sensors Article

Electrochemical Aptasensor for Myoglobin-Specific Recognition Based on Porphyrin Functionalized Graphene-Conjugated Gold Nanocomposites Guojuan Zhang 1,2 , Zhiguang Liu 1 , Li Wang 1 and Yujing Guo 1, * 1 2

*

Institute of Environmental Science, Shanxi University, Taiyuan 030006, China; [email protected] (G.Z.); [email protected] (Z.L.); [email protected] (L.W.) College of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China Correspondence: [email protected]; Tel.: +86-138-3422-9215

Academic Editor: Huangxian Ju Received: 9 September 2016; Accepted: 22 October 2016; Published: 28 October 2016

Abstract: In this work, a novel electrochemical aptasensor was developed for sensitive and selective detection of myoglobin based on meso-tetra (4-carboxyphenyl) porphyrin-functionalized graphene-conjugated gold nanoparticles (TCPP–Gr/AuNPs). Due to its good electric conductivity, large specific surface area, and excellent mechanical properties, TCPP–Gr/AuNPs can act as an enhanced material for the electrochemical detection of myoglobin. Meanwhile, it provides an effective matrix for immobilizing myoglobin-binding aptamer (MbBA). The electrochemical aptasensor has a sensitive response to myoglobin in a linear range from 2.0 × 10−11 M to 7.7 × 10−7 M with a detection limit of 6.7 × 10−12 M (S/N = 3). Furthermore, the method has the merits of high sensitivity, low price, and high specificity. Our work will supply new horizons for the diagnostic applications of graphene-based materials in biomedicine and biosensors. Keywords: graphene; TCPP; AuNPs; myoglobin

1. Introduction 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 [9,10]. 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

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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 [13,14]. 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 [12,16,17], high surface-to-volume ratio, and remarkable chemical stability [18]. The unique characteristics of graphene make it an excellent material for electrochemical sensors [19,20]. 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 [23–25], 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 [27,28]. 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 [32–34]. 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.

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The human myoglobin-binding aptamer (MbBA): 50 -SH–CH2)6–CCC TCC TTT CCT TCG ACG TAG ATC TGC TGC GTT GTT CCG A–Fc-30 [8,35] 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 Hummer0 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).

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3. Results and Discussion 3.1. The Principle of the Aptasensor The procedure for the electrochemical sensing platform was shown in Scheme 1. Firstly, TCPP–Gr Sensors 2016, 16, 1803 4 of 12 was prepared by a simple wet-chemical reaction under alkaline conditions. TCPP, a negatively charged water-soluble molecule with a large planar aromatic surface, could serve asserve a stabilizer for graphene charged water-soluble molecule with a large planar aromatic surface, could as a stabilizer for through π–π stacking interactions. Then, AuNPs wereAuNPs incorporated into TCPP–Gr fresh NaBH graphene through π–π stacking interactions. Then, were incorporated into by TCPP–Gr by 4 reduction and 4the MbBAand wasthe modified on the TCPP–Gr/AuNPs/GCE surface by Au–Sbybond fresh NaBH reduction MbBA was modified on the TCPP–Gr/AuNPs/GCE surface Au–S[37]. Thebond end of theThe MbBA with thiolwith wasthe combined AuNPs, and the other of theend MbBA with [37]. end of thethe MbBA thiol waswith combined with AuNPs, and end the other of the MbBA was withattached ferroceneto was to the electrode surface.served Ferrocene served as anfor indicator for the of ferrocene theattached electrode surface. Ferrocene as an indicator the detection detectionIn ofthe myoglobin. In myoglobin, the absence ferrocene of myoglobin, ferrocene exhibited stronger in of myoglobin. absence of exhibited stronger signal. Whilesignal. in the While presence the presence of myoglobin, the MbBA on the electrode surface recognized the target specifically and myoglobin, the MbBA on the electrode surface recognized the target specifically and the conformation the conformation of MbBA changed; simultaneously, ferrocene far away from electrode. As of MbBA changed; simultaneously, ferrocene was far away from was the electrode. As athe result, the electron a result, the electron transfer rate was hindered, leading to the decrease of ferrocene signal [32–34]. transfer rate was hindered, leading to the decrease of ferrocene signal [32–34]. The more myoglobin The more myoglobin captured onto the electrode, the lower the peak current obtained will be. The captured onto the electrode, the lower the peak current obtained will be. The decreased peak current decreased peak current is associated with the concentration of myoglobin. Consequently, an is associated with the concentration of myoglobin. Consequently, an electrochemical aptasensor for electrochemical aptasensor for myoglobin detection has been constructed. myoglobin detection has been constructed. HAuCl4 N2H4 , NH3.H2O

