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Electrochemiluminescent Imaging for Multi-immunoassay Sensitized by Dual DNA Amplification of Polymer Dot Signal Ningning Wang, Yaqiang Feng, Yawei Wang, Huangxian Ju, and Feng Yan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01610 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Analytical Chemistry

Electrochemiluminescent

Imaging

for

Multi-immunoassay

Sensitized by Dual DNA Amplification of Polymer Dot Signal Ningning Wang,† Yaqiang Feng,† Yawei Wang,† Huangxian Ju,*,† and Feng Yan*,‡ †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry

and Chemical Engineering, Nanjing University, Nanjing 210093, P.R. China ‡

Department of Clinical Laboratory, Nanjing Medical University Cancer Hospital &

Jiangsu Cancer Hospital, 42 Baiziting Road, Nanjing 210009, P.R. China

___________________________ * Corresponding author. Phone/Fax: +86-25-89683593. E-mail: [email protected], [email protected]

1

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ABSTRACT: A true-color electrochemiluminescent (ECL) imaging strategy was designed for multi-immunoassay of proteins by coupling highly efficient polymer dots (Pdots) with dual DNA amplification. The Pdots were prepared by nanoprecipitation of poly[(9,9-dioctylfuorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1’,3}-thiadiazole)]

in

the

presence of poly(styrene-co-maleic anhydride), and functionalized with DNA1 that hybridized with black hole quencher-labelled DNA2 to self-quench the ECL emission. The Pdots modified Au/ITO electrode showed 100-fold stronger ECL emission than Pdots modified ITO electrode. After the capture antibody immobilized on Au/ITO slide recognized the target protein and then reacted with biotin-labeled antibody, streptavidin and biotin-labeled oligonucleotide, respectively, a large number of DNA1 functionalized Pdots could be introduced onto the slide surface by rolling circle amplification of the oligonucleotide to trigger the enzymatically cyclic release of the Pdots from the self-quenched probes to solution in the presence of Exo III. The dual DNA amplification produced greatly amplified ECL signal for true-color ECL imaging. Using carcinoembryonic antigen, cytokeratin-19-fragment and neuron-specific enolase as a lung cancer-specific biomarker panel, the ECL imaging-based multi-immunoassay exhibited excellent performance with a linear range of 1 pg mL−1 to 500 ng mL−1 and limits of detection of 0.17 pg mL−1, 0.12 pg mL−1 and 0.22 pg mL−1, respectively. The proposed method could accurately detect these biomarkers in clinical human serum samples for lung cancer screening. The Pdots-based true-color ECL imaging approach possessed the advantages of visual analysis along with wide detection range and high sensitivity, and thus has great potential in clinical application.

Keywords: Electrochemiluminescence imaging; Polymer dots; Signal amplification; Multi-immunoassay; Lung cancer

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Electrochemiluminescence (ECL) is an electrochemically initiated energy-relaxation process with the features of electrochemical controllability and low background due to the separation of excitation mode from signal detection.1-6 Due to the relatively convenient temporal and spatial control of ECL technique, ECL imaging has been rapidly developed for multiplex targets analysis with a charge coupled device (CCD) camera for signal acquisition, and broadly applied in genotoxins screening,7 immunoassays,8,9 fingerprint analysis,10,11 cell detections,12,13 and reaction mechanism research.14 Typically, several ECL imaging methods have been proposed for recolor ECL immunoassays of multiple proteins.9,15,16 Nonetheless, most of ECL imaging methods contain a process of false color that are not easy to implement visualized detection at real time. Nowadays, true-color ECL imaging systems have attracted more attention for the simultaneous detection of protein biomarkers with spectrum distinct luminophores and the study of electron transfer pathways via manipulating the applied potentials.17,18 These systems show tremendous superiorities of spatial discrimination and visualized detection in on-site testing.19 However, the current true-color ECL imaging is usually achieved with classical luminophores such as ruthenium and iridium complexes, and the insolubility of some complexes in aqueous solution limits its application in bioanalysis.20 Therefore, searching for preeminent ECL luminophores with good biocompatibility and high ECL efficiency for true-color ECL imaging is indispensable. Recently, semiconductor polymer nanoparticles or polymer dots (Pdots) have been quickly developed and used in single particle tracing,21,22 cellular labeling,23,24 and drug delivery25,26 due

