Ultra-Fast Mapping of Subcellular Domain via Nanopipette Based

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Ultra-Fast Mapping of Subcellular Domain via Nanopipette Based Electroosmotically Modulated Delivery into a Single Living Cell Ruo-Can Qian, Jian Lv, and Yi-Tao Long Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04159 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Analytical Chemistry

Ruo-Can Qian#, Jian Lv#, and Yi-Tao Long* Key Laboratory for Advanced Materials, School of Chemistry and Molecular Engineering, East China University of Science & Technology, 200237 Shanghai, P. R. China KEYWORDS: Electroosmosis, Nanopipette, Single cell imaging, Subcellular detection ABSTRACT: Recently, a variety of strategies have been developed for single-cell detection. However, the precise probing of the given area at single cell level is still a challenge. Here, we put forward a rapid and targeted imaging approach for the mapping of subcelluar domain, which realizes the precise injection of multi-fluorescence into a single living cell via an ultra-small quartz capillary nanopipette (~100 nm), and can successfully transport different fluorescent probe molecules to the pointing subcellullar area around the tip in the cytoplasm within 20 seconds. This method is also applied for monitoring the loss of intracellular mitochondrial membrane potential under the treatment of metformin in the single MCF-7 breast cancer cell. The major driven force in the nanopipette, electroosmotic flow, is evaluated by theory calculation method and finite element simulations, and the solution indicates a confined solute distribution profile around the tip within the working range. Overall, the nanopipette approach realizes the precise and simultaneous delivery of multiple probe molecules into the single living cell through the electroosmotically modulated, non-destructive, and one-step injection, which is especially powerful and convenient for the multi-channel single cell imaging and monitoring, indicating favorable potential for understanding, monitoring, and controlling the biological processes from the single cell to subcellular organelles.

Currently, single cell research is of great interest in analyzing important biochemical processes, cellular signal pathways, medicine delivery and disease diagnosis.1-5 Significant advances have been achieved in the development of new strategies for single-cell detection, particularly the techniques for single cell imaging.6-8 Among these various methods, singlecell fluorescence imaging is especially favorable due to its preferable imaging effect and convenient operation.9-11 To go further, attempts to develop tools for the imaging of subcellular areas have also been made.12,13 However, in most of the methods, the imaging probes enter into the cells through the endocytosis process,14,15 which usually takes a long incubation time and may cause interference to the probes. This inspired us to design a precise transportation platform for the effective and rapid delivery of fluorescent probes targeting to designated subcellular domain in the single living cell.16-18 Nanoscale devices have showed great potential for cellular research because of their high spatial and temporal resolution. Among them, the glass nanopipettes have been considered as a promising tool due to their application potential in cell research, which is convinient to fabricate and simple to use.19-21 Nanopipettes are also characterized by the nanoscale size of the opening pore of the tip, which provides favorable ability to perform localized measurements in ultrasmall areas, especially in individual cell samples.22-27 Therefore, integration of nanopipettes with maneuvering control devices may allow ultrafast and real-time sensing and imaging of subcellular structures and organelles.28-30 In this work, we use a quartz capillary pulled ultra-small nanopipette (tip diameter: ~100 nm) to realize the one-step injection of multiple fluorescent probe molecules to the pointing subcellullar area in the single living

cell, and study its potential application in observing and regulating the subcellular biological processes. As shown in Figure 1a, the nanopipette was filled with different fluorescent probe molecules and then inserted through the membrane into a living breast cancer cell (MCF-7). With the tip diameter less than 100 nm, the nanopipette could be easily inserted into the living cell with little damage. An Ag/AgCl electrode was inserted into the nanopipette as the working electrode, and another Ag/AgCl reference electrode was immersed in the cell culture medium. A positive DC voltage was added to the working electrode to induce the electroosmotic flow to drive the imaging probes into the cytoplasm under the force of the electrical field. By changing the voltage applied on the working electrode, the magnitude of the electric field inside the nanopipette could be adjusted to generate a certain outflow. By changing the injecting time, the injected volume could be regulated. Therefore, the nanopipettebased controllable injection was achieved. The whole process was performed under the inversion fluorescence microscope (Figure S1a). The nanopipettes were prepared by stretching a quartz capillary (Figure S1b). Under the microscope, the coneshaped tip of the nanopipette could be seen clearly (Figure 1b, c). The tip hole was extremely small with a diameter less than 100 nm in the scanning electron microscope (SEM) image (Figure 1d). Thus, a nanopipette suitable for subcellular research was obtained successfully. Before cellular injection, the nanopipette was filled with fluorescent solutions Cy5 and fluorescein isothiocyanate (FITC) at the tip, respectively (Figure 1e, f) and then inserted into a living cell carefully. As shown in Figure 1g, the tip of the nanopipette passed through the cell membrane. Thus, a robust subcellular delivery platform has been established driven by pure electrical force.

