Hollow Porous Polymeric Nanospheres of a Self-Enhanced

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Hollow Porous Polymeric Nanospheres of Self-Enhanced Ruthenium Complex with Improved Electrochemiluminescent Efficiency for Ultrasensitive Aptasensor Construction Anyi Chen, Min Zhao, Ying Zhuo, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02003 • Publication Date (Web): 04 Aug 2017 Downloaded from http://pubs.acs.org on August 4, 2017

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Analytical Chemistry

Hollow Porous Polymeric Nanospheres of Self-Enhanced Ruthenium Complex with Improved Electrochemiluminescent Efficiency for Ultrasensitive Aptasensor Construction Anyi Chen, Min Zhao, Ying Zhuo,∗ Yaqin Chai, 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, China

∗

Corresponding authors at: Tel.: +86 23 68252277, fax: +86 23 68253172.

E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan). 1

ACS Paragon Plus Environment


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ABSTRACT

Although Ru(II) complex-based bulk nanomaterials have received considerable concern in the electrochemiluminescent (ECL) assays owing to their strong ECL signals, the ECL efficiency of these nanomaterials was quite low since the bulk nanomaterials brought about serious inner filter effect and excess inactive emitters. Herein, hollow porous polymeric nanospheres of self-enhanced ruthenium complex (abbreviated

as

Ru-HPNSs)

were

prepared

with

precursor

of

polyethyleneimine-ruthenium complex to greatly decrease the inner filter effect and minimize inactive emitters, which significantly improved the ECL efficiency. Based on the novel Ru-HPNSs as efficient ECL tags and target-catalyzed hairpin hybridization as signal amplification strategy, an ultrasensitive ECL aptasensor was constructed for the detection of mucin 1 (MUC1), which showed excellent linear response to a concentration variation from 1.0 fg/mL to 100 pg/mL with the limit of detection down to 0.31 fg/mL. It was worth mentioning that this work opened a new avenue for developing high-performance ECL nanomaterials as well as ultrasensitive ECL biosensors for clinical and biochemical analysis. Keywords: hollow porous polymeric nanospheres, self-enhanced ruthenium complex, electrochemiluminescent efficiency, aptasensor, MUC1

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INTRODUCTION Electrochemiluminescent (ECL) methodology, as the unique characteristics of excellent controllability, high sensitivity and low-cost instruments 1-5, has attracted extensive research interest in the fields such as food analysis 6, environmental monitoring

5

and clinical diagnosis

8, 9

. To improve the sensitivity of ECL

methodology, conventional practices were developing nanomaterials as carriers to immobilize ECL emitters 10-12. Nevertheless, some popularly used nanomaterials (e.g. gold nanoparticles, graphene) would increase the risk of ECL quenching 13-14 and the capacity of immobilization was restricted to the surface of the carriers. Bard’s group first prepared single crystalline nanobelts of water-insoluble tris(2,2'-bipyridyl) ruthenium(II) (Ru(bpy)32+) derivative utilizing its reprecipitation in aqueous solution 15

. The prepared nanobelts achieved relatively strong ECL signal as they avoided the

ECL quenching of carriers and maximized the immobilization of ECL emitters. Recently, in our previous works, self-enhanced Ru(bpy)32+ derivatives containing intramolecular

coreactive

groups

were

used

as

precursor

to

prepared

nanorods/nanoparticles, which further improved the ECL intensity and stability16,17. However, the ECL efficiency of these bulk nanomaterials was still limited as the tight molecular arrangement of the ECL emitters unavoidably increased inner filter effect and impeded the electrochemical activation of the internal ECL emitters. Ru-complex-based hollow porous nanomaterials were a better choice rather than bulk nanomaterials to improve ECL efficiency as they decreased the inner filter effect and minimized inactive ECL emitters. 3



