Single molecule delivery into living cells

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform which uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform the nanopipette is used as both a scanning ion conductance microscope (SICM) scanning probe and as an injection probe. The SICM is used to position the nanopipette above the cell surface, before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modelling, we also show that macromolecular crowding in the cell increases the signal to noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.


Main
The cell, the fundamental unit of life, is a microscopic chemical reactor in which thousands of processes happen simultaneously to bring about biological function, all within a highly crowded and compartmentalised environment 1 .The manipulation of cells enabled by the controlled delivery of biological macromolecules is an indispensable tool to study these cellular processes 2,3 .For this an array of methods has been developed to deliver molecules into mammalian cells, including encapsulation into lipid vesicles and viral-like particles, chemical transfection, electroporation and microinjection [2][3][4] .
Microinjection uses glass micropipettes with micron size tips to mechanically penetrate the plasma membrane, enabling molecules to be delivered [2][3][4] .However, the large size of the micropipette's tip (typically ranging from 0.5-75 µm in external diameter) 5 relative to the cell can cause significant perturbation during injection, disrupting the actin cytoskeleton and deforming the morphology of the cell 6 .One solution is to reduce the probe size, and several approaches have been developed that use nanoscale probes, including fluidic force microscopy (FluidFM) 7,8 , nanostraws [9][10][11] , carbon-nanotube cellular endoscopes 6,12 , nanofountain probe electroporation 13,14 , nanopore electroporation 15,16 , and electrowetting nanoinjection 17,18 .Herein, we use nanopipettes as a nanoscale injection probe.Nanopipettes can be easily fabricated at the bench with tuneable pore diameter down to 10 nm (a nanopore) and by fitting nanopipettes with electrodes, the translocation of molecules into the cell is stimulated by the application of a suitable voltage [19][20][21] .Moreover, the small size of the nanopipette's tip size relative to the cell mean that cell survival is much improved compared to microinjected cells [22][23][24][25] .
The translocation of a single macromolecule through the pore at the tip of a nanopipette results in a detectable alteration in the measured ionic current, which can be used to characterise and quantify the number of molecules that pass through the nanopore [26][27][28][29][30][31][32][33] .In recent work we have shown that the detection of nucleic acids and proteins is enhanced by their translocation into a polymer-electrolyte bath containing poly(ethylene) glycol (PEG) 27,30 , and have shown that this arises from a combination of unique ion transport behaviour and the interaction between the translocating molecule and the polymer-electrolyte interface 32,33 .Inspired by these observations, we here describe the development of a nanoinjection platform, in which nanopipettes are used to perform the quantitative delivery of biological macromolecules into the highly crowded interior of mammalian cells that induce different cellular responses.
In our nanoinjection platform, a nanopipette is integrated into a scanning ion conductance microscope (SICM), where it functions both as a scanning probe and an injection probe [34][35][36] .
The SICM enables the automated positioning of the nanopipette's tip on the surface of a living cell with nanometer resolution 36 .The nanopipette can then be inserted into either the nucleus or cytoplasm, and the delivery of molecules into the cells is then triggered by the application of an appropriate voltage.We demonstrate that the nanoinjection platform can be used for both cell lines and primary cells to perform quantitative delivery of DNA, globular proteins, and protein fibrils, all at single molecule resolution, and that the macromolecules retain function after delivery into the cells.Furthermore, we show that the ionic current signatures are enhanced for single molecule delivery into the macromolecular crowded environment of either the cell interior or an electrolyte solution with high concentration of bovine serum albumin (BSA).By using computational modelling, we demonstrate that this enhancement is caused by an increased concentration of analyte at the nanopipette opening after translocation and the displacement of crowding molecules near the nanopore.