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Figure 1. Schematic illustration of the electoosmotic delivery of multi-fluorescent molecules into a single living cell. (a) Electroosmotically modulated imaging in a single MCF-7 cell via the nanopipette-based controllable injection (right: the solute distribution profile around the tip with finite element mesh; color bar from up to down: c/c0 from 1 to 0). (b) & (c) Microscopic images of the nanopipette (BF: bright field; DF: dark field). (d) Image showing the pore on the tip of the nanopipette. (e) Fluorescent image of nanopipette filled with Cy5 at the tip. (f) Fluorescent image of nanopipette filled with FITC at the tip. (g) Bright field of the nanopipette inserted into a cell. Scale bars in (b) & (c): 1 μm; scale bar in (d): 100 nm; scale bars in (e), (f) & (g): 10 μm.

Chemicals and Materials. All reagents are analytical grade, and solvents were purified by standard procedures. All solutions were prepared by Milli-Q ultrapure water with resistance of 18.2 MΩ cm (EMD Millipore, TONDINO, Shanghai). Cy5, FITC, metformin and MCF-7 cells were obtained from MoXi Biotech. Co. Ltd. (Shanghai, China). JC-10 (5,6-dichloro1,1’,3,3’-tetraethyl-imidacarbocyanine iodide) was obtained from BBI Life Sciences Co. Ltd. (Shanghai, China). Sodium dihydrogen phosphate and disodium hydrogen phosphate were purchased from Aladdin, which was used for preparing the buffer solution. Cy5, FITC and JC-10, as fluorescence molecules, were dissolved by buffer solution (10 mM NaCl, 2.0 mM NaH2PO4, 8.0 mmol Na2HPO4, pH=7.4). Quartz capillaries (O.D.: 1.0 mm; I.D.: 0.70 mm; 7.5 cm length) were purchased from Sutter Instrument. The quartz capillaries were cleaned first by ethanol, and then washed successively in acetone and pure water by 30 min sonication. The obtained nanopipettes were dried with nitrogen gas. Apparatus. The scanning electron microscope (SEM) observations of the nanopipettes were performed with a field-emission scanning electron microscope (Ultra 55, Carl Zeiss Ltd.,

Germany). We used an inverted microscope (eclipse Ti-U, Nikon, Japan) to observe the fluorescence images using a mercury lamp as the light source (100 W Epi illuminator). Nanopipettes were made by a P-2000 laser puller (Sutter Instrument, Novato, CA) using a single pull cycle (parameters: heat=660; fil=3; vel=30; del=170; pul=205). The nanopipette is fixed under the microscope by a holder (Axon Instruments, Union City, CA) connected to the Axopatch 700B low-noise amplifier (Molecular Devices, Sunnyvale, CA) for electrical control and measurement. A MP-285 micromanipulator (Sutter Instrument, Novato, CA) was used for the coarse control of the nanopipette positioning in the X, Y, and Z directions, and a Nanocube piezo actuator (Physik Instrument, Irvine, CA) was used for the fine control. A PCIe-7851R Field Programmable Gate Array (FPGA) (National Instruments) was used for hardware control of the system. Preparation of Nanopipettes. The quartz capillaries were cleaned and then pulled using a Sutter P-2000 laser-based pipette puller using optimized parameters. Typically, quartz nanopipettes presented an aperture diameter of ~100 nm. Afterwards, the nanopipettes were filled with dye solution using


