture was incubated for 1 h at room temperature (RT), and then the cTnI-magnetic bead complexes were washed twice with the binding buffer to remove unbound proteins. A ssDNA library that consists of 40 random and 50 binding sequences with two primers at both 5′ and 3′ ends was incubated in the binding buffer at 90 °C for 3 min and cooled down to 4 °C. The ssDNA library was maintained on ice for 1 h to keep the thermodynamically stable structures. The ssDNA library (500 pmol) was then incubated with the cTnI-immobilized magnetic beads at RT for 1 h, and unbound ssDNAs were separated using an external magnet. The concentration of unbound ssDNAs was monitored by ultraviolet−visible spectroscopy to calculate the amount of bound ssDNAs. The bound ssDNAs were eluted with an elution buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol, 0.1% Tween-20, 5 mM MgCl2, and 300 mM imidazole), and the eluted ssDNAs were recovered by ethanol precipitation. For the next round of selection, the eluted ssDNAs were amplified by PCR using the forward primer and the biotin-modified reverse primer. The amplified double-stranded DNA (dsDNA) was incubated with the streptavidin-coated magnetic beads in a coupling buffer (5 mM Tris-HCl, pH 7.5, 1 M NaCl, 0.5 mM EDTA, and 0.0025% (v/v) Tween-20) at RT for 1 h. The magnetic beads were washed twice to remove unbound dsDNAs and incubated with 100 mM NaOH to separate ssDNAs from dsDNAs. The isolated ssDNAs were used in the next round of selection, and every round was carried out using the same procedure as described above. Measurement of Binding Affinities of Aptamer Candidates by SPR. After the 10th round of SELEX, the selected ssDNAs were amplified by PCR using unmodified primers for sequence analysis. The amplified dsDNAs were cloned using a pENTR directional TOPO cloning kit (Invitrogen). The plasmid DNAs containing the ssDNA sequences were purified utilizing a Hybrid-Q plasmid rapid prep kit (GeneAll, South Korea) and sequenced (Cosmo Genetech, South Korea). To measure the binding affinities of each aptamer candidate, the dissociation constants (Kd) were determined by SPR. The His-tagged cTnI and the cardiac Troponin complex were immobilized on a Ni2+-charged Biacore NTA chip by injecting 20 μL of 200 nM protein at a flow of 10 μL/min at RT. After washing with an eluent buffer (10 mM HEPES, pH 7.4, 150 mM NaCl, 50 μM EDTA, and 0.005% Tween-20), various concentrations of each aptamer were injected into the chip at a flow rate of 30 μL/min. Each Kd value was calculated using the BIA Evaluation software (Biacore). For the measurement of Kd for a cTnI antibody, the same procedure as described above was carried out. Synthesis of Ferrocene-Modified Silica Nanoparticles. Fumed silica (1 g) was suspended in 25 mL of toluene with stirring at 130 °C, and then 1 mL of (3-aminopropyl) trimethoxysilane (APTMS) was added. The silica mixture was refluxed with stirring for an additional 18 h. After cooling to RT, the mixture was centrifugally washed with toluene four times, and the amine-functionalized SiNPs were vacuum-dried at RT for 4 h. To immobilize the electrochemically active probes (Fc) onto the particles by an amine-carboxylic acid coupling reaction, 1 g of amine-modified SiNPs was resuspended in 30 mL of N,N-dimethylformamide (DMF) by sonication, followed by the reaction with 3 mmol of Fc-COOH, 3.2 mmol of N-hydroxybenzotriazole (HOBt), and 3.2 mmol of

