Macromolecular Crowding Enhances the Detection of DNA and Proteins by a Solid-State Nanopore

Nanopore analysis of nucleic acid is now routine, but detection of proteins remains challenging. Here, we report the systematic characterization of the effect of macromolecular crowding on the detection sensitivity of a solid-state nanopore for circular and linearized DNA plasmids, globular proteins (β-galactosidase), and filamentous proteins (α-synuclein amyloid fibrils). We observe a remarkable ca. 1000-fold increase in the molecule count for the globular protein β-galactosidase and a 6-fold increase in peak amplitude for plasmid DNA under crowded conditions. We also demonstrate that macromolecular crowding facilitates the study of the topology of DNA plasmids and the characterization of amyloid fibril preparations with different length distributions. A remarkable feature of this method is its ease of use; it simply requires the addition of a macromolecular crowding agent to the electrolyte. We therefore envision that macromolecular crowding can be applied to many applications in the analysis of biomolecules by solid-state nanopores.


DNA linearization
The pMaxGFP plasmid from the Cell Line Nucleofector TM Kit V (VVCA-1003; Lonza) was used as a model plasmid. The plasmid was linearized by restriction digestion using the enzyme Kpn I (R0142S; New England Biolabs). 1 µg of the circular and linearized plasmids were analysed by gel electrophoresis with a 1% agarose gel with 1X SYBER safe (S33102; Thermo Fisher). 1 µg of TrackIt TM 1 Kb Plus DNA Ladder (10488085; Thermo Fisher) was run alongside the plasmid samples. The DNA concentration was measured by UV absorbance at 260 nm.

Protein purification and amyloid fibril formation
The β-galactosidase (G5635; Sigma-Aldrich) was further purified by gel filtration using a calibrated Superdex 200 10/300 GL column (GE Healthcare Life Sciences). The column was calibrated by three separate analyses of protein standard mixes composed of: vitamin B12 (V2876; Sigma-Aldrich) and blue dextran (D5751; Sigma-Aldrich); cytochrome C (C2506; Sigma-Aldrich), bovine serum albumin (A7030; Sigma-Aldrich) and ferritin (F4503; Sigma-Aldrich); and ovalbumin (A5503; Sigma-Aldrich) and alcohol dehydrogenase (A7011; Sigma-Aldrich). β-galactosidase eluted at the expected elution volume for a tetramer and it was concentrated using a Vivaspin protein concentrator (Z614092; Sigma-Aldrich). The concentration of the protein was determined by UV absorbance at 280 nm using an extinction coefficient of 241,590 M -1 cm -1 [1], snap frozen and stored at -80˚C for future use. BL21(DE3) competent Escherichia coli (E. coli) was transformed with pET23a encoding a codon optimised gene encoding full length human α-synuclein. Protein expression was induced by using 1 mM isopropyl β-D-1-thiogalactopyranosides at an OD600 of 0.6. The purification of monomeric α-synuclein was performed as described previously [2], with an additional step of size exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare Life Sciences) prior to lyophilisation.
The generation of the fragmented fibrils was performed as described previously [3].To generate the fragmented α-synuclein amyloid fibrils the lyophilised monomeric protein was rehydrated in Dulbecco's PBS (D8537; Sigma-Aldrich) at a concentration of 500 μM and filtered through a 0.22 µm syringe. 500 μl of the filtered monomers were transferred to a 2 ml glass vial (27267-U; Sigma-Aldrich), a sterile magnetic stirrer bar was added before the vial was sealed. The vial was then put inside a mineral oil bath on top of a heated magnetic stirrer (N2400-3010; STARLAB) and stirred for 3 days at 1500 rpm at 40˚C. The fragmented fibrils were diluted to 200 µM monomeric equivalent concentration, snap frozen and stored at -80˚C for future use.
The thawed fragmented fibrils Eppendorf tubes were sonicated inside a water bath sonicator at maximum power at 25 ºC (U500H; Ultrawave) for 5 minutes prior use.
To generate the elongated fibrils, lyophilised monomeric α-synuclein was rehydrated with Dulbecco's PBS at a concentration of 200 µM and filtered through 0.22 µM syringe filter. Freshly defrosted sonicated fragmented fibrils were mixed with filtered monomeric α-synuclein such that the final solution contained 20% (v/v) fragmented fibrils and 100 µM of α-synuclein monomers in 500 µl inside a 1.5 ml Eppendorf tube. The sample was then incubated at 37˚C under intermittent shaking conditions (600 rpm shaking for 5 seconds followed by every 4:55 minutes without shaking) for 5 days using the Thriller shaking incubator (91-7010; Peqlab). The resultant fibrils were stored at room temperature for future use.