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Analytical Chemistry a microloader (Eppendorf). First, about 10 µl of the dye solution (2 mM) was drawn into the microloader, and then the microloader was inserted carefully into the back end of the nanopipette. The nanopipette was filled with solution by slowly pushing the solution while continuously drawing the microloader back. The as-prepared nanopipettes were centrifuged by 5 min, 8000 rpm to make sure that the dye solution reached the end of the tip (Centrifuge 5430, Eppendorf). Preparation of electrodes. Silver wire was cut up into 4 cm segments and incubated in FeCl3 solution overnight for the preparation of Ag/AgCl electrodes. Cell Culture. MCF-7 breast cancer cells were cultured by RPMI-1640 (GIBCO), with fetal bovine serum (10%, Sigma), streptomycin (100 μg mL-1), and penicillin (100 μg mL-1). The culture dished were placed in a humid atmosphere at 37 °C, with 5% CO2. Cell numbers were obtained using a cell counter (Petroff-Hausser). The imaging methods. One Ag/AgCl electrode was inserted into the nanopipette as the working electrode while another Ag/AgCl electrode immersed in the culture medium was used as the reference electrode. After applying a positive voltage for a certain time, the nanopipette was pulled out. The whole process was observed under a microscope. Finite Element Simulations. Numerical simulations of the electroosmotic flow inside the nanopipette were performed by COMSOL Multiphysics 5.2a (COMSOL AB, Stockholm, Sweden) to show the detailed profiles of the flow velocity and solvent concentration in this study. A 2D axisymmetric model was used for the cone-shaped nanopipette based on electrostatics module and creeping flow module. Navier-Stokes equations were used to describe the laminar flow of the solution. After the axial velocity profile of the solution flow was calculated, the velocity outside the tip of the nanopipette was simulated for the following simulation of the concentration distribution in the cell. The cell mimicking was also established on a 2D axisymmetric model using creeping flow module and transport of diluted species model. The mesh of the simulations was set as extremely fine.

Figure 2. Microscopic images of MCF-7 cells treated under different voltages for 20 s. From left to right: 300 mV, 400 mV, 600 mV and 800 mV. BF: bright field; Red: red channel fluorescent image; Dis R: Color-coded fluorescence intensity of red channel; Green = green channel fluorescent image; Dis G = Color-coded fluorescence intensity of green channel. Scale bar: 10 μm. Color bar left to right: intensity form 0 to 180.

Figure 3. Microscopic images of MCF-7 cells injected with two dyes. (a) MCF-7 cells treated under different voltages for 20 s. From up to down: 300 mV, 400 mV, 600 mV and 800 mV. BF: bright field; Red: red channel fluorescent image; Green: green channel fluorescent image; Merge: mixed image of red and green channels; Dis R: Color-coded fluorescence intensity of red channel; Dis G: Color-coded fluorescence intensity of green channel. Scale bar: 10 μm. Color bar up to down: intensity form 0 to 180. (b) & (c) Channel intensities along the mid-vertical line in the corresponding fluorescence images in (a).



Intracellular Delivery in Single MCF-7 Cells by Nanopipette Injection. After successful preparation of the nanopipette, cell imaging experiments were performed. Before inserting the cell, the nanopipette was immobilized on the microoperation holder for the precise movement control using the electric driving system. Then, a culture dish holding MCF-7 cells were placed on the microscopic platform. Using the fine tuning mode, the tip of the nanopipette was carefully moved closing to the selected cell until the tip was inserted into through the plasma membrane. First, nanopipettes filled with Cy5 or FITC solution were tested respectively. Before applying any voltage, no fluorescence could be observed in the cell area, which proved that the dye molecules would not leak from the tip if no voltage was applied (Figure S2). A positive voltage of 300 mV was added to the nanopipette filled with FITC. The fluorescence images were taken after different times. As shown in Figure S3, the fluorescence signal increased with the time, which obtained a strong fluorescence at 20 seconds. The dye’s safety was checked by injecting the cell with the mixed dye solution of Cy5 and FITC (2.5 mM each), and good cell morphology could be observed 30 min after the injection (Figure S4). Afterwards, different voltages were used for the cell injection of 20 s. For better illustration of the fluorescence distribution in the cells, we used Matlab to extract the fluorescence intensity of green and red channels for each pixel within the cell area (Figure S5). As shown in Figure 2, the intracellular fluorescence increased with growing potential, and the intensity profile showed a concentrated luminescence around the tip of the nanopipette. Further up, a nanopipette filled with the mixed solution of Cy5 and FITC (2.0 mM each) was used to perform the cell injection experiments. As shown in Figure 3a, fluorescence signals of red channel (Cy5) and green channel (FITC) could be seen clearly

