Electrochemical Aptasensor for Ultralow Fouling Cancer Cell

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Electrochemical Aptasensor for Ultralow Fouling Cancer Cell Quantification in Complex Biological Media Based on Designed Branched Peptides Nianzu Liu, Jingyao Song, Lu Yanwei, Jason J Davis, Fengxian Gao, and Xiliang Luo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01129 • Publication Date (Web): 23 May 2019 Downloaded from http://pubs.acs.org on May 24, 2019

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Analytical Chemistry

Electrochemical Aptasensor for Ultralow Fouling Cancer Cell Quantification in Complex Biological Media Based on Designed Branched Peptides

Nianzu Liu†, Jingyao Song†, Yanwei Lu†, Jason J. Davis‡, Fengxian Gao†, Xiliang Luo†,*

†

Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE;

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. ‡ Department

of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom

ABSTRACT The rapid, convenient and selective assaying of clinical targets directly in complex biological media brings with it the potential to revolutionise diagnostics. One major hurdle to impact is retention of selectivity as and a tight control of nonspecific surface interactions or biofouling. We report herein, the construction of an antifouling interface through the covalent attachment of designed branched zwitterionic peptides onto electrodeposited polyaniline film. The antifouling capability of the designed branched peptide significantly outperforms that of the commonly used PEG and linear peptides. The interfaces modified with branched peptides are exceptionally effective in reducing nonspecific protein and cell adsorption as verified by electrochemical and fluorescent characterization. The derived sensors with mucin1 protein (MUC1) aptamer as the recognition element detect MUC1-positive MCF-7 breast cancer cells in human serum with high sensitivity and selectivity. The linear response range of the cytosensor for the MCF-7 cell is from 50 to 106 cells/mL, with a limit of detection as low as 20 cells/mL. More importantly, the assaying performances remains unchanged in human serum owing to the presence of branched antifouling peptide, indicating feasibility of the cytosensor for practical cancer cell quantification in complex samples.

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INTRODUCTION Cancer is one of the most serious threats to global public health and quality of life, and remains too commonly associated with high mortality despite substantial development in treatment.1 Specifically, breast cancer is one of the most common forms of cancer worldwide. According to the U.S. National Cancer Institute (NCI), for example, approximately 252,000 women in the United States were diagnosed with breast cancer, with more than 40,000 of them ultimately dying of the disease.2 Approximately 1 in 8 women will be diagnosed with breast cancer sometime in their lives.3 A reliable, noninvasive and early screen for cancer cells buried within the circulating ocean of normal and benign cells could underpin a step change in our ability to dramatically improve this picture and to guide effective treatment.4 In recent years a broad range of cancer cell assays based on colorimetric detection,5-7 chemiluminescence,8-9 electrochemical platforms,10-12 and fluorescence have been presented.13-14 Of these electrochemical analysis brings with it a particularly favourable mix of sensitivity, convenience and low-cost.15 By default these are label free assays operating through an electrode interface restricted recognition process, where real clinical applications, in a complex matrix, can be challenging. A general way to mitigate this challenge is to integrate target recognition into an otherwise very antifouling surface, ideally one which is easily chemically tuned, has good biocompatibility and high chemical stability. Polyethylene glycol (PEG)-based adlayers are commonly used but oxidize under physiological conditions and are not readily modified or otherwise chemically tuned. Peptides are natively zwitterionic, exhibit outstanding biocompatibility and a range of zwitterionic peptide-based antifouling film have been reported.16-17 For example, a zwitterionic peptide sequence composed of alternating positively charged lysines (K) and negatively charged glutamic acid (E) exhibited high resistance to protein adsorption ( 97%) was obtained from Equitech-Bio (Kerrville, TX). Fetal bovine serum (FBS), globulin (GLO), carcinoembryonic antigen (CEA), human serum albumin (HSA), hemoglobin (Hb), bovine serum albumin (BSA), lysozyme (Lys) were purchased from Beijing Bo Yang Hongda Technology Co. Ltd. (Beijing, China). Human serum samples were provided by the Eighth People’s Hospital of Qingdao (Qingdao, China). Other chemicals were of analytical grade and ordered from Aladdin Reagent Co., Ltd. (Shanghai, China). Millipore water with a resistivity greater than 18 MΩ cm was used in all experiments.