Atomic Force microscopy (AFM) and sample preparation
1 µl of the 100 µM monomeric equivalent fibril samples was added to 99 µl of 1M MgCl2 and incubated for 15 minutes, then the solution was deposited onto a freshly cleaved mica (AGG250-1; Agar Scientific) and incubated for 30 minutes. The solution was drained, and the mica surface was washed 3 times with ddH2O and dried with streams of nitrogen, followed by overnight drying at room temperature. The α-synuclein fibrils were imaged using a Dimension Fastscan (Bruker) in air tapping mode using the Fastscan A Silicon Nitride cantilever (FASTSCAN-A; Bruker). The probes were driven at resonance (1400 kHz in air). Images were acquired with a scan rate of 8-15 Hz (1024 X 1024 pixels) and processed with Nanoscope Analysis v1.9.

Nanopipette fabrication and ion current measurement
Quartz capillaries with outer and inner diameter of 1.0 mm and 0.5 mm respectively were used to fabricate nanopipettes (QF100-50-7.5; Sutter Instrument). Nanopipettes were fabricated by using the SU-P2000 For the translocation experiments, the nanopipettes were filled with 1X PBS, 0.01% (w/v) Tween-20 containing the analytes at the stated concentration. The tip of the nanopipette was immersed into either a 1X PBS, a glycerol PBS, PEG 4000 PBS or a PEG 8000 PBS bath with a Ag/AgCl working electrode connecting the inside of the nanopipette to the outer bath solution. The reference electrode was grounded and immersed into the bath solution. Application of a negative potential 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 patch-clamp amplifier (Molecular Devices) in voltage-clamp mode. The signal was filtered using low pass filter at 10 kHz and digitized with a Digidata 1550B at a 100 kHz (interval 10 µs) sampling rate and recorded using the software pClamp 10 (Molecular Devices). The presence of Tween-20 in the sample buffer allowed molecules to translocate through the nanopipette pore readily with high event frequencies [4]. The current trace was analysed using a custom written MATLAB script provided by Dr Aleksandar P. Ivanov and Prof Joshua B. Edel (Imperial College London). For translocation events analysis, the threshold level was defined at least 5 sigma away from the baseline, only events that were above this threshold were identified as positive event signals.

Scanning electron microscopy
The nanopores of the nanopipettes were imaged by scanning electron microscopy (Leo 1530 FEG-SEM; Zeiss). Nanopipettes were first sputter coated with a gold layer of a few nanometres in thicknesses. The nanopipettes were then mounted onto the sample holder and tilted to an angle of 60 and above for imaging.
The nanopipettes were imaged at between 2 and 3 kV at a working distance of 5 mm and below at an aperture size of 30.00 m using a InLens detector.

Statistical analysis
All data were tested for normality and data that had less than 40 points was assumed to be normally distributed. For a normally distributed data set, a parametric test (Welch's t test for 2 groups and One-way ANOVA for multiple groups) was used to test the difference between columns. A non-parametric test was used for not normally distributed data (Mann-Whitney test for 2 groups and Kruskal-Wallis test for multiple groups). All analyses were performed in GraphPad Prism 8.

