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Multiparameter Analysis-based Electrochemiluminescent Assay for Simultaneous Detection of Multiple Biomarker Proteins on a Single Interface Wenbin Liang, Chenchen Fan, Ying Zhuo, Yingning Zheng, Chengyi Xiong, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00878 • Publication Date (Web): 11 Apr 2016 Downloaded from http://pubs.acs.org on April 13, 2016

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Analytical Chemistry

Multiparameter

Analysis-based

Electrochemiluminescent

Assay

for

Simultaneous

Detection of Multiple Biomarker Proteins on a Single Interface Wenbin Liang,†,‡ Chenchen Fan,† Ying Zhuo,† Yingning Zheng,† Chengyi Xiong,† Yaqin Chai,*,† and Ruo Yuan*,† †

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University),

Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China ‡

Department of Clinical Biochemistry, Laboratory Sciences, Southwest Hospital, Third Military

Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, PR China

*

Corresponding Author

E-mail: [email protected] (Yaqin Chai), [email protected] (Ruo Yuan). Tel.: +86-23-68252277; Fax: +86-23-68253172

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ABSTRACT: Electrochemiluminescent (ECL) assay with high sensitivity has been considered as one of the potential strategies to detect multiple biomarker proteins simultaneously. However, it was essential but full of challenges to overcome the limitation caused by cross reactions among different ECL indicators. Herein, the multiparameter analysis of ECL-potential signals demonstrated by multivariate linear algebraic equations was firstly employed in the simultaneous ECL assay to realize multiple detection of biomarker proteins on a single interface. Additionally owing to the exponential amplification of self-synthesized nucleotide dendrimer by hybridization chain reaction (HCR) and rolling circle amplification (RCA), the developed simultaneous ECL assay showed improved sensitivity and satisfactory accuracy for the detection of N-terminal of the prohormone brain natriuretic peptide (BNPT) and cardiac troponin I (cTnI). Furthermore, a self-designed magnetic beads-based flow system was also employed to improve the feasibility and analysis speed of the simultaneous ECL assay. Importantly, the proposed strategy enabled simultaneous detection of multiple biomarker proteins simply, which could be readily expanded for the multiplexed estimation of various kinds of proteins and nucleotide sequence also, revealing a new avenue for early disease diagnosis with higher efficiency. KEYWORDS: biomarker protein, electrochemiluminescence, multiple detection, nucleotide amplification

INTRODUCTION Electrochemiluminescent (ECL) assay as a powerful analytical technique with low background, high sensitivity, wide dynamic range, and cost-effectiveness has attracted considerable attentions in pharmaceutical analysis, clinical diagnosis and environmental 2


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analysis.1-3 Generally, ECL assays were preformed based on the ECL indicators with an electrochemically triggered optical radiation process produced by the energy relaxation of excited species. Significant advantages have been achieved in sensitive detection of antigens and nucleotide sequence, especially ECL assays with luminol (LUM), tris (2, 2'-bipyridyl-4, 4'dicarboxylato) ruthenium (II) (Ru(dcbpy)32+ or RU) and their derivatives as ECL indicators, which were considered as “artificial ECL indicators” due to their high ECL efficiency, controllable ECL reaction and good electrochemical stability. 4-8 However, to the best of our knowledge, most of these established works just performed single-target detection on a single interface, which could not satisfy the demands for clinical diagnosis now, especially on the selectivity and accuracy for early diagnosis of acute diseases. It was highly valuable but full of challenges to develop simultaneous ECL assays for multiple detection. With the goal to develop simultaneous ECL assays for multiple detection, it was essential to label ECL indicators with different ECL-potential responses onto the specific recognition elements (e.g. antibodies, nucleotide sequence), but this approach was hard to be employed in simultaneous ECL assay due to the unavoidable cross reactions among these ECL indicators.9-13 Jiang and co-workers have tried to overcome this limitation based on recording ECL responses of LUM and RU in different detection solutions, where excess ECL indicator in the detection solution was employed to trigger enough cross reaction signals to cover up the cross reaction signals on the sensor surface.14 The ECL assay for multiple detection could be realized based on this approach with tedious processes, but actually, it was not the common-sense simultaneous ECL assay with more simple process and higher efficiency, which was normally performed the multiple detections on single interface in one detection solution. Considering the challenges and

