Single Cell Transfection with Single Molecule Resolution Using a

Letter pubs.acs.org/NanoLett

Single Cell Transfection with Single Molecule Resolution Using a Synthetic Nanopore Volker Kurz,† Tetsuya Tanaka,‡ and Gregory Timp*,§ †

Department of Electrical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States Departments of Chemical and Biomolecular Engineering and Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States § Departments of Electrical Engineering and Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States ‡

S Supporting Information *

ABSTRACT: We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient but also transfects with single molecule resolution at low voltage (1 V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precisionno other method offers such capabilities.

KEYWORDS: Nanopore, electroporation, transfection, single molecule, single cell

S

nitride membranes 325 pA are shown in blue. The inset shows all events with ΔI > 100 pA on a log time scale. Alongside the scatter plot are histograms of the dwell time and the blockade current. Events are automatically tallied using a threshold current of 275 pA and a rearm current of 5 pA.

rearm of 5 pA. These values for ΔI were consistent with measurements of a single dsDNA molecule translocating through a pore33 for similar electric fields, and the dwell time was consistent with YOYO-dsDNA translocating through a pore this size.15 Blockades with ΔI >175 pA have a log-normal distributed dwell time characterized by median τ = 1.07 ms and an interarrival time of Δt = 249 ± 35 ms; these were attributed to actual translocations with 95% confidence. When a cell was positioned over the pore with optical tweezers (Figures 2d-f), the unambiguous identification of a blockade with an actual translocation was complicated by excess noise. Figures 2d,e show typical current traces measured under the same conditions as in Figure 2a but with an MDA-MB-231 607

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the position of the synthetic pore in the cross-sectional image in Figure 3a even though the membrane was hardly perceptible. The cell was hardly perceptible at t = 1 min also, but as time progressed to 4 and 7 min and more DNA entered the cell, the fluorescence increased, eventually showing the outline of the cell. Thus, a cell in close proximity to the pore can be electroporated with 1 V. To establish the correspondence between the blockades in the pore current and the number of molecules transfecting a cell, the fluorescence associated with YOYO-1 intercalated 20 kbp linearized DNA plasmids was measured after transfection into U937 human leukemic monoblasts (Figure 3b). To measure the fluorescence, cells were conveyed to the nanopore using tweezers, one cell was transfected whereas the others were not, and then the cells were repositioned 170 pA and return to ΔI < 5 pA. In order to estimate the error within the event count, two additional tallies were made: one using a 10% smaller and one with a 10% larger threshold. Using the smaller threshold, noise in the signal could be large enough to be counted as a translocation, whereas using the larger threshold tests for an inappropriate association of a translocation with a blockade depth. Correspondingly, 33 translocations were tallied in Figure 3b; for the entire 260 s duration of the voltage pulse, 259 blockades were tallied in total, a number that is identical to the value obtained from the fluorescence measurements. The same experiment was repeated on multiple cells and cell types with similar results; the close correspondence between the blockade count and the fluorescence (Figure 3d) supports the contention that a cell can be transfected with single molecule accuracy. Below 50 molecules, the relative error associated with the fluorescence measurement precludes an accurate count; however, the blockade count can be extended to single molecule sensitivity (Figure 3d, inset). The close correspondence between the tally of blockades and the fluorescence indicates that all the molecules transferred across the nitride membrane entered the cell, which suggests that the gap between the cell and nitride membrane was miniscule. As a preliminary gauge of size of the gap, an attempt to electroporate similar cells was made after coating the same membrane used to produce the data in Figure 3 with Pluronic F108 (PF108), an antiadhesive for cells. PF108 is a nonionic triblock copolymer, composed of two hydrophilic poly ethylene oxide (PEO) chains with an intervening hydrophobic poly propylene oxide (PPO) domain that readily adsorbs to

