Coreactant-Free and Label-Free Eletrochemiluminescence

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Coreactant-free and Label-free Eletrochemiluminescence Immunosensor for Copeptin Based on Luminescent Immuno-Gold Nanoassemblys Zhili Han, Jiangnan Shu, Qiaoshi Jiang, and Hua Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05406 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

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Analytical Chemistry

Coreactant-free and Label-free Eletrochemiluminescence Immunosensor for Copeptin Based on Luminescent Immuno-Gold Nanoassemblys Zhili Han, Jiangnan Shu, Qiaoshi Jiang and Hua Cui* CAS Key Laboratory of Soft Matter Chemistry, iChEM (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China * Hua Cui, Fax: +86-551-63600730, E-mail: [email protected] ABSTRACT: In this work, the eletrochemiluminescence (ECL) behavior of Cu2+/cysteine complexes and N-(aminobutyl)-N(ethylisoluminol) (ABEI) functionalized gold nanoparticles combined with chitosan (Cu2+-Cys-ABEI-GNPs-CS) were studied by cyclic voltammetry and a double-step potential, which exhibited excellent ECL properties without any coreactant. It was found that the ECL intensity of Cu2+-Cys-ABEI-GNPs-CS could increase at least one order of magnitude compared with that of Cu2+-CysABEI-GNPs. Furthermore, a coreactant-free and label-free ECL immunosensor has been established for the determination of early acute myocardial infarction biomarker copeptin based on luminescent immuno-gold nanoassemblys consisting of Cu2+-Cys-ABEIGNPs-CS and immuno-gold nanoparticles prepared by connecting copeptin antibody with trisodium citrate stabilized gold nanoparticles. In the presence of copeptin, an obvious decrease in ECL intensity was observed due to the formation of antibody-antigen immunocomplex, which could be used for the determination of copeptin in the range of 2.0×10-14-1.0×10-11 mol/L with a detection limit of 5.18×10-15 mol/L. The detection limit of the ECL immunosensor is at least two orders of magnitude lower than that of sandwich immunoassays based on labeling technology. And the ECL immunosensor does not need any coreactant, and avoids complicated labeling and purification procedure. It is ultrasensitive, simple, specific and low-cost. This work reveals that the proposed luminescent immuno-gold nanoassemblys are ideal nanointerfaces for the construction of immunosensors. The proposed strategy may be used for the determination of other antigens if corresponding antibodies are available.

Acute myocardial infarction (AMI) is one of the most vital disease. Although general diagnostic technique has been significantly improved over the past decade, especially great effort has been increasingly deployed in the region of AMI diagnosis and therapy, death rate has not significantly decreased yet, mainly due to the lack of proper and rapid diagnostic technique for AMI.1 Among the various biomarkers of AMI evaluation, cardiac troponin I (cTnI) has been recognized as the gold standard biomarker for AMI diagnosis.2 However, it is difficult to make a definite diagnosis for AMI according to the level of cTnI within two hours of symptom onset.3 In recent years, arginine vasopressin (AVP) was found to play an important role in theoretical diagnosis of cardiovascular disease.4 However, its clinical detection was limited by the short half-life in the circulation.5 Copeptin is the C-terminal portion of the AVP precursor peptide and more stable than AVP, which could act as the surrogate biomarker for the release of AVP.6,7 The amount of copeptin has been reported to dramatically rise more than 14 pmol/L within two hours of AMI symptom onset.3 Therefore, copeptin has shown to provide amendatory diagnostic information for early discrimination of acute AMI in combination with cardiac troponin I.8 To date, the detection of copeptin mainly depends on immunoassays based on labeling technique, such as enzyme-linked immunosorbent assay,9 chemiluminescence (CL) sandwich immunoassays.6 However, these methods need complicated labeling and purification steps, which are tedious and time-consuming. Therefore, the development of a rapid, simple, sensitive, spe-

cific and low-cost immunosensor for copeptin is highly desired. Label-free immunosensor based on the direct detection of changes in optical, electrochemical and mass signal during the interactions between antigen and antibody without the second antibody have attracted great attention.10-13 They have inherent merits that are simple, fast and low-cost. Up to now, various label-free eletrochemiluminescence (ECL) immunosensors with high sensitivity have been developed to determine kinds of targets. For example, Guo’s group constructed an ECL immunosensor for the determination of vibrio parahaemolyticus based on N-(4-aminobutyl)-N-ethylisoluminol (ABEI).14 Ma’s group developed an ECL immunosensor to detect anti-cyclic citrullinated peptide antibody based on graphite-like carbon nitride.15 However, these ECL-based strategies usually need coreactants to achieve high ECL efficiency and sensitivity, such as H2O2, K2S2O8, Na2SO3, tripropylamine. Most of coreactants are not stable, which may affect analytical accuracy. They are also not environment-friendly and are not suitable for the determination of ECL signals with a thin-layer ECL cell such as ECL immunoassays based on microfluidic devices, microchips and screen-printed electrodes since the coreactants could be easily exhausted in working buffer with thin-layer ECL cell. Up to now, coreactant-free ECL immunosensors have rarely reported. In our previous work, Cu2+/cysteine complexes and ABEI functionalized gold nanoparticles (Cu2+-Cys-ABEI-GNPs)

