Myocardial infarction has become a leading cause of death in most industrialized nations. The prevention or treatment of acute myocardial infarction (AMI) for people has become especially urgent, and accurate diagnosis of the patients is essential coequally [1
]. It is worth mentioning that myoglobin is the main marker of early acute myocardial infarction as the concentration of myoglobin could indicate myocardial damage [2
]. Generally, the normal concentration of myoglobin in the human body ranges from 0.48 nM to 0.90 nM, which is much lower compared to that in cardiac vascular disease patients. Myoglobin is quickly released into circulation within several hours after AMI onset, and myoglobin concentration will ultimately be elevated to 4.8 μM [3
]. Therefore, sensitive and convenient detection of myoglobin is of great value. To date, methods that have been reported for measuring myoglobin include mass spectrometry [4
], surface plasmon resonance [5
], and electrochemical [6
] and colorimetric biosensors [7
]. Although most of the methods possess high sensitivity and selectivity, there still exist some limitations, such as expensive equipment and complicated operation [8
]. In order to surmount these shortcomings, scientists are making efforts to develop simple, rapid, and efficient analytic techniques.
Electrochemical sensors have the advantages of simplicity, low cost, and the possibility to be constructed as portable devices for on-site determination [9
]. Therefore, an increasing number of electrochemical sensors have been developed for myoglobin detection, including the three-dimensional reduced graphene oxide and gold composite with carbon ionic liquid electrode (3D RGO–Au/CILE) composite sensor [6
], molecularly imprinted polymer/gold on screen printed electrode (MIP/Au–SPE) devices [11
], and peptide-based electrochemical myoglobin [12
]. Among them, electrochemical aptasensors and immunosensors are much superior due to their high specificity and sensitivity resulting from high affinity between aptamers/antibodies and the corresponding targets [13
]. However, antibodies are usually costly, difficult to synthesize, and unstable in the long term. Thus, their applications in the construction of biosensors are limited. Compared with antibodies, aptamers can be synthesized chemically with ease and extreme accuracy at low cost. Furthermore, they are easy to modify and show excellent stability for long-term storage. Therefore, they have been widely used to construct biosensors recent years [15
The other important factor to improve the sensitivity of the biosensors is the material of the modified electrode. Graphene, a two-dimensional carbon crystal with only one atom thickness, has drawn much attention because of its excellent electrical conductivity [12
], high surface-to-volume ratio, and remarkable chemical stability [18
]. The unique characteristics of graphene make it an excellent material for electrochemical sensors [19
]. In particular, the combination of graphene with nanoparticles has been exploited in the development of various biosensing platforms because they have displayed synergic effects with graphene and other nanoparticles [21
]. Among the diverse nanoparticles, gold nanoparticles (AuNPs) have obtained the most extensive and in-depth study due to their unique properties including excellent biocompatibility [22
], high electrical conductivity and catalytic activity [23
], easy preparation, and good stability [26
]. Furthermore, upon introducing AuNPs into the graphene system, the aptamer will be able to bind on the electrode surface by forming a Au–S bond between thiolated aptamer and AuNPs [27
]. So, the incorporation of AuNPs with graphene will not only enhance the electron transfer and amplify the electrochemical signals, but also provide binding sites for thiolated aptamer immobilization [29
In this study, meso-tetra (4-carboxyphenyl) porphyrin functionalized graphene conjugated gold nanoparticles (TCPP–Gr/AuNPs) were synthesized via a simple wet-chemical strategy and self-assembly approach. TCPP is one of the anion porphyrins which can bind graphene through π–π stacking and improve the solubility and stability of graphene [30
]. Moreover, it can be used as catalyst to enhance electrochemical reactions [31
]. Based on the obtained nanocomposite, a sensitive electrochemical aptasensor (MbBA/TCPP–Gr/AuNPs) was constructed for myoglobin detection through myoglobin-specific binding with aptamers on the surface of the modified electrode. The aptamer with ferrocene can be regarded as an electrochemical probe for target analysis. It attached to the electrode without a target. In the presence of myoglobin, myoglobin-binding aptamer (MbBA) on the electrode surface could recognize the target by the conformational change [32
]. Therefore, ferrocene increased its distance from the electrode surface. The electron transfer rate was decreased, resulting in reduced ferrocene signal. From the changes of the peak current in the absence and presence of myoglobin, a convenient electrochemical aptasensor for myoglobin was developed with high sensitivity and specificity and a broad linear range. This method has great potential for the determination of myoglobin in clinical diagnostics.