to

their

nontoxic

features,

photostability

and

size/surface-trap

controlled

luminescence.27,28 The Pdots, including poly(phenylene vinylene),29,30 polyfluorene31 and fluorene-based copolymers,32 can act as new ECL emitters with fantastic features such as biocompatibility and tunable optical properties through changing of monomer and easy functionalization.33 However, resulting from the relatively lower ECL efficiency of most 3



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Pdots than general inorganic emitters, these Pdots have been rarely used for the development of

ECL

imaging

technology.

Our

previous

work

firstly

used

poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(1-cyanovinylene-1,4-phenylene)] (CN-PPV) Pdots to achieve true-color imaging via the chelating interaction of metal ions and Pdots for the analysis of iron (III) ion.29 Nevertheless, the deficiency in specificity and ECL efficiency limits its application in the complex samples. To achieve highly sensitive true-color ECL imaging for specific detection of multiplex proteins

in

complex

biological

samples,

here

we

used

highly

efficient

poly[(9,9-dioctylfuorenyl-2,7-diyl)-alt-co-(1,4-benzo-{2,1’,3}-thiadiazole)] (PFBT) Pdots as biocompatible ECL emitter to design a dual DNA amplification strategy. The strong ECL emission of PFBT Pdots at gold evaporated indium tin oxide slide (Au/ITO) inducted us to use the substrate for preparation of immunosensing array. The dual DNA amplification for obtaining greatly amplified ECL signal was achieved on the array by rolling circle amplification (RCA) of oligonucleotide strands and then enzymatically cyclic release (ECR) of PFBT Pdots from self-quenched probes, following the affinity recognition of streptavidin to biotin-labelled antibody and biotin-labelled oligonucleotide (Scheme 1). The self-quenched probe was designed by functionalizing the Pdots with DNA1 and then binding a black hole quencher (BHQ)-labelled DNA2 to DNA1 (Scheme 1A). Although the RCA and ECR have been well known for signal amplification, here the RCA reaction triggered linear periodic assembly of a large number of Pdots-DNA134,35 on immunosensors array and then the Exonuclease III (Exo III) catalyzed release of the Pdots into detection solution, leading to greatly amplified “Off-On” signal to achieve the powerful ECL imaging with Pdots as emitter. The practicability of the ECL imaging method for screening of cancer diseases was demonstrated with carcinoembryonic antigen (CEA), cytokeratin-19-fragment (CY211) and neuron-specific enolase (NSE) as specific biomarker panel for lung cancer.36 The excellent 4


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performance of the proposed ECL imaging array along with the dual DNA amplification strategy and high ECL efficiency of PFBT Pdots for multi-immunoassay of three protein biomarkers in clinical serum samples indicated its promising application in clinical diagnosis and cancer screening.