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were successfully synthesized, which exhibited excellent CL activity.16 In this work, a nanocomposite of Cu2+-Cys-ABEIGNPs with chitosan (Cu2+-Cys-ABEI-GNPs-CS) was prepared and characterized. The ECL behavior of Cu2+-Cys-ABEIGNPs-CS were studied by cyclic voltammetry and a doublestep potential, which exhibited excellent ECL properties without any coreactant. It was found that the ECL intensity of Cu2+-Cys-ABEI-GNPs-CS could increase at least one order of magnitude compared with that of Cu2+-Cys-ABEI-GNPs. On this basis, a coreactant-free and label-free ECL strategy for copeptin immunosensor was developed based on luminescent immuno-gold nanoassemblys consisting of Cu2+-Cys-ABEIGNPs-CS and immuno-gold nanoparticles. The immuno-gold nanoparticles were obtained by connecting copeptin antibody (Ab) with trisodium citrate stabilized gold nanoparticles (citGNPs). In this strategy, Cu2+-Cys-ABEI-GNPs-CS was firstly assembled onto the surface of indium-doped tin oxide (ITO) electrode to form Cu2+-Cys-ABEI-GNPs-CS modified ITO electrode, then the immuno-gold nanoparticles were further coated on the modified ITO electrode via electrostatic interaction and Au-N bond to form luminescent immuno-gold nanoassemblys. The luminescent immuno-gold nanoassemblys can not only obtain strong and stable ECL signal, but also provide the recognition site for copeptin. The assembly process of ECL immunosensor were characterized by ECL, electrochemical impedance spectra (EIS) and scanning electron microscope (SEM). The assembly mechanism is proposed. The conditions for the determination of copeptin were optimized. The analytical performance of the proposed ECL immunosensor was studied. Finally, the applicability of the proposed immunosensor for the determination of copeptin in serum samples was explored.

EXPERIMENTAL SECTION Chemicals and materials. Stock solution of chloroauric acid (HAuCl4) (6.0 mM) were obtained via dissolving 1.0 g of HAuCl4•4H2O (Shanghai Reagent, China) in 412 mL of ultrapure water and stored at 4 °C. A 4.0 mM stock solution of ABEI were obtained by dissolving ABEI (TCI, Japan) in 0.1 M NaOH solution and stored at 4 °C. Cysteine was purchased from Shanghai Sangon Biotechnology Co. Trisodium citrate was purchased from Sinopharm Chemical Reagent Co., Ltd. Double sided polyimide tapes (DSPT) with silicone adhesive coating on two sides were purchased from KAIZHEN (Shenzhen, China). 1 % (w/w) CS (95% deacetylation) was obtained by dissolving chitosan in 2 % acetic acid solution, removing the insoluble substance and adjusting the pH to 5.0 with 1.0 M NaOH solution. Human immunoglobulin G (IgG) and bovine serum albumins (BSA) were obtained from Solarbio (Beijing, China). Cardiac myoglobin (Mb) and human serum albumin (HSA) were purchased from Sigma (St. Louis, MO). Copeptin was purchased from Genscript (Nanjing) and dissolved in ultrapure water. The sequence of copeptin was as follows: AlaSer-Asp-Arg -Ser-Asn-Ala-Thr-Gln-Leu-Asp-Gly-Pro-AlaGly-Ala-Leu-Leu Leu-Arg-Leu-Val-Gln-Leu-Ala-Gly-AlaPro-Glu-Pro-Phe-Glu-Pro-Ala-Gln-Pro-Asp-Ala-Tyr. The sequence of Y-H, 3Y-H and HGGG were as follows: NapPhe-Phe(F)-Glu-Tyr-Ile-OH, Nap-Phe-Phe(CF3)-Glu-Tyr-IleOH, (Trt)His-Gly-Gly-Gly, respectively. Fatty acid binding proteins (FABP) and copeptin antibody were provided by the First Affiliated Hospital of Nanjing Medical University. Serum samples were provided by the Hospital of University of Science and Technology of China. All of the other reagents

