Improved device for analyzing nucleic acid molecules

Abstract

The invention proposes a device (1) for analyzing a nucleic acid molecule (M), comprising: a bead (20) on which one end of the molecule can be anchored; a surface (520) on which the other end of the molecule can be anchored; an actuator (30) adapted to move the bead in one direction relative to the surface; a sensor (50) adapted to measure the distance between the bead and the surface; the device further comprising a well (11) having an axis (X-X) extending in the direction of movement of the bead and a bottom (110) formed by the surface, the well being filled with an electrically conductive solution (40) and receiving the bead (20); and the sensor being adapted to measure the impedance of the well, which depends on the distance between the bead and the surface, to determine the distance between the bead and the surface.

Description

[0001] This application is a divisional application of Chinese invention patent application 201680040179.0, filed on May 6, 2016, entitled "An improved apparatus for analyzing nucleic acid molecules". Technical Field

[0002] This invention relates to an apparatus and method for analyzing molecules. The invention is particularly applicable to the analysis of nucleic acid molecules such as DNA or RNA. Background Technology

[0003] If a nucleic acid molecule is attached to a micrometer-sized bead, information about the nucleic acid's structure can be inferred by manipulating the bead and tracking its position at high resolution. Under specific experimental conditions, real-time tracking of the bead's position can be used to generate useful information about the structure of the DNA or RNA molecules attached to it.

[0004] This can then be used to determine the overall organization and structure of a molecule, its base sequence, the presence of biochemical modifications to nucleic acid bases, and the interactions between the molecule and proteins such as polymerases, helicases, and topoisomerases.

[0005] The apparatus for performing such nucleic acid molecular analysis is described in document US 2003 / 0027187. This apparatus includes optical devices to determine the location of beads attached to the molecules.

[0006] More specifically, the beads are illuminated from above and observed through a microscope objective using images captured on a high-definition camera. The position of the beads is tracked in real time by a tracking algorithm that measures the x and y coordinates from the bead images, while the z-position is determined by the light diffraction pattern around the bead image, which determines the height of the bead relative to the surface to which the molecules are attached.

[0007] In fact, the light illuminating the beads is scattered from the beads and produces a diffraction pattern through interference with the directly incident light. To use this diffraction pattern to infer the z-position of the beads, a set of diffraction images is acquired by holding the beads in a fixed position while precisely moving the objective lens at different distances from the objective lens focal point. These images are used independently to calibrate the system for each bead.

[0008] Afterward, the objective lens is held in a fixed position with great precision, and the distance of the bead from the focal point along the optical z-axis can then be tracked using a ring diagram by cross-referencing the calibration set.

[0009] This optical method for determining bead position is effective. However, it has many drawbacks that affect the method's cost and scalability.

[0010] In practice, analyzing nucleic acid molecules requires near-single-base resolution, within the range of a 1-nanometer bead movement, because 1 nanometer equals the distance increased each time a dsDNA base pair opens in a depolymerized state. This distance is the sum of the extensions of the two complementary ssDNA mononucleotides that have already opened. The situation is similar for RNA molecules.

[0011] This level of resolution can be achieved using the optical methods disclosed above. However, such a system requires a very complex mechatronic setup and has a relatively small field of view, which limits the number of beads that the system can analyze simultaneously. For example, a 30x objective lens has a field of view of approximately 300 by 300 micrometers, which only allows the analysis of about 1,000 beads.

[0012] Conversely, specific applications that require the simultaneous analysis of a very large number of molecules are preferred; for example, up to 10. 9 Each molecule.

[0013] Furthermore, even if this optical method allows for the simultaneous analysis of approximately 1000 beads, it requires generating a set of calibration images for each bead, which is time-consuming and computationally intensive. Therefore, achieving higher throughput using the same optical method is extremely complex. Summary of the Invention

[0014] One object of the present invention is to overcome the above-mentioned disadvantages of the prior art by providing a system and method for analyzing nucleic acid molecules, which allows for the simultaneous analysis of a greater number of molecules than the prior art.

[0015] Another objective of this invention is to maintain at least the same resolution as the prior art for accurate molecular analysis.

[0016] For this purpose, an apparatus for performing nucleic acid molecular analysis is disclosed, comprising:

[0017] - Beads, where one end of a molecule can be anchored.

[0018] -On the surface, the other end of the molecule can be anchored to it.

[0019] - An actuator adapted to move the bead relative to the surface in one direction of motion.

[0020] - A sensor adapted to measure the distance between the bead and the surface, the device being characterized in that it further includes an aperture having an axis extending along the direction of movement of the bead and a bottom formed by the surface, the aperture being filled with a conductive solution and the bead being received in the aperture, and wherein the sensor is adapted to measure the impedance of the aperture, the impedance depending on the distance between the bead and the surface, thereby determining the distance between the bead and the surface based on the measured impedance.

[0021] In some embodiments, the device may include at least one of the following features:

[0022] -The sensor may include:

[0023] The main electrode, located at the top of the orifice and in contact with the conductive solution, is at a known potential.

