Zhong
Chen
a,
Shubao
Xie
a,
Li
Shen
a,
Yu
Du
a,
Shali
He
a,
Qing
Li
a,
Zhongwei
Liang
a,
Xin
Meng
a,
Bo
Li
a,
Xiaodong
Xu
a,
Hongwei
Ma
b,
Yanyi
Huang
b and
Yuanhua
Shao
*a
aBeijing National Laboratory for Molecular Sciences, Institute of Analytical Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing, 100871, China
bCollege of Engineering, Peking University, Beijing, 100871, China. E-mail: yhshao@pku.edu.cn; Fax: +86-10-62751708; Tel: +86-10-62759394
First published on 29th July 2008
The interactions between Hela cells and silver nanoparticles (AgNPs) have been studied by scanning electrochemical microscopy (SECM) with both IrCl62−/3− and Fe(CN)63−/4− as the dual mediators. IrCl62−, which can be produced in situ and react with AgNPs, is used as the mediator between the AgNPs on the cells and the SECM tip. Another redox couple, Fe(CN)63−/4−, which has a similar hydrophilicity to IrCl62−/3−, but cannot react with AgNPs, is also employed for the contrast experiments. The cell array is cultured successfully onto a Petri dish by microcontact printing (μCP) technique, which can provide a basic platform for studying of single cells. The approach curve and line scan are the two methods of SECM employed here to study the Hela cells. The former can provide the information about the interaction between Hela cells and AgNPs whereas the later gives the cell imaging. The permeability of cell membranes and morphology are two main factors which have effects on the feedback mode signals when K3Fe(CN)6 is used as the mediator. The permeability of the cell membranes can be ignored after interaction with high concentration of AgNP solution and the height of the Hela cells is slightly decreased in this process. The kinetic rate constants (k0) between IrCl62− and Ag on the Hela cell can be evaluated using K3IrCl6 as the mediator, and they are increased with the higher concentrations of the AgNP solutions. The k0 is changed about 10 times from 0.43 ± 0.04 × 10−4 to 1.25 ± 0.07 × 10−4 and to 3.93 ± 1.9 × 10−4 cm s−1 corresponding to 0, 1 and 5 mM of AgNO3 solution. The experimental results demonstrate that the AgNPs can be adsorbed on the cell surface and detected by SECM. Thus, the amount of AgNPs adsorbed on cell membranes and the permeability or morphology changes can be investigated simultaneously using this approach. The dual mediator system and cell array fabricated by μCP technique can provide better reproducibility because they can simplify experiments, and provide a platform for further single cell detection.
It is usually difficult to study cells or bacteria quantitatively at the single-cell level by SECM when the diameter of the SECM tip is comparable with the size of an individual cell.20 To make further progress in the development of the SECM based cellular sensing systems, it will be undoubtedly required to place cells in predetermined locations with defined shapes and sizes.27 In this way, it is easier to eliminate the effect of other cells around or near the cell under studied, and obtain more reliable information. Another approach is to employ a much smaller size of SECM tip, for instance, Mirkin et al. reported recently the electrochemical behavior of mammalian cells by SECM with nanoelectrodes as the tip.28 Microfabrication combined with surface chemistry and material science has provided a new possibility for further investigating the interactions of anchorage-dependent cells with their environments.29 A versatile ensemble of methods was developed during the last decade. For example, micro-contact printing (μCP), a soft lithography technique, is commonly used to create chemical structures on surfaces for controlling cell-substrate interactions. This method uses an elastic stamp with micrometre-scale features, which made from poly(dimethylsiloxane) (PDMS), for making monolayer patterns of blocking effect to the adsorption of extracellular matrix (ECM) proteins. Chilkoti and coworkers have reported a simple and generic method to micro-patterning surfaces with an amphiphilic comb polymer presenting short oligoethylene glycol side chains, which can prevent the non-specific adsorption of ECM proteins during cell culture.30 Single cell arrays can be easily obtained using such an approach through variation of the size of pattern.