Figures
Supporting Figure 1. The properties and the current-voltage plot of the nanopipettes. The nanopore of a representative nanopipette was imaged by scanning electron microscopy showing a nanopore of approximately 10 nm in diameter (A). 0.01% (v/v) Tween-20 in PBS was used to fill the nanopipette before immersion into different electrolyte containing various percentages of either glycerol (v/v) (B) or PEG 8000 (w/v) (C) in PBS. Three nanopipettes per condition were tested. The I-500mV/I500mV ratio and resistance for the nanopipette measured were calculated and shown in (D).
Supporting Figure 2. Plasmid translocation at different voltages. 60 s traces of the translocation of the circular plasmid at -300, -500 and -700 mV in PBS. Increasing the voltage to -500 mV or -700 mV caused an increase in molecule count when compared to -300 mV. A nanopipette with a larger nanopore was used here with an estimated size of c.a. 20 nm. A one-way ANOVA test was used here. Asterisks indicate the P-values (***, P<0.01). Error bars are S.E.M.
Supporting Figure 3. The 60s current traces for plasmid DNA in different concentrations of PEG 8000 and glycerol. 1.33 pM of circular DNA plasmid was used to fill the nanopipette prior to immersion into either PBS (A), or PBS with various concentrations of PEG 8000 (B) or glycerol (C). A potential difference of -700 mV was used to drive molecules out of the nanopipette. N refers to the total number of events recorded. The right panel shows one representative event peak from each condition, solid horizontal line represents the calculated baseline, green and red dotted vertical lines represent the beginning and the end of the event respectively.
Supporting Figure 4. The effect of the 50% (w/v) PEG 8000 on the detection of plasmid DNA by the nanopipette is reversible. A nanopipette containing 1.33 pM of plasmid DNA was first immersed into the PBS bath and -700 mV was applied to drive the plasmids into the bath. Without pausing the recording or changing the voltage, the Faraday cage was opened followed by transferring the nanopipette and the reference electrode into the 50% (w/v) PEG 8000 bath. The Faraday cage was closed for approximately 15s, the cage was then opened again to transfer the nanopipette and the reference electrode back into the PBS bath. The pronounced and reversible change in the current baseline and the peak amplitudes of the plasmid between the PBS and 50% (w/v) PEG 8000 bath can both be observed.
Supporting Figure 5. PEG 8000 translocations are not detected by the nanopipette. Nanopipettes were filled with 0.01% Tween-20 PBS and immersed into the 50% (w/v) PEG 8000 bath and -700 mV potential difference applied. No event peaks were observed throughout the 60s. Each panel shows the current trace from one nanopipette. Four different nanopipettes were tested.
Supporting Figure 6. Gel electrophoresis of circular and linearized DNA plasmids. 1 µg of either circular or linearized (following digestion with Kpn I) plasmid DNA was resolved by agarose gel electrophoresis. Two bands are observed for the circular plasmid, the upper band corresponds to the relaxed state and the lower band to supercoiled plasmid DNA. The linearized plasmid runs as a single band at approximately 3.5 kbp, consistent with the plasmid's size of 3,486 bp.
Supporting Figure 7. The detection of circular and linearized plasmid in either PBS or in 50% (w/v) PEG 8000 in PBS electrolyte baths. The 60s current traces for circular and linearized plasmid in either 50% (w/v) PEG 8000 (A) or in a PBS bath (B). A potential difference of -700 mV was used to drive molecules out of the nanopipette. The right panel shows one representative event peak from each trace, solid horizontal line represents the calculated baseline, green and red dotted vertical lines represent the beginning and the end of the event respectively. The population scatter for the circular and linearized plasmid detection in PBS is shown (C). N refers to the total number of events recorded.
Supporting Figure 8. Size exclusion chromatography of β-galactosidase. β-galactosidase was purified using a Superdex 200 10/300 GL column. (A) The elution profile of β-galactosidase. The bracket indicates the volume (4 ml) collected for use. The β-galactosidase elution profile (red) is overlaid on top of the elution profiles of protein standard mixes for size exclusion chromatography (see Methods). The β-galactosidase eluted at the expected elution volume for a tetramer. (B) The calibration curve for the Superdex 200 10/300 GL column, the void volume is at 7.6 ml. The calculated molecular weight for the β-galactosidase is 491 kDa, whereas the molecular weight of the tetramer is 465 kDa.
Supporting Figure 10. The 60s traces for β-galactosidase detected by the nanopipette in different concentrations of PEG 8000 or glycerol. 1 µM of β-galactosidase was used to fill the nanopipette prior to immersion into a bath containing either PBS (A), various concentrations of PEG 8000 (B), or glycerol (C). A potential difference of -700 mV was used to drive molecules out of the nanopipette. N refers to the total number of events recorded. The right-hand panels show one representative event peak from each condition, solid horizontal line represents the calculated baseline, green and red dotted vertical lines represent the beginning and the end of the event respectively.