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the promising applications, herein, we proposed an simultaneous ECL assay for multiple detection on single interface in one detection based on multiparameter analysis with LUM and RU as ECL indicators. Concretely, the cross reactions among these ECL indicators in the mixed system were related with both of their concentrations, and thereby the ECL signal of one ECL indicator was not only related with its concentration but also with the other ECL indicator's concentration. Herein, the quantitative multiparameter analysis was for the first time employed in the simultaneous ECL assay. With this approach, the ECL signals of these ECL indicators could be demonstrated as = ( , ) and = ′ ( , ), where IRU and ILUM were the ECL intensities of LUM and RU, and cRU and cLUM were the concentrations of LUM and RU respectively. Given these facts, it was possible to calculate cLUM and cRU with ILUM and IRU based on these associated multivariate functions. Importantly, this strategy could reveal the details of cross reactions among these ECL indicators and relationship between ECL responses and concentration of ECL indicators, providing novel insights into simultaneous ECL assay on a single interface for multiple detection. In order to improve the sensitivity of the simultaneous ECL assay, a novel amplification approach with exponential amplification efficiency was presented based on the self-synthesized nucleotide dendrimer for the detection of biomarker proteins. It was well known that there was no method to amplify proteins by itself. In contrast, various approaches could be applied to amplify nucleotide sequence. Therefore, it was possible to improve the detection sensitivity for biomarker proteins by labeling the secondary antibody with nucleotide sequence and introducing nucleotide amplification.15-19 Hybridization chain reaction (HCR) and rolling circle amplification (RCA) as widely used approaches in nucleotide amplification, could extend from an initiator

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(short specific nucleotide sequence) into a long repeated nucleotide sequence with cascade amplification.20-26 A remarkably enhanced signals could be obtained by attaching large number of signal indicators on these repeated nucleotide sequence. Recently, Zhu and coworkers have demonstrated an efficient amplification strategy by combining both HCR and RCA (specifically, introducing HCR on the long repeated nucleotide sequence prepared by RCA reaction), which was employed successfully for sensitive chemiluminescent assay.27 However, improvements were still needed to overcome the limitation due to the limited amplification efficiency of HCR reactions on the folded single strand nucleotide sequence introduced by RCA reactions. Taking the advantages and challenges of these nucleotide amplification into consideration, a novel nucleotide dendrimer was synthesized for the first time with not just cascade but exponential amplification efficiency. Briefly, the nucleotide was prepared by extending an initiator into a long repeated nucleotide sequence by HCR and further extending these repeated nucleotide sequence by RCA into nucleotide dendrimer with extremely large amount of repeated nucleotide sequences to attach ECL indicators labeled complementary nucleotide sequence. Thus, great potentials could be expected to amplify ECL responses and thereby improve detection sensitivity efficiently. Herein, a simultaneous ECL assay was proposed by overcoming the limitation from unavoidable cross reactions with multiparameter analysis and amplifying ECL responses exponentially with self-synthesized nucleotide dendrimer for the multiple detection of biomarker proteins. N-terminal of the prohormone brain natriuretic peptide (BNPT) and cardiac troponin I (cTnI), as efficient and widely used biomarker proteins for acute myocardial infarction,28-30 were employed as models to investigate its application (Scheme 1). In addition, a self-designed

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magnetic beads-based flow system was also employed to improve the feasibility of the simultaneous ECL assay. Concretely, the biotinylated primary antibodies for BNPT and cTnI were attached onto streptavidin coated magnetic beads (MB@SA), which could capture nucleotide dendrimer labeled secondary antibodies for BNPT and cTnI by antibody-antigen interactions in the presence of the BNPT and cTnI. Thereby, exponentially amplified amount of LUM and RU labeled complementary nucleotide sequence could be combined onto the surface of magnetic beads to perform enhanced ECL emission and employed to indicate the concentrations of BNPT and cTnI simultaneously with high-sensitivity. The success in the establishment of the simultaneous ECL assay offered an efficient strategy for the multiple detection of biomarker proteins without any expensive setup and tedious processes, which could be readily expanded for the multiplexed estimation of various kinds of proteins and nucleotide sequence also, revealing a new avenue for early disease diagnosis with higher efficiency.

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Scheme 1. Schematic diagrams of the simultaneous ECL assay. (A) preparation of the nucleotide dendrimer labeled secondary antibodies; (B) schematic diagrams of the simultaneous ECL assay system; (C) Image and mechanical design drawing of self-designed magnetic flow system; (D) cathodic ECL responses of the asproposed ECL assay for multiple detection of BNPT and cTnI. (Abbreviations, bio-PAb, biotinylated primary antibody; CT1-2, circular template for RCA reaction; H1-4, hairpin nucleotide for HCR reaction; NI1-2, nucleotide initiator; SP1-2, signal probe).

