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A Compatible Sensitivity Enhancement Strategy for Electrochemiluminescence Immunosensors Based on the Biomimetic Melanin-Like Deposition Hongmin Ma, Yanhua Zhao, Yuanyuan Liu, Yong Zhang, Dan Wu, He Li, and Qin Wei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04397 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 28, 2017

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Analytical Chemistry

A Compatible Sensitivity Enhancement Strategy for Electrochemiluminescence Immunosensors Based on the Biomimetic Melanin-Like Deposition

Hongmin Ma, Yanhua Zhao, Yuanyuan Liu, Yong Zhang, Dan Wu, He Li, Qin Wei*

Key Laboratory of Interface Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China

Corresponding author: Tel: 86-531-82767872 Fax: 86-531-82765969 E-mail: [email protected] (Qin Wei)

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ABSTRACT In this work, a compatible strategy was demonstrated for the enhancement of detection

sensitivity

of

sandwich-type

electrochemiluminescence

(ECL)

immunosensors. The enhanced signal response was based on the combination of biomimetic melanin-like deposition with the effective ECL quenching ability of quinone-rich biopolymers. Gold nanoparticle-loaded horseradish peroxidase (HRP) was used as a catalytic label for the secondary antibodies. The intrinsic catalytic property of HRP towards hydrogen peroxide (H2O2) generates reactive oxygen species, which highly promote the auto-polymerization of catecholamines. The resulting

fast

deposition

of

quinone-rich

biopolymers

approaching

the

luminophor-incorporated sensing platform achieves an obvious ECL quenching. A broad-spectrum tumor marker alpha fetoprotein (AFP) was selected as a model analyte to demonstrate the feasibility of the proposed strategy. Under optimal conditions, a very low detection limit of 0.056 pg·mL-1 was obtained. Two orders of magnitude enhancement was achieved in contrast to the signal response without the step of catalytic biopolymer deposition. The combination of compatible HRP labeling with unique melanin-like deposition has potential as a universal strategy in other ECL bioassays.

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Introduction With the combination of traditional electrochemistry and chemiluminescence, electrochemiluminescence (ECL) has been well recognized as a charming technique in analytical chemistry for the detection of metal ions, small molecules, biomarkers and cancer cells.1-3 Although the real application of ECL biosensors in clinical diagnosis is still limited, this technique shows promising potential due to the simple instrumentation requirement and high sensitivity. The separation of the output optical signal from the input electrochemical signal enables the detection with very low background signal. The well-established sensitivity enhancement innovations or signal amplifying strategies in electrochemistry, optical sensor and other analytical technologies can be easily transplanted to the sensing platform especially for the solid state ECL biosensors, which involves the incorporation of luminophors into electrode surfaces.4-6 Generally there are two sensing mode, that is off-on and on-off, for the response of the analyte. The on-off sensing mode depends on the signal decrease resulting from the biorecognition events and the increase of the steric hindrance. While the steric hindrance induced signal depression is very limited, various ECL quenching systems have been proposed for the enhancement of detection sensitivity. For example, nanomaterials-based resonance energy transfer has been adopted broadly to develop ECL quenching systems.7-9 Although great innovations have been achieved,10 the development of simple sensitivity enhancement strategy compatible with the current biological and medical methods will promote the future application of ECL biosensors. Polydopamine (PDA) produced from oxidation of dopamine is a synthetic insoluble melanin-like polymer and possess some intriguing physicochemical properties to mimic the natural pigments in living organism.11 The unique mussel-inspired material-independent adhesion behavior has enabled the wide application of PDA in the field of material science, nanomedicine and bioelectronics.12 The bioconjugation property of PDA enables the

facile

immobilization of biomolecules on different nanoparticles and surfaces,13 showing promising application in biosensors. PDA and its derivative materials have been used 3



as

grafting

materials

and

label

carriers

to

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develop

electrochemical,14

photoelectrochemical,15 and colorimetric biosensors.16 While these quinone-rich polymers have been well exploited as energy harvesting and storage materials,17-18 the physicochemical properties are less utilized for the development of biosensors. Recently, the ﬂuorescence quenching ability of PDA and melanin-like nanostructures have been demonstrated and used to create sensing platforms.19-21 However, the sensitivity enhancement strategy based on the quenching ability towards ECL is less reported. Considering the strong free radical scavenging activity of quinone structure,22 oxidative polymerization of melanin-like structures holds great promise for the ECL sensing technologies.

