Strong Electrochemiluminescence from MOF Accelerator Enriched

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Strong Electrochemiluminescence from MOFs Accelerator Enriched Quantum Dots for Enhancing Sensing of Trace cTnI Xia Yang, Yan-qing Yu, Ling-Zhi Peng, Yan-Mei Lei, Ya-Qin Chai, Ruo Yuan, and Ying Zhuo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05137 • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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Analytical Chemistry

Strong Electrochemiluminescence from MOFs Accelerator Enriched Quantum Dots for Enhancing Sensing of Trace cTnI Xia Yang, Yan-Qing Yu, Ling-Zhi Peng, Yan-Mei Lei, Ya-Qin Chai, Ruo Yuan∗, Ying Zhuo∗. 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

∗

Corresponding authors: Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (R. Yuan); [email protected](Y. Zhuo). 1

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ABSTRACT Sensitive and feasible electrochemiluminescence (ECL) bioassay relies on the achievement of the ECL signal tags with high and stable signal intensity. In this work, strong electrochemiluminescence (ECL) emission was achieved from metal organic framework (MOFs) accelerator enriched Quantum Dots (CdTe), which was applied as an efficient ECL signal tag for trace biomarker detection. It is particularly worth noting that a novel mechanism to drastically enhance the ECL intensity of CdTe is established because the isoreticular metal organic framework-3 (IRMOF-3) with 2-amino terephthalic acid (2-NH2-BDC) as organic ligand is not only devoted for loading large amount of CdTe via encapsulating effect and internal/external decoration, but also as a novel co-reactant accelerator for promoting the conversion of co-reactant S2O82−

into

the

sulfate

radical

anion

(SO4•−),

further

boosting

the

electrochemiluminescence (ECL) emission of CdTe. Based on the simple sandwich immunoreaction model, the cardiac troponin-I antigen (cTnI), a kind of biomarker related with myocardial infarction, was chosen as detection model using the IRMOF-3-enriched CdTe labeled antibody as signal probe. This immunosensor demonstrated the desirable assay performance for cTnI with a wide response range from 1.1 fg mL-1 to 11 ng mL-1 and a very low detection limit (0.46 fg mL-1). This strategy suggested the IRMOF-3-enriched CdTe nanocomposite can integrate the co-reactant accelerator and luminophore to significantly enhance the ECL intensity and stability, providing a direction for promising ECL tag preparation in broad applications. Keywords: isoreticular metal organic framework-3, co-reactant accelerator, CdTe 2


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quantum dots, immunosensor, cardiac troponin-I

INTRODUCTION Electrochemiluminescence (ECL) analysis has attracted considerable attention due to its high sensitivity, controllability, low background, and low cost, which has been widely employed in clinical diagnosis and pharmaceutical analysis1-3. Admittedly, the efficient ECL signal probe was vital for sensitivity improvement of biosensor since it decided the output signal of the target4,5. Up to now, in order to improve the analytical performance of signal probe, signal amplification strategies were widely used which mainly involved nanomaterials-based amplification6, enzymatic signal amplification7, 8

, and nucleic acid amplification9,10. However, the enzyme or nucleic acid was usually

employed by labeling them on the surface of bio-recognition element, resulting in some negative factors of complicated operation and high cost11. Thus, the nanomaterials-based amplification has become the preferred signal amplification strategy to improve the sensitivity because of their large specific surface area, strong electronic transmission capability and convenience of labeling12. Usually, the amplification effect of the nanomaterials could be attributed to the enhancement of the signal molecule or biological probe loading13. Nevertheless, this single function for signal amplification might not meet the requirement for trace bioanalysis. Considering that the ECL signal output was based on the light emitting phenomenon of luminophore excited species, the promotion of the electrochemical reaction efficiency between luminophore and co-reactant was the basic and crucial approach for signal 3