NaBH4

GCE

TCPP

AuNPs

MbBA

MCH

myoglobin

Scheme 1. The procedure for the synthesis of meso-tetra (4-carboxyphenyl) porphyrin-functionalized Scheme 1. The procedure for the synthesis of meso-tetra (4-carboxyphenyl) porphyrin-functionalized graphene-conjugated gold nanoparticles (TCPP–Gr/AuNPs) nanocomposites and electrochemical graphene-conjugated gold nanoparticles (TCPP–Gr/AuNPs) nanocomposites and electrochemical aptasensor forfor myoglobin detection. aptasensor myoglobin detection.

3.2. 3.2. Characterization of of thethe TCPP–Gr/AuNPs Characterization TCPP–Gr/AuNPs TCPP–Gr/AuNPs characterized UV–vis spectroscopy. As shown in Figure TCPP–Gr/AuNPswere were firstly firstly characterized byby UV–vis spectroscopy. As shown in Figure 1, GO 1, absorption at 230 nmnm andand a shoulder at 290–300 nm,nm, GOdispersion dispersion(curve (curvea)a)exhibits exhibitsa amaximum maximum absorption at 230 a shoulder at 290–300 corresponding π–π* transitionofofaromatic aromatic C–C bonds of of thethe C=O corresponding to to thethe π–π* transition bonds and andthe then–π* n–π*transition transition C=O bonds [38], respectively. TCPP (curve b) displays a narrow absorption at 414 nm which is the typical bonds [38], respectively. TCPP (curve b) displays a narrow absorption at 414 nm which is the typical characteristic peak owingtotothe theSoret Soret band band of of TCPP TCPP and from 500500 nmnm to to characteristic peak owing and aaseries seriesofofweak weakpeaks peaks from which attributedtotothe theQ-bands Q-bands of of TCPP. TCPP. After hydrazine to form 700 700 nmnm which areare attributed After GO GOwas wasreduced reducedbyby hydrazine to form TCPP–Gr, the absorption peak of GO shifted from 230 to 269 nm and a peak at 448 nm is observed, TCPP–Gr, the absorption peak of GO shifted from 230 to 269 nm and a peak at 448 nm is observed, which corresponds to the Soret band of TCPP with a large bathochromic shift (34 nm). This illustrated which corresponds to the Soret band of TCPP with a large bathochromic shift (34 nm). This illustrated that there were strong π–π interactions between graphene and TCPP [30]. After decorating AuNPs that there were strong π–π interactions between graphene and TCPP [30]. After decorating AuNPs on the surface of TCPP–Gr, a new strong peak appeared at 530 nm, which was consistent with the on the surface of TCPP–Gr, a new strong This peakdemonstrated appeared atthat 530 TCPP–Gr/AuNPs nm, which was were consistent with the surface plasmon of absorption of AuNPs. successfully surface plasmon of absorption of AuNPs. This demonstrated that TCPP–Gr/AuNPs were successfully synthesized (curve d). synthesized (curve d).

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Figure 1. UV–vis spectra of graphite oxide (GO) suspension (a); TCPP (b); TCPP–Gr suspension (c); Figure Figure1.1. UV–vis UV–visspectra spectraof ofgraphite graphiteoxide oxide(GO) (GO) suspension suspension (a); (a); TCPP TCPP (b); (b); TCPP–Gr TCPP–Gr suspension suspension (c); (c); and TCPP–Gr/AuNPs TCPP–Gr/AuNPs suspension (d). and and TCPP–Gr/AuNPs suspension (d).

