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Enhancing Luminol Electrochemiluminescence by Combined Use of Cobalt-based MOF and Silver Nanoparticles and its Application in Ultrasensitive Detection of Cardiac Troponin I Shanshan Wang, Yangyang Zhao, Minmin Wang, Haijuan Li, Muhammad Saqib, Chunhua Ge, Xiangdong Zhang, and Yongdong Jin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05443 • Publication Date (Web): 25 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019
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Enhancing Luminol Electrochemiluminescence by Combined Use of Cobalt-based MOF and Silver Nanoparticles and its Application in Ultrasensitive Detection of Cardiac Troponin I Shanshan Wanga,b, Yangyang Zhaoa, Minmin Wanga,c, Haijuan Lia*, Muhammad Saqiba, Chunhua Geb, Xiangdong Zhangb, and Yongdong Jina,c* a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China b College of Chemistry, Liaoning University, Shenyang 110036, People’s Republic of China c University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ABSTRACT: Electrochemiluminescence (ECL) has been emerged as one of the most important methods for in vitro diagnosis and detection, but it is still limited in sensitivity for ultrasensitive bio-detections. While fast and ultrasensitive detection of biomolecules is critical, especially for the clinical detection of cardiac troponin I (cTnI) for cardiac infarction diagnosis. In this study, an effective tactic was developed to enhance ECL efficiency of the luminol system, by combined use of Co 2+-based metal organic frameworks (MOF), zeolitic imidazolate frameworks (ZIF-67) and luminol-capped Ag nanoparticles (luminol-AgNPs). The integration leads to a pronounced ~ 115-fold enhancement in luminol ECL. Based on this fascinating sensing platform, a robust label-free ECL immunosensor was constructed for ultrasensitive detection of cTnI, the main marker of myocardial infarction, with good stability and a detection limit as low as 0.58 fg mL-1 (S/N=3).
INTRODUCTION Electrochemiluminescence (ECL) has been emerged as one of the most important electrochemical methods for in vitro diagnosis and biodetection1-5 because of its high sensitivity, easy controllability, rapidness and wide dynamic range. Due to high ECL efficiency and low excitation potential of luminol, it has been widely used in ECL studies.6 Several studies have proved that the generation of oxygen-related radicals can greatly enhance the efficiency of luminol ECL.7-10 Noble metal nanoparticles, metal ions, enzymes, and hemin that can accelerate the decomposition of reactant H2O2 to generate OH•, O2•−, and other radical derivatives, have therefore been adopted to enhance the ECL of luminol system.7-10 Although numerous efforts have been made, it is still highly desired to find new methods to further improve the ECL sensitivity of luminol system for ultrasensitive bio-detections. Metal organic frameworks (MOFs), consists of metal ions and organic linkers, represent a charming kind of crystalline porous materials with ordered crystalline pores, adjustable functions, and tunable chemical compositions, have been widely used in the area of catalysis.11,12 The atomically dispersed metal sites in the MOF structures play a key role in the catalysis reactions. Their porous structures make all the active sites accessible and their open channels render the diffusion of the substrate and products easily. Recently, researchers have tried to take advantage of high catalytic activity of MOFs to enhance the efficiency of ECL.13-17 For example, Yuan et al. found that the introduction of isoreticular metal organic framework-3 can greatly enhance the ECL of the CdTe/S2O82− system by
enhancing the conversion of S2O82− coreactant into the sulfate radical anion (SO4•−).16 Herein, we found that Co-based MOF, zeolitic imidazolate frameworks (ZIF-67), in which Co2+ are atomically dispersed, can greatly enhance the ECL of luminol system. In this work, luminol-capped Ag nanoparticles (luminol-AgNPs) were decorated on the surface of ZIF-67 (Scheme 1) by electrostatic interaction. The integration of AgNPs and ZIF-67 has greatly enhanced the ECL of luminol system by ~ 115-fold. Furthermore, the luminol-AgNPs@ZIF-67 system was successfully explored for ultrasensitive label-free detection of the acute myocardial infarction marker cTnI. Scheme 1. Schematic illustration of the integration of Cobased MOF and AgNPs for the enhancement of luminol ECL.
