Talanta 132 (2015) 65–71
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A luminol electrochemiluminescence aptasensor based on glucose oxidase modified gold nanoparticles for measurement of platelet-derived growth factor BB Jing-Jing Zhang a, Jun-Tao Cao a, Gui-Fang Shi a, Ke-Jing Huang a, Yan-Ming Liu a,n, Shu-Wei Ren b a b
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, PR China Xinyang Central Hospital, Xinyang 464000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 13 July 2014 Received in revised form 19 August 2014 Accepted 21 August 2014 Available online 30 August 2014
A sandwich-type luminol electrochemiluminescence (ECL) aptasensor for highly sensitive and selective detection of platelet-derived growth factor BB (PDGF-BB) is fabricated. For this proposed ECL aptasensor, a multilayered AuNPs-electrochemically reduced graphene (AuNPs-EG) nanocomposite film was formed on the GCE surface as the base of the aptasensor through a co-electrodeposition method. The AuNPs-EG composites possess high conductivity to promote the electron transfer at the electrode interface and good biocompatibility and large surface area to capture large amounts of primary aptamer (Apt1), thus amplifying the detection response. Moreover, glucose oxidase (GOD) functionalized AuNPs labeled secondary aptamer (GOD–Apt2–AuNPs) was designed as the signal probe for the sandwiched aptasensor. Enhanced sensitivity was obtained by in situ generation of H2O2 from reaction between GOD and glucose and the excellent catalytic behavior of AuNPs to the ECL of the luminol–H2O2 system. Under the optimal conditions, the as-prepared ECL aptasensor exhibited excellent analytical property for the detection of PDGF-BB in the range from 1.0 10–13 to 5.0 10 10 mol L 1 with a detection limit of 1.7 10–14 mol L 1 (S/N¼3). The application of the present protocol was demonstrated by analyzing PDGF-BB in human serum and human urine samples with the recoveries from 85.0% to 110%. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Aptasensor PDGF-BB Glucose oxidase (GOD) Luminol
1. Introduction Diabetic nephropathy (DN) is a major chronic microvascular complication of diabetes mellitus (DM), which is an important cause of increased morbidity and mortality in patients with DM [1]. Strong evidence suggests that several growth factors may contribute to the initiation and progressive fibrosis of DN. Platelet derived growth factor (PDGF), one of the critical growth factors found in human platelets, has increased in importance due to its role in the regulation of cell growth and division [2]. It is composed of two disulfide-linked polypeptide chains, designated A and B, occurs in three isoforms: PDGF-BB, PDGF-AB and PDGF-AA [3]. Among these isoforms, PDGF-BB is known to be directly implicated in the cell transformation process and in tumor growth and progression [4]. Moreover, PDGF-BB is considered as a good predictor for early deterioration of renal function in DN [5]. Thus, precise and sensitive evaluation of PDGF-BB in biological samples will be substantial for disease diagnosis.
n
Corresponding author. Tel./fax: þ 86 376 6392889. E-mail address: [email protected] (Y.-M. Liu).
http://dx.doi.org/10.1016/j.talanta.2014.08.058 0039-9140/& 2014 Elsevier B.V. All rights reserved.
Detection of PDGF-BB has been attempted including the traditional antibody based radioisotropic method and ELISA [6,7]. However, the antibody used in these methods is temperaturesensitive, irreversibly denatured, and has a limited shelf life. These drawbacks limit the applications of the antibody-based assay. Aptamer is a specific DNA or RNA strand obtained from randomsequence nucleic acid libraries by SELEX (systematic evolution of ligands by exponential enrichment) technology [8]. They have been widely used as recognition elements for biochemical analysis due to their advantages of simpler synthesis, easier storage, lower cost and higher specificity [9]. Several aptasensors have been developed for detecting PDGF-BB based on electrochemistry (EC) [10], fluorescence [11,12], colorimetric colorimetry [13], chemiluminescence (CL) [14], and electrochemiluminescence (ECL) [15]. Among all the aptasensors, ECL-based methods have attracted intensive and extensive research interests, as they integrate the advantages of EC detection and chemiluminescent techniques, such as high sensitivity, low background, cost-effectiveness and high specificity [16]. Zhu et al. [17] reported an aptamer-based bio bar code immunomagnetic separation and ECL detection of PDGF2þ BB. ½RuðbpyÞ3 (TBR)-Au bio bar code-labeled aptamer was used as an ECL nanoprobe in the method. Recently, our group [15]
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developed an ECL aptasensor featuring MoS2–AuNPs composites as a matrix and aptamer modified CdSe–ZnS quantum dots as a signal probe for PDGF-BB detection. Luminol, one of the most widely used efficient ECL luminophores, has some excellent inherent properties such as inexpensive, nontoxic and high light-emission yield [18,19], and luminolbased ECL studies have caught scientists' eyes in biochemical analysis. To achieve a sensitive detection for biochemical samples, many efforts have been devoted to amplifying the ECL signal of luminol. Generally, dissolved oxygen and hydrogen peroxide are used as a coreactant for the luminol ECL reaction to enhance the ECL intensity [20–22]. Nanoparticles with the merits of good biocompatibility, large surface area, excellent electrocatalytic activity and conductivity, could act as a versatile role for biochemical analysis, such as a good matrix for electrode modification or biological labels decorated with enzyme, aptamer or protein [23–26]. Moreover, some of the nanoparticles could catalyze H2O2 to generate a great amount of reactive oxygen species which could greatly accelerate the oxidization of luminol, leading to an amplified ECL signal. Cao et al. [27] developed an ECL immunosensor for ultrasensitive detection of carcinoembryonic antigen with a detection limit of 0.03 pg mL 1 using Pd and Pt nanoparticles decorated reduced graphene oxide (Pd&PtNPs@rGO) and glucose oxidase (GOD) labeled secondary antibody as the trace label. The sensitivity was enhanced by in situ generation of hydrogen peroxide with GOD and the catalysis of Pd&PtNPs to the ECL reaction of the luminol–H2O2 system. The synergetic effects of enzyme and nanopartciles for amplifying the luminol ECL signal in immunoassay provide an ingenious idea for the construction of aptasensors. Herein, we developed a novel sandwich-type luminol ECL aptasensor for PDGF-BB detection using GOD decorated Au nanoparticles as labels. In this protocol, AuNPs-electrochemically reduced graphene (AuNPs-EG) composites were firstly electrodeposited on the glassy carbon electrode (GCE) as an aptasensor platform which could amplify the ECL signal of the luminol–H2O2 system for its fascinating conductivity and served as carrier to immobilize primary aptamers (Apt1) through chemical absorption between AuNPs and –NH2 of aptamers [28,29]. Then, secondary aptamers (Apt2) and GOD decorated AuNPs were designed as the
signal probe for the sandwiched aptasensor. The proposed ECL aptasensor exhibited a sensitive and stable response for the detection of PDGF-BB and achieved a wider linear range and lower detection limit. The proposed ECL aptasensor was applied to the detection of PDGF-BB in human serum samples and human urine samples.
