Design of a nanoprobe based on its binding with amino acid residues

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Design of a nanoprobe based on its binding with amino acid residues on cell surface and its application to electrochemical analysis of cells Juan Zhang, Hong Chen, Ya Cao, Chang Feng, Xiaoli Zhu, and Genxi Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04247 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Analytical Chemistry

Design nanoprobe based on its binding with amino acid residues on cell surface and its application to electrochemical analysis of cells Juan Zhanga,#, Hong Chena,#, Ya Caoa, Chang Fengb, Xiaoli Zhua,*, Genxi Lia,b, * a

Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, P. R. China. b State Key Laboratory of Pharmaceutical Biotechnology and Collaborative Innovation Center of Chemistry for Life Sciences, Department of Biochemistry, Nanjing University, Nanjing 210093, P. R. China. ABSTRACT: Nanoprobe usually plays a vital role in the development of new electrochemical methods for cell analysis. However, nearly all the currently used versatile probes are prepared by using lectin. So, a new kind of universal nanoprobe is designed and fabricated in this work for electrochemical cell analysis. Specifically, p-sulfonatocalix[4]arene modified sliver nanoparticles (pSC4AgNPs) are synthesized and explored as a universal nanoprobe. In this probe, pSC4 can recognize and bind to various amino acid residues on the membrane protein, and AgNPs can give a sensitive electrochemical signal. Therefore, by using the pSC4-AgNPs as a probe, a variety of cells can be well detected, and the testing results are comparable, showing that the probe has good versatility. At the same time, the detection sensitivity reaches five cells, which is much better than other methods for electrochemical analysis of cells, showing its application prospect in trace cell analysis. In view of the advantages including simplicity of synthesis, stability of pSC4 moiety, and redox capability of AgNPs, the nanoprobe pSC4-AgNPs show a great potential in the development of electrochemical methods for cell detection.

specifically recognizes the mannosyl groups on cell surface11, 14, 15 . So, the versatility of the probe type is not ideal because of the significant differences in glycosylation on different cell surfaces, such as the high expression of glycoprotein MUC1 on the surface of certain cancer cells16-20 and the low expression of glycoprotein MUC1 in most normal cells21-23, which will results in different detection sensitivity and directly incomparable data for different cell types. Therefore, it is highly required to design the more versatile probes. Compared to glycosyl residues, the amino acid composition of cell membrane proteins is relatively stable, which provides the possibility for the development of versatile probes. Numerous researches have shown that p-sulfonatocalix[4]arene (pSC4) as a supramolecular compound, can bind to residues of various amino acids such as Arg, Lys, Glu, Phe, etc, and their methylated product 24-29. For example, the water-soluble pSC4 can bind the lysine-rich cytochrome C at three different sites with binding constant of ~ 103 M-1 27; The amino acid residues such as Arg, Lys, Glu, Ala, Phe, etc. present in the human insulin, show good binding propensity towards pSC4 (~ 105 M1 26 ) . The high binding constant is beneficial to improve the sensitivity of detection. Meanwhile, the combination of multiple amino acid residues is beneficial to eliminate the difference originating from different types of cells and make the detection more versatile. Based on these considerations, pSC4 modified sliver nanoparticles (pSC4-AgNPs) have been synthesized and explored as a universal nanoprobe for cell analysis in this work. For this probe, in addition to the advantages

1 Introduction It is well known that cell analysis is of importance for early diagnostic of diseases1-3. Up to now, cells have been analyzed by diverse methods including ultra-violet visible spectroscopy4, fluorescence spectroscopy5-6, and electrochemical techniques7,8. Among them, various electrochemical methods have been extensively explored in view of the simplicity and economy of electrochemical device7,9. For the development of electrochemical methods for cell analysis, many nanoprobes have been designed and prepared. For example, Zhang et al designed a nanoprobe by noncovalent assembly of concanavalin A (Con A) on CdTe quantum dots - labeled silica nanospheres with poly(allylamine hydrochloride) as a linker and further established an electrochemical platform for ultrasensitive detection of apoptotic cells and early evaluation of therapeutic effects10; Wang et al developed a multiple layer CdS quantum dots-functionalized polystyrene microspheres to ultra- sensitively detect tumor cells11; Chen et al used the ALP and Con A coated gold nanoparticles nanoprobes to test cancer cells and glycan expression profiling12; Chen et al fabricated Con Aintegrating gold-nanoparticle-modified Ru(bpy)32+-doped silica nanoprobe for in situ and dynamically evaluating cell surface N-glycan expression13. Although nanoprobes have received more and more interests, nearly all the universal probes are prepared by using lectin. The binding of these probes with target cells mainly depends on the occurrence of Con A, which