amplification14. Recently, we have demonstrated a type of substance named co-reaction accelerator which could promote the electrochemical reaction of co-reactant to produce more active intermediates, achieving the significant improvement of the reaction efficiency between luminophore and co-reactant for ECL signal amplification11. For example, semicarbazide (Sem) could act as a co-reaction accelerator to react with persulfates (S2O82−) to produce more intermediates SO4•−, which further boosted the response signal of CdTe quantum dots (CdTe)15. Then, Fe3O4-CeO2 nanocomposites were employed as a co-reaction accelerator to amplify the signal from silver nanoclusters/S2O82− ECL system16, which further demonstrated that the co-reaction accelerator features great efficiency and universality in many ECL systems. In the above works, the semiconductor nanocrystalline and co-reaction accelerator are divided into two parts, which may enlarge the distance between each other and decrease the efficiency of accelerator. In this work, we integrate the luminophore and co-reaction accelerator into one nanostructure, which aims to enhance the ECL intensity. As a kind of luminescent nanomaterial, quantum dots (QDs) have been extensively studied in ECL biosensing due to their narrow emission spectra, high quantum yields and low cost17. Nevertheless, there are still an apparent gap for construction of QD-based ECL sensors for sensitive detection due to the limited ECL efficiency of QDs, which lead to a low ECL intensity comparing with the conventional ECL reagents of Ru(bpy)32+ 18. Alternatively, metal-organic frameworks (MOFs), which employed metal ion as the connection point and organic ligands as the support, were a 4


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class of crystalline microporous materials and have emerged as promising carrier to enhance the amount of QDs loading, resulting in the amplification of the QDs signal intensity. For example, Huo’ group have combined CdTe with the cavities of zeolitic imidazolate framework to obtain the enhanced photoluminescence intensity19. Jin and co-workers have employed the surface modification of porphyrin-based MOFs with core shell CdSe@ZnS QDs for enhancing light harvesting by direct binding using a suitable linking group20. Thus, it can be seen that MOFs could act as a desirable carrier for enrichment of QDs due to their unique property of mesoporous cages which could not only act as the anchor carrier for QDs loading, but also effectively reduce the inner filter effect to improve ECL efficiency. Herein, we reported the functionalization of isoreticular metal organic framework-3 (IRMOF-3) with CdTe via encapsulation and surface decoration for enhancement of ECL intensity. It is worth noting that the IRMOF-3 can increase the immobilization amount of CdTe and further exhibited a new function as a novel co-reaction accelerator to enhance the ECL of QDs/S2O82− system because the organic ligand of 2-amino terephthalic acid (2-NH2-BDC) in IRMOF-3 could promote the conversion of co-reactant S2O82− into the sulfate radical anion (SO4•−), leading to enhanced ECL intensity in comparison to the other quantum dot aggregates. Consequently, due to the multifunctions of IRMOF-3, the IRMOF-3-enriched CdTe nanocomposite could act as an efficient and integrated ECL signal tag to produce a strong signal. In the present work, a sensitive ECL immunosensor was constructed by using the IRMOF-3 accelerator enriched Quantum Dots (CdTe@IRMOF-3@CdTe) as a signal 5



probe for detection of trace cTnI, a standard marker of myocardial infarction in clinical diagnosis21. The schematic diagram of preparation for the immunosensor and the ECL response principle was shown in Scheme 1. Briefly, the CdTe encapsulated in IRMOF-3 (CdTe@IRMOF-3) with rich amino groups were synthesized via the method of “intra-filling”, which were further used as carriers for more CdTe loading by the methods of “inner surface modification” and “outer surface modification” to obtain the carboxyl groups modified CdTe@IRMOF-3@CdTe composites. After that, with the coupling agents of EDC and NHS, the CdTe@IRMOF-3@CdTe composites were further used to label detection antibody (Ab2) to form the multifunctional signal probe of CdTe@IRMOF-3@CdTe labeled Ab2. Besides, AuNPs modified TiO2 (Au@TiO2) nanoneedles were employed to fabricate the electrode for immobilizing first antibody (Ab1). When the immunosensor was incubated with cTnI and the CdTe@IRMOF-3@CdTe labeled Ab2 signal probe successively based on a sandwiched format, the ECL signal increased with the increased concentration of cTnI. Comparing with the corresponding bulk CdTe, the obtained CdTe@IRMOF-3@CdTe nanocomposite exhibit efficient ECL intensity, which may inspire more application of MOFs for ECL sensing strategy.

6


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Scheme 1 Schematic diagrams of the ECL immunosensor. (A) Preparation of CdTe@IRMOF-3@CdTe labeled signal probe. (B) The possible enhanced mechanism of IRMOF-3 accelerator for cTnI detection in CdTe/S2O82− system.