Figure 3. Using a nanopore to transfect a mammalian cells via electroporation with molecular precision. (a) A series of confocal (x-z) slices showing the accumulation of 7 kbp YOYO-1 intercalated circular plasmids in an MDA-MB-231 cell positioned over a 20.5 nm diameter pore. As time progresses from t = 1 to 7 min, more DNA enters the cell and the fluorescence increases, eventually showing the outline of the cell. The nitride membrane is hardly perceptible, but the fluorescent DNA in the nanopore is easily visualized. (b) Reconstruction of the confocal data acquired after the electroporation of a U937 cell, recording the emission of YOYO-1 dye intercalated in 20 kbp linear dsDNA; the outline of the membrane window fluoresces brightly because DNA sticks there. Optical tweezers were used to push the cell against a 30 nm thick nitride membrane with a 4.0 × 3.4 ± 0.2 nm cross-sectioned pore in it, where it was subsequently transfected. The same cell was then moved off the membrane window and immobilized using a photopolymerized hydrogel. Control cells (with faint autofluorescence) that were not electroporated were likewise positioned adjacent to it. The specular green fluorescence below the membrane is associated with fluorescent DNA. (c) Measurement of the pore current during transfection. Blockades that appear in the current indicate the translocation of a single dsDNA molecule across the silicon nitride membrane through the nanopore. Events attributed to the translocation of one DNA are marked with blue diamonds. (d) The correspondence between events counted using the blockade current and the resulting fluorescence from accumulated YOYO-1 intercalated in DNA in 14 transfected cells. Cells were transfected with 56−1534 molecules. Inset: The relative error in the molecular count using the blockade current.

human breast cancer cell with 1 V applied. Fluorescence from the circular plasmids, intercalated with YOYO-1, illuminated 608

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hydrophobic surfaces. The attachment of the PPO to the hydrophobic surface leaves the PEO chains extending away from the surface, forming a brushlike film that prevents cell adhesion.38,39 At physiological pH, the PF108 provides a steric hindrance on the scale of the film thickness, which is about 12− 15 nm.39 In the absence of a cell, under the same conditions as in Figure 3b, the pore conductance was found to be 16.5 ± 0.5 nA/V, similar to the value obtained before the application of F108. However, when another U937 cell was conveyed in KTE to the trans-side of the pore, forced against the membrane with tweezers and the voltage was scanned from 0 to 1 V for 10 min, no transfection was evident in the fluorescence. Although inconclusive, this result suggests that a small gap (∼15 nm) is required for electroporation. To establish a capability to transfect cells with nucleic acids and reprogram their functionality while maintaining viability, cells were transfected with 6,968 bp circular plasmids40 that constitutively allow expression of DsRed by the CAG promoter, which is used to drive high levels of gene expression in mammalian cells.41 On the cis-side of the membrane 5 pM circular plasmids were placed in a 100 mM KCl solution at pH 8, and then a single cell in PBS was positioned with tweezers on the trans-side of the membrane over a nanopore with a 15.2 × 13.2 ± 0.2 nm cross-section. The bias voltage was set to 1 V and maintained for 10 min, while the pore current was measured. A current trace recorded during electroporation (Figure 4a) frequently showed current blockades ΔI > 500 pA (ranging from 11.8 < I < 12.3 nA), which were interpreted as plasmids occluding the pore volume and lowering the electrolytic current through it. According to this interpretation, the portion of the current trace highlighted red indicates an interval in which a single circular plasmid was attempting to translocate through the pore into the cell. Once the current returns to the open-pore value, a single translocation was tallied. In the elapsed time between 0 and 2.5 s, six translocations were counted. In total, 380 events with a duration of >82.5 ms were tallied over the 10 min duration of the experiment, which were interpreted as 380 molecules transfecting the cell. Confocal images of the same cell revealed the development of the fluorescence with time post-transfection from 0.2 to 90 h (Figure 4b). The red fluorescence intensified in 17 h and reached a plateau level. The relative, cumulative fluorescence levels in the transfected cell were quantified (Figure 4c), which uses the integrated value obtained at t = 0.2 h as a reference guide. The red fluorescence increased 5-fold in 17 h posttransfection relative to the fluorescence level at t = 0.2 h. The observed fluorescence was attributed to the 380 plasmids delivered through a nanopore. Thus, these data indicate that it is feasible to induce transgene expression and precisely count the number of biomolecules transfecting a single cell using a nanopore without compromising viability of the cell. Moreover, cell viability is preserved even though a voltage is applied for 10 min. The duration of electroporation is extraordinary and likely testifies to a minimal perturbation of the cell membrane. In contrast to gene induction, post-transcriptional gene silencing by short-interfering RNAs (siRNAs) was used to inhibit specific gene expression and to further demonstrate the viability of transfected cells after electroporation using a nanopore.42 siRNA-mediated gene silencing was carried out using transgenic mESCs in which EGFP and DsRed were independently and constitutively expressed.26,27,40 The siRNAs against EGFP and GAPDH (Life Technologies, Carlsbad, CA)