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recorded through a FT-IR spectrometer (Vertex 70, Bruker Co. Ltd., Bergisch Gladbach, Germany). 2.4 Cell analysis 2.4.1 Cell culture HepG2, SMMC-7721, Caco-2, and MCF-7 cells were cultured in DMEM medium supplemented with 10% FBS and 80 U/mL penicillin and 0.08 mg/mL streptomycin. MDA-MB231 and L-02 cells were cultivated in RPMI-1640 medium supplemented with 10% FBS, 80 U/mL penicillin and 0.08 mg/mL streptomycin. All cells were maintained in a humidified incubator at 37°C with 5% CO2 and 80% relative humidity, and they were detached from the culture surface by trypsinization and collected by centrifugation at 2000 rpm for 5 min. After washing with PBS buffer twice, the cell sediment was dispersed in the buffer at various concentrations. The number of cells was acquired by using TC20TM Automated Cell Counter (Bio-Rad, America). 2.4.2 Cell imaging analysis To prove the recognition ability of pSC4-AgNPs on different cells, the labeled cells were imaged by using a LSM 710 confocal laser scanning microscope (Zeiss, Germany). For samples for imaging, cell nucleuses were firstly stained with DAPI for 15 min, followed by the staining of cell membrane with Dio for 10 min. Then samples were mixed with pSC4-AgNPs for 30 min, followed by the addition of Cyanine 3 amine and subsequent incubation for 15 min. The images were obtained with 345 nm, 488 nm, and 550 nm of excitation wavelengths and 478 nm, 511 nm, and 610 nm of the corresponding emission wavelengths. 2.5 Specificity analysis For specificity analysis of electrochemical biosensor, five different samples including Caco-2, MCF-7, MDA-MB-231, L02, and SMMC-7721 cells were used instead of HepG2 cell for experiment. The concentrations of all cells were 5 × 106 cells/mL. Moreover, PBS buffer containing FBS (9.09%), Dglucose (25 mM), and L-glutamine (4 mM) was used for electrochemical detection of cells. 2.6 Electrochemical detection The gold electrode (3.0 mm diameter) was firstly purified with piranha solution (98% H2SO4 : 30% H2O2 = 3 : 1) for 5 min to eliminate other impurities and then rinsed with distilled water. After that, the electrode was polished discreetly with 1, 0.3, and 0.05 µm alumina slurry, respectively. The residual alumina powder was removed by sonicating the electrode unceasingly in both ethanol and distilled water for 10 min in sequence. Subsequently, the electrode was again soaked in piranha solution for 10 min, followed by electrochemical cleaning with 0.5 M H2SO4 to remove any remaining impurities. After being dried with mild stream of nitrogen, the electrode was incubated with 0.2 μM SH-TLS11a apatmer or SHZY8 aptamer in TE buffer solution at 4°C overnight. After 16 h, the electrode was rinsed with distilled water and subsequently soaked in 1 mM MCH for 1 h in dark to obtain wellaligned DNA monolayer. All electrochemical measurements were carried out on an electrochemical workstation (CHI-660C, CH Instruments,