7



EXPERIMENTAL METHOD Preparation of Au@TiO2 Nanoneedles Titanium dioxide (TiO2) nanoneedles were synthesized according to the previous method

22

with some modifications. 475.6 mg of Me4NOH was added to a 250 mL

two-necked flask containing 150 mL ultrapure water. Then the flask was placed in a large crystallizing dish with ice filling on top of a magnetic stirrer. Meanwhile, 3.264 mL of TTIP solution was diluted with 6.736 mL of IPA solution to obtain the TTIP/IPA solution. When the temperature of the Me4NOH solution has reached 2 °C, 0.333 mL of TTIP/IPA solution was added dropwise while the mixture was stirred vigorously. After the addition of TTIP/IPA solution, the mixture was kept stirring for 10 min, then the ice bath was replaced by heating equipment and the mixture was heated at 100 °C and refluxed for 15 h. The resulting suspension was equally distributed into two 100 mL teflon-lined autoclaves and heated to 175 °C for 4 h. After cooling to room temperature naturally, the milk white material was obtained via centrifugation (8,000 rpm, 10 min), washed with ultrapure water for three times, then re-dispersed in 50 mL of ultrapure water and stored in 4 °C refrigerator for further use. Prior to use, the as-synthesized TiO2 nanoneedles were coated with the positively charged PDDA to get the PDDA functionalized TiO2 nanoneedles. Then, the Au@TiO2 nanoneedles were synthesized by decorating the negatively charged AuNPs on the positively charged PDDA functionalized TiO2 nanoneedles via electrostatic

8


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interaction. Briefly, 0.75 mL of PDDA (3.2%) was injected into 2 mL as-synthesized TiO2 solution and slightly stirred for 12 h at 4 °C, followed by centrifugation and washing with ultrapure water to remove the excess PDDA. Afterwards, 1 mL negatively charged AuNPs was added into the PDDA functionalized TiO2 solution under stirring and the Au@TiO2 nanoneedles were obtained after 6 h. Then, the Au@TiO2 nanoneedles were separated by centrifugation and washed. The resultant precipitates were finally dispersed in 3 mL ultrapure water for biosensing interface construction. Preparation of CdTe The synthesis of CdTe was referred to a classical method reported previously23. Briefly, 73.8 mg of CdCl2·2.5H2O were dissolved in 100 mL ultrapure water. Then, trisodium citrate dehydrate (100 mg), Na2TeO3 (0.02 M, 1 mL), MPA (66 µL), and NaBH4 (200 mg) were added and dissolved in the above solution under stirring. The resultant solution was transferred to a 250 mL flask and refluxed at 130 °C for 8 h. Finally, the CdTe were washed with ethanol, centrifugated 3 times (10,000 rpm, 4 °C, 5 min), and then equally distributed into 8 mL ethanol and 8 mL ultrapure water. The corresponding TEM image of the CdTe was shown in Figure S1 (see the Supporting Information). Preparation of CdTe Encapsulated IRMOF-3 (CdTe@IRMOF-3) Typically, 0.20 g PVP was added into a solution containing 18 mL of DMF and 11 mL of ethanol, and then 1 mL as-prepared CdTe ethanol solution was added drop by drop 9



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into the above mixture under stirring. Meanwhile, 68.9 mg of Zn(NO3)2·6H2O and 16.5 mg of 2-NH2-BDC dissolved into 6 mL DMF were added into the above mixed solution. After that, the resultant solution was treated under ultrasonic conditions for 30 min. Subsequently, the mixture was transferred into a 50 mL Teflon-lined stainless steel autoclave and heated at 100 °C for 20 h. After reaction, the product was collected by centrifugation at 8,000 rpm for 5 min. The precipitate was washed with DMF, collected via centrifugation three times (8,000 rpm, 5 min). Finally, the obtained CdTe@IRMOF-3 was dried at 80 °C for 3 h. The IRMOF-3 was prepared using the same method as shown in the Supporting Information. Preparation of Ab2 Signal Probe Labeled by the Multilayered CdTe Decorated IRMOF-3 (CdTe@IRMOF-3@CdTe Labeled Ab2 Signal Probe) A general preparation of CdTe@IRMOF-3@CdTe labeled Ab2 signal probe was described as follows. Firstly, 1 mL of as-synthesized CdTe were added into 200 µL of EDC (0.35 M) and NHS (0.1 M) mixed solution and gently stirred for 40 min at 4 °C to convert the −COOH groups of CdTe to amine reactive N-succinimidyl esters. Then, 500 µL of CdTe@IRMOF-3 (2 mg mL-1) were added in the above solution and kept stirring for 12 h at 4 °C. After reaction, the precipitates were collected by washing with

ultrapure

water

and

centrifugation.