[0024] The secondary electrode located at the bottom of the aperture carries molecules that can be anchored to its surface, and

[0025] An electronic circuit suitable for measuring the current between electrodes.

[0026] -The electronic circuit may include:

[0027] The current connected to the secondary electrode is sent to the voltage amplifier.

[0028] A voltmeter suitable for measuring the output voltage of a voltage amplifier from current, and

[0029] A calculation circuit adapted to calculate the impedance of the orifice from the measured voltage.

[0030] - The cross-sectional area of ​​a hole that is transverse to its axis can increase strictly from the bottom to the top of the hole.

[0031] - The area of ​​the cross-section at the bottom of the hole can be greater than the area of ​​the maximum cross-section of the bead.

[0032] - The hole can have the shape of a truncated cone.

[0033] - The cross-sectional area of ​​the hole can increase linearly with the distance from the bottom of the hole.

[0034] - The cross-section of the hole can be, for example, circular at its bottom and increases linearly with distance from the bottom of the hole in a direction perpendicular to the hole axis.

[0035] The actuator may include at least one magnet mounted to be displaceable along the axis of the hole, and the bead is made of a paramagnetic material and is inserted between the bottom of the hole and the magnet.

[0036] The device may include a plurality of identical beads and a plurality of identical holes, each hole adapted to receive one bead. Alternatively, in one embodiment, the sensor includes a main electrode, secondary electrodes, and electronic circuitry adapted to measure the current between the electrodes. The sensor may include a plurality of secondary electrodes, each disposed at the bottom of a corresponding hole and forming a surface on which the corresponding bead is anchored. The electronic circuitry may then include a plurality of current-to-voltage amplifiers, each connected to a corresponding secondary electrode. The computational circuitry is also adapted to simultaneously measure the output voltage of the current-to-voltage amplifiers and calculate the impedance of the corresponding hole.

[0037] The device may include an electrically insulating material plate in which the holes are formed, all the holes opening at their top surface, and the actuator may also include a plurality of rods made of magnetic material, each rod disposed on the top surface and extending between two adjacent holes.

[0038] - The length of each rod can be less than 10 μm, and the diameter of each bead can be less than or equal to 1 μm.

[0039] A method for analyzing nucleic acid molecules is also disclosed, the method being carried out by the apparatus described above, and including at least one step: measuring the distance between a bead and the bottom of a well, each measurement step including measuring the impedance of the well to determine the position of the bead in the well.

[0040] In one embodiment, the analytical method may further include a preparatory step of anchoring at least one molecule to the bottom of the bead and the pore, the step comprising:

[0041] - Position at least one bead with molecules anchored thereon in a solution that fills the pores.

[0042] - A first potential difference is applied between the primary and secondary electrodes of the sensor to drive the bead into contact with the bottom of the hole, and

[0043] - Reverse the potential difference between the electrodes.

[0044] The apparatus according to the invention enables the analysis of molecules connected at one end to microbeads and at the other end to the bottom of a pore. By monitoring the impedance change of the pore based on the position of the bead within the pore, its position can be measured with great precision.

[0045] In fact, the overall conductivity of a pore corresponds to the conductivity of the solution filling the pore. When a bead moves within the pore and occupies a portion of the latter, it reduces the cross-section of the pore filled with the conductive solution, thereby altering the pore's conductivity.

[0046] Therefore, by determining the exact shape of the hole, especially when the cross-sectional area of ​​the hole increases with the distance from the bottom of the hole, the position of the bead in the hole can be easily determined.

[0047] This device can be multiplexed to simultaneously analyze a large number of nucleic acid molecules without reducing resolution. In practice, when the device includes multiple wells and corresponding beads, all beads can be monitored by sensors. For each well, the sensor includes a secondary electrode and a current-to-voltage amplifier, enabling precise monitoring of the position of each bead within its corresponding well. Attached Figure Description

[0048] The features and advantages of the present invention will become apparent from the following more detailed description of certain embodiments of the invention and from the accompanying drawings, wherein:

[0049] - Figure 1a An exemplary embodiment of a device for analyzing nucleic acid molecules is shown.

[0050] - Figure 1b and Figure 1c Cross-sectional and perspective views of the possible shapes of the holes in the device are shown respectively.

[0051] - Figure 2 It shows Figure 1a In the implementation scheme, the resistance of the pore is determined based on the position of the bead.

[0052] - Figure 3 The electrical equivalent circuit of a device according to one embodiment of the present invention is schematically shown.

[0053] - Figure 4a and 4b This is a schematic diagram of the analysis device based on two implementation schemes.

[0054] - Figure 5a and Figure 5b The magnetic field gradient applied to the beads in an exemplary analytical apparatus is shown.

[0055] - Figure 6 The main steps of an analytical method according to one embodiment of the present invention are shown. Detailed Implementation Plan

[0056] Device for analyzing molecules

[0057] Overall description of the device

[0058] refer to Figure 1a The diagram illustrates an apparatus 1 for analyzing nucleic acid molecules. These molecules may, in particular, be DNA or RNA molecules. Preferably, as shown, the molecules may be hairpin double-stranded molecules.

