WO2009022152A1

WO2009022152A1 - Single molecule spectroscopy using nanoporous membranes

Info

Publication number: WO2009022152A1
Application number: PCT/GB2008/002776
Authority: WO
Inventors: Joshua Edel
Original assignee: Imperial Innovations Limited
Priority date: 2007-08-16
Filing date: 2008-08-15
Publication date: 2009-02-19
Also published as: GB0716005D0

Abstract

Apparatus for characterizing molecules comprises a barrier (10, 14) between a first volume (24) and a second volume (32), the barrier having a pore (18) through it, a pair of electrodes (36, 38) arranged to generate an electrical potential difference between the first and second volumes to drive molecules from the first volume to the second volume, and optical sensing means (44, 40, 42) arranged to detect light from the pore thereby to detect passage of a molecule through the pore.

Description

SINGLE MOLECULE SPECTROSCOPY USING NANOPOROUS

MEMBRANES

FIELD OF THE INVENTION The present invention relates to the characterization of molecules. It can be used in the characterization of many types of molecules, but has particular application in the characterization of large molecules such as DNA.

BACKGROUND TO THE INVENTION In the post-genomic era much effort is still focused on identifying genes responsible for specific biological functions (or diseases) and determining the DNA sequence bearing the information. The need to perform such processes in a rapid and efficient manner has dictated the creation of a new generation of experimental tools. Of particular note has been the considerable progress in the development of microfluidic systems for use in the chemical and biological sciences (deMello, AJ. ; Nature 2006, 442, 394-402). Interest in such technology has been driven by a range of fundamental features that accompany system miniaturization. These features include the ability to process and handle small volumes of fluid, improved analytical performance when compared to macroscale analogues, reduced instrumental footprints, low unit costs, facile integration of functional components and the exploitation of atypical fluid dynamics to control molecules in both time and space. Based on these beneficial characteristics, microfluidic chip devices have been used to good effect in a wide variety of applications including nucleic acid separations, protein analysis, small-molecule organic synthesis, DNA amplification, immunoassays, DNA sequencing, cell manipulations, and medical diagnostics ^Dittrich, P. S.; Tachikawa, K.; Manz, A.; Analytical Chemistry 2006, 78, (12) , 3887 - 3908).

More recently, significant effort has centered on scaling down microfluidic systems to create features such as channels and pores with cross-sectional dimensions most easily measured in nanometers. Such 'nanofluidic' devices open up new opportunities for fundamental and applied studies of chemical and biological phenomena. In basic terms, nanofluidics describes fluid flow in and around structures with dimensions smaller than a few hundred nanometers. A number of defining characteristics separate fluid flow at the nanoscale from flow in larger environments. First, flow occurs in structures which are comparable to natural scaling lengths, e.g. the Debye length in electrolyte solutions. Second, internal surface area-to-volume ratios can be enormous. Third, diffusion becomes an extremely efficient mass transport mechanism at this scale and finally the ultra- small volumes associated with nanofluidic environments allow effective confinement of analyte molecules to defined regions and thus theoretically allow perfect detection efficiencies in analytical applications. All of these characteristics can be exploited to significant advantage when processing or analyzing chemical and biological systems. For example, single molecule-based fragment sizing methods are known using devices involving small capillaries and flow cytometry ⁽Yan, X.; Grace, W. K.; Yoshida, T. M.; Habbersett, R. C; Velappan, N.; Jett, J.

H.; Keller, R. A.; Marrone, B. L. Analytical Chemistry 1999, 71, (24), 5470-5480). Moreover, single molecule DNA fragment sizing is also known in microchannel environments ⁽Eijkel, J. C. T.; van den Berg, A.; Manz, A. Electrophoresis 2004, 25, (2), 243-252). In these situations, channel dimensions are still relatively large, and thus restrictions are imposed on the optimal resolution that can be obtained, achievable analytical throughput and non-perfect molecular detection efficiencies. For example, large channel dimensions require relatively large observation windows for uniform illumination of the entire channel width. Therefore, only slow flow speeds, or low sample concentrations, can be employed to avoid multiple molecular occupancies. Second, the larger the channel the greater noise contributions become from background interferences. By reducing channel dimensions to the nanometer scale, all analyte molecules can be maneuvered through a defined detection region and can be analyzed rapidly and with high signal-to-noise ratios.