Overview of the nanoinjection platform
The nanoinjection platform comprises five components: (1) a SICM, (2) a nanopipette which acts as the SICM scanning probe, a nanoinjection tool and as single molecule counter(S.Figure 1), (3) microstepper motors for coarse positioning of the nanopipette in the region of interest (4) piezoelectric actuator for positioning the nanopipette in 3 dimensions with nanoscale precision and (5) a spinning disk confocal microscope for brightfield and fluorescence imaging before and after nanoinjection (Figure 1A).In the nanoinjection procedure, brightfield and fluorescence microscopy are used to identify the cell for injection and the microstepper motor performs the initial coarse positioning of the nanopipette at the site of injection.Fine positioning of the nanopipette uses the SICM control software and piezoelectric actuators (Figure 1B).SICM relies on the measurement of the ionic current between an Ag/AgCl electrode inserted in the nanopipette, and a reference electrode immersed in an electrolytic solution where the sample is placed [34][35][36] .By applying a voltage between the two electrodes, an ionic current flows through the nanopore at the tip of the nanopipette.When the nanopipette approaches a surface, the measured ionic current drops.This current drop is proportional to the separation between the nanopore and the sample and can be used as active feedback to maintain the nanopipette-sample distance constant (S.Figure 2) 36 .A topographic image of the cell surface can be obtained, both pre-and post-injection, by recording the vertical position of the probe over each scanned pixel 36 .Integration of the nanopipette with piezoelectric actuators allows the positioning of the probe in the three dimensions with nanoscale precision.Thus, by determining the height of the plasma membrane by SICM, the nanopipette can then be inserted into the cell to a pre-determined depth (Figure 1B).Once inserted, the application of an appropriate voltage drives the movement of molecules from inside the nanopipette into the cell.By measuring the disruption in the ionic current flow caused when individual molecules of sufficient size pass through the pore at the nanopore's tip, the number of molecules delivered into the cell can be quantified (Figure 1B).The nanopipette is then retracted from the cell and, as appropriate, the cell can then be imaged by microscopy and/or SICM to examine the cellular effect of molecule delivery by nanoinjection after different lengths of time.

Quantitative delivery of DNA into HeLa cells and primary neurons
Nanopipettes have been used hitherto for quantitative detection and characterisation of biological macromolecules in vitro with single molecule resolution, including globular proteins, amyloid fibrils, nucleic acids, ribosomes and nanostructures [26][27][28][29][30][31][32][33] .To demonstrate that nanopipettes can also be used for the quantitative delivery of macromolecules at single molecule resolution into cell and that this results in a demonstrable phenotypic effect we used the nanoinjection platform to deliver a DNA plasmid into the nucleus of the human cervical epithelial HeLa cell line.We used the subsequent formation of the encoded fluorescent protein both as a readout for the successful delivery of the plasmid and for the functional integrity of the nanoinjected cell to transcribe and translate the encoded green florescent protein (GFP) (Figure 2).First, to confirm that the nanoinjection platform can perform site specific delivery, we used the nanopipette to introduce a 70 kDa fluorescein dextran conjugate into either the nucleus or cytoplasm of HeLa cells expressing the nuclear localised protein pmCherry-NLS (HeLa RNuc) (S.Figures 3-6).Upon insertion of the nanopipette into the cell, we observed a ~25% reduction in the baseline current (S.Figure 7).This is consistent with an increased resistance to ion flow due to the plasma membrane acting as a permeability barrier 37 and provides additional feedback to the user that the nanopipette tip is inside the cell 23,24 .The fluorescein dextran was then delivered by the application of -700 mV into either the nucleus or cytoplasm, resulting in nuclear and cytoplasmic localisation respectively (S.Figure 5-6).Moreover, SICM imaging of the injection site immediately after retraction of the nanopipette revealed no residual damage to the plasma membrane, although the height of the apical plasma membrane was increased by up to ~0.5 µm (S.Figure 8).
Next, for nanoinjection of DNA the 3.5 kbp pMaxGFP plasmid, which encodes Pontella mimocerami GFP, was used as a model analyte (Figure 2A).The nanopipette containing the plasmid in a solution of phosphate buffered saline (PBS), was inserted into the nucleus of a HeLa RNuc cell and delivery triggered by the application of -500 mV.The translocation of individual DNA molecules into the nucleus resulted in alterations of the ionic current flow through the nanopipette, with a total of 132 events detected over 20 sec (Figure 2B and C).In contrast, less than 5 events were recorded over 60 sec when nanopipettes containing a mixture of PBS and ATTO 488 dye were inserted into the nuclei of HeLa RNuc cells (S.Figure 9).These data are consistent with the majority of events recorded for DNA nanoinjection resulting from the translocation of the plasmid into the nucleus, although a small number of the events may be caused by the translocation of intracellular molecules into the nanopipette 38 .24 hours post nanoinjection the HeLa cell had divided and both daughter cells expressed GFP (Figure 2B).This demonstrates successful delivery of the plasmid and also shows that nanoinjection is well tolerated by the cell, as least as judged by its ability to grow, divide and produce the plasmidencoded GFP.This was replicated for two additional cells, in which 37 and 44 translocation events were detected, respectively and, for each, the cells had also divided, with both daughter cells expressing GFP (S. Figure 10, 11).Thus, these we have shown quantitative nanoinjection of DNA, with single molecule resolution, into living cells in culture.
Whereas the transfection of immortalised cell lines, such as HeLa, is routine, the transfection of primary cells such as neurons can be more challenging.Using the nanoinjection platform we performed quantitative delivery of the GFP plasmid into the nuclei of two primary dorsal route ganglion (DRG) neurons (Figure 2D and E, S.Figure 12).41 and 44 translocation events were detected, respectively, and GFP expression observed in both cells 24 hours later (Fig 2D and E, S.Figure 12).Thus, the nanoinjection platform can also be used to perform quantitative delivery of plasmid DNA into primary neurons and this results in a phenotypic change with the cells expressing a protein encoded by the plasmid.

