Ratiometric Electrogenerated Chemiluminescence Cytosensor Based

Subscriber access provided by University of Winnipeg Library

Article

Ratiometric Electrogenerated Chemiluminescence Cytosensor Based on Conducting Polymer Hydrogel Loaded with Internal Standard Molecules Caifeng Ding, Yunxia Li, Lei Wang, and Xiliang Luo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04116 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Ratiometric

Electrogenerated

Chemiluminescence

Cytosensor Based on Conducting Polymer Hydrogel Loaded with Internal Standard Molecules Caifeng Ding*, Yunxia Li, Lei Wang，Xiliang Luo* Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education; Shandong Key Laboratory of Biochemical Analysis; Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong; College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China.

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract A sensitive and reliable bimodal electrochemiluminescent (ECL) system based on CdTe Quantum Dots (QDs) and luminol as double luminophores is constructed. CdTe QDs tagged with the aptamer (CdTe-Apt 2) of cancer cells are used as the detection signal, while luminol molecules are used as internal standards. The electrodeposited polyaniline based conducting polymer hydrogel (CPH) on the electrode surfaces improves the biocompatibility and conductivity of the sensing interfaces effectively. Furtherly, electron transfer is probably much easier when luminol and coreactant potassium persulfate (K2S2O8) immobilized in the CPH compared to that in solution. Cancer cells are captured to the electrode surface by another aptamer linked to the Au nanoparticles immobilized in the CPH through Au-S bonds. In the developed bimodal ECL system, internal standard method is used to quantify cancer cells by comparing the differences in sensitivity of the double-peak ECL signals with that of target analytes. The internal standard method of ECL strategy can provide very accurate detection results in complex environment because interferences in the system can be eliminated through the self-calibration of two emission spectra. A linear relation is found based on the ∆ECLCdTe/∆ECLluminol against the concentration of cancer cells within 100 to 6500 cells mL-1 under optimized conditions. The developed ratiometric ECL cytosensor with internal standard can significantly improve the accuracy and reliability of cell assay in complex biological media, demonstrating promising applications in healthcare monitoring and clinical diagnostics. Keywords: Electrogenerated Chemiluminescence, Cytosensor, Conducting Polymer Hydrogel, Internal standard method, Cancer cells

2


Page 2 of 21

Page 3 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introductions Hydrogels originating from hydrophilic polymers either natural or synthesized ones have threedimensional (3D) networks. Due to the swollen network structures with high water content, hydrogels can resemble biological soft tissues, showing great potential in biomedical applications.1-6 With unique biocompatibility, flexible synthesis methods, and suitable physical properties, hydrogels have attracted great attention in many biotechnology applications, including drug delivery, tissue engineering, regenerative medicine, molecular diagnostics, and wearable biosensors.7-14 Hydrogel shows responses to a specific external biochemical stimulus after simple modulation, and target molecules can be monitored through observing the gel formation or deformation.15, 16 Targetresponsive hydrogel biosensors have been constructed accordingly to detect various biomolecules, including antibodies, aptamers, and enzymes.17-19 However, most hydrogel materials are nonconductive, which has limited the performance of hydrogel-based electrochemical biosensors. Conducting polymers (CPs) with a conjugated double-bond backbone formed by dopants are organic polymers that can conduct electricity as semiconductors or metals. The conductivity of CPs can be tuned during the polymerization process. Conducting polymer hydrogels (CPHs) have been attracting growing attention due to their high permeability to biomolecules, biocompatibility, and rapid electron transfer and thus have been widely used as interfacial materials for fabricating functional electrochemical biosensors 20-24. Applications of CPHs in energy storage devices, medical electrodes, and bio-sensing platform25-29 have been reported. Attention to electrochemiluminescent (ECL) has been growing as a result of its excellent advantages, such as controllability, high sensitivity, and low-cost. Yuan and co-workers have done much work on biomolecules detection with ECL technology.30-36 To eliminate interferences, a large 3