of pSC4, the high signal-to-noise ratio of AgNPs and lowpotential electrochemical signals also offer the possibility for highly sensitive analysis of cells and even live cell analysis. 2 Materials and methods 2.1 Materials and reagents All oligonucleotides were synthesized and purified by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). paraSulfonatocalix[4]arene (pSC4) was purchased from TCI Development Co., Ltd. (Shanghai, China). NaBH4 (98%), AgNO3 (99%), sodium 4-hydroxybenzenesulfonate (HBS), Tris(2carboxyethyl)phosphine hydrochloride (TCEP), and mercaptohexanol (MCH) were obtained from Sigma-Aldrich (Shanghai, China). H2O2 was purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). DIO was purchased from Head (Beijing) Biotechnology Co., Ltd. (Beijing, China). Cyanine 3 amine was purchased from Beijing QiWei YiCheng Tech Co., Ltd. (Beijing, China). HepG2, SMMC-7721, Caco2, MDA-MB-231, L-02 cells, and MCF-7 cells were purchased from the Institute of Biochemistry and Cell Biology of Chinese Academy of Science (Shanghai, China). DMEM medium and RPMI-1640 medium were purchased from Gibco Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was purchased from Gemini Co., Ltd. (Woodland, America). DAPI and Trypsin-EDTA Solution were purchased from Jiangsu Keji Biotechnology Co., Ltd. (Nanjing, China). All solutions were prepared with Milli-Q water (18.2 MΩ cm−1) from a Milli-Q purification system (Millipore Corp, Milford, MA, America). All other chemicals were of analytical reagent grade. 2.2 Preparation of pSC4-AgNPs pSC4-AgNPs were prepared through one-pot synthesis according to the method reported previously6. In brief, 2.04 × 104 M AgNO3 aqueous solution (98 mL) was mixed with 10-2 M pSC4 aqueous solution (2 mL), followed by stirring for 20 min. Then NaBH4 (8.8 mg) was added with continually stirring for 10 min at room temperature. The mixture was subsequently centrifuged at 10000 rpm for 10 min. The precipitate obtained was re-dispersed in deionized water to give pSC4AgNPs which was kept at 4°C in dark for further use. 2.3 Characterization of pSC4-AgNPs 2.3.1 Transmission electron microscopy (TEM) pSC4-AgNPs in aqueous solution were dropped on 200 mesh Formvar/carbon-coated Cu grids and dried at room temperature. TEM images were obtained at an accelerating voltage of 200 kV on a Hitachi H-8000 transmission electron microscope (Hitachi, Tokyo, Japan). 2.3.2 Ultra-violet visible spectroscopy The UV-vis spectra were detected from 200 nm to 800 nm by using a UV-vis spectrophotometer (Shimadzu UV-2450, Kyoto, Japan). 2.3.3 Fourier transform infrared (FT-IR) spectroscopy The solid pSC4-AgNPs were obtained through centrifugation and further dried at room temperature. The FT-IR spectra were


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Shanghai, China) at room temperature. For the electrochemical experiment, the modified gold electrode was utilized as the working electrode with saturated calomel electrode (SCE) as the reference electrode and platinum wire electrode as the counter electrode. Linear sweep voltammetry was performed in the potential range from -0.2 V to 0.3 V at a scan rate of 100 mV/s with 1 M KCl solution as the electrolyte solution. 2.7 Statistical analysis All tests were conducted three time. Mean values and standard deviations were calculated. The mean separation was carried out by LSD (p ≤ 0.05) with the performance of variance analysis through the software of SPSS 13.0 program (SPSS Inc., IL, USA) 3 Results and discussions 3.1 Recognition of a novel electrochemical probe pSC4AgNPs on cell

anism for gold electrode (AuE) at the different modification stages ((a) AuE/TLS11a/HepG2; (b) AuE/TLS11a/HepG2/AgNPs; (c) AuE/TLS11a/HepG2/pSC4-AgNPs). (D) Linear sweep voltammograms for AuE/TLS11a/HepG2/pSC4-AgNPs with different concentrations of pSC4-AgNPs ((a) 0 μM, (b) 30 μM, and (c) 60 μM). Cell concentration is 5  106 cells/mL. (E) Influence of aptamer on the binding of pSC4-AgNPs with HepG2 cells and (Inset) the corresponding peak current values. Columns labeled by different letters indicate significantly statistical differention (p ≤ 0.05).

It has been confirmed that pSC4 can well bind with amino acid residues such as Arg, Lys, Glu, Ala, Phe, etc., and their methylated products 26-29. Considering the fact that these residues exist at the surface of cell, so pSC4 around AgNPs can coordinate with cell immobilized on the interface of electrode, and pSC4-AgNPs can be served as a universal and sensitive electrochemical cell probe considering the favorable redox property of AgNPs and its capability to facilitate electron transfer reactivity of some biological molecules31. In order to confirm recognition of pSC4-AgNPs on cell, three different fluorescent dyes, DAPI, Dio, and Cyanine 3 amine, have been utilized for fluorescent image. DAPI and Dio can insert into DNA and lipid bilayer of cell membrane, respectively. pSC4-AgNPs can bind with amino group of Cyanine 3 amine26-29, leading to happening of red fluorescence at the surrounding of cell. In absence of pSC4-AgNPs, only blue and green fluorescene can be detected (a1 and a2, Figure 1(A)), due to no existence of Cyanine 3 amine around cell. With the increase of pSC4-AgNPs from 30 μM to 60 μM, blue and green fluorescenes keep unchanged (from a1 to c1 for blue fluorescences and from a2 to c2 for green fluorescences, Figure 1(A)). Inversely, red fluorescence becomes brighter (from b3 to c3, Figure 1(A)). It can be explained for the incremental coordination of pSC4-AgNPs with HepG2 cell. These results well confirm that pSC4-AgNPs can bind to the surface of cell.