Quantitative delivery of proteins into primary endothelial cells and neurons
The intracellular delivery of proteins, for example CRISPR-Cas9 39,40 , fluorescent proteins 41 and antibodies 42,43 , into cells typically uses pressure-based microinjection or electroporation 2,3 .However, proteins can misfold and aggregate under high shear pressure losing their biological function 44 , and there is little control of the number of proteins delivered into the cell when using these techniques.In previous work we have shown that individual β-galactosidase molecules can be detected by a nanopipette when delivered into a polymer-electrolyte bath using a nanopipette 27 .Building on these observations, we used the nanoinjection platform to deliver purified Escherichia coli β-galactosidase into cells and to quantify the number of molecules delivered.
E. coli β-galactosidase is a 465 kDa tetrameric globular protein, with a pI of 4.61, hence the protein is negatively charged at neutral pH 45 .Since E. coli β-galactosidase is enzymatically active only as a native tetramer 46,47 , the use of this enzyme as a test substrate enabled us to determine whether the nanoinjection platform can deliver proteins without disrupting their structure and function.Mammalian cells have endogenous β-galactosidase activity in lysosomes 46,47 , therefore to distinguish between exogenous E. coli β-galactosidase delivered by nanoinjection and endogenous lysosomal β-galactosidase, we performed nanoinjection of the nucleus.For these experiments we used primary human umbilical vein endothelial cells (HUVEC), whose nuclei are easily identified because they protrude above the rest of the cell surface 48 and can thus be identified without the need for fluorescent dyes or proteins.HUVEC cell nuclei were nanoinjected with β-galactosidase (Figure 3B and C, S.Figure 13 -16) by applying a voltage of -700 mV.The number of translocation events recorded ranged from 100 to 1000 (Figure 3C, S.Figure 13 -16).For example, for the cell in Figure 3B, nanoinjection of β-galactosidase resulted in the detection of 439 single molecule translocation events (Figure 3C).β-galactosidase enzymatic activity was detected using the membrane-permeable fluorescent substrate SPiDER-βGal 49 (Figure 3A and B, S.Figure 17 -18) and after nanoinjection there was an increase in the fluorescence of SPiDER-βGal throughout the nucleus (Figure 3B and S.Figure 13 -16).By determining the corrected total cell fluorescence (CTCF) value for the nucleus of the injected cell and comparing with a neighbouring noninjected cell (Methods), we confirmed that the injected cell had a greater fold increase in SPiDER-βGal nuclear fluorescence throughout its nucleus (Figure 3D).Conversely, no increase in SPiDER-βGal fluorescence in the nucleus was observed, relative to a neighbouring cell, when the nanoinjection platform was used to deliver a fluorescent dye into the nucleus (S.Figure 19).These data are therefore consistent with the delivery of E. coli β-galactosidase, in its enzymatically active form, into HUVEC nuclei.Next, we explored whether nanoinjection platform can perform quantitative delivery of protein fibrils into cells.For this we used α-synuclein amyloid fibrils generated in vitro (Methods) 50 .We have shown previously, by using a polymer-electrolyte bath, that delivery of α-synuclein amyloid fibrils can be detected by a nanopipette 27 .To enable visualisation of the fibrils delivered into cells, the A90C α-synuclein fibrils were labelled with Alexa Fluor 594 on the cysteine residue (Methods).AFM imaging revealed that the labelled fibrils had an average length of 69±2 nm (Figure 4A and B, S.Figure 20).The fibrils were nanoinjected into the cytoplasm of primary rat cortical neurons (Figure 4C and S.Figure 21 -22).In the example shown in Figure 4C, 628 events were detected (Figure 4D), and this resulted in a corresponding increase in Alexa Fluor 594 fluorescence in the injected neuron (Figure 4C).Two additional neurons were nanoinjected with 305 and 426 events detected, respectively, and the neurons were observed to have increased cell-associated fluorescence after the injection (S. Figure 21 -22).Thus, we have demonstrated that nanoinjection can perform quantitative delivery, with single molecule resolution, of fibrous proteins into primary cells.