2. Experimental 2.1. Reagents and apparatus Human platelet-derived growth factor-BB (PDGF-BB) was purchased from Peprotech (USA). The aptamers for PDGF-BB (Apt1: 50 NH2-(CH2)6-TTT TTT TTT TCA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-30 , Apt2: 50 -SH-(CH2)6-TTT TTT TTT TCA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-30 ), glucose oxidase (GOD) and 10 mmol L 1 phosphate buffer saline (PBS, pH 7.4) were purchased from Sangon Biological Co., Ltd. (Shanghai, China). The aptamer and protein solutions were prepared with 10 mmol L 1 pH 7.4 PBS, containing 137 mmol L 1 NaCl, 2.7 mmol L 1 KCl, and 1 mmol L 1 MgCl2 and were stored at 4 1C until being used. Hydrogen tetra chloroaurate (III) trihydrate (HAuCl4 3H2O) was obtained from Alfa Aesar (A Johnson Matthey Company, Ward Hill, MA), luminol from Sigma-Aldrich (St. Louis, MO, USA) and bovine serum albumin (BSA) from Siobio Biotechnology Inc. (Shanghai, China). All reagents were of analytical reagent grade and used without further purification. Ultrapure water was used throughout the experiments. ECL emission was monitored with a model BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were carried out with a RST 5200 electrochemical workstation (Zhengzhou Shiruisi Technology Co., Ltd.). A conventional three-electrode system was used with a GCE (Φ ¼3 mm) as the working electrode, an Ag/AgCl (sat. KCl) as the reference electrode and a platinum electrode as the counter electrode. The morphologies of the samples were recorded on a Hitachi S-4800 scanning electron microscope (SEM) and a Tecnai G2 F20 transmission electron microscope (TEM).
Scheme 1. The procedure of the ECL aptasensor preparation.
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2.2. Preparation of GOD–Apt2–AuNPs bioconjugates AuNPs were prepared according to the previously described procedure [30]. The concentration of the AuNPs was calculated to be about 1.4 10 9 mol L 1. The GOD–Apt2–AuNPs bioconjugates was prepared according to the literature reported previously [31,32]. 4 μL 10% (v/v) Tween 80 was firstly added into 1 mL AuNPs, and then the mixture was incubated for 30 min at 25 1C under gentle stirring. Subsequently, 50 μL of GOD (1 mg mL 1) and 50 μL of Apt2 (5 μmol L 1) were added into the as-mixture. After incubation at 25 1C under shaking for 4 h and keeping overnight at 4 1C, the resulted solution was isolated by centrifugation at 10,000 rpm for 15 min to remove the unconjugated aptamer and GOD. The GOD–Apt2–AuNPs hybrid nanoprobes were obtained by re-dispersing the conjugates in pH 7.4 0.1 mol L 1 PBS containing 0.05% (v/v) Tween 80 and 0.1% BSA and stored at 4 1C. 2.3. Assembly of sandwich-type ECL aptasensor Scheme 1B shows the strategy for constructing the ECL aptasensor. First of all, the GCE was carefully polished by 1.0, 0.3, and 0.05 mm alumina powder respectively to a mirror finish and then sonicated in ethanol and ultrapure water in turn. A multilayered AuNPs-EG composite film was formed on the GCE surface through a co-electrodeposition method in a dispersion containing 1.0 mg mL 1 graphene oxide (GO) and 100 mmol L 1 HAuCl4, while applying scan potential between 1.5 and 0.6 V and scan rate at 25 mV s 1 as previously demonstrated by Zhu et al. [31]. After deposition, the electrode was rinsed with H2O. Then, 6 μL of 0.1 μmol L 1 capture
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Apt1 of PDGF-BB was applied to the AuNPs-EG modified electrode and incubated overnight at room temperature according to the literature [10]. After incubation, the Apt1/AuNPs-EG/GCE was rinsed with PBS (pH 7.4) carefully, immersed in 1% BSA solutions for 30 min at 37 1C to block the nonspecific binding sites. Followed by washing thoroughly with PBS, the modified GCE was incubated in PDGF-BB solution for 1 h at 37 1C (abbreviated as PDGF-BB/BSA/Apt1/AuNPsEG/GCE). At last, as a sandwich format, the resultant electrode was immersed in the GOD–Apt2–AuNPs solution. Thus the proposed aptasensor was constructed successfully. 2.4. Measurement procedure The aptasensors incubated with different PDGF-BB concentrations were placed in an ECL detector cell containing 3 mL PBS with 1.0 10 4 mol L 1 luminol and an appropriate concentration of glucose to record the change of ECL signals. ECL signals were obtained by CV scans with potential varying from 0.2 to 0.8 V and scan rate at 100 mV s 1. The CV and ECL curves were recorded simultaneously. 2.5. Preparation of real-world samples Human blood samples and human urine samples were from volunteers in Xinyang Central Hospital (Xinyang, China). The complete ethical approval has been obtained, and all the female volunteers gave written informed consent. The study was approved by the Institutional Review Board of Xinyang Central Hospital. The fresh blood samples were centrifuged immediately at 1000 rpm for 15 min to obtain serum. These serum samples were
Fig. 1. SEM image of AuNPs-EG nanocomposite (A) and TEM images of bare AuNPs (B), AuNPs-aptamer (C), and GOD–Apt2–AuNPs (D).
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stored at 20 1C before use. Urine samples were detected without pretreatment.