EXPERIMENTAL SECTION Preparation of PFBT Pdots and Self-quenched Probe. PFBT Pdots were prepared in aqueous solution through nanoprecipitation according to a previous work.37 In brief, the mixture stock solution of 50 µg mL−1 PFBT and 10 µg mL−1 PSMA was firstly prepared in 2 mL THF. After ultrasonically degassed for 20 minutes, the mixture was quickly added to 10 mL Milli-Q water at bath sonicator (120 W, 37 kHz) for 3 min. The THF solvent was removed by rotary evaporation under vacuum followed by filtration through a 0.22-µm poly(ether sulfones) syringe filter to obtain carboxyl PFBT Pdots dispersion. The PFBT Pdots were functionalized with DNA1 utilizing EDC catalytic reaction between carboxyl on Pdots and the amine group of DNA1.38 In a typical conjugation reaction, Pdots dispersion (0.18 mg mL−1, 3 mL), HEPES buffer (1 M, 60 µL) and PEG (5% w/v, Mw 3350, 60 µL) were mixed completely and adjusted pH to 7.1 with 0.1 M NaOH. Then, DNA1 (100 µM, 60 µL) and EDC (5 mg mL−1, 600 µL) were added to the mixture and vibrated for 3 h at room temperature. The resulting Pdots-DNA1 conjugate was separated by ultrafiltration for three times to remove free DNA. Finally, the purified Pdots-DNA1 hybridized with molar equivalent BHQ-DNA2 in hybridization buffer (10 mM PBS, 0.25 M NaCl, pH 7.4) at 37 °C for 30 min to obtain the self-quenched probe, which was stored at 4 °C for further use. Preparation of ECL Immunosensors Array. The Au/ITO (5 nm chrome followed by 50 nm gold) slide was fabricated with electron beam evaporation technique (DE Tec. Ltd, USA), and pasted with a porous sticker with 3×7 well array (diameter : 2 mm, depth : 1 mm) as 5



working electrode array (Figure S1). Then, 5 mM MPA was dropped on the electrodes for 8 h at room temperature to form the self-assembled monolayer (SAM), and followed by incubation in 20 mM EDC solution for 3 h at 4 °C to activate the carboxyl group of MPA. Afterward, 10 µg mL−1 capture antibody (Ab1) was added, and incubated overnight at 4 °C. The remaining activated groups were deactivated by addition of 1 M ethanolamine, and the array was incubated in 5% BSA for 30 min to block the unspecific sites. After the array was placed vertically to wash row by row for three times by gently dropping washing buffer (10 mM PBS, pH 7.4), the immunosensors array was ready for multi-immunoassay with the proposed ECL imaging method. ECL Imaging Detection. After 2 µL of antigen solution or sample was added into each well at 37 °C for 1 h, the resulting immunosensors were washed with washing buffer to remove the excess antigens, and 10 µg mL−1 biotinylated detection antibody and SA were dropped into the wells in sequence and incubated for 30 min at 37 °C in turn (Scheme 1B), which were followed by washing with washing buffer. To initiate the RCA reaction, 5 µM biotin conjuncted primer DNA was added into each well to recognize the SA at 37 °C for 30 min, and the mixture of 10×reaction buffer (2.5 µL), phi29 DNA polymerase (25 U), dNTPs (1 µL, 100 mM), circle DNA template (7.5 µL, Supporting Information) and 15 µL ultrapure water was injected into these wells quickly to incubate for 1 h at 37 °C, and followed by washing. The ECR of PFBT Pdots was then performed by adding 2 µL self-quenched probe and Exo III (0.5 U mL−1) into the wells for 2 h at 37 °C. The reaction mixture was finally dried at 37 °C for 40 min to form a film on the immunosensor surface, which was used to perform the ECL imaging by applying a constant potential of +1.2 V for 2 s in 0.1 M pH 7.4 PBS containing 25 mM TPrA solution. ECL spectra. After 200 µL of 0.36 mg mL−1 PFBT Pdots and 10 mM Ru(bpy)32+ were coated at carbon/ITO electrodes, respectively, the modified electrode was immersed in 0.1 M 6


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pH 7.4 PBS containing 0.1 M KNO3 and 0.1 M TPrA to apply a constant potential of +1.2 V for 9 s. CHI 660D electrochemical workstation conjugated with F-7000 fluorescence spectrometer under the closed shutter was used to record the ECL spectra of PFBT Pdots and Ru(bpy)32+. The relative ECL efficiency was calculated using the equation:

ΦECL = Φ °ECL(I/Q)/(I°/Q°)

(1)

where Q and Q° are the consumed charges (integrating current vs. time), I and I° are the integrated ECL intensities (integrating ECL spectrum vs. wavelength), ΦECL and Φ °ECL are the ECL efficiency of the sample and standard, respectively. In this work, 10 mM Ru(bpy)32+ was used as the standard with the Φ °ECL value of 1.