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Porous polymers, due to their properties of high specific surface area, well defined porosity, easy processibility, and light weight,18,19 are satisfactory nanocarriers used in extensive areas such as gas storage and separation,20-25 drug delivery,25,26 catalysts,23, 27-28

and sensors29-30. To synthesize porous polymers, layer-by-layer (LbL) assembly

was the most popular strategy due to its powerful control and easy process31. In typical syntheses of porous polymers, oppositely charged moieties polymers, such as anionic poly(styrenesulfonate) (PSS) vs. cationic poly(allylamine hydrochloride) (PAH) 32 and anionic polyacrylic acid (PAA) vs. cationic polyethyleneimine (PEI), etc. 33

, were the most used raw materials, which were successively deposited on the

surface of templates with the help of electrostatic interaction. It is worthy to point out, with large amount of tertiary amines in the structure, PEI is an efficient coreactant of Ru(bpy)32+ ECL system. Thus, the self-enhanced ECL complex was synthesized by covalently cross-linking PEI with Ru(bpy)32+ derivative (PEI-Ru), which exhibited excellent ECL efficiency as well as characteristics of positively charged polymer 34. Inspired by the above unique property of self-enhanced PEI-Ru complex, anionic PAA and cationic PEI-Ru were chosen as the fascinating precursors to synthesize Ru(II) complex-based hollow porous polymeric nanomaterials, which were expected to improve ECL efficiency by reducing inner filter effect and inactive ECL emitters. Herein, hollow porous polymeric nanospheres of self-enhanced PEI-Ru complex (Ru-HPNSs) were synthesized with precursor of PEI-Ru and employed as signal tags in ECL aptasensor for ultrasensitive detection of MUC1, an important biomarker associated with many types of cancer.35, 36 First, self-enhanced PEI-Ru was obtained 4


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by

covalently

crosslinking

Bis(2,2-bipyridyl)(4-methyl-4-carboxypropyl-2,2-bipyridyl)

PEI

with ruthenium(II)

[Ru(mcbpy)(bpy)22+] via amido linkage. As show in Scheme 1, the positively charged PEI-Ru and the negatively charged PAA were successively coated on SiO2 nanoparticles via electrostatic interaction. After the layers were crosslinked to strengthen the skeleton, the SiO2 nanoparticles was etched away by HF, resulting the expected product of Ru-HPNSs. With an aptamer-embedded hairpin motif probe (H1) as recognition element, the Ru-HPNSs were used as ECL signal tags in an aptasensor for MUC1 detection. In the presence of target MUC1, H1 recognized the target MUC1 with structure-switching, which further catalyzed the hybridization of the capture hairpin motif (H2, immobilized on the electrode) and the label hairpin motif (H3, labeled with Ru-HPNSs). As a result, the Ru-HPNSs were immobilized on the electrode surface to generate ECL signal in response to the target MUC1. Owing to hollow porous structure, the as-synthesized Ru-HPNSs exhibited high ECL efficiency as the significant decrease of inner filter effect and inactive emitters. Meanwhile, the Ru-HPNSs contained large amount of Ru(II) complex molecules, thus they exerted strong ECL intensity. Employing the novel Ru-HPNSs as ECL tags and the target catalyzed hairpin hybridization for signal amplification, the ECL aptasensor achieved ultrasensitive detection of MUC1. This method using Ru(II) complex-based hollow porous polymeric nanomaterials to decrease the inner filter effect and minimize inactive emitters opened a new avenue for the improvement of ECL efficiency and a versatile strategy for monitoring other biomolecules (e.g., DNA, small molecule, 5



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metal ion, peptide, etc.).

Scheme 1 Schematic illustration of the aptasensor: (A) Synthesis of the Ru-HPNSs. (B) Working principle of the aptasensor for MUC1 detection.