3. Results and discussion 3.1. Characterization of the AuNPs-EG nanocomposite, AuNPs, AuNPs–aptamer, and GOD–Apt2–AuNPs Fig. 1 shows the SEM image of AuNPs-EG nanocomposite and TEM images of bare AuNPs (B); AuNPs–aptamer (C) and GOD– Apt2–AuNPs (D). Fig. 1A reveals that AuNPs (with an average diameter of 18 nm) of AuNPs-EG nanocomposite were uniformly distributed between the graphene sheets. As for the bare AuNPs to prepare the GOD–Apt2–AuNPs nanoprobe, the average diameter was about 20 nm (Fig. 1B). As seen in Fig. 1C and D, AuNPsaptamer and GOD–aptamer–AuNPs were surrounded by a transparent ring compared to the bare AuNPs images. 3.2. The characterization of ECL aptasensor 3.2.1. Electrochemical characterization The stepwise ECL aptasensor assembly process was monitored by CV measurements. Fig. 2A presents the CV curves of the modified electrodes at different stages in the presence of a 5 mmol L 1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture containing 0.1 mol L 1 KCl. It can be seen that the bare GCE produced relatively low CV intensity (curve a). Compared with the electrode only electrodeposited with AuNPs (curve b), the peak current of the electrode co-electrodeposited with AuNPs-EG nanocomposites further increased (curve c), which suggested the AuNPs-EG nanocomposites film significantly increased the surface area and electro-conductivity of the electrode interface and thus facilitated the interfacial electron transfer process. After Apt1 functionalization (curve d), the peak current significantly decreased due to the fact that aptamer severely blocked the electron transfer tunnel and reduced effective area of electron transfer. It demonstrated that Apt1 has been immobilized successfully. When the aptasensor was blocked with BSA (curve e) and incubated with PDGF-BB (0.1 nmol L 1) (curve f), the CV responses were declined in succession for the hindrance of BSA and aptamer–PDGF-BB complex. Finally, the attachment of the hybrid nanoprobes onto the PDGF-BB surfaces further decreased the peak current (curve g) owing to the barrier effect of the conjugates against the electron communication between [Fe(CN)6]3 /4 and the electrode surface. As an effective method to investigate the interface properties of modified electrodes, EIS was also carried out to study the stepwise
assembly of the aptasensor. The semicircle diameter which equals the electron-transfer resistance, reflects the restricted diffusion of the redox probe through the multilayer system related directly to film permeability. As can be seen from Fig. 2B, the results of EIS are consistent with the CV characterization. 3.2.2. ECL characterization In the case of the luminol–H2O2 system, ROSs such as superoxide anion (O2 ) and hydroxyl radical (OH) reacts with luminol to produce excited 3-aminophthalate dianion and then emit lights [33]. According to the literatures [34], the probable mechanism is shown as follows: (LH is the deprotonated luminol and Ap2 ∗ is 3-aminophthalate) LH e -LHd-L d þH þ
(1)
GOD
ð2Þ
H2 O2 þ L d ⟹ Ap2 n þ products
AuNPs
ð3Þ
Ap2 n -Ap2 þ hv
ð4Þ
Glucose þO2 ⟹Gluconic acid þ H2 O2
The fabrication process of the ECL aptasensor was characterized by ECL in 0.1 mol L 1 PBS contain 1.0 10 4 mol L 1 luminol and 10 mmol L 1 glucose solution. As shown in Fig. 3, the bare GCE produced low ECL intensity (curve a). After electrodepositing of AuNPs, a sharp increase of the ECL intensity was observed (curve b). Such improvement reflects that AuNPs can catalyze ECL of luminol. The ECL intensity further improved at AuNPs-EG/GCE
Fig. 3. ECL profiles of the stepwise modification. Bare GCE (a); AuNPs/GCE (b); AuNPs-EG/GCE (c); Apt1/AuNPs-EG/GCE (d); BSA/Apt1/AuNPs-EG/GCE (e); PDGFBB/BSA/Apt1/AuNPs-EG/GCE (f); GOD–Apt2–AuNPs/PDGF-BB/BSA/Apt1/AuNPs-EG/ GCE (g).
Fig. 2. CVs (A) and EISs (B) of electrodes at different stages in 5.0 mmol L 1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 mol L 1 KCL. Bare GCE (a); AuNPs/GCE (b); AuNPs-EG/ GCE (c); Apt1/AuNPs-EG/GCE (d); BSA/Apt1/AuNPs-EG/GCE (e); PDGF-BB/BSA/Apt1/AuNPs-EG/GCE (f); GOD–Apt2–AuNPs/PDGF-BB/BSA/Apt1/AuNPs-EG/GCE (g). cPDGF-BB: 0.1 nmol L 1.
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Fig. 4. Effect of the glucose concentration (A) and incubation time (B) on ECL response. Working solution: 0.1 mol L 1 pH 7.4 PBS containing 1.0 10 4 mol L 1 luminol and different concentrations of glucose, scan rate: 100 mV s 1, scan range: 0.2–0.8 V.
Fig. 5. ECL intensity–time curves of the aptasensor with different concentrations of PDGF-BB (A). The calibration plot against the corresponding log cPDGF-BB (B). Other conditions are as in Fig. 4.
(curve c), which is attributed to the AuNPs-EG nanocomposite films increased the surface area and electrical conductivity and facilitated the reduction of O2 to generate Od2 in the solution dissolved with trace oxygen thus increase the ECL intensity. Then the ECL intensity dropped with the immobilization of Apt1 (curve d), blocking with BSA (curve e) and incubating with PDGF-BB (curve f) because of the formation of less conductive layers, which slowed down the electron transfer speed significantly and led to the decrease of ECL signal. However, when the GOD–Apt2–AuNPs bioconjugates were immobilized onto the modified electrode surface, an obvious enhancement of the ECL signal (curve g) appeared. The enhanced signal could be ascribed to the in situ generated H2O2 from reaction of GOD with glucose and the excellent catalytic behavior of AuNPs to the ECL emission of the luminol–H2O2 system.
3.3. Optimization of experimental conditions The glucose concentration is an important factor for the ECL intensity. Fig. 4A displays the influence of the glucose concentration. With the increase of the glucose concentration, the ECL signal increased and then reached a plateau in 10 mmol L 1. Hence, 10 mmol L 1 was selected. The effect of the incubation time of aptamer with PDGF-BB on the ECL signal was also investigated (Fig. 4B). The ECL emission was found to gradually increase with the increase of time and reach a constant value at 1 h. Therefore, 1 h was chosen.