The creation of nanochannels or nanopores in thin membranes is also known and is of interest due to the potential to isolate and sense single DNA molecules while they translocate through the highly confined channels (Akeson, M.; Branton, D.; Kasianowicz, J. J.; Brandin, E.; Deamer, D. W. Biophysical Journal 1999, 77, (6), 3227-3233). Short translocation lengths make decontamination of the nanopores facile whilst maintaining effective spatial confinement for single molecule detection. Nanopores for such applications can be fabricated in insulating lipid membranes (Meller, A.; Nivon, L.; Brandin, E.; Golovchenko, J.A.; Branton, D.; Proceedings of the National Academy of Sciences 2000, 97, 1079-1084), silicon dioxide membranes (Storm, A. J.; Chen, J. H.; Zandbergen, H. W.; Dekker, C. Physical Review E 2005, 71, (5)) and silicon nitride membranes (Li, J.; Stein, D.; McMullan, C; Branton, D.; Aziz, M. J.; Golovchenko, J. A. Nature 2001, 412, (6843), 166-169, and Striemer, C. C; Gaborski, T. R.; McGrath, J. L.; Fauchet, P. M. Nature 2007, 445, (7129), 749-753) and thus in principle may be integrated into monolithic analysis systems. Generally the detection of translocation events is performed electrically by measuring an applied ionic current (Li, J. L.; Gershow, M.; Stein, D.; Brandin, E.; Golovchenko, J. A. Nature Materials 2003, 2, (9), 611- 615). In simple terms, molecules translocating through a nanopore will momentarily perturb the ionic current, with the duration of the perturbation and the magnitude of the current blockade providing more detailed information about molecular shape and structure.

SUMMARY OF THE INVENTION

The present invention provides apparatus for characterizing molecules comprising a barrier between a first volume and a second volume, the barrier having a pore through it, a pair of electrodes arranged to apply an electrical potential between the first and second volumes to drive molecules from the first volume to the second volume, and optical sensing means arranged to detect light from the pore thereby to detect passage of a molecule through the pore.

The molecules may be DNA or RNA, proteins or other molecules. The pore is preferably a nano-pore, and may have a diameter of from 1 to lOOOnm, preferably from 5 to 500nm. However in some cases other sizes of pore can be used.

The barrier may include a light shielding layer which is at least partially opaque. This may be formed of a metal, such as aluminium, gold, chromium or platinum, and is preferably opaque at the operational wavelengths of the optical sensing means.

The apparatus may further include a light source arranged to direct light at the pore. The light source may include one or more mirrors or one or more lenses. The light source may be located on the opposite side of the barrier to the optical sensing means, but is preferably on the same side of the barrier as the optical sensing means.

One of the electrodes may be transparent, at least at the operational wavelengths of the optical sensing means, and located between the pore and the optical sensing means.

One of the volumes may be partly defined by a wall which includes the transparent electrode, which may for example be of indium tin oxide (ITO). The first volume may be formed, at least partly, as a pore in one or more layers of material. The one or more layers of material have a further pore therethrough connected to the second volume.

This can provide a convenient structure in which solutions can be introduced into and removed from first and second volumes quickly and efficiently. These pores may be formed as micro-pores, for example of diameter from 1 to 1000 microns, i.e. of greater diameter than the nano-pores. This helps to enable control of the contents of the first and second volumes.

The wall may be on one side of the barrier and the one or more layers of material on the other side of the barrier. This allows the introduction and removal of the solutions from one side of the apparatus, and the optical sensing to take place on the other. This is useful if a large array of such devices is used, as common sheets of material can be used for all of the devices.

The barrier may have a plurality of pores through it and the optical sensing means may be arranged to detect light from some or all of the pores. The barrier may be supported on a support layer, which may for example be silicon. The support layer may have an aperture through it exposing a region of the barrier which has the pore, or the plurality of pores. The aperture may therefore form part of the first or second volume. The apparatus may further comprise a control electrode the potential of which can be varied so as to vary the electrical field in the pore. The control electrode may extend around the pore, for example it may be formed on the barrier. Indeed it may be formed as part of the optical shielding layer, which may be electrically isolated from the rest of that layer.

The present invention further provides a method of characterizing a molecule comprising passing the molecule through a pore in a barrier from a first volume and a second volume, wherein the movement of the molecule is controlled by a pair of electrodes arranged to apply an electrical potential between the first and second volumes, and detecting light from the pore thereby to detect passage of a molecule through the pore.