Effects of the intracellular environment on single molecule translocation
We have shown previously that the signal to noise ratio (SNR) for the detection of molecules by nanopipettes is dependent on the composition of the electrolyte solution into which the molecule is translocated, and can be enhanced by using the synthetic polymer PEG 27,[30][31][32] .The intracellular environment is a complex mixture of macromolecules, small molecules and ions, densely packed and crowded, and hence is very different to the electrolyte baths typically used for single molecule detection by nanopipettes 51 .We therefore investigated whether the intracellular environment affects the ionic current signatures of the single molecule translocations.For this we compared the translocation of a model analyte, a linear 7kbp dsDNA molecule (Methods), delivered into cells with that of the DNA translocated into an electrolyte bath of either the electrolyte solution PBS or PBS containing 30% w/v bovine serum albumin (PBS BSA), as a simple mimic of the intracellular crowded environment 52 (Figure 5).The DNA was delivered sequentially into a HeLa RNuc cell, into a PBS electrolyte bath, and then finally into a 30% (w/v) BSA PBS bath (Figure 5A).Crucially, by using the same nanopipette for all three conditions, we could discount any differences in the nanopipette's geometry and dimensions on the translocation signatures of the DNA.To confirm the cell had been injected, the nanopipette also contained the small molecule fluorescent dye ATTO 488.
Translocation events were detected for the DNA in each instance (Figure 5B and S.Figure 23 -26).An increase in both the current peak amplitude and the dwell time for the events recorded for the delivery of the DNA into either the cell or 30% (w/v) BSA PBS bath was observed compared with delivery into PBS (Figure 5C).This was reflected in the integrals of the area calculated for each translocation event, which showed a shift from under 100 fC for PBS, to close to ~200 fC for the cell and PBS-BSA (Figure 5D).These results imply that the cellular environment enhances the detection of DNA into cells, and that macromolecular crowding in the cell maybe responsible for the increased SNR.To gain further insight into the effects of macromolecular crowding on the translocation of DNA, we employed coarse-grained simulation to simulate the moment a DNA molecule reached the nanopore at the tip of the nanopipette, and its translocation into a standard electrolyte environment versus a macromolecular crowded environment composed of 30% (w/v) BSA.
Coarse-grained simulations modelled the translocation of 2.7 kbp DNA molecules through a model of a nanopipette with a 10 nm aperture, with and without Lennard-Jones particles representing BSA molecules under a -600 mV applied bias (Figure 6A).The ionic current was estimated from the translocation trajectories, allowing an enhancement relative to the open-pore current to be computed (Figure 6B).Each ensemble, with and without BSA molecules, consisted of 24 simulations (Supplementary Movies 1-6).For each simulation, the translocation process could be monitored (Figure 6C) and the elapsed time between the first and last DNA beads exiting the pore was obtained (Figure 6D), revealing that the presence of BSA molecules slowed the translocation of the DNA and increased the current.
The simulations allowed qualitative investigation of multiple factors that cause the current enhancement during DNA translocation in the presence of BSA at the aperture of the nanopore opening.We observed that the DNA was initially more compact as it was being translocated in either the presence or absence of DNA, as characterized by its radius of gyration.By the end of the translocation process the DNA swelled ~10% more in the absence of BSA than it did when translocated in the presence of BSA (Figure 6E).Moreover, in the presence of BSA, the DNA continued to be spread out more slowly after the last base pair exited the pore, likely contributing to a slower recovery of the current towards the baseline (Figure 6F).
The enhancement in the ionic current caused by BSA can be explained by two mechanisms.
