Versatile High-Performance Electrochemiluminescence ELISA

Jun 17, 2019 - However, the preparation of an ECL probe-labeled Ab2 is complex, ... enzyme amplification in ELISA, making it more sensitive than ECLIA...
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Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

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Versatile High-Performance Electrochemiluminescence ELISA Platform Based on a Gold Nanocluster Probe Huaping Peng,† Zhongnan Huang,† Weihua Wu,† Mingkai Liu,§ Kaiyuan Huang,† Yu Yang,† Haohua Deng,*,† Xinghua Xia,‡ and Wei Chen*,†

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†

Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Department of Pharmaceutical Analysis, Fujian Medical University, Fuzhou 350004, China ‡ State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China § School of Chemistry & Materials Science, Jiangsu Key Laboratory of Green Synthetic Chemistry for Functional Materials, Jiangsu Normal University, Xuzhou 221116, China S Supporting Information *

ABSTRACT: This report outlines a versatile high-performance electrochemiluminescence (ECL) enzyme-linked immunosorbent assay (ELISA) platform, which combines the merits of high-quantum-yield Au nanocluster (AuNC) probe-based ECL technology, the efficient ECLresonance energy-transfer (ECL-RET) strategy, and highly sensitive and specific ELISA technology. The ECL detection procedure was performed on a recyclable MnO2/AuNC-modified glassy carbon electrode interface by taking advantage of the ECL-RET between the AuNC probe and MnO2 nanomaterials (NMs) to quench the ECL intensity. The etching of MnO2 NMs by the product of ALP-based ELISA recovers the ECL signal. Notably, the ELISA process and the ECL detection procedure in this system are independent. Thus, the ECL-ELISA system can effectively avoid the influence of complex biological samples, and the ECL efficiency of the AuNC probe can be used readily. As demonstrated on TNF-α, because of the abovementioned characteristics, the ECL-ELISA platform presented an extremely wide dynamic range, with a detection limit of 2 orders lower than ELISA. Moreover, the system was also applicable for ultrahigh sensitive detection of various disease-related proteins and able to detect trace biomarkers in real serum samples. Therefore, this multifunctional ECL assay platform is versatile, facile, ultrasensitive, recyclable, and sufficiently straightforward for trace biomarker detection in complex biological samples. This approach not only enriches the foundational study of ECL devices but also greatly expands the potential application of ECL sensors in biological testing and clinical high-throughput diagnosis. KEYWORDS: electrochemiluminescence, enzyme-linked immunosorbent assay, Au nanocluster, MnO2 nanomaterial, biomarker

1. INTRODUCTION The development of a high-performance multifunctional assay for the detection of trace levels of disease markers plays a vital role in early clinical diagnosis, prognosis, and disease treatment. Enzyme-linked immunosorbent assay (ELISA) is one of the most effective assays for detecting and identifying biomolecules, which is mainly based on the hyper-specific antigen−antibody recognition and highly efficient biocatalytic property of the enzymes.1 This technique has been widely used in a large range of applications, such as clinical diagnosis, food test, environmental analysis, and laboratory research.2−5 Nonetheless, because of the unsatisfactory sensitivity of conventional ELISA, improving ELISA is critical for detecting trace amounts of samples in various areas. Electrochemiluminescence (ECL) technology is a powerful technique in sensing and detecting trace amounts of samples because of its prominent features, such as low-light back© 2019 American Chemical Society

ground, high sensitivity, excellent controllability, low cost, simplicity, ease of use, and portability.6,7 In the most widespread sandwich-type of electrochemiluminescence immunoassay (ECLIA), the immunocomplex is formed by binding the primary antibody (Ab1) that is immobilized on the electrode surface and the secondary antibody (Ab2) that is modified with an ECL probe with the antigen by immunoreaction. The target molecule detection can be realized by measuring the signal of the ECL probe.8 ECLIA integrates the excellent sensitivity of ECL and the specific antibody−antigen recognition of immunoreaction and provides a powerful analytical tool to help overcome the limitations of ELISA with a simplified optical setup. However, the preparation of an Received: May 22, 2019 Accepted: June 17, 2019 Published: June 17, 2019 24812

DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

Research Article

ACS Applied Materials & Interfaces ECL probe-labeled Ab2 is complex, making it difficult to realize commercialization. Furthermore, the complex biological matrix can foul the electrode surface and influence the ECL behavior of the emitter because of the coexistence of immunocomplexes on the interface of the same electrode.9,10 Moreover, the ECL signal can be influenced by the increased electron-transfer distance between the ECL probe and the electrode, which is caused by the binding of immunocomplexes on the surface of the electrode and the weakened recognition ability of the antibody caused by steric hindrance from labeled luminophore. Thus, the ECL-ELISA technology, which consists of two independent moieties of ECL and ELISA, has been proposed to effectively avoid these shortcomings.11 First, the ECLsensing platform does not come into contact with the complex samples in the whole detection procedure, as to the interfering substance is discarded during the well washing process. Furthermore, only the substrate and product are introduced to the ECL-sensing platform because the immunocomplex is immobilized on the surface of the ELISA plate. Moreover, this interface can minimize the decrease in the ECL intensity of the luminophore because it does not employ luminophore-labeled antibodies and thus avoids the accompanying steric hindrance during the ECL process. Besides, ECL-ELISA takes advantage of the enzyme amplification in ELISA, making it more sensitive than ECLIA. Therefore, ECL-ELISA exhibits higher sensitivity, better stability, and excellent anti-interference performance.12,13 As an emerging analysis technology, ECL-ELISA shows great prospect to meet the current requirements for biological and medical application. Over the past decade, exploring novel luminophore species that would deal with the problem of low-sensitivity biosensing has been one of the key aims in ECL research.14−16 Thus far, various types of ECL probes have been developed. Apart from other ECL species, such as Ru(bpy)32+ and luminol,13−15 quantum dot probes, including semiconductor nanocrystals and carbon nanodots, have proven extremely promising.17−19 Moreover, gold nanoclusters (AuNCs) have attracted extensive attention in photoluminescence and ECL field because of its superior stability, low cost, good biocompatibility, and versatile molecule-like structures.20−23 However, in the field of ECL sensing, because of the low ECL quantum efficiency and indistinct mechanism, the further applications of the ECL of AuNCs has been hindered. Although various methods have been proposed to improve the efficiency of AuNC ECL, these methods have not achieved a notable impact.24,25 Aiming at these shortcomings, our group has previously developed a highly effective strategy for preparing AuNC-based ECL probes by valence regulation, which greatly improved the ECL quantum efficiency of AuNCs.26,27 Thus, we believe that the aforementioned AuNCs probe is highly suitable for designing and developing an ECL-sensing platform to satisfy the needs of various fields. In the present study, we first propose a novel and efficient ECL-resonance energy-transfer (ECL-RET) strategy, with the high-performance AuNC probe as the ECL donor and MnO2 nanomaterials (NMs) as the ECL acceptor. Subsequently, an ECL-ELISA platform based on ELISA product-triggered ECL turn-on of the MnO2/AuNC system has been successfully constructed (Scheme 1). This novel ECL-based ELISA strategy presents several advantages. First, it can effectively avoid the influence of complex biological samples because the independence of the ELISA and ECL procedures separates the MnO2/AuNC sensing interface architecture from the complex

Scheme 1. Schematic Diagram for the (A) Conventional ECL Immunoassay and (B) ECL Immunoassay Based on the ECL-ELISA Platform

practical samples. Furthermore, high-sensitivity detection can be achieved thanks to the combination of the dual-signal amplification of both the ECL technology and the enzymecatalyzed signal amplification technique. Moreover, the proposed MnO2/AuNC RET-based ECL platform can markedly enhance the detection performance compared to other conventional detection methods. Notably, the ECL efficiency of the AuNC probe can be used effectively because of the separate interface of the AuNC probe-modified electrode, bypassing the need to use luminophore-labeled antibodies and thus effectively avoiding the accompanying effect of steric hindrance. As expected, by employing TNF-α as an analysis model, the detection limit of the proposed ECL sensor was as low as 36 fg/mL, which was 2 orders lower than the sensitivity of ELISA kits. More importantly, the multifunctional ECL assay platform is versatile, facile, recyclable, and sufficiently straightforward for detecting trace biomarkers in complex serum samples. Thus this study not only greatly expands the applications of the high-performance AuNC probe but also provides a universal prospective strategy for disease marker determination in clinical diagnosis.