The present invention in some aspects can therefore provide a system for optically detecting DNA translocation events through one, or an array of, solid state nanopores which allows for ultra high-throughput, parallel detection at the single molecule level. In some cases optical probing of fluorescently labeled DNA molecules translocating through sub-wavelength pores within a thin aluminum/silicon nitride membrane can be used. The opaque aluminum layer in this case may act as an optical barrier between the illuminated region and the analyte reservoir. In these conditions high contrast imaging of single molecule events can be performed.

The present invention will now be described by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figures Ia and Ib are schematics of a nanoporous membrane device according to an embodiment of the invention showing different steps of its fabrication;

Figure Ic is a scanning electron microscope image of the nanopore array of the device of Figures Ia and Ib;

Figure 2 is a schematic of a molecular spectroscopy apparatus including the device of Figure 1;

Figure 3 is a set of fluorescence images of two DNA translocation events occurring in an apparatus according to a further embodiment of the invention;

Figure 4 is a set of graphs showing variation of detected fluorescence as a function of time from four different pores of an apparatus according to a further embodiment under different applied voltages;

Figure 5 is a set of graphs showing fluorescence signals from a single pore acquired using an avalanche photodiode detector under different applied voltages; and

Figure 6 is a schematic of a nanopore/electrode architecture according to a further embodiment of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to Figure 1, a nanoporous device 8 comprises free standing silicon nitride membrane 10 supported on a silicon support substrate 12. The top surface of the membrane 10 is coated with a layer of aluminium 14. The support substrate 12 has a micropore 16 formed through it and the membrane 10 extends over the micropore 16. The free-standing part of the membrane which extends over the micropore 16 has an array of nanopores 18 extending through it and the aluminium coating 14 connecting the volume above the membrane with the volume within the micropore 16.In this case the silicon nitride membrane is 200 nm thick and is fabricated using standard photolithographic techniques and KOH wet etching as described by Kim et al. (Kim, M. J.; McNaIIy, B.; Murata, K.; Meller, A. Nanotechnology 2007, 18, (20)). The membrane 10 is then coated with the 100 nm thick aluminum layer 14 by thermal evaporation. Subsequently, the sub-micron sized holes are milled in a sequential fashion using a focused ion beam (FIB) at 30 kV and 20 pA to form the nanopores 18. These nanopores provide nanofluidic channels with a diameter between 250-300 nm. Figure Ic shows typical results after milling nine 300 nm wide pores in the membrane. The pores 18 have an aspect ratio close to unity.

Referring to Figure 2, in an apparatus for molecular analysis, the nanoporous device 8 is supported within a support structure 20. This support structure 20 comprises spacers 22 which form the sides of a cavity 24, with a lower cover 26 covering the underside of the cavity 24 and an upper cover 28 covering the top of the cavity 24. The upper cover 28 comprises a glass layer 28a and a spacer layer 28b. The lower cover comprises a glass layer 26a coated with ITO 26b on its inner side facing the cavity 24. The upper cover 28 has a first aperture 32 through it of approximately the same diameter as the micropore 16 in the nanopore device, and the nanopore device is supported against the upper cover so that the micropore 16 is sealed to the aperture 32. Further apertures 34 are provided in the upper cover 28 which open directly into the cavity 24 to the side of the nanopore device 8. The coated SiN membrane is spaced from the lower cover 26 and therefore form a porous barrier between the volume within the micropore 16, which is connected to the first aperture 32 and forms with it a first reservoir, which with the device orientated as shown is an upper reservoir, and the volume within the main part of the cavity 24, which is connected to the further apertures 34, and which forms a second reservoir which, the device orientated as shown, is a lower reservoir. An analyte solution containing the molecules to be analyzed and a buffer solution are fed into these two volumes or reservoirs so that the molecules can migrate from the analyte solution to the buffer solution through the nanoporous membrane.

Electrodes 38, 38 are provided to produce an electrical potential difference between the two volumes. In this case one electrode 36 is connected to the ITO coating to apply one potential to the solution within the cavity 24, and the other electrode 38 is provided in the aperture 32 in the upper cover 28 to apply a different potential to the solution on the other side of the membrane 10.

An imaging system, in this case in the form of an electron multiplying CCD (emCCD) camera 40 (Cascade II, Photometries) is arranged to image the porous membrane 10, and an image processing system 42 is arranged to analyze the images generated by the camera. The image the camera generates will therefore include each of the pores 18 in the porous membrane. The image processing system is arranged to identify a region of the image associated with each of the pores 18 and to monitor the variation in intensity in each of those regions over time. A light source 46 is arranged to illuminate the pores 18, in this case from the same side as the camera 40 being directed at the aluminium layer 14 of the porous membrane.