First, due to the closer proximity of the DNA to the nanopore aperture, the DNA directly increases the ionic current through a direct effect on the conductivity of the solution near the nanopore 53,54 .Second, the DNA displaces a small number of BSA molecules near the nanopore aperture that would otherwise be sterically blocking the ionic current flow, thus resulting in an increase in the ionic current.Herein we demonstrate the development of a nanoinjection platform in which a nanopipette is used both as an SICM scanning probe and as an injection probe.We describe the application of this device in the precise manipulation of living cells, demonstrating that the platform can perform the delivery of DNA, globular proteins, and protein fibrils into different cellular locations with single molecule resolution.Moreover, we demonstrate that the injection process is well tolerated by different cell lines and primary neurons and show that the delivery of biological macromolecules into cells can result in a demonstrable phenotypic change.
Several different approaches have been developed over recent years using nanoscale probes for delivery of materials into cells 3 .Examples include using hollow nanoelectrodes for the intracellular delivery of individual gold nanoparticles that could be monitored by enhanced Raman scattering 55 .Solid-state nanopores coupled with optical tweezers have also been used for proof-of-concept experiments in single cell transfection, but did not demonstrate protein injection or the ability to manipulate primary cells 56 .Pandey et al. employed a multifunctional nanopipette for the intracellular delivery of single entities including a model protein, ferritin, and PEGylated gold nanoparticles 57 .Our study provides a direct demonstration that biological macromolecules can be delivered intact into defined cellular locations (herein nucleus vs cytoplasm) visualized by expression of plasmid encoded genes, detection of β-galactosidase enzymatic activity and imaging of fluorescent α-synuclein fibrils.Crucially, the finding that cells divide after injection, produce plasmid-encoded GFP, and enable detection of enzymatically active β-galactosidase, demonstrate that cellular function is not perturbed and that protein structure is not affected by the nanoinjection procedure.
We demonstrate intracellular delivery of plasmid DNA, β-galactosidase and α-synuclein amyloid fibrils, all of which we have shown previously to be detected by nanopipette when translocated into a polymer-electrolyte bath 27 .It also potentially opens the door for detailed investigation into concepts such as the proteotoxicity associated with amyloid fibril formation and screening for the effects of small molecules or molecular chaperones on cellular proteotoxicity of amyloid aggregates.Given that the self-assembly of α-synuclein and other proteins involved in neurodegeneration occur intracellularly 58 , the introduction of preformed fibrils into neurones offers a route to determine directly the effects of protein fibrils on cellular homeostasis, to compare with results obtained by the commonly adopted practice of adding preformed fibrils to culture medium [59][60][61][62][63] .Nanoinjection of α-synuclein aggregates into neurons also enables quantification of the number of aggregates delivered, enabling the direct comparison of the effects of different numbers of fibrils on a cellular response.Finally, and importantly, given that different fibril structures are associated specifically with different diseases, even when formed from very similar or even identical sequences 64 , the nanoinjection platform developed here will enable the cellular effects of individual fibril types to be directly and quantitatively compared.
One intriguing observation we report here, is that translocation of macromolecules into the cell increases the sensitivity of the nanopipette, with an enhanced SNR compared to delivery of macromolecules into an electrolyte bath.This signal enhancement was also observed for an electrolyte bath containing BSA, suggesting that the SNR is increased due to macromolecular crowding in the cell.For translocation into a macromolecular crowded BSA bath, coarse-grained simulation showed that the DNA after translocation displaced BSA molecules around the nanopore and that the DNA remained near the nanopipette opening resulting in an increase in the ionic current flow.We propose that a similar phenomenon is likely for the observed SNR enhancement for the translocation of DNA and protein molecules from the nanopipette into cells, with macromolecular crowding being responsible, at least in part, for the increased SNR, thus enhancing the sensitivity of the instrument for use in cells.