were all analytical grade. Ultrapure water was obtained by a Milli-Q system (Milli-pore, France) and used throughout the experiment. Apparatus. ECL was performed with a homemade ECL/electrochemical cell system, including an H-type electrochemical cell (homemade), a CHI 760B electrochemistry workstation (Chenhua, China) and a RFL-1 luminometer (Ruimai, China) equipped with a CR-105 photomultiplier tube (PMT) (Bingsong, China). EIS experiment was performed with CHI 760B electrochemistry workstation (Chenhua, China). The temperature of the reaction was controlled by A PST60 HL plus Thermo Shaker (Biosan, Latvia). Transmission electron microscope (TEM) images of the prepared Cu2+-CysABEI-GNPs, Cu2+-Cys-ABEI-GNPs-CS and cit-GNPs were recorded on an electronic microscopy (Jeol, JEM-2010F, Japan). The assemble process of the immunosensor was characterized on a JEM-2010 scanning electron microscope (Hitachi, Japan). The zeta potential was detected by using a zeta potential analyzer (Nano ZS90 Zetasizer, Malvern). Circular dichroism (CD) spectra were record on a Jasco J-810 spectrometer (Jasco, Japan). The PMT voltage was set as -600 V in the whole work. Preparation and ECL property of Cu2+-Cys-ABEIGNPs-CS. Cu2+-Cys-ABEI-GNPs were prepared as described previously with some modifications (Supporting Information, section S1).16 First, ABEI-GNPs were synthesized according to our previous work.17 Then cysteine and Cu2+ complexes were assembled on the surface of the ABEI-GNPs to form Cu2+-Cys-ABEI-GNPs. The obtained Cu2+-Cys-ABEI-GNPs were dispersed in 0.01 M NaOH solution and then combined with 1.0 % chitosan solution (pH = 5) as 1:1 (V/V). 20 µL of Cu2+-Cys-ABEI-GNPs-CS were dropped into the reservoir of the ITO electrode and dried in air atmosphere naturally. The ECL property of Cu2+-Cys-ABEI-GNPs-CS modified electrode was studied by cyclic voltammetry and a double-step potential under air atmosphere. Preparation and CD spectra of copeptin antibody conjugated cit-GNPs. Cit-GNPs were synthesized according to a reference.18 Then copeptin antibody conjugated cit-GNPs were prepared as described previously.19 CD spectra were used to characterize the copeptin antibody conjugated cit-GNPs (Figure S2). The results demonstrated that copeptin antibody was successfully immobilization on the surface of cit-GNPs and still kept activity (Supporting Information, section S2). Fabrication of ECL immunosensor. The ITO glass slide were used for working electrode and pretreated as described in a reference.19 The conductive area of ITO electrode was settled by DSPT with a punched round hole of 6 mm in diameter, which was used as a reservoir for subsequent assembly. The assemble steps are as follows. To start with, the Cu2+-CysABEI-GNPs dispersed in 0.01 M NaOH solution were combined with 1.0 % (w/w) CS solution (pH = 5). 20 µL of Cu2+Cys-ABEI-GNPs-CS were dropped into the conductive surface of an ITO electrode and dried in air atmosphere naturally. A homogeneous Cu2+-Cys-ABEI-GNPs-CS layer was formed on the surface of ITO electrode. Then, 40 µL of copeptin antibody conjugated with cit-GNPs (Ab-GNPs) solution were dropped on the Cu2+-Cys-ABEI-GNPs-CS/ITO electrode and incubated at 4 ℃ for 9 h. Next, 40 µL 1% (w/w) BSA in 0.01 M phosphate buffered saline (PBS, containing 0.01 M Na2HPO4/NaH2PO4 and 0.02 M NaCl, pH = 7.4) were used to block non-specific binding sites for 40 minutes at 37 ℃. It was