[0059] A hairpin is a double-chained helix in which the 5' end of one chain is physically connected to the 3' end of the other chain via an unpaired loop. This physical connection can be covalent or non-covalent, but is preferably covalent.

[0060] Therefore, the hairpin consists of a double-chain stem and an unpaired single-chain ring.

[0061] Device 1 includes an electrically insulating material plate 10. For example, the plate may be made of silicon, glass, a non-conductive polymer, or a resin.

[0062] At least one hole 11 is formed in the plate, each hole extending along a main axis XX, the main axis XX being perpendicular to the plane to which the plate extends. Each hole opens at the top surface 101 of the plate.

[0063] In addition, each hole 11 has a bottom 110, which preferably extends perpendicular to the axis XX.

[0064] A pore is also known as a micropore because its size (depth, maximum length of cross-section) is on the order of approximately 1 μm or approximately 0.1 μm. For example, the depth of a pore along the XX axis can be several micrometers, including, for example, 1 to 10 micrometers, or, for example, equal to 8 μm.

[0065] The maximum length of the hole in the cross-section of a plane perpendicular to the XX axis can range from hundreds of nanometers to several micrometers, for example, about 4 or 5 μm, as shown in Figure 1.

[0066] Existing techniques capable of producing such holes include, for example, a technique known as orbital etching, which involves irradiation with heavy ions to form latent tracks followed by chemical etching. More details on techniques for producing these holes can be found in, for example, the work of the Siwy Research Laboratory (website: http: / / www.phvsics.uci.edu / ~zsiwy / fab.html) or in the publication M. Davenport, K. Healy, M. Pevarnik, N. Teslich, S. Cabrini, A.P. Morrison, Z.S. Siwy and S.E.L.Tant, "The Role of PoreGeometry in Single Nanoparticle Detection", in ACSNANO, vol. 6, no. 9, 8366-8380, 2012.

[0067] The device further includes at least one bead 20. Preferably, the device includes a plurality of beads 20, the number of which is equal to the number of holes.

[0068] The number of holes and beads can preferably be greater than 1,000, for example greater than 10,000, such as about 100,000 or 1,000,000.

[0069] Each bead is spherical and has a diameter of no more than 5 μm. For example, the diameter of bead 20 can be about 1.5 μm or 1 μm. Preferably, the beads can be smaller and have a diameter of less than 1 μm, for example, 0.3 μm.

[0070] As a non-limiting example, the following beads may be used as a reference:

[0071] -MyOne, manufactured by Invitrogen, has a diameter of 1.04μm.

[0072] -M270, manufactured by Invitrogen, has a diameter of 2.8 μm.

[0073] -M450, manufactured by Invitrogen, has a diameter of 5.5 μm.

[0074] -Ademtech 500, manufactured by Ademtech, has a diameter of 0.5μm.

[0075] -Ademtech 300, manufactured by Ademtech, has a diameter of 0.5μm.

[0076] For the analysis of nucleic acid molecules, one end of a molecule M is anchored to bead 20, and the other end is anchored to the surface of the bottom of pore 110.

[0077] To anchor molecules to the bottom surface of beads and pores, the beads and surfaces can be coated with specific materials suitable for bonding with the ends of molecules.

[0078] For example, one end of a DNA or RNA molecule can be labeled with biotin and the other end with digoxigenin, and the beads can be coated with streptavidin to bind to the labeled (e.g., biotin) end of the DNA / RNA hairpin molecule, and the bottom of the pore 110 can be further coated with anti-Dig antibody to bind to the Dig-labeled end of the DNA / RNA molecule, see, for example, Hunter MM, Margolies MN, Ju A, Haber E, "High-affinity monoclonal antibodies to the cardiac glycoside, digoxin, Journal of Immunology, 1982 Sep; 129(3):1 165-1 172.

[0079] Therefore, the beads are connected to the bottom 110 of the hole 11 by molecules.

[0080] Furthermore, the bead 20 is free to move relative to the bottom 110 of the hole 11. In particular, the bead 20 can move along the XX axis. To control the movement of the bead 20 along this axis, the device 1 also includes an actuator 30 adapted to move the bead 20 parallel to the axis.

[0081] According to a preferred embodiment, the movement of the beads can be controlled by a magnetic force applied to the beads 20 by the actuator 30. In this case, the beads are made of a paramagnetic material, such as a superparamagnetic material. For example, the beads can be made of ferrite-doped latex and coated with streptavidin to anchor the molecule M.

[0082] The actuator 30 may include at least one permanent magnet 31, which can be controlled to move parallel to the XX axis. Preferably, as shown in FIG1, the actuator 30 may include two permanent magnets 31, which are located at equal distances along the XX axis and whose magnetic poles are aligned perpendicular to the XX axis, with the north pole of one magnet facing the south pole of the other.

[0083] Bead 20 is located between the bottom 110 of hole 11 and magnet 31.

[0084] These magnets enable the application of force to the beads, and thus to the molecules they are anchored to. By moving the magnets closer to or further away from the beads 20 in the XX-axis direction, the magnetic field can be altered, thereby controlling the magnitude of the force applied to the beads and thus controlling the elongation of the sample in the XX-axis direction.