Figure 1 (A) Fluorescence images of HepG2 with different pSC4-AgNPs concentrations ((a) 0 μM, (b) 30 μM, and (c) 60 μM) separately using DAPI, Dio, and Cyanine 3 amine as molecular probes with blue, green, and red fluorescence, respectively. (B) Cyclic voltammograms of AgNPs and pSC4-AgNPs. (C) Linear sweep voltammograms and (Inset) schematic illustration of mech-

pSC4-AgNPs can be further served as a universal electrochemical probe for the detection of cell. Similar to that of AgNPs, pSC4-AgNPs show an electrochemical oxidation and reduction in KCl solution (Figure 1(B)), which are attributed to oxidation of Ag to AgCl and reduction of AgCl back to Ag, respectively32-35. It suggests that pSC4-AgNPs can be utilized as an electrochemical probe. To further verify the binding of pSC4-AgNPs with cells on the surface of electrode, HepG2 is immobilized with the aid of aptamer. As exhibited in Figure 1(C), an obvious current value can be observed with peak potential of 0.06 V for AuE/TLS11a/HepG2/pSC4-AgNPs (curve c) in comparison with AuE/TLS11a/HepG2 (curve a) and AuE/TLS11a/HepG2/AgNPs (curve b), which demonstrates the coordination between pSC4-AgNPs and HepG2 cell. Moreover, a low peak current value is given for AuE/TLS11a/HepG2/AgNPs, which can be attributed for nonspecific adsorption of AgNPs on the cell surface. Additionally, the peak current increases along with the increasing concentrations of pSC4-AgNPs from 30 μM to 60 μM (curves b-c, Figure 1(D)). The results are in accordance with the enhanced fluorescence intensities for cells stained by Cyanine 3 amine (from b3 to c3, Figure 1(A)). Furthermore, it can be observed

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that aptamer almost has no influence on the recognition of pSC4-AgNPs on amino acid residues on the cell outer membrane, because there is no significantly statistical difference and between AuE/TLS11a/HepG2/pSC4-AgNPs AuE/HepG2/pSC4-AgNPs (Figure 1(E)). 3.2 Mechanism investigation of electrochemical method for cell analysis The mechanism for cell assay has been illustrated in Figure 2 (A). Apatmer modified gold electrode can specifically capture cell onto its surface. As a general electrochemical probe, pSC4-AgNPs can recognize amino acid residues on the cell surface, giving the electrochemical signal. On the contrary, pSC4-AgNPs can not be immobilized onto the surface of modified electrode without cell as a linker, leading to the appearance of a negligible current value. Hence, the amount of pSC4AgNPs captured by the modified electrode surface can be decided by the number of cell, and a novel electrochemical method with utilization of pSC4-AgNPs as a universal probe, can be exploited.

Figure 2 (A) Schematic illustration for mechanism of cell electrochemical detection by using pSC4-AgNPs as a universal and sensitive electrochemical probe. (B) Linear sweep voltammograms with the inserted schematic illustration for the detection of cell at the different concentrations: (a) 0 and (b) 5  106 cells/mL. (C) Complex plane plot for the electrochemical impedance measurements and (Inset) schematic illustration of the gold electrodes with gradual modification: (a) Bare AuE, (b) AuE/TLS11a, (c) AuE/TLS11a/HepG2, and (d) AuE/TLS11a/HepG2/pSC4-AgNPs. 5 mM [Fe(CN)6]3−/4− was used as electrochemical species in 0.1 M KCl electrolyte with biasing potential of 0.224 V and frequency range from 0.01 Hz to 10 kHz.