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Analytical Chemistry worth to note that each step of the assembly needed to be washed by PBS buffer for three times. Finally, the BSA/AbGNPs/Cu2+-Cys-ABEI-GNPs-CS/ITO electrode was successfully obtained and could be directly used for the sensing of copeptin. Copeptin detection. 40 µL of copeptin solutions with various concentrations were dropped on the above modified electrode and incubated at 37 ℃ for 50 minutes, respectively, followed by washing with PBS buffer. As copeptin was dissolved in ultrapure water, 40 µL of ultrapure water was used for the blank experiment. ECL measurements were performed by the homemade ECL system (Supporting Information, section S3). The difference value of ECL intensity (∆I) in the absence and presence of copeptin under a double step potential was used for quantitative determination of copeptin. RESULTS AND DISCUSSION Characterization of Cu2+-Cys-ABEI-GNPs-CS. Cysteine and Cu2+ complexes were assembled on the surface of the ABEI-GNPs to form Cu2+-Cys-ABEI-GNPs as described previously with some modifications (Supporting Information, section S1). Considering the good biocompatibility, filmforming and easy assembly on the surface of electrode, Cu2+Cys-ABEI-GNPs-CS was prepared by mixing Cu2+-CysABEI-GNPs with CS. The zeta potential of Cu2+-Cys-ABEIGNPs and Cu2+-Cys-ABEI-GNPs-CS was -33.2 and 28.8 mV, respectively, indicating strong electrostatic interaction between positively charged CS and negatively charged Cu2+Cys-ABEI-GNPs. The morphology of Cu2+-Cys-ABEI-GNPs and Cu2+-Cys-ABEI-GNPs-CS were characterized by TEM. As shown in Figure 1, Cu2+-Cys-ABEI-GNPs were spherical nanoparticles with an average size of approximate 18 nm (Figure 1A) and Cu2+-Cys-ABEI-GNPs could disperse well in CS film (Figure 1B). It was found that the interparticle distance of Cu2+-Cys-ABEI-GNPs-CS (Figure 1B) was obviously increased compared with that of Cu2+-Cys-ABEI-GNPs (Figure 1A). It was reported that CS could act as the stabilizing agent for gold nanoparticles due to electrostatic repulsions of ammonium.20 Cu2+-Cys-ABEI-GNPs-CS with zeta potential of 28.8 mV rendered sufficient

charge to make gold nanoparticles disperse well. The molecular weight of CS used in this work was 20,000 Da. Hence, the CS might be present in an unfolded conformation the surface of Cu2+-Cys-ABEI-GNPs.21 In conclusion, the enlarged interparticle distance of Cu2+-Cys-ABEI-GNPs-CS were attributed to the electrostatic and mechanical barrier property of CS. ECL Property of Cu2+-Cys-ABEI-GNPs-CS. The ECL properties of Cu2+-Cys-ABEI-GNPs-CS were explored by dropping it on the surface of an ITO electrode in 0.02 M phosphate buffer (PB) (pH=11.8) using cyclic voltammetry and a double-step potential without any coreactants. Figure 2A shows IECL–E curves of Cu2+-Cys-ABEI-GNPs-CS with initial positive scan direction. Two ECL peaks (ECL-1 and ECL-2) were observed in positive potential scan. The ECL spectra of ECL-1 and ECL-2 showed an emission at about 440 nm (Figure 2B), indicating that both of ECL-1 and ECL-2 were related to ABEI.22 The ECL property of Cu2+-Cys-ABEI-GNPs was also studied by cyclic voltammetry under the same conditions to explore the ECL effect of CS in Cu2+-Cys-ABEI-GNPs-CS. As shown in Figure 2A, two ECL peaks (ECL-1´ and ECL-2´) of Cu2+-Cys-ABEI-GNPs with weak intensity were observed. By contrast, the ECL intensity of Cu2+-Cys-ABEI-GNPs-CS was over one order of magnitude higher than that of Cu2+-CysABEI-GNPs, indicating CS could enhance the ECL emission. Willets and coworkers revealed that polymer film could reduce the diffusion of the radicals and prolong the excited state lifetime of luminophores in the ECL reaction.23 Accordingly, CS as a natural polymer may follow similar mechanism to enhance ECL emission by reducing the diffusion of the radicals generated on the surface of gold nanoparticles and prolonging excited state lifetime of the excited-state oxidation product N-(aminobutyl)-N-(ethylphthalate) (ABEI-ox*) in the CS film.

Figure 2. (A) IECL–E curves of Cu2+-Cys-ABEI-GNPs and Cu2+Cys-ABEI-GNPs-CS, (B) ECL spectra of ECL-1, ECL-2 in the IECL–E curve (Figure 2A) and ABEI obtained using a spectrofluorophotometer with lamp off, (C) CV of Cu2+-Cys-ABEIGNPs-CS in the initial positive scan direction in 0.02 M PB (pH 11.8). Inset shows the enlarged semidifferential voltammogram from 0.50 to 0.70 V in (C), (D) ECL signals of Cu2+-Cys-ABEIGNPs-CS on an ITO electrode under pulse potential in 0.02 M PB (pH 11.8). Initial potential 0 V; pulse time 0.05 s; pulse period 20 s; pulse potential 1.1 V. Figure 1. TEM image of (A) Cu2+-Cys-ABEI-GNPs and (B) Cu2+Cys-ABEI-GNPs-CS. Scale bar, 200 nm.