FT-IR spectra (Figure 2) of TCPP–Gr (a) and TCPP (b) also provide an obvious verification for FT-IR spectra spectra (Figure (Figure 2) 2) of of TCPP–Gr TCPP–Gr (a) (a) and and TCPP TCPP (b) (b) also also provide provide an an obvious obvious verification verification for for FT-IR −1 the successful synthesis of TCPP–Gr hybrids. The peak of 1701 cm in TCPP is attributed to νν (C=O) −1 − 1 the successful synthesis of TCPP–Gr hybrids. The peak of 1701 cm in TCPP is attributed to (C=O) the successful synthesis of TCPP–Gr hybrids. The peak of 1701 cm −1 in TCPP is attributed to ν (C=O) vibrations of –COOH, whereas in TCPP–Gr itit moved to 1572 cm due to stacking vibrations of of –COOH, –COOH, whereas whereas in in TCPP–Gr TCPP–Gr it movedto to1572 1572cm cm−−11 which which to the the π–π π–π stacking stacking vibrations moved which due due to the π–π interactions between TCPP and graphene [30]. The spectrum of TCPP–Gr also demonstrated a interactionsbetween betweenTCPP TCPPand and graphene spectrum of TCPP–Gr also demonstrated interactions graphene [30].[30]. TheThe spectrum of TCPP–Gr also demonstrated a broad,a −1 broad, band at 3422 cm corresponded to O–H vibrations of the O–H −1 which broad, intense intense cm−1 which which corresponded tostretching O–H stretching stretching vibrations thegroup O–H group group intense band atband 3422 at cm3422 corresponded to O–H vibrations of the of O–H from from carboxyl as well as remaining water. In brief, FT-IR spectra illustrated that TCPP molecules are from carboxyl as well as remaining water. In brief, FT-IR spectra illustrated that TCPP molecules are carboxyl as well as remaining water. In brief, FT-IR spectra illustrated that TCPP molecules are attached attached to the surface of on graphene. attached to the of on graphene. to the surface ofsurface on graphene.

Transmittance/ /a.u. a.u. Transmittance

aa 661.69 661.69

1076.61 1076.61 1407.20 1407.201571.75 1571.75

bb 3422.47 3422.47

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Figure 2. The Fourier-transformation infrared (FT-IR) spectra spectra of TCPP–Gr TCPP–Gr (a) and and TCPP (b). (b). Figure Figure 2. 2. The The Fourier-transformation Fourier-transformation infrared infrared (FT-IR) (FT-IR) spectraof of TCPP–Gr(a) (a) andTCPP TCPP (b).

TEM carried out to characterize the morphology of TCPP–Gr/AuNPs nanocomposites. As TEM was nanocomposites. As TEM was carried out out to to characterize characterizethe themorphology morphologyofofTCPP–Gr/AuNPs TCPP–Gr/AuNPs nanocomposites. displayed in Figure 3, AuNPs evenly scattered on the surface of TCPP–Gr. The mean size of the displayed in Figure 3, AuNPs evenly scattered on the surface of TCPP–Gr. The mean size of the As displayed in Figure 3, AuNPs evenly scattered on the surface of TCPP–Gr. The of the AuNPs was estimated to be 18 nm. AuNPswas wasestimated estimatedto tobe be18 18nm. nm. AuNPs

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50 nm 50 nm Figure TheTEM TEMimage image of of TCPP–Gr/AuNPs. Figure 3.3.The TCPP–Gr/AuNPs. Figure 3. The TEM image of TCPP–Gr/AuNPs.