As shown in Figure 2 (B), a slight peak current can be tested in absence of HepG2 cell (curve a) because of almost no occurrence of pSC4-AgNPs on the surface of electrode. On the contrary, an obvious electrochemical current can be observed with HepG2 cell (curve b). It can be ascribed to coordination of pSC4-AgNPs with amino acid residues on the surface of

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cells. Meanwhile, in the presence of HepG2 cell, the occurrence of the evident peak current value can be attributed for the beneficial electron transfer ability of AgNPs. As exhibited in Figure 2(C), the addition of pSC4-AgNPs evidently decreases resistance so as to enhance electron transfer for AuE/TLS11a/HepG2/pSC4-AgNPs (curve d) in the comparison with AuE/TLS11a (curve b) and AuE/TLS11a/HepG2 (curve c). 3.3 Specificity and anti-interference capability of the established electrochemical method In order to investigate the specificity of the developed method, different cells including colonic adenocarcinoma Caco-2 cell, hepatoma carcinoma SMMC-7721 cell, breast cancer MCF-7, MDA-MB-231 cells, and normal hepatocyte L-02, are used instead of HepG2 cell, and the corresponding results are given in Figure 3 (A). The obvious increase of current values can not be observed with the separate addition of the cells except for HepG2. It can be ascribed to nearly no appearance of pSC4-AgNPs on the electrode surface. On the contrary, high current value can be found with HepG2 cell, as a result of specific recognition of cell by TLS11a and its subsequent linkage effect for binding of pSC4-AgNPs on the electrode surface.

Figure 3 (A) Linear sweep voltammograms with inserted peak current values for the detection of different cells including HepG2, Caco-2, SMMC-7721, MCF-7, MDA-MB-231, and L-02. All cell concentration is 5  104 cells/mL. (B) Linear sweep voltammograms with inserted peak current values for the detection of HepG2 cells in the presence of different interfering substances including glucose (25 mM), glutamine (4 mM), and FBS (9.09%). Error bars are obtained by calculating the values from three independent measurements. Columns labeled with different alphabet indicate significantly statistical difference (p ≤ 0.05).

We further inspect the anti-interference capability of the established electrochemical method, and the results are shown in Figure 3 (B). Same as that of HepG2 cell, almost unchangeable peak currents can be obtained for the mixtures prepared through separate addition of glucose, glutamine, and FBS into HepG2 cell, and these results well confirm the good antiinterference of the method. The anti-interfering ability of the established electrochemical method can be ascribed to high binding capability between aptamer and cell36,37. It has been confirmed that TLS11a can specifically recognize HepG2 cell with Kd value in the nanomolar range38,39. 3.4 Establishment of electrochemical method for cell analysis


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Cells with different amounts have been analyzed by linear sweep voltammetry and the corresponding results are given in Figure 4 (A). When cell amounts increase from 0 to 2.5  105, the peak current values increase. It suggests the increased accumulation of pSC4-AgNPs onto electrode surface. The aptamer TLS11a located on the surface of electrode, can specifically capture HepG2 cells which can bind with pSC4-AgNPs due to happening of amino acid residues at cell surface. Hence the increasing amount of the cells can result in the correspondingly increasing binding of pSC4-AgNPs onto the cell surface, in accordance with the results obtained through confocal laser scanning microscope (Figure 4 (C)). When cell amount increases from 5  104 to 5  105, the change of peak current becomes slight, signifying the nearly total binding of aptamer with HepG2 cells followed by maximal coordination of pSC4AgNPs onto the surface of the modified electrode.

Figure 4 (A) Linear sweep voltammograms and (inset) images for the detection of HepG2 cells with different amounts: 0, 5, 25, 2.5  102, 2.5  103, 2.5  104, and 2.5  105. (B) Peak current values for different amounts of cells. Inset: The linear relationship between peak current values and the logarithmic values of cell amounts. (C) Fluorescence images of HepG2/pSC4-AgNPs with different cell amounts (10, 50, 5  102, 5  103, 5  104, and 5  105) using DAPI, Dio, and Cyanine 3 amine as molecular probes with blue, green, and red fluorescence, respectively.