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EXPERIMENTAL SECTION Preparation of the H3/Ru-HPNSs bioconjugate. The self-enhanced PEI-Ru(II) complex was prepared by covalently crosslinking PEI with Ru(mcbpy)(bpy)22+ in the presence of EDC and NHS in ethanol solution. Ru(mcbpy)(bpy)2Cl2 (5 mM) was dissolved in absolute ethanol. After the addition of EDC (40 mM) and NHS (10 mM), the complex was stirred for 2 h at 4 °C. The final solution of PEI-Ru was obtained after the addition of 1% PEI and sequential stirring at room temperature overnight. 1.0 mg SiO2 nanoparticles (the preparation of SiO2 nanoparticles was showed in Supporting Information) were dispersed in 10 mL absolute ethanol, followed by the addition of PEI-Ru solution (100 µL) and stirring for 30 min to obtain the PEI-Ru coated SiO2 nanoparticles (SiO2@PEI-Ru). The SiO2@PEI-Ru was collected by centrifugation (8000 rpm, 2 min), followed by a double washing with ethanol to remove the residual PEI-Ru. Then 1 mL PAA (1%) was dropped into the solution with vigorously stirring for 30 min. After double washing with ethanol, the nanoparticles were encapsulated with PEI-Ru, PAA, and PEI-Ru successively as above-mentioned protocol to obtain SiO2@PEI-Ru/PAA/PEI-Ru/PAA/PEI-Ru nanoparticles. Finally, EDC (40 mM) and NHS (10 mM) were added into the solution of SiO2@PEI-Ru/PAA/PEI-Ru/PAA/PEI-Ru nanoparticles and stirred at 4 °C for 2 h to crosslink the PAA and PEI-Ru layers and strengthen the skeleton of the polymeric capsule. The resultant SiO2@PAA-PEI-Ru core-shell nanoparticles were collected by

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centrifugation (8000 rpm, 5 min), which was then dispersed in 10 mL deionized water and stored at 4 °C for further use. The Ru-HPNSs were obtained by etching away the SiO2 core using HF. Briefly, 0.1 M HF (100 µL) was added into the solution of SiO2@PAA-PEI-Ru core-shell nanoparticles and stirred for 15 min. The Ru-HPNSs were collected by centrifugation (12000 rpm, 5 min). After washed with deionized water and ethanol for 3 times, the Ru-HPNSs were dispersed in 1 mL PBS (0.1 M, pH 7.4). Then, H3 (200 nM), EDC (40 mM) and NHS (10 mM) were supplied into the dispersion and stirred for 2 h at 4 °C to form H3/Ru-HPNSs bioconjugate via amide crosslinking. After collected by centrifugation (8000 rpm, 5 min) and washed with 0.1 M PBS (pH 7.4) for 3 times, the final product H3/Ru-HPNSs bioconjugate was dispersed in 1 mL PBS (0.1 M, pH 7.4) and stored at 4 °C. Fabrication of the Aptasensor. The glassy carbon electrode (GCE, Φ = 4 mm) was polished and cleaned referring to the reported works.37 Then platinum nanoparticles (Pt NPs) was electrodeposited on the electrode surface in 1% H2PtCl6 solution via potential stripping method at -0.25 V for 30 s to obtain Pt NPs modified GCE (Pt NPs/GCE). After rinsing with deionized water and dried in the nitrogen atmosphere, 10 µL of H2 solution (1 µM) was dropped on the electrode surface and incubated overnight at room temperature. Thus, H2 was immobilized on the electrode through Pt-S bond (H2/Pt NPs/GCE). Finally, MCH (1