Scheme 1. The signal response principle of the proposed ECL immunosensor. Besides the auto-polymerization of catecholamines in aerated solutions, the production of melanin-like polymers can also be achieved by enzymatic polymerization using peroxidase/H2O2 combination.22 Dramatic enhancement of detection-sensitivity in immunoassays has been demonstrated based on the combination of enzymatic fast PDA deposition with the above-mentioned bioconjugation capabilities.23 In this work, the enzymatic fast biopolymer deposition was firstly combined with the ECL quenching ability to enhance the detection sensitivity of ECL biosensors (Scheme 1). The other highlight of the proposed strategy includes: 1) the use of gold nanoparticles (Au NPs) as carriers enables not 4


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only the facile bioconjugation but also the multi-labeling of enzyme. 2) Epinephrine (EP) was exploited as an alternative for dopamine to avoid the undesired deposition through the covalent interaction between amino groups and gold-containing elements, which are usually used for the construction of ECL sensing platform. 3) The ECL of the semiconductor luminophor was efficiently quenched and high detection sensitivity was obtained. The simple combination of biomimetic melanin-like deposition with ECL quenching may reveal a universal approach for ECL-based bioassays.

Figure 1. TEM images of g-C3N4@PANI-Au hybrids (A and B) with different amplification. The arrows in A indicate the presence of irregular-shaped g-C3N4 nanosheets. The arrows in B indicate the margin of a dumbbell-like PANI-Au nanocomposite. Results and Discussion Graphite-like carbon nitride (g-C3N4) nanosheets (Figure 1A and Figure S1A) was used as an ECL luminophor due to the high stability and emission efficiency. An asymmetric dumbbell-like polyaniline-gold (PANI-Au) nanocomposite (Figure 1B and Figure S1B)was exploited as a matrix to improve the interfacial stability of g-C3N4 and immobilize the capture antibodies. The improved ECL behaviors of g-C3N4@PANI-Au hybrids (Figure S3) based on the utilization of asymmetric dumbbell-like PANI-Au nanocomposite was reported elsewhere.24 The sensing platform was fabricated by modifying glass carbon electrode (GCE) using g-C3N4@PANI-Au hybrids. For a standard sandwich-type immunosensor, bovine serum albumin (BSA) was used to block the nonspecific biding sites on the sensing platform. Citric acid capped Au NPs with narrow size distribution and an average diameter of 14 nm (the size distribution and the surface plasmon resonance spectrum 5



were shown in Figure S2) were used to load HRP and label the detection antibodies. The interfacial immunological recognition induces the incorporation of HRP to the electrode surface. Before the ECL measurement, the unique catalytic effect of HRP on H2O2 enables the fast polymerization of EP and generates melanin-like poly-epinephrine (PEP). It has been demonstrated that the presence of biomacromolecules on solid surface promotes the local deposition of PDA.23 The immobilized capture antibody and the blocking BSA on the GCE also create the circumstance for the deposition of PEP on the electrode (Scheme 1). After the step of PEP deposition, the ECL signals will be significantly suppressed due to the strong free radical scavenging activity of melanin-like biopolymers.25 Thus a greatly enhanced signal response was achieved for the detection of the biomarker.

Figure 2. (A) The absorbance measured at 400 nm for the EP solutions with and without the presence of H2O2 or HRP: 5 mg mL-1 EP, 20 mmol L-1 H2O2, 0.01 µg mL-1 HRP, pH 8.0. (B) The effect of pH on the rate of oxidative polymerization of EP. (C) The quenching effect of PEP deposition on the ECL of g-C3N4. (a) ECL signal of a g-C3N4@PANI-Au modified electrode, (b) ECL signal of the electrode after the modification with Au NP-loaded HRP. (c) ECL signal of the electrode after the further deposition of PEP. The deposition time for PEP in (B) and (C) was 1 min.