3.4. Performance of the proposed ECL aptasensor Fig. 5 depicts ECL signals of the aptasensor in response to PDGF-BB of varying concentrations under the optimal conditions. The calibration plot reveals that the ECL intensity of the aptasensor increases linearly with the logarithm of PDGF-BB concentration in the range of 1.0 10 13–5.0 10 10 mol L 1. The linear regression equation is I¼ 40,959.0 þ 1361.1 logc with a correlation coefficient of R¼0.9931. The detection limit was calculated to be 1.7 10 14 mol L 1 (S/N ¼ 3). Furthermore, the comparison of the proposed method with previous reports was listed in Table 1. Compared with other methods, the proposed ECL aptasensor exhibits high sensitivity and a wide linear range. 3.5. Specificity, reproducibility, and stability of the ECL aptasensor Specificity is an important criterion for ECL aptasensor. To investigate the specificity of the aptasensor, some proteins such as human serum albumin (HSA), human immunoglobulin G (hIgG) and thrombin were used as the interferences to evaluate the specificity of the ECL aptasensor. The concentrations of the interfering substances were 0.6 mmol L 1, 50 μmol L 1, and 1.0 nmol L 1 respectively and the results are illustrated in Fig. 6A. In the absence of target protein, no apparent change of the ECL intensity was observed compared with that of the testing blank solution. Furthermore, the ECL signals of the mixture samples containing 0.01 nmol L-1 PDGF-BB, 0.6 mmol L 1 HSA, 50 μmol L 1 hIgG, and 1.0 nmol L 1 thrombin presented neglectable changes compared with that of the determination of 0.01 nmol L 1 PDGF-BB only. These results clearly indicated that the as-constructed sensor had an excellent sensing specificity
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Table 1 Comparison of different methods for PDGF-BB detection. Analytical methods
Linear range (nmol L 1)
Detection limit (nmol L 1)
References
Sandwich-type colorimetric aptasensor Sandwich-type ECL aptasensor Fluorescence aptasensor Chemiluminescence aptasensor Sandwich-type electrochemical aptasensor ECL aptasensor Sandwich-type ECL aptasensor
10–100 0.00001–0.1 1–50 0.0001–1.0 0.01–35 0.001–10 0.0001–0.5
6 0.0000011 0.37 0.0001 0.008 0.001 0.000017
[13] [15] [35] [14] [10] [17] This work
Fig. 6. The specificity of the ECL aptasensor towards different targets (A). cHSA: 0.6 mmol L 1, chIgG: 50 μmol L 1, cthrombin: 1.0 nmol L 1, cPDGF-BB: 0.01 nmol L 1. The stability of the ECL aptasensor under consecutive cyclic potential scans for 36 cycles (B). cPDGF-BB: 1.0 pmol L 1.
Table 2 Analytical results of human serum and urine samples (n¼ 3). Type of samples
No. of samples
Human serum
1 2 3
Human urine
4 5 6
PDGF-BB in patients (pmol L 1) 6.31 6.76 5.0 46.2 52.8 11.5
RSDs (%, n¼ 3)
No. of samples
PDGF-BB in healthy people (pmol L 1)
RSDs (%, n¼3)
3.2 4.1 5.2
7 8 9
0.14 – –
3.6 – –
4.9 3.7 5.3
10 11 12
0.43 0.31 0.12
3.4 2.9 5.7
-: not detected
consecutive cyclic potential scans for 36 cycles, the RSD for the change of the ECL signal is less than 1.0%, manifesting that the sensor had good stability.
Table 3 Recovery of PDGF-BB in human serum and urine samples. Samples
Found (pmol L
Added 1
)
(pmol L
Total found 1
)
(pmol L
1
)
Recovery (%)
Human serum
5.0
1.0 10.0 100.0
5.9 15.2 94.7
90.0 102 89.7
Human urine
52.8
1.0 10.0 100.0
53.9 61.3 143.9
110 85.0 91.1
and was feasible for the determination of PDGF-BB in complex samples. The reproducibility of the aptasensor was investigated. The inter-assay precision of the aptasensor was evaluated with the application of five electrodes. The relative standard deviation (RSD) is less than 5.0%. The stability of the ECL aptasensor was also recorded and the results are presented in Fig. 6B. Under
3.6. Analytical application It has been reported that PDGF might play a very important role in the initiation and progression of DN [1]. Determination of PDGF-BB in human body fluid could be used for early diagnosis of diabetic renal dysfunction. To demonstrate the applicability of the proposed method, the contents of PDGF-BB in human urine and human serum samples collected from DN patients and healthy people at Xinyang Central Hospital (Xinyang, China) were determined and the results are listed in Table 2. The results show that the value of DN in six patients (5.0– 6.76 pmol L 1 for human serum, 11.5–52.8 pmol L 1 for human urine) is much higher than that in six healthy persons (0.14 pmol L 1 for human serum, ;0.12–0.43 pmol L 1 for human urine). The recovery of the aptasensor was also estimated by standard addition method (Table 3). Three different concentrations of PDGF-BB (1.0, 10, and 100 pmol L 1) were added into human serum and urine samples with recoveries from 89.7–102% for human serum sample and 85.0–110% for human urine sample, respectively.