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EXPERIMENTAL SECTION
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Materials Luminol, AgNO3 were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Co(NO3)2 and 2-methylimidazole (MeIM) were obtained from Aladdin. Biotin-anti-cTnI and cTnI were both obtained from Shanghai Linc-Bio Science Co. LTD (Shanghai, China). Bovine serum albumin (BSA), IgG, Mb, Dbiotin were purchased from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China). 0.1 M phosphate buffered saline (PBS) with pH of 7.4 was used for incubation. The H2O2 solution were freshly prepared daily from 30% H2O2 (Xinke Electrochemical Reagent Factory, Bengbu, China). All the reagents used in the experiment were of analytical grade and water was purified with a Millipore system.
Instrument The electrodes were cleaned by the novascan PSD Pro Series Digital UV Ozone System. Transmission electron microscopy (TEM) images were taken from Hitachi 600 transmission electron microscope at an accelerating voltage of 100 kV (Hitachi, Tokyo, Japan). The concentrations of the Co 2+ in ZIF-67 were obtained by ICP-OES (Thermo Scientific iCAP6300). Xray photoelectron spectroscopic (XPS) measurements were carried out on Escalab 250Xi, Thermo Fisher Scientific. CV characterizations of each step of the modification of gold electrode were performed on an electrochemical workstation (CHI660E) using three electrodes system. The Zeta potential values of the luminol-AgNPs and ZIF-67 were obtained by a Zeta sizer Nano-Z system (Malveren Instruments). The UV−Vis absorption spectra were obtained using a Lambda 750 spectrophotometer (Perkin−Elmer). ECL experiments were carried out by a model MPI A capillary electrophoresis ECL system (Xi’an Remex Electronics Co. Ltd., Xi’ an, China).
Synthesis of luminol-AgNPs@ZIF-67 Nanocomposite. Luminol-functionalized AgNPs (luminol-AgNPs) were synthesized by the reduction of AgNO3.18 The details can be summarized as follows: firstly, 9 mL of absolute ethanol and 5 mL of ultrapure water were mixed under continuous stirring, then 3 mL of 5 mM AgNO3 aqueous solution was added. After a few seconds, 0.5 mL of 0.01 M luminol containing 0.1 M NaOH was added to the previous solution rapidly which changed the color from colorless to light yellow. All of the above mentioned experiments were conducted at room temperature under continued magnetic stirring for about 2 hours. When the color becomes dark yellow, it indicated that the luminolAgNPs were synthesized successfully. Finally, the mixture was centrifuged at 10000 rpm for 10 minutes. After aspirate the supernatant, the soft sediment was obtained and ready for the next step. It is noted that the synthesized nanoparticles were stable and no precipitations were observed in few months. ZIF-67 particles was synthesized according to the literature report.19 In procedure, 0.182 g of cobalt nitrate hexahydrate (Co(NO3)2·6H2O) was dissolved in 10 mL methanol to form a pink solution, and 0.205 g of MeIM was dissolved in another 10 mL methanol solution. To mix the two solutions together, the latter was added to the pink solution at a fast speed and the color changed to purple quickly. After stirring for 30 s, the mixture was kept static for 24 h at room temperature. Then the mixture was centrifuged and washed with methanol for five times.19
As confirmed by the dynamic light scattering experiment, luminol-AgNPs and ZIF-67 particles were oppositely charged. The luminol-AgNPs@ZIF-67 nanocomposite was then prepared via the electrostatic interaction by adding ZIF-67 into the luminol-AgNPs colloid and shaking vigorously.
Fabrication of Label-Free cTnI ECL Immunosensor. Prior to the experiment, the gold electrode was cleaned by the following steps. Firstly, polished the surface with the 1.0 μm, 0.3 μm Al2O3 in turn, then ultrasonicated in ultra-pure water for 5 min and rinsed with ultra-pure water. After drying with inert gas argon, the gold electrode was finally cleaned in 1 M H2SO4 using cyclic voltammetry (CV) on a CHI660E electrochemical workstation (Chenhua, China) between -0.4 and 1.6 V versus Ag/AgCl at a scan rate of 0.5 V/s until a reproducible cyclic voltammogram was obtained. In order to remove the organics on the surface of the gold electrode before the modification, the electrode was continuous lighted by the UV ozone cleaning lamp for 30 min, and waited for 10 more minutes to let the ozone released. After the gold electrode was cleaned, 1,3-propanedithiol was assembled on the surface of the electrode by incubating the electrode in 1,3-propanedithiol solution overnight. After rinsing with ethanol and ultrapure water in turn, 1 μL of the luminolAgNPs@ZIF-67 suspension was dropped on the gold electrode and dried at room temperature. Afterwards, 2 μL of 100 μg·mL−1 biotin-anti-cTnI was dropped onto the electrode and incubated for 12 h at 4 °C to get the biotin-anti-cTnI/luminolAgNPs@ZIF-67 bioconjugates. In order to block the nonspecific binding sites, 2 μL of 1% BSA was added and incubated at 4 °C for 1 h. After it was rinsed thoroughly with 0.1 M PBS buffer (pH 7.4) to remove the physically absorbed conjugates, the label-free ECL immunosensor was fabricated and stored at 4 °C for cTnI detections.