The understanding of interactions between metal nanoparticles and living systems is of fundamental and practical interest due to metal particles in the nanometre size range that exhibit unique physical and chemical properties, different from both the ion and the bulk material themselves. Although the silver ion has long been known to have strong toxicity to a wide range of micro-organisms,31 the cytotoxicity of AgNPs has only been studied recently owing to the demands of the application of nanotechnology to biosystems. The practical application of the analysis of nucleolar organizer regions (NORs) in tumor pathology began in 1986, when a simple argyrophilic technique (silver-stained nucleolar organizer regions, AgNOR) to detect proteins associated with NORs was described.32 At present, the evaluation of AgNOR quantity can be regarded as an indicator of prognosis.33
The interactions between the AgNPs and the bacteria or cancer cells could be partially identified in three ways:33–35 (1) attaching to the surface of the cell membrane and disturbing its proper function, like permeability; (2) penetrating into the cell and causing further damage by possibly interacting with sulfur- and phosphorus-containing compounds such as DNA or NORs; (3) releasing silver ions, which will have an additional contribution to the bactericidal effect of the AgNPs. In previous work, scanning electron microscopy (SEM) was a common tool, which can be employed to investigate the change of cell morphologies under the attachment of AgNPs on the surface.36 However, it is hard to obtain the further information like the permeability of the cell membranes by this way. Girault's group has shown that the silver stained proteins adsorbed on poly(vinylidene difluoride) (PVDF) membranes can be imaged by SECM and developed a readout tool for detection of proteins with high sensitivity.37,38 This method actually provides a basis for the current studying of the interaction between cells and AgNPs by SECM.
In this work, we have tried to study the interaction between AgNPs and Hela cells by SECM with both K3Fe(CN)6 and K3IrCl6 as mediators in the same solution. A cell-resistant polymer, an amphiphilic comb polymer, was patterned onto a Petri dish using the μCP method, and then the Hela cell array could be obtained when culturing the Hela cells in proper experimental conditions. IrCl62−/3−, which can react with AgNPs, serves as the mediator to study the effect of the amount of AgNPs adsorbed on the cell membranes on the kinetic rate constants, whereas Fe(CN)63−/4−, which cannot react with AgNPs, has been used as the mediator to study the varying of the permeability or morphology of cell membranes when adding AgNP solution to the cell array. We have demonstrated that both mediators can be used simultaneously and have no interaction with each other during the detection. This will benefit the investigation because one does not need to change the solution in the process, which will make the results more compatible and reproducible.
Comb polymers were synthesized according to the previously reported procedures, consisting of methyl methacrylate (MMA), poly(ethylene glycol methyl ether methacrylate) (referred herein as hydroxyl-poly(oxyethylene methacrylate) (HPOEM)), and poly(oxyethylene methacrylate) (POEM) via free-radical polymerization with a range of compositions.39
The Hela cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units mL−1penicillin, 100 μg mL−1streptomycin, at 37 °C in 5% CO2. The cells were incubated for 24 h, and after that we could obtain the cell array because the micropatterned comb polymer is cell-resistant.
A Nikon TE-2000 inverted microscope (Nikon Co., Japan) was used to observe the coverage and growth status of the Hela cells. SECM and cyclic voltammetric (CV) measurements were performed with a CHI 910B workstation (CH Instrument Co., Austin, TX) which mounted on the stage of the inverted microscope. The three-electrode system consisted of a 12.5 μm radius Pt working electrode, an Ag/AgCl electrode as the reference electrode, and a Pt wire as the auxiliary electrode.
Before the measurement, the physiological saline solution was replaced by 150 mM NaNO3, and then added the newly synthesized AgNP solution. The interaction of AgNPs and cells was limited to 10 min, and then the cells were washed using 150 mM NaNO3 solution twice carefully to repulse the excessive silver nanoparticles in the solution, and detected by SECM in 150 mM NaNO3 solution with 1 mM K3IrCl6 and 1 mM K3Fe(CN)6 as the mediators.