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Synthesis of PANI films PANI films were electrodeposited through a galvanostatic technique onto the GCE, which was prior successively polished with 0.3 and 0.05 μm alumina powder followed by an ultrasonic washing in absolute ethanol and water for 2 min, respectively. The electropolymerization was performed at a constant current density of 0.01 mA/cm2 at room temperature for 1 h, and the typical electrolyte was 1.0 M HClO4 solution containing 0.1 M aniline and 0.01 g/mL PSS. The deposited PANI containing amino groups can be used for the following covalent immobilization of peptides and aptamers, and its electrochemical redox peaks can be used as the sensing signal of the constructed biosensor. Immobilization of aptamers and peptides As shown in Scheme S1, electrodes modified with PANI films (PANI/GCE) were incubated in 2 mM sulfo-SMCC as cross-linker for 1 h prior to incubation in a mixed PBS (0.2 M, pH 7.4) solution containing 1.0 µM MUC1-aptamers and 1.0 µM branched zwitterionic peptides for 60 min at room temperature. During this process, the cys-terminal thiols were covalently bonded with PANI’s amino groups, achieving the attachment of aptamers and peptides onto the PANI/GCE. The obtained biosensors were washed with PBS and stored in PBS when not in use. Finally, the fabricated surfaces of peptide-aptamer/PANI/GCE were equilibrated in PBS (0.2 M, pH 7.4) for 24 h at room temperature after extensively washing with PBS (0.2 M, pH 7.4) prior to analysis. Characterization of the peptides. Hydrophilicity characterizations were carried out by a static water contact angle (WCA) measurement. Water drops (10 μL) were equilibrated in contact with surfaces for 10 s prior to data collection. Circular dichroism (CD) spectra were used to elucidate the secondary structure of branched zwitterionic peptides, recording on 0.3 mg/mL peptides solution over a wavelength range of 190-270 nm with a step resolution of 0.5 nm. Buffer spectra were subtracted from the peptides sample spectra. Surface specificity assessments To assess the antifouling performance of different modified electrodes, relatively complex media fetal bovine serum was tested. DPV technique was utilized to monitor the nonspecific adsorption and the responses of electrode surfaces before and after soaking in FBS (30 min) were recorded in PBS (0.2 M, pH 7.4). FBS samples were diluted with PBS (10 mM, pH 7.4) to different dilution ratio (V/V) solutions. Cell culture and fluorescence imaging The target MCF-7 cells (human breast carcinoma cell line), the control HepG2 cells (human hepatocellular carcinoma cell line) and Hela cells (human cervical cancer cells) used in this study were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, St. Louis, MO) supplemented with 10% (v/v) FBS and 1% antibiotics (penicillin/streptomycin). Cell culture environment was maintained in a humidified 5.0% CO2 incubator at 37 °C. Cells were detached by trypsinization with 0.25% trypsin and 0.02% EDTA in PBS buffer. Before measurement, cells were collected and separated from the DMEM medium through centrifugation at 800 rpm for 5 min. Then the cells were washed with PBS (0.2M, pH 7.4). The cell number was measured using a blood counting chamber. For fluorescence imaging, FDA (Sigma-Aldrich, St. Louis, MO) was used to stain the MCF-7 cells 4


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cultured on the samples for investigation by fluorescence microscopy using an excitation wavelength at 488 nm. FDA in acetone (10.0 mg/mL) was prepared as stock solution. Then PBS containing a final concentration of 50.0 μg/mL FDA was prepared as the freshly working solution to incubate MCF-7 cells in the dark for 5 min at room temperature and washed with PBS. The samples were placed on a glass slide for fluorescence microscopy measurement. Laser radiation at a wavelength of 488 nm was used to excite the dye.

MCF-7 quantification The peptide-aptamer/PANI modified surfaces were incubated in different concentrations of MCF-7 cell solution in PBS (0.2 M, pH 7.4) for 60 min at 37 °C. Spiking experiments were conducted to evaluate the application potential of the proposed format to assay within real biological samples. A specific number of MCF-7 cells were spiked into 10% (V/V) human serum and then subjected to the electrochemical analysis. The signal suppression as the ratio before and after hybridization was measured by DPV in the range of -0.4 to 0.6 V.

RESULTS AND DISCUSSION Investigation of designed branched zwitterionic peptides. The nonspecific adsorption of proteins to a surface that causes fouling is mainly ascribed to the hydrophobic interaction and charge attraction. Hydrophilic surface will suppress the adsorption of protein to the surface through hydrophobic interaction, and the surface that is neutral in charge can prevent protein adsorption to the surface through charge attraction. Therefore, a high performance antifouling molecular film should be highly hydrophilic and charge neutral, and this rationale was followed in designing antifouling peptides. Here the low fouling segment of the branched zwitterionic peptide sequences (-EK2(EK)4(EK)), contains seven positively charged lysine residues (K) and seven negatively charged glutamic acid residues (E). The isoelectric point (pI) of the peptide is 6.78 and the measured zeta potential close to 0.0 mV (Figure S2). The secondary structure of peptides was characterized using circular dichroism (CD), where a strong negative ~200 nm band and a weaker positive band at 220 nm, indicated the presence of an extended polyproline-generated helix conformation (Figure 1a), one that aids film density.25

Scheme 1. Schematic Illustration of the Preparation of a PANI Supported Branched Peptide-Based Amperometric Cell Sensor. C: cysteine; P: proline; E: glutamic acid; K: lysine.