ECL Detection of cTnI. For the detection of cTnI, different concentrations of cTnI (1 fg mL-1, 10 fg mL-1, 100 fg mL-1, 1 pg mL-1, 10 pg mL-1, 100 pg mL-1, 1 ng mL-1, 10 ng mL-1, 100 ng mL-1, and 1 μg mL-1) in 0.1 M PBS buffer (pH=7.4) were dropped on the surface of the electrode and incubated with the modified electrode for half an hour until the electrode was dried. Then the electrode was thoroughly washed by 0.1 M PBS (pH=10.5). Three-electrode detection system consisting of modified Au electrode as the working electrode, a platinum wire as the counter-electrode, and Ag/AgCl with saturated KCl as the reference electrode was used to detect cTnI. The ECL signal was detected by the MPI-A ECL analyzer (Ruimai, Xi-an) at room temperature. The detection electrolyte contained 0.1 mM H2O2 and 0.1 M PBS (pH=10.5). Since, high potential will facilitate the generation of oxygen radicals, which can improve the ECL intensity, so we chose a high voltage of -1.3 V and 1.3 V in this study. The calculation of recovery. The recovery of as-fabricated immunosensor was estimated by R=(V2C2)/(VsCs)×100 % (V2 and C2 are the volume and concentration after adding standard sample, Vs and Cs are the original sample add in the standard sample), as the V2 and Vs are the same, the recovery is calculated by R=C2/Cs, with the result of 97.8~104.8 %.
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Analytical Chemistry The calculation of Limit of Detection (LOD). LOD was calculated according to Harris’ method.20 The minimum detectable signal, ydl, is defined as Signal detection limit: ydl = yblank + 3s. (s: standard deviation) The corrected signal, ysample - yblank, is proportional to sample concentration: Calibration line: ysample - yblank = m × sample concentration where ysample is the signal observed for the sample and m is the slope of the linear calibration curve. The minimum detectable concentration, also called the detection limit, is obtained by substituting ydl for ysample: Detection limit: Minimum detectable concentration = 3s/m.
exhibited three peaks at ~ 355 nm, 425 nm and 600 nm, corresponding to the absorbance of luminol, AgNPs and ZIF-67 particles, respectively. And as shown in the XPS spectra of luminol-AgNPs@ZIF-67, the presence of Ag peak and pyridinic N peak indicates the successful adsorption of luminol-AgNPs on the surface of ZIF-67 particles. As calculated from the TEM images, when the ratio of ZIF-67 to luminol-AgNPs was 0.3, the surface coverage of luminol- AgNPs on ZIF-67 was about 60-70 luminol-AgNPs per one ZIF-67 nanoparticle. That is, about 36.7~46.5% of the surface of ZIF-67 was covered by luminol-AgNPs.
Figure 1. TEM images of the as-prepared luminol-AgNPs (A), ZIF-67 particles (B), and luminol-AgNPs@ZIF-67 (C). (D-I) TEM image and elemental mapping analyses of a single luminolAgNPs@ZIF-67 particle.