Subsequently,

the

obtained

CdTe@IRMOF-3@CdTe were re-dispersed in 2 mL ultrapure water, then 100 µL Ab2 solution was added into the CdTe@IRMOF-3@CdTe solution and gently stirred for 12 h at 4 °C. Then the dispersion was collected by centrifugation and dispersed in PBS (pH 7.4). Nextly, 20 µL of BSA (0.5%) was added to the mixture for 1 h to block 10


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the non-specific adsorption. The CdTe@IRMOF-3@CdTe labeled Ab2 signal probe was stored at 4 °C. Different Ab2 signal probes (Probe A: CdTe labeled Ab2; Probe B: CdTe encapsulated IRMOF-3 (CdTe@IRMOF-3) labeled Ab2; Probe C: IRMOF-3 of outer surface modified with CdTe (IRMOF-3@CdTe) labeled Ab2) was prepared in the Supporting Information. Fabrication of the Sandwich-type ECL Immunosensor First, 5 µL solution of Au@TiO2 nanoneedles was coated onto the GCE surface and dried. Then, the modified electrode (Au@TiO2/GCE) was incubated with 10 µL Ab1 (1 µg mL-1) for 12 h at 4 °C. Following this, the resultant electrode (Ab1/Au@TiO2/GCE) was incubated with 5 µL of 0.25% BSA for 40 min. Then the resultant electrode was extensively rinsed with PBS (pH 7.4). The prepared immunosensor was kept at 4 °C until use. Measurement Procedure A sandwich immunoassay was fabricated for detection of cTnI antigen as follows: Initially, the prepared immunosensors were incubated with 10 µL cTnI solutions at different concentrations for 40 min, followed by incubation of 20 µL CdTe@IRMOF-3@CdTe labeled Ab2 signal probe for 80 min. After washed with PBS (pH 7.4) to remove the unbound CdTe@IRMOF-3@CdTe labeled Ab2 signal probe, the immunosensors were tested with the MPI-A analyzer in air-equilibrated 10 mM S2O82− solution (3 mL, pH 7.4) with the potential ranging from -1.5 V to 0 V at 200 mV s-1. The measurement of clinical serum samples were performed with the 11



same procedures mentioned above without any other treatments. RESULTS AND DISCUSSION TEM, XPS and SEM Characterization of the Different Nanomaterials As shown in Figure 1A, the IRMOF-3 was clearly seen from the TEM image where the diameter was about 500 ± 50 nm with similar-spherical structure, which was consistent with the previously reported24. Meanwhile, the analysis of elemental composition of IRMOF-3 was characterized with XPS as displayed in Figure 1B, which showed the characteristic peaks of C1s, O1s, N1s and Zn2p of IRMOF-3. After imbedded CdTe, shown in Figure 1C, the TEM image of CdTe@IRMOF-3 displayed well-demarcated sphere structure with good dispersity, and the particle size was about 1 µm. Meanwhile, the TEM image of CdTe@IRMOF-3 composites clearly revealed that CdTe@IRMOF-3 was well dispersed. As seen from the TEM image of the CdTe@IRMOF-3@CdTe composite (Figure 1D), many dots were covered on the surface of the globular solid structures. From the enlarged TEM mages (Figure 1E, F), the crystalline structure of CdTe with the existence of well-resolved lattice planes was apparently observed on the surface of CdTe@IRMOF-3 and the CdTe size were mainly 5 nm, indicating the loading of CdTe onto the surface of CdTe@IRMOF-3. As shown in Figure 1G, the characteristic peaks of Cd and Te elements appeared which verified that CdTe was decorated on the IRMOF-3. Figure 1H and Figure 1I showed the SEM of TiO2 and Au@TiO2 nanocomposites, respectively. As displayed in Figure 1H, the typical SEM image of TiO2 nanostructures showed a large quantity of

12


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needle-like structures with widths of 50 ± 10 nm and lengths of 400 ± 50 nm, which was consistent with previous reports19. As shown in Figure 1I, small bright spots with size of 16 ± 2 nm were distributed uniformly on the TiO2 surfaces, demonstrating that TiO2 surface were modified with lots of AuNPs.