EXPERIMENTAL METHODS Reagents and Materials. 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), LUM, biotin N-hydroxysuccinimide ester (NHS-biotin), tripropylamine (TPrA) and streptavidin coated magnetic beads (MB@SA) were purchased from Sigma Chemical Co. (MO, USA). RU was obtained from Suna Technology Inc. (Suzhou, China). T4 ligase and Phi29 polymerase were purchased from Vazyme Biotech Co., Ltd. 7



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(Nanjing, China). Exconuclease I and Exconuclease III were purchased from Thermo Fisher Scientific Inc. (Shanghai, China). Deoxynucleotides (dNTPs) was purchased from Genview Scientific Inc. (IL, USA). phosphate buffered saline (PBS) and phosphate buffered saline with tween-20 (PBST) were received from Beyotime Institute of Biotechnology Inc. (Jiangshu, China). Ultrapure water with a resistivity of 18.2 MW/cm was used throughout this study. All oligonucleotides were custom-synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The sequences information was listed as Table 1. Before using, the hairpin nucleotides were heated to 95 °C for 2 min and then cooled to room temperature to form stem-loop structure. The circular templates were mixed with the associated primers, heated to 65 °C for 10 min, and then reacted with T4 ligase after cooling to room temperature to link 5' and 3' end of these circular templates. The non-reacted circular templates and primers were cleaned by adding 5 µL Exconuclease I and 2 µL Exconuclease III and heating to 37 °C for 30 min. The Exconuclease I and Exconuclease III were deactivated on 90 °C for 15 min. Table 1. The oligonucleotides list for the preparation of nucleotide dendrimer. Name nucleotide initiator 1 hairpin nucleotide 1

hairpin nucleotide 2

circular template 1

Sequence (5'→3') HOOC-TTT TTA GTC TAG GAT TCG GCG TGG GTT AA PO4-ATT CGA TCT TAA CCC ACG CCG AAT CCT AGA CTC AAA GTG ATT CGA TCA GTC TAG GAT TCG GCG TG PO4-ATT CGA TCA GTC TAG GAT TCG GCG TGG GTT AAA TCC TAG ACT GAT CGA ATA CTT TGT CGG CGT G PO4-CAC GCC GAG ATC GAA TCA CTT AGG ACG TAG TGA AGC AGG AA

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primer1

TTC GAT CTC GGC GTG TTC CTG CTT CAC TAC

signal probe 1

NH2-TTT TTA GGA CGT AGT GAA GCA GG

nucleotide initiator 2

HOOC-TTT TTC CTA GAG CTA AGC TAT GAC CTG GT



circular template 2

PO4-GCT AAG CTA TGA CCT GGT CCA TGA CGA CCT TGA CCA GGT CAT AGC TTA GCT CTA GGC CAT GAC G PO4-GCT AAG CTC AAG GTC GTC ATG GAC CAG GTC ATC CTA GCT AAG CTA TGA CCT GGT CCA TGA CG PO4-CGT CAT GGA GCT TAG CAT GAC GGT GTG ATA TGA GCG TAT GA

primer2

CTA AGC TCC ATG ACG TCA TAC GCT CAT ATC

signal probe 2

HOOC-TTT TTA CGG TGT GAT ATG AGC GTA T

Apparatus. Electrochemistry and electrochemiluminescence (ECL) measurements were performed with a model MPI-E II multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remax Electronic Science and Technology Co., Xi’an, China). A conventional three-electrode system was employed with a Ag/AgCl (saturated KCl) as the reference electrode, a platinum wire as auxiliary electrode, and a glassy carbon plate (GCP) with/without magnetic adsorption of magnetic beads as the working electrode, respectively. The element distribution mapping was performed with energy dispersive X-ray spectroscope (EDS, Oxford Instruments, Palo Alto, CA) combined with JSM-7800 Fextreme-resolution analytical field emission scanning electron microscope (SEM, Jeol Inc., MA, USA) at an acceleration voltage of 15 kV. The characterizations of the ECL materials labeled nucleotide sequences were performed on Ultimate 3000 high performance liquid chromatography system (HPLC, Thermo Scientific, PA, USA). The photophysical characterizations were carried out with a RF-5301PC

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fluorescence spectrophotometer (Shimadzu Co., Tokyo, Japan) with standard quartz cuvettes (open-top type, 10 mm optical path length and 3.5 mL volume). Mechanism of Signal Amplification based on Nucleotide Dendrimer. The nucleotide initiator 1 on the secondary antibodies of BNPT could pair with the sticky end of hairpin nucleotide 1, open the hairpin structure via the strand-displacement interaction, and expose two new terminuses. One of the new terminuses could be used for further reaction with hairpin nucleotide 2 and the other of which could be further employed to achieve RCA reaction. And then, the opened hairpin nucleotide 2 could expose two new terminuses for further reactions with hairpin nucleotides 1 and achievement of RCA amplification similar with hairpin nucleotide 1. In this way, each nucleotide initiator 1 can trigger HCR reaction to form a long nucleotide polymer with large amount of terminuses as initiator for RCA, which could initiate RCA reaction to form nucleotide dendrimer 1 with massive repeated sequences. Due to the high efficiency of HCR and RCA, a large amount of RU labeled signal probe could be associated onto these repeated sequences on nucleotide dendrimer 1, leading to enhanced ECL signals of the sensing system. The procedure and mechanism of nucleotide initiator 2 was similar with that of nucleotide initiator 1 to form nucleotide dendrimer 2 with LUM labeled secondary antibodies of cTnI. Preparation of Nucleotide Dendrimer Labeled Secondary Antibodies. The secondary antibodies for BNPT (BNPT SAb) were firstly labeled with nucleotide initiator 1 by EDC/NHS reaction. Briefly, 20 µL BNPT SAb (1 mg/mL) was mixed with 100 µL nucleotide initiator 1 with continues stirring for 10 min. After addition of 50 µL EDC (0.20 M) and 50 µL sulfo-NHS (0.05 M), the mixture was stirred at room temperature for 2 h to label nucleotide initiator 1 onto BNPT SAb. The non-reacted nucleotide initiator 1 and related chemicals were removed by