RESULTS AND DISCUSSION Characterization of luminol-AgNPs@ZIF-67. Luminol-AgNPs were synthesized at room temperature by a one-pot method by using ethanol, luminol and AgNO3 as coreagents.18,21 As shown in the TEM image (Figure 1A and Figure S1A&B), the diameter of the resulting luminol-AgNPs is about 30 nm. The complex NPs exhibited two UV-Vis absorbance peaks at 365 nm and 450 nm (Figure S2), corresponding to the absorbance of luminol and AgNPs, respectively. The ZIF-67 particles were synthesized according to the literature procedures.19 As shown by TEM (Figure 1B and Figure S1C&D) and SEM images (Figure S3), the as-prepared ZIF-67 particles exhibited dodecahedron structure with very smooth surface and the side length was about 400 nm, which is consistent with the literature reported.19 Since the surfaces of luminol-AgNPs and ZIF-67 particles were oppositely charged as confirmed by Zeta potential measurements (Figure S4), luminol-AgNPs can be easily adsorbed on the surface of ZIF-67 particles by electrostatic interaction. This was confirmed by TEM images and energy-dispersive X-ray spectroscopy (EDX) analyses (Figure 1, C-I). The successful assembly of luminol-AgNPs on ZIF-67 was further proved by UV-Vis absorbance spectra (Figure S2) and X-ray photoelectron spectra (XPS) analysis (Figure S5). The UV-Vis absorbance spectrum of luminol-AgNPs@ZIF-67
Figure 2. CV curves (A) and ECL kinetics curves (B) of luminolAgNPs, luminol-AgNPs with free Co2+, and luminol-AgNPs@ZIF67 with 0.1 M PBS (pH=7.4) containing 0.1 μM H2O2. (C) ECL kinetics curves of luminol-AgNPs@ZIF-8, luminol-AgNPs with free Zn2+, and luminol-AgNPs with 0.1 M PBS (pH=7.4) containing 0.1 mM H2O2, PMT 800 V.
Electrochemical and ECL Behavior of LuminolAgNPs@ZIF-67.
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luminol and H2O2, after the addition of 0.13 mg/mL Ag+, showed only ~ 25% enhancement in the ECL intensity (Figure S7). Thus, compared with the 115-fold enhancement by the nanosystem, Ag+ played a negligible role in enhancing the ECL. Herein, luminol-AgNPs act as carriers of ECL reagent which can amplify the ECL signal as reported in previous studies.18,21 Therefore, the pronounced ECL enhancement in the case of ZIF-67 may attributed to its porous and ordered crystalline structure and the atomically dispersed Co2+, which facilitate the generation of oxygen radicals.19 Besides, the high surface area of the ZIF-67 particles makes it a good platform to support luminol-AgNPs, avoiding the agglomeration of AgNPs. The ECL spectra of luminol-AgNPs@ZIF-67 was shown in Figure S8, the ECL maximum emission wavelength was centered at about 470 nm, which corresponded to the light emission of the excited state oxidation product of luminol at 470 nm.23 As shown in Figure S9, the maximum ECL intensity reached at about 0.55 V, inconsistent with the low excitation potential of luminol.6 The possible ECL emission mechanism was proposed in Figure S10. Firstly, luminol was oxidized to luminol anion (eq 2 in Figure S10) upon anodic potential scanning on the surface of the electrode. Meanwhile, H2O2 was oxidized to O2˙‾on the surface of electrode (eq 3 in Figure S10). Then, luminol anion reacts with O2˙‾and produces the excited-state luminol anion, which emits light.24
Strategy for label-free immunosensor.
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Cyclic voltammetry (CV) was used to study electrochemical behaviors of the as-prepared luminol-AgNPs@ZIF-67 in 0.1 M PBS solution (pH=7.4) containing 0.1 mM H2O2, the ECL signal was generated at the interface of the modified electrode and solution. As shown in Figure 2A, for the luminol-AgNPs electrode, an oxidization peak at ~ 0.42 V and redox peaks at ~ 0.05 V were corresponding to the oxidation and reduction of AgNPs.22 For the ZIF-67 electrode, only a redox peak at about -0.05 V was observed which can be ascribed to the reduction of H2O2. While for the luminol-AgNPs@ZIF-67 electrode, the reduction peak of H2O2 was greatly enhanced, indicating that the integration of ZIF-67 and AgNPs enhances the reduction of H2O2. To investigate ECL enhancement mechanism of ZIF-67 on luminol, the ECL of the luminol-AgNPs, luminolAgNPs@ZIF-67 and control luminol-AgNPs with free Co2+ in solution were conducted. As shown in Figure 2B, the ECL signal of the luminol-AgNPs (Co2+-free) was about 30 au; while for the luminol-AgNPs@ZIF-67, the ECL signal was significantly raised to 3500 au which was probably 115-fold stronger than that of the luminol-AgNPs. To investigate the possible effect of free Co2+ on the ECL of luminol, control experiment was performed using luminol-AgNPs but adding Co2+ with the same content of that in ZIF-67 (obtained by ICP-MS) in the electrolyte solution (0.26 mg/mL). The result showed only ~ 4-fold enhancement in ECL signal. ECL Intensity / A. U.