Figure 1 TEM image (A) and XPS analysis (B) of IRMOF-3, TEM image of (C) CdTe@IRMOF-3. TEM images of (D) CdTe@IRMOF-3@CdTe composite, (E) individual CdTe@IRMOF-3@CdTe from the circle image in D, and (F) CdTe with crystalline structure from the enlarged image in E. XPS analysis for (G) CdTe@IRMOF-3@CdTe composite. SEM images of (H) TiO2 and (I) Au@TiO2. The Possible ECL Mechanism of the CdTe/IRMOF-3/S2O82− Ternary System In order to identify the luminophore and further reveal the ECL reaction mechanism 13



of IRMOF-3 in the CdTe/S2O82− system, the corresponding ECL and CV measurements were exhibited in Figure 2. At first, the ECL spectra of (a) S2O82−, (b) S2O82− + IRMOF-3, (c) CdTe + S2O82− and (d) CdTe + S2O82− + IRMOF-3 solution (where the concentrations of CdTe, S2O82− and IRMOF-3 were 0.3 mg mL-1, 10 mM, and 0.1 mg mL-1 in PBS (pH 7.4), respectively) were tested by using the optical filter, respectively. In Figure 2A, curve a and b depicted the typical ECL emission spectra of S2O82− without and with IRMOF-3, respectively. Both of them showed the maximum wavelength at 575 nm, which is due to the emission of the singlet state oxygen (1(O2)2*)25 in the two ECL systems. This also indicates that IRMOF-3 can enhance the ECL signal of O2/S2O82− system. The ECL emission spectrum of CdTe in S2O82− solution indicated a maximum emission wavelength at 650 nm (Figure 2A, curve c), which was expectedly in full agreement with the attribution of the emitted light to the CdTe based excited state (CdTe*) and S2O82− as the co-reactant in the CdTe/S2O82− system26. Interestingly, the maximum emission wavelength of the CdTe + S2O82− + IRMOF-3 solution also located at 650 nm (Figure 2A, curve d), which was same as that of CdTe + S2O82− solution. This result implied that the luminophore of CdTe/IRMOF-3 ECL system was also CdTe not the 1(O2)2* in S2O82− solution. With a comparison of the CdTe + S2O82− solution (Figure 2A, curve c), an enhanced ECL was obtained for the CdTe + S2O82− + IRMOF-3 solution (Figure 2A, curve d), which indicated that IRMOF-3 could boost the intensity of the CdTe/S2O82− ECL system. Thus, we suspected that IRMOF-3 can act as a co-reaction accelerator for CdTe/S2O82− in this ternary system. 14


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To further prove the role of IRMOF-3 in the CdTe/IRMOF-3/S2O82− ternary system, the ECL and CV curves with GCE in the following four different solutions (a) CdTe , (b) CdTe + IRMOF-3, (c) CdTe + S2O82−, and (d) CdTe + S2O82− + IRMOF-3 (where the concentrations of CdTe, S2O82− and IRMOF-3 were 0.3 mg mL-1, 10 mM, and 0.1 mg mL-1 in PBS (pH 7.4), respectively) were tested to evaluate the possible reaction of CdTe, S2O82−, and IRMOF-3. As shown in Figure 2B, a weak cathode ECL signal about 200 a.u. was observed on GCE in CdTe solution (Figure 2B, curve a) and it exhibited no remarkable changes compared with that with IRMOF-3 (Figure 2B, curve b), indicating that IRMOF-3 had no direct effect on CdTe ECL emission. However, when in CdTe + S2O82− solution, the ECL signal was noticeably raised to 2062.2 a.u. from 200 a.u., which indicated that S2O82− was the co-reactant of CdTe to improve the ECL response of the CdTe/S2O82− ECL system (Figure 2B, curve c). Interestingly, when IRMOF-3 was introduced in CdTe + S2O82− solution, a highest ECL emission of 8293 a.u. was obtained (Figure 2B, curve d), which was about 4 times as that from CdTe + S2O82− solution. Similarly, the CV responses of the four different solutions were also presented in Figure 2C. A pair of redox peaks appeared in CV of CdTe solution (Figure 2C, curve a). After IRMOF-3 was added in CdTe solution, the currents and potentials of the redox peaks from CdTe had no obvious changes (Figure 2C, curve b) although a redox peak at Ep,a = −1.15 V from IRMOF-3 was obviously observed27, suggesting that IRMOF-3 had no direct reaction with CdTe. When GCE was measured in CdTe + S2O82− solution, curve c showed a pair of reduction and oxidation peaks of QDs at 15