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dialysis with centrifugal filter devices (50 KD). And then, the nucleotide dendrimer was synthesized based on the HCR and RCA reactions from nucleotide initiator 1. Briefly, the prepared nucleotide initiator labeled BNPT SAb was incubated with 1 µM hairpin nucleotide 1 and hairpin nucleotide 2 in 6.67 mM Tris-HCl buffer (pH 8.0) containing 66.67 mM MgCl2 for 8 h. And then, the resulted product, BNPT SAb labeled with a long repeated nucleotide sequence based on HCR reactions, was further reacted with the prepared circular template 1 in TE buffer with 1 µL Phi29 polymerase and 5 µL dNTPs, and the reaction was remained for 6 h at 37 °C to form nucleotide dendrimer onto BNPT SAb (BNPT SAb/ND1) based on RCA reaction. The RU labeled signal probe 1 was further bonded onto the prepared nucleotide dendrimer 1 through hybridization between signal probe 1 and associated sequences on the nucleotide dendrimer 1 to form RU labeled BNPT SAb with nucleotide dendrimer (BNPT SAb/ND1/SP1@RU). The prepared BNPT SAb/ND1/SP1@RU was dispersed in PBST and stored at -20 °C when no use. The LUM labeled secondary antibodies for cTnI by nucleotide dendrimer (cTnI SAb/ND2/SP2@LUM) were prepared with the similar protocol as described above. Furthermore, ECL materials conjugated secondary antibodies with different approaches, including secondary antibodies labeled with RU directly without any amplification (SAb@RU) and just with amplification of HCR or RCA (SAb/HCR/SP@RU or SAb/HCR/SP@RU), were prepared with the similar protocols, and the details were shown in the supporting information. Preparation of the Biotinylated Primary Antibodies. The primary antibodies for BNPT (BNPT PAb) were biotinylated according to the manufacturer’s instructions. Briefly, the appropriate volume of NHS-biotin solution was added into the PBS solution with BNPT PAb (1 mg/mL). After complete mixing, the resulted mixture was incubated at 37 °C for 2 h for the

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reaction between NHS-biotin and amine groups on the antibodies. Finally, the resulted solution was desalted to remove non-reacted NHS-biotin and obtain the biotinylated BNPT PAb (bioBNPT PAb). The same protocol was employed to prepare biotinylated primary antibodies for cTnI (bio-cTnI PAb). Preparation of the ECL materials labeled nucleotide sequences. The particular nucleotide sequences were labeled with ECL materials (LUM or RU) by EDC/NHS reaction to react as signal probes. Briefly, 20 µL nucleotide sequences (50 µM) was mixed with 100 µL RU or LUM (0.10 M), 50 µL EDC (0.20 M) and 50 µL sulfo-NHS (0.05 M) firstly, and the mixture was stirred at room temperature for 2 h for complete reaction. The non-reacted RU or LUM and related chemicals were removed by purification with high performance liquid chromatography (HPLC). The purity of these ECL materials labeled nucleotide sequences were characterized by HPLC and the purities of the RU and LUM labeled nucleotide sequence were above 95%, indicating the acceptable applicability of these nucleotide sequences. Measurement Procedure. Before the ECL measurement, the GCP was carefully polished with alumina slurries (0.3, 0.05 µm), ultrasonicated in deionized water and dried with nitrogen. The ECL measurement was based on a sandwich-type assay in tube as shown in Scheme 1. Briefly, the bio-BNPT PAb, bio-cTnI PAb, BNPT SAb/ND1/SP1@RU and cTnI SAb/ND2/SP2@LUM were firstly added into the solution containing SA coated magnetic beads. Followed with adding test samples and shaking gently, the mixture was incubated at 37 °C for 40 min. Then, the mixture was flowed into the magnetic flow system. Unconjugated antibodies were removed by washing twice with PBST. Finally, the ECL emission was investigated with a MPI-E II multifunctional electrochemical and chemiluminescent analytical system in the detection 12