■

MATERIALS AND METHODS Preparation of cTnI and Troponin Complex. The cardiac Troponin I, Troponin T, and Troponin C genes were amplified from HEK293 cDNA by polymerase chain reaction (PCR). The amplified Troponin I and Troponin C genes were inserted into pET28a vectors containing a (His)6-tag and a TEV protease site, whereas the Troponin T gene was inserted into a pET21a vector (details in the Supporting Information). To purify cTnI, the pET28a vector containing the Troponin I gene was transformed into E. coli strain BL21 (DE3) and cTnI was overexpressed. The cTnI-expressing cells were resuspended in lysis buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 0.5 mM βmercaptoethanol, and 0.1% Tween-20) and disrupted by sonication on ice. The supernatant containing soluble cTnI was applied to a Ni-NTA column (GE Healthcare) and further purified via a Superdex peptide gel filtration column (GE Healthcare). The purified cTnI was stored in a storage buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 5 mM βmercaptoethanol, and 0.1% Tween-20) with a concentration of 20 μM at −80 °C until use. For the purification of the troponin complex (cTnI-cTnT-TnC), the cTnI and cTnT plasmid genes were cotransformed into E. coli strain BL21CodonPlus (DE3)-RIG for coexpression, and the cTnC plasmid gene was transformed into BL21 (DE3). Each expressed protein was purified using the Ni-NTA column, and the three proteins were mixed together and incubated on ice for 1 h. The troponin complex was further purified by a MonoQ ion exchange column (GE Healthcare) and a Superdex 200 HL gel filtration column (GE Healthcare). The purified troponin complex (20 μM) was stored in the storage buffer as described above (details under Supporting Information). In Vitro Selection of cTnI Aptamer. For the selection of cTnI aptamer, magnetic bead-based SELEX was utilized. Dynabeads His-tag Isolation & Pulldown (Invitrogen) was washed three times with a binding buffer (20 mM Tris, pH 8.0, 300 mM NaCl, 5 mM β-mercaptoethanol, 0.1% Tween-20, and 5 mM MgCl2), and His-tagged cTnI (100 μL of 5 μM in the B

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry N,N′-diisopropylcarbodiimide (DIC) with stirring at RT for 24 h. The resulting particles were centrifugally washed four times with DMF and consecutively four times with MeOH, followed by air-drying at RT. The Fc-modified silica nanoparticles (0.3 g) and potassium tert-butoxide (KOtBu, 1 mmol) were resuspended in 30 mL of tetrahydrofuran (THF) under stirring, and the mixture was refluxed at 65 °C for 5 min. An excess amount (2 mmol) of 1,3-propane sultone was dissolved in 5 mL of THF solution and added to the above suspension. The mixture was further refluxed with stirring at 65 °C for 12 h. After cooling to RT, the particles were washed with THF four times and with MeOH four times to remove unreacted materials. The negatively charged Fc-SiNPs were dried and stored under vacuum conditions until use. Characterization of Fc-SiNPs. The surface charge of FcSiNPs was determined using a Zetatrac zeta potential analyzer (Microtrac Inc.). The particles (0.2 mg/mL) were resuspended in phosphate buffered saline (PBS) buffer, and the ζ potentials were measured. The average size of Fc-SiNPs was measured by dynamic light scattering measurements using a Zetasizer Nano series instrument (Malvern Instruments, Malvern, U.K.). The morphological characteristics of Fc-SiNPs were analyzed utilizing a TECNAI G2 20 S-Twin microscope (FEI). The samples were prepared by drying sample droplets onto a Formvar/carbon 400 mesh Cu grid and then examined under the microscope. In addition, the electrochemical property of FcSiNPs was determined using a three-electrode configuration with a CHI 760D potentiostat (CH Instrument), consisting of a disk-type gold electrode (A = 0.0314 cm2) as a working electrode, an Ag/AgCl reference electrode, and a platinum wire as a counter electrode. After ultrasonication in PBS for 5 min, the CV for electrochemically active Fc-SiNPs was performed. Quantification of cTnI Using Fc-SiNPs Based on Square Wave Voltammetry. A polyethylene terephthalate (PET) substrate (acrylic coated; Sigma-Aldrich) was placed under a mask with an appropriate aperture pattern. A rectangularly shaped working electrode (3.5 mm × 1.5 mm) was connected to a lead line (2 mm × 8 mm) with a rectangular electrical contact end (4 mm × 8 mm). To formulate a reference electrode on the masked PET, a silver chloride paste (Acheson; Hatfield, U.K.) was screen-printed using an LS-150 semiautomatic screen-printer (Newlong Seimitsu Kogyo, Japan). The substrate was cured at 130 °C for 12 min. A rectangular gold working electrode was then deposited on the PET substrate using a plasma sputter (NCVS-580S; Nuricell, Korea). Prior to the quantification of cTnI by square wave voltammetry (SWV), the affinity of cTnI-binding aptamers was estimated via CV. The 5′-thiol modified Tro4 aptamer (20 μM) was preincubated in a reaction solution (PBS, pH 7.4, 10 mM NaCl, 5 mM KCl, and 1 mM MgCl2) at 30 °C for 5 min, then dropped onto the surface of the gold working electrode, and incubated at RT for 1 h. After washing with PBS, the gold surface was treated with 20 μL of 6-mercapto-1-hexanol (1 mM in ethanol) for 20 min to block nonspecific binding. After washing with PBS, different concentrations of cTnI ranging from 10−12 to 10−8 M were applied to the working electrode for 20 min at 37 °C. Finally, unbound cTnI was washed thoroughly with PBS, and the CV measurements were performed. To generate the electrochemical signals, negatively charged FcSiNPs were used as electrochemically active probes. The particle suspension of Fc-SiNPs (2 mg/mL in PBS, pH 7.0) was prepared via ultrasonication for 30 min. All electrochemical