The effect of air-saturated, oxygen and nitrogen atmospheres on ECL emission of Cu2+-Cys-ABEI-GNPs-CS was examined (Supporting Information Figure S3). Figure S3 shows the IECL–E curves of Cu2+-Cys-ABEI-GNPs-CS in 0.02 M PB (pH 11.8) under various atmospheres using the initial positive scan. Compared with the ECL peaks under an air atmosphere, the intensity of ECL-1 increased under an oxygen atmosphere and decreased obviously under a nitrogen atmosphere, which is opposite for ECL-2. These results suggested that O2 was involved in the reactions of ECL-1.When ECL-1 is strong, ECL-2 became weak. It seems that ECL-2 is related to the remaining amount of ABEI after ECL-1 process. ECL-1 started from 0.5 V and reached its maximum at 0.8 V, which corresponded to the oxidation of ABEI (inset in Figure 2C) to ABEI•− radicals.24 ECL-2 peaked at 1.1 V. Dimethyl sulfoxide (DMSO) and p-benzoquinone are effective radical scavengers of hydroxyl radical (OH•) and superoxide radical (O2•−).25,26 As shown in Figure S4A, DMSO did not show obvious effect on the intensity of both ECL peaks. The relative standard deviation (R.S.D.) of ECL-1 and ECL-2 intensity in the presence of different concentration of DMSO were 2.3 % and 4.6 %, respectively, indicating that OH• was not involved in the ECL1and ECL-2 process. Both of ECL-1 and ECL-2 intensity decreased with increasing the concentration of p-benzoquinone (Figure S4B), indicating that O2•− was involved in the ECL1and ECL-2 process. Moreover, the intensity of ECL-2 dramatically increased with increasing the pH of working buffer (Figure S5), which indicated that OH– was also involved in the ECL-2 process. Based on the above results, the reaction mechanism of ECL-1 and ECL-2 were described as follows. ECL-1 might be due to that ABEI•− electro-oxidized by ABEI in alkaline solution could react with O2 to form O2•−, which further interacted with ABEI•− to generate light emission.27,28 For ECL-2 process, on one hand, OH– could be oxidized to oxygen under the oxygen evolution potential in alkaline solution,26 which could react with ABEI•− electro-oxidized by ABEI to generate O2•−. Then O2•− further reacted with ABEI•– to generate light emission. On the other hand, ABEI•− might be also converted into ABEI-ox* via direct electrooxidation, accompanying by light emission.27,29 As shown in Figure 2D, when a double-step potential (pulse time 0.05 s, pulse period 20 s, pulse potential 1.1 V) was applied to the modified ITO electrode, pulse ECL signals were obtained and the ECL signals for fifteen times circulation were quite stable and strong. Under the same condition, the ECL intensity of Cu2+-Cys-ABEI-GNPs-CS was compared with other ECL functionalized nanomaterials, including ABEI functionalized GNPs (ABEI-GNPs), 2-[Bis[2-[carboxymethyl[2-oxo-2-(2sulfanylethylamino)ethyl]amino]ethyl]amino]acetic acid/Co2+ complexes and ABEI bifunctionalized GNPs (DTDTPA/Co2+ABEI-GNPs) and Cu2+-Cys-ABEI-GNPs as shown in Figure S6. As a result, the ECL intensity follows the order: Cu2+-CysABEI-GNPs-CS > DTDTPA/Co2+-ABEI-GNPs > Cu2+-CysABEI-GNPs > ABEI-GNPs. Accordingly, Cu2+-Cys-ABEIGNPs-CS exhibited excellent ECL properties and may be used as a nanointerface for bioassays. Self-assembly strategy for ECL immunosensor. The ECL immunosensor for the determination of copeptin is schematically described as shown in Scheme 1. Cu2+-Cys-ABEI-GNPsCS was assembled on the surface of an ITO electrode via

Scheme 1. Schematic illustration of proposed coreactant-free and label-free ECL immunosensor for copeptin based on luminescent immuno-gold nanoassemblys.