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solution (PBS with 25 mM TPrA and 2 mM H2O2) with the self-designed magnetic flow system. After each determination, the magnetic flow system could be employed for the next determination after a simple regeneration by washing twice with PBST. Details of the magnetic flow system were shown in the supporting information. Computational Method for Mechanism of the Cross Reactions. In this work, the ground states, the lowest singlet and triplet excited states geometries of LUM and RU were fully optimized together with the polarizable continuum model (PCM) using the density functional theory (DFT) with the hybrid exchange-correlation functional O3LYP, which has been proved to be more accurate for this system.31, 32 To obtain the absorption and emission spectra, the timedependent density functional theory (TD-DFT) calculations associated with PCM were performed by several different representative functions on the basis of optimized S0, S1, and T1 geometries. In the TD-DFT calculations, it was found that the same functional O3LYP could be more accurate in reproducing the experimental absorption spectra and the emission spectra of LUM. However, the hybrid exchange-correlation functional wB97XD was the most suitable function to simulate the emission spectra of RU. Meanwhile, the 6-31G* basis set was employed for all the light atom (C, H, O and N) and the LANL2DZ basis set was adopted for the heavy atom Ru.33, 34 All the above calculations were performed in Gaussian 09 program package.35 RESULTS AND DISCUSSION Electrochemical and Electrochemiluminescence of LUM and RU. With aim for simultaneous detection of multiple biomarker proteins, the separated ECL-potential responses were needed firstly. As shown in Figure 1A, it could be found that there were two significant ECL responses for LUM and RU with peak intensity on -1.5 V and 1.26 V, which were associated with the ECL 13



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generations of LUM and RU respectively. On -1.5 V, the LUM could be reacted to 3aminophthalate in an excited state and then produced the characteristic “LUM” emission with coreactant of H2O2. On 1.26 V, RU could be reacted to oxidized state of RU and the TPrA could be oxidized to form radical cations. Immediately, the oxidized RU could react with the radical cation of TPrA to excited state of RU and then back to RU with ECL emission. Herein, the well separated ECL responses on different potentials could act as good indicators for simultaneous detection. However, cross reactions between LUM and RU made the simultaneous detection difficult to indicate their concentration correctly based on the ECL responses.

Figure 1. Characterizations of the ECL materials (LUM and RU) and nucleotide dendrimer. (A) cyclic voltammogram (a, dot line) and ECL-potential (b, solid line) results of LUM and RU in PBS with coreactant (H2O2 and TPrA); (B) Polyacrylamide gel electrophoresis characterization of nucleotide dendrimer and the related nucleotides: lane 1, nucleotide initiator 1 (2 µM); lane 2, hairpin nucleotide 1 (2 µM); lane 3, hairpin nucleotide 2 (2 µM); lane 4, circular template 1 (2 µM); lane 5, primer1 (2 µM); lane 6, signal probe 1 (2 µM); lane 7, nucleotide dendrimer 1 (2 µM); lane a, nucleotide initiator 2 (2 µM); lane b, hairpin nucleotide 3 (2

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µM); lane c, hairpin nucleotide 4 (2 µM); lane d, circular template 2 (2 µM); lane e, primer2 (2 µM); lane f, signal probe 2 (2 µM); lane g, nucleotide dendrimer 2 (2 µM).

Characterization of Nucleotide Dendrimer. All of the materials used to prepare nucleotide dendrimer were characterized by polyacrylamide gel electrophoresis (Figure 1B). The distant places of UV band from the notch indicated the molecular weight of the studied nucleotide. The emission bands of the nucleotides for the preparation of nucleotide dendrimer were shown in Figure 1B, lane 1-6 and a-f, respectively, including nucleotide initiator 1-2 (lane 1 and a), hairpin nucleotide 1-4 (lane 2-3 and b-c), circular template 1-2 (lane 4 and d), primer 1-2 (lane 5 and e) and signal probe 1-2 (lane 6 and f). The nucleotide initiator could trigger HCR reaction to form a long nucleotide polymer with large amount of terminuses as primer for RCA, and then initiate RCA reaction to form nucleotide dendrimer with massive repeated sequences. As shown in Figure 1B, lane 7 and g, the emission bands of high-molecular weight structures could be observed, indicating the successful preparation of nucleotide dendrimer based on the HCR and RCA reactions. Notably, the strand length of nucleotide dendrimer was different and tailing phenomenon could be observed, because the HCR and RCA system would finally reach the equilibrium state in terms of kinetics and thermodynamics. Herein, the average molecular weight of the nucleotide dendrimer was associated with amount of nucleotide initiator, so one nucleotide initiator could form a nucleotide dendrimer with massive repeated sequences to attach a large amount of signal probes for the exponential enhancement of ECL signals. Importantly, the amount of nucleotide dendrimer and attached signal probes were associated with nucleotide initiator, thereby the ECL signals could be employed to indicate the concentration of nucleotide initiator with improved sensitivity and efficiency.