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Figure 3. (A) Effect of the ratio of ZIF-67 to luminol-AgNPs in 0.1 M PBS buffer (pH=7.4) containing 1 mM H2O2. (B) Effect of pH value of the solution on ECL, with 0.1 M PBS containing 0.1 μM H2O2 (n=3).
In order to disclose the enhancement mechanism of ZIF-67 on the ECL of luminol, another zinc ion based MOF (ZIF-8) material was investigated. As shown in Figure S6, ZIF-8 particles exhibited dodecahedron structure which was very similar with those of ZIF-67, with side length of ~ 100 nm, which was smaller than that of ZIF-67. As shown in Figure 2C, the ECL intensity of the luminol-AgNPs@ZIF-8 showed only ~ 2-fold enhancement in ECL intensity as compared with free Zn2+ (in solution) of same concentration, indicating the key role of ZIF67 in the ECL enhancement of the nanosystem. As Zn2+ was found have negligible effect on the ECL of luminol, the moderate 2-fold enhancement in the case of ZIF-8 may be caused merely by the porous structure of the MOF nanomaterials. As shown in the CV curve of the luminol-AgNPs (cf. Figure 2A), the AgNPs have been oxidized during the ECL tests. The AgNPs@ZIF-67 particles with the same amount of those immobilized on the surface of the electrode was dissolved in 700 μL aqua regia, and the concentration of Ag+ detected by ICPMS was ~ 0.17 mg/mL; while after one CV cycling, the concentration of Ag+ in the electrolyte solution (700 μL PBS) was detected (by ICP-MS) to be ~ 0.13 mg/mL, which means about 76% AgNPs has been oxidized. Therefore, the effect of Ag+ on the enhancement of ECL was also investigated. Control experiments of a bare gold electrode with the same concentrations of
To investigate the practical use of the luminol-AgNPs@ZIF67, a label free sensor for cTnI was constructed. To achieve the optimal experimental performance, the ratio between luminolAgNPs and ZIF-67 particles and the pH of the reaction solution were investigated (Figure 3). The ratio of ZIF-67 to luminolAgNPs was adjusted from 0.2:1 to 2:1. As shown in Figure 3A, best ECL response was achieved when the ratio of ZIF-67 to luminol-AgNPs was 0.3, since MOF materials often encountered the problem of poor conductivity,11 too much ZIF-67 (when the ratio is higher than 0.3), the ECL intensity may decrease attributed to the poor conductivity of ZIF-67. While when the ratio is lower than 0.3, the cobalt ions may be not enough to catalyze the ECL reaction. So that the highest ECL signal was obtained at the ratio of 0.3. As the literatures reported, the ECL behavior of luminol was a pH dependent process. Therefore, in our study, the pH of the reaction solution was optimized in the range of 7.4 to 11.5, with the highest ECL achieved at pH 10.5 (Figure 3B), which was in accordance with previous study.18 Therefore, the 0.3:1 ratio of luminol-AgNPs to ZIF-67 and solution pH of 10.5 was chosen as the optimized conditions for constructing the ECL immunosensor for cTnI detection. The pulse potential was optimized in the range of 0-1.4 V, and the ECL intensity reached its highest value at 1.3 V (Figure S11A), this may be due to that at a higher potential, luminol can be electrochemically oxidized to luminol anion at a high speed.25 But when the potential was above 1.3 V, the ECL signal began to decline because the Au-S bond between gold electrode and 1, 3-propanedithiol molecules would be broken at a high potential.26 The initial potential was optimized in the range of 0.8 V to -1.3 V and the ECL intensity reached its highest value at -1.3 V (Figure S11B). This may be due to that at negative potential, H2O2 and the dissolved oxygen can be electrochemically reduced by ZIF-67 and AgNPs to generate superoxide radicals (O2•–) and hydroxyl radicals (•OH), which can react with