under positive potentials, and the intensity increased with the applied potential. Corresponding fluorescence intensity along the mid-vertical line of the fluorescence images showed that the signal was highest at the center area of the cell (Figure 3b, c), which indicated that the dye molecules concentrated in the peripheral zone around the tip of the nanopipette. Considering the safety of cells, it is better to use lower potential for the injection, in order to minimize the cell damage, while at the same time to ensure the fluorescence signal is strong enough for detection. In this case, 300 mV was chosen as the working potential for the following cell injection experiments. The above results demonstrated the successful delivery of the dye molecules by the nanopipette injection. The whole process was rapid and facile. Imaging of Subcellular Organelles. Mitochondria are important subcellular organelles providing necessary energy for various cellular processes, and the health state of mitochondria is especially important for the survival of cells.31-33 Therefore, we chose mitochondria to test the feasibility of the injection method. JC-10, a dye showing red fluorescence in activated mitochondria and green fluorescence in inactivated mitochondria, was used as the imaging probe, and metformin, a biguanide agent with anti-cancer effect was used for the treatment of MCF-7 cells, while the activity of intracellular mitochondria would be changed. 34-37 Before the drug treatment, MCF-7 control cells were incubated with JC-10 directly (without using nanopipette) for different times. As shown in Figure 4a, there was no fluorescence within 20 seconds, and weak fluorescence signal emerged after 20 min. Therefore, the traditional incubation method needed a much longer time for the internalization of dye molecules compared with the nanopipette injection (less than 20 s). After that, cells were imaged by the JC-10-filled nanopipette (Figure 4b, c).

Figure 4. Imaging of mitochondria in MCF-7 cells. (a) JC-10 (10 μg mL-1) incubated MCF-7 control cells without nanopipette for 20 s, 20 min and 30 min from up to down. The nanopipette inserted (b) control and (c) metformin (Met, 10 mg mL-1, 3 h) -treated MCF-7 cells injected under 300 mV for 20 seconds. From left to right: Schematic illustration; BF: bright field; Red: red channel fluorescent image; Green: green channel fluorescent image; Merge: mixed image of red and green channels; Dis R: Color-coded fluorescence intensity of red channel; Dis G: Color-coded fluorescence intensity of green channel. Scale bar: 10 μm. Color bar up to down: intensity form 0 to 50. Histogram showing the average (d) red channel and (e) green channel fluorescence intensities within the cell area in (a), (b) and (c).


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Analytical Chemistry

After applying a positive voltage of 300 mV for 20 s, the control cell showed strong red fluorescence with negligible green fluorescence; while the metformin treated cell showed strong green fluorescence. From the fluorescence distribution in the cell area, we could clearly see that most of the mitochondria in the control cell displayed red fluorescence, indicating good activity of the mitochondria. In contrast, the metformin treated cell exhibited much stronger green fluorescence than red fluorescence, indicating the inhibited activity of the mitochondria and the depletion of mitochondrial membrane potential. In addition, the cell shape of the drug treated cell was also impaired, with part of the injected JC-10 molecules leaking out of the cell membrane. The average fluorescence intensity within the cell area was shown in Figure 4d, e. The above results confirmed the metformin induced mitochondria inactivation and cell growth impediment. Significantly, we have further expanded the application of our nanopipette-based method for investigating the changes of subcellular organelles under drug treatment and its related cell pathways at the single cell level. Evaluation of the Electroosmotic flow in the nanopipette. A cone-shaped nanopipette model was shown in Figure 5a.