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Cross Reactions between LUM and RU. Fluorescence spectra as a power photophysical measurement was employed to demonstrate the cross reactions between LUM and RU. As shown in Figure 2A, there were single characteristic maximum excitation/emission peaks for pure LUM and RU on 359/437 nm (curve a and b) and 494/591 nm (curve c and d), respectively. For the mixture of LUM and RU (Figure 2B), the similar excitation response (curve e) with that of pure LUM when emission wavelength was set as 437 nm, and the similar emission response (curve h) with that of pure RU when excitation wavelength was set as 494 nm. Whereas, double emission peaks on 411 nm and 605 nm could be obtained (curve f) when the excitation light was set as 359 nm, and double excitation peaks on 377 nm and 533 nm could be obtained (curve g) when the emission light was set as 591 nm for the mixture of LUM and RU, indicating the cross reactions from LUM to RU. To confirm these interactions quantitatively, further study was investigated by the fluorescence spectra on excitation light of 359 nm for the mixture of 250 nM LUM with different concentrations of RU ranged from 0 to 450 nM (Figure 2D), which was compared with the fluorescence spectra of pure RU with the same concentration range (Figure 2C). Obviously, the fluorescent emission of RU was increased and the fluorescent emission of LUM was decreased with increasing concentration of RU, and the fluorescent emission of RU was increased more than that without LUM (Figure S2 A and B). In addition, the fluorescent emission of RU and LUM was both increased with increasing concentration of LUM, when the concentration of RU was fixed as 1 µM (Figure 2F), in which the fluorescent emission of LUM was increased less than that without RU (Figure S2 C and D). The similar results could be obtained in the ECL measurements. All of these results indicated the increased energy transfer with increasing concentration of RU and LUM. The mechanism of the cross reaction was

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calculated and shown in Figure 2G. For the fluorescent emission, the energy of LUM (S1 state) could be transferred to RU (S8 state) via external conversion. The mechanism of the cross reaction of ECL was a little different with that of fluorescence, in which the energy of LUM could be transferred from LUM (S1 state) to LUM (T1 state) via internal conversion and then to RU (T1 state) via external conversion with emission of RU, or from LUM (S1 state) to RU (S8 state) via external conversion, to RU (S1 state) via vibration relaxation, and then to RU (T1 state) via internal conversion with emission of RU.

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Figure 2. Photoluminescence spectra and ECL-time of LUM and RU. (A) normalized photoluminescence spectra of LUM and RU respectively; (B) normalized photoluminescence spectra of the mixture of LUM and RU; (C) normalized photoluminescence spectra of RU with concentration ranged from 0 to 450 nM; (D) normalized photoluminescence spectra of 450 nM LUM with different concentration of RU ranged from 0 to 450 nM; (E) normalized photoluminescence spectra of LUM with concentration ranged from 0 to 45 nM; (F) normalized photoluminescence spectra of 1 µM RU with different concentration of LUM ranged from 0 to 45 nM; (G) singlet and triplet energies of LUM and RU, and the potential mechanism of the energy transfer from LUM to RU. (VR, vibration relaxation; IC, internal conversion; EC, external conversion; FC, fluorescence emission; PE, photophorescence emission; S0, ground-state; Sn, singlet state; T1, triplet state).

Normally, the relationship between the ECL signal and the concentration of an ECL indicator could be demonstrated as a single-parameter linear algebraic equation, such as = ( ) in the ECL system just using LUM as ECL indicator. Whereas, in the mixture of LUM and RU, the ECL response of LUM would be influenced by the concentration of RU due to the cross reactions. Thus, the concentration of RU should be added as a influence factor to the ECL signal of LUM, and the relationship between the ECL signal of LUM and concentration of LUM and RU would be demonstrated as a multivariate linear algebraic equation, = ( , ). Based on the same mechanism, the ECL emission of RU would be demonstrated as = ′ ( , ). Although the cross reactions in multiple detection were puzzled and difficult to be solved, it would be possible to calculate cLUM and cRU based on multi-parameter analysis with these multivariate linear algebraic equations and related ECL responses (ILUM and IRU), which would be helpful and efficient in the simultaneous ECL assays for multiple detection. Comparison of Different Amplification Approaches. To investigate the efficiency of amplification with nucleotide dendrimer used in the as-proposed simultaneous ECL assay,