2. Materials and Methods
2.1. Materials and Instruments
Graphite was purchased from Alfa Aesar, and TCPP from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Hydrazine hydrate solution (80 wt %) and ammonia solution (30 wt %) were obtained from the Third Chemical Reagent Factory (Tianjin, China). MCH (6-mercapto-l-hexanol), tris (2-carboxyethyl) phosphine hydrochloride (TCEP) and poly (dimethyl diallyl ammonium chloride) (PDDA, Mw = 400,000–500,000, 20 wt % in water) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) HAuCl4
was obtained from Beijing Chemical Reagent Factory (Beijing, China). Myoglobin protein (from human heart tissue) was purchased from Abcam (USA). Bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb), and carcinoembryonic antigen (CEA) were obtained from Sigma Co. (St. Louis, MO, USA). Blood samples were provided by a local hospital. The human myoglobin-binding aptamer (MbBA): 5ʹ-SH–CH2)6–CCC TCC TTT CCT TCG ACG TAG ATC TGC TGC GTT GTT CCG A–Fc-3ʹ [8
] was purchased from Shanghai Sangon Biotech Co., Ltd.,(Shanghai, China) and dissolved in T-buffer (20 mM Tri-HCl, 0.10 M NaCl, 5.0 mM MgCl2
, 10 mM TECP, pH = 7.4). All reagents were analytical pure, and water used throughout all experiments was purified with the Millipore system.
UV–vis absorption spectra were recorded on a U-3010 spectrometer (Hitachi, Ltd., Tokyo, Japan). Fourier-transformation infrared (FT-IR) spectra were recorded on a Shimadzu 8400S spectrometer (Shimadzu, Kyoto, Japan). The morphology of the nanocomposite was recorded on a JEOL-2100 TEM (Electron Ltd, Tokyo, Japan) with an accelerating voltage of 200 kV. X-ray photoelectron spectroscopy (XPS) measurement was performed on an ESCALAB-MKII 250 photoelectron spectrometer (VG Co., New York, NY, USA) with Al Kα X-ray radiation as the X-ray source for excitation. Electrochemical measurements were conducted on a CHI660C Electrochemical Workstation (Chen Hua Instruments Co., Shanghai, China) with a conventional three-electrode system composed of a bare or modified glassy carbon electrode (GCE, 3.0 mm in diameter) as the working electrode, and a platinum wire and a saturated calomel electrode (SCE) as the counter electrode and the reference electrode, respectively.
2.2. Synthesis of the TCPP–Gr and TCPP–Gr/AuNPs
The graphite oxide (GO) was synthesized from natural graphite powder by a modified Hummer′s method [36
]. TCPP–Gr were prepared as follows: 2.5 mL of 1.0 mg/mL GO was mixed with 2.5 mL 1.0 mg/mL TCPP, then 100 μL of ammonia solution and 10 μL of hydrazine solution were added. The mixture was vigorously shaken for a few minutes and heated at 60 °C for 3.5 h. The stable black dispersion was obtained. The dispersion was filtered with a nylon membrane (0.22 μm) to obtain TCPP–Gr that can be dispersed readily in water by ultrasonication.
The TCPP–Gr/AuNPs were synthesized as follows: 125 μL of PDDA was added into 4.0 mL of 0.12 mg/mL TCPP–Gr aqueous dispersion in a 15 mL reaction system, stirred with a magnetic stir bar for 15 min, then 200 μL of 10 mg/mL HAuCl4 was added into the vial. After several minutes, 2.0 mL of 20 mM fresh NaBH4 solution was added to the mixing solution at room temperature, followed by another 30 min stirring. The obtained TCPP–Gr/AuNPs nanocomposites were accumulated by centrifugation and washed two times with deionized water.
2.3. Fabrication of the Sensing Interface
GCE was polished to a mirror-like surface with 0.05 μm alumina powder before modification, and then rinsed with ethanol and re-distilled water. The cleaned GCE was dried with high-purity nitrogen gas. Then, 8.0 μL of 0.25 mg/mL TCPP–Gr/AuNPs dispersion was carefully dropped on the surface of the pretreated GCE and dried under an infrared lamp to obtain the modified TCPP–Gr/AuNPs/GCE. Subsequently, the TCPP–Gr/AuNPs/GCE was dipped in 30 μL of 2.5 μM MbBA solution including 10 mM TCEP for 4 h at 4 °C in a refrigerator [15
]. Then, the complex was immersed in 200 μL of 1.0 mM MCH for 0.50 h to block possible remaining active sites and avoid nonspecific adsorption [23
]. Thus, the aptasensor of MbBA/TCPP–Gr/AuNPs/GCE was obtained. Then, the electrode was rinsed with ultrapure water and PBS for the measurement of myoglobin.
2.4. Electrochemical Detection of Myoglobin
The well-prepared electrode was incubated in different concentration of myoglobin solutions for 45 min at room temperature. Differential pulse voltammetry (DPV) was employed to express the response for the myoglobin by measuring the peak current changes. DPV measurements were performed by scanning the potential from 0 V to 0.70 V with the amplitude of 50 mV, pulse width of 0.050 s. All the electrochemical measurements were performed in 0.10 M PBS (pH 7.4).