Figure 4. Using a nanopore to induce transgenes (a-c) in a cancer cell and silence (d,e) gene expression in an embryonic mouse stem cell. (a) Blockade current traces corresponding to a 2.5 s-long interval of transfection via a nanopore with a 15 × 13 nm2 cross-section. Dashed lines indicate open pore current that is 12.3 nA. Current blockade events shown in red during the interaction of the 7 kb circular plasmids with the pore. According to the blockade count, six plasmids translocated across the nitride membrane and transfected the cell. (b) Perspective iso-surfaces reconstructed from bright field images outline the silicon nitrite membrane and are added to the confocal data of an MDA-MB-231 cell transfected with 380 plasmids, permitting expression of DsRed over time up to 90 h post transfection. (c) Relative red fluorescence levels in the transfected cell in (b), the value at 0.2 h post-transfection is set as 1.0. (d) Confocal images of a dualreporter mouse embryonic stem cell. Short-interfering (si) RNAs against EGFP were transfected through a nanopore. Fluorescence images of EGFP (left) and DsRed (right) were taken 0 (top) and 15 (bottom) h after transfection. Both EGFP and DsRed were independently and constitutively expressed in this stable cell line. (e) Relative levels of EGFP and DsRed fluorescence in siRNAtransfected single cells (green and red bars) were analyzed at 1, 15, and 22 h post-transfection. As a control, EGFP fluorescence in nontransfected single cells (blue bar) was measured over the same time period during which the cells did not divide. The results are the average of three replicates (three cells). The value of fluorescence at 1 h is set to 1. Error bars indicate a standard deviation.

were labeled using a Cy3-labeling kit according to the manufacturer (Life Technologies). Initially, the efficacy of siRNA used in this investigation in silencing EGFP expression was tested with a standard liposome-based transfection method (SI, Figure S5). Transfection of siRNA was carried out with Lipofectamin siRNAMax (Life Technologies). The final concentration of siRNA in the culture was 10 nM, which was determined as the minimum concentration required to observe bright Cy3 expression and effective knockdown of EGFP. Figures 4d,e relate data obtained from a dual-fluorescence mESC transfected with siRNA against EGFP using a 2.5 nm diameter nanopore. By applying a 1 V transmembrane voltage, it is possible to force the siRNA through the pore. EGFP (left) and DsRed (right) fluorescence was observed at 0 h (top) and 609

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persistence length, occurs only after extended interaction with the pore, which likely results in highly bent sections of DNA. Likewise, the translocation of a plasmid across the permeabilized cell membrane is expected to be frustrated, because of the small (1−10 nm) pore diameters there. It has been posited that the electrophoretic force is also responsible for driving DNA into the cell.46 A comparison of the dwell time associated with the same plasmid (6,968 bp) in a 20 ± 0.2 nm diameter nanopore in the silicon nitride membrane without a cell and one with a permeabilized cell membrane positioned over it (SI, Figure S4) suggests that the permeabilized cell membrane presents the severest limitation on the molecular transfer rate. In summary, because the transcription factors that dictate cell fate are translated from fewer than a thousand transcripts, a method for precisely conveying a biologically relevant number of distinct molecules into a cell is required to modify its genetic code and modulate gene expression in it to create a homogeneous population of one phenotype. This study establishes a nanopore as a gene delivery tool that offers unprecedented control over transfectionreprogramming cells with nucleic acids with single molecule resolution with very high efficiency without compromising viability. It has also been established the maximum threshold for permeabilizing the membrane is 1 V. Moreover, a cell located precisely over the nanopore remains viable even after 10 min exposure to 1 V, demonstrating unequivocally a minimally invasive transfection tool. The successful application of this method to the transfection of nucleic acids also could be extended easily to accommodate other charged analytes, such as proteins,16 by adjusting the pH on the cis-side to be above the pI so that the molecule carries a net negative charge. However, to be practical, the limitations on throughput, the most severe of which may be the molecular transfer rate of a circular plasmid, remain to be relieved.