Ep,c = −1.05 V and Ep,a = −0.83 V, respectively, which shifted negatively compared with pure CdTe solution (Figure 2C, curve a), owing to the increasing of the over-potential produced by the interaction between S2O82− and CdTe. When IRMOF-3 was introduced into the CdTe + S2O82− system, the redox peak current of CdTe increased remarkably (Figure 2C, curve d), indicating that the IRMOF-3 could promote the oxygenation between CdTe and S2O82−. In conclusion, IRMOF-3 was indeed defined as the co-reaction accelerator to interact with S2O82− rather than CdTe for enhancing the ECL intensity in CdTe/S2O82− system. The possible ECL mechanisms of CdTe/S2O82−/IRMOF-3 system were shown as Figure 2D.

Figure 2 ECL spectra of GCE (A) in (a) S2O82− solution, (b) S2O82− + IRMOF-3 solutions, (c) CdTe + S2O82− solution, (d) CdTe + S2O82− + IRMOF-3 solution with a series of optical filters (from 350 to 825 nm with interval of 25 nm). ECL (B) and CV responses (C) of GCE in (a) CdTe solution, (b) CdTe + IRMOF-3 solution, (c) CdTe + S2O82− solution and (d) CdTe + S2O82− + IRMOF-3 solution. (D) The possible 16


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enhanced ECL mechanism of IRMOF-3 in CdTe/S2O82− system. CV and EIS of the Fabrication of the ECL Immunosensor The assembly steps of the immunosensor were investigated by CV measurements in 0.1 M PBS with 5.0 mM [Fe(CN)6]3−/4− acting as redox probe (pH 7.4)at a scan rate of 100 mV s-1. In Figure 3A, a pair of symmetric redox peaks could be observed (curve a), which was corresponding to the reversible redox reaction of [Fe(CN)6]3−/4− on the bare GCE. When Au@TiO2 was casted onto the bare GCE, the oxidation and reduction

peak

currents

decreased

obviously

because

of

the

weak

electron-transporting capability of Au@TiO2 (Figure 3A, curve b). After Ab1 coated on the surface of Au@TiO2, the peak current decreased (Figure 3A, curve c) owing to the electron inert feature of Ab1. Subsequently, when non-electroactive BSA was used to block nonspecific sites, the peak currents decreased further (Figure 3A, curve d). Obviously decreased peak current could be observed after the modification of cTnI onto the BSA/Ab1/Au@TiO2/GCE, because the formation of a huge formula weight immune complex could also block the electron transfer of the redox probe (Figure 3A, curve e). Electrochemical impedance spectroscopy (EIS) was another approach for monitoring the changes in the surface features of the electrodes 28. The semicircle diameter of EIS indicated the electron transfer resistance, Ret, which directly controlled the electron transfer kinetics of the redox-probe at the electrode interface. A small semicircle (Figure 3B, curve a) indicated a clear GCE was obtained for the construction of biosensor. The Ret increased when the electrode was modified with Au@TiO2 (Figure 17



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3B, curve b). After Ab1 bonded on the Au@TiO2 film via Au-S or Au-N bonds, leading to an increase of Ret due to the electron inert feature of Ab1 which hindered the electron transfer of redox probe (Figure 3B, curve c). Then with incubation in BSA to block residual active sites, the Ret increased, which could be attributed to insulating property of protein molecules (Figure 3B, curve d). Subsequently, when cTnI standard solution was incubated onto the surface, the Ret further increased, which might ascribe to the fact that the formation of immunocomplex as coat largely hampered the electron transport from bulk solution to electrode interface (Figure 3B, curve e). The EIS results showed the same tendency of electron transfer with that of CVs.

