IMPROVEMENTS RELATING TO PURIFICATION OF BIOMOLECULES

The present invention relates to an apparatus for, and methods of, improving purification, separation and/or analysis of analytes or molecules and fragments thereof using electrophoresis. The apparatus and methods of the present invention are particularly useful in the manipulation of nanoparticles and oligonucleotides, and in methods of DNA origami fabrication, functionalisation, and purification the invention includes inter alia a kit of parts.

BACKGROUND Electrophoresis is a standard laboratory technique commonly used to separate charged molecules such as DNA, RNA and proteins according to their size. An electric current is applied across a gel thereby establishing a positively charged end and an opposing negatively charged end. The charged molecules placed into the gel move towards the opposite charge side of the gel. Smaller molecules migrate through the gel quickly travelling further than larger molecules that migrate more slowly and therefore will travel a shorter distance. Hence molecules are separated by size.

While gel electrophoresis is often used in a purely analytical manner it is also possible to refine/purify samples directly from bands identified in the gel itself. This exploits agarose gel electrophoresis’s ability to efficiently separate species within any given sample in a very identifiable manner. However, while it is possible to purify directly from electrophoresis it is demonstrably inefficient. Kits that do so require other equipment, such as centrifuges, to facilitate them. Other methods forgoing electrophoresis completely are very expensive, time consuming/labour intensive, and complex. The device of the present invention, termed “Electrophoresis Matrix Alternator” or EMA provides a solution to all of these issues by providing a simple method to purify charged molecule strands directly from agarose gel in high throughput. Furthermore, it allows purification directly, in solution, of individual species within a given sample by exploitation of any difference in physical properties, foregoing any use of agarose within the electrophoresis setup up bar its use as a capping material.

BRIEF SUMMARY OF THE DISCLOSURE

According to a first aspect of the invention there is provided an apparatus comprising a hollow insulating channel having first and second opposing ends, the first end comprising an open loading port for receiving a capping material which, in use, seals the loading port, the second end being capped with a porous dielectric capping membrane, wherein both the capping material and the capping membrane permits an electrophoretic force/ electrophoretic field to permeate the apparatus and allows passage of a sample contained within the insulator channel into a collection reservoir positioned at the second end. The volume of the being dictated by the volume of the capping material in relation to the overall volume of the insulating channel, or, if the loading port is sealed by a membrane or exterior cap, the volume of the hollow channel itself.

The second end is capped by a membrane described below in a manner that retains the sample. Preferably, the capping membrane is fixed or fixable.

Preferably, the capping material is either agarose gel from a pre-run gel containing the sample or is molten agarose gel. Alternatively, the capping material maybe any other material that is capable of providing a seal to the loading port and that will permit passage of an electric field through the insulator channel while retaining the sample.

Preferably, the porous dielectric, insulator or conducting capping membrane, is chosen for the specific origami/nano-particles to be fabricated and/or purified, using the specific pore size, permittivity and conductivity to allow separation of the desired product from unwanted residual DNA and nano-particles. The typical physical parameters of are, pore size between membrane would be chosen from 0.1 - 1000 nanometers, relative permittivity (e _G ) with a magnitude between 0 < e _G < 10 ² , and conductivity between (s) 0 < s < 3x10 ⁶ (W ¹ cm ^-1 ).

Preferably, the reservoir portion further includes a vertical channel perpendicular to the insulator channel and parallel to an applied electric field. The vertical channel permits direct access to the collection reservoir. The vertical channel may be extended out from the device creating a chimney which maybe composed of the same material as the insulator channel.

Preferably, the collection reservoir portion includes a first buffer that creates a bridge between the gel in the insulator channel and the porous dielectric capping membrane and the analytes or molecules collect in said collection reservoir portion for recovery. According to a further aspect of the invention there is provided a method of purifying an analyte or molecule in a sample in the sub 100 nanometer range, the method comprising:

(i) sealing the loading port with a capping material or sample containing capping material if it is not sealed by a fixed membrane creating a reservoir volume proximal to the membrane where the sample can be inserted and/or collected;

(ii); inserting and securing the apparatus in an electrophoresis tank so that it is parallel to the direction of an electric field;

(iii) collection buffer or sample containing first buffer is inserted into the reservoir;

(iv) filling the electrophoresis tank with a second buffer to a selected level;

(v) applying the electric field for a selected period of time;

(vi) collecting the eluted or purified sample from the reservoir.