The constituent and oxidation states of TCPP–Gr/AuNPs nanocomposites were further analyzed The constituent and oxidation of TCPP–Gr/AuNPs nanocomposites were further analyzed by XPS, and results are shown instates Figure Compared to the graphene nanosheets, the survey of The constituent and oxidation states of4. TCPP–Gr/AuNPs nanocomposites were further analyzed by XPS, andand results areare shown ininFigure to the graphenenanosheets, nanosheets, the survey TCPP–Gr/AuNPs (Figure 4A) showed the 4. presence of Auto and Ngraphene originating from AuNPs TCPP, by XPS, results shown Figure 4. Compared Compared the theand survey of of TCPP–Gr/AuNPs (Figure 4A) the presence Au and from AuNPs TCPP, respectively. Figure 4B shows Au 4f7/2 and 4f5/2 peaks appeared atoriginating 84.4 eV and 87.8 eV, respectively. TCPP–Gr/AuNPs (Figure 4A)showed showed the Au presence ofofAu and NN originating from AuNPs and and TCPP, Figure 4C showed N 1s spectrum appeared at 402.5 eV, which originated from TCPP [39]. The C 1s respectively. Figure 4B 4B shows Au 4f4f Au 4f 4f5/2 peaks appeared at 84.4 eV and eV, respectively. respectively. Figure shows Au 7/2 and and Au peaks appeared at 84.4 eV and 87.887.8 eV, respectively. 7/2 5/2 spectrum of TCPP–Gr/AuNPs has five kindsat carbon (284.6from eV), C–O (286.7 eV), C=O Figure showed spectrum appeared eV, which originated TCPP [39].[39]. The The C 1sC 1s Figure 4C 4C showed NN 1s1s spectrum appeared atof402.5 402.5 eV,bonds: whichC–C originated from TCPP (287.7 eV), C=N (285.7 eV), andhas O–C=O (288.7 (Figure 4D) [40,41]. All(284.6 the C–O above illustrated the eV), spectrum TCPP–Gr/AuNPs five ofeV) carbon bonds: C–C (284.6 eV), (286.7 eV), C=O spectrum of of TCPP–Gr/AuNPs has fivekinds kinds of carbon bonds: C–C eV), C–O (286.7 formation of TCPP–Gr/AuNPs. eV), C=N (285.7 eV),eV), andand O–C=O (288.7(288.7 eV) (Figure 4D) [40,41]. All theAll above the C=O(287.7 (287.7 eV), C=N (285.7 O–C=O eV) (Figure 4D) [40,41]. the illustrated above illustrated formation of TCPP–Gr/AuNPs. the formation of TCPP–Gr/AuNPs.

7.5

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Binding Energy (ev) Binding Energy (ev) Figure 4. The X-ray photoelectron spectroscopy (XPS) spectra of TCPP–Gr/AuNPs (A); Au 4f (B); N 1s4.(C); CX-ray 1s photoelectron (D). Figure 4. and The photoelectronspectroscopy spectroscopy (XPS) (XPS) spectra (A);(A); Au 4f Figure The X-ray spectraofofTCPP–Gr/AuNPs TCPP–Gr/AuNPs Au(B); 4f (B); N 1s (C); and C 1s (D). N 1s (C); and C 1s (D).

3.3. Characterization of the Aptasensor 3.3. Characterization of the Aptasensor DPV was employed to characterize the aptasensor. As can be seen from Figure 5A, there was 3.3. Characterization of the Aptasensor no redox at the bare (line a) the andaptasensor. TCPP–Gr/AuNPs/GCE (linefrom b) inFigure the potential range. DPV peak was employed to GCE characterize As can be seen 5A, there was DPV was employed to characterize As can be(line seenb)from Figure 5A, range. there was no redox peak at the bare GCE (line a) the and aptasensor. TCPP–Gr/AuNPs/GCE in the potential

no redox peak at the bare GCE (line a) and TCPP–Gr/AuNPs/GCE (line b) in the potential range.

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After modified a well-defined oxidation peak After modifiedMbBA MbBAwas wason onthe thesurface surfaceof ofthe theTCPP–Gr/AuNPs/GCE, TCPP–Gr/AuNPs/GCE, a well-defined oxidation peak appeared at 0.33 V, which attribute to the ferrocene modified on the end of MbBA. The result indicated appeared at 0.33 V, which attribute to the ferrocene modified on the end of MbBA. The result indicated that ofMbBA/TCPP–Gr/AuNPs/GCE MbBA/TCPP–Gr/AuNPs/GCE was successfully constructed. thatthe thesensing sensing interface interface of (line c)(line wasc) successfully constructed. After −7 M After 10− M of myoglobin was added into the above buffer solution and incubated for 45 min, 4.404.40 × 10× of7 myoglobin was added into the above buffer solution and incubated for 45 min, the peak current obviously decreased (line d) d) duedue to the impediment of electron transfer by myoglobin. the peak current obviously decreased (line to the impediment of electron transfer by myoglobin. Potential / V