number of ratiometric ECL sensors have been constructed for detecting protein, cancer cells, microRNA, DNA, and metal ions.37-42 Chen et al. reported a double-potential ratiometric ECL biosensor based on poly(9,9-dioctylfluorene) (PFO) dots and took advantage of RGO-CdTe QDs as the anodic and cathodic emitters without ET. This approach has been used to detect organophosphorus pesticides.43 Recently, Sojic’s group has synthesized the prototypical thin redox hydrogel films based on polyalkylacrylamide derivatives covalently incorporated to Ru(bpy)32+ luminophores through electrochemically assisted free radical polymerization. It has been demonstrated that the swelling-to-collapse transition in thermoresponsive hydrogels provokes a huge amplification of the ECL emission.44-46 A ratiometric ECL cytosensor platform based on Au nanoparticle (AuNP)-modified CPH electrode is constructed in the present work. Besides excellent electrochemical performance of the conducting polymer hydrogel, reliability and accuracy of the measurement can be ensured without the interferences induced by external factors. The developed CPH-based cytosensor platform presents good sensing performance upon exposing to cancer cells with a wide linear range (100 to 6500 cells mL-1), high sensitivity and low sensing limit. With good biocompatibility and simple fabrication process, CPH-based cytosensor platform demonstrates promising applications in healthcare monitoring and clinical diagnostics. EXPERIMENTAL SECTION Reagents and Materials. Aniline, chloroauric acid (HAuCl4), N, N’-Methylenebisacrylamide (MBAA), acrylic acid (AA), chromium chloride (CdCl2), tellurium powder, luminol, sodium borohydride (NaBH4), bovine serum albumin (BSA) and glutathione (GSH) were obtained from Sigma-Aldrich. Sulfuric acid (H2SO4, 95.0-98.0%) and potassium persulfate (K2S2O8) were 4


Page 4 of 21

Page 5 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


purchased from Chengdu Kelong Chemical Reagent Co. (Chengdu, China). Indium Tin Oxide (ITO) coated glass slide was obtained from NanoSci Inc. (China). MCF-7 cells, Hela cells and U87-MG cells were purchased from KeyGEN BioTECH Co. (China). DMEM medium, Fetal Bovine Serum and penicillin-streptomycin were obtained from HyClone Co. (China). Clinical human serum samples were supplied by the Eighth People’s Hospital of Qingdao (Qingdao, China). The DNA oligonucleotides were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. (China). The sulpher-labeled MUC1 aptamer and the AS1411 aptamer have the sequence as follows: 5'-SH-(CH2)6-GGGAGACAAGAATAAACGCTCAAGCAGTTGATCCTTTGGA TACCCTGGTTCGACAGGAGGCTCACAACAGGC-3'. 5'-GGTGGTGGTGGTTGTGGTGGTGGTGGAAAAATCGGGCGTAC-3'. Apparatus. The ECL emission was detected using an MPI-E multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remax Electronic Science and Technology Co., Ltd., Xi’an, China). Cyclic voltammetric (CV) and Electrochemical impedance spectra (EIS) measurements were conducted with a CHI 660C electrochemistry workstation (Shanghai CH Instruments, China). All experiments were performed with a modified ITO electrode as the working electrode, an Ag/AgCl (saturated KCl) as reference electrode, and a platinum wire as counter electrode. The scanning electron micrographs were taken with Scanning Electron Microscope (SEM, S-4800, Hitachi). Transmission Electron Micrograph (TEM) images were recorded on a JEOL-JEM 200 CX transmission electron microscope (Hitachi Instrument, Japanese). Fabrication of the CPH based Sensing Interface. First, a typical electrochemical polymerization was conducted by scanning 30 cycles from - 0.2 V to + 0.8 V with a scan rate of 5



100 mV s-1 in a mixed aqueous solution containing 0.2 M aniline, 0.5 M H2SO4, 0.12 M K2S2O8, 0.09 M AA, 0.03 M MBAA, and 0.3125× 10-4 M luminol. The CPH modified electrode was obtained after drying at 25 °C. An amount of 100 μL AuNPs suspension was coated onto ITO electrode (3.0 cm × 1.0 cm) and dried at 25 °C. Then, 30 μL MUC1 aptamer (30 μM) was dropped onto the functionalized electrode. After incubated for 4 h at 4 °C, an amount of 10 μL 1% BSA was deposited on the modified electrode sequently for 2 h at room temperature. At last, the ECL sensing interface was rinsed with 0.1 M PBS solution (pH 7.4). Cell culture. Human breast carcinoma cells (MCF-7 cells), malignant glioma cells (U87-MG cells), and cervical cancer cells (Hela cells) were incubated under 5% CO2 atmosphere at 37 °C . The culture medium (DMEM medium or 1640 medium) was supplemented with 10% fetal bovine and 1% penicillin-streptomycin. Before measurement, MCF-7 cells, U87-MG cells, and Hela cells were separated from the culture medium and collected by centrifugation at 850 rpm for 5 min. And then the cells were re-suspended in 0.1 M PBS (pH 7.4) for the following experiment. ECL Detection for cells. CPH and aptamer functionalized cytosensor surface was incubated with cell suspension at 25 °C for 3 h, and then CdTe-Apt 2 nanoprobe solution were dropped onto the CPH sensing interface for 3 h under 25 °C. After each step, the modified electrode was thoroughly rinsed with 0.1 M PBS buffer (pH 7.4) to remove physically adsorbed species. After that, the fabricated cytosensor was placed in an electrochemical cell. The voltage of photomultiplier tube was set at 800 V, the scanning potential range was set from +0.8 V to −1.5 V in 0.1 M PBS solution (pH 7.4) to detect cells. RESULTS AND DISCUSSION Response Mechanism of the Ratiometric ECL Signal System. Scheme 1 shows the principle of 6