[0085] Another embodiment of actuator 30 may include a permanent magnet and a strip covered with a magnetizable material, positioned at a fixed location approximately a few micrometers relative to aperture 11. By bringing the permanent magnet closer to or further away from the strip covered with the magnetizable material, the magnetic field exerted on the bead by the strip can be altered (see also the embodiment disclosed in the section on configuring multiple apertures below).

[0086] Other methods can be used to control the movement of beads 20, such as optical or acoustic tweezers, the latter meaning applying sound waves to the beads, see, for example, G. Sitters, D. Kamsma, G. Thalhammer, M. Ritsch-Marte, EJG Peterman and GLJ Wuite, "Acoustic Force spectroscopy", in Nature Methods, Vol. 12, No. 1, Jan. 2015 or X. Ding, ZSLin, B. Kiraly, H. Yue, S. Li, I. Chiang, J. Shi, SJ Benkovic and TJ Huang, "On-Chip Manipulation of single microparticles, cells, and organisms using surface acoustic waves", PNAS, July 10, 2012, vol. 109, no. 28, 11 105-11 109.

[0087] Pore ​​impedance variation with bead position

[0088] Finally, by monitoring the impedance of the hole, especially the resistance (or conductivity), the device 1 is able to determine the distance between the bead 20 and the bottom 110 of the corresponding hole 11, which corresponds to the length of the molecule anchored to the bead and the bottom 110 of the hole.

[0089] Therefore, each hole 10 is filled with a conductive solution 40.

[0090] The conductivity of conductive solution 40 is preferably 10. -7 S / cm and 10 1 Between, preferably in the range of 10 -3 and 10 -2 Between S / cm.

[0091] For example, solution 40 could be a solution with a concentration of 100 mmol / m 3 Solution 40 may contain a 100 mM aqueous solution of sodium chloride. Alternatively, solution 40 may contain a buffer compatible with the preservation of DNA molecules, such as an aqueous buffer containing 10 mM Tris HCl, 0.1 mM EDTA, and 100 mM sodium chloride. The buffer may also contain a divalent cation compatible with enzyme activity, such as 10 mM MgCl2. In some embodiments, the buffer should support electrophoresis (e.g., Tris borate EDTA buffer).

[0092] like Figure 1aAs seen, the conductive solution 40 completely fills each hole 11 and covers the top surface 101 of the plate.

[0093] The beads 20 must have a conductivity different from that of the solution. The beads are preferably electrically insulating.

[0094] Furthermore, the cross-section of hole 11 varies with z, where z is the distance from the bottom of the hole along the XX axis. This distance can be measured from the nearest point from the bead to the bottom of the hole, or from the center of the bead.

[0095] Preferably, the cross-sectional area of ​​the hole 11 transverse to the XX axis increases strictly with the distance z from the bottom of the hole (z axis is shown in the figure). Figures 1a to 1c In the middle, the origin of the shaft is located at the center of the bottom of the hole.

[0096] Therefore, since the size of bead 20 is constant, it occupies different proportions of the internal volume of the hole.

[0097] For example, if the bead is very close to the bottom of the hole, it occupies the majority of the portion P of the hole extending around the bead. The portion P is defined as the volume of the hole extending between the cross-sections occupied by the lowest and highest points of the bead. Therefore, the resistance of this portion increases dramatically because the remaining space left for the conductive solution is very small. Since the overall resistance of the hole is the integral of the resistance along the entire depth of the hole (along the XX axis), the overall resistance also increases dramatically.

[0098] Conversely, if the bead is close to the top surface of the hole, it occupies a small portion of the portion P of the hole extending around the bead. Therefore, the resistance of this portion increases more slowly than in the previous example, and the overall resistance of the hole is smaller than in the previous example.

[0099] Therefore, for example in Figure 2 As shown, a curve can be generated that illustrates the resistance of the pore as a function of the distance between the bead and the bottom of the pore, where each resistance value corresponds to a single distance value between the bead and the bottom of the pore. Therefore, by measuring the resistance value of the pore, the corresponding distance between the bead and the bottom of the pore can be inferred with good accuracy.

[0100] To achieve good accuracy in determining the position of bead 20 within hole 11, the minimum cross-sectional area of ​​the hole is preferably larger than the maximum cross-sectional area of ​​the bead. Specifically, in a preferred embodiment, the area of ​​the cross-sectional area of ​​the hole transverse to its axis increases strictly with distance from the bottom, with the minimum cross-sectional area located at the bottom 110 of the hole. This fact that the cross-sectional area is larger than the bead allows the bead to reach the bottom of the hole and thus enables the measurement of all positions of the bead within the hole, including positions where the distance between the bead and the hole is zero.

[0101] Holes can come in various shapes. First, a hole can be rotationally symmetrical about an X-axis.

[0102] For example, such as Figure 1b As shown, the hole can have the shape of a truncated cone, with its smallest part corresponding to its bottom 110.