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Concentration / μM

Figure 5. (A) Differential voltammetry of the bare electrode a), TCPP–Gr/ Figure 5. (A) Differential pulsepulse voltammetry (DPV)(DPV) of the bare electrode (curve a), (curve TCPP–Gr/AuNPs/GCE AuNPs/GCE (curve b), MbBA–Fc/TCPP–Gr/AuNPs/GCE (curve c), and MbBA–Fc/TCPP–Gr/AuNPs/ (curve b), MbBA–Fc/TCPP–Gr/AuNPs/GCE (curve c), and MbBA–Fc/TCPP–Gr/AuNPs/GCE in −7 M myoglobin solution (curve d); (B) The relationship between the peak current and in−4.4 × 10 7 M 4.4GCE × 10 myoglobin solution (curve d); (B) The relationship between the peak current and the incubated time; (C) The effect of the concentration of TCPP–Gr/AuNPs on the peak current; (D) The the incubated time; (C) The effect of the concentration of TCPP–Gr/AuNPs on the peak current; effect of the concentration of MbBA on the peak current (Amplitude: 50 mV, pulse width: 0.050 s). (D) The effect of the concentration of MbBA on the peak current (Amplitude: 50 mV, pulse width: 0.050 s).

3.4. Optimization of Experimental Conditions 3.4. Optimization of Experimental Conditions 3.4.1. Influence of the Incubation Time 3.4.1. Influence of the Incubation Time Myoglobin should be captured on the surface of MbBA/TCPP–Gr/AuNPs/GCE by specific Myoglobin should be captured on the surface of MbBA/TCPP–Gr/AuNPs/GCE by specific binding with the aptamer, and the incubation time is an important factor that affects the sensitivity. binding with the aptamer, and the incubation timeinto is an1.47 important that affects theDPV sensitivity. When MbBA/TCPP–Gr/AuNPs/GCE was immerged × 10−10 Mfactor myoglobin solution, was −10 M myoglobin solution, When MbBA/TCPP–Gr/AuNPs/GCE was immerged into 1.47 × 10 used to monitor the changes of the signal. As shown in Figure 5B, the peak currents decreased evidently DPV was to monitor changes of showed the signal. As shownof in Figure 5B, the peak within 45used min and reached athe plateau, which the saturation bioaffinity between MbBAcurrents and decreased evidently within 45 min and reached a plateau, which showed the saturation of bioaffinity myoglobin. As a result, 45 min was selected as the optimal incubation time for myoglobin detection. between MbBA and myoglobin. As a result, 45 min was selected as the optimal incubation time for 3.4.2. Influence of the TCPP–Gr/AuNPs Concentration myoglobin detection. To optimize the amount of TCPP–Gr/AuNPs, 8.0 μL of TCPP–Gr/AuNPs solution with different 3.4.2. Influence of the TCPP–Gr/AuNPs Concentration concentrations was dropped onto the surface of GCE, respectively. Then, 30 μL of 2.5 μM MbBA To optimize the amount of TCPP–Gr/AuNPs, 8.0 µLpeak of TCPP–Gr/AuNPs different solution was spread on the TCPP–Gr/AuNPs/GCE. The current increased solution with thewith increasing concentration of TCPP–Gr/AuNPs fromsurface 0.125 mg/mL to respectively. 0.25 mg/mL and then the concentrations was dropped onto the of GCE, Then, 30decreased µL of 2.5when µM MbBA concentration of TCPP–Gr/AuNPs exceeded 0.25 mg/mL (Figure 5C). Thus 8.0 μL of 0.25 mg/mL solution was spread on the TCPP–Gr/AuNPs/GCE. The peak current increased with the increasing TCPP–Gr/AuNPs solution was employed in themg/mL following concentration of TCPP–Gr/AuNPs from 0.125 to experiments. 0.25 mg/mL and then decreased when the concentration of TCPP–Gr/AuNPs exceeded 0.25 mg/mL (Figure 5C). Thus 8.0 µL of 0.25 mg/mL TCPP–Gr/AuNPs solution was employed in the following experiments.