Preferably, the reservoir portion is filled with the first buffer so that it contains the required volume of elution buffer and also creates a constant bridge between the agarose filling the insulator channel and the membrane.

The method may use any buffers under which electrophoresis of samples may be conducted. This also includes the use of separate/distinct buffers within the device reservoir/reservoirs and the tank itself, e.g. a sample loading buffer or first buffer within the device reservoir such as standard TBE, with another second buffer (e.g. containing magnesium chloride, TBE MgCL ₂ ) in the electrophoresis tank on the outside, so DNA is unable to elute out from the membrane under the MgCL ₂ ionic conditions and is trapped in the recovery reservoir to be collected.

In one embodiment of the invention the method is used for purification of between sub 5 to 10 nanometer particles.

It will be appreciated that the methods of the present invention are particularly well suited to the purification of DNA origami and nanoparticles, and other functional groups, used for its decoration. DNA origami is a nanofabrication technique which can produce arbitrary shaped structures with sub 5 nanometer feature size in high throughput. This technique has been suggested as a route for the fabrication of nanotechnology, such as optical metamaterials, which are difficult to mass produce by conventional methods. Use of DNA origami as nanocircuitry requires functionalisation with metalisation and/or semiconducting material of the DNA scaffold. A common method of functionalisation is the complementation of DNA sequences attached to metallic/semiconducting nanoparticles to site specific regions on origami. Contemporary purification protocols are inefficient for the purification of sub 10 nanometer nanoparticles in the high concentration required for the functionalisation of complex designs with metals and/or semiconducting materials.

The present invention provides methods for the purification of thiol-DNA functionalised nanometer gold nanoparticles, and semiconducting nanoparticles, to the level required for high throughput origami fabrication, including sub 10 nanometer particles, in greater than 90% yield. Additionally, the invention also provides a method for the manipulation and concentration of sub 10 nanometer functionalised nanoparticles, from native agarose gel electrophoresis, without the further use of ultra-centrifuge. Finally, the system also shows effectiveness in recovering small DNA sequences in higher throughput than existing techniques.

According to a yet further aspect of the invention there is provided a porous dielectric capping membrane in purifying an analyte or molecule in the sub 10 nanometer range from an electrophoretic gel.

According to a yet further aspect of the invention there is provided a kit of parts comprising at least one apparatus according to a first aspect of the invention and instructions for use.

The kit provides a way of collecting oligonucleotide sequences and purifying small (sub 10 nanometer) nanoparticles without the use of centrifuge/ultracentrifuge/further equipment/materials. The only required kit is the native tank used to analyse the samples.

It will be appreciated that preferred features ascribed to one aspect of the invention applies mutatis mutandis to each and every aspect of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 shows the basic components of the EMA columns of the present invention. Figure 1A shows a side cross-sectional view of the insulator channel and reservoir portion, Figure 1B shows a plan view of Figure 1A. Figure 1C shows the same view as Figure 1 A with the semi-permeable dielectric membrane cap. Figure 1 D shows the plan view of Figure 1 C.

Figure 2 shows a schematic diagram of one embodiment of the method of the present invention in a purification technique. Figure 3 shows a schematic diagram of further embodiment of the method of the present invention in an elution purification technique.

Figure 4 shows a schematic diagram of further embodiment of the device of the present invention including a vertical channel perpendicular to the EMA column body. Figure 4A shows the basic EMA with respect to the buffer level, Figure 4B shows an agarose gel example and Figure 4C shows an alternative embodiment of Figure 4A.

Figure 5 shows a schematic diagram of further embodiment of the device of the present invention including a tab. Figure 5A shows an agarose gel example, Figure 5B shows a side loading schematic diagram and Figure 5C shows a schematic top-loading embodiment with tab.

Figure 6 shows examples of recovery of nanometre gold particles from a conjugated DNA sequence. Figure 6A shows the first step wherein excess thiolate DNA is separated from the sample of Interest. Figure 6B shows the excess band of interest inserted into the EMA column of the present invention. Figure 6C shows resumption of electrophoresis and sample egress.