simply dropped and dried in air naturally. Secondly, due to the good biocompatibility, large surface area and conductivity property of cit-GNPs, copeptin antibody were conjugated with cit-GNPs to form immuno-gold nanoparticles. Thirdly, AbGNPs were immobilized on the surface of Cu2+-Cys-ABEIGNPs-CS modified ITO electrode. On the one hand, the zeta potential values of Cu2+-Cys-ABEI-GNPs-CS and Ab-GNPs were 28.8 mV and-4.03 mV, respectively, indicating that AbGNPs could be connected with Cu2+-Cys-ABEI-GNPs-CS by electrostatic interact-tion. On the other hand, as CS has a large number of amino groups,20 Cu2+-Cys-ABEI-GNPs-CS modified ITO electrode could form an amino-rich film, which could interact with Ab-GNP to generate covalent Au-N bond. Finally, the non-specific binding sites on the assembled ITO electrode were blocked by BSA to form BSA/Ab-GNPs/Cu2+-CysABEI- GNPs-CS modified ITO electrode, i.e., immunosensor. When a pulse potential was applied to the immunosensor without any coreactant, strong ECL emissions could be obtained. In the presence of copeptin, ECL signal decreased greatly due to the formation of antibody-antigen complexes. The ECL signals in the absence and presence of copeptin, were recorded as I0 and I, respectively. The decreased ECL signal ∆I (∆I = I0 - I) could be used for quantitative determination of copeptin. Characterization of the fabricated ECL immunosensor. To monitor the fabrication of ECL immunosensor during dif-

Figure 3. (A) ECL signals under pulse potential obtained on (a) bare ITO electrode, (b) Cu2+-Cys-ABEI-GNPs-CS/ITO electrode, (c) Ab-GNPs/Cu2+-Cys-ABEI-GNPs-CS/ITO electrode, (d) BSA/Ab-GNPs/Cu2+-Cys-ABEI-GNPs-CS/ITO electrode, (e) copeptin/BSA/Ab-GNPs/Cu2+-Cys-ABEI-GNPs-CS/ITO electrode (copeptin 2.0×10-13 mol/L). (B) ECL signals of immunosensor on an ITO electrode under pulse potential in 0.02 M PB (pH 11.8). Pulse potential 1.1 V, pulse time 0.05 s, pulse period 20 s, 0.02 M PB (pH 11.8).


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Analytical Chemistry Table 1. A comparison of ECL immunosensor with other reported immunosensors for determination of copeptin. Analytical method

Sensing platform

Linear range (pM)

Detection limit (pM)

Commercial Kits or Ref.

ELISAa

HRP-TMBb

24-558

24

Phoenix Pharmaceutical

a

b

15.6-1000

3.9

CUSABIO

ELISA

HRP-TMB

CL

Acridinium ester

2.25–1215

2.25

30

ECL

Cu2+-Cys-ABEI-GNPs-CS

0.02-10

0.00518

This work

a

ELISA: Enzyme-linked immunosorbent assay b HRP-TMB: Horseradish peroxidase - 3,3',5,5'-Tetramethylbenzidine

ferent steps, the assembled ITO electrodes were characterized by ECL, EIS and SEM. As shown in Figure 3A, no ECL signals were observed on a bare ITO electrode (curve a), which exhibited a small electron-transfer resistance value (Figure S8, curve a). When Cu2+-Cys-ABEI-GNPs-CS were assembled on the surface of an ITO electrode, strong ECL signals were observed (curve b). Due to the poor conductivity of CS, the Cu2+- Cys-ABEI-GNPs-CS modified electrode showed a remarkable increased electron transfer resistance (Figure S8, curve b). When the Ab-GNPs were further assembled on the modified ITO electrode, the ECL emissions were greatly increased (curve c). In comparison, when the cit-GNPs were connected to the surface of Cu2+-Cys-ABEI-GNPs-CS modified ITO electrode (Figure S7b), the ECL intensity was stronger than that of Cu2+-Cys-ABEI-GNPs-CS modified ITO electrode (Figure S7a). In addition, the electron transfer resistance of Ab-GNPs/Cu2+-Cys-ABEI-GNPs-CS decreased significantly compared with that of Cu2+-Cys-ABEI-GNPs-CS modified ITO electrode from the result of EIS (Figure S8, curve c). Therefore, the increased ECL emission of AbGNPs/Cu2+-Cys-ABEI-GNPs-CS modified electrode compared with that of Cu2+-Cys-ABEI-GNPs CS modified electrode was due to good conductivity property of cit-GNPs. After binding with BSA, ECL intensity was obviously decreased (Figure 3A, curve d) since protein could inhibit the electron transfer (Figure S8, curve d). Similarly, the ECL signals further decreased after reacting with copeptin, indicating the successful combination of antibody and antigen since peptide could also inhibit the electron transfer (Figure S8, curve