RESULTS AND DISCUSSION Characterization of Self-quenched Probe. The PFBT Pdots were prepared using PFBT polymer as a luminescent precursor and PSMA as carboxyl-functionalized copolymer to produce carboxyl groups on the surface of Pdots via the hydrolysis of maleic anhydride (Scheme 1A). The TEM images of PFBT Pdots showed the spherical and monodisperse feature with a diameter around 23 nm (Figure 1A), which was close to the hydrodynamic diameter of 25 nm with a size distribution of 20 to 35 nm from DLS measurement (Figure S2). The ECL spectrum of PFBT Pdots modified carbon/ITO electrode in the presence of TPrA as an anodic co-reactant showed a wide emission ranging from 490 to 680 nm (Figure 1B, curve a). The relative ECL efficiency was calculated to be 120% (vs. Ru(bpy)32+ modified carbon/ITO electrode, Figure 1B, curve b and Table S1), which was 10.8 times higher than the ECL efficiency of CN-PPV Pdots/TPrA system.29 The ECL emission peak of PFBT Pdots occurred at the same wavelength as that of the fluorescence spectrum (Figure 1B, curve c), indicating that the ECL was generated from the band gap emission of PFBT Pots.3 7



After PFBT Pdots were functionalized with DNA1, the Pdots-DNA1 conjugate showed three absorption peaks at 270 nm, 323 nm and 460 nm (Figure 1C, curve a). The absorption peaks at 323 nm and 460 nm were coincident with those of PFBT Pdots (Figure 1C, curve b), while the absorption peak at 270 nm was the merge of another absorption peak of PFBT Pdots at 273 nm and the absorption peak of DNA1 at 260 nm (Figure 1C, curve c), and thus was much stronger than that of PFBT Pdots, indicating the successful conjugation of DNA1 on Pdots surface. The conjugation of DNA1 led to negative increase of Zeta-potential from −18.3 mV of Pdots to −42.2 mV of Pdots-DNA1 (Figure S3). The selection of quencher was performed by hybridizing PFBT Pdots-DNA1 with ferrocene (Fc)- or BHQ-labeled DNA2. The hybridization of BHQ-labeled DNA2 led to a quenching of 93.8% ECL emission of PFBT Pdots (Figure S4) due to the overlap between the absorption of BHQ (inset in Figure 1D) and fluorescent emission of PFBT Pdots (Figure 1B, curve c), which resulted in resonance energy transfer from excited Pdots to BHQ. The resonance energy transfer could also be observed from the fluorescent spectrum of the probe (Figure 1D, curve a), which showed much lower fluorescent intensity than the mixture of PFBT Pdots and BHQ-DNA2 at the same concentration of Pdots (Figure 1D, curve b). Fc-labeled DNA2 decreased the ECL emission of PFBT Pdots by 59.4% (Figure S4) through the charge transfer between Fc and excited Pdots.39 Thus the probe was prepared with BHQ-labeled DNA2 and possessed low background for subsequent imaging detection. Characterization of Immunosensors Array. To obtain high imaging quality, the Au/ITO slide was employed as the substrate for preparation of immunosensing array since TPrA is more easily oxidized on gold surface,40 which led to obvious oxidation wave of TPrA at potentials more positive than +0.8 V (inset in Figure 2A) and about 100-fold stronger ECL emission of PFBT Pdots modified Au/ITO electrode at +1.04 V than PFBT Pdots modified ITO electrode (Figure 2A). 8