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4. Conclusions The work reported here develops a novel sandwich-type luminol ECL aptasensor for PDGF-BB detection based on in situ generated H2O2 as coreactant with GOD anchored AuNPs labeling. The high sensitivity and wide linear range obtained should be taken into account for the following reasons: (1) the hierarchical assembly of AuNPs-EG composite films and aptamer functionalization on the GCE surface provide not only a highly conductive and biocompatible electrode interface for ECL sensing but also an analog of extracellular matrix for the specific target recognition and adhesion; (2) AuNPs acted as an versatile platform not only to provide biocompatible surface area for the immobilization of abundant GOD and Apt2, but also to catalyze the ECL reaction of the luminol–H2O2 system; (3) amplified ECL of luminol response was obtained by in situ production of H2O2 with GOD as coreactant and the catalysis of AuNPs. The applicability of the aptasensor was successfully demonstrated in the determination of PDGF-BB in human serum and human urine samples. With high sensitivity, good selectivity and stability, the asproposed ECL aptasensor provided great potential in biochemical analysis. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant 21375114, U1304214), the Project of Science and Technology Development of Henan Province (142300410197) and the Foundation of Henan Educational Committee (14A150013). References [1] [2] [3] [4]
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A luminol electrochemiluminescence aptasensor based on glucose oxidase modified gold nanoparticles for measurement of platelet-derived growth factor BB Jing-Jing Zhang a, Jun-Tao Cao a, Gui-Fang Shi a, Ke-Jing Huang a, Yan-Ming Liu a,n, Shu-Wei Ren b a b
College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, PR China Xinyang Central Hospital, Xinyang 464000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 13 July 2014 Received in revised form 19 August 2014 Accepted 21 August 2014 Available online 30 August 2014
A sandwich-type luminol electrochemiluminescence (ECL) aptasensor for highly sensitive and selective detection of platelet-derived growth factor BB (PDGF-BB) is fabricated. For this proposed ECL aptasensor, a multilayered AuNPs-electrochemically reduced graphene (AuNPs-EG) nanocomposite film was formed on the GCE surface as the base of the aptasensor through a co-electrodeposition method. The AuNPs-EG composites possess high conductivity to promote the electron transfer at the electrode interface and good biocompatibility and large surface area to capture large amounts of primary aptamer (Apt1), thus amplifying the detection response. Moreover, glucose oxidase (GOD) functionalized AuNPs labeled secondary aptamer (GOD–Apt2–AuNPs) was designed as the signal probe for the sandwiched aptasensor. Enhanced sensitivity was obtained by in situ generation of H2O2 from reaction between GOD and glucose and the excellent catalytic behavior of AuNPs to the ECL of the luminol–H2O2 system. Under the optimal conditions, the as-prepared ECL aptasensor exhibited excellent analytical property for the detection of PDGF-BB in the range from 1.0 10–13 to 5.0 10 10 mol L 1 with a detection limit of 1.7 10–14 mol L 1 (S/N¼3). The application of the present protocol was demonstrated by analyzing PDGF-BB in human serum and human urine samples with the recoveries from 85.0% to 110%. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Aptasensor PDGF-BB Glucose oxidase (GOD) Luminol
1. Introduction Diabetic nephropathy (DN) is a major chronic microvascular complication of diabetes mellitus (DM), which is an important cause of increased morbidity and mortality in patients with DM [1]. Strong evidence suggests that several growth factors may contribute to the initiation and progressive fibrosis of DN. Platelet derived growth factor (PDGF), one of the critical growth factors found in human platelets, has increased in importance due to its role in the regulation of cell growth and division [2]. It is composed of two disulfide-linked polypeptide chains, designated A and B, occurs in three isoforms: PDGF-BB, PDGF-AB and PDGF-AA [3]. Among these isoforms, PDGF-BB is known to be directly implicated in the cell transformation process and in tumor growth and progression [4]. Moreover, PDGF-BB is considered as a good predictor for early deterioration of renal function in DN [5]. Thus, precise and sensitive evaluation of PDGF-BB in biological samples will be substantial for disease diagnosis.
n
Corresponding author. Tel./fax: þ 86 376 6392889. E-mail address: [email protected] (Y.-M. Liu).
http://dx.doi.org/10.1016/j.talanta.2014.08.058 0039-9140/& 2014 Elsevier B.V. All rights reserved.
Detection of PDGF-BB has been attempted including the traditional antibody based radioisotropic method and ELISA [6,7]. However, the antibody used in these methods is temperaturesensitive, irreversibly denatured, and has a limited shelf life. These drawbacks limit the applications of the antibody-based assay. Aptamer is a specific DNA or RNA strand obtained from randomsequence nucleic acid libraries by SELEX (systematic evolution of ligands by exponential enrichment) technology [8]. They have been widely used as recognition elements for biochemical analysis due to their advantages of simpler synthesis, easier storage, lower cost and higher specificity [9]. Several aptasensors have been developed for detecting PDGF-BB based on electrochemistry (EC) [10], fluorescence [11,12], colorimetric colorimetry [13], chemiluminescence (CL) [14], and electrochemiluminescence (ECL) [15]. Among all the aptasensors, ECL-based methods have attracted intensive and extensive research interests, as they integrate the advantages of EC detection and chemiluminescent techniques, such as high sensitivity, low background, cost-effectiveness and high specificity [16]. Zhu et al. [17] reported an aptamer-based bio bar code immunomagnetic separation and ECL detection of PDGF2þ BB. ½RuðbpyÞ3 (TBR)-Au bio bar code-labeled aptamer was used as an ECL nanoprobe in the method. Recently, our group [15]
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developed an ECL aptasensor featuring MoS2–AuNPs composites as a matrix and aptamer modified CdSe–ZnS quantum dots as a signal probe for PDGF-BB detection. Luminol, one of the most widely used efficient ECL luminophores, has some excellent inherent properties such as inexpensive, nontoxic and high light-emission yield [18,19], and luminolbased ECL studies have caught scientists' eyes in biochemical analysis. To achieve a sensitive detection for biochemical samples, many efforts have been devoted to amplifying the ECL signal of luminol. Generally, dissolved oxygen and hydrogen peroxide are used as a coreactant for the luminol ECL reaction to enhance the ECL intensity [20–22]. Nanoparticles with the merits of good biocompatibility, large surface area, excellent electrocatalytic activity and conductivity, could act as a versatile role for biochemical analysis, such as a good matrix for electrode modification or biological labels decorated with enzyme, aptamer or protein [23–26]. Moreover, some of the nanoparticles could catalyze H2O2 to generate a great amount of reactive oxygen species which could greatly accelerate the oxidization of luminol, leading to an amplified ECL signal. Cao et al. [27] developed an ECL immunosensor for ultrasensitive detection of carcinoembryonic antigen with a detection limit of 0.03 pg mL 1 using Pd and Pt nanoparticles decorated reduced graphene oxide (Pd&PtNPs@rGO) and glucose oxidase (GOD) labeled secondary antibody as the trace label. The sensitivity was enhanced by in situ generation of hydrogen peroxide with GOD and the catalysis of Pd&PtNPs to the ECL reaction of the luminol–H2O2 system. The synergetic effects of enzyme and nanopartciles for amplifying the luminol ECL signal in immunoassay provide an ingenious idea for the construction of aptasensors. Herein, we developed a novel sandwich-type luminol ECL aptasensor for PDGF-BB detection using GOD decorated Au nanoparticles as labels. In this protocol, AuNPs-electrochemically reduced graphene (AuNPs-EG) composites were firstly electrodeposited on the glassy carbon electrode (GCE) as an aptasensor platform which could amplify the ECL signal of the luminol–H2O2 system for its fascinating conductivity and served as carrier to immobilize primary aptamers (Apt1) through chemical absorption between AuNPs and –NH2 of aptamers [28,29]. Then, secondary aptamers (Apt2) and GOD decorated AuNPs were designed as the
signal probe for the sandwiched aptasensor. The proposed ECL aptasensor exhibited a sensitive and stable response for the detection of PDGF-BB and achieved a wider linear range and lower detection limit. The proposed ECL aptasensor was applied to the detection of PDGF-BB in human serum samples and human urine samples.