[0103] Or, in Figure 1a In the process, the radius r of the hole wall is determined by the distance z from the bottom using the following formula:

[0104] z = I0 tanh 2(r-r0)

[0105] Where I0 is the height of the hole, and r0 is the bottom radius of the hole. The hole shape is given... Figure 2 The resistance curve.

[0106] According to another different embodiment, preferably, the cross-sectional area of ​​the hole increases linearly with distance from the bottom of the hole. Therefore, the difference between the cross-sectional area of ​​the hole and the cross-sectional area of ​​the bead is proportional to the distance of the bead from the bottom of the hole.

[0107] Therefore, the resistance of the pore decreases linearly as the distance between the bead and the bottom of the pore increases.

[0108] For example, such as Figure 1c As shown, the hole can have an elliptical cross-section, which is formed by a circular cross-section at the bottom 110 of the hole, and increases linearly with z along the direction perpendicular to the XX axis of the hole.

[0109] Therefore, it is even easier to infer the position of the bead from the measurement of the pore impedance. In particular, Figure 2 The curve shown in the figure—its shape is based on the position of the bead in the hole (geometry shown in the figure) Figure 1a The resistance of the hole becomes a straight line.

[0110] Impedance sensor

[0111] Reference Figure 3 The device 1 also includes a sensor 50 adapted to measure the impedance of the hole 11 and infer the distance z between the bead 20 and the bottom surface 110 of the hole 11 based on the measurement.

[0112] Sensor 50 includes a main electrode 51, which is capable of setting the conductive solution 40 to a reference voltage V0. For example... Figure 1a As shown, the main electrode 51 is in contact with the conductive solution 40. Preferably, the main electrode 51 can be located on the top surface of the plate 10. Since the conductive solution extends beyond the top surface, it contacts the main electrode 51. The main electrode 51 is, of course, connected to a voltage source (not shown), preferably a DC voltage source.

[0113] The voltage V0 is preferably a constant voltage. To avoid electrolysis within the orifice, it is preferably below 0.5V. Preferably, the voltage V0 includes between 10 and 500mV, and even more preferably between 50 and 300mV. For example, the voltage V0 can be equal to 0.25V.

[0114] Furthermore, the sensor 50 includes at least one secondary electrode 52. More specifically, the sensor 50 includes a number of secondary electrodes 52 equal to the number of holes 11, which is also the number of beads 20. Each secondary electrode 52 may be a gold or platinum electrode.

[0115] The secondary electrode 52 forms the bottom 110 of the hole and has a surface 520, which forms the surface to which the molecule is anchored.

[0116] exist Figure 3 In the cylindrical conductive solution 40 with corresponding beads of variable position, the resistance of the cylinder is equal to that of resistors R1, R2, and R3. Each resistor has one pole in contact with the main electrode 51 and another pole in contact with the secondary electrode 52 at the bottom of the hole.

[0117] Sensor 50 also includes electronic circuitry 53 that measures the resistance of the orifice by measuring the current flowing through it. For this purpose, according to one embodiment, the electronics may include a defined resistor connected in series with a secondary impedance. The potential difference at the resistor electrodes can be measured to infer the current flowing through the resistor.

[0118] However, since the current in each hole is very low, this implementation may be inaccurate. Therefore, electronic circuit 53 preferably includes a current amplifier in the hole.

[0119] For this purpose, electronic circuit 53 preferably includes a current-to-voltage amplifier 530 for each secondary stage.

[0120] The current-to-voltage amplifier includes an operational amplifier whose inverting input is connected to the secondary electrode 52, its non-inverting input is connected to ground, and its output is connected to the inverting input via a feedback resistor of known value.

[0121] The current i1 between the main electrode 51 and the secondary electrode 52 is given by the following equation:

[0122]

[0123] Where V- is the potential at the inverting input terminal of the operational amplifier, R i It is the resistance value of the i-th hole.

[0124] Since the two input terminals are theoretically at the same potential: V - =0V, therefore:

[0125]

[0126] Furthermore, in the feedback loop, the current i l This can be expressed by the following equation:

[0127]

[0128] Where R l It is the resistance value of the feedback loop, V i It is the current flowing to the output potential of the voltage amplifier 530.

[0129] Therefore, by measuring the potential V i The value of the current ii in the i-th hole can be obtained using the following equation:

[0130]

[0131] Furthermore, the resistance value of the hole can then be obtained:

[0132]

[0133] Therefore, electronic circuit 53 also includes components suitable for measuring potential V. i The device 531, such as a voltmeter, and the computing circuit 532, including, for example a processor, control the acquisition measurement of the device 531 and infer the resistance value from the measured potential. The processor is preferably adapted to run a dedicated program comprising a set of instructions for controlling the sensor (in particular the measuring device 531), inferring the resistance value of each hole and the corresponding distance of each bead from the bottom of the corresponding hole.

[0134] Preferably, the measurement is performed at a sampling rate of approximately 50 Hz across the entire device (measurements performed in parallel at the sampling rate across all orifices of the device). The measured potentials should be integrated or averaged between two measurements to reduce noise or suppress parasite signals. In this regard, the sampling rate can be a multiple of the main power frequency. Specific Implementation

[0136] An implementation example is described in detail with reference to the accompanying drawings. Figure 1a The case of a single micropore 11 is shown, and its curve is described by the following equation:

[0137] z = I0 tanh 2(r-r0)

[0138] Where I0 = 8 μm is the height of the pore, and r0 = 2 μm is the bottom radius of the pore. The pore is partially sealed by a bead with a diameter of d = 1.5 μm.