Figure 3 The electrochemical characterization of the stepwise modified electrodes: CV

(A)

and

EIS

Ab1/Au@TiO2/GCE

(B)

of

(curve

GCE c),

(curve

a),

Au@TiO2/GCE

BSA/Ab1/Au@TiO2/GCE

(curve

(curve

b),

d),

and

cTnI/BSA/Ab1/Au@TiO2/GCE (curve e) measured in 0.1 M PBS (pH 7.4) containing 5.0 mM [Fe(CN)6]3−/4− (acting as redox probe) and 0.1 M KCl at a scan rate of 100 mV s-1.

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ECL with Different Ab2 Signal Probes To demonstrate the advantages of the CdTe@IRMOF-3@CdTe signal probe, the ECL intensities of the immunosensor were measured with different Ab2 signal probes (Probe A: CdTe labeled Ab2; Probe B: CdTe@IRMOF-3 labeled Ab2; Probe C: IRMOF-3@CdTe labeled Ab2; Probe D: CdTe@IRMOF-3@CdTe labeled Ab2) under optimized condition (Figure S2, see the Supporting Information). In Figure 4A, the ∆IECL of the immunosensor with probe A was raised about 2102.8 a.u. compared with blank values, owing to the introduction of CdTe for ECL emission. When the probe A was changed to probe B, ∆IECL was about 5780.0 a.u. emission (Figure 4B), which was about 3 times as that of probe A. The reason was that IRMOF-3 was not only as the carrier of CdTe, but also as an effective co-reaction accelerator to significantly promote the ECL emission of CdTe. And the signal of probe C was also demonstrated this viewpoint (Figure 4C). When the immunosensor was incubated with the as-prepared probes of CdTe@IRMOF-3@CdTe labeled Ab2 (probe D), the ∆IECL noticeably raised to 14,806 a.u., which increased 129.7%~138.8% than that of probe B (Figure 4B) or probe C (Figure 4C), indicating that the co-reaction accelerator role of IRMOF-3 in CdTe/S2O82− system and the proposed biosensors with the probe D exhibited best sensitivity. Moreover, the detection limit and liner range of four different probes have also been evaluated and the results have been presented in Figure S3 (see the Supporting Information). Thus, the comparison results showed that the CdTe@IRMOF-3@CdTe labeled Ab2 probe could be effectively utilized to detect cTnI for its good performance of ECL signal amplification. 19



Figure 4 ECL profiles of the different sandwich format immunosensors without (a, blue line) and with (b, red line) cTnI by different Ab2 signal probes: (A) CdTe labeled Ab2 signal probe, (B) CdTe@IRMOF-3 labeled Ab2 signal probe, (C) IRMOF-3@CdTe labeled Ab2 signal probe, and (D) the proposed probe was CdTe@IRMOF-3@CdTe labeled Ab2. The concentration of cTnI was 11 ng mL-1. Working solution, PBS (pH 7.4) containing 10 mM S2O82−. Scan rate was 200 mV s-1. Analytical Performance of the ECL Immunosensor To evaluate the detecttion ability of the ECL immunoassay, cTnI standards with different concentrations were tested based on the developed protocol. From Figure 5, the ECL signal obviously increased with the increasing concentration of cTnI (Figure 5A, curves a-i) and presented an excellent linear relationship with the logarithm of cTnI concentration (Figure 5B). The linear equation was I=1953.6lg(c/pg mL-1) + 20


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8408.0 with a correlation coefficient of 0.9986, where I was the ECL intensity and c was the concentration of cTnI. The estimated limit of detection (LOD) was 0.46 fg mL-1, which was estimated by LOD = 3σb/k29 (σb was the standard deviation of the blank and k was the slope of the corresponding calibration curve). In addition, compared with the previous reports30-33, the prepared immunosensor showed better performance, as it was shown in Table 1, in which the proposed biosensors exhibited best sensitivity. Table 1 Comparison of our present work with other methods for cTnI detection Measurement protocol