measurements were performed in a standard three-electrode configuration using a CHI 760D potentiostat with the gold working electrode, an Ag/AgCl reference electrode, and a platinum counter electrode. Furthermore, after the optimization of the concentration of the aptamer and Fc-SiNPs, square wave voltammograms were obtained at different concentrations of cTnI (at a scan range from 0 to +0.7 V, a frequency of 15 Hz, a step potential of 4 mV, and an amplitude of 25 mV). The oxidation peak currents (Ipa) of Fc-SiNPs were observed at 0.39 V (vs Ag/AgCl), and the calibration curve for cTnI was constructed. Verification of the Selectivity of the cTnI Detection System. To evaluate the selectivity of this system, the designed sensor was applied to various protein solutions (1 nM), such as cTnT, cTnC, human serum albumin (HSA), Mb, and B-type natriuretic peptide (BNP). The measurements were carried out as described above. In addition, the concentration of cTnI was detected in the reaction buffer containing HSA to confirm the applicability of the proposed detection system to clinical diagnosis. Furthermore, human serum (human male AB plasma, Sigma) was added to the reaction buffer, and then the selectivity of the proposed system was demonstrated.

■

RESULTS AND DISCUSSION In Vitro Selection of Specific DNA Aptamers for cTnI. To prepare the highly purified proteins, recombinant cTnI and cardiac Troponin complex were overexpressed using a bacterial expression system and purified via fast protein liquid chromatography. As shown in Figure S1, both purified cTnI and Troponin complex showed a final purity greater than 95%. The cTnI specific aptamers were selected by magnetic beadbased SELEX, which offers rapid and simple screening of aptamers. During the SELEX procedure, the percentage of binding of ssDNAs did not noticeably change until the ninth round. After negative SELEX using magnetic beads without cTnI, the binding ratio of ssDNAs abruptly increased up to 63% in the 11th round but did not increase thereafter (Figure S2). After the 11th round, ssDNAs were eluted and amplified by PCR for cloning, followed by TOPO cloning. As a result of sequence analysis, six representative sequences were selected as aptamer candidates for cTnI (Table 1). All aptamer candidates contained similar sequences as highlighted in the table, suggesting that the colored sequences played a significant role in the specific binding to cTnI. Characterization of cTnI Aptamer Candidates. The selected ssDNAs were divided into three groups (Table 1). Whereas “TTT-T” and “TCCC” sequences were found in the first group (Tro1 and Tro2), the second group (Tro3, Tro4) had a “TTT-TCA” sequence (a single stem-loop) and a conserved “CCCTC” sequence at similar distance from the “TTT-TCA” sequence. The last group (Tro5, Tro6) had a single stem-loop containing “AA-GT” and a conserved “CCTC” sequence. These results indicate that the cTnI aptamer candidates in each group have a single stem-loop and a repeated purine sequence in common. The binding affinity of aptamer candidates was evaluated as Kd values, which were measured by SPR. As represented in Table 1, overall the candidates showed great binding affinity toward cTnI. The Kd value of the Tro4 aptamer (270 pM) was the lowest among the aptamer candidates (Figure S3A). In addition, the Kd values for the Troponin complex were measured and the lowest Kd value was 3.10 nM for Tro4, as expected (Figure S3B). The secondary structure of the Tro4 C