Figure 7 shows direct visual comparison of recovery efficiency between the EMA of the present invention and Freeze and Squeeze methods. Figure 8A (equivalent to Figure 6A) shows a gold nanoparticle sample in agarose inserted into the EMA device. The collection reservoir is visible at the bottom of the device distinct from the agarose above it. Figure 8B (equivalent to Figure 6B) shows the sample post electrophoresis. The nanoparticle fraction is completely eluted into the reservoir. The remaining agarose is clear. Figure 8C shows an agarose sample post elution using the Freeze and Squeeze method. Nanoparticle residue is still clearly visible. Figure 8D shows the eluted nanoparticle sample using the Freeze and Squeeze method.

Figure 8 shows: lane 1) 10 nanometer; lane 2) 3.5 nanometer; lane 3) 5 nanometer gold spherical nucleic acids run through a 1% agarose gel.

Figure 9 Left gel electrophoresis of an un-purified functionalised gold nanoparticle samples. The white smear below the band shows high levels of unconjugated DNA in the solution. Right the purified sample shows significantly lower levels of unconjugated DNA.

Figure 10 shows recovered images of metalized origami “C” boards.

Figure 11 UV illumination of agarose gel electrophoresis of left un-purified and right EMA purified DNA origami fabrication solutions.

Figure 12 AFM images of samples recovered via left freeze and squeeze and right purified by EMA applied to a mica sheet and dried completely under nitrogen. The mica sheets were then washed with ultra- purified water, and dried again, before examination.

Figure 13 shows recovery of 250, 500, 750 and 1000 base pair bands using the method of the present invention versus a kit and Freeze and Squeeze methods.

Figure 14 shows recovery of sub 100 base pair DNA sequences using the method of the present invention.

Figure 15 shows a general explanation of the device and its operation 1. A. The devices insertion channel. B. The insulator, hollow, body of the device. C. Insertion/Withdrawal window into device reservoir. D. Task specific membrane capping body of device. 2. The body of the device must be filled. This is done by either insertion of raw agarose or agarose containing a sample of interest to be eluted. 3. The reservoir is filled with electrophoresis buffer and electrophoresis initiated. The sample diffuses out of the agarose into the reservoir where it can be collected directly. 4. A sample for purification is inserted directly into the reservoir. Electrophoresis is initiated and a sub species within the sample is eluted while the other is retained.

Figure 16 shows the device of the present invention for the controlled elution of pre purified nanoparticles into a known volume with easy recovery. The recovery opening allows the collection reservoir to be filled and the sample to be collected post electrophoresis. The reservoir is capped on the side by the agarose filled body of the device. It is this area in which sample is inserted while the agarose used to fill the device is still molten. The end of the column is capped with a fixed regenerated cellulose membrane. This prevents sample escaping while permitting the electric field to propagate through the device. The insulated backplate prevents sample reaching the bottom of the column and drifting back into the agarose cap. Addition of the insulated region also causes sample to collect there allowing quick concentration using the device.

Figure 17 shows elution of sample from agarose into a buffer filled capture well. Figure 17.1 shows excised agarose shards containing purified nanoparticles of interest as described in Figure 4.2, the shards are encased within agarose in the body of the device. Figure 17.3 shows electrophoresis is engaged and the nanoparticles are eluted from the shard they are inserted within, into the agarose filling the body of the device, into the recovery reservoir.

DETAILED DESCRIPTION

Conventional scaffolded DNA origami fabrication, and the more emergent field of DNA brick based construction, are techniques which employ the manipulation of 100s of sub 100 base pair nucleic acid sequences to create nanoscale architectures in high throughput. For visible light/EM applications or biomedical uses, the DNA origami itself is not a functional unit but a scaffold to which interactive units, typically spherical nucleic acids/functionalised nanoparticles, are attached. Gold nanoparticles are commonly used, as well as other materials, in a variety of sizes.

One method of attaching a functional group to a DNA origami is to conjugate it to a DNA sequence which then binds to a site-specific region on the origami. In the case of gold nanoparticles, a material commonly conjugated to origami, this is done by securing thiol terminated DNA sequences to the nanoparticles surface. If nanoparticles are not sufficiently ‘coated’ in DNA sequences during this process they will not be stable under the conditions required for further purification or origami fabrication. This means in practice, even for small nanoparticles, a 100-fold ratio of DNA sequence to nanoparticle is used. Typical DNA nanofabrication methodologies share the commonality of excess components being used to ensure successful fabrication. This leads to stepwise purification methodology. For instance, gold nanoparticles must be purified of excess thiolated DNA before they are attached to structures, and the fabricated origami structures must be purified completely of all excess DNA components before this attachment takes place.