Page 6 of 21

Page 7 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the ratiometric ECL aptasensor based on CPH. The electrode is modified with CPH film by electropolymerization to improve the biocompatibility of the cytosensor. Then AuNPs are coated onto CPH film to strengthen conductivity and increase specific surface area of the cytosensor. The MUC1 aptamer self-assembled onto AuNPs surfaces can capture MCF-7 cells through the thiol anchor. Compared with reported methods for cell capturing,47,48 the present approach based on aptamer is easier, cheaper without demanding complex instrument. To avoid the influences caused by the remaining active sites, BSA is deposited dropwise onto the sensing interface. After the combination of MUC1 aptamer and the target cells, the CdTe-Apt 2 is used to label MCF-7 cells to introduce the ECL signal probe. Since MUC1 is overexpressed not only in MCF-7 cell, but also in almost all human epithelial cell adenocarcinomas, including breast, gastric, colorectal, lung, prostate, ovarian, pancreatic, and bladder carcinomas just as the reviewer concerned. In our method, two aptamers (MUC1 aptamer and nucleolin aptamer) are used together to improve the recognition efficiency of targeted MCF-7 cell. As one of recognized molecules, MUC1 aptamer can identify MUC1 protein overexpressed in MCF-7 cell, so MCF-7 cells can be captured to the sensor surface. The nucleolin aptamer tagged with CdTe QDs can recognize nucleolin protein that is also overexpressed in the MCF-7 cell-surface and give the signal. Compared the recognition with single aptamer, sensing platform with double aptamers can further increase the selectivity and accuracy of identification. We have to point out that the electrochemical polymerization treatment is performed in H2SO4 solution containing coreactant K2S2O8 and luminophore luminol. Luminous efficiency has improved efficiently compared with the normal ECL reactions. As in this work, electron-transfer is much easier when luminophore and coreactent entrapped in the CPH, while normally the coreactant is dispersed in the detection solutions. Moreover, the coreactant immobilized in CPH film can be 7



reused until the hydrogel film is destroyed completely. When the operation experimental conditions are fixed, ECLCdTe signal varies with the amount of cells, while the ECLluminol remains constant. Quantity of the cells can be obtained accurately by analyzing the changes of (∆ECLCdTe/∆ECLluminol) using luminol as an internal standard. A linear relationship between the concentration of the target cell and ∆ECLCdTe/∆ECLluminol is found, which enables it to do quantitative measurement. ∆ECLCdTe and ∆ECLluminol are the ECL intensity variations relative to the signal baseline for CdTe probe and luminol respectively. ECLCdTe varies with the cell concentration, while ECLluminol remains the same.

Scheme 1. Schematic illustration of the ratiometric ECL cytosensor working principle. Characterization of CdTe QDs, AuNPs and CPH based electrode. The morphology and size of the prepared CdTe QDs NPs and AuNPs were characterized with Transmission Electron Mmicroscopy (TEM). The CdTe NPs have an average diameter of about 6 nm as shown in Figure S1A. And the final AuNPs prepared by this method have an average diameter of approximately 15 nm as shown in Figure S1B. The morphology of bare ITO electrode, CPH/ITO electrode, and AuNPs/CPH/ITO electrode is characterized by SEM, and results are shown in Figure 1. Compared to bare ITO (Figure 1A), a network structure is found on the ITO electrode surface (Figure 1B), suggesting successful 8


Page 8 of 21

Page 9 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


deposition of fiber-like CPH on the ITO glass slide through the electropolymerization. Figure 1C and Figure 1D show that AuNPs (small white spherical particles) are evenly distributed on CPH film surface. The result demonstrates the successful decoration of CPH film with gold nanoparticles. The mesoporous structure of the CPH would facilitate electron and mass transfer within CPH.