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3.4.3. Influence of the MbBA Concentration 3.4.3. Influence of the MbBA Concentration The relationship between the peak current and the concentration of MbBA was also evaluated. relationship between the peak the concentration of MbBA increased was also evaluated. WhenThe fixing the volume of MbBA to current 30 μL, and the peak current of ferrocene with the When fixing the volume of MbBA to 30 µL, the peak current of ferrocene increased withthen the concentration of MbBA, which increased from 0.6 μM to 2.5 μM. The peak current of ferrocene concentration MbBA,increase which increased from 0.6 µMoftoMbBA 2.5 µM. The peak of ferrocene decreased withoffurther of the concentration (Figure 5D), current indicating that less then decreased with further increase of the concentration of MbBA (Figure 5D), indicating that aptamer led to poor sensitivity and too much MbBA made it difficult for ferrocene to get close to less the aptamer led to poor sensitivity and too much MbBA made it difficult for ferrocene to get close to the electrode surface, resulting in hindered electron transfer. From the plot of the current response versus electrode surface,of resulting electron transfer. From was the plot of the current versus the concentration MbBA, in thehindered optimized amount of aptamer determined as 2.5 response μM. the concentration of MbBA, the optimized amount of aptamer was determined as 2.5 µM. 3.5. Analytical Application of the Aptasensor 3.5. Analytical Application of the Aptasensor Under the optimum conditions, the electrochemical sensing performance of the MbBA/TCPP– Under the optimum the electrochemical performance of the Gr/AuNPs/GCE towards conditions, the myoglobin was studied bysensing DPV. After incubating theMbBA/TCPP–Gr/ sensing interface AuNPs/GCE towards the myoglobin was studied by DPV. After incubating the sensing in in different concentrations of myoglobin in PBS (pH = 7.4), the DPV signals of ferrocene wereinterface recorded. different of myoglobin in displayed PBS (pH =a7.4), the DPVrelationship signals of ferrocene recorded. As seen inconcentrations Figure 6, the calibration curve good linear betweenwere the decreased As seen in Figure 6, the calibration curve displayed a good linear relationship between the decreased −11 peak current and the logarithm concentrations of myoglobin in the range from 2.0 × 10 M to 7.7 × 10−7 −11 M to peak and thelinear logarithm concentrations in+the range from × 10 M (R2 current = 0.9941). The regression equation isofΔimyoglobin = 0.2882 lgc 3.833. Here, Δi 2.0 is the decreased 7.7 ×current 10−7 Mand (R2 c= is 0.9941). The linear regression equation is ∆i current = 0.2882decreased lgc + 3.833. Here, ∆i is the peak the concentration of myoglobin. The peak with the increase decreased peak current and c is the concentration of myoglobin. The peak current decreased with of myoglobin concentration. The reason was that the electron transfer rate was hindered after more the increase of myoglobin concentration. The reason was that the electron transfer rate was hindered targets were combined with MbBA. The limit of detection (LOD) was evaluated to be 6.7 × 10−12 M (S/N more targets were combined with MbBA. Thecompared limit of detection was evaluated tofor be =after 3), indicating a sensitive electrochemical aptasensor to most of(LOD) the previous biosensors −12 M (S/N = 3), indicating a sensitive electrochemical aptasensor compared to most of the 6.7 × 10 myoglobin detection, as shown in Table 1. The excellent electrochemical-sensing performance may previous biosensors foramounts myoglobin as shown in Table The excellent be attributed to large of detection, binding sites provided by 1. AuNPs and theelectrochemical-sensing great electrocatalytic performance may be attributed to large amounts of binding sites provided by AuNPs and the great activity of graphene and AuNPs. electrocatalytic activity of graphene and AuNPs.

B 2.0

10.5 0M

1.5 9.0

7.72E-7 M

ΔI / μA

Current / μA

A

1.0 7.5

0.2

0.3

0.4

0.5

1E-11

1E-10

Potential / V

1E-9

1E-8

1E-7

1E-6

c/M

−11 M, Figure 6. 6. (A) (A) DPVs DPVs of of the the biosensor Figure biosensor for for different different concentrations concentrationsofofmyoglobin: myoglobin:0.00 0.00M, M,2.00 2.00×× 10 10−11 M, − 10 − 10 − 9 − 8 − 8 −−7 7 M, 1.00 ××1010 2.50 × 1.25 10 × M, × 10× 10−8M, 10−7 M, M,and 3.09 × ×1010 −10 M, M, −10 10 −9 M, 1.00 5.005.00 × 10× M, 2.50M, × 10 10−81.25 M, 6.18 M,6.18 3.09×× 10 7.72 M 7 M in 5.0 mL 0.10 M PBS (pH = 7.4); (B) The calibration curve between the peak current and 10−M in 5.07.72 mL×0.10 PBS (pH = 7.4); (B) The calibration curve between the peak current changes and the changes andconcentration the myoglobin concentration The measurements repeated 3 timesthe to myoglobin (logarithm). The(logarithm). measurements were repeatedwere 3 times to obtain obtain the standard deviation. (Amplitude: 50 mV, pulse width: 0.050 s). standard deviation. (Amplitude: 50 mV, pulse width: 0.050 s).