e). Therefore, the ECL immunosensor was successfully obtained and could be used for the quantitative detection for copeptin. The stability of ECL immunosensor was explored as shown in Figure 3B. The R.S.D. of ECL signals in 15 periods was 1.984 %, indicating excellent stability of the ECL immunosensor. Figure 4 displays the typical SEM images of the modified electrode at each immobilization step. The morphology of the Cu2+-Cys-ABEI-GNPs-CS (A) exhibited a film with rough structure. After reacting with Ab-GNPs for 9 hours, the Cu2+Cys-ABEI-GNPs-CS film was covered with a large amount of well-dispersed Ab-GNPs as shown in Figure 4B, demonstrating the successful connection of Ab-GNPs on the Cu2+-CysABEI-GNPs-CS modified electrode. When BSA was incubated with the modified electrode, the morphology of the modified ITO electrode changed (Figure 4C), indicating the coating of BSA. In the presence of copeptin, the SEM image became blurry (Figure 4D), indicating that copeptin was connected to the surface via specific interaction between antigen and antibody since the cover of copeptin led to a decrease in concavity and convexity surface of the modified ITO electrode. The SEM results together with the EIS results revealed that the electrodes were fabricated as expected. Analytical performance of the label-free ECL immunosensor. Under the optimized conditions (pH 11.8, pulse potential 1.1 V, pulse time 0.05 s, pulse period 20 s and incuba- tion time 50 minutes; Supporting Information, section S8), the quantitative performance of the as-prepared immunosensor for copeptin was evaluated by measuring the values of ∆I on the concentration of copeptin. The calibration curve for the detection of copeptin is shown in Figure 5. The ECL intensity de-

Figure 4. Representative SEM images of (A) Cu2+-Cys-ABEIGNPs-CS, (B) Ab-GNPs/Cu2+-Cys-ABEI-GNPs-CS, (C) 2+ BSA/Ab-GNPs/Cu -Cys-ABEI-GNPs-CS, (D) copeptin/BSA/Ab-GNPs/Cu2+-Cys-ABEI-GNPs-CS modified ITO electrode.

Figure 5. Linear relationship between ECL response and logarithm of copeptin concentration. Initial potential, 0 V; pulse potential, 1.1 V; pulse time, 0.05 s; pulse period, 20 s. All ECL signals were measured in 0.02 M PB (pH 11.8) solution.



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Table 2. Quantitative determination of copeptin in healthy human serum samplesa

a

Mean

Serum sample

Copeptin found (pM)

Copeptin added (pM)

Total copeptin detected (pM)

Recovery (%)

1

0.056 ± 0.0052

0.15

0.220 ± 0.014

109.3

2

0.049 ± 0.0054

1.0

1.023 ± 0.046

97.4

3

0.076 ± 0.0081

1.5

1.666 ± 0.035

106

value

±

SD

of

three

creased with the increase of copeptin concentration and exhibited linear variation with the logarithm of copeptin concentration in the range of 2.0×10-14 to 1.0×10-11 mol/L. The regression equation was ∆I = 4499.94+274.35×log C (unit of C is mol/L) and its correlation coefficient is 0.994. ∆I was the relative ECL intensity calculated by I0－I, the value of I0 and I were the ECL intensity in the absence and presence of copeptin, respectively. The detection limit with a signal-to-noise ratio of 3 was 5.18×10-15 mol/L. The R.S.D. of seven replicate detections of copeptin at1.0×10-12 mol/L within a day (intraday precision, n = 7) was 1.44 %, which was 4.93 % in 7 days (inter-day precision, n = 7). The result demonstrated that the ECL immunosensor had good reproducibility. A comparison of the ECL immunosensor with other immunoassays based on labeling technique for the determination of copeptin is list in Table 1. The detection limit of the proposed immunosensor is three orders of magnitude lower than that obtained by commercial ELISA kit (3.9×10-12 mol/L) and over two orders of magnitude lower than previously reported CL sandwich immunoassays.30 The results show that immunosensor is superior to the reported immunoassays based on labeling technique in sensitivity. And the immunosensor does not need any coreactant and labeling procedure, which is much faster and simpler than that of labeling technique. Selectivity of the ECL immunosensor. The developed immunosensor was supposed to be selective toward copeptin detection since the sensing depended on antibody-antigen recognition. Thus, interference experiment was conducted to explore the specificity of copeptin immunosensor. Seven polypeptides and proteins including Y-H, 3Y-H, HGGG, MB, HAS, FABP and IgG were used for the immunosensor instead of copeptin (Figure 5, inset). The concentration of these interferents was one order of magnitude higher than that of copeptin. The results indicated that only copeptin demonstrated the strong ∆I, whereas other interferents including Y-H, 3Y-H, HGGG, MB, HAS, FABP and IgG showed very weak ∆I. The ECL response from the mixture of copeptin with Y-H, 3Y-H, HGGG, MB, HAS, FABP and IgG was also examined, which was close to that of copeptin solution. All these results exhibited that the developed immunosensor could selectively detect copeptin. Determination of copeptin in healthy human serum samples. To explore the practicability of the ECL immunosensor, the ECL immunosensor was used to determine copeptin in real human serum samples. The healthy human serum samples were obtained from the Hospital of University of Science and Technology of China and stored in -20 ℃. The samples were diluted 100 times with 0.01 M PBS (pH = 7.4) and did not need other pretreatment prior to determination. The results acquired from the proposed immunosensor is shown in Table 2. The content of copeptin in the serum samples are in range of healthy person (1-13.8 pmol/L).6 The good recoveries (97.4%-109.3%) demonstrated that serum sample matrices did not affect the detection of copeptin, indicating