Figure 1. (A) SEM image of the bare ITO electrode, (B) CPH / ITO electrode, (C) AuNPs / CPH / ITO electrode, and (D) the zoom-in image of circled zone in Figure C. Water content (Q) of the hydrogel samples is evaluated from the equation: Q = (Ws-Wd) / Ws. Ws and Wd are the weight of the fully swollen sample and dry sample separately. The dry weight of CPH was obtained by freeze-drying each sample for 10 h. The average water content is about 50%. Such high water content can prevent the leakage of the film and facilitate the fast transport of electrons from the coreactant (K2S2O8) and the internal standard molecules (luminophore luminol) electro-polymerized in the conductive polymer hydrogel. Moreover, the hydrogel is generally biocompatible and help to keep the activity of biomolecules which demands water environment. Biocomplibility

and

Electrochemistry

Properties

of

the

Modified

Electrode.

Biocompatibility of the sensing interfaces is investigated with fluorescence labeling technology. As shown in Figure 2A and Figure 2B, more cells are found on CPH/ITO and AuNPs/CPH/ITO 9



electrode than on bare ITO surface or the culture dish. As reported in the references, conducting polymers, such as polyaniline, show good biocompatibility for nerve and muscle cell growth and regeneration.49,50 While hydrogels are particularly suitable candidates as scaffolds for cell and tissue growth because of their hydrophilic properties, their mass transport properties, and their tissue-like mechanical properties.49 So conducting polymer hydrogels (CPH) are more suitable for cells to adhere and proliferate than the cell culture dish which is composed of treated polystyrene, and more cells survive on AuNPs/CPH/ITO or CPH/ITO electrode than culture dish. These results suggest the improved biocompatibility with the modification of CPH and AuNPs.

Figure 2. (A) Fluorescence image of HeLa cells incubated with (a) bare ITO electrode, (b) culture dish, (c) CPH/ITO electrode and (d) AuNPs/CPH/ITO electrode. (B) Fluorescence image of MCF7 cells incubated with (a) bare ITO electrode, (b) culture dish, (c) CPH/ITO electrode and (d) AuNPs/CPH/ITO electrode. In addition, the specific capacitance values are calculated from the discharge curve of the charge– discharge cycles as shown in Figure S2. Specific capacitance as high as 229 F g−1 at 1 A·g−1 current density is obtained using the following equation: Cs = I td / m ∆V Where Cs is the specific capacitance in F·g−1, m is the mass of electroactive material in gram, ΔV 10


Page 10 of 21

Page 11 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


is the potential window in volts (Vf −Vi), I is the current in ampere, and td is the discharge time in seconds. The galvanostatic charge–discharge cycles obtained in chronopotentiometric measurements indicate the good electrochemical stability. Electrical Characterization of Stepwise Fabrication of Sensing Interface. The ECL-sensing platform was characterized by Cyclic Voltammetry (CV) and Electrochemical Impedance Spectrum (EIS) measurements according to the reference.42 From Figure 3A we can see that a pair of redox peaks can be observed at 0.12 V and 0.37 V for bare ITO (curve a). After the electrode surface is electropolymerized with hydrogel compounds (curve b) or modified with AuNPs (curve c), the redox peak current of both samples increase, which is attributed to the increase of the surface active area of the electrode. When the electrode surface is modified with MUC1 aptamer, the peak current decreases (curve d). The redox peak current decreases lower with the subsequent capture of MCF7 cells as shown in curve e. After MCF-7 cells are modified with CdTe-APT 2 nanoprobes, redox peak current decreases furtherly (curve f), these results can be ascribed to the insulation effects of aptamers and MCF-7 cells. Moreover, Figure 3B shows the results of impedance spectroscopy of the different modified electrodes. Curves (a) is the EIS of bare ITO electrode in the electrolyte solution, after electropolymerized with hydrogel compounds, the EIS of the CPH showes a lower Ret (curves b, Ret ≈ 40 Ω), which proves that CPH is beneficial to the electrons transfer. After AuNPs are loaded on the CPH/ITO, the Ret decreases (curves c, Ret ≈ 24 Ω) implying that the adsorption of AuNPs on CPH surface. When the electrode is modified with MUC1 aptamer, MCF7 cells and CdTe-APT 2 nanoprobes, the diameter of the semicircle increases (curves d, e, f). All the results suggest the successful fabrication of the multiplex cytosensor.