8


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mM) was incubated with the modified electrode to block the nonspecific binding sites forming MCH/H2/Pt NPs/GCE. ECL Measurement of the Aptasensor. For the analysis of the MUC1, the sample solution (5 µL) with different concentrations was dropped on the sensing surface of the aptasensor together with H1 solution (5 µL) and H3/Ru-HPNSs bioconjugate solution (5 µL). After incubated for 2 h, the aptasensor was rinsed with 0.1 M PBS (pH 7.4) to remove unbound reagents. Then the potential from 0.2 to 1.2 V with a scan rate of 200 mV/s was applied to the aptasensor for ECL measurement (PMT high-voltage 800 V, magnitude 3) in 0.1 M PBS (pH 7.4). RESULTS AND DISCUSSION Morphology Characterization of the Materials. TEM was employed to verify the formation of the core-shell and hollow porous polymeric nanostructures of Ru-HPNSs. Figure 1A demonstrated the TEM image of as-prepared SiO2 nanoparticles, which exhibited a homogeneous solid sphere with a diameter of about 200 nm. After the LbL assembly of PEI-Ru and PAA, the core-shell nanostructure was clearly observed where the SiO2 nanoparticles were encapsulated with cotton-like polymer shells (Figure 1B), suggesting PEI-Ru and PAA were fabricated on the surface of SiO2 nanoparticles as expected. Finally, the Ru-HPNSs were obtained by utilizing HF to etch the SiO2 core of the SiO2@PAA-PEI-Ru core-shell nanostructure. As shown in Figure 1C, the polymers formed a balloon-like 9



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nanostructure, manifesting the successful synthesis of the Ru-HPNSs. Figure S1 demonstrated the TEM image at lower magnification, suggesting the good dispersibility of the Ru-HPNSs.

Figure 1 TEM image of (A) SiO2 nanoparticles, (B) SiO2@PAA-PEI-Ru core-shell nanoparticles, and (C) as-synthesized Ru-HPNSs.

Spectral Characteristics of the Ru-HPNSs.

The optical properties of the as-synthesized Ru-HPNSs were profiled by UV-vis absorption spectra, fluorescence spectra and ECL spectra. As shown in Figure 2A, SiO2 nanoparticles exhibited a smooth absorption curve which presented decreasing absorbance with the increase of wavelength (curve a). PEI-Ru exhibited two absorption peaks at 290 nm and 470 nm which was assigned to the n→π* transition of amide groups and π→π* transition of bipyridyl ligands (curve b). Apparently, the two specific absorption peaks could be observed in the absorption spectra of SiO2@PAA-PEI-Ru core-shell nanoparticles (curve c), indicating the successful modification of PEI-Ru on SiO2 nanoparticles. After the SiO2@PAA-PEI-Ru core-shell nanoparticles treated with HF, the two peaks at 290 nm and 470 nm became more prominent (curve d), as the removal of SiO2 core reduce the background absorbance. Figure 2B demonstrated the FL spectra of Ru(mcbpy)(bpy)22+, PEI-Ru, 10


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and Ru-HPNSs. A distinct emission peak was observed with PEI-Ru at 603 nm (curve b), which exhibited little evolution towards Ru(mcbpy)(bpy)22+ (curve a). By contrast, the emission peak of Ru-HPNSs was located at 593 nm (curve c). The distinct blue shift could be attributed to the reinforcing of rigidity caused by the assembly of PEI-Ru and PAA, which could reduced the collisional energy loss.

Figure 2 (A) The UV-vis absorption spectra of (a) SiO2 nanoparticles, (b) PEI-Ru complex, (c) SiO2@PAA-PEI-Ru core-shell nanoparticles, and (d) Ru-HPNSs. (B) The FL spectra of (a) Ru(mcbpy)(bpy)22+, (b) PEI-Ru, and (c) Ru-HPNSs.