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry Table 1. Aptamer Sequences and Dissociation Constantsa

of the antibody was 20.8 nM (Figure S3C), which was higher than those of all aptamer candidates, suggesting that the binding affinities of selected aptamers were stronger than that of the antibody. Therefore, the developed aptamers could serve as substitutes for the anti-cTnI antibody for the highly sensitive diagnosis of AMI. Design of Aptasensor for the Detection of cTnI. To enhance the performance of biosensors, it is imperative to develop a highly sensitive and label-free detection method. Various strategies such as fluorometry, electrochemical techniques, quartz crystal microbalance, and SPR spectroscopy have been utilized to achieve label-free immunosensing.29−31 Herein, we have newly designed an electrochemical and labelfree biosensor for cTnI using aptamer and Fc-SiNPs. In the proposed technique, the aptamer and Fc-SiNPs act as the detection probe for cTnI and electrochemically active probes for electron transfer, respectively. First, Fc-SiNPs were synthesized (Figure 2A). Briefly, the Fcmoieties were immobilized onto the silica surface via

a

Green and red colors indicate similar nucleotide sequences in each group.

aptamer was predicted as shown in Figure 1. It is expected that the structure of Tro4 is composed of a stem portion and a loop portion, which are key structures for the binding to cTnI. The binding strengths of selected aptamers were compared with that of a commercially available antibody against cTnI. The Kd value Figure 2. (A) Synthetic diagram of Fc-SiNP. (B) Schematic illustration of the detection of cTnI. Following the immobilization of the cTnI aptamer on the surface of the gold working electrode, cTnI is added. The concentration of cTnI was quantified based on the decrease of SWV signal.

carbodiimide coupling between ferrocenecarboxylic acid and the amine functional groups.32,33 The Fc-modified SiNPs were further modified with 1,3-propane sultone to introduce the anionic sulfonate group (−SO3−) on the SiNPs via the ringopening reaction.34 The size of newly synthesized Fc-SiNPs measured by TEM imaging was 20 ± 5 nm (Figure S4) and their surface charge was −44.1 ± 3.1 mV, indicating that the negatively charged Fc-SiNPs were homogeneously fabricated. Such highly negative surface charge makes the particles greatly stable in aqueous solutions (PBS, pH 7.0) for at least 2 days without noticeable precipitation of the particles (data not shown), eventually resulting in the excellent electron transfer propensity. Using Fc-SiNPs, cTnI was detected as illustrated in Figure 2B. In the absence of cTnI, only the aptamer-self-assembled monolayer layer allows easy access of Fc-SiNPs to the surface of the electrode, resulting in a huge current response. On the other hand, in the presence of cTnI, the specific interaction between cTnI and aptamers prevents the approach of FcSiNPs. Therefore, an oxidation peak current (Ipa) of a square

Figure 1. Secondary structure of the Tro4 aptamer as predicted by the Mfold program. Similar sequences compared with other aptamer candidates are shown in the red and green colors. D