11



Figure 3. (A) CV and (B) EIS characterization of the sensing interfaces in 0.1 M PBS (pH 7.4) containing 0.1 M KCl and 5.0 mM [Fe(CN)6]3−/4−: (a) bare ITO electrode; (b) CPH modified ITO electrode; (c) AuNPs and CPH modified ITO electrode; (d) MUC1 aptamer, AuNPs, and CPH modified ITO electrode; (e) MCF-7 cells, MUC1 aptamer, AuNPs, and CPH modified ITO electrode; (f) CdTe-APT 2, MCF-7 cells, MUC1 aptamer, AuNPs, and CPH modified ITO electrode. Feasibility of CPHs Based Ratiometric ECL Cytosensor. In the present ratiometric ECL cytosensor, K2S2O8 and luminol are all entrapped in the CPH modified electrode surfaces to construct the detection interface. ECL signals generated from the system containing K2S2O8 in the test solution and that with K2S2O8 immobilized on the electrode surface (electropolymerized from the solution containing the same concentration of K2S2O8 as in the test solution) are compared, as shown in Figure 4A. The result shows that the coreactant on the electrode surface can give stronger ECL signal. Such higher efficiency is probably due to the easier electron-transfer when the coreactant entrapped in the hydrogels on the electrode. Furthermore, the positive role of luminol as an internal standard signal is evaluated by changing the photomultiplier tube voltage as shown in Figure 4B. From the result, we can see that the ECL intensities of CdTe QDs and luminol all increase when photomultiplier tube voltage increases from 650V to 850 V. However, the ratio of the two signals is almost the same as shown in the inset of Figure 4B. The result demonstrates that the external factors such as photomultiplier tube voltage could influence the detecting result if only one luminophore (CdTe QDs) is used in the system. 12


Page 12 of 21

Page 13 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


When another luminophore (luminol) is used as an internal standard ECL signal, the capability of the cytosensor for quantifying cancer cells is improved with high accuracy.

Figure 4. (A) ECL intensity-time curves obtained on CdTe QDs and luminol with MCF-7 cell concentrations of 3300 cells/mL in 0.1 M PBS (pH 7.4). a: with K2S2O8 in the test solution. b: with K2S2O8 immobilized on the electrode surface. (B) ECL intensity-time curves of CdTe QDs and luminol with different photomultiplier tube voltage (black, 650V; red, 700V; blue, 750V; purple, 800V; and green, 850V) with MCF-7 cell concentrations of 3300 cells/mL in 0.1 M PBS (pH 7.4). Inset is the ratio of the two signals. Optimization of the Proposed Cytosensor. The ECL performance of the proposed cytosensor is influenced by many factors. To obtain high sensitivity, experiments are performed to optimize the concentration of each component (Figure S3). And the optimum conditions are found as follows: 0.09 M K2S2O8, 0.09 M AA, and 0.03 M MBAA. Details can be referred to Figure S3A, S3B, and S3C. And the optimized electropolymerization time of CPH is 600 s (Figure S3D). Quantification of target Cells. The as-prepared ECL cytosensor is employed to detect the amount of MCF-7 cells by the CdTe-APT 2 nanoprobes labeled on the surface of the cells, which are captured by MUC 1 aptamer on the sensing interface. Figure 5A is the ECL signals of the luminol and CdTe QDs, which shows the relation between the ECL intensity and the cells concentration ranging from 100 to 6500 cell mL-1. The ECL intensity of CdTe QDs increases with the increase of cell concentration while the ECL intensity of luminol almost keeps constant, which 13



is the same as the internal standard method. ECL intensity of CdTe QDs is used as an analytical signal, and ECL intensity of luminol is used as an internal standard signal in the developed system. As shown in Figure 5B, the ratio of ∆ECLCdTe and ∆ECLluminol is in linear relationship with the target cell concentration within the range from 100 to 6500 cell mL-1. The linear regression equation is ∆ECLCdTe/∆ECLluminol = 3.47× 10-4 Ccell + 0.163 with a correlation coefficient (R) of 0.993. The detection limit (80 cell mL-1 where S/N = 3) is lower than that of many previous reports (Table S1).51-57 The result indicates that the high bioaffinity of the sensing interface towards the target cells and it can detect MCF-7 cells at a very low level.