The requisite for complete purification of fabrication components is due to the small size and relatively high ratio of excess DNA sequences used in fabrication and the efficiency and predictability of Watson-Crick base pairing. If small sequences are unattached and still present, they will bind, and block, complementing sites on structures more rapidly than those correctly attached to larger groups. Incorrect or incomplete purification is typically associated with protocol failure.

There are several purification protocols associated with both the purification and recovery of gold nanoparticles, and other similar functional groups, in preparation for conjugation with DNA origami. The ‘Freeze and Squeeze’ is a method commonly used for the recovery of both DNA origami and functionalised nanoparticles.

The Freeze and Squeeze method incorporates a pre-purification electrophoresis step: A sample containing different constituent components is divided into individual species within an agarose medium. These species, separated into bands dictated by mass or charge, can then be identified and recovered. This is useful as it allows complete separation of species within a sample before extraction takes place. A section of agarose, containing the sample of interest, is removed from the gel and placed in a recovery column incorporating a fine cellulose mesh. When the column is centrifuged the mesh retains the agarose component of the sample, while the running buffer and a fraction of the sample it contains, is eluted. Freeze and Squeeze is effective at recovering uniform or heavily purified samples: the electrophoresis step allowing very high selectivity in the fraction of the original sample recovered. However, the method is associated with low recovery rates due to sample being retained in the agarose lattice when the centrifuge step is conducted.

A contrasting purification technique is serial centrifugation cycles. The centrifugal technique relies on the fractional separation of species within a solution by only their relative mass. As such it is only practical to separate 2 distinct species within a sample in a single step. Typically, in DNA based nanotechnology fabrication a spherical nucleic acid is purified from excess DNA strands which remain in solution and are discarded. For nanoparticles over 10 nanometers in size this method does allow for almost complete recovery of sample.

As the mass of the nanoparticle lowers the gravities required to bring the sample out of solution increase. For sub 10 nanometer nanoparticles this requires the use of industrial ultra-centrifuge. This is practically ineffective when dealing with the microliter scaled samples prevalent in origami fabrication.

The Electrophoresis Matrix Alternator (EMA) method allows recovery and/or purification of sample directly from agarose/buffer. This means it can be used to recover defined species in a similar manner to Freeze and Squeeze or forego the initial purification in native agarose gel electrophoresis, that the Freeze and Squeeze method requires, and purify a sample directly in solution. EMA also allows almost complete recovery of sample in a controllable volume/concentration allowing purification on a similar scale to centrifuge. However, unlike centrifuge-based purification, small nanoparticles, down to at least the 3.5 nanometre level, can be effectively purified/collected in a tank in a single step.

EMA operates by either channelling a sample directly from agarose gel into its collection reservoir portion or by inserting a sample in solution directly into the collection reservoir portion itself. The difference between these methods is collection and size separation and is illustrated in Figures 2 and 3 described hereinafter. The gel is a standard electrophoresis gel and can be agarose, polyacrylamide or variant thereof or a starch, selection of which is not intended to limit the scope of the application. To enable collection of samples the buffer inside the reservoir portion is typically filled with standard Tris-borate-EDTA buffer (TBE) and the buffer filling the tank itself is TBE MgCh. It will be appreciated that other buffers commonly used for nucleic acids such as Tris-acetate- EDTA buffer (TAE) would be equally applicable. As DNA is unable to elute out from the porous dielectric membrane under these ionic conditions, it is trapped in the recovery reservoir portion and can be subsequently eluted. When purification is conducted standard TBE is used in both the recovery reservoir portion and the tank itself. DNA is free to elute out of the sample past the porous dielectric membrane but anything larger, or with a charge preventing it passing the membrane, is retained. This allows effective separation of DNA from nanoparticles. This method can also be used to concentrate sub 10 nanometre nanoparticles. If sub 10 nanometre nanoparticles are run through an agarose gel they can be purified and then inserted into the EMA device in agarose. Because the device allows elution into an arbitrary volume very large samples of agarose can be eluted into very small volumes (~10 microliters). This provides a protocol that would be inefficient without the use of ultracentrifuge.