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Fig. 1 The cyclic voltammograms obtained by the Pt microelectrode in the 150 mM NaNO3 solution as the blank (dash line) or including 1 mM K3IrCl6 (b) and 1 mM K3Fe(CN)6 (a) (solid line) as the mediators. The scan rate was 10 mV s−1, and Ag/AgCl served as the reference electrode. The arrows indicate the tip voltage for each mediator during the SECM experiments. |
Using the same solution containing K3Fe(CN)6 and K3IrCl6, two series of data for SECM lateral scans over the same position with each of them as mediator have been obtained (see the ESI‡). The signals based on K3IrCl6 are about five times larger than those based on K3Fe(CN)6. This is because the Fe(CN)64− (generated by the tip reaction) cannot react with the AgNPs, which only act as an insulating substrate, while IrCl62−, which has a more positive standard potential, can oxidize the AgNPs into Ag+ and cause the enhancement of the SECM feedback signals:
Tip: IrCl63− − e → IrCl62− | (1) |
Substrate: Ag(s) + IrCl62− → Ag+(l) + IrCl63− | (2) |
Girault et al. have demonstrated that SECM can be employed as a sensitive and quantitative readout method for the detection of proteins adsorbed on PDVF membranes after stained by AgNPs, based on similar reactions.37
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Fig. 2 (a) The cell array cultured onto the Petri dish, pictured by the inverted microscopy. The diameter of cell island is about 20 μm and the distance between two nearest islands is about 30 μm. (b) The TEM image of AgNPs, and the diameters of them were between 30–50 nm. |
After finding a Hela cell and positioning the SECM tip above it, with the help of the optical microscope, the current–distance (approach) curves can be obtained, which are shown in Fig. 3, and the relevant kinetic rate constants can be obtained by fitting the experimental curves to the theoretical ones (see Table 1). Fig. 3A shows the approach curve for the case when K3IrCl6 is used as the mediator. Positive feedback is observed when approaching the AgNP clusters adsorbed on PVDF membrane, which is consistent with the line scan results in the previous section (see ESI‡). The effective heterogeneous kinetic rate constants (k0) of reactions between K3IrCl6 and different substrates are evaluated and listed in Table 1. The rate constant for the cell without AgNPs is similar to that of the PVDF as the substrate, and negative feedback is obtained. Fig. 3B shows the case when K3Fe(CN)6 is used as the mediator. All the feedback curves are almost overlapping each other, which indicates that the reaction between the mediator and cell or AgNPs is very slow.
Substrate | Rate constant (k0) × 10−4/cm s−1 | |
---|---|---|
Mediator (1 mM) | ||
K3IrCl6 | K3Fe(CN)6 | |
PVDF | 0.45 ± 0.07 | 0.45 ± 0.05 |
PVDF + AgNPs | 50.0 ± 3.0 | 0.85 ± 0.07 |
Cell | 0.43 ± 0.04 | 0.45 ± 0.07 |
Cell + AgNPs (low concentration) | 1.25 ± 0.07 | — |
Cell + AgNPs (high concentration) | 3.93 ± 1.9 | 0.55 ± 0.06 |
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Fig. 3 Normalized approach curves. Five curves are shown in (A) using K3IrCl6 as the redox mediator, from top to the bottom are: (1) PVDF + AgNPs, (2) Hela cell + AgNPs (high concentration), (3) Hela cell + AgNPs (low concentration), (4) Hela cell, (5) PVDF. Four curves are shown in (B) using K3Fe(CN)6 as the redox mediator, from top to the bottom are (1) PVDF + AgNPs, (2) Hela cell + AgNPs (large concentration), (3) Hela cell, (4) PVDF. The tip potential was held at 0.65 V for K3IrCl6 and 0.05 V for K3Fe(CN)6 (vs.Ag/AgCl). The approach rates are 10 μm s−1. |
According to our results and previous reports, there are at least three factors influencing the feedback signals in SECM experiments using Fe(CN)64− and IrCl62− as redox mediators for the same cell array: (1) the reaction between the mediators and AgNPs adsorbed on the cell membrane, (2) the permeability of the cell membrane for the mediators, (3) the reaction between the mediators and intracellular redox species (see Fig. 4). The permeability is depended mainly on the hydrophilicity of mediator and the perforation in cell membrane after interaction with AgNPs. Since the hydrophilicity is similar for Fe(CN)64− and IrCl62−, and the interaction process between cell and AgNPs is identical in our experiments, so the permeability for the two mediators should be similar. From Fig. 3B and Table 1, the rate constants for the Hela cell before and after the interaction with AgNPs have no significant difference when K3Fe(CN)6 is used as the mediator. Therefore, the change of permeability of the cell membrane can be ignored in this process and there is no perforation in these concentrations of AgNPs. It has been reported that after treatment with AgNPs, perforation appears in the bacteria cell membranes,36 which will change the cell permeability, but the constitution and configuration of the membranes in bacteria and cancer cells are quite different from each other, so that the perforation may not appear in the current studies. That is, such hydrophilic mediators will still hardly be able to pass through the cell membrane and react with the intracellular redox species. Hence the signal differences between the two mediators are mainly attributed to the interactions between the SECM tip and AgNPs deposited on the Hela cells or species on the cells’ surfaces, and processes 2 and 3 shown in Fig. 4 can be ignored in the present work. After interaction with AgNPs, the Hela cell shows a larger rate constant using K3IrCl6 as mediator, but still smaller than AgNPs clusters adsorbed on PVDF as the substrate. This is probably because the density of AgNPs adsorbed on the cell membrane is smaller than that adsorbed on the PVDF. The cell after interaction with a higher concentration of AgNP solution shows a higher rate constant than the one with a smaller concentration, which means the amount of AgNP adsorbed on the cell membranes is increased with the higher concentration of AgNP solution. These experimental results demonstrate that we can investigate simultaneously the permeability of cell membranes using K3Fe(CN)6 as the mediator and the effect of the amount of AgNPs adsorbed on the Hela cell membranes using K3IrCl6 as the mediator.