2. EXPERIMENTAL SECTION 2.1. Chemicals and Materials. Chloroauric acid (HAuCl4· 4H2O), sodium borohydride (NaBH4), and N-acetyl-L-cysteine (NAC) were bought from Aladdin Reagent Company (Shanghai, China). Potassium permanganate (KMnO4) was obtained from Lisheng Chemical Reagent Co. Ltd. Potassium peroxydisulfate (K2S2O8) and ascorbic acid (AA) were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). L-ascorbic acid 2phosphate trisodium salt (AAP), alkaline phosphatase (ALP), superoxide dismutase, catalase, pyrophosphohydrolase, acid phosphatase (ACP), proteinase K, lysozyme, α-glucosidase, and urease were purchased from Sigma-Aldrich Chemical Co. Ltd. (Shanghai, China). Streptavidin-ALP, cancer antigen 15−3 (CA15-3), tumor necrosis factor α (TNF-α), β2-microglobulin (β2-MG), carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), prostate-specific antigen (PSA), human epidermal growth factor receptor 4 (HE4), and human serum albumin (HSA) were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). Phosphate buffer solution (0.1 M, pH 7.4) containing 0.1 M K2S2O8 was used as the electrolyte in electrochemistry and ECL analysis. All other regents were of analytical grade and used as received without further purification. 2.2. Apparatus and Characterization. Scanning electron microscope (SEM) images and energy-dispersive X-ray spectroscopy (EDXS) were executed with an SU8010 SEM instrument (JEOL, Tokyo, Japan). UV−vis absorption spectra were collected using a UV2450 UV−vis spectrophotometer (Shimadzu, Kyoto, Japan). X-ray 24813

DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

Research Article

ACS Applied Materials & Interfaces

Figure 1. SEM images and EDXS of CR-AuNC/GCE (A, D), MnO2/CR-AuNC/GCE (B, E), and MnO2/CR-AuNC/GCE treated with 0.1 M AA (C, F). α (50 μL) standard solution were added to the anti-TNF-α antibodycoated polystyrene microwells and incubated at 37 °C for 90 min. Following this step, 100 μL of biotinylated antibody was added and incubated at 37 °C for 1 h. Afterward, 100 μL of 1:100 streptavidinALP solution was added to each microwell, and the solution was incubated at 37 °C, with gentle shaking, for 1 h. After washing the microwells, the following enzymatic reaction was performed: 100 μL of 4 mM AAP solution (10 mM Tris-HCl buffer solution, pH 8.0) was added to the microwells and allowed to react at 37 °C, in the dark, for 30 min. Afterward, the resultant solution in each well was transferred to a centrifuge tube and the MnO2/CR-AuNC/GCEs were placed in each centrifuge tube for 4 min. Finally, the treated electrodes were rinsed with water and dried with N2 immediately for ECL measurements. Other proteins were tested using a procedure similar to the one described above. 2.6. Real Sample Analysis. Blood samples were kindly provided by the Second Hospital of Fuzhou. The pretreatment process of the sample was as follows: 10 μL of blood sample was diluted to 100 mL by Tris-HCl buffer (20 mM, pH 8.0) for AA and ALP assays. The detection procedure of the prepared blood samples was as described above. For the ECL-ELISA analysis, clinical serum samples from four healthy people and two patients who suffered from inflammation were collected from the Second Hospital of Fuzhou. The ECL-ELISA procedure referred to that for TNF-α, substituting the standard solution of TNF-α for the diluted human serum. 2.7. ECL Measurement Procedure. The ECL measurement was carried out using the MPI-B ECL analyzer by SP in PBS (0.1 M, pH 7.4) containing 0.1 M K2S2O8. The potential steps were set up during 0 V and −2 V. The change in ECL intensity (ΔI) was defined as ΔI = I − I0, where I0 stands for the ECL intensity of MnO2/CR-AuNC/ GCE and I stands for the ECL intensity of MnO2/CR-AuNC/GCE treated with the product of ALP-based ELISA.