[0139] The pore was filled with a concentration of 100 mol / m³. 3An aqueous solution of sodium chloride corresponds to a conductivity σ = 10. -2 S / cm.

[0140] For the voltage across the orifice V, the current in the orifice is I = V / R, where R is the equivalent resistance of the orifice filled with solution 40 and partially sealed by the beads. The resistance is shown in... Figure 2 In the curve, the resistance is a function of the distance from the center of the bead to the bottom of the hole. It can be seen that by measuring the resistance of the hole, the position of the bead can be inferred by reporting the resistance value on the curve and determining the corresponding distance.

[0141] The electronic current noise measured over bandwidth Δf can be attributed to two sources:

[0142] Johnson noise, also known as thermal noise, is electronic noise generated by the thermal agitation of electrons. This noise is represented by the following equation:

[0143]

[0144] Where T is the ambient temperature, kB is the Boltzmann constant, and

[0145] - Shot noise (related to the random flux of the fundamental electron charge, and is represented by the following equation):

[0146]

[0147] Where I is the signal current in the hole, and e is the elementary charge (absolute value) carried by the electron.

[0148] It can be seen that for values ​​above 2k b The voltage V of T / e, which is approximately 50 mV at an ambient temperature of about 293 K, is dominated by shot noise associated with the signal current. Furthermore, this shot noise increases with the resistance of the aperture. Therefore, to maintain the highest possible signal-to-noise ratio, the aperture size may not be too small; for example, apertures on the order of 1 nm (“nanopores”) provide noise that is too great to be utilized.

[0149] Returning to this example, the voltage applied by the main electrode 51 is equal to 0.25V. According to... Figure 2 For the electrolyte solution in this example, the maximum resistance of the pore is approximately 2500 kΩ. For such a voltage, the current is I = V / R = 100 nA, and the current noise is... (For a bandwidth Δf = 100Hz).

[0150] The impedance ΔR changes by approximately 300 kΩ over a distance δI = 5 μm. This means that to resolve an impedance change of δR = 0.06 kΩ over a distance of approximately 1 nm (corresponding to a single base pair in a 2500-base-pair double-stranded DNA molecule), it is necessary to be able to resolve an impedance change of δR = 0.06 kΩ. This corresponds to a change in current δ1 = I. δR / R = 2.5 pA, which corresponds to a signal-to-noise ratio of approximately 1.

[0151] An average of Δf = 1 Hz or higher enables the detection of single base pairs with a suitable signal-to-noise ratio of 10.

[0152] Configure multiple holes

[0153] As described above, the device 1 disclosed above preferably includes a plurality of holes 11 and the same number of beads 20, which are packaged on the chip at a high density, for example, a density of 10. 5 Up to 10 8 Hole / cm 2 Between these values, for example, if the maximum cross-section of each micropore device (at the top surface of the plate) is 5 × 5 micrometers, then approximately 4.10 per square centimeter should be obtained. 6 One hole.

[0154] In this configuration, by providing an equal number of secondary electrodes 52 and current to voltage amplifiers 530, sensor 50 can be easily multiplexed, enabling parallel measurement of the aperture's conductivity in a manner similar to that of a CMOS camera containing multiple pixels.

[0155] However, the computing unit 532 is common to all holes 11.

[0156] To minimize the computational requirements of the computing unit 532, all beads and all holes are preferably identical, allowing for tolerances introduced by the manufacturing process. Furthermore, since a single main electrode 51 is in contact with a conductive solution 40 that fills all holes and extends beyond the top surface 101 of the plate 10, the single main electrode 51 is common to all holes 11, and the same voltage V0 is applied to all holes.

[0157] In addition, when multiple holes 11 are arranged in the common plate 10, the actuator 30 may preferably include a pair of main macroscopic permanent magnets 31 that can move parallel to the XX axis and substantially cover all surfaces of the plate 10 in order to generate a uniform magnetic field.

[0158] Actuator 30 also includes multiple components made of magnetic materials (e.g., alloys of iron and nickel, known by trade name...). The rod 32, made of [material name], is fixed to the plate 10.

[0159] like Figure 4a and 4bAs shown, each rod 32 is located on the plate 10, on its top surface 101. Each rod 32 extends between two adjacent holes such that the extreme ends 320 of the rod 32 are flush with the edges of the holes. Therefore, the rods 32 are very close to the beads 20. It should be understood that when the holes are packed in the plate 10 at a high density, the distance between two adjacent holes is on the order of several μm or tens of μm. Therefore, the magnetic rods 32 have a length of the same order of magnitude.

[0160] according to Figure 5a The example shown, for instance, has a magnetic rod 32 that is 8 μm long, 1 μm wide, and spaced apart at a distance of 2 μm.