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Analytical Chemistry luminol anion and emit light.27 Therefore, -1.3 V and 1.3 V were chosen as the initial potential and pulse potential.28 The sensing mechanism and sensor fabrication procedures were depicted in Scheme 2. Luminol-AgNPs@ZIF-67 was immobilized on the gold electrode by 1,3-propanedithiol, after that, biotin-anti-cTnI was adsorbed on the silver nanoparticles by Ag-S bond, and BSA was used to block the residual nonspecific binding sites, and the modified electrode is ready for the detection of cTnI. CV curves obtained in 100 mM KCl solution containing 5.0 mM Ru(NH3)6Cl3 were used to characterize the modification processes and monitor the interfacial changes of the modified electrode (Figure 4A). After the assembly of 1,3-propanedithiol, the redox peak current of Ru(NH3)6Cl3 was decreased slightly, indicating the retardation of electron transfer after the assembly of 1,3-propanedithiol on Au electrode. After the modification of electrode with luminol-AgNPs@ZIF-67, a new pair of peaks appeared at ~ 0.15 V and -0.05 V which can be ascribed to the redox of AgNPs.22 With the sequential combination of biotin-anti-cTnI and BSA, the current density decreased gradually, ascribed to the block of electron transfer by the proteins. ECL responses with respect to each electrode modification step were also recorded in 0.1 M PBS and 0.1 mM H2O2 solution (Figure 4B). For bare gold electrode and the 1,3propanedithiol-modified electrode, there was no detectable ECL signal. After the modification of electrode with luminolAgNPs@ZIF-67, a high ECL signal was obtained, and then ECL signal declined after the modification of biotin-anti-cTnI and BSA, indicating that the adoption of proteins hampered the electron transport across the electrode. After incubating with cTnI, the ECL signal declined dramatically, indicating successful construction of the ECL immunosensor for cTnI detection. Scheme 2. Sensing mechanism and fabrication procedures of the label-free ECL immunosensor for the detection of cTnI.
SH-(CH2)3-SH
concentration of cTnI from 1 fg mL−1 to 1 μg mL−1 (Figure 4C), with a regression equation of I = −213.36 log (c / ng mL-1) + 822.59 (n = 3), and the correlation coefficient of 0.996. The ECL intensity is proportional to log[analyte], which is in consistent with literature reports on ECL sensing with MOFs.16,29 A limit of detection (LOD) of 0.58 fg mL-1 (S/N=3) was obtained, which is much lower than those obtained by other methods and also previously reported label-free cTnI ECL immunosensors (Table S1).
Luminol-AgNPs@ZIF-67
Au electrode bio-anti-cTnI
cTnI
BSA
As fast and ultrasensitive detection of cTnI is of critical clinical significance for cardiac infarction diagnosis, we investigated the effect of the incubation time of the immunosensor with sample on ECL signal. As shown in Figure S9, after the incubation of cTnI with modified electrode for ~ 0.5 h, the ECL signal reached a steady state, so that the full detection can be finished within ~ 30 min. Under the optimal conditions, the ECL intensity showed a linear decrease with increasing the
Figure 4. (A) CV characterizations of each modification step on gold electrode in 0.1 M KCl and 5.0 mM Ru(NH3)6Cl3 aqueous solution, (a) bare gold electrode (b) gold electrode/1,3-propanedithiol, (c) gold electrode/1,3-propanedithiol/luminol-AgNPs@ZIF67, (d) gold electrode/1,3-propanedithiol/ luminol-AgNPs@ZIF67/biotin-anti-cTnI, (e) gold electrode/1,3-propanedithiol/ luminolAgNPs@ZIF-67/biotin-anti-cTnI/BSA, (B) corresponding ECL responses of each electrode modification step in 0.1 M PBS buffer (pH=10.5) containing 0.1 mM H2O2: (a) gold electrode/1,3-propanedithiol/luminol-AgNPs@ZIF-67, (b) gold electrode/1,3-propanedithiol/ luminol-AgNPs@ZIF-67/biotin-anti-cTnI, (c) gold electrode/1,3-propanedithiol/ luminol-AgNPs@ZIF-67/biotinanti-cTnI/BSA, (d) gold electrode/1,3-propanedithiol/ luminolAgNPs@ZIF-67/biotin-anti-cTnI/BSA/cTnI, (e) bare gold electrode, (f) gold electrode/1,3-propanedithiol. (C) Calibration curve of cTnI detection (n=3).