3.6. The The Reproducibility, Reproducibility, Stability Stability and and Specificity Specificity of the Aptasensor 3.6. 10 M myoglobin −1010 The reproducibility reproducibilityofofthe theaptasensor aptasensor was examined measuring The was examined byby measuring 4.9 4.9 × 10× M−myoglobin three three times. The relative standard deviation (RSD) was 1.9%, indicating reasonable reproducibility. times. The relative standard deviation (RSD) was 1.9%, indicating reasonable reproducibility. After the After the electrode modified was electrode stored in refrigerator at 10 4 ◦ days, C for 10 days, 98% of thepeak initial peak modified storedwas in refrigerator at 4 °C for 98% of the initial current current remained, which be ascribed to the goodof stability of the electrode. remained, which could becould ascribed to the good stability the electrode.

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1. The comparison of different analytical methods for myoglobin detection. Sensors 2016, 16, xTable FOR PEER REVIEW

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Detection Detection Table 1. The comparison of differentLinear analytical methods for myoglobin detection. Platform Specificity Method Range (nm) Limit (nm) Detection Linear Detection 3D RGO–Au/CILE CV 200–36,000 60 No Platform Specificity Method Range (nm) Limit (nm) Hemin/G-quadruplet/AuNPs Colorimetric 0–1000 2.5 Antibody 3D RGO–Au/CILE CV 200–36,000 60 No Myoglobin specific Hemin/G-quadruplet/AuNPs Colorimetric 0–1000 2.5 Antibody Au/DSP/Peptide CV 1.1–105 58 binding peptide Myoglobin specific Au/DSP/Peptide CV 1.1–105 58 rGO/CNT CV 0.058–235 0.020 Aptamer binding peptide rGO/CNT CV 0.058–235 0.020 Aptamer POC PGM 0–200 0.050 aptamer POC PGM 0–200 0.050 aptamer CQDs Fluorescence 0.059–5.9 0.059 Anti-Mb-aptamer CQDs Fluorescence 0.059–5.9 0.059 Anti-Mb-aptamer TCPP–Gr/AuNPs DPV 0.020–770 0.0067 Aptamer TCPP–Gr/AuNPs DPV 0.020–770 0.0067 Aptamer

Reference [6] Reference [7] [6] [7] [12] [12] [42] [42] [8] [8] [43] [43] Thiswork work This

For evaluating the specificity of of the the developed developed aptasensor, aptasensor, some nontargets such as bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb), (BSA), (Hb), and carcinoembryonic carcinoembryonic antigen antigen (CEA) (CEA) were tested. As shown in Figure 7, compared with the DPV DPV signal signal of of the the proposed proposed aptasensor aptasensor immersed 9M into 1.6 myoglobin, thethe peak current changes areare negligible after incubating thethe aptasensor in 1.6 ××10 10−9−M myoglobin, peak current changes negligible after incubating aptasensor the mixture of myoglobin with the above four nontargets (50 times of the myoglobin concentration), in the mixture of myoglobin with the above four nontargets (50 times of the myoglobin concentration), respectively. It indicated the excellent specificity specificity of of the the proposed proposed aptasensor aptasensor for for myoglobin. myoglobin.

8

without any protein Mb Mb+CEA Mb+Hb Mb+HSA Mb+BSA

A

B 1.2

0.8

ΔI / μA

Current / μA

10

6 0.4

0.0

4 0.3

0.4

Potential / V

Mb

b

+H Mb

A

S +B Mb

A CE

+ Mb

SA +H Mb

Figure 7. 7. (A) (A) DPVs DPVs of of the the aptasensor aptasensor proposed proposed in in the the absence absence and and presence presence of of unspecific unspecific proteins; proteins; Figure (B) Specificity Specificity of of the the aptasensor for myoglobin myoglobin detection. detection. Error Error bars bars show show the the standard standard deviations deviations of of (B) aptasensor for measurements taken taken from from three measurements three times times (Amplitude: (Amplitude: 50 50 mV, mV, pulse pulse width: width: 0.050 0.050 s). s).