ECL efficiency of the Ru-HPNSs

The ECL quantum efficiency was defined as the number of photons per electron transferred, which was calculated using the relation below38-40:

d d ∅ ∅

1 d d

∅st is the ECL quantum efficiencies of [Ru(bpy)3]2+ (1 mM and 0.1 M (TBA)PF6/acetonitrile) via annihilation, taken as 5.0 %, I is ECL intensity, i is current value, and x is the sample. To demonstrate the inner filter effect of Ru(mcbpy)(bpy)22+,

11



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the relative ECL quantum efficiencies were calculated in the concentrations range from 1.0 mM to 20 mM. Defining the ECL quantum efficiency of Ru(mcbpy)(bpy)22+ (1 mM) as ∅0, the quotient of ∅x and ∅0 can be calculate as the following equation:

d d ∅

2 ∅ d d

As shown in Figure 3A, the value of Q decreases with the increasing concentration, indicating the decrease of the ECL quantum efficiency. The results suggested that inner filter effect was significant in Ru(II) complex of high concentrations and seriously limited the ECL efficiency. Figure 3B demonstrated the ECL spectra of 1 mM Ru(mcbpy)(bpy)22+ (curve a), PEI-Ru (curve b), Ru(mcbpy)(bpy)2Cl2 particles (curve c, see SEM image in Figure S3) and Ru-HPNSs (curve d). Apparently, the emission peak of Ru(mcbpy)(bpy)22+ and PEI-Ru were both located at about 625 nm, suggesting that the crosslinking of Ru(mcbpy)(bpy)22+ and PEI exerted little influence on the ECL emission spectrum. While the emission peak of Ru(mcbpy)(bpy)2Cl2 particles shift to 640 nm. The notable red shift could be ascribed to the strong inner filter effect of Ru(mcbpy)(bpy)2Cl2 particles. However, even though Ru(mcbpy)(bpy)22+

was

concentrated by LbL assembly, the ECL emission peak of Ru-HPNSs exhibited little evolution comparing to PEI-Ru in low concentration. The ECL spectra verified low level of the ECL inner filter effect in Ru-HPNSs, indicating that the hollow porous polymeric nanostructure could significantly decrease the inner filter effect and improve the ECL efficiency of Ru(II) complex-based bulk nanomaterials. The ECL efficiencies of different Ru(II) complex-based nanomaterials were calculated based on 12


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equation 1 and listed in Table 1, suggesting that the hollow porous polymeric nanostructure could remarkably improve the ECL efficiency by reducing inner filter effect.

Figure 3 (A) The relative ECL efficiencies of Ru(mcbpy)(bpy)2Cl2 in different concentrations. (B) The ECL spectra of (a) Ru(mcbpy)(bpy)22+ (1 mM), (b) PEI-Ru, (c) Ru(mcbpy)(bpy)2Cl2 particles and (d) Ru-HPNSs. Table 1 ECL efficiencies of different Ru(II) complex-based nanomaterials. Sample

ECL efficiency

Ru(bpy)3Cl2/1 mM (annihilation)

5.0 %

Ru(mcbpy)(bpy)2Cl2/particles

1.2 %

SiO2@PAA-PEI-Ru

7.1 %

Ru-HPNSs

7.2 %

Characterization of the Nucleic Acid Amplification Strategy.

Native polyacrylamide gel electrophoresis (PAGE) assay was implemented to confirm that MUC1 could catalyze the assembly of the designed two hairpin DNA probes (H2 and H3) with the assistant of the aptamer probe (H1). As shown in Figure 4, three

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single bands were observed in lane 1, 2, and 3, which stood for the samples of H1, H2, and H3, respectively. As the relatively more bases (48 bases), H1 presented slower migration than H2 (38 bases) and H3 (38 bases). The bands in lane 2 and 3 exhibited similar migration due to their identical base numbers. The mixture of H1, H2 and H3 was loaded in lane 4, which displayed a band of H1 and a band of H2/H3, indicating that the assembly of H2 and H3 was forbidden in the absence of MUC1. However, in lane 5, where MUC1 was added into the mixture of H1, H2 and H3, a remarkable band was observed with much slower migration than H1, manifesting the hybridization of H2 and H3. The PAGE result verified that the assembly of H2 and H3 was catalyzed in the presence of the target MUC1.