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various amplification approaches including labeling ECL indicators directly onto the antibodies and just with HCR or RCA amplification were employed as controls. Here, the same batches incubated with BNPT were prepared firstly, and then incubated with SAb without or with different amplifications including SAb@RU (Figure 3A), SAb/HCR/SP@RU (Figure 3B), SAb/RCA/SP@RU (Figure 3C) and SAb/ND/SP@RU (Figure 3D), respectively. It could be seen in Figure 3A that the ECL response of ECL assay with SAb@RU as signal indicator was about 130 a.u. compared with the background value. As shown in Figure 3B and C, enhanced ECL responses, about 706 a.u. and 1047 a.u. were received by the ECL assay with SAb/HCR/SP@RU and SAb/RCA/SP@RU, respectively, which were attributed to the increased amount of RU based on HCR or RCA amplification. For the as-proposed ECL assay with amplification based on nucleotide dendrimer (SAb/ND/SP@RU), a further enhanced ECL response about 4002 a.u. was obtained due to significantly increased RU on nucleotide dendrimer, indicating the great efficiency of our proposed amplification with nucleotide dendrimer, which could be utilized for ultrasensitive assay.

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Figure 3. Comparison of various amplification approaches based on ECL profiles, including SAb/RU (A), SAb/HCR/SP@RU (B), SAb/RCA/SP@RU (C) and SAb/ND/SP@RU (D), respectively.

Morphology and Characterizations of the Reactions on the Magnetic Beads. SEM was a simple and efficient technology to characterize the morphology of the nano-/micro-materials and reactions on the nano-/micro-materials. Furthermore, SEM with EDS mapping could provide elemental distribution images of the studied samples, which has been widely employed for the characterizations of various materials with elemental analysis. As shown in Figure 4A, the magnetic beads presented a spherical structure with an average diameter of 1.23 µm. After the binding of bio-BNPT PAb, BNPT and BNPT SAb/ND@SP@RU, the average diameter increased with some strip-like edge on the magnetic beads surface (Figure 4B). Further characterizations 21



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by EDS mapping were shown in the insert of Figure 5. There was no significant differences for the EDS mapping of Fe element between the streptavidin coated magnetic beads (Figure 4A2) and the magnetic beads after reaction with bio-BNPT PAb, BNPT and BNPT SAb/ND@SP@RU (Figure 4B2). Compared with the EDS mapping of Ru element in streptavidin coated magnetic beads (Figure 5A3), an obvious EDS signals for Ru could obtained after reaction with bio-BNPT PAb, BNPT and BNPT SAb/ND@SP@RU (Figure 4B3), which was matched well with the SEM image. In this case, the Ru element could be identified clearly by SEM as well as EDS mapping, indicating the RU labeled signal DNA could be employed as signal probes by specific attachment onto magnetic beads surface to indicate the concentration of their associated targets.

Figure 4. SEM images for the morphology characterization for the streptavidin coated magnetic beads (A) and the magnetic beads after reaction with bio-BNPT PAb, BNPT and BNPT SAb/ND@SP@RU. Insert: SEM image (1) , EDS element mapping of Fe (2) and EDS element mapping of Ru (3) for the streptavidin coated magnetic beads (A) and the magnetic beads after reaction with bio-BNPT PAb, BNPT and BNPT SAb/ND@SP@RU (B).

ECL Analysis of the Proposed Simultaneous ECL Assay. To estimate the performance of the as-proposed simultaneous ECL assay, the quantitative measurement was employed by detecting 22


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different concentration of BNPT and cTnI. As shown in Figure 5, the ECL signals increased linearly with increasing concentrations of BNPT and cTnI in the range from 0.1 pg/mL to 8 ng/mL and from 0.2 pg/mL to 1 ng/mL respectively. The linear relationship between the ECL signals and concentrations of BNPT and cTnI could be demonstrated as IRU = 1006 lgcBNPT + 1344 lgccTnI – 916.1 and ILUM = -141.7 lgcBNPT + 219.3 lgccTnI +267.2, respectively, where I represented the ECL signals (a.u.) of LUM and RU, and c represented the concentration (pg/mL) of BNPT and cTnI respectively. Thus, cBNPT and ccTnI could be calculated based on multiparameter analysis with ECL signals of LUM and RU and the multivariate linear algebraic equations. These results indicated that the as-proposed simultaneous ECL assay could be used to detect multiple biomarker proteins quantitatively.

Figure 5. ECL responses of the as-proposed ECL assay for BNPT (A) and cTnI (B) in the presence of different concentrations of BNPT and cTnI in the range from 0.1 pg/mL to 8 ng/mL and from 0.2 pg/mL to 1 ng/mL, respectively.