[0161] Each magnetic material rod 32 is magnetized under the influence of the magnetic field generated by the main magnet 31. Therefore, placing the main magnet 31 closer to or further away from the plate 10 will increase or decrease the magnetic field density between the rods, which in turn will increase or decrease the force applied to the beads.

[0162] This embodiment is particularly preferred because the magnetizing rod 32 is smaller than the main magnet 31 and is closer to the bead 20. By reducing the size of the magnet, the magnetic field gradient near the bead 20 increases, thus increasing the magnetic force applied to the bead.

[0163] In fact, Figure 5a and 5b The simulation results show that when the magnetic material rods 32 are exposed to a uniform magnetic field of 0.088T generated by the main magnet 31, the magnetic field between the rods 32 is enhanced, which produces a significant magnetic field gradient along the XX axis. (As shown in...) Figure 5b As observed, the maximum value of this magnetic field gradient is approximately 0.09 T / μm, which is 90.10. 3 T / m.

[0164] The magnetic force applied to the bead depends on the magnetic field gradient. Specifically, the maximum magnetic force (in N) that can be applied to the bead can be expressed as follows:

[0165]

[0166] Where m s This is the saturation value of the bead's magnetic moment, measured in Am. 2 dB / dz is the gradient of the magnetic field, with units of T / m.

[0167] Here are the saturation values ​​of the magnetic moment of the beads based on the examples given above:

[0168] -Invitrogen's MyOne: 13, 2.10-15A.m2

[0169] -Invitrogen M270: 57, 5.10-15A.m2

[0170] -Invitrogen M450: 238, 5.10-15A.m2

[0171] -Ademtech 500: 4,7.10-15A.m2

[0172] -Ademtech 500: 1,017.10 -15 Am 2

[0173] Therefore, for MyOne type beads, whose structure is shown in Figure 5, the magnetic force applied to the beads can, for example, exceed 1000 pN.

[0174] Conversely, without the magnetizing rod 32 inserted between the plate 10 and the main magnet 31, the maximum magnetic field gradient generated by the magnet is approximately 1.6610. 3 T / m. For MyOne type beads, this maximum magnetic field gradient gives a maximum force of approximately 22 pN applied to the beads. Therefore, the presence of the magnetizing rod allows for a magnetic force of approximately 55 times greater on the beads than without the magnetizing rod.

[0175] This increase in the magnetic force applied to bead 20 allows for a reduction in bead size while maintaining a value at least equal to, or even greater than, that of the bead. Figure 1a The force applied to the bead by the magnet is shown.

[0176] The reduction in bead size allows for a reduction in pore size, thereby increasing pore density and device throughput. Therefore, the number of molecules that can be analyzed simultaneously can reach 5 × 10⁻⁶. 7 molecule / cm2 (assuming bead diameter is 300 nanometers).

[0177] like Figure 4a and 4b As shown, holes can be stacked in plate 10 according to different configurations to achieve different densities. For example, in Figure 4a In the middle, it is shown that... Figure 4b The pores have a lower density. In the latter, the pores 11 extend in one dimension perpendicular to the XX axis, which makes the conductivity more linear with the position of the beads and also allows for a reduction in the overall unit size, since the cross-section of the pores increases in only one direction.

[0178] Methods for analyzing nucleic acid molecules

[0179] refer to Figure 6This document illustrates the main steps of a method for analyzing nucleic acid molecules such as DNA or RNA molecules. The analysis includes, for example, determining the nucleic acid sequence, i.e., deciphering the actual base sequence in the nucleic acid, and determining other informational fragments on the nucleic acid sequence, such as detecting specific sequences in a nucleic acid molecule, detecting differences between sequences of two different nucleic acid molecules, or the binding of a protein to a specific sequence, see, for example, WO 2011 / 147931; WO 2011 / 1147929; WO 2013 / 093005.

[0180] As mentioned earlier, the analyzed molecule M is a hairpin molecule. In a hairpin molecule, the ends of the two strands not bound to the loop are attached to the bottom of the bead and the pore, respectively, and can therefore be pulled apart during bead movement. It is even possible to completely open a hairpin double-stranded nucleic acid molecule by pulling each end of the molecule with a defined force.

[0181] The method is described for a single bead in a single hole, but it can be applied to any number of holes.

[0182] The method includes a first step 100: anchoring the bead to the bottom of the pore using a nucleic acid molecule M, i.e., anchoring the first end of molecule M to bead 20 and the second end of molecule M to the bottom of pore 11.

[0183] According to a preferred embodiment, the above-described structure of the device enables this step, particularly when the device comprises a large number of pores densely packed in a plate. According to this embodiment, step 100 includes a first sub-step 110: placing beads with attached nucleic acid molecules into the device, in a solution 40 above the pores 11.

[0184] The second sub-step 120 then involves applying a potential difference between the main electrode 51 and the secondary electrode, thereby driving the beads toward the electrode 52 at the bottom of the pore via electrophoresis. The potential difference applied between the electrodes is approximately a few V / cm. For more information on the electrophoretic mobility of the beads, see B. Xiong, A. Pallandre, I. le Potier, P. Audebert, E. Fattal, N. Tsapis, G. Barratt and M. Tavema, "Electrophoretic mobility measurement by laser Doppler velocimetry and capillary electrophoresis of micrometric fluorescent polystyrene beads", in Analytical. Methods, 2012, 4, 183.