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry wave voltammogram significantly decreases depending on the concentration of cTnI. In this work, the highly sensitive and selective detection for cTnI was conducted. Electrochemical Detection of cTnI. For the detection of cTnI, the Tro4 aptamer was chosen because of its strong binding affinity toward cTnI as well as the Troponin complex. The 5′ thiol and six additional thymine nucleotides (T6) as a spacer were introduced to the Tro4 aptamer to immobilize the aptamer on the gold surface and diminish the hindrance effect. The modified aptamer was attached directly on the surface of the working electrode and 6-mercapto-1-hexanol was also utilized to prevent nonspecific binding by nontarget proteins. The surface of the sensor was analyzed via X-ray photoelectron spectroscopy (Theta Probe AR-XPS System, Thermo Fisher Scientific, U.K.). After the treatment of the aptamer, a N 1s peak was observed, implying that the Tro4 aptamer was wellimmobilized on the working electrode (Figure S5). Before constructing the calibration curve for cTnI, the designed aptasensor was characterized by CV. As exhibited in Figure S6A, distinct anodic and cathodic potential peaks were observed in the absence of cTnI, revealing that synthesized FcSiNPs present typical electrochemical behaviors of ferrocene such as one-electron and chemically reversible reaction. Furthermore, to confirm the validity of the proposed sensor for the quantification of cTnI, various concentrations of cTnI were applied to this system and incubated with the working electrode in the binding buffer (PBS, pH 7.4, 10 mM NaCl, 5 mM KCl, and 1 mM MgCl2) and then cyclic voltammograms were obtained (Figure S6B). The calibration curves of cTnI were constructed by monitoring both the oxidation and reduction peak currents (Ipa and Ipc, respectively) (Figure S6C,D). Although both calibration plots did not display a perfect linear relationship between currents and cTnI concentrations (insets in Figure S6C,D), the signal deviation of the calibration curve based on Ipa was smaller than that of the Ipc curve, implying that the oxidation current of this system was more reliable. Furthermore, the quantification of cTnI was accomplished precisely by SWV. Prior to the quantification, the concentrations of the aptamer and Fc-SiNPs were optimized (Figure S7). The concentrations of the aptamer and nanoparticles did not cause significant fluctuations in the currents, indicating that same concentrations with CV (20 μM for aptamer and 2 mg/ mL for Fc-SiNPs) are appropriate for this work. The oxidation peak currents (Ipa) significantly decreased in proportion to the concentration of cTnI (Figure 3). The developed aptasensor in this work exhibited excellent analytical performance with a detection limit of 1.0 pM (24 pg/mL; S/N = 3) and a wide linear range of 1−10 000 pM (corresponding to 0.024−240 ng/ mL; 4 orders of magnitude, slope = 0.23 μA/decade, r2 = 0.9901). Although the cutoff levels of cTnI vary according to the patients and detection methods, it is known that the 99th percentile and clinical cutoff level for cTnI are 70 and 400 pg/ mL, respectively.35,36 Also, this detection limit is much lower than other values reported in the previous work such as ELISA and RIA (350 pg/mL to 1.7 ng/mL).7−10 Therefore, this aptasensor is sufficiently sensitive to be utilized for the detection of cTnI. Verification of the Specificity of the Aptasensor. Diagnostic sensors must possess high specificity for target molecules compared to other interfering materials. Therefore, the specificity of the designed sensor was evaluated using nonspecific binding proteins, including cTnT, cTnC, HSA, Mb,

Figure 3. Electrochemical detection of cTnI in the buffer. Square wave voltammograms at various concentrations of cTnI are shown (a) 10−12 M, (b) 10−11 M, (c) 10−10 M, (d) 10−9 M, (e) 10−8 M). The inset represents the calibration curve. All experiments were performed in triplicate, and the error bars were indicated on the curve.