The EMA device of the present invention advantageously allows manipulation of sub 10 nanometre diameter nanoparticles such as gold nanoparticles functionalised with DNA. The EMA has demonstrated concentration of 3.5 nanometre gold nanoparticles in a small electrophoresis tank. The EMA can also be used to purify excess DNA sequences from gold nanoparticles with negligible loss of gold sample. A further advantage of the EMA apparatus and methods of the present invention is in the field of archeogenetics where samples are usually both small and the initial throughput is very much a limiting factor. Using EMA enables a high throughput and as such provides a significant contribution to DNA nanotechnology.

Reference herein to a “channel” refers to the function of the main body of the device of the present invention in that it provides a passageway for fluidic movement and an electric field between two opposing ends, the channel may also be referred to as a pipe, tube, column or cylinder. It may in cross-sectional diameter be circular, oval, square, oblong or any other geometric shape the selection of which is not intended to limit the scope of the invention nor are its dimensions. Accordingly, the channel may be millimetres, centimetres or even meters in length or in cross-sectional area.

Reference herein to an “insulator” refers to a material having high resistivity and whose internal electric charges do not flow freely and through which very little electric current will pass even under the influence of an electric field. An insulator effectively provides electrical obstruction within the electrophoresis system. Suitable insulating materials for the insulator channel of the EMA column, include but are not limited to plastics, polyurethanes, polyurethane derivatives, glass, rubber and so on.

A range of insulator materials can be used, this list includes but is not limited to; mica, glass, resin, polytetrafluoroethylene, rubber, wax, perfluoroalkoxy, polyethylene, polyvinyl chloride, silicone and other composite polymer materials Reference herein to “semi-permeable” is synonymous with “porous” and refers to the physical properties of the membrane itself. It is intended to encompass that the membrane functions to allow certain moieties to pass through by diffusion but to prevent others from so doing. The pore size of the membrane can be selected according to a user’s requirements, the pore size acts to determine what analyte/molecule is retained within the channel and what is permitted to run out the end of the body into the collection well.

The membrane could be fabricated from a range of materials either individual elements, compounds or composites, this list includes but is not limited to; regenerated cellulose, cellulose ester, copper, steel, nickel, silver, gold, iron, silicon, Glass, PTFE, polyethylene, polyimide, polypropylene, polystyrene, titanium dioxide, strontium titanate, barium strontium titanate, barium, conjugated calcium copper titanate, polysulfone, polyethersulfone, etched polycarbonate and collagen. Reference herein to “dielectric” is with reference to the physical molecular property inherent in materials capable of impeding electron movement and hence creating polarization within the substance, when exposed to an external electric field. The dielectric membranes of the present invention allow an electric field to pass through it.

The dielectric, insulator or conducting membrane, is chosen for the specific origami/nano particles to be fabricated and/or purified, using the specific pore size, permittivity and conductivity to allow separation of the desired product from unwanted residual DNA and nano-particles. The typical physical parameters a membranes would be chosen from are, pore size between 0.1 - 1000 nanometers, relative permittivity (e _G ) with a magnitude between 0 < e _G < 10 ² , and conductivity between (s) 0 < s < 3x10 ⁶ (W ¹ cm ^-1 ).

Reference herein to a “reservoir” refers to an area at one end of the channel defining dimensions for containing the buffer into which the sample is eluted. The reservoir comprises a collection well which is in contact with the porous dielectric membrane. The collection well, in use, may contain a buffer volume that is not a limiting feature of the invention.

If will be appreciated that the apparatus and methods of the present invention are suitable for separation and purification of any molecule or analyte that can hold a charge for example DNA, RNA, proteins, conjugated proteins and analytes or molecules bound to, for example and without limitation beads, microspheres or metals. Nanoparticle Purification

The insulator channel of the EMA device is backfilled with agarose containing nanoparticles purified via native gel electrophoresis, leaving the required area free in the reservoir portion free for insertion of recovery buffer for their collection, or, the device is backfilled with agarose leaving the required volume free proximal to the membrane that caps the device for sample insertion. The EMA device is then inserted, and secured, in an electrophoresis tank parallel to the direction of the electric field. The tank is filled just below the lowest point of the reservoir portion in the EMA device. The sample, or buffer for recovery of sample contained in the agarose capping material is inserted directly into the reservoir portion. Electrophoresis is typically conducted at 50V for 30 minutes but any combination of voltage or time the tank permits could be used. The sample can be collected directly from the reservoir portion via the channel in the device and is ready to be used in origami fabrication/further experimentation. • Recovery of 100-300 base pair sequences from 1KB DNA ladder (Band of interest protocol)