Figure 4 Native PAGE image of the samples. Lane 1: 2 µM H1; Lane 2: 2 µM H2; Lane 3: 2 µM H3; Lane 4: mixture of H1, H2, and H3 (500 nM each); Lane 5: the mixture of H1, H2, H3 (500 nM each) and MUC1 (100 ng/mL). The samples were incubated at 25 °C for 2h before PAGE. 14


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Electrochemical Characterizing of the Aptasensor. The fabrication process of the aptasensor was confirmed by CV (Figure 5A) and EIS (Figure 5B) in the presence of 5.0 mM [Fe(CN)6]3-/4- solution containing 0.1 M KCl. As shown in Figure 5A, a pair of reversible redox peaks of [Fe(CN)6]3-/4-(curve a) were observed on the bare GCE. After the GCE was electrochemically deposited with PtNPs, significantly, the peak currents increased (curve b), indicating the superior electron transfer acceleration capacity of the PtNPs. When H2 was fabricated on the electrode, the peak currents decreased back to the level approximate to the bare GCE (curve c), suggesting the electron transfer was impeded by the DNA monolayer. After incubated with MCH, the peak current decreased (curve d) as the assembled MCH further impeded the electron transfer. Figure 5B demostrated the EIS of the electrode at different modification stage. With the bare GCE, the electron transfer resistances (Ret) was 220 Ω (curve a). The resistances dramatically decreased to 50 Ω when PtNPs were deposited on the bare GCE (curve b), as the PtNPs accerlated the electron transfer. After the modification of H2 on the PtNPs/GCE, the value of Ret increased to approximate 70 Ω. Then MCH was incubated with H2/PtNPs/GCE to block unspecific binding sites, the value of Ret further increased to 140 Ω, indicating that electron transfer became mor difficult.

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Figure 5 (A) CV curves of (a) bare GCE, (b) PtNPs/GCE, (c) H2/PtNPs/GCE, (d) MCH/H2/PtNPs/GCE. (B) EIS plots of (a) bare GCE, (b) PtNPs/GCE, (c) H2/PtNPs/GCE, (d) MCH/H2/PtNPs/GCE in 0.1 M PBS (pH 7.4) containing 5.0 mM [Fe(CN)6]3-/4- and 0.1 M KCl.

Performance Comparison of different ECL Tags

To confirm the superiority of the as-constructed Ru-HPNSs tags in ECL intensity, a contrast experiment was implemented by comparing the performance of Ru-HPNSs tags to that of PEI-Ru and SiO2@PAA-PEI-Ru. As shown in Figure 6A, only a weak ECL signal (291 a.u) was detected when PEI-Ru was labeled on H3 for signal probe. Figure

6B

demonstrated

the

ECL

intensity

of

the

aptasensor

using

SiO2@PAA-PEI-Ru core-shell nanoparticles as ECL tags, which exhibited an ECL intensity of 4239 a.u.. However, when Ru-HPNSs was preferred to act as ECL tags, a remarkably higher ECL signal (6335 a.u.) was observed by the aptasensor, indicating that the hollow nanostructure possessed better ECL properties. The increase of ECL intensity was ascribed to the hollow nanostructure of Ru-HPNSs, which activated the ECL emitters in the inner layers. 16


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Figure 6 ECL responses of the aptasensors with ECL tags of (A) PEI-Ru, (B) SiO2@PAA-PEI-Ru

core-shell nanoparticles

without

etching,

and

(C)