3.7. Real Sample Analysis The validity and feasibility feasibility of the proposed electrochemical electrochemical aptasensor aptasensor was was evaluated evaluated by recovery experiments using using standard standardaddition additionmethods. methods.Briefly, Briefly,50 50μL µL of of human human serum serum was was diluted diluted to to 5.0 5.0 mL experiments with 0.10 M PBS (pH == 7.4), which is analogous analogous to the the patient patient serums. Then, different concentrations of myoglobin were were added added in diluted human serum samples. samples. The The detection detection steps steps were were the the same as those myoglobin described above. above. As Asshown shownin inTable Table 2, 2, the the recoveries recoveriesare arein in the therange rangeof of96.8%–105.8%, 96.8%–105.8%,illustrating illustrating good described accuracy of the aptasensor for myoglobin analysis in real clinical samples. Table 2. The recovery of myoglobin from human serum samples. Table

Samples (nM) Samples Added Added (nM) 1 0.152 1 0.152 2 2 1.56 1.56 3 3 9.88 9.88

Found(nM) (nM) Found

Recovery/% Recovery/%

0.159 0.159 1.51 1.51 10.5 10.5

104.6 96.896.8 106.3 106.3

104.6

4. Conclusions In summary, we have successfully designed a sensitive and selective electrochemical aptasensor for myoglobin based on the newly synthesized TCPP–Gr/AuNPs. The nanocomposites were synthesized by a simple wet-chemical strategy and possessed the advantages of large surface area,

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4. Conclusions In summary, we have successfully designed a sensitive and selective electrochemical aptasensor for myoglobin based on the newly synthesized TCPP–Gr/AuNPs. The nanocomposites were synthesized by a simple wet-chemical strategy and possessed the advantages of large surface area, good biocompatibility, excellent electric conductivity, and high stability. The attachment of TCPP on the graphene surface made the graphene disperse in aqueous solution uniformly. AuNPs provided a large number of binding sites for conjugating MbBA through a Au–S bond. The proposed aptasensor had a wide linear range and lower detection limit. It also showed good sensitivity, stability, and specificity. Besides, this method was cost-effective and easy to fabricate. In addition, the developed electrochemical aptasensor is promising for myoglobin detection in real clinical samples. Acknowledgments: This research work was supported by the National Natural Science Foundation of China (No. 21275093), the Young “Sanjin” Scholar Foundation of Shanxi Province, 2016 and the Returned Overseas Scholar Foundation of Shanxi Province (No. 205586801011). Author Contributions: Yujing Guo and Guojuan Zhang conceived and designed the experiments; Guojuan Zhang performed the experiments; Guojuan Zhang, Zhiguang Liu and Li Wang analyzed the data; Yujing Guo contributed reagents/materials/analysis tools; Guojuan Zhang and Yujing Guo wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations The following abbreviations are used in this manuscript: AMI AuNPs BSA CEA DPV FT-IR GCE GO HAS HAuCl4 HB LOD MbBA MCH PBS PDDA RSD SCE TCEP TCPP TCPP-Gr TEM UV-vis XPS

Acute Myocardial Infarction Gold Nanoparticles Bovine Serum Albumin Carcinoembryonic Antigen Differential Pulse Voltammetry Fourier Infrared Spectrometer Glassy Carbon Electrode Graphene Oxide Human Serum Albumin Chloroauric Acid Hemoglobin Limit of Detection Myoglobin Binding Aptamer 6-mercapto-l-hexanol Phosphate Buffered Solution Poly Dimethyl Diallyl Ammonium Chloride Relative Standard Deviation Saturated Calomel Electrode Tris (2-carboxyethyl) Phosphine Hydrochloride Meso-tetra (4-carboxyphenyl) Porphyrin Meso-tetra (4-carboxyphenyl) Porphyrin Functionalized Graphene Composite Transmission Electron Microscopy Ultraviolet Visible Absorption Spectrum X-ray Photoelectron Spectroscopy

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