When a voltage, ΔΨ, was added on the electrode over the nanopipette, the potential variation inside the conical nanopipette could be written as following:38-41

d   z    Id  ( z )  

I

  R0  z tan  

2

dz

(1)

Where  is the resistance of the nanopipette, z is the distance from the tip of the nanopipette,  is the conductivity of the solution inside the nanopipette, R0 is the radius of the nanopipette tip (R0 = 50 nm), and  is the half-cone angle of the inside wall. Since R0 is much smaller than the length of the nanopipette, the resistance of the nanopipette is then:



1  R0 tan 

(2)

The magnitude of the electric field inside the nanopipette, E, has the following equation:

E( z) 

R0 tan   ( R0  z tan  )2

(3)

Figure 5. Schematic illustration of the electroosimotic flow in the nanopipette. (a) Geometry of the nanopiette (Lz: the distance between the tip and the top of the nanopipette;  : the half-cone angle of the inside wall;  : the zeta potential of the wall; a positive electric field was added from the top to the tip). (b) Averaged electroosmotic velocity along the axis at different potentials. (c) Electric field distribution along the axis at different potentials. (d) The simulated axial velocity of the flow inside the nanopipette when 300 mV potential was applied at the top. (e) The simulated velocity of the flow outside the tip of the nanopipette at 300 mV (vertical section). (f) The theoretical concentration distribution outside the tip of the nanopipette at 300 mV after (i) R0. Diffuse electric double layer model was used to describe the inside wall of the nanopipette. Set the thickness of the adsorptive layer to be  at the interface between the nanopipette and the solution, then the electronic force per unit area at the interface is then: F E (4) Where  is the charge-density at the interface. The reversed shear resistance of the liquid at the interface is given by:

f 

dv v  dx 

(5)

Where  is the viscosity of the bulk solution inside the nanopipette (  = 1 mPa s), v is the velocity driven by electroosmosis and x is the vertical distance. After velocity of the solution became a constant, F = f, therefore:

v

E

(6)



Since the charge distribution at the interface could be seen analogous to the situation of a plate capacitor, the capacity could be written by:

C Where S is the area,



Q





S r  0

is the zeta potential of the nanopipette

r

is the relative permit-

tivity of the solution (  r = 80 for water), and

0

is the permit-

tivity of vacuum, and k is electrostatic constant (k = 9.0 × 109 N m2/C2). From equation (7),  is given by:



Q  r  0  S 

(8)

From equation (6) and equation (8), the electroosmotic velocity is obtained as following:

v

E r  0

(9)



In addition, the electroosmotic mobility could be determined by: (10) eo   r  0∕ The total flow (QΔΨ) leaving the pipette due to the electroosmosis is then:

Q  eo R0 tan   

(11)

QΔΨ at different ΔΨ and electroosmotic velocity at the tip (v0) were listed in Table 1 as following: Table 1. Electroosmotic flow at different applied potentials. ΔΨ (mV)

The flow of molecules in the nanopipette (seen as steady flow), J, could be written as:

J   Dc  (v p  veo  vep )c

300

400

600

800

QΔΨ (fL/s)

11.0

14.7

22.0

29.3

v0 (μm/ms)

1.40

1.87

2.80

3.73

(12)

Where D is the diffusivity, c is the concentration, vp is the velocity caused by pressure, veo is the velocity driven by electroosmosis, and vep is the velocity driven by electrophoresis. In our system, no pressure was applied, thus vp is ignored. The dyes are dissolved by buffer solution (pH=7.4). For FITC, the charge is zero, thus the vep of FITC could be ignored. For Cy5, the charge is one. According to the following equation for electrophoretic mobility: (13)  ep  qD / kBT Where q is the charge, kB is the Boltzmann factor, and T is the temperature. Assuming the diffusivity of Cy5 is 1×10-10 m2/s, μep is estimated to be ~0.4×10-8 m2/V s. Sinceμeo is 1.4×10-8 m2/V s according to equation (10), the effect of electroosmosis for Cy5 is larger than that of electrophoresis here. In this case, the total velocity of Cy5 is larger than FITC, while the concentration profile is the same. Using FITC as the example, only veo is considered as the main velocity field. The concentration distribution over a spherical shell (radius R) is then:

(7)



wall (  = -20 mV for SiO2 surface),

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c  c0 (1  e

Q 4 DR

)

(14)

Where c is the concentration profile out the tip of the pipette, c0 is the concentration of molecules in the nanopipette, and Q is the flow leaving the pipette. The averaged electroosmotic velocity along the axis at different potentials were exhibited in Figure 5b, and the corresponding electric field distribution along the axis were shown in Figure 5c, indicating dramatically increased velocity and electric field intensity at the tip of the nanopipette. So far, analytic solutions of the electroosmotic flow inside the nanopipette have been obtained. Furthermore, numerical simulations based on COMSOL Multiphysics were performed to show the detailed profiles of flow velocity and solvent concentration (Figure S6). First, a 2D axisymmetric model for the cone-shaped nanopipette was established using electrostatics module and creeping flow module based on Navier-Stokes equations. Boundary conditions: Same pressure at both ends, potential drop 300 mV (the left more positive), surface charge 1 mC/m2. The axial velocity profile of the solution flow inside the nanopipette was shown in Figure 5d, where the velocity increased from the left end to the tip, and dropped from the center to the inner wall for the radical distribution. The velocity of the flow outside the tip of the nanopipette was shown in Figure 5e. Afterwards, the concentration profile of the solvent outside the tip was firstly calculated by equation (13). As shown in Figure 5f, the concentration decreased quickly along the radical direction, and the steady state was reached less than 0.5 ms. The diffused concentration increased over time (Figure 5f, i-iii), with most of the solvents concentrated around the tip with the 20 s injection. Next, simulations based on a 2D axisymmetric model mimicking the tip inserting into a living cell was established using creeping flow module and transport of diluted species model. Boundary conditions: velocity 1.40 μm /ms at the tip, zero velocity at the cell boundary. The concentration profile outside the tip was shown in Figure 5g, tending to be high in the middle area around the tip, which was consistent with the experiment results of cell injection. The confined solute distribu-

Thus, the injected volume was about 0.22 pL under 300 mV for 20 s.


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Analytical Chemistry tion profile at the tip indicated the advantage of the electroosmotic delivery for the precise probing of the well-defined subcellular domain.

In conclusion, a rapid and targeted imaging method based on the glass nanopipette is developed to realize the precise mapping in the single living cell. This imaging platform uses electroosmotic flow to realize the simultaneous delivery of multiple fluorescent probe molecules in less than 20 seconds. Based on MCF-7 cells as the model, the practicality of this method has been proved by monitoring the activity of intracellular mitochondria under the drug treatment in single cells. The transport situation of the solute from the nanopipette into a cell is evaluated by finite element simulations, which shows a confined concentration profile at the working conditions. Thus, this work provides an ultra-fast, robust and non-destructive approach for single cell detection through the electroosmotically modulated one-step injection, which will potentially be further applied for the investigation of intracellular biomolecules and pathways.

The experimental details for the electroosmotic injection, image data extraction, and setup for numerical simulations. This material is available from the ACS Publications website.

*Phone/fax: 86-21-64252339 *E-mail: [email protected]

R.-C.Q. and J.L. contributed equally to this work.

Yi-Tao Long: 0000-0003-2571-7457 Ruo-Can Qian: 0000-0002-1039-9866

The authors declare no competing financial interest.

This research was supported by the National Natural Science Foundation of China (21421004, 21605048, 21327807), Innovation Program of Shanghai Municipal Education Commission (2017-01-0700-02-E00023), the Program of Introducing Talents of Discipline to Universities (B16017), Chenguang Program (16CG35) and the Fundamental Research Funds for the Central Universities (222201717003, 222201718001).

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Analytical Chemistry for TOC only


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Ultra-Fast Mapping of Subcellular Domain via Nanopipette Based

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