Firstly, the ladder is separated, 5ul of 1 KB GeneRuler™ ladder is inserted into a loading well of 1% 0.5 TBE agarose gel. The gel is then typically run at 100V for an hour. The EMA device can be inserted into the gel directly thus encapsulating the band of interest. Alternatively, the band of interest can be excised (Typically this process would be conducted if the band of interest is wider than the EMA device) and inserted into the device. The device can then be backfilled with molten agarose. Whichever pathway is used the reservoir portion must be filled in a manner such that it contains the required volume of elution buffer and also creates a constant bridge between the agarose filling the insulator channel and the porous dielectric membrane. The device should be inserted and secured in the electrophoresis tank parallel to the direction of the electric field. Once the device is secured the entire tank should be inserted into an ice bath. The tank should be filled, to the level just below the lowest point of the reservoir portion, typically with 0.5X TBE 1mM MgCI2. The reservoir portion should be filled with 0.5X TBE. The sample can then be collected directly from the reservoir portion.

Figures 1A to 1D shows side and plan views of the basic components of an EMA column according to the present invention. The column comprises a hollow insulator channel (1) having opposing open ends “A” and “B” within the same plane of orientation. The EMA column is basically an open-ended channel or hollow body composed of an insulating material that is also chemically inert or resistant to chemicals in the buffer contained within the buffer tank. The hollow insulator channel (1) can be filled with or inserted directly into an agarose gel thereby completely trapping a sample therein. The insulator channel is filled at end “A”, the loading port end with agarose either from a pre-run gel containing sample or molten agarose to act as a capping material and prevent diffusion of sample. The end of the channel “B”, comprising the reservoir or opening and the porous dielectric membrane is not filled with agarose in this manner, rather it is left as a vacant reservoir to allow the insertion of a collection buffer. Once prepared the EMA is then inserted into a tank, where the reservoir portion or opening is always above the running buffer level, and electrophoresis is then conducted. At end “B” there is provided a reservoir portion (2) or well whose function is to collect a sample directly or to retain an inserted sample in solution in the collection buffer there within. In the Figures, the reservoir portion (2) is depicted as “V” shaped however it will be appreciated that this is a non-limiting design feature and that other reservoir portion shapes such a “U” would be equally appropriate so long as the reservoir portion retains its purpose as a collection/retaining well for the sample. It is important that, in use, the reservoir portion is above surface of the buffer level within the buffer tank. Once a sample has been loaded it is sealed within the reservoir portion at end “B” by means of a porous dielectric membrane (3). The porous dielectric membrane (3) is fixed at end “B” so as to create a cap over the reservoir portion (2). The porous dielectric membrane (3) permits passage of an electric field, once applied during electrophoresis, to permeate through the insulator channel (1). The membrane retains certain moieties or species within any given sample whilst allowing others to elute.

The present invention advantageously provides for simple purification of fragile, precious or very small electrophoresis samples without the use of chemical reactions, centrifugation or freezing. In principle, any moiety that is robust enough to be electrophoresed can also be recovered by the methods of the present invention. It will be appreciated that the buffer used in the EMA device can be interchanged/selected to affect the migration of samples. Figure 2 illustrates schematically one embodiment of the method of the present invention in which a sample “S” such as a DNA/nanoparticle is embedded within agarose gel (4). The insulator channel (1) is inserted into the agarose gel so as cut out the sample section (5). End “A” of the insulator channel (1) can therefore also function as a cutting means for removal of a sample within the agarose gel and advantageously provides a sample section (5) with dimensions commensurate with the inner dimensions of the insulator channel (1) thereby acting as a plug within the insulator channel. The sample section (5) is then urged forward through opening (6) at end “A” towards end “B” and guided within the insulator channel (1) until the space between the sample section (5) and end “B” is equal to the volume of buffer required. The distance between sample section (5) and end “B” is termed the reservoir “C” and this is filled with buffer. An electric field is then applied in the same direction parallel to ends “A” to “B” so as to initiate electrophoresis. The sample (7) is then eluted out of the agarose gel and into the buffer contained between the sample section (5) and end “B” i.e. the reservoir “C”, from there the sample travels towards area “D” which comprises the reservoir portion (2) and the fixed terminal porous dielectric membrane cap (8). The porous dielectric membrane (8) allows the electric field to effectively permeate the insulator channel while the sample is retained. Area “D” is shown in plan view in area Έ”. The reservoir portion is provided with a window which sits above the level of the buffer in the buffer tank. Sample (7) is then eluted into buffer in the reservoir portion and may be directly accessed or collected therefrom. In an alternative embodiment of the method of the present invention as depicted schematically in Figure 3, electrophoresis can be conducted on any small components in the sample so that they are free to elute through the porous dielectric membrane (8) fixed over end “B”. In this version of the method, agarose gel (4) in the form of a disc is used to block or cap off end “A” so as to prevent the sample “S” from escaping from the insulating channel (1). Sample “S” is inserted directly into the insulating channel containing a volume of buffer “C”. During electrophoresis small components (10) can cross the membrane (8) whilst large sample constituents (9) are retained.