and BNP (1 nM). Although there are some signal fluctuations (Figure 4), there was no significant alteration of the current

Figure 4. Specificity test of the developed aptasensor in the buffer. Each protein was treated to the aptasensor at a concentration of 1 nM (cTnT, cardiac troponin T; cTnC, cardiac troponin C; HSA, human serum albumin; Mb, myoglobin; BNP, B-type natriuretic peptide). The “blank” represents the signal of the aptasensor in the absence of cTnI. All experiments were performed in triplicate, and each error bar was evaluated.

signals in the presence of each protein, compared to the blank signal of the aptasensor. However, the cTnI protein produces a noticeable reduction of the signal. These results confirm that the developed aptasensor has a high specificity toward cTnI. In addition, the specificity of this system was further demonstrated via electrochemical impedimetry (details under the Supporting Information). Aptamer-immobilized sensor exhibited relatively high sensitivity toward cTnI in both buffer condition and Fetal Bovine Serum-supplemented condition and also high selectivity E

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

expected that the highly sensitive, selective, and label-free cTnI aptasensor is readily applicable for the diagnosis of AMI.

for only cTnI (Figure S8). These results also verify the specificity of the developed aptasensor. Demonstration of the Applicability of the Aptasensor for Clinical Diagnosis. For the clinical application of the proposed sensor to the early diagnosis of AMI, the sensitivity of this system in a biochemical environment similar to blood must be validated. First, the detection of cTnI was carried out in a HSA solution. It is known that the concentration of HSA similar to human serum environments is approximately 6 × 10−4 M. Therefore, the aptamer-functionalized gold electrode was incubated with various concentrations of cTnI (10, 1.0, 0.1, and 0.01 nM; corresponding to 240, 24, 2.4, and 0.24 ng/mL, respectively) in the reaction solution pretreated with 6 × 10−4 M of HSA (PBS, pH 7.0) and then the calibration curve was constructed (Figure 5). Only two data points (0.24 and 240

■

CONCLUSIONS In the present study, highly sensitive and selective ssDNA aptamers against cTnI, a diagnostic biomarker for AMI, were developed through SELEX. The aptamers were selectively bound to both cTnI and the cardiac Troponin complex, and their dissociation constants were lower than that of an anticTnI antibody. In addition, to our knowledge for the first time, we designed the aptamer-based sensor for cTnI using Fc-SiNPs. The convergence of high sensitivity of the aptamer and great electrochemical activity of Fc-SiNPs allowed for the effective determination of cTnI. As a result, cTnI was detected at a concentration of 1.0 pM (24 pg/mL) in a buffer condition. This detection limit is low enough in comparison with the cutoff value of cTnI for AMI. Furthermore, the test of the specificity of the aptasensor clearly showed that the proposed sensor identified only cTnI. Moreover, cTnI was successfully detected in HSA solution and human serum solution with the great detection limit. To employ this system for the clinical diagnosis of AMI, we have made an attempt at surmounting several difficulties, such as a disturbance of many blood cells, the instability of DNA aptamers in the blood, and a miniaturization of this system for a detection kit. It is anticipated that the highly sensitive and selective biosensor using target-specific aptamer will become a novel diagnostic platform for not only AMI but also other diseases through further study.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b02312.

Figure 5. Determination of the concentrations of cTnI in the solution containing human serum. The calibration curve was constructed using the solution supplemented with HSA. Various concentrations of cTnI ranging from 0.24 to 240 ng/mL (corresponding to 10−11 and 10−8 M) were added into the human serum solution, and each current was measured. The concentrations of cTnI were calculated from the current values using the calibration curve. The dashed lines indicate the original concentration of cTnI added to the serum solution. The error bars represent the standard deviations of triplicate measurements.