photoelectron spectroscopy (XPS) was performed using an ESCALAB 250XI electron spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with a focused monochromatic Al Kα X-ray source for excitation and a spherical section analyzer. ECL measurements were obtained using an MPI-B ECL analyzer (Xi’an Rimex Analysis Instrument Co. Ltd. Xi’an, China). All ECL measurements and electrochemical techniques were carried out with a three-electrode system, including the modified glass carbon electrode (GCE) working electrode, an Ag/AgCl (saturated KCl solution) reference electrode, and a Pt wire counter electrode. The ECL signals were generated by the step potential (SP) method.26 The photomultiplier tube was biased at 750 V. All experiments were carried out at room temperature. 2.3. Preparation of the ECL-Sensing Interface. The NAC− AuNCs were first prepared according to our previous method.28 The modified electrodes were developed according to our previous study.26 The CR-AuNC/GCE modified with MnO2 was fabricated by electrodepositing the CR-AuNC/GCE in the solution of 10 mM KMnO4 and 45 mM H2SO4 (v/v = 1:1) at −0.2 V for 300 s by chronoamperometry and then thoroughly rinsed with water. The resulting electrode was termed MnO2/CR-AuNC/GCE and employed as the ECL biosensing platform for further study. 2.4. Procedures for AA and ALP Detection. For the AA assay, the MnO2/CR-AuNC/GCE was simply immersed in various concentrations of AA with 0.1 M phosphate-buffered saline (PBS) (pH 8.0) for 4 min. Then, the AA-treated electrodes were taken out, rinsed with water, and dried with N2 immediately for ECL measurements. For the ALP activity assay, 37.5 μL of different concentrations of ALP and 337.5 μL of pH 8.0 Tris-HCl buffer containing 4 mM AAP were incubated at 37 °C for 30 min. Subsequently, the MnO2/CR-AuNC/GCEs were then soaked in the reactant solution for 4 min at room temperature. Afterward, the treated electrodes were rinsed with water and dried with N2 for ECL measurements. 2.5. Procedures for Immunoassay. For the ECL-ELISA, a 5 mg/mL dopamine solution (pH 8.5) was first added into polystyrene microwells at 4 °C for 2 h. Thus, polydopamine (PDA)-coated polystyrene microwells were obtained because of the self-polymerization of dopamine. Then, the wells were rinsed with a 10 mM TrisHCl buffer solution (pH 7.3) three times after removing the solution in the polystyrene microwells. The above process was repeated after each subsequent step until the enzyme reaction. After that, 100 μL of 0.2 mg/mL anti-TNF-α antibody was added on the surface of PDAcoated polystyrene microwells and incubated at 4 °C for 12 h. Then, 100 μL of 1% bovine serum albumin (BSA) was injected and incubated for 1 h before rinsing. Different concentrations of the TNF-

3. RESULTS AND DISCUSSION 3.1. Characterizations of the Modified Electrodes. The surface morphology of the modified electrodes was investigated by SEM. As shown in Figure 1A, the CR-AuNC/ GCE showed many pearl-like nanoclusters distributed uniformly on the surface of the GCE. EDXS indicated that Au was the only element present on the CR-AuNC/GCE (Figure 1D). The ECL behavior of the modified electrodes was performed by the SP method using K2S2O8 as the cathodic coreactant, with potential steps from 0 to −2 V. As shown in Figure 2, bare GCE had a weak ECL emission, while an 24814

DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

Research Article

ACS Applied Materials & Interfaces

Figure 2. (A) ECL curve of bare GCE (a), NAC−AuNC/GCE (b), CR-AuNC/GCE (c), MnO2/CR-AuNC/GCE (d), and MnO2/CR-AuNC/ GCE treated with 0.1 M AA (e) in PBS (0.1 M, pH 7.4) containing 0.1 M K2S2O8. (B) Schematic illustration of the AuNC/S2O82− ECL-quenching mechanism by MnO2 NMs.