Conclusions
In summary, we demonstrate a nanoinjection platform for the quantitative delivery of macromolecules into both cell lines and primary cells with single molecule resolution.The platform is universal and could also enable the injection of ribosomes, DNA origami and viral RNAs 28,30,31 for precise cellular manipulation.We also believe this approach will provide a wide range of applications for the nanoinjection platform, enabling new insights into structure-function relationships of protein and protein complexes in the cell.
confocal system coupled with ANDOR iQ3 live cell imaging system (Oxford Instrument), allowing fluorescence and brightfield imaging.The confocal microscope was fitted with a 455, 488 and 561 nm laser and emission filter set that enables the visualisation of a wide range of fluorescent dye.The nanopipette tip was aligned with the microscope and positioned next to a cell of interest for scanning or nanoinjection.Unless stated otherwise, all fluorescent images were captured by the ANDOR iQ3 live cell imaging system with appropriate excitation laser and emission filter combinations.

Nanoinjection
For all nanoinjection procedures, the ion current trace was recorded by pClamp 10 (Molecular Devices).All fluorescent images were captured by the ANDOR iQ3 live cell imaging system with appropriate excitation laser and emission filter combinations.The nanopipette was lowered down at 10 µm/s during cell penetration.Detailed information on the composition of the nanoinjection analyte can be found in the Supplementary information.

Single molecule detection
For the translocation experiments, the nanopipettes were filled with analyte of interest diluted into 1X PBS, the nanopipette was fitted with a Ag/AgCl working electrode.The tip of the nanopipette was then immersed into the electrolyte bath of choice with a grounded Ag/AgCl reference electrode, thus establishing a complete electric circuit between the inside of the nanopipette to the outer bath solution.Depending on the polarity of the analyte, the application of a voltage to the working electrode caused molecules from inside of the nanopipette to translocate through the nanopore and into the bath solution.The ionic current was measured using a MultiClamp 700B (Molecular Devices) patch-clamp amplifier in voltage-clamp mode.
Unless specified, the signal was filtered using Bessel filter at 10 kHz and digitized with a Digidata 1550B (Molecular Devices) at a 100 kHz sampling rate (every 10 µs) and recorded using the software pClamp 10 (Molecular Devices).Translocation event current analysis was carried out with a custom MATLAB script (provided by Prof Joshua B. Edel, Imperial College, London, UK).The MATLAB script identifies individual events in given ion current trace using defined thresholds, at least 5 standard deviations above baseline noise.The baseline is tracked via asymmetric least square smoothing algorithm and fit determined by Poisson probability distribution function.