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After MPA monolayer was assembled on Au/ITO slide, the electron-transfer resistance Rct obviously increased (Figure 2B, curves a and b). The Rct successively increased upon binding of capture antibody and blocking of BSA (Figure 2B, curves c and d). After the immunosensor was incubated with target and then biotin-labelled antibody as well as streptavidin and biotin-labelled oligonucleotide to perform RCA reaction, the Rct further increased (Figure 2B, curve e). This result confirmed the successful RCA reaction, which introduced negatively charged DNA strand on immunosensor surface to repel the electron transfer of K3Fe(CN)6/K4Fe(CN)6. The binding of capture antibody onto MPA monolayer modified Au/ITO slide increased the surface roughness, which was much more greatly increased after immunoreactions and successful RCA reaction due to the presence of more protein molecules and produced long DNA strands on immunosensor surface (Figure S5). Feasibility of ECL Imaging at Immunosensors. Although the probe showed 93.8% quenching on ECL emission of PFBT Pdots due to the presence of BHQ (Figure S4), the mixing of PFBT Pdots with BHQ-DNA2 did not quench the ECL emission (Figure 2C), as observed from the fluorescent spectra (Figure 1D). Thus the presence of free BHQ or BHQ-DNA2 in the detection system did not influence the ECL imaging of PFBT Pdots film modified Au/ITO slide (Figure 2C, inset), which led to an “Off-On” imaging method with low background for monitoring the release of PFBT Pdots from the self-quenched probe. The release of PFBT Pdots could be achieved with an ECR strategy following the RCA reaction on immunosensor surface. After the long DNA product was formed, it could hybridize a large number of Pdots-DNA1, which led to the ECR in the presence of Exo III and the probe to release PFBT Pdots and then the formation of PFBT Pdots film on immunosensor surface. As shown in Figure 2D, the successive immunoreactions, streptavidin-biotin recognition, RCA, probe binding and ECR led to strong ECL emission for imaging of the formed PFBT Pdots film, while any lack of these processes exhibited very 9



weak or negligible ECL emission, which further verified the feasibility of the proposed ECL imaging strategy for multi-immunoassay of proteins. Optimization of Detection Conditions. As the co-reactant ECL emission of PFBT Pdots film depended on the presence of gold coating evaporated on ITO (Figure 2A), the thickness of gold coating was firstly optimized. The ECL signal increased with the augment of gold coating thickness, and reached the maximum value at 50 nm (Figure 3A). The decrease of ECL signal at greater thickness might attribute to the nonradiative energy dissipation, which quenched the ECL emission.41 Thus, 50-nm gold coating was evaporated on ITO to obtain high ECL signal. With the increasing PFBT Pdots concentration for preparation of the self-quenched probe, the ECL imaging signal increased and treaded to the maximum value, and the optimal concentration was 0.18 mg mL-1 (Figure S6). The amount of capture antibody immobilized on immunosensor surface was an important parameter to affect the ECL response, which depended on the amount of MPA assembled on Au/ITO surface. At 100 ng mL−1 CEA, the ECL imaging signal quickly increased with the increasing MPA concentration used for preparation of the immunosensor and trended to a maximum value at 5 mM (Figure 3B), which was thus selected as the optimum concentration of MPA. The time of RCA reaction decided the strand length or the number of repeated sequence of RCA product for binding Pdots-DNA1, thus it affected the efficiency of ECR for obtaining the amplified ECL signal. The ECL signal increased with the extension of reaction time and tended to a constant value at 1 h (Figure 3C). Thus, the RCA reaction was performed for 1 h, which produced the DNA strands with high molecular weight (Figure S7) to bind Pdots-DNA1. The hybridization of DNA1 with the repeated sequence of RCA product was demonstrated by the PAGE image of the mixture of 1 µM DNA3 and 3 µM DNA1, which showed only one 10


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lane with larger molecular weight than both DNA1 and DNA3 (Figure S8, lanes 1, 3 and 5). Meanwhile, the presence of DNA3 could release DNA2 from the hybridization product of DNA1 and DNA2 to form the hybrid of DNA1 and DNA3 (Figure S8, lanes 2, 4, 5 and 6). In the presence of Exo III, DNA1 in the hybrid of DNA1 and DNA3 could be cleaved, which led to the lane of only DNA3 (Figure S8, lanes 3 and 7). After Exo III was deactivated at 85 o