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Fig. 4 The operating principle of SECM to detect the interaction between AgNPs and Hela cells. When the tip approaches the cell, the IrCl63− is regenerated in three ways: (1) IrCl62− reduced by the AgNPs adsorbed on the cell membranes; (2) IrCl63−, which is plentiful in the bulk solution, may penetrate into cells and diffuse away from the tip; (3) IrCl62− penetrates into the cells and is reduced by intracellular redox species or the AgNPs in the NORs. |
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Fig. 5 Feedback currents for the lateral scanning of the Hela cell arrays using (a) K3IrCl6 and (b) K3Fe(CN)6 in 150 mM NaNO3. There were no silver nanoparticles (A) or different concentrations (B: low, C: 10 times greater than that of B) of AgNPs interacting with the Hela cells. The scan rate was 30 μm s−1. |
Although Fe(CN)64− cannot oxidize the silver as we have demonstrated in the previous sections, the negative feedback currents using K3Fe(CN)6 as a mediator are a little decreased with the increasing of the concentration of AgNP (the ratios of peak current and background current are 0.78 ± 0.07 for Fig. 5B and 0.90 ± 0.04 for Fig. 5C, see Table 2). The permeability of cell membranes or the heights of the cell are two factors which will influence the feedback signals during a constant height scan above the cell arrays. A similar method was used by Baur et al.42,43 to study the morphology of PC12 cells. In the previous section, we demonstrated that the permeability has little change after interaction with AgNPs, so this difference is probably due to the height of the Hela cells being decreased at higher concentrations of AgNPs.
Current ratio | Mediators (1 mM) | |
---|---|---|
K3IrCl6 | K3Fe(CN)6 | |
A | 0.81 ± 0.06 | 0.85 ± 0.05 |
B | 0.87 ± 0.06 | 0.78 ± 0.07 |
C | 1.0 ± 0.02 | 0.90 ± 0.04 |
SECM can also be employed for electrochemical mapping of redox activity of individual living cells. Constant height and constant distance are the two commonly used modes for the SECM imaging. Schuhmann and coworkers have introduced a shear-force based constant-distance approach to control the SECM tip precisely,44,45 but the constant height mode is technically simple and easy to be used. Fig. 6A and C show images of the cell array obtained by the SECM constant height mode. The redox activity maps obtained by SECM are compared with the optical micrographs captured for the same fields (Fig. 6B and D). Pure negative feedback imaging of the Hela cell array without adding AgNPs can be obtained in the presence of K3IrCl6. On the contrary, positive feedback imaging can be also obtained after the interaction with higher concentration of AgNPs and cells. These phenomena are consistent with the lateral scan of the cell array. We noticed that the sizes of cells imaged by SECM were slightly larger than those obtained by the optical method. This is probably because the electrochemical signals of the mediators detected by the tip were in a diffusion field, so that the results were larger than the real size. After the interaction with AgNPs with high density, the activity of cells decreased and some of them were easy to glide from “cell islands” indicating by the red circles in Fig. 6C. This may also be the damage caused by the addition of AgNPs. It is hard to obtain higher spatial resolution with the present micro-sized SECM tip. A study of using a nanometre-sized SECM tip to analyse the local electrochemical reactivities is being undertaken in our laboratory.
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Fig. 6 Optical micrographs (B and D) and SECM images (A and C, 200 μm × 100 μm) of island patterns of Hela cells formed onto the Petri dish. The red circles in C indicate the lateral excursion of the cells after the interaction with AgNPs, which stands for the decreasing of the activities. |
Footnotes |
† This paper is part of an Analyst themed issue highlighting Chinese science, with guest editor Mengsu (Michael) Yang. |
‡ Electronic supplementary information (ESI) available: Fig. S1, experiment for the lateral scanning over the silver nanoparticles spots with different mediators. See DOI: 10.1039/b807057a |
This journal is © The Royal Society of Chemistry 2008 |