the

as-constructed Ru-HPNSs, respectively. The ECL measurements were executed with 100 pM MUC1 by the aptasensor. Detection of MUC1 with the Developed Aptasensor. The sensitivity of the aptasensor was investigated by monitoring the ECL intensity with MUC1 standard samples with different concentrations. As shown in Figure 7A, the ECL intensity increased in response to the increasing concentration of MUC1. The calibration plot showed that a good linear relationship was achieved between the ECL response and the logarithmic value of MUC1 concentrations range from 1.0 fg/mL to 100 pg/mL with a correction coefficient of 0.9977 (Figure 7B). The regression equation is I=1087.7lg[c/(fg/mL)]+794.13 with a limit of detection of 0.31 fg/mL (the calculation was shown in Figure S8). Moreover, the developed ECL aptasensor for MUC1 analysis was compared with some recently reported aptasensors. Table S2 demonstrated the application of the aptasensor in clinical samples and achieved recovery range from 91% ~ 105%, indicating that the proposed sensor could determine MUC1 in human serum. Comparing to recent works, as shown in Table S3, it could be seen that this aptasensor achieved prominent sensitivity, manifesting the 17



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good performance of Ru-HPNSs as ECL tags.

Figure 7 (A) ECL response of the aptasensor with different concentrations of MUC1. (B) Calibration plot to the ECL intensity and the logarithm of MUC1 concentrations. Selectivity and Stability of the Aptasensor. Three different proteins, including human serum albumin (HSA), human alpha-fetoprotein (AFP), carcino-embryonic antigen (CEA) and prostate specific antigen (PSA), were employed as interfering substances to estimate the performance of the aptasensor in distinguishing MUC1 from other proteins. As shown in Figure 8A, HSA (10 ng/mL), AFP (10 ng/mL), CEA (10 ng/mL) and PSA (10 ng/mL) were used to replace the target MUC1, respectively, to execute the contrast experiments. We could see that no obvious ECL signal increase could be observed with HSA, AFP, CEA and PSA comparing to the blank. Moreover, HSA (10 ng/mL), AFP (10 ng/mL), CEA (10 ng/mL) and PSA (10 ng/mL) were added into MUC1 sample (100 pg/mL), which exhibited ECL intensity approximate to that of the standard MUC1 sample (100 pg/mL). The result suggested that HSA, AFP, CEA and PSA had almost no influence on the response to MUC1, manifesting the good selectivity and specificity 18


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of the aptasensor. The stability was of great significance which estimated the performance of the aptasensor adapting to long testing processes. The continuous ECL scans of 19 cycles were monitored to study the stability of the aptasensor (with 10 pg/mL MUC1) in 0.1 M PBS (pH 7.4). As shown in Figure 8B, no obvious fluctuation (RSD = 0.73%) was observed with the ECL peak intensity during the continuous scanning, indicating favorable stability of the proposed biosensor.

Figure 8 (A) Performances of the aptasensors tested with different interferences. (B) Stability test of the aptasensor by continuous scanning. CONCLUSION In summary, the as-prepared Ru-HPNSs with hollow porous polymeric structure effectively decreased the inner filter effect and minimize inactive emitters, which achieved notable improvement of ECL efficiency comparing to Ru(II) complex-based bulk nanomaterials. With the novel Ru-HPNSs as ECL tags, the aptasensor achieved ultrasensitive detection of MUC1. In view of these advantages, this work provided a

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new avenue to improve ECL efficiency and a versatile method to design high-performance ECL nanomaterials, which exhibited a great application potential in bioanalysis and early clinical diagnose of cancer. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org/. Sequence information for the nucleic acids, TEM image of the Ru-HPNSs, UV-vis absorption spectra of the reagents, SEM image of Ru(mcbpy)(bpy)2Cl2 particles, fluorescent intensities and emission spectra of Ru(mcbpy)(bpy)2Cl2 in different concentrations, elemental analysis of the Ru-HPNSs, optimization of the layers of {PEI-Ru/PAA}n, calculation of the limit of detection, analysis of MUC1 in clinical samples, and comparison of different methods for MUC1 detection. AUTHOR INFORMATION *Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (Y. Zhuo), [email protected] (R. Yuan). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the NNSF of China (21575116, 20


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51473136, 21675129, 21675130) and the Fundamental Research Funds for the Central Universities (XDJK2016E056), China.

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For TOC only

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Hollow Porous Polymeric Nanospheres of a Self-Enhanced

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