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Fabrication of the electrochemical MCF-7 cell biosensor. The operational strategy is summarised schematically in Scheme 1. PANI films were electrodeposited through a galvanostatic method onto the GCE and used herein as both a conductive support and a transducing electrochemical signal without the need to add additional redox probes. MUC1 aptamers to capture any MCF-7 cells, and branched zwitterionic peptides to reduce nonspecific adsorption, were covalently coupled onto the PANI film through the reaction between the Cys-terminal thiols and PANI’s amino groups with the assistance of sulfo-SMCC heterobifunctional cross-linker (see Scheme S1 for details). The covalent attachment of peptides and aptamers onto PANI was verified using FTIR (Figure 1b). The FTIR spectrum of PANI (curve a) resolves clear benzenoid and quinoid ring stretching bands (C=C) at 1472 and 1558 cm-1, respectively. Bands at 1121 and 1299 cm-1 are ascribed to the C-N stretching of the secondary aromatic amine, and the bands at 745 and 801 cm-1 are ascribed to the out-of-plane bending of C-H. These characteristics are as expected for PANI.26-28 When peptide and aptamer are covalently attached together onto PANI film (curve b), new bands appear at 1636 cm-1, which are ascribed to the generated amides.15, 29-30

Figure 1. (a) CD spectra of branched peptide in PBS at a concentration of 0.3 mg mL−1. (b) FTIR spectrum of PANI (curve a) and peptide-aptamer/PANI (curve b). (c) SEM of the PANI film, and inset shows the magnified part, with the particles are in the size of 50-100 nm. (d) Contact angles images of (A) bare GCE, (B) PANI, (C) peptide/PANI coated GCE surfaces. (e) DPV and CV curves recorded in PBS (0.2 M, pH 7.4). (a: the bare GCE; b: PANI/GCE; c: peptide-aptamer/PANI/GCE; d: MCF-7 cells/peptide-aptamer/PANI/GCE).

Interface Characterization. The micromorphology of the PANI film was evaluated by SEM. As shown in Figure 1c, a layer of PANI is the formed on the electrode surface, and it shows a particulate microstructure (particles of 50-100 nm). Wetting characteristics were measured by static water contact angle; as shown in Figure 1d and Table S1, the contact angle of the electropolymerized polymer 6


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surface (PANI/GCE) is about 47.5°, which is smaller than that of the bare GCE (55.7°). Subsequent peptide coupling (peptides/PANI/GCE) is associated with a significant decrease in contact angle (21.3°), indicating a notable hydrophilicity consistent with reports of other peptide films.15, 17, 20 Hydrophilic and Charge neutral interfaces are exposed to aqueous solutions, water molecules will accumulate onto the surface to form a hydration layer, which can inhibit the proteins and other contaminants from interacting with the surface.31 DPV and CV were used to characterize film formation process in the absence of a solution phase redox probe (Figure 1e). It is worth noting that the electrodeposited PANI film (curve b) shows a marked DPV response compared with the bare GCE (curve a), with a peak potential at -0.03 V corresponding to the transition of leucoemeraldine/emeraldine forms of PANI. The peak currents of the PANI and peptide-aptamer/PANI modified electrode presented a linear relationship with the scan rate, indicating that the redox processes of the coated electrodes were surface confined processes with fast electron transfer kinetics (Figure S3.). The integration of peptides and aptamers predictably decrease signal as they significantly impede the electron transfer (curve c). Subsequent capture of target MCF-7 cells results in a further decrease in current response as the electrolyte exposed surface becomes increasingly sterically congested with ow dielectric material (curve d). The results of non-Faradaic EIS are in line with CV and DPV measurement (Figure S4).

Investigations of nonspecific adsorption. Linear or straight zwitterionic peptides and PEG (“gold standard” of antifouling polymers), have been applied in a range of biosensors to circumvent non-specific adsorption or fouling.15, 31 In order to investigate the antifouling ability of different surfaces, DPV responses of different electrodes modified various antifouling materials, PEG, straight peptide and branched peptides, were compared before and after incubation in FBS (for 30 min) (Figure 2a). The DPV current decrease can directly reflect the nonspecific adsorption (at this stage these surfaces are receptor-free) (Figure S5). Notably, even after incubation in 20% FBS, the current response of the branched peptides-based film is modulated by less than 5.0%, indicating an excellent antifouling performance.32-33 A comparative assessment of performance over more prolonged periods in 20% FBS is shown in Figure 2b. Clearly, the branched architecture peptides consistently outperform both linear peptides and PEG modified interfaces, features we ascribe to their combined steric repulsion and water permeability,24 where inter- and intramolecular cavities promote the water transport.36

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Figure 2. (a) Comparative antifouling characteristics of the bare and modified electrode interfaces in fetal bovine serum samples (V/V). (b) Antifouling analysis of the peptide modified surface in 20% FBS over 1-6 h.