Figure 5. (A) ECL intensity-time curves obtained on CdTe nanoprobes with MCF-7 cell concentrations of 100, 900, 1700, 2500, 3300, 4100, 4900, 5700, 6500 cells mL-1. (B) Calibration curve of the MCF-7 cells and ∆ECLCdTe/∆ECLluminol. (Note: ∆ECL is referred to the change of the ECL intensity compared to the baseline. For luminol, ∆ECLluminol keeps constant with the variation of cell concentrations.) Selectivity, Stability and Reproducibility of the Electrochemical Biosensor. In order to investigate the selectivity of the proposed cytosensor, the system is exposed to different cells composition: Hela cells, U87-MG cells, MCF-7 cells, a mixture of any of the above two cells and a mixture of the three cells. As shown in Figure 6A, the significant ECL signal is only observed in the presence of MCF-7 cells, either only with MCF-7 cells or the mixture containing MCF-7

14


Page 14 of 21

Page 15 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


samples. Hela cells, U87-MG cells, or the mixture of the two types of cells present weak signal. Such results indicate the high selectivity of the cytosensor towards the MCF-7 cells. Prominent stability is a crucial factor for the practical application of the ECL cytosensor. As shown in Figure 6B, the cytosensor displays good reproducibility and no obvious ECL intensity changes are observed through continuous cyclic potential scans for 10 cycles with the presence of 1.0×105 cells mL−1 MCF-7 cells. The results suggest the satisfying stability of the multiplex cytosensor. Additionally, the reproducibility of the sensing system is also evaluated. Five modified electrodes were used to detect the same concentration of MCF-7 cells (3300 cells/mL). This electrochemical cytosensor exhibits a RSD = 5.2% for CdTe QDs and RSD = 4.9% for luminol, indicating good reproducibility.

Figure 6. (A) Selectivity of the aptasensor after incubation with different kinds of cells: MCF-7 cell; the mixture of MCF-7 cell and Hela cell; the mixture of MCF-7 cell and U87-MG cells; the mixture of MCF-7 cell, Hela cell, and U87-MG cell; Hela cell; U87-MG cell; the mixture of Hela cell and U87-MG cell. (B) ECL intensity vs. time: the aptasensor under continuous scanning for 10 cycles in 0.1 M PBS solution (pH = 7.4). Detection of MCF-7 cancer cells in human serum. In order to evaluate the potential application of the MCF-7 cell biosensor in complex real sample, three different concentrations of MCF-7 cells are spiked in the human serum. As shown in Table S2, the recovery values (98.0, 102.8, and 100.2) 15



indicate the good applicability of the method for analying cancer cells, demonstrating the capatility of the constructed biosensor in MCF-7 breast cancer cell detection within complicated sample matrix. Conclusion In conclusion, a ratiometric ECL cytosensor based on CPH loaded with internal standards in the sensing surface is constructed. In combination with the excellent electrical conductivity of the conductive polymer and the perfect physical properties of hydrogel, the ratiometric ECL cytosencor has shown good stability and the electrochemical conductivity. This ECL assay can be applied to detect MCF-7 cells accurately by using QDs as signal probes and luminol as the internal standard. Moreover, the co-reactant K2S2O8, as the internal standards of luminol, is also entrapped in the CPH on the sensing interface during the electropolymerization process. In this way, the sensitivity of the ECL has been improvedand luminous efficiency has been enhanced compared to reported ECL reactions. As a soft material with unique features such as good conductivity and biocompatibility, CPH based sensing interfaces demonstrate great potential in the healthcare monitoring and clinical diagnostics. ACKNOWLEDGMENTS The authors greatly acknowledge support from the Project Fund for Shangdong Key R&D Program (2017GGX20121), the National Natural Science Foundation of China (Grants 21422504), and the Taishan Scholar Program of Shandong Province of China (Grant ts20110829). *S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.XXX. 16