We have further used the current values to quantitatively detect HepG2 cells, and the corresponding results are given in Figure 4 (B). The current values linearly raise with the increased logarithmic values of HepG2 cell amounts from 5 to 2.5  105, wider than previous reports40,41. The linear fitting equation of I = 2.31598 lgCell - 0.49081 (R2 = 0.99082) can be

obtained with detection limit of 5 cells, lower than previously reported values by using electrochemical method42-46. Moreover, based on three independent electrochemical measurement, we have assessed the detection precision by using the slopes of regression equations when cell amounts change from 5 to 2.5  105, giving 2.77% of RSD value. The result indicates that the established electrochemical method owns the acceptable reproducibility and precision. 3.5 Universal investigation of signal probe pSC4-AgNPs To study universality of signal probe pSC4-AgNPs for cell detection, SMMC-7721 has been chosen instead of HepG2 for fluorescence image analysis. Almost no red fluorescence can be observed without pSC4-AgNPs (a3, Figure 5 (A)), as a result of no occurrence of Cyanine 3 amine around the cell. In contrast, the increasing red fluorescence can be given in the presence of pSC4-AgNPs (b3 and c3, Figure 5 (A)), confirming the binding of pSC4-AgNPs with cell. These results suggest pSC4-AgNPs can be used as a general signal probe for cell analysis.

Figure 5 (A) Fluorescence images of SMMC-7721 cells with different pSC4-AgNPs concentrations ((a) 0 μM, (b) 30 μM, and (c) 60 μM) using DAPI, Dio, and Cyanine 3 amine as molecular probes with blue, green, and red fluorescence, respectively. (B) Linear sweep voltammograms for the detection of SMMC-7721 cells with different amounts: 0, 5, 50, 5  103, 5  104, 5  105, and 5  106. (C) Peak current values against different cell amounts. Inset: The linear relationship between peak current values and the logarithmic values of cell numbers. (D) The three dimension column graph for the specific binding of aptamer with cell.

In order to further investigate universality of pSC4-AgNPs as an electrochemical probe and the established electrochemical



method, aptamer ZY8 and SMMC-7721 have been utilized instead of TLS11a and HepG2 cell, respectively. As exhibited in Figure 5 (B), along with the increase of cell amounts from 0 to 5106, the peak current values increase, which suggests the incremental quantity of pSC4-AgNPs on the electrode surface. It well demonstrates universality of pSC4-AgNPs which can not only recognize amino acid residues located on the surface of HepG2 cell but also those on SMMC-7721 cell. Moreover, we have further utilized the peak current values to quantitatively test SMMC-7721 cell (Figure 5 (C)). Along with the increased cell amounts from 5103 to 5106, the peak current values are linearly increased. Meanwhile, the regression equation of I = 1.3995 lgCell + 0.3804 (R2 = 0.9522) is given. Moreover, the RSD value of the three slopes of the regression of enzyme (from 0.01 U/mL to 1.9 U/mL) is 3.07%, confirming good precision and reproducibility of the current method using pSC4-AgNPs as an electrochemical probe. Meanwhile, the electrochemical method not only exhibits excellent versatility deriving from the universality of signal probe, but also shows high selectivity. As depicted in Figure 5 (D), high peak current values can be obtained for SMMC-7721 cell using aptamer ZY8 as recognition molecule or HepG2 cell with TLS11a as captured unit. On the contrary, for SMMC7721 cell detected by using TLS11a modified gold electrode or HepG2 cell tested through ZY8 functionalized electrode, the low current values can be given. These results well demonstrate the specificity of the electrochemical method, resulting from the high selectivity of cell-SELEX aptamer for the corresponding cell. 4 Conclusions In conclusion, pSC4-AgNPs has been explored as a universal and simple nanoprobe for the electrochemical detection of cell in this work. Compared with current electrochemical probes based on lectin, pSC4-AgNPs will have a great potential for the electrochemical assay of cell counting in view of its simplicity of synthesis, stability of pSC4 moiety, and well redox capability of AgNPs, so it can well serve as a universal electrochemical probe for the development of cytosensor.

ASSOCIATED CONTENT Supporting Information. Sequences for aptamers, characterization of pSC4-AgNPs, investigation of stability for pSC4AgNPs as an universial electrochemical probe, optimization of experimental conditions, and quantitative analysis of SMMC7721 using pSC4-AgNPs as a probe. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected] (G. Li); * [email protected] (X. Zhu). Author Contributions

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The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. / #These authors contributed equally.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant Nos. 31671923, 31571763, 81772593) and Shanghai Pujiang Program (Grant No. 18PJD016).

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Design of a nanoprobe based on its binding with amino acid residues

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