Cell adhesion investigation. The antifouling and specific binding ability of the peptide and aptamer modified surfaces were further confirmed by fluorescence microscopy. Figure 3a and b shows typical images of MCF-7 cells adsorbed on a PANI support layer before and after peptide integration. Notably, there was very few cell adhesions on branched peptides-coated surface (Figure 3b). By contrast, higher cell density and strong fluorescence was observed on the aptamer and peptide modified surface (peptide-aptamer/PANI surface, Figure 3c), confirming specificity of recruitment and the antifouling role of the peptide.

Figure 3. Representative fluorescence microscopy images of PANI (a), peptides/PANI (b), and peptide-aptamer/PANI (c) modified electrodes after incubation in 104 cells/mL MCF-7 cells. The scale bar in images is 100 µm.

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Detection of MCF-7 cells. After optimization of the sensor fabrication and sensing conditions (see Figure S6), an assessment of the aptasensor response to MCF-7 cells is shown in Figure 4, with the transducing PANI oxidation peak responding across 50 to 106 cells/mL. The corresponding regression, △i/i0 = 4.36 logC - 2.54, with a correlation coefficient (R2) of 0.996. The limit of detection (LOD) of this cytosensor (20 cells/mL, S/N = 3) is lower than that of many reported cytosensors or assays for cancer cells (Table S2).8, 37-44 Such a low LOD may be ascribed to the relatively high surface area of the supporting PANI film (generate sensitive electrochemical signal) and the excellent biocompatibility of the branched peptide (provide biocompatible environment for the aptamer). In order to evaluate the applicability of these sensor interfaces to real biological samples, spiking experiments were carried out by adding specified numbers of target MCF-7 cells into 10% (V/V) human serum (Figure 3b) and resolved (△i/i0 = 4.30 logC - 2.01 (R2 = 0.995)) a retention of highly specific and broad linear response. Clearly, even in the presence of complex serum, the sensing performances of the aptasensor remain nearly unchanged (similar calibration curves in Figure 4 a and b), which is hard to achieve for most electrochemical sensors. Specificity, stability and regeneration of the sensing system. The specificity of the peptide-based interface was measured by monitoring its exposure to a broad range of proteins of different molecular weight and pI/charge (Figure 4c). Even at 1.0 mg/mL interferential proteins and 104 cells/mL of other cells, the interfacial amperometric responses are significantly lower than that of the specific response to MCF-7 cells, indicating excellent cytosensor specificity. An analysis of response stability, carried out by continuous CV measurement in PBS (0.2 M, pH 7.4) with the potential range from -0.4 to 0.4 V,15, 21 demonstrates both a largely unchanged peak current/potential profile after 50 cycles (Figure S7) and a retention of transducing signal across 8 days, shows a retention of >98% baseline signal (Figure 4d). The regenerability of the aptasensor was evaluated by immersing the modified electrode into 1.0 M NaOH at 4 °C for 10 min, and then used to detect MCF-7 cells again (104 cells/mL). Fig. S8 shows the current values of the aptasensor during twelve regeneration runs, where only a slight current decrease, indicating the aptasensor can be facilely regenerated.

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Figure 4. Calibration curves of the cytosensor for MCF-7 cells in the absence (a) and presence (b) of 10% human serum (V/V). (c) Responses of the MCF-7 cell biosensor to 1.0 mg mL-1 of Lys, Hb, HSA, CEA, BSA, IgG, GLO, and 104 cells/mL HepG2, Hela cell, mixture (Mix) of different types of cells (MCF-7, HepG2 and Hela cell), respectively. (d) Stability of the peptide-aptamer/PANI/GCE sensor interface response as stored in PBS (0.2 M, pH 7.4) over eight days.

CONCLUSIONS In integration of a branched zwitterionic peptide into a conducting and transducing polymer interface underpins the generation of a label free, ultrasensitive and antifouling electrochemical cytosensor that supports the quantification of MCF-7 cells in human serum across a 6 orders dynamic range. The peptide component possesses an antifouling capability consistently and significantly superior to that observed with linear peptides and PEGs. We believe the interface and design principles presented herein represent a very promising advance along the road to fabricate target selective biosensors with robust operation in complex media.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION 10


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Corresponding Author * E-mail: [email protected]; Tel: + 86 532 84022860; Fax: + 86 532 84023681

ACKNOWLEDGMENTS This research is supported by the National Natural Science Foundation of China (21675093, 21422504), and the Taishan Scholar Program of Shandong Province of China (ts20110829).

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Electrochemical Aptasensor for Ultralow Fouling Cancer Cell

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