Referring to Figures 4, there is shown a further embodiment of the apparatus of the present invention in which the EMA device sits semi submerged in the buffer tank. The sample must be gated in a manner that prevents it eluting into the surrounding buffer at either end “A” or “B” but leaves it accessible to a user. Figure 4A shows an EMA comprising an insulating channel (1) with a reservoir portion (2) adjacent the fixed membrane (3) The maximum height of the sample (12) must be lower than the buffer level (11) in the buffer tank. If the buffer level is higher than the lowest point of the reservoir portion then the sample will no longer be trapped and would leak out. Figure 4B shows an example of an EMA column with respect to the buffer level and the maximum height (13) of the sample. By including a vertical channel (14) or chimney perpendicular to the EMA column body, allows the EMA column to be completely submerged and operable below the buffer level (11). It is envisaged that this embodiment would advantageously facilitate use of the device in the methods of the present invention.

In a yet further embodiment of the apparatus of the present invention depicted in Figures 5, there is shown in Figure 5A an EMA column filled with agarose gel.

Currently the EMA device must be carefully filled with agarose and a well or reservoir portion created at end “B” for either collecting or inserting the sample in solution this reservoir portion is next to the porous dielectric membrane (3). Figure 5A shows an EMA device (15) filled with agarose gel. In Figure 5B the filling of insulator channel (1) is filled in a sideways fashion in the direction of arrow (16). However, should a user wish to fill a column in a vertical direction in the direction of arrow (17) it will be necessary to protect the dielectric porous membrane (3) from the agarose gel itself. Protection of the membrane and creation of a reservoir portion can be achieved using a removable tab (18). Tab (18) can be inserted at end “B” of insulator channel (1) into the area where currently a sample is inserted or withdrawn. In practise the device may be filled completely with sample and agarose, allowed to set, and then the tab removed.

It has been observed that by inserting an insulator wall, proximal to the recovery well, and perpendicular to the flow of the electric field, that sample eluted from agarose will collect in this region overtime (During electrophoresis), and is unable to escape due to electrophoretic force. This provides an efficient method of collecting samples in a known volume and can be used to concentrate several samples into a single volume. This process can be seen in figures 16 and 17. EXAMPLE 1

Studies were conducted to compare conventional standard agarose gel electrophoresis purification kits kits to the EMA of the present invention. EMA was compared to Thermo Fisher™ Purelink Quick Gel extraction kit, and Bio-Rad Freeze and Squeeze Columns, for the extraction/purification of sub 1000 base pair DNA sequences. Results from experiments carried out recovering DNA of differing lengths using commercial kits, Freeze and Squeeze™ and the EMA of the present invention are shown in Table 1 and Figure 13.

Table 1: Comparison of EMA to commercial kits for the extraction and purification of sub 1000 base pair DNA sequences

Results indicate that for standard sequences the EMA of the present invention offers a significantly (in the region of 10X) higher recovery than standard kits/methods. EXAMPLE 2

There is a broad trend that as the size of the sequence to be recovered/purified becomes smaller the methods available to do so, and their yield, become smaller. Figure 14 contrasts the EMA system and the Freeze and Squeeze method, in terms of yield, in eluting 40 base pair length DNA sequences directly from agarose. Typically, an origami scaffold is bound by similar sized staples and they are also of appropriate size for use in bio brick fabrication.

There are currently few methods available for the purification of sub-100 base pair DNA sequences from gel electrophoresis. Experiments were conducted comparing the amount of sub-100 base pair DNA recovered using conventional Freeze and Squeeze™ as opposed to the EMA of the present invention. Table 2 and Figure 14 show that, even in situations where only 1 contemporary kit provides a solution, EMA still exhibits about a 10- fold higher efficiency than the commercial solution.