C for 10 min, the remained DNA3 could further hybridize with DNA1, while the active Exo

III did not affect the presence of the hybrid of DNA1 and DNA2 (Figure S8, lanes 4, 5, 8 and 9), demonstrating the feasibility of the ECR strategy. The amount of released PFBT Pdots depended on the ECR time. After the ECR mixture was dried on immunosensor surface, the ECL imaging signal increased with the increasing ECR time, which reached a platform at about 1 h (Figure 3D), indicating the maximum release of PFBT Pdots from the probe. Hence, 1-h ECR was applied in the multi-immunoassay via ECL imaging. Performance of ECL Imaging for Multi-immunoassay. Under optimal conditions, the immunosensors array was used for ECL imaging of three proteins, CEA, CY211 and NSE, as a lung cancer-specific biomarker panel.36,42 The ECL brightness increased with the increasing protein concentration, while the array showed extremely low background (Figure 4A). The plots of ECL intensity integrated from the marked area vs the logarithm of protein concentration showed good linearity in the concentration range of 1 pg mL−1 to 500 ng mL−1 with the correlation coefficients of 0.9956, 0.9950 and 0.9957, respectively (Figures 4B-4D). The limits of detection were 0.17 pg mL−1, 0.12 pg mL−1 and 0.22 pg mL−1 for CEA, CY211 and NSE at a signal-to-noise ratio of 3, respectively, which were significantly lower than the previous true-color ECL immunoassay with inorganic complexes as the ECL emitters (Table S2).20,43 The selectivity of this strategy was further evaluated by comparing the ECL intensity in 11



the presence of different proteins, including alpha fetoprotein (AFP), CA125, CEA, CY211 and NSE. As expected, the array only showed the ECL response to corresponding target proteins (Figure 5). Overall, the good selectivity, wide detection range and high sensitivity of the proposed ECL imaging array indicated its potential application in clinical diagnosis and cancer screening. Real Sample Analysis. The practicability of the ECL imaging method was firstly evaluated with seven clinical serum samples from lung cancer patients. All these samples showed positive responses of three proteins at the immunosensors array (Figure 6A). From the ECL intensity and calibration curves (Figures 4B-4D), the concentrations of three proteins in these samples could be obtained, which were comparable with the reference results obtained from commercial electrochemiluminescent testing (Figures 6B-6D), indicating the reliability of the proposed method. The relative standard deviations (RSD) of parallel detection with three immunosensors arrays were less than 7.3%, indicating good fabrication repeatability of the arrays and good detection precision. Moreover, owing to the appropriate and wide detection range, no dilution was required for the detection of serum samples, which was more direct and convenient than the previous recolor ECL assays (Table S2).9,15,16 The proposed method was further used for multi-immunoassay of seven random clinical serum samples which were demonstrated to be CEA, CY211 and NSE positivity. Again, the results obtained were almost in agreement with those obtained from the commercial method (Figure S9), indicating high specificity and good accuracy in practice. To further prove the feasibility of the proposed method for screening of lung cancer, 21 random clinical serum samples were detected. As shown in Figure S10, seven samples (no. 3, 7, 9, 11, 15, 17 and 20) showed the positivity of all three biomarkers, which was in good agreement with commercial testing, indicating the value of the ECL imaging strategy and 12


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immunosensors array for cancer screening.