Because the catecholamines can self-polymerize in aerated solutions especially under basic conditions, the auto-polymerization of EP without the HRP promotion effect will generate background signal response. Due to the chromophoric behavior of catecholamine oxidation, the polymerization rate of EP can be easily monitored using UV-vis absorption spectra. The polymerization of EP under aerated conditions, in the 6


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presence of H2O2, and in the presence of HRP was investigated (Figure 2A). The combination of HRP with H2O2 significantly promotes the polymerization rate of EP. The promoted fast polymerization enables the establishment of the sensitivity enhancement strategy using HRP as a catalytic label to increase the signal response. The background signal response from the auto-polymerization of EP can be effectively suppressed by controlling the reaction time. In present work, 1 min was selected as the deposition time for PEP while the background signal response can be ignored. The effect of pH value of the deposition solution on the polymerization rate of EP was also investigated (Figure 2B). It can be seen that the maximum polymerization rate was obtained at the pH of 8.0, while the auto-polymerization of EP is still weak. At higher pH values, the auto-polymerization of EP becomes prominent and the rate of catalytic polymerization is decreased, possibly due to the decreased catalytic activity of HRP at higher pH values. The quenching effect of deposited PEP on ECL of g-C3N4 was confirmed by the modification with Au NP-loaded HRP and the following PEP deposition (Figure 2C). A contrast experiment in the absence of H2O2 was also performed and no obvious signal response was obtained, indicating the necessary of H2O2 for the fast deposition of PEP. However, when dopamine was used for the contrast experiment, big background signal response (ECL quenching) was observed in the case of H2O2 absence. This was attributed to the adsorption of dopamine to the gold-containing sensing platform due to the presence of amino group in its molecular structure. EP not only possesses the polymerization behavior of catecholamine and the free radical scavenging activity of melanin but also avoid the background signal response. To more specifically illustrate the signal response principle of proposed strategy, the corresponding ECL-potential profiles for the stepwise construction process of the ECL sensor was recorded (Figure S4A). While the bare GCE has very weak ECL signal (curve a), a remarkable strong ECL signal was obtained after the modification with g-C3N4@PANI-Au hybrids (curve b). As the primary anti-AFP was immobilized, the ECL intensity was decreased (curve c). The site blocking using BSA, the 7



recognition of AFP, and the binding of labeled detection anti-AFP further induce the decreasing of ECL (curve d-f) because of the impeded electron transfer and diffusion of the reagent to the surface of the electrode.26 Additional ECL decrease was achieved after the deposition of PEP (curve g). The fabrication process of the immunosensor and the successful PEP deposition can be well confirmed by the electrochemical impedance spectroscopy (EIS) (Figure S4B). In addition, the CV peak currents of the above modified electrodes were significantly reduced compared to bare GCE (Figure S5), which also indicated the successful construction of the sensor.

Figure 3. ECL intensity profiles (A) and calibration curve (B) for the determination of AFP with different concentrations. From a to i, the concentration of AFP was 0, 0.0001, 0.001, 0.01, 0.02, 0.05, 0.1, 0.5, and 1.0 ng mL-1. (∆I = I0 - I, I0 and I are the ECL intensity of the immunosensor before and after PEP, S/N = 3). Error bar = SD (n= 5)

The detection conditions including the concentration of co-reactant K2S2O8 and the amounts of PANI-Au nanocomposite for the preparation of g-C3N4@PANI-Au hybrids were investigated for the achievement of maximum signal response (Figure S6). Under the optimized conditions, the analytical performance of the proposed sensing strategy was demonstrated for the detection of AFP. In order to reveal the enhancement of detection sensitivity, the ECL response to AFP without the step of PEP deposition (the labeled detection antibody was also attached) was also recorded (Figure S7). A linear response range of 0.01~15 ng mL-1 was obtained with the calculated detection limit of 5.4 pg mL-1. In contrast, after applying the PEP deposition strategy, down-shifted linear response range from 0 to 1 ng mL-1 was 8