having a broad absorption spectrum (Figure S3). These results clearly indicated that Förster resonance energy transfer played only a minor role in the ECL quenching process. Afterward, cyclic voltammetry (CV) was performed to investigate the current changes in CR-AuNC/GCE and MnO2/CR-AuNC/ GCE in S2O82− systems (Figure S4). Compared with CRAuNC/GCE, there was no significant difference in the peak current at MnO2/CR-AuNC/GCE. This indicated that MnO2 NMs do not impede electron transfer of S2O82− and SO4•− at the electrode. Finally, the contribution of RET between the MnO2 NMs and the AuNCs was explored. The highest occupied molecular orbital (HOMO) (EHOMO) and the lowest unoccupied molecular orbital (LUMO) energy levels (ELUMO) of AuNCs were investigated by CV (see calculation details in the Supporting Information, Figure S5) and calculated to be −4.9 and −3.3 eV, respectively.35 Meanwhile, the EHOMO and ELUMO for MnO2 NMs were −4.8 and −6.9 eV, respectively.36,37 Because the ELUMO of AuNCs was higher than the ELUMO of MnO2 NMs, the electrons could transfer from AuNCs to MnO2 NMs,38 indicating that the ECL-RET dominated the process of MnO2 NMs quenching the ECL of AuNCs.39,40 The ECL quenching mechanism of MnO2 NMs to AuNC/S2O82− system is illustrated as Figure 2B. Interestingly, when the electrode modified with MnO2 was immersed in 0.1 M AA for 4 min, it was found that MnO2 was almost completely etched (Figure 1C). The EDXS result demonstrated that Mn and O were negligible and that Au was nearly the only element on the electrode surface (Figure 1F), which convincingly validated the etching effect of AA on MnO2 via the redox reactions between AA and MnO2. These results indicated that the electrode surface can be restored nearly to its original status. As expected, the ECL signal was significantly restored after the MnO2/CR-AuNC/GCE was further treated with AA (Figure 2, curve e), which could be ascribed to the etching of MnO2 NMs by AA.41 The etching mechanism could be described with the following redox reaction42

evident ECL emission was observed for the NAC−AuNC/ GCE (Figure 2, curve b), indicating that the origin of the ECL signal was NAC−AuNCs. Afterward, CR-AuNC/GCE exhibited a significantly enhanced ECL signal because of the valence state effect (Figure 2, curve c). The ECL efficiency (ΦECL) of CR-AuNC/GCE was calculated to be 4.11%, which was correspondence with our previous work.26 After electrodeposition in the solution of KMnO4 and H2SO4, a monolayer of flowerlike NMs was distributed uniformly on the CRAuNC/GCE surface (Figure 1B). The EDXS measurement reveals the existence of Mn, O, and Au, confirming the successful formation of MnO2 on the electrode surface (Figure 1E). Valence states of MnO2/CR-AuNC/GCE were characterized by XPS (Figure S2). The Au 4f spectrum exhibited a binding energy of 83.98 eV, which could be attributed to Au(0), which demonstrated that the CR-AuNCs were successfully prepared. It is worth noting that the Mn (2p) XPS spectra showed two peaks at 642.3 eV and 654.1 eV, which were characteristic of Mn (2p3/2) and Mn (2p1/2), respectively.29−31 The XPS spectrum of O revealed a dominant peak at 532.28 eV that could be fitted in the band between Mn and O.32,33 These results further verified the successful deposition of MnO2. Notably, an obvious decrease in ECL signal was observed after electrodeposition of the MnO2 NMs (Figure 2, curve d), indicating efficient AuNC ECL quenching by the MnO2 NMs. On the basis of the above observations, the following four possible quenching mechanisms were speculated: (1) the valence state of Au(0) changed to Au(I) under the influence of MnO2 NMs; (2) MnO2 NMs, owing to their broad absorption spectrum, absorbed the ECL of the AuNC/S2O82− system;34 (3) the MnO2 NMs hindered the electronic transfer of S2O82− and SO4•− on the electrode surface in the AuNC/S2O82− system; and (4) the RET between the MnO2 NMs and the AuNCs occurred because of their matching energy levels. To understand the exact mechanism responsible for the MnO2 NM-mediated ECL quenching of the AuNC/S2O82− system, the following experiments were carried out. First, the valence state of Au was assessed. The binding energy of 83.98 eV of Au 4f spectrum indicated the existence of Au(0) without any valence change occurring (Figure S2B). Thus, the MnO2 did not affect the valence of Au(0). Meanwhile, a maximum ECL emission of the CR-AuNCs was observed at ∼700 nm. Compared with the ECL spectrum of AuNC/GCE, the intensity of UV−visible absorption of MnO2 NMs/indium tin oxide could almost be ignored, in spite of MnO2 NMs