CONCLUSION This work uses biocompatible PFBT Pdots as ECL emitter to design a self-quenched probe and develop a true-color ECL imaging strategy for multi-immunoassay. The excellent performance of this imaging strategy results from the designed dual DNA amplification along with the substrate of gold evaporated ITO to sensitize the ECL emission. The PFBT Pdots show 10.8 times higher ECL efficiency than previously reported CN-PPV Pdots/TPrA system, and the evaporation of gold on ITO can enhance the ECL emission by 100 folds. The RCA reaction provides a lot of repeated sequence to hybridize with Pdots-DNA1 for separation from the quencher, and followed ECR process leads to cascade release of Pdots from the probe. The high quenching efficiency of BHQ provides low background for “Off-On” imaging. The proposed strategy shows distinct brightness for immunoassay of different targets with high sensitivity, good selectivity, excellent accuracy and acceptable precision. The reliability has been demonstrated by testing the serum samples from lung cancer patients and clinically demonstrated positive patients. This method can conveniently been used for cancer screening through ECL imaging of the biomarker panel. These results indicate its great potential in clinical application.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and reagents, Apparatus, Preparation of circle DNA template, gel electrophoresis analysis, electrolytic cell, DLS and Zeta-potential of Pdots, ECL-time curves and AFM images of modified electrodes, immunoassay results of 7 positive 13



samples and 21 random serum samples, relative ECL efficiency, and performance comparison of the proposed imaging method with previous ECL imaging immunoassays (PDF).

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected]. E-mail: [email protected] ORCID Huangxian Ju: 0000-0002-6741-5302 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We acknowledge the financial support of the National Natural Science Foundation of China (21635005 and 21361162002).

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FIGURE CAPTIONS Scheme 1. Schematic Diagram of (A) Preparation of Signal Probe and (B) Stepwise Construction of ECL Imaging Array and Immunoassay Procedure. Figure 1. (A) TEM images of PFBT Pdots. (B) ECL spectra of PFBT Pdots (curve a) and Ru(bpy)32+ (curve b) modified carbon/ITO electrode in 0.1 M pH 7.4 PBS containing 0.1 M TPrA at +1.2 V, and FL spectrum of 0.36 mg mL−1 PFBT Pdots (curve c). (C) UV-vis spectra of Pdots-DNA1 (a), PFBT Pdots (b) and DNA1 (c). (D) FL spectra of probe (a) and mixture of PFBT Pdots and BHQ-DNA2 (b) at the same concentrations of Pdots and DNA, respectively. Inset: UV-vis spectrum of BHQ. Figure 2. (A) ECL curves of PFBT Pdots modified Au/ITO and ITO electrodes in 0.1 M pH 7.4 PBS containing 0.1 M KNO3 and 25 mM TPrA. Inset: corresponding CV curves. (B) EIS of Au/ITO (a), MPA modified Au/ITO (b), CEA-Ab1/Au/ITO (c), BSA/CEA-Ab1/Au/ITO (immunosensor) (d) and immunosensor after incubation with 100 ng mL−1 CEA and biotin-antibody, streptavidin-biotin recognition and RCA reaction successively (e). (C) ECL-time curves of Au/ITO modified with probe (a) and mixture of PFBT Pdots and BHQ-DNA2 (b). Inset: ECL image. (D) ECL image of immunosensors array with different detection procedures (“+” presence, “-” absence). Figure 3. Effects of (A) gold coating thickness and (B) MPA concentration for CEA immunosensor preparation, and (C) RCA and (D) ECR time for dual DNA amplification on ECL imaging signal for CEA at 100 ng mL-1 (n = 3). Figure 4. (A) ECL image of three targets at different concentrations. (B-D) Calibration curves for detection of (B) CEA, (C) CY211 and (D) NSE (n = 3). Figure 5. Specificity of the proposed immunosensors array for imaging detection of CEA, 20


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CY211 and NSE at 50 ng mL−1, AFP at 50 ng mL−1 and CA 125 at 50 U mL−1. Figure 6. Immunoassay results of clinical serum samples using the proposed ECL imaging method and reference ECL method. (A) ECL image for three proteins, and (B-D) comparison for (B) CEA, (C) CY211 and (D) NSE (n = 3).

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 6

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