In one experiment, double stranded λ-DNA (48 kbp in length) duplexes were labeled with YOYO-I (Molecular Probes) at a ratio of five base pairs per dye molecule and the solution diluted to yield a concentration of 10 pM. The top reservoir 32 was used to contain the analyte solution, with the lower reservoir 24 containing buffer (50 mM phosphate, 200 mM KCl, pH = 7.4). Translocation of DNA through pores 18 was initiated electrokinetically by applying a voltage between the upper and lower reservoirs using the electrodes 36, 38. Assuming there is no potential drop at the interface between the electrodes and the solution, the effective electric field E across the pores would be equal to V/l, where V is the applied voltage and / the channel length (300 nm in this case). For instance, a voltage of 200 mV corresponds to a field on the order of 6.7 x 10⁵ V.m^"1. The distance between the aluminum nanopores and the ITO layer 26b was set to be 30 μm by using a thin space insuring complete isolation. However, in other embodiments, this could be from a few nanometers up to, for example, lOOμm, depending on the type of optical sensing system that is to be used. ITO was used in this embodiment as it is optically transparent and allows production of a uniform electric field at the bottom of the lower reservoir 24. The latter feature is crucial in allowing DNA to be transported quickly away from the detection probe volume, or image area associated with each pore, subsequent to detection. Prior to addition of the buffer solution, the nanopore substrate is treated with oxygen plasma to ensure the pore surfaces are hydrophilic in nature. Additionally, the plasma serves to grow a thin oxide layer on the aluminum which inhibits native aluminum dissolution under basic conditions.

The camera 40 and imaging system 42 used for detection of DNA was in this case a custom-built confocal microscope with imaging capabilities (Huebner, A.; Srisa- Art, M.; Holt, D.; Abell, C; Hollfelder, F.; deMello, A. J.; Edel, J. B. Chemical Communications 2007, (12), 1218-1220. Briefly, the excitation light from the light source 46 which in this case was a mercury lamp (filtered for 488 nm excitation) is focused onto the aluminum membrane surface 14 using a high numerical aperture objective (6Ox). Since silicon nitride is optically transparent in the visible region of the electromagnetic spectrum, the aluminum layer is used to block any excitation light from illuminating the top reservoir, thus eliminating background cross-talk between the upper and lower reservoirs. This feature is important in removing all residual fluorescence associated with the top reservoir and hence maximizing the signal-to-noise ratio associated with translocation events. Fluorescence originating from translocating DNA molecules is then collected by the objective 44 of the imaging device 40 and directed to an electron multiplying CCD (emCCD) camera (Cascade II, Photometries). The camera 40 has a pixel size of 16 μm, however when used in conjunction with the 6Ox objective and a further 3.3x post objective magnification, this generates an effective pixel size of 81 run. This is significantly below the diffraction limit resulting in translocation events being registered on an array of pixels with a fluorescence signature which is Gaussian in nature.

It will be appreciated that the arrangement described with simultaneous measurement of light intensity from a number of pores with a single camera is an efficient method for high though-put systems. A variant of this setup was used to serially probe one pore at a time and provide superior time resolution than CCD imaging (microsecond versus millisecond resolution). In this configuration an excitation laser beam is focused onto an individual pore and the fluorescence signal is acquired using an avalanche photodiode detector (AQR-141, EG&G, Perkin- Elmer). Such a system can be used for more accurate measurements in lower volume systems.