Additional experimental details (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +82-54-940-5627. *E-mail: [email protected]. Phone: +82-54-279-2127. Fax: +82-54-279-5840.

ng/mL) are noted in the figure. Although the slope is slightly smaller than that of Figure 3 (0.21 vs 0.23 μA/decade), there is a good linear relationship between the decrease of the Ipa values and the increase of the concentration of cTnI. Furthermore, the commercially available human serum (from human male AB plasma, sterile-filtered) was applied to this system. The serum was diluted 10-fold with PBS (pH 7.0), and several serum samples were prepared by adding various concentrations of cTnI ranging from 1.2 (sample A) to 120 ng/mL (sample G). Each measurement was performed in triplicate, and then the averaged cTnI concentrations were determined from the calibration curve (Table S1). The data were evaluated using SigmaPlot (Systat Software; San Jose, CA) and presented as the means ± SD (P ≤ 0.001). As represented in Figure 5, the calculated concentrations of cTnI showed a good correlation with the actual concentrations (dashed lines) and the detection limit was considerably lower than the cutoff values of cTnI as mentioned previously. It is

Author Contributions §

H.J. and H.G. contributed equally to this work. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the National Research Foundation of Korea (Grants NRF-2015027587 and NRF-2014029301) and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number HI12C1852). In addition, this work has been also conducted by the Grant of Kwangwoon University (2014) to J.H.S. F

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

■

(34) Choi, N. S.; Lee, Y. M.; Lee, B. H.; Lee, J. A.; Park, J. K. Solid State Ionics 2004, 167, 293−299. (35) Altinier, S.; Mion, M.; Cappelletti, A.; Zaninotto, M.; Plebani, M. Clin. Chem. 2000, 46, 991−993. (36) Chapelle, J. P.; Aldenhoff, M. C.; Pierard, L.; Gielen, J. Clin. Chem. 2000, 46, 1864−1866.