Linear range

Detection limit

Reference

Electrochemical

0.2 ~ 1.0 ng mL-1

0.2 ng mL-1

30

-1

-1

31

Fluorescence

0.1 ~ 100 ng mL

0.1 ng mL

Colorimetric

0~ 30 ng mL-1

0.01 ng mL-1

ECL ECL

-1

-1

0.05 pg mL ~ 0.1 ng mL -1

-1

1.1 fg mL ~ 11 ng mL

-1

0.017 pg mL

-1

0.46 fg mL

32 33 Present work

Performance of the Proposed Immunosensor The stability of the immunosensor was estimated under consecutive test after the immunosensor was incubated with 1.1 ng mL-1 cTnI. As shown in Figure 5C, the immunosensor has an excellent stability with relative standard deviations (RSD) of 0.92% of ECL peaks in 15 cycles. The non-target proteins of cardiac troponin-T (cTnT), human serum albumin (HSA), hemoglobin (Hb), carcinoembryonic antigen (CEA) and α-1-fetoprotein (AFP) as the interfering substances was used to check the performance of the immunosensor. According to Figure 5D, the developed immunosensors with these interfering substances exhibited almost the same response 21



as blank solution. Less than 2.6% difference were obtained in solution of cTnI comparing to that in cTnI with interfering substances, which indicated that the immunosensor possessed good selectivity and specificity for the detection of cTnI.

Figure 5 (A) ECL-time plots of the proposed immunosensor incubated with cTnI at different concentrations: (a) 0, (b) 1.1 fg mL-1, (c) 11 fg mL-1, (d) 0.11 pg mL-1, (e) 1.1 pg mL-1, (f) 11 pg mL-1, (g) 0.11 ng mL-1, (h) 1.1 ng mL-1 and (i) 11 ng mL-1. (B) Calibration plots of the ECL intensity vs. the logarithm of cTnI concentration. (C) The stability of the proposed biosensor incubated with 1.1 ng mL-1 cTnI under consecutive cyclic potential scans for 15 cycles. (D)The selectivity of the biosensors with different interfering and target proteins: HSA (1.1 ng mL-1), cTnT (1.1 ng mL-1), CEA (1.1 ng mL-1), Hb (1.1 ng mL-1), AFP (1.1 ng mL-1), cTnI (0.11 ng mL-1), and a mixture (HSA + cTnT + CEA + Hb + AFP + cTnI). 22


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Preliminary Analysis of Real Samples A recovery experiment was performed to investigate the feasibility of the ECL immunosensor for clinical application. Different concentrations of cTnI were added in the human serum and then were measured by using the proposed immunosensor. The ratio between the actual concentration (defined as added) and the detected concentration based on the linear equation (defined as found) was the recovery. According to Table 2, the recoveries are 93.6% to 109.7%, which demonstrated the good potential application of the obtained immunosensor in clinical determination. Table 2 Determination of cTnI in normal human serum with the proposed immunosensor Sample number

added/(ng mL-1)

found/(ng mL-1)

recovery/%

1

1.00

1.097

109.7

2

0.50

0.468

93.6

3

0.10

0.0955

95.5

4

0.05

0.0478

95.6

5

0.01

0.0105

105.0

CONCLUSION In this work, a signal-amplified ECL immunosensor was fabricated based on the multifunctional signal probe of CdTe@IRMOF-3@CdTe. More importantly, this strategy not only employed the IRMOF-3 to act as co-reaction accelerator and carries of luminophore for enhancing ECL, which could meet the demand for high sensitivity with simple and convenient process, but also broadened the application of IRMOF-3 in biosensor areas. Thus, the as-constructed ECL immunosensor showed ultralow detection limit, high sensitivity, broad linear range and acceptable stability in the cTnI 23



analysis. In view of these advantages, the co-reaction accelerator amplification had great potential for the development of the low-intensity luminophore-based platform in ultrasensitive bioanalysis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org/. Material and Reagents, Apparatus, Preparation of IRMOF-3, Preparation of Different Ab2 Signal Probes for Comparison,The Optimization of Detection Conditions, TEM image of the CdTe, The optimization of experimental parameter,Calibration curve of biosensors with different Ab2 signal probes.

AUTHOR INFORMATION *Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (R. Yuan), [email protected] (Y. Zhuo). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (21675130, 21575116, 21675129, 51602263), the Fundamental Research Funds for the Central Universities (XDJK2015A002), China Postdoctoral Science Foundation (2015M572427, 2016T90827) and Chongqing Postdoctoral Research 24


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Project (xm2015019).

25



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Strong Electrochemiluminescence from MOF Accelerator Enriched

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