C6H8O6 + MnO2 + 2H+ → C6H6O6 + Mn 2 + + 2H 2O (1)

Thus, this approach provided an efficient ECL platform for ECL turn-on detection of AA and offered the possibility of its related reaction-based assay. 3.2. ECL Assay of AA and ALP Activity. According to the above findings, the presented ECL-sensing platform was first employed to analyze the activity of AA and ALP. To assess the 24815

DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

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ACS Applied Materials & Interfaces

showed that the AAP, ALP, and serum could barely affect the ECL behavior of the CR-AuNC/GCE and MnO2/CR-AuNC/ GCE and the sensing performance of ALP, which indicated that the ECL sensor exhibits satisfactory specificity for ALP assay in real serum samples. Interestingly, this ECL-sensing platform showed surprisingly strong recyclability, even after 12 rounds of recycling of the proposed ECL platform by immersing the MnO2/CR-AuNC/ GCE in AA solution or by electrodeposition of MnO2 NMs on the surface of CR-AuNC/GCE (Figure 4C), indicating its quick refreshing property. Encouraged by the excellent performance of the proposed ECL turn-on assay approach, the application of the ECL sensing platform in biological samples was explored by detecting ALP in fresh diluted human serum (0.01%). A ttest and an F-test showed that there was no significant difference between the proposed method and the PNPP test (Table S3), indicating that the proposed method is applicable for clinical diagnosis. 3.3. ECL-ELISA Application. ALP is one of the most commonly used enzyme labels in ELISA. Inspired by this, we attempted to combine ELISA with our proposed highperformance ECL technology and further investigate the potential application of the MnO2/CR-AuNC ECL-sensing platform to ALP-labeled ECL-ELISA for detection of the target antigen (Scheme 1). Herein, TNF-α was chosen as an example for this ECL-ELISA platform. In this process, the target TNF-α was first captured with specific antibodies that preimmobilized on a polystyrene microwells plate. Then, secondary antibodies against TNF-α labeled with biotin were further immobilized via antigen−antibody-specific recognition, and streptavidin− AP solution was conjugated to the immunocomplex via the biotin−avidin affinity reaction. Subsequently, AAP was added, allowing ALP to hydrolyze it into AA. When the MnO2/CRAuNC/GCE was immersed into the above hydrolysis product, the produced AA could induce the etching of MnO2 on the electrode surface, accompanied by an increase in the signal. To verify our design, a quantitative TNF-α detection experiment was executed through the proposed ECL-ELISA system. As expected, the ECL signals gradually increased with the increase of TNF-α concentrations. The above phenomena may be due to the following reasons: as the concentration of TNF-α increased, more TNF-α molecules could be conjugated to the antibodies, and more resulting ALP-labeled antigen−antibody immunocomplex could lead to the increase in AA concentration, thus ECL signals recovery can be promoted. As shown in Figure 5A, owing to the above principles, this ECL-ELISA

capability of the prepared MnO2/CR-AuNC/GCE platform, we have optimized several experimental conditions, such as deposition time of MnO2 NMs, pH, reaction time between MnO2 NMs and AA, and AAP concentrations. We found that 5 min of deposition of MnO2 NMs, pH 8.0, 4 min of reaction time between MnO2 NMs and AA, and 4 mM of AAP were the optimal values for the subsequent experiments (Figure S6). Under the optimal conditions, ΔI rose linearly with the concentration of AA from 10−9 to 10−2 M (Figure 3A). The

Figure 3. Linear calibration plots for AA (A) and ALP (B) detection.