Reproducibility, Stability and Specificity of the Proposed Simultaneous ECL Assay. The reproducibility as one of the important properties was evaluated by variation coefficients (CV%) for five duplicate measurements of intra-assays and inter-assays. Both of the relative standard 23



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deviations (RSD) for intra-assays and inter-assays were not more than 5%, indicating the acceptable reproducibility of the proposed simultaneous ECL assay. The specificity and stability of the proposed simultaneous ECL assay were investigated under consecutive cyclic potential scans in the presence of components which may be present in the detection sample, including 1 µg/mL human serum albumin (HSA), human immunoglobulin G (IgG), α-fetoprotein (AFP), carcino-embryonic antigen (CEA) or prostate specific antigen (PSA). As depicted in Figure 6, the interferences exhibited no significant ECL responses compared with that of 100 pg/mL BNPT or 10 pg/mL cTnI, indicating a satisfactory specificity of the proposed simultaneous ECL assay. Besides, the relative standard deviations (RSD) for the ECL signals in these consecutive cycles were 2.60% and 2.69% for the detecion of BNPT and cTnI respectively, which indicated the satisfactory stability of the proposed simultaneous ECL assay.

Figure 6. Specificity evaluation of the proposed ECL assay for BNPT (A) and cTnI (B).

Application of the As-proposed Simultaneous ECL Immunosensor for the Detection of BNPT and cTnI. To investigate the feasibility of as-proposed simultaneous ECL assay, the lab study results of human serum samples were compared with that obtained by the commercial ECL 24


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immunoassay used in clinical diagnosis. As shown in Figure 7 for the determination of BNPT and cTnI respectively, both the correlation coefficient and slope were close to 1, which indicated the lab study results by as-proposed simultaneous ECL assay were in good agreement with that by the commercial ECL immunoassay (insert: the concentration range of BNPT and cTnI obtained by the as-proposed simultaneous ECL assay and the commercial ECL immunoassay). These results demonstrated that as-proposed simultaneous ECL assay with well performance provided an efficient approach to detect BNPT and cTnI in clinical diagnosis.

Figure 7. Concentrations of BNPT (A) and cTnI (B) in human serum samples detected by the as-proposed simultaneous ECL assay and commercial ECL immunoassay. Insert: The concentration range for the concentration of BNPT (a) and cTnI (b) respectively by box chart (box, 25%-75% value range; ☆, mean value; ▲, 99% percentile; ■, 1% percentile; CR; clinical results; LSR, lab study results).

CONCLUSION In summary, a novel simultaneous ECL assay for the detection of multiple biomarker proteins on a single interface was developed by utilizing multiparameter analysis and amplification with nucleotide dendrimer. Based on the proposed ECL assay, the concentrations of multiple 25



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biomarker proteins could be demonstrated simply based on multiparameter analysis with multivariate linear algebraic equations and related ECL responses. Furthermore, compared with the approach based on ECL indicator labeled antibodies directly without any amplification, about 30 folds amplification was received based on the nucleotide dendrimer prepared simply by HCR and RCA reactions. Using BNPT and cTnI as models, the results from the proposed simultaneous ECL assay were in good agreement with that from the commercial ECL immunoassay. The success in the establishment of the simultaneous ECL assay could provide a simple method for the detection of multiple targets on single interface and an efficient amplification approach for ultrasensitive biomolecules diagnostics. More importantly, the proposed approach for simultaneous ECL assay could be readily expanded for the multiplexed estimation of more than two multiple targets based on multiparameter analysis with multivariate linear algebraic equations and related detection signals, revealing a new avenue for early disease diagnosis with higher efficiency. ASSOCIATED CONTENT Supporting Information. Experimental details for the preparation of secondary antibodies with amplification of HCR or RCA, the magnetic flow system and supplementary figures and tables. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Yaqin Chai), [email protected] (Ruo Yuan). Notes

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The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation (NNSF) of China (81301518, 21575116, 51473136 and 21275119), and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China. REFERENCES (1) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. (2) Miao, W. J. Chem. Rev. 2008, 108, 2506-2553. (3) Miao, W. J.; Choi, J. P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478-14485. (4) Li, G. X.; Yu, X. X.; Liu, D. P.; Liu, X. Y.; Li, F.; Cui, H. Anal. Chem. 2015, 87, 1097610981. (5) Wang, D. F.; Li, Y. Y.; Lin, Z. Y.; Qiu, B.; Guo, L. H. Anal. Chem. 2015, 87, 5966-5972. (6) Feng, Q. M.; Shen, Y. Z.; Li, M. X.; Zhang, Z. L.; Zhao, W.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2016, 88, 937-944. (7) Yu, Y. C.; Lu, C.; Zhang, M. N. Anal. Chem. 2015, 87, 8026-8032. (8) Dong, Y. P.; Chen, G.; Zhou, Y.; Zhu, J. J. Anal. Chem. Just accepted. DOI: 10.1021/acs.analchem.5b04379. (9) Wu, M. S.; Shi, H, W.; Xu, J. J.; Chen, H. Y. Chem. Commun. 2011, 47, 7752-7754. (10)

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