Table 2: Comparison of EMA to the only commercial kit for the extraction and purification of sub-100 base pair DNA sequences

EXAMPLE 3

A sample of interest (in this case 3.5 nanometer gold nanoparticles conjugated to 20 base pair long thiolated DNA sequences) is run until excess thiolated DNA is completely separated from the sample of interest (Figure 6A).

Subsequently, the band of interest is excised from the gel and cut into small chunks using a scalpel and then directly inserted into the EMA device. The insulator channel of the EMA column is then filled with a molten agarose and left to set (Figure 6B). The EMA device is then secured in an electrophoresis tank and the reservoir filled with buffer. The tank is then also filled with buffer until the device is submerged but the recovery channel above the water line.

Electrophoresis is then resumed. Overtime the sample egresses from the gel into the buffer (Figure 6C), potentially increasing in relative concentration as it does so if the volume of the reservoir is smallerthan the volume of the gel it is recovered from. It is then collected directly for further use as appropriate.

Analysis was conducted on a sample which had undergone purification via native agarose gel electrophoresis. Elution of the sample from agarose, via the device, was characterised and directly compared to a common contrasting method of sample recovery. Gold nanoparticles, 3.5 nm in diameter, were functionalised, divided into 2 equal volumes and underwent agarose gel electrophoresis. A section of agarose containing each sample was excised with a scalpel. Each half was eluted with either our device or a Freeze and Squeeze column purchased commercially. Fig 7 Provides a direct visual comparison of recovery efficiency between the EMA and Freeze and Squeeze methods. The final recovery volume via EMA was ~25 microlitres with almost complete purification from agarose (Fig 7 A and 7 B). In contrast, the Freeze and Squeeze method recovered into ~80 microlitres (Fig 7 D). There is also significant residue remaining in the agarose the sample was eluted from (Fig 1 C).

Fig 8 shows that when nanoparticles are sub 10 nanometer the size of the band required for recovery increases dramatically. If the relative concentration of nanoparticle in solution is important for its further use (as in origami fabrication) then recovery of larger quantities of sample in a less concentrated solution is not appropriate.

EXAMPLE 4

A gold nanoparticle sample functionalised with excess thiolated DNA sequences was purified. The sample is inserted directly into a capped EMA device reservoir in solution that is compatible with electrophoresis, the device placed in a tank, and the buffer level of the tank filled to below the water line of the recovery channel, and electrophoresis initiated. Over time the smaller thiolated DNA sequences are free to permeate through the membrane away from the sample. The functionalised nanoparticles are retained and over time purified of all excess thiolated DNA sequences that are not correctly attached to their surface.

Efficiency of purification was first observed directly via UV visualisation of unconjugated DNA in both un-purified and purified samples. High level of mobile unconjugated DNA were observed leaving the un-purified gold nanoparticle, with trace amounts noticeable on the post purified sample Figure 9. An effective test indicating confirmation of any given method is fabrication using the sample post purification. To test the nanoparticles for their suitability for use in DNA origami decoration they were adhered to a 50nm x 50 nm DNA origami board and AFM was conducted using a Bruker Dimension Icon QNM tapping in air. Figure 10 is an AFM measurement of a structure fabricated using the purification method described above. The designs are all decorated in ‘C’ motifs dictated by binding sites on the boards.

EXAMPLE 5

A DNA origami solution containing all fabrication components was purified of excess staples. The sample is inserted directly into a capped EMA device reservoir in solution that is compatible with electrophoresis, the device placed in a tank, and the buffer level of the tank filled to below the water line of the recovery channel, and electrophoresis initiated. Overtime the smaller staple DNA sequences are free to permeate through the membrane away from the sample. The larger DNA origami are retained and over time purified of all excess staple DNA sequences.

Efficiency of purification was first observed directly via UV visualisation of unconjugated DNA in both un-purified and purified samples. High level of mobile staple DNA were observed leaving the un-purified DNA origami sample, with trace amounts noticeable on the post purified sample Figure 11.

Purification of DNA origami boards throughput was compared to recovery of purified DNA origami boards from native agarose gel electrophoresis via a freeze and squeeze column Figure 12 - left freeze and squeeze recovery right EMA purification.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.