Page 16 of 21

Page 17 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Preparation of AuNPs and CdTe-Apt 2 Nanoprobes; characterization of CdTe NPs and Au NPs (Figure S1); electrochemistry properties of the modified electrode (Figure S2); optimization of the experimental condition of the proposed cytosensor (Figure S3); comparison of sensing performances for the MCF-7 cell detection (Table S1); detection of MCF-7 cancer cells in human serum (Table S2). Reference (1) Annabi, N.; Tamayol, A.; Uquillas, J. A.; Akbari, M.; Bertassoni, L. E.; Cha, C. Adv. Mater. 2014, 26, 85-124. (2) Singh, A.; Peppas, N. A. Adv. Mater. 2015, 26, 6530-6541. (3) Tanaka, Y.; Kuwabara, R.; Na, Y. H.; Kurokawa, T.; Gong, J. P.; Osada, Y. J. Phys. Chem. B. 2005, 109, 1155911562. (4) Elliott, J. E.; Macdonald, M.; Nie, J.; Bowman, C. N. Polymer. 2004, 45, 1503-1510. (5) Krepker, M. A.; Segal, E. Anal. Chem. 2013, 85, 7353-7360. (6) Peppas, N. A.; Hilt, J. Z.; Khademhosseini, A.; Langer, R. Adv. Mater. 2006, 18, 1345-1360. (7) Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A. Adv. Mater. 2010, 21, 33073329. (8) Drury, J. L.; Mooney, D. J. Biomaterials. 2003, 24, 4337-4351. (9) Piver, M. S.; Marchetti, D. L.; Parthasarathy, K. L.; Suraj Bakshi, M. D.; Peter Reese, J. D. Cancer. 1985, 56, 76-80. (10) Ouasti, S.; Donno, R.; Cellesi, F.; Sherratt, M. J.; Terenghi, G.; Tirelli, N. Biomaterials. 2011, 32, 6456-6470. (11) Deligkaris, K.; Tadele, T. S.; Olthuis, W. Sens. Actuators, B. 2010, 147, 765-774. (12) Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A. Adv. Mater. 2010, 21, 33073329. 17



(13) Ding, H.; Zhong, M.; Kim, Y. J.; Pholpabu, P.; Balasubramanian, A.; Hui, C. M. Acs. Nano. 2014, 8, 43484357. (14) Chen, H.; Müller, M. B.; Gilmore, K. J.; Wallace, G. G.; Li, D. Adv. Mater. 2010, 20, 3557-3561. (15) Huang, X.; Liu, Y.; Yung, B.; Xiong, Y.; Chen, X. Acs. Nano. 2017, 11, 5238-5292. (16) Zhu, Z.; Wu, C.; Liu, H.; Zou, Y.; Prof, X. Z.; Kang, H. Angew. Chem., Int. Ed. 2010, 49, 1052-1056. (17) Kim, J.; Singh, N.; Lyon, L. A. Angew. Chem., Int. Ed. 2006, 45, 1446-1449. (18) Hu, J.; Ni, P.; Dai, H.; Sun, Y.; Wang, Y.; Jiang, S. Analyst. 2015, 140, 3581-3586. (19) Feng, L.; Zhu, A.; Shi, H. Sensors. 2013. 13, 13928-13948. (20) Soohyun, P.; Jung, K. Y.; Sheereen, M. Adv. Mater. 2015, 27, 7583-7619. (21) Chen, L.; Hong, Z.; Li, G. Sista, S.; Chen, L. M. Adv. Mater.

2009, 21, 1434-1449.

(22) Gustafsson, G. Cao, Y.; Treacy, G. M. Klavetter, F.; Colaneri, N.; Heeger, A. J. Nature, 1992, 357, 477-479. (23) Hao, G. P.; Hippauf, F.; Oschatz, M.; Wisser, F. M.; Leifert, A.; Nickel, W. Acs. Nano. 2014, 8, 7138-7146. (24) Chen, Q.; Zhao, L.; Chun, Li. A.; Shi, G. J. phys. chem.C. 2007, 111, 18392-18396. (25) Abraham, S.; Brahim, S.; Ishihara, K.; Guiseppi-Elie, A. Biomaterials, 2005, 26, 4767-4778. (26) Ghosh, S.; Inganäs, O. Adv. Mater. 1999, 11, 1214-1218. (27) Li, L.; Wang, Y.; Pan, L.; Shi, Y.; Cheng, W.; Shi, Y. Nano. Lett. 2015, 15, 1146-1152. (28) Zhang, J.; Zhao, Z.; Xia, Z.; Dai, L. Nat. Nanotechnol. 2015, 10, 444-452. (29) Fekete, Z. Sens. Actuators, B: Chem. 2015, 215, 300-315. (30) Jie, G.; Zhang, J.; Wang, D.; Cheng, C.; Chen, H. Y.; Zhu, J. J. Anal. Chem. 2018, 80, 4033-4039. (31) Zhuo, Y.; Liao, N.; Chai, Y. Q.; Gui, G. F.; Zhao, M.; Han, J. Anal. Chem. 2014, 86, 1053-1060. (32) Ma, M. N.; Zhuo, Y.; Yuan, R.; Chai, Y. Anal. Chem. 2015, 87, 11389-11397. (33) Xu, S.; Liu, Y.; Wang, T.; Li, J. Anal. Chem. 2011, 83, 3817-3823. 18