Coarse-grained simulation
The coarse-grained mrDNA model 65 was used to simulate translocation of linear 2.7 kbp dsDNA molecules through a nanopipette represented by a grid-based potential using the ARBD simulation engine 66 .Twenty-four simulations were performed per solvent condition-with or without Lennard-Jones spheres (3.9 nm ܴ ୫୧୬ ; 0.1 kcal mol ିଵ ߳) at a concentration of 4.5 mM representing crowding BSA molecules.DNA-BSA interactions were computed by attributing by setting ܴ ୫୧୬ to 1.1 nm for DNA beads and ߳ to 0.05ൈ ܰ ୬୲ kcal mol ିଵ where ܰ ୬୲ is the number of nucleotides represented by a DNA bead.BSA molecules were assigned a damping coefficient of 215 ns ିଵ , and a 40 fs timestep was used to advance the configuration of the system while Langevin forces maintained a 291 K temperature.In all simulations, the nanopore was represented by the electrostatic potential obtained from a previously described 67 finite element COMSOL model of a nanopipette, with geometry adapted to a 10-nm-diameter aperture with a 600 mV applied electrostatic potential ejecting the DNA from the pipette.The electrostatic COMSOL axisymmetric solution was sampled at regular lattice sites in cylindrical coordinates before being interpolated onto a 3D grid using a custom Python script.A steric grid potential to prevent the DNA from entering the pore walls was obtained using a custom Python script to compute the distance ݀ of a given voxel from the nearest voxel with a valid solution, with the steric potential at a given voxel set to ଵ ଶ ݇݀ ଶ , where ݇ ൌ 2 kcal mol ିଵ Å ିଶ .The steric potential acting on the center of BSA spheres was obtained by convolving a mask with voxels lacking an electrostatic solution having values of ten kcal mol ିଵ with a normalized 3D kernel computed from a linear ramp ranging from one to zero for distances 4.5 to 2.5 nm from the kernel center, which was added to the harmonic steric potential also applied to DNA.The initial DNA configurations were obtained from previously performed simulation with the voltage reversed.
The twenty-four simulations in each ensemble consisted of three subgroups of eight simulations each with the DNA end nearest the aperture initially placed around 100, 185 or 270 nm.BSA beads were initially randomly distributed through the system.
Each simulation system was equilibrated for 50-100 ߤs in the presence of the steric potentials and absence of an electrostatic potential.After equilibration, the electrostatic potential was turned on until the DNA was fully translocated from the nanopipette.A steric exclusion model (SEM) 68 was used to process each trajectory to obtain an estimate of the ionic current as previously described 67 using data obtained from atomistic simulations of the monovalent ion enhancement around DNA to estimate the associated current enhancement near a DNA molecule in 170 mM KCl solution.Before calculating the ionic current, the ionic mobility map including the DNA enhancement was modulated by a BSA-distance-dependent function.Briefly at each site in a discretized grid, the distance to the nearest BSA molecule was computed, and the mobility was scaled by a linear ramp from zero to one between distances of 2.5 and 4 nm.
Eight simulations without DNA lasting 80-100 ߤs each were used to estimate the bulk ion conductance without DNA used to compute the modulated current.

Figure 1 .
Figure 1.The nanoinjection platform.(A) SICM integration into the platform.The position of the nanopipette is controlled by the SICM through a piezoelectric actuator.(B) The quantitative nanoinjection procedure.The nanopipette approaches the surface of the cell membrane through the spatial control of the SICM, then the nanopipette is moved downward by a predefined distance to penetrate the cell, finally, the delivery of materials will be triggered by electrophoretic forces via the application of a suitable voltage.During delivery, the current is monitored in