independent

experiments,

n

=

3.

that the ECL immunosensor is of great potential for rapid and accurate determination of copeptin in patients with suspected AMI.

CONCLUSIONS A coreactant-free and label-free ECL immunosensor has been established for the determination of early AMI biomarker copeptin based on luminescent immuno-gold nanoassemblys consisting of Cu2+-Cys-ABEI-GNPs-CS and immuno-gold nanoparticles. The luminescent immuno-gold nanoassemblys on the surface of an ITO electrode exhibited excellent ECL property, immuno-activity and good stability. In the presence of copeptin, the ECL signal decreased due to inhibition of the electron transfer by peptides. According to the decreased ECL intensity, the concentration of copeptin could be determined in range of 2.0×10-14-1.0×10-11 mol/L with extremely low detection limit of 5.18×10-15 mol/L, which was at least 2-3 orders of magnitude lower than that of obtained by commercial ELISA kit and CL sandwich immunosensor. And the proposed immunosensor does not need any coreactant, and avoids complicated labeling and purification procedure. Besides, the ECL immunosensor is also simple, specific and low-cost, which can be used for the determination of copeptin in human serum samples. This work reveals that the proposed luminescent immuno-gold nanoassemblys are ideal nanointerfaces for coreactant-free and label-free immunoassays. The proposed strategy may be used for the determination of other antigens if corresponding antibodies are available. Furthermore, the coreactant-free ECL immunoassay is of great application potential in ECL immunoassays based on microfluidic devices, microchips and screen-printed electrodes.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text (S1. Preparation and of ABEI-GNPs and Cu2+-Cys-ABEI-GNPs, S2. Preparation and circular dichroism spectra of copeptin antibody conjugated citGNPs, S3. ECL measurements, S4. Effect of N2, O2 atmospheres, radical scavengers and pH on IECL-E curves of Cu2+-Cys-ABEIGNPs-CS, S5. An ECL intensity comparison of Cu2+-Cys-ABEIGNPs-CS with other ECL functionalized nanomaterials, S6. An ECL intensity comparison of Cu2+-Cys-ABEI-GNPs-CS with citGNPs/Cu2+-Cys-ABEI-GNPs-CS and Ab-GNPs/Cu2+-Cys-ABEIGNPs-CS, S7. Electrochemical impedance spectra of the ECL immunosensor, S8. Optimization of experimental conditions. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Fax: +86-551-63600730. E-mail: [email protected]


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Analytical Chemistry ORCID Hua Cui: 0000-0003-4769-9464

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

(25) Stylidi, M.; Kondarides, D. I.; Verykios, X. E. Applied Catalysis B: Environmental 2004, 47, 189-201. (26) Bardouki, H.; Da Rosa, M. B.; Mihalopoulos, N.; Palm, W.U.; Zetzsch, C. Atmospheric Environment 2002, 36, 4627-4634. (27) Cui, H.; Zou, G.-Z.; Lin, X.-Q. Anal. Chem. 2003, 75, 324331. (28) Arai, K.; Takahashi, K.; Kusu, F. Anal. Chem. 1999, 71, 22372240. (29) Heng, A. J. C. X. C. Chinese. J. Anal. Chem. 1988, 2, 007. (30) Dobša, L.; Cullen Edozien, K. Biochem. Med. 2013, 23, 172190.

ACKNOWLEDGMENT The support of this research by the National Key Research and Development Program of China (Grant No. 2016YFA0201300) and the National Natural Science Foundation of China (Grant Nos. 21475120 and 21527807) are gratefully acknowledged.

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