Page 18 of 21

Page 19 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(34) Gui, G. F.; Zhuo, Y.; Chai, Y. Q.; Xiang, Y.; Yuan, R. Anal. Chem. 2014, 86, 5873-5880. (35) Zhuo, Y.; Liao, N.; Chai, Y. Q.; Gui, G. F.; Zhao, M.; Han, J. Anal. Chem. 2014, 86, 1053-1060. (36) Liang, W.; Zhuo, Y.; Xiong, C.; Zheng, Y.; Chai, Y.; Yuan, R. Anal. Chem. 2015, 87, 12363-12371. (37) Zhang, H. R.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2013, 85, 5321-5325. (38) Wu, P.; Hou, X.; Xu, J. J. Nanoscale. 2016, 8, 8427-8442. (39) Lei, Y. M.; Huang, W. X.; Zhao, M.; Chai, Y. Q.; Yuan, R..; Zhuo, Y. Anal. Chem. 2015, 87, 7787-7794. (40) Zhang, H. R.; Wu, M. S.; Xu, J. J.; Chen, H. Y. Anal. Chem. 2014, 86, 3834-3840. (41) Cheng, Y.; Huang, Y.; Lei, J.; Zhang, L.; Ju, H. Anal. Chem. 2014, 86, 5158-5163. (42) Zhou B., Qiu Y. Y., Wen Q. Q., Zhu M. Y., Yang P. H. ACS Appl. Mater. Interfaces 2017, 9, 2074-2082. (43) Fu, X.; Tan, X.; Yuan, R.; Chen, S. Biosens. Bioelectron. 2017, 90, 61-68. (44) Pinaud, F.; Russo, L.; Pinet, S.; Isabelle, G.; Valérie, R.; Neso, S. J. Am. Chem. Soc. 2013, 135, 5517-5520. (45) Wang, J.; Li, W.; Zhu, J. Polymer. 2014, 55, 1079-1096. (46) Milutinovic, M..; Sallard, S.; Manojlovic, D.; Mano, N.; Sojic, N. Bioelectrochemistry. 2011,82, 63-68. (47) Ying Y. L.; Hu Y. X.; Gao R.; Yu R. J.; Gu Z.; Lee L. P.; Long Y. T. J. Am. Chem. Soc. 2018, 140, 5385-5392. (48) Yu Z.; Zhou L.; Zhang T.; Shen R.; Li C. X.; Fang X.; Griffiths G.; Liu J. ACS Sens. 2017, 2, 626-634. (49) Luo, X. L., Weaver, C. L., Zhou, D.D., Greenberg, R., Cui, X. T. Biomaterials 2011, 32, 5551-5557. (50) Shi, Z. J., Gao, X., Ullah, M. W., Li, S. X., Wang, Q. Yang, G. Biomaterials 2016, 111, 40-54. (51) Hua, X.; Zhou, Z. X.; Yuan, L.; Liu, S. Q. Anal. Chim. Acta. 2013, 788, 135-140. (52) Jia, S.; Li, P.; Koh, K.; Chen, H. Microchimica Acta, 2016, 183, 683-688. (53) Wang, K.; Zhang, R.; Sun, N.; Li, X.; Wang, J.; Cao, Y. Acs Appl. Mater. Interfaces. 2016, 8, 25834-25839. (54) Li, T.; Fan, Q.; Liu, T.; Zhu, X.; Zhao, J.; Li, G. Biosens. Bioelectron. 2010, 25, 2686-2689. (55) Soleymani, J.; Hasanzadeh, M.; Somi, M. H.; Shadjou, N.; Jouyban, A. Biosens. Bioelectron. 2018, 115, 61-69. (56) Guo, Q.; Li, X.; Shen, C.; Zhang, S.; Qi, H.; Li, T. Microchimica Acta. 2015, 182, 1483-1489. 19



(57) He, Y.; Li, J.; Liu, Y. Anal. Chem. 2015, 87, 9777-9785.

20


Page 20 of 21

Page 21 of 21 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only

21


Ratiometric Electrogenerated Chemiluminescence Cytosensor Based

Recommend Documents