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achieved (Figure 3). A very low detection limit of 0.056 pg·mL-1 was obtained which was two orders of magnitude enhancement for the detection sensitivity and was better than most of previously reported methods for the detection of AFP (Table S2). The significantly enhanced sensitivity was ascribed to the excellent ECL quenching abilities of deposited PEP.

Figure 4. (A) The selectivity of the fabricated ECL immunosensor without the step of PEP deposition. (B) The selectivity of the proposed ECL immunosensor after the PEP deposition. (C) The reproducibility of the ECL immunosensor applying PEP deposition. The concentration of AFP is 0.5 ng mL-1 and the concentration of interfering proteins is 5 ng mL-1. Error bar = SD (n= 5).

While the detection sensitivity was greatly enhanced through the biomimetic melanin-like deposition, the selectivity of the immunosensor was another major concern about the utilization of the sensing strategy. For the examination of the selectivity towards the model analyte, carcinoembryonic antigen (CEA), human immunoglobulin antigen (H-IgG), squamous cell carcinoma antigen (SCCA), prostatic specific antigen (PSA) were used as interfering proteins. It can be seen from Figure 4A that the immunosensor without the deposition of PEP shows good selectivity towards the AFP. For the immunosensor with the strategy of PEP deposition, only AFP induced a big signal response (Figure 4B), indicating that the selectivity of the immunosensor was well preserved. The repeatability of the immunosensor utilizing the ECL quenching strategy was also examined by five freshly prepared electrodes for the detection of AFP (0.5 ng mL-1). As shown in Figure 4C, the RSD value of the detected signals was 3.05%. The 9



results showed that the proposed sensing strategy also had a good repeatability. The signal stability of the prepared immunosensor was investigated at different construction steps: electrode modification with the g-C3N4@PANI-Au hybrids (curve a in Figure S8), after the recognition of AFP (curve b in Figure S8), and after the PEP deposition (curve c in Figure S8). Stable signals were obtained at each construction step of the immunosensor. The storage stability of the immunosensor for 2 days in 4℃ is also acceptable (RSD < 5%). The potential application of the proposed sensing strategy for practical samples was examined by detecting the AFP in serum samples. Standard addition method was carried out by adding different concentrations of AFP in serum samples (0.2, 0.5 and 0.7 ng mL-1). The average recoveries of sensing strategy in these fortified samples were observed in the range of 94.0 - 101.0% with the RSD of 3.1 - 4.5% (Table S3), which further indicating that the fabricated immunosensor has a reliable and satisfactorily application in clinical determination of AFP. Conclusion In summary, the biomimetic melanin-like deposition was exploited to develop a sensitivity enhancement strategy for ECL immunosensors. A very low detection limit at the level of sub-picogram was achieved based on the excellent strong free radical scavenging activity of the deposited melanin-like polymers, which shows promising application for the determination of low abundance protein biomarker in bioassays. The detailed quenching mechanism needs a further research for the future application of the biomimetic melanin-like deposition on ECL immunosensors. Considering the wide application of HRP in biosensors, the biomimetic melanin-like deposition may find universal application in aptasensors and other biosensors. Supporting Information. Synthesis of nanomaterials, preparation of the ECL immunosensor, EIS spectrogram and the detection conditions were supplied as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author 10


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E-mail: [email protected] (Qin Wei) Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (21675063, 21575050), the National Key Scientific Instrument and Equipment Development Project of China (No. 21627809), the China Postdoctoral Science Foundation (2016M590609), and the Science and Technology Planning Project of Higher Education of Shandong Province (J16LC23). Q. Wei thanks the Taishan Scholar Foundation of Shandong Province (No. ts20130937).

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