detection limit was 0.26 nM (S/N = 3), which was much lower than that of recent ECL sensors (Table S1). These results could be ascribed to the high sensitivity of the AuNC-based ECL platform and the large surface area of MnO2 with many reaction sites for AA. Thus, the high-sensitivity detection of AA provided a good prerequisite for ALP detection. As expected, when the MnO2/CR-AuNC/GCE was dipped into the ALP catalytic reaction solution, the hydrolytic product AA recovered the ECL of CR-AuNC/GCE. The recovery of ECL intensity correlated well with the activity of ALP in the ranges of 0.1−2 × 105 mU/L (R = 0.998) with detection limit as low as 0.015 mU/L (S/N = 3) (Figure 3B), both of which were superior to those of the reported sensors and the standard, p-nitrophenyl phosphate (PNPP)-based chromogenic method (Table S2). To investigate the selectivity of the proposed method, the impact of interfering enzymes was investigated. A remarkable change of ECL signal was detected for ALP, whereas other enzymes showed negligible effects in the acceptable range, even at concentrations 10 times higher than that of ALP (Figure 4A). Therefore, the proposed ECL sensing platform showed potential applicability to the detection of complex samples, such as human serum. We then investigated the effects of AAP, ALP, and serum on the MnO2/CR-AuNC/ GCE and CR-AuNC/GCE interfaces (Figure 4B). The results

Figure 4. (A) Selectivity study of the proposed sensor for ALP assay: (1) ALP, (2) horseradish peroxidase, (3) superoxide dismutase, (4) catalase, (5) pyrophosphohydrolase, (6) proteinase K, (7) lysozyme, (8) ACP, (9) α-glucosidase, (10) urease, and (11) blank. The ALP concentration was 20 U/L, and the concentration of all others was 200 U/L. (B) Effect of AAP (40 mM) (1), ALP (2000 U/L) (2), and serum (0.01%) (3) on the ECL signal of MnO2/CR-AuNC/GCE; the effect of serum (0.01%) on the ECL signal of CR-AuNC/GCE (4); and the effect of serum (0.01%) with 20 U/L ALP and 4 mM AAP on the ECL signal of MnO2/CR-AuNC/GCE (5). (C) Recyclability of the developed ECL-sensing platform. 24816

DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

Research Article

ACS Applied Materials & Interfaces

Figure 5. (A) Relationship between the recovery of ECL signal and the concentration of TNF-α. (B) Selectivity of the proposed method for TNFα determination.

A high-performance technique for immunoassay in human blood samples is of great importance in clinical diagnosis. Inspired by the excellent performance of the proposed approach for TNF-α analysis, the feasibility of the proposed method in human serum sample detection was investigated. The recovery was in the range of 91.3−108.3% with RSD ranging from 1.0 to 4.6% (Table S4), indicating satisfactory potential applicability of this ECL sensor for testing clinical samples. Therefore, we further assayed five different human serum samples from three healthy adults and two patients with sepsis. The results obtained from patients of this assay corresponded with those from the standard ELISA (Table S5), and the RSD values were no more than 7%. Notably, the commercial kits could not analyze the concentration of TNF-α in normal human serum because of their unfavorable sensitivity. Thus, the proposed approach greatly expands the applicability of the AuNC-based ECL-ELISA sensing platform.

platform showed an excellent linear response to TNF-α concentration in the range of 0.06−31 pg/mL (R = 0.999). The detection limit was 36 fg/mL (S/N = 3), which was 2 orders of magnitude lower than the result of ELISA kits, and prominently more sensitive than other reported approaches for the detection of TNF-α (Table 1). Table 1. Comparison of the Analytical Performance of Different Techniques in TNF-α Assay method EIS EA ECL SWV EA EA ECD AM DI ECL

dynamic linear range (g/mL) −12

1.00 × 10 to 1.00 1.00 × 10−10 to 1.00 1.00 × 10−10 to 2.00 1.00 × 10−12 to 1.00 0.00 to 5.00 × 10−9 7.60 × 10−11 to 5.00 0 to 5.00 × 10−8 0 to 4.00 × 10−10 − 6.10 × 10−14 to 3.13