2. Experimental 2.1. Reagents and apparatus Human platelet-derived growth factor-BB (PDGF-BB) was purchased from Peprotech (USA). The aptamers for PDGF-BB (Apt1: 50 NH2-(CH2)6-TTT TTT TTT TCA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-30 , Apt2: 50 -SH-(CH2)6-TTT TTT TTT TCA GGC TAC GGC ACG TAG AGC ATC ACC ATG ATC CTG-30 ), glucose oxidase (GOD) and 10 mmol L 1 phosphate buffer saline (PBS, pH 7.4) were purchased from Sangon Biological Co., Ltd. (Shanghai, China). The aptamer and protein solutions were prepared with 10 mmol L 1 pH 7.4 PBS, containing 137 mmol L 1 NaCl, 2.7 mmol L 1 KCl, and 1 mmol L 1 MgCl2 and were stored at 4 1C until being used. Hydrogen tetra chloroaurate (III) trihydrate (HAuCl4 3H2O) was obtained from Alfa Aesar (A Johnson Matthey Company, Ward Hill, MA), luminol from Sigma-Aldrich (St. Louis, MO, USA) and bovine serum albumin (BSA) from Siobio Biotechnology Inc. (Shanghai, China). All reagents were of analytical reagent grade and used without further purification. Ultrapure water was used throughout the experiments. ECL emission was monitored with a model BPCL ultraweak luminescence analyzer (Institute of Biophysics, Chinese Academy of Science, Beijing, China). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements were carried out with a RST 5200 electrochemical workstation (Zhengzhou Shiruisi Technology Co., Ltd.). A conventional three-electrode system was used with a GCE (Φ ¼3 mm) as the working electrode, an Ag/AgCl (sat. KCl) as the reference electrode and a platinum electrode as the counter electrode. The morphologies of the samples were recorded on a Hitachi S-4800 scanning electron microscope (SEM) and a Tecnai G2 F20 transmission electron microscope (TEM).
Scheme 1. The procedure of the ECL aptasensor preparation.
J.-J. Zhang et al. / Talanta 132 (2015) 65–71
2.2. Preparation of GOD–Apt2–AuNPs bioconjugates AuNPs were prepared according to the previously described procedure [30]. The concentration of the AuNPs was calculated to be about 1.4 10 9 mol L 1. The GOD–Apt2–AuNPs bioconjugates was prepared according to the literature reported previously [31,32]. 4 μL 10% (v/v) Tween 80 was firstly added into 1 mL AuNPs, and then the mixture was incubated for 30 min at 25 1C under gentle stirring. Subsequently, 50 μL of GOD (1 mg mL 1) and 50 μL of Apt2 (5 μmol L 1) were added into the as-mixture. After incubation at 25 1C under shaking for 4 h and keeping overnight at 4 1C, the resulted solution was isolated by centrifugation at 10,000 rpm for 15 min to remove the unconjugated aptamer and GOD. The GOD–Apt2–AuNPs hybrid nanoprobes were obtained by re-dispersing the conjugates in pH 7.4 0.1 mol L 1 PBS containing 0.05% (v/v) Tween 80 and 0.1% BSA and stored at 4 1C. 2.3. Assembly of sandwich-type ECL aptasensor Scheme 1B shows the strategy for constructing the ECL aptasensor. First of all, the GCE was carefully polished by 1.0, 0.3, and 0.05 mm alumina powder respectively to a mirror finish and then sonicated in ethanol and ultrapure water in turn. A multilayered AuNPs-EG composite film was formed on the GCE surface through a co-electrodeposition method in a dispersion containing 1.0 mg mL 1 graphene oxide (GO) and 100 mmol L 1 HAuCl4, while applying scan potential between 1.5 and 0.6 V and scan rate at 25 mV s 1 as previously demonstrated by Zhu et al. [31]. After deposition, the electrode was rinsed with H2O. Then, 6 μL of 0.1 μmol L 1 capture
67
Apt1 of PDGF-BB was applied to the AuNPs-EG modified electrode and incubated overnight at room temperature according to the literature [10]. After incubation, the Apt1/AuNPs-EG/GCE was rinsed with PBS (pH 7.4) carefully, immersed in 1% BSA solutions for 30 min at 37 1C to block the nonspecific binding sites. Followed by washing thoroughly with PBS, the modified GCE was incubated in PDGF-BB solution for 1 h at 37 1C (abbreviated as PDGF-BB/BSA/Apt1/AuNPsEG/GCE). At last, as a sandwich format, the resultant electrode was immersed in the GOD–Apt2–AuNPs solution. Thus the proposed aptasensor was constructed successfully. 2.4. Measurement procedure The aptasensors incubated with different PDGF-BB concentrations were placed in an ECL detector cell containing 3 mL PBS with 1.0 10 4 mol L 1 luminol and an appropriate concentration of glucose to record the change of ECL signals. ECL signals were obtained by CV scans with potential varying from 0.2 to 0.8 V and scan rate at 100 mV s 1. The CV and ECL curves were recorded simultaneously. 2.5. Preparation of real-world samples Human blood samples and human urine samples were from volunteers in Xinyang Central Hospital (Xinyang, China). The complete ethical approval has been obtained, and all the female volunteers gave written informed consent. The study was approved by the Institutional Review Board of Xinyang Central Hospital. The fresh blood samples were centrifuged immediately at 1000 rpm for 15 min to obtain serum. These serum samples were
Fig. 1. SEM image of AuNPs-EG nanocomposite (A) and TEM images of bare AuNPs (B), AuNPs-aptamer (C), and GOD–Apt2–AuNPs (D).
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stored at 20 1C before use. Urine samples were detected without pretreatment.