Translocation events of DNA molecules through the array of pores can be observed on a millisecond time scale. Figure 3 shows a sequence of images of typical translocation events for 4 pores under an applied voltage of 0.45 V (1.5 x 10⁶ V.m^" ^l) as captured by the CCD camera. The images are taken at times t=0 (a), t=45 ms (b), t=210 ms (c) and t=360 ms (d). Each pixel represents an area of 81x81 nm². The dotted circles in frame (a) indicate the location of the pores. Illustrations below each image frame provide an indication of the progression of DNA through the pore and the illumination plane (dotted line). It should be noted that although the CCD consists of 512 x 512 pixels, the area was cropped to a more defined region of interest of 200 x 200 pixels. This enables simultaneous probing of 9 pores at a frame rate of approximately -15 ms (67 Hz). Figure 3b illustrates the onset of a translocation event in the top left-hand pore. DNA passage through the field of view is complete after approximately 360 ms. Interestingly, the duration of the event is approximately 2 orders of magnitude greater than what has been reported by Smeets et al. using ionic current as a detection mechanism within a 10 run wide and 60 ran thick pore (Smeets, R.M.M; Keyser, U.G.; Krapf, D; Wu, M. Y.; Dekker, N.H.; Dekker, C; Nano Letters 2006, (6) 89-95). In the experiment described using this embodiment, the membrane thickness of 300 nm results in a greater entropic barrier to initiate complete translocation. Perhaps more importantly, as the technique is optical, fluorescence is continuously being detected from the onset of translocation, until the DNA strand has completed translocation and department from the field of view of the focal plane via a combination of electrokinetic and diffusive methods. This entire process is significantly longer in duration than measuring a blockage current. When the DNA is in the pore, fluorescence is isolated to the aluminium / pore interface, as the DNA strand translocates through the pore 18 the fluorescence signature is broadened and detected over a larger pixel range on the CCD. Once the translocation is completed, the molecule is no longer confined and can move from the axis of the pore as shown in Figure 3c. By diffusing freely in the illuminated reservoir, the molecule gradually works its way outside of the focal plane resulting in a progressive decrease in fluorescence intensity as shown in Figure 3d. A simultaneous translocation event is also observed on the bottom right pore in

Figures 3c and 3d.

The high contrast between the background and translocation events is directly attributable to the opaque properties of the aluminum layer 14. A thicker aluminum layer would in principle result in higher contrast; however, there is a compromise between the total length of the pore (in this case 300 run) and the potential for pore blockage. Having said this, at a low applied voltage this configuration allows discrimination of the successive translocation events whilst maintaining an acquisition area that is large enough for monitoring an array of 3x3 pores with 5 μm spacing between them. Higher density arrays are clearly possible and the pore spacing can be decreased at least down to 1 μm whilst maintaining sufficient optical resolution to resolve independent translocation events without cross talk between the pores. Once translocation is complete the DNA strand is driven towards the lower electrode resulting in no increase background fluorescence. It should also be emphasized that at the onset of experiments the lower reservoir 24 contains simply buffer. As the experiment progresses the overall DNA concentration in the lower reservoir 24 gradually increases, resulting in the possibility for a concomitant increase of the fluorescence background. This was not observed in the experiments described as the whole reservoir 24 is under illumination, and as a result molecules are readily photobleached whilst being illuminated outside of the detection window. The ionic strength of the buffer solution plays a crucial role in determining the threshold voltage at which the translocation occurs. In some cases experiments were carried out with a buffer solution containing 50 mM phosphate without the addition of monovalent electrolytes. For these experiments the voltage needed to accomplish DNA translocation was in excess of 2V, which induced sufficient Joule heating to create microscopic bubbles in the solution. Addition of 200 mM of KCl allowed for operation at sufficiently low voltages to negate this effect. At low salt concentrations (< 100 mM), the effect of the negatively charged walls sets in, resulting in translocations being affected by the counter ions which screen the walls (Dekker, C. Nature Nanotechnology 2007, 2, (4), 209-215).

It is also found that the translocation behavior of the DNA molecules within the channels greatly depends on the hydrophilic state of the surface. On injection of the DNA solution immediately after oxygen plasma treatment, DNA molecules can diffuse freely through pores without the application of voltage. Although lambda- DNA molecules are larger than the diameter of the pores (with a radius of gyration between 700 nm - 850 nm), the concentration gradient across the pores is sufficient to induce free translocations. Because the current oxygen plasma treatment has only a transitional effect, pores lose their hydrophilic character after a few hours. Accordingly, to ensure that the membrane did not allow any translocation without the application of a drive voltage, pores were wetted immediately after treatment and left for 4 hours before injection of the DNA solution. By recording a sequence of images and analyzing the intensities from the center of the pores, for example using custom written Matlab algorithms, it is possible to monitor the intensity at each pore as a function of time. Figures 4a to 4c each illustrate the intensity values from four pores, with a different applied potential in each figure. The applied voltages are 0.20V (a), 0.30V (b), and 0.40V (c). The vertical axis is arbitrary and the four signals have been shifted vertically for convenience. All data were measured simultaneously.