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five ECL immunosensors were fabricated and examined by incubation of 0.1 pg mL-1 cTnI in 0.1 M PBS buffer (pH 10.5) containing 0.1 mM H2O2. As shown in Figure 5B and 5C, the relative standard deviation (RSD) of the tested five electrodes was ~ 5%, tested in 5 days, which confirms the reliability of our method the good stability of the immunosensor. Recovery experiments were further conducted by standard addition methods to investigate the practical use of the immunosensor. Different concentrations of cTnI were added to the human serum, and the sample was diluted by ten times by 0.1 M PBS (pH=7.4). The prepared sample solution was incubated with the modified electrode to test the ECL signal. As shown in Table 1, the as-fabricated immunosensor displayed great performance with the recovery of 97.8~104.8 %, which indicated that the designed ECL immunosensor can be applied to detect cTnI in human serum samples. Table 1. Quantitative Determination of cTnI in Healthy Human Serum Samples sample number
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The practical application of the ECL immunosensor was further investigated by detecting cTnI in the serum of myocardial infarct patients. The concentrations of cTnI in the diluted serum sample determined with our label-free sensor were consistent with those obtained by ELISA in hospital, as shown in Table 2. These results indicated that the developed method could be applied to the determination of cTnI in human plasma samples. Table 2. Quantitative Determination of cTnI in Patient Samples
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275.6
261.13±15.15
Time / day Figure 5. (A) Selectivity of the ECL immunosensor, measured at pH 10.5 in blank, IgG (10 ng mL-1), Mb (10 ng mL-1), D-biotin (10 ng mL-1), PSA (10 ng mL-1), BSA (10 ng mL-1), cTnI (1 ng mL-1), and the mixture (containing all the above analytes) (n=3). (B) Differences between five ECL immunosensors incubated with 0.1 pg mL-1 cTnI in 0.1 M PBS buffer (pH=10.5) containing 0.1 mM H2O2. Relative standard deviation (RSD) = 3% (n=5). (C) Stability of the ECL immunosensor tested in 5 days (n=3).
Analytical Performance of the Label-Free ECL Immunosensor. To check the selectivity of the cTnI ECL immunosensor, interfering proteins such as IgG, Mb, D-biotin, PSA, BSA were introduced during the detection. The concentration of all the interfering proteins were chosen as 10 ng mL-1 while cTnI was chosen as 1 ng mL-1 which is much higher than those in healthy peoples’ serum.30 As shown in Figure 5A, only the ECL responses of cTnI and its mixture were obviously decreased, while the introduction of IgG, Mb, D-biotin PSA or BSA has a negligible effect on the ECL signal. This indicates that the asprepared ECL immunosensor has excellent selectivity towards cTnI detection. Moreover, to investigate inter-electrode errors,
CONCLUSION In conclusion, an effective tactic was developed in this study to enhance ECL efficiency of the luminol system, by combined use of Co2+-based MOF (ZIF-67) and luminol-capped AgNPs. Due to the ordered crystalline structure, porosity and the atomically dispersed Co2+, the integrated MOF-based luminol nanosystem can facilitate the generation of oxygen radicals and greatly improve its ECL performance (with ~ 115-fold enhancement). Based on the fascinating sensing platform, a robust label-free ECL immunosensor was also constructed for ultrasensitive detection of cTnI, the main marker of myocardial infarction, with good stability and a detection limit as low as 0.58 fg mL-1 (S/N=3). This report is a successful attempt of the application of MOF for ECL area, pushing forward the detection sensitivity of ECL towards ultrasensitive bioassay.
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Analytical Chemistry Additional experimental procedures, characterizations and supplementary Figures S1-9 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author
[email protected];
[email protected] Author Contributions All authors have given approval to the final version of the manuscript.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by the National Key Research and Development Program of China (Grant No. 2016YFA0201300), the National Natural Science Foundation of China (grant Nos. 21475125 and 21675146), and the Instrument Developing Project of the Chinese Academy of Sciences (Grant No. YZ201666). We are pleased to acknowledge the generous support of ECL instrument by Prof. Guobao Xu, and the kind help from Prof. Liansheng Wang and Dr. Qiming Wang from First Affiliated Hospital of Nanjing Medical University for providing Patient samples and hospital test reports.
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