Figure 2 .
Figure 2. Quantitative nanoinjection of DNA plasmids into living cells.(A) Schematic of the nanoinjection of GFP plasmids (pMaxGFP) into the nucleus and the transfection of the cell.(B) The transfection of a HeLa cell expressing the nuclear localised mCherry-NLS (HeLa RNuc) with pMaxGFP plasmid through quantitative nanoinjection.HeLa RNuc cells were cultured on a grided dish to enable identification of the cell after nanoinjection.pMaxGFP plasmids were quantitatively nanoinjected into the nucleus of the cell (arrow).24 hours later, the two daughter cells were imaged to confirm the expression of GFP from the injected pMaxGFP plasmids.(C) A snapshot of the current trace (20 seconds) recorded during the nanoinjection.Based on peak counting, a total of 132 pMaxGFP plasmids were nanoinjected into the HeLa RNuc cell.(D) The transfection of a DRG primary neuron with pMaxGFP plasmid through quantitative nanoinjection.24 hours later, the neuron was imaged to confirm expression of GFP.(E) The current trace (20 seconds) recorded during the nanoinjection step.A total of 41 pMaxGFP plasmids were delivered into the DRG neuron.The dotted line in (C) and (D) indicated the threshold for events search.

Figure 3 .
Figure 3. Quantitative nanoinjection of β-galactosidase into cells.(A) The dye SPiDER-βGal was used to detect β-galactosidase enzymatic activity inside the cell.Endogenous β-galactosidase is localised to lysosomes in the perinuclear cytoplasm.The nanoinjection of E. coli β-galactosidase into the nucleus causes the nucleus to become fluorescent.A target cell's nucleus nanoinjected with E. coli β-galactosidase shows an increase in nuclear overall fluorescence.(B) A snapshot of the current trace (100 seconds) during the nanoinjection.Based on peak counting, a total of 439 β-galactosidases were nanoinjected into the cell.(C) The Corrected Total Cell Fluorescence (CTCF) of the nucleus area before and after the nanoinjection were calculated and plotted against the molecule count for 8 independent experiments.The dotted line in (B) indicated the threshold for events search.

Figure 4 .
Figure 4.The quantitative nanoinjection of α-synuclein fibrils into rat primary cortical neurons.(A) A representative image of the α-synuclein fibrils and (B) their associated length distribution of 69±2 nm (standard error of the mean, 628 fibrils traced).(C) The primary neuron before and after the nanoinjection of the α-synuclein fibrils.(D) A snapshot of the current trace (100 seconds) during the nanoinjection.Based on peak counting, a total of 153 αsynuclein fibrils were nanoinjected into the cell.

Figure 5 .
Figure 5. Analysis of the effects of the intracellular environment on single molecule translocation of DNA.(A) The same nanopipette was filled with 5 nM 7 kbp dsDNA in PBS mixed with 10 µM ATTO 488 fluorescent dye, a voltage of -500 mV was used to drive the dsDNA from the nanopipette to either the HeLa RNuc cell, PBS or 30% (w/v) BSA PBS.The cell turns into fluorescently green after the injection due to the ATTO 488.(B) The 5 sec current traces of the translocation of the dsDNA into either PBS, 30% (w/v) BSA PBS or cell.(C) The population distribution of the translocation event of the 7 kbp dsDNA, both 30% (w/v) BSA PBS and cell shows a wider distribution on the dwell time.(D) The equivalent charge of the translocation events were plotted, a clear shift was observed between PBS and 30% (w/v) BSA PBS and cell.For (C) and (D) a total of 500 events were randomly sampled and plotted.

Figure 6 .
Figure 6.Coarse-grained simulations of DNA translocation.(A) Coarse-grained simulation systems consisting of a 2.7 kbp dsDNA molecule (orange) driven out of a nanopipette (gray) by an applied electric potential into an electrolyte solution with and without BSA proteins (blue).Each case (with or without BSA) consisted of an ensemble of 24 independent simulations.(B) Ionic current enhancement as the DNA moved through the pore, averaged over each ensemble.Here and throughout the figure, solid lines depict the ensemble average whereas shaded regions depict the standard deviation among the simulations.(C) Number of base pairs having left the pore during the simulations, averaged over each ensemble.(D) Scatter plot showing the elapsed time between first and last base pair being translocated through the pore in each simulation against the average current enhancement during that time interval.(E) Radius of gyration of DNA having been translocated through the pore, plotted against the number of translocated base pairs (left) or the time since the last base pair was translocated (right).(F) Distance of the center of