Observable peaks in each plot indicate simultaneous detection of translocation events through multiple pores. It is apparent that a higher voltage induces more translocation events to occur per unit time. Optical translocation events occur on a timescale between 100 - 800 ms depending on the applied voltage. For example, at an applied potential of 200 mV as shown in Figure 4a a single translocation (taking 500 ms) was observed for 4 pores over a period of 10 seconds. Increasing the applied potential by 100 mV results in the observation of 6 events over the same time period and pore window as shown in Figure 4b. A further increase by 100 mV results in 25 translocations being registered in Figure 4c. These experiments demonstrate successfully that a thin aluminum membrane containing nanometre sized holes can be used for parallel array detection (and ultimately high-throughput analysis) while maintaining the benefits of single molecule resolution and high levels of molecular confinement. For example, in a conventional solution-based single molecule detection experiment, approximately 10³-10⁴ molecules are detected within a 60 second time frame (1 molecule every 6-60 ms). (Stavis, S. M.; Edel, J. B.; Li, Y. G.; Samiee, K. T.; Luo, D.; Craighead, H. G. Nanotechnology 2005, 16, (7), S314-S323) Using synthetic nanopores as described in the embodiments above this value increases proportionally with the number of pores being analyzed simultaneously. By modifying the current chip configuration and optical components it is expected to be possible to detect up to 10² pores simultaneously; this results in a 100 fold improvement in throughput whilst ensuring all analyte molecules pass through the detection probe volume. Importantly it should be stressed that all fluorescent molecules that translocate are detected (100 % detection efficiency) meaning that these devices will prove useful in applications such as rare event detection and diagnostics.

Referring back to Figure 1, it is possible to provide high numbers, such as 100, pores in the Al and SiN layer 10, 14 all located over a single aperture 16 in the Si support layer. However, in other embodiments a plurality of apertures can be made in the support layer, each with a number of micro-pores formed over it. In this case it may be necessary to have a number of optical sensing devices, for example one for each aperture in the support layer. This can provide even higher through-put.

In a further embodiment, to further characterize translocation events, a single pore was probed using confocal optics (this results in a diffraction limited Gaussian spot at the aluminum / pore interface). Although this approach reduces the overall throughput, there is a significant advantage in terms of time-resolution and signal- to-noise ratio, enabling more efficient characterization of translocation events at higher potentials. Consequently, a separate experiment was performed with the same membrane but using an APD to probe the exit of a single pore under different applied voltages of 1.50V (a), 1.60V (b), 1.70V (c), 1.90V (d). Data was acquired at a time resolution of 50 μs and then resampled at 5 ms for plotting. The results are summarized in Figures 5a to 5d and are in excellent agreement with the translocation times measured using the emCCD. Each peak corresponds to a single translocation event. For a potential of 1.5 V 14 peaks register above the background threshold as defined by Poisson counting statistics. Increasing the potential to 1.7 V resulted in 33 peaks over the same 60 s acquisition time. Increasing the voltage further resulted in a drastic increase in the overall baseline implying that the rate of DNA translocation is sufficiency high that single molecule events could no longer be resolved. Burst width and areas were determined to be on average 600 ms and 5900 photons respectively for an applied potential of 1.6 V. Increasing the potential resulted in not only shorter translocation times but also fewer registered photon counts attributed to the shorter residence times within the pore.

These examples demonstrate that optical detection of DNA translocation events through an array of solid state nanopores has a number of advantages. Results indicate that it is possible to obtain high spatial resolution DNA analysis whilst independently controlling the applied voltage that drives the molecules into nanopore. An advantage of the systems described is the possibility of parallelizing molecular analysis by probing an entire array of nanopores under uniform illumination. Operations such as fragment sizing of DNA molecules are potentially achievable on timescales significantly shorter than with single pore devices. Referring to Figure 6 a system according to a further embodiment of the invention comprises a device similar to that of Figures 1 and 2 with a silicon nitride membrane 110 support over an aperture or window 116 in a support 112. The membrane 110 only has one nanopore in this example. A gating electrode 119 is formed on the membrane 110 around the pore 118 and an electrical supply arranged to apply a variable electrical potential to the gating electrode 119. This nanopore/electrode architecture, integrated in a microfluidic structure, allows characterizing and controlling the translocation of DNA by local, frequency- modulated gating fields, as will be described, as it can be used to apply a varying electric field component in addition to the field provided by the two electrodes 36, 38 in the lower and upper reservoirs.