3. Results and discussion 3.1. Characterization of the AuNPs-EG nanocomposite, AuNPs, AuNPs–aptamer, and GOD–Apt2–AuNPs Fig. 1 shows the SEM image of AuNPs-EG nanocomposite and TEM images of bare AuNPs (B); AuNPs–aptamer (C) and GOD– Apt2–AuNPs (D). Fig. 1A reveals that AuNPs (with an average diameter of 18 nm) of AuNPs-EG nanocomposite were uniformly distributed between the graphene sheets. As for the bare AuNPs to prepare the GOD–Apt2–AuNPs nanoprobe, the average diameter was about 20 nm (Fig. 1B). As seen in Fig. 1C and D, AuNPsaptamer and GOD–aptamer–AuNPs were surrounded by a transparent ring compared to the bare AuNPs images. 3.2. The characterization of ECL aptasensor 3.2.1. Electrochemical characterization The stepwise ECL aptasensor assembly process was monitored by CV measurements. Fig. 2A presents the CV curves of the modified electrodes at different stages in the presence of a 5 mmol L 1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture containing 0.1 mol L 1 KCl. It can be seen that the bare GCE produced relatively low CV intensity (curve a). Compared with the electrode only electrodeposited with AuNPs (curve b), the peak current of the electrode co-electrodeposited with AuNPs-EG nanocomposites further increased (curve c), which suggested the AuNPs-EG nanocomposites film significantly increased the surface area and electro-conductivity of the electrode interface and thus facilitated the interfacial electron transfer process. After Apt1 functionalization (curve d), the peak current significantly decreased due to the fact that aptamer severely blocked the electron transfer tunnel and reduced effective area of electron transfer. It demonstrated that Apt1 has been immobilized successfully. When the aptasensor was blocked with BSA (curve e) and incubated with PDGF-BB (0.1 nmol L 1) (curve f), the CV responses were declined in succession for the hindrance of BSA and aptamer–PDGF-BB complex. Finally, the attachment of the hybrid nanoprobes onto the PDGF-BB surfaces further decreased the peak current (curve g) owing to the barrier effect of the conjugates against the electron communication between [Fe(CN)6]3 /4 and the electrode surface. As an effective method to investigate the interface properties of modified electrodes, EIS was also carried out to study the stepwise
assembly of the aptasensor. The semicircle diameter which equals the electron-transfer resistance, reflects the restricted diffusion of the redox probe through the multilayer system related directly to film permeability. As can be seen from Fig. 2B, the results of EIS are consistent with the CV characterization. 3.2.2. ECL characterization In the case of the luminol–H2O2 system, ROSs such as superoxide anion (O2 ) and hydroxyl radical (OH) reacts with luminol to produce excited 3-aminophthalate dianion and then emit lights [33]. According to the literatures [34], the probable mechanism is shown as follows: (LH is the deprotonated luminol and Ap2 ∗ is 3-aminophthalate) LH e -LHd-L d þH þ
(1)
GOD
ð2Þ
H2 O2 þ L d ⟹ Ap2 n þ products
AuNPs
ð3Þ
Ap2 n -Ap2 þ hv
ð4Þ
Glucose þO2 ⟹Gluconic acid þ H2 O2
The fabrication process of the ECL aptasensor was characterized by ECL in 0.1 mol L 1 PBS contain 1.0 10 4 mol L 1 luminol and 10 mmol L 1 glucose solution. As shown in Fig. 3, the bare GCE produced low ECL intensity (curve a). After electrodepositing of AuNPs, a sharp increase of the ECL intensity was observed (curve b). Such improvement reflects that AuNPs can catalyze ECL of luminol. The ECL intensity further improved at AuNPs-EG/GCE
Fig. 3. ECL profiles of the stepwise modification. Bare GCE (a); AuNPs/GCE (b); AuNPs-EG/GCE (c); Apt1/AuNPs-EG/GCE (d); BSA/Apt1/AuNPs-EG/GCE (e); PDGFBB/BSA/Apt1/AuNPs-EG/GCE (f); GOD–Apt2–AuNPs/PDGF-BB/BSA/Apt1/AuNPs-EG/ GCE (g).
Fig. 2. CVs (A) and EISs (B) of electrodes at different stages in 5.0 mmol L 1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 mol L 1 KCL. Bare GCE (a); AuNPs/GCE (b); AuNPs-EG/ GCE (c); Apt1/AuNPs-EG/GCE (d); BSA/Apt1/AuNPs-EG/GCE (e); PDGF-BB/BSA/Apt1/AuNPs-EG/GCE (f); GOD–Apt2–AuNPs/PDGF-BB/BSA/Apt1/AuNPs-EG/GCE (g). cPDGF-BB: 0.1 nmol L 1.
J.-J. Zhang et al. / Talanta 132 (2015) 65–71
69
Fig. 4. Effect of the glucose concentration (A) and incubation time (B) on ECL response. Working solution: 0.1 mol L 1 pH 7.4 PBS containing 1.0 10 4 mol L 1 luminol and different concentrations of glucose, scan rate: 100 mV s 1, scan range: 0.2–0.8 V.
Fig. 5. ECL intensity–time curves of the aptasensor with different concentrations of PDGF-BB (A). The calibration plot against the corresponding log cPDGF-BB (B). Other conditions are as in Fig. 4.
(curve c), which is attributed to the AuNPs-EG nanocomposite films increased the surface area and electrical conductivity and facilitated the reduction of O2 to generate Od2 in the solution dissolved with trace oxygen thus increase the ECL intensity. Then the ECL intensity dropped with the immobilization of Apt1 (curve d), blocking with BSA (curve e) and incubating with PDGF-BB (curve f) because of the formation of less conductive layers, which slowed down the electron transfer speed significantly and led to the decrease of ECL signal. However, when the GOD–Apt2–AuNPs bioconjugates were immobilized onto the modified electrode surface, an obvious enhancement of the ECL signal (curve g) appeared. The enhanced signal could be ascribed to the in situ generated H2O2 from reaction of GOD with glucose and the excellent catalytic behavior of AuNPs to the ECL emission of the luminol–H2O2 system.
3.3. Optimization of experimental conditions The glucose concentration is an important factor for the ECL intensity. Fig. 4A displays the influence of the glucose concentration. With the increase of the glucose concentration, the ECL signal increased and then reached a plateau in 10 mmol L 1. Hence, 10 mmol L 1 was selected. The effect of the incubation time of aptamer with PDGF-BB on the ECL signal was also investigated (Fig. 4B). The ECL emission was found to gradually increase with the increase of time and reach a constant value at 1 h. Therefore, 1 h was chosen.