REFERENCES

(1) O’Gara, P. T.; Kushner, F. G.; Ascheim, D. D.; Casey, D. E., Jr.; Chung, M. K.; de Lemos, J. A.; Ettinger, S. M.; Fang, J. C.; Fesmire, F. M.; Franklin, B. A.; Granger, C. B.; Krumholz, H. M.; Linderbaum, J. A.; Morrow, D. A.; Newby, L. K.; Ornato, J. P.; Ou, N.; Radford, M. J.; Tamis-Holland, J. E.; Tommaso, C. L.; Tracy, C. M.; Woo, Y. J.; Zhao, D. X.; Anderson, J. L.; Jacobs, A. K.; Halperin, J. L.; Albert, N. M.; Brindis, R. G.; Creager, M. A.; DeMets, D.; Guyton, R. A.; Hochman, J. S.; Kovacs, R. J.; Kushner, F. G.; Ohman, E. M.; Stevenson, W. G.; Yancy, C. W. Circulation 2013, 127, e362−e425. (2) Guzy, P. M. West. J. Med. 1977, 127, 455−460. (3) Rotenberg, Z.; Weinberger, I.; Sagie, A.; Fuchs, J.; Sperling, O.; Agmon, J. Clin. Chem. 1987, 33, 1419−1420. (4) McComb, J. M.; McMaster, E. A.; MacKenzie, G.; Adgey, A. A. Heart 1984, 51, 189−194. (5) Davies, K. R.; Gelb, A. W.; Manninen, P. H.; Boughner, D. R.; Bisnaire, D. Br. J. Anaesth. 1991, 67, 58−63. (6) Korff, S.; Katus, H. A.; Giannitsis, E. Heart 2006, 92, 987−993. (7) Cummins, B.; Auckland, M. L.; Cummins, P. Am. Heart J. 1987, 113, 1333−1344. (8) Bodor, G. S.; Porter, S.; Landt, Y.; Ladenson, J. H. Clin. Chem. 1992, 38, 2203−2214. (9) Zaninotto, M.; Altinier, S.; Lachin, M.; Carraro, P.; Plebani, M. Clin. Chem. 1996, 42, 1460−1466. (10) Apple, F. S.; Falahati, A.; Paulsen, P. R.; Miller, E. A.; Sharkey, S. W. Clin. Chem. 1997, 43, 2047−2051. (11) Kong, T.; Su, R.; Zhang, B.; Zhang, Q.; Cheng, G. Biosens. Bioelectron. 2012, 34, 267−272. (12) Guo, Q.; Kong, T.; Su, R.; Zhang, Q.; Cheng, G. Appl. Phys. Lett. 2012, 101, 093704. (13) Jayasena, S. D. Clin. Chem. 1999, 45, 1628−1650. (14) Centi, S.; Tombelli, S.; Minunni, M.; Mascini, M. Anal. Chem. 2007, 79, 1466−1473. (15) Medley, C. D.; Smith, J. E.; Tang, Z.; Wu, Y.; Bamrungsap, S.; Tan, W. Anal. Chem. 2008, 80, 1067−1072. (16) Maehashi, K.; Katsura, T.; Kerman, K.; Takamura, Y.; Matsumoto, K.; Tamiya, E. Anal. Chem. 2007, 79, 782−787. (17) Song, K. M.; Cho, M.; Jo, H.; Min, K.; Jeon, S. H.; Kim, T.; Han, M. S.; Ku, J. K.; Ban, C. Anal. Biochem. 2011, 415, 175−181. (18) Drummond, T. G.; Hill, M. G.; Barton, J. K. Nat. Biotechnol. 2003, 21, 1192−1199. (19) Rodriguez, M. C.; Kawde, A. N.; Wang, J. Chem. Commun. 2005, 4267−4269. (20) Min, K.; Cho, M.; Han, S. Y.; Shim, Y. B.; Ku, J.; Ban, C. Biosens. Bioelectron. 2008, 23, 1819−1824. (21) Ahammad, A. J. S.; Choi, Y. H.; Koh, K.; Kim, J. H.; Lee, J. J.; Lee, M. Int. J. Electrochem. Sci. 2011, 6, 1906−1916. (22) Darain, F.; Park, D. S.; Park, J. S.; Shim, Y. B. Biosens. Bioelectron. 2004, 19, 1245−1252. (23) Takahashi, S.; Anzai, J. Materials 2013, 6, 5742−5762. (24) Wang, J.; Zhu, X.; Tu, Q.; Guo, Q.; Zarui, C. S.; Momand, J.; Sun, X. Z.; Zhou, F. Anal. Chem. 2008, 80, 769−774. (25) Zhuo, Y.; Chai, Y. Q.; Yuan, R.; Mao, L.; Yuan, Y. L.; Han, J. Biosens. Bioelectron. 2011, 26, 3838−3844. (26) Qiu, J. D.; Guo, J.; Liang, R. P.; Xiong, M. Electroanalysis 2007, 19, 2335−2341. (27) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818−822. (28) Tuerk, C.; Gold, L. Science 1990, 249, 505−510. (29) Lee, J. O.; So, H. M.; Jeon, E. K.; Chang, H.; Won, K.; Kim, Y. H. Anal. Bioanal. Chem. 2008, 390, 1023−1032. (30) Cho, E. J.; Lee, J. W.; Ellington, A. D. Annu. Rev. Anal. Chem. 2009, 2, 241−264. (31) Iliuk, A. B.; Hu, L.; Tao, W. A. Anal. Chem. 2011, 83, 4440− 4452. (32) Budny, A.; Novak, F.; Plumere, N.; Schetter, B.; Speiser, B.; Straub, D.; Mayer, H. A.; Reginek, M. Langmuir 2006, 22, 10605− 10611. (33) Miklan, Z.; Szabo, R.; Zsoldos-Mady, V.; Remenyi, J.; Banoczi, Z.; Hudecz, F. Biopolymers 2007, 88, 108−114. G

DOI: 10.1021/acs.analchem.5b02312 Anal. Chem. XXXX, XXX, XXX−XXX