The microfluidic structure, solvent, and ions are not shown for clarity in Figure 6. The Si₃N₄ membrane 110 separates the two reservoirs; DNA translocates through a nanometre pore in the centre of a ring electrode 110, which may be formed separately on the membrane, or may be formed as part of an aluminium layer corresponding to that of Figure 1, electrically isolated from the rest of that layer. A pair of electrodes 136, 138 are provided in the upper and lower reservoirs to cause translocation through the pore. A controller 150 controls the electrical potentials provided to each of the electrodes 110, 136, 138. Typically the two electrodes 136, 138 are kept at constant potentials, and the controller is arranged to vary the potential of the ring electrode 119 so as to vary the electrical field in and around the pore. In a bi-potentiostatic setup one working electrode will be used to drive electrokinetic transport, while the other gate electrode, which in this case is the small, micrometer-size ring electrode 119, with a fast response time will be employed to modulate the transport characteristics of the pore structure by application of an AC or other controlled field.

This enables the translocation to be controlled in a number of ways. As the strength of the electric field in the region of the nano-pore will determine which molecules are forced through the nano-pores, the potential applied to the gate electrode 119 can be controlled and varied to select only one or some out of a number of different types of molecule which will translocate through the nano-pore. This allows the membrane to be used as a charge sensitive dialysis membrane which can perform molecular sieving.

Translocation events can be monitored in two independent ways, namely electrochemically by measuring the blockade current through the solid-state nanometre pore, for example using a pair of electrodes formed on a surface of the membrane, and optically by employing single-fluorophore imaging as described above.

This approach enables detailed studies of DNA translocation, the effect of frequency and amplitude of the time-dependent gate field, the relative contributions of "surface current" and "pore current", and a simple and straightforward way to modulate the translocation speed of DNA strands. The latter may be of particular importance with a view to improved precision for DNA fragment sizing or sieving applications.

Claims

1. Apparatus for characterizing molecules comprising a barrier between a first volume and a second volume, the barrier having a pore through it, a pair of electrodes arranged to generate an electrical potential difference between the first and second volumes to drive molecules from the first volume to the second volume, and optical sensing means arranged to detect light from the pore thereby to detect passage of a molecule through the pore.

2. Apparatus according to claim 1 wherein the barrier includes a light shielding layer which is at least partially opaque.

3. Apparatus according to claim 2 wherein the light shielding layer is opaque at the operational wavelengths of the optical sensing means.

4. Apparatus according to any foregoing claim further including a light source arranged to direct light at the pore.

5. Apparatus according to claim 4 wherein the light source is located on the same side of the barrier as the optical sensing means.

6. Apparatus according to any foregoing claim wherein one of the electrodes is transparent at the operational wavelengths of the optical sensing means and located between the pore and the optical sensing means.

7. Apparatus according to claim 6 wherein one of the volumes is partly defined by a wall which includes the transparent electrode.

8. Apparatus according to any foregoing claim wherein one of the volumes is formed as an aperture in one or more layers of material.

9. Apparatus according to claim 8 wherein the one or more layers of material have a further aperture therethrough connected to the other of the volumes.

10. Apparatus according to claim 9 when dependent on claim 7 wherein the wall is on one side of the barrier and the one or more layers of material are on the other side of the barrier.

11. Apparatus according to any foregoing claim wherein the barrier has a plurality of pores through it and the optical sensing means is arranged to detect light from all of the pores.

12. Apparatus according to claim 11 wherein the optical sensing means includes imaging means arranged to image a plurality of the pores and processing means arranged to identify a region of the image associated with each of the pores and to monitor the intensities of the regions.

13. Apparatus according to any foregoing claim further comprising a control electrode and control means arranged to vary the potential of the control electrode so as to vary the electrical field in the pore.

14. Apparatus according to claim 13 wherein the control electrode extends around the pore.

15. Apparatus according to claim 14 wherein the electrode is formed on the barrier.

16. A method of characterizing a molecule comprising passing the molecule through a pore in a barrier from a first volume and a second volume, wherein the movement of the molecule is controlled by a pair of electrodes arranged to generate an electrical potential difference between the first and second volumes, and detecting light from the pore thereby to detect passage of a molecule through the pore.

17. A method according to claim 16 wherein a further electrode is provided to control the electric field in the region of the pore, a solution containing a plurality of different types of molecule is placed in the first volume, and the potential of the electrode is controlled so as to cause only one or only some of the types of molecule to pass through the pore.