3.4. Performance of the proposed ECL aptasensor Fig. 5 depicts ECL signals of the aptasensor in response to PDGF-BB of varying concentrations under the optimal conditions. The calibration plot reveals that the ECL intensity of the aptasensor increases linearly with the logarithm of PDGF-BB concentration in the range of 1.0 10 13–5.0 10 10 mol L 1. The linear regression equation is I¼ 40,959.0 þ 1361.1 logc with a correlation coefficient of R¼0.9931. The detection limit was calculated to be 1.7 10 14 mol L 1 (S/N ¼ 3). Furthermore, the comparison of the proposed method with previous reports was listed in Table 1. Compared with other methods, the proposed ECL aptasensor exhibits high sensitivity and a wide linear range. 3.5. Specificity, reproducibility, and stability of the ECL aptasensor Specificity is an important criterion for ECL aptasensor. To investigate the specificity of the aptasensor, some proteins such as human serum albumin (HSA), human immunoglobulin G (hIgG) and thrombin were used as the interferences to evaluate the specificity of the ECL aptasensor. The concentrations of the interfering substances were 0.6 mmol L 1, 50 μmol L 1, and 1.0 nmol L 1 respectively and the results are illustrated in Fig. 6A. In the absence of target protein, no apparent change of the ECL intensity was observed compared with that of the testing blank solution. Furthermore, the ECL signals of the mixture samples containing 0.01 nmol L-1 PDGF-BB, 0.6 mmol L 1 HSA, 50 μmol L 1 hIgG, and 1.0 nmol L 1 thrombin presented neglectable changes compared with that of the determination of 0.01 nmol L 1 PDGF-BB only. These results clearly indicated that the as-constructed sensor had an excellent sensing specificity
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Table 1 Comparison of different methods for PDGF-BB detection. Analytical methods
Linear range (nmol L 1)
Detection limit (nmol L 1)
References
Sandwich-type colorimetric aptasensor Sandwich-type ECL aptasensor Fluorescence aptasensor Chemiluminescence aptasensor Sandwich-type electrochemical aptasensor ECL aptasensor Sandwich-type ECL aptasensor
10–100 0.00001–0.1 1–50 0.0001–1.0 0.01–35 0.001–10 0.0001–0.5
6 0.0000011 0.37 0.0001 0.008 0.001 0.000017
[13] [15] [35] [14] [10] [17] This work
Fig. 6. The specificity of the ECL aptasensor towards different targets (A). cHSA: 0.6 mmol L 1, chIgG: 50 μmol L 1, cthrombin: 1.0 nmol L 1, cPDGF-BB: 0.01 nmol L 1. The stability of the ECL aptasensor under consecutive cyclic potential scans for 36 cycles (B). cPDGF-BB: 1.0 pmol L 1.
Table 2 Analytical results of human serum and urine samples (n¼ 3). Type of samples
No. of samples
Human serum
1 2 3
Human urine
4 5 6
PDGF-BB in patients (pmol L 1) 6.31 6.76 5.0 46.2 52.8 11.5
RSDs (%, n¼ 3)
No. of samples
PDGF-BB in healthy people (pmol L 1)
RSDs (%, n¼3)
3.2 4.1 5.2
7 8 9
0.14 – –
3.6 – –
4.9 3.7 5.3
10 11 12
0.43 0.31 0.12
3.4 2.9 5.7
-: not detected
consecutive cyclic potential scans for 36 cycles, the RSD for the change of the ECL signal is less than 1.0%, manifesting that the sensor had good stability.
Table 3 Recovery of PDGF-BB in human serum and urine samples. Samples
Found (pmol L
Added 1
)
(pmol L
Total found 1
)
(pmol L
1
)
Recovery (%)
Human serum
5.0
1.0 10.0 100.0
5.9 15.2 94.7
90.0 102 89.7
Human urine
52.8
1.0 10.0 100.0
53.9 61.3 143.9
110 85.0 91.1
and was feasible for the determination of PDGF-BB in complex samples. The reproducibility of the aptasensor was investigated. The inter-assay precision of the aptasensor was evaluated with the application of five electrodes. The relative standard deviation (RSD) is less than 5.0%. The stability of the ECL aptasensor was also recorded and the results are presented in Fig. 6B. Under
3.6. Analytical application It has been reported that PDGF might play a very important role in the initiation and progression of DN [1]. Determination of PDGF-BB in human body fluid could be used for early diagnosis of diabetic renal dysfunction. To demonstrate the applicability of the proposed method, the contents of PDGF-BB in human urine and human serum samples collected from DN patients and healthy people at Xinyang Central Hospital (Xinyang, China) were determined and the results are listed in Table 2. The results show that the value of DN in six patients (5.0– 6.76 pmol L 1 for human serum, 11.5–52.8 pmol L 1 for human urine) is much higher than that in six healthy persons (0.14 pmol L 1 for human serum, ;0.12–0.43 pmol L 1 for human urine). The recovery of the aptasensor was also estimated by standard addition method (Table 3). Three different concentrations of PDGF-BB (1.0, 10, and 100 pmol L 1) were added into human serum and urine samples with recoveries from 89.7–102% for human serum sample and 85.0–110% for human urine sample, respectively.
J.-J. Zhang et al. / Talanta 132 (2015) 65–71
4. Conclusions The work reported here develops a novel sandwich-type luminol ECL aptasensor for PDGF-BB detection based on in situ generated H2O2 as coreactant with GOD anchored AuNPs labeling. The high sensitivity and wide linear range obtained should be taken into account for the following reasons: (1) the hierarchical assembly of AuNPs-EG composite films and aptamer functionalization on the GCE surface provide not only a highly conductive and biocompatible electrode interface for ECL sensing but also an analog of extracellular matrix for the specific target recognition and adhesion; (2) AuNPs acted as an versatile platform not only to provide biocompatible surface area for the immobilization of abundant GOD and Apt2, but also to catalyze the ECL reaction of the luminol–H2O2 system; (3) amplified ECL of luminol response was obtained by in situ production of H2O2 with GOD as coreactant and the catalysis of AuNPs. The applicability of the aptasensor was successfully demonstrated in the determination of PDGF-BB in human serum and human urine samples. With high sensitivity, good selectivity and stability, the asproposed ECL aptasensor provided great potential in biochemical analysis. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grant 21375114, U1304214), the Project of Science and Technology Development of Henan Province (142300410197) and the Foundation of Henan Educational Committee (14A150013). References [1] [2] [3] [4]
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