15 h (bottom) after transfection (Figure 4d). Apparently, the green fluorescence is down-regulated, while the red fluorescence changes little over the same time interval in the same cell. The relative levels of fluorescence in siRNA-transfected single cells (green and red bars) were analyzed at 1, 15, and 22 h after transfection along with EGFP fluorescence in nontransfected single cells (blue bar) as a control. Nontransfected, single mESCs that stably express EGFP showed a steady increase of green fluorescence (Figure 4e).26 The results, which represent the average of three replicates (three cells), indicate that ∼100 siRNA molecules are required to down-regulate green fluorescence in 12 h post-transfection, as opposed to a commonly accepted lipofection method that required 10 nM siRNA or about 20,000 molecules/cell for detection of complete knockdown of green fluorescence in 40 h posttransfection (SI, Figure S3). Finally, to be practical, transfection will have to be accomplished with high throughput to produce a large population of transfectants commensurate with specifications for tissue engineering or industrial-scale production in which cells are used as a foundry to produce molecules. There are several limitations on throughput with this method, however, among them is the use of a single nanopore for transfection; the use of a single (array) of tweezers for single cell placement; and the molecular transfer rate (as indicated by comparison of Figures 2 c and f, by Figure 4a, and Figure S4). To overcome this first limitation, what is required is an array of nanopores to deliver the expression vector to a group of cells all at the same time, which can be accomplished by relying on the same methods used to fabricate a single nanopore in a membrane (SI, Figure S5). However, each nanopore element in the array would have to be addressed independently by the microfluidics used to convey the cells to the pore, and by the wiring that is used to create the electric field to permeabilize the cell membrane, and detect the translocating molecules. It is now practical to create such complex microfluidic circuits with integrated valves43 that, in combination with time-multiplex optical tweezers,13 could be used to manipulate hundreds of cells simultaneously into position over the nanopores for transfection and recovery of the transfectants. The electroporation process may present a more fundamental limitation, however. The critical relationships between the gap and the threshold voltage, and the pulse width and voltage amplitude and the permeability of the cell membrane and the corresponding molecular transfer rate have yet to be established. In particular, the extended duration of each translocation event associated with a circular plasmid (Figure 4a), especially when compared to the current blockade associated with the linear plasmids (Figure 2c), presumably reflects the time required to electrophoretically transport a single circular plasmid through a pore smaller than the bending radius or persistence length of the DNA and is likely to adversely affect throughput. For a circular plasmid to close, it has to be long enough to bend into a circle with correct number of bases so the ends are properly rotatedin multiples of 10.4 base pairsto allow them to bond. Therefore, the minimum length for a circular DNA plasmid is supposed to be ∼416 base pairs (or about 141 nm). This value is also consistent with the persistence length of duplex DNA, which ranges from 44−55 nm, with variation due to electrolyte concentration and chemical constituency.44,45 Thus, the translocation of the DNA through a 15 nm diameter pore like that used to produce the data in Figure 4, which is smaller than the bending radius or

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Finite element calculations of the electric field distribution in the vicinity of a nanopore, synthesis of hydrogels, viability assay after exposure to the optical tweezers, transfection controls for the gene knockdown using siRNA transfected with lipofection, a comparison of molecular transfer rates with and without a cell over the nanopore, and an example of an array of nanopores sputtered in a silicon nitride membrane. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported through the National Science Foundation (DBI-1256052). REFERENCES

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