× × × ×

−10

10 10−6 10−6 10−8

× 10−9

× 10−11

a

LOD (g/mL)

1.00 5.00 7.00 5.00 4.40 3.80 4.10 5.80 1.00 3.60

× × × × × × × × × ×

10−12 10−11 10−12 10−13 10−11 10−11 10−9 10−12 10−11 10−14

references 43 44 45 46 47 48 49 50 51 this work

4. CONCLUSIONS In this study, a high-performance ECL-ELISA platform has been constructed by integration of the merits of high-quantumyield AuNC probe-based ECL technology with highly sensitive and specific ELISA technology. Interestingly, taking advantage of the ECL-RET between AuNCs and MnO2 NMs and the etching of MnO2 NMs by the ALP-based immunoreaction product AA, the proposed ECL biosensing interface exhibited high sensitivity and excellent recyclability. Moreover, the proposed ECL-ELISA platform can effectively nullify the influence of complex biological samples, and the ECL efficiency of the AuNC probe can be used readily because of the separate and simple interface of the AuNC probe-modified electrode. On the basis of these advantages, the presented platform exhibited a superwide dynamic range and an extreme low detection limit value that was 2 orders lower than that of commercial ELISA kits. In addition, the system could be effectively applied to other proteins, indicating that this platform allows for facile and versatile immunoassays. Finally, we have successfully detected trace biomarkers in real serum samples. We believe that this ECL-ELISA platform can find other wide applicability and can also hold great prospects for commercialization.

a

LOD, limit of detection (S/N = 3). EIS, electrochemical impedance spectroscopy; EA, electrochemical assay; ECL, electrochemiluminescence; SWV, square wave voltammetry; ECD, electrochemical detection; AM, amperometric magnetoimmunoassay; DI, diffusometric immunosensor.

To verify the versatility of the ECL-ELISA sensing platform, other proteins, such as CEA, β2MG, PSA, CA15-3, CA125m and HE4, were analyzed (see the Supporting Information, Figure S6). The results showed that the presented ECL-ELISA platform exhibited excellent performance for other biomolecule assays, indicating that a versatile high-performance ECLELISA platform has been successfully developed. To verify the imaging ability of the assay in real samples, high specificity toward the target antigen TNF-α was necessary. Therefore, we challenged the ECL-ELISA platform with other nonspecific proteins, such as HSA, BSA, human IgG, AFP, PSA, HE4, and CEA. The results showed that the interferences were negligible (Figure 5B), demonstrating excellent specificity of this ECL immunoassay. The repeatability of the ECL-ELISA system was investigated by measuring the ECL signal of 1.0 pg/mL of TNF-α for 6 times. The relative standard deviation (RSD) was as low as 1.6%. Furthermore, the ECL signal response still remained at 91% after 30 days of storage. The above results validate the excellent repeatability and stability of the proposed AuNCbased ECL-ELISA sensor.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b08819. 24817

DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819

Research Article

ACS Applied Materials & Interfaces

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ECL efficiency of Met-AuNC/GCE; HOMO and LUMO energy levels; characterization of NACAuNCs; TEM and HRTEM images of NAC-AuNCs; XPS spectrum of MnO2/CR-AuNCs; UV−visible absorption spectrum; cyclic voltammograms; optimization of experimental conditions; relationship between the restored ECL intensity and the concentration of different proteins; comparison of the analytical performance; analysis of ALP activity; and recovery experiment of TNF-α (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (H.D.). *E-mail: [email protected] (W.C.). ORCID

Mingkai Liu: 0000-0001-9060-848X Xinghua Xia: 0000-0001-9831-4048 Wei Chen: 0000-0003-3233-8877 Author Contributions

H.P. and Z.H. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (21874020, 21804021, and 51703087), Fujian Province Health Commission Young and Middle-Aged Talent training project (2018ZQN-62), the Natural Science Foundation of Jiangsu Province (BK20150238 and BK20170240), the Natural Science Foundation of Fujian Province (2017J01575, 2019J01305, and 2016J05031), and the Program for Fujian Top-notch Innovative Personnel.

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DOI: 10.1021/acsami.9b08819 ACS Appl. Mater. Interfaces 2019, 11, 24812−24819