[0185] When the bead contacts the bottom of the hole, at least one bead bonds with it. Preferably, each hole is designed such that only one bead can bond with its bottom, i.e., the cross-section of the bottom of the hole is less than twice the cross-section of the bead.

[0186] Then, the anchoring step 100 includes a third sub-step 130: reversing the voltage between the electrodes. As a result, all unbonded beads are expelled from the hole.

[0187] These sub-steps 120 and 130 can be repeated to maximize the overall loading efficiency of the holes.

[0188] Finally, sub-step 140 can be performed: measuring the conductivity of at least one pore to determine whether there are bonded beads in the pore.

[0189] The method then includes at least one step 200: actuating the bead 20 via the actuator 30 to change the distance between the bead 20 and the bottom 110 of the hole 11, thereby applying tension to both ends of the molecule; and at least one step 300: measuring the distance between the bead and the bottom of the hole. This distance is measured by measuring the resistance of the hole 11. As already referenced Figure 3 As described in detail, the measurement itself is performed by measuring the current to the output voltage of the voltage amplifier 530.

[0190] The method can be implemented in a variety of different ways, but it preferably includes a series of multiple steps 200: actuating beads at different distances from the bottom of the hole.

[0191] like Figure 6 As shown, in that case, step 300: measuring the distance between the bead 20 and the bottom 110 of the hole 11 is preferably performed continuously during all the actuation steps 200.

[0192] Alternatively, measurement step 300 can be performed simultaneously with each actuation step.

[0193] As a non-limiting example, the method can be performed, for instance, according to the order disclosed in document EP2390351, and further details regarding the implementation of the order can be found in that reference. This order includes:

[0194] - In the first pair of steps 200 and 300, during which the actuated bead is used to separate the two chains of the hairpin molecule M by applying a tension of about 15 pN or greater, for example, equal to 18 pN, to the molecules. The distance between the bead and the bottom 110 of the hole 11 is measured, which corresponds to the total length of the opened hairpin molecule.

[0195] - Step 400: Hybridize a single-stranded nucleic acid fragment with one strand of molecule M.

[0196] - The second pair of steps 200 and 300, wherein the actuation step 200 causes the beads to release the tension applied to the molecules.

[0197] Then, the nucleic acid molecule M is recompressed to reform the hairpin.

[0198] However, the presence of a single-stranded nucleic acid molecule that hybridizes with one of the nucleic acid strands in step 400 causes the hairpin to pause during rehybridization (or recompression). Detecting this pause indicates that the single-stranded nucleic acid molecule contains a sequence complementary to a portion of the hairpin molecule M. Furthermore, continuous measurements of the molecule length during hairpin rehybridization, including measurements of the molecule length during pauses when the hairpin molecule partially rehybridizes, enable the determination of the position of said sequence within the molecule. In fact, a comparison between the length of the molecule at the pause and the total length of the molecule allows the precise location of the hybridized nucleic acid molecule to be inferred, thereby allowing the inference of the sequence of molecule M at said location.

[0199] According to another non-limiting embodiment, the method can be carried out in the order disclosed in document EP 2 390 350, for further implementation details of which can be found in that document.

[0200] The apparatus and methods disclosed above demonstrate key improvements to optical detection systems known in the prior art.

[0201] First, the position of each bead is directly derived from the impedance change of the simple aperture structure. This avoids the need for complex optical components.

[0202] Secondly, as mentioned earlier, the size of the beads and the holes can be reduced. Therefore, the holes can be packed in high density on a single chip, and the impedance of the holes can then be read in parallel.

[0203] Since the relationship between the current in the hole and the position of the bead requires very little calculation, the above-mentioned device also reduces the computational requirements.

[0204] Finally, refer to Figure 4a and 4b As shown, magnetic materials capable of actuating beads can now be directly incorporated into the plate. This results in a greater force applied to the beads, and thus allows for a reduction in the size of the beads and holes.

Claims

1. An apparatus for analyzing nucleic acid molecules, comprising: A plate having a top surface and a bottom surface, the plate including a hole formed therein, the hole extending from the top surface along an axis perpendicular to the bottom surface into the plate, the hole opening on the top surface and closing on the bottom surface; Nucleic acid molecules disposed in the wells, the nucleic acid molecules being anchored at their first ends to the bottom surface of the wells defined by the plate; Beads arranged in the pores, the beads being anchored to the second end of nucleic acid molecules, the beads being configured to move within the pores along the axis of the pores, the pores having a cross-sectional area measured perpendicular to the axis, the cross-sectional area increasing between the bottom surface of the pores defined by the plate and the top surface of the plate; An actuator configured to move a bead along the axis of the hole; and A sensor is configured to measure the impedance of the hole, which depends on the distance between the bead and the bottom surface of the hole defined by the plate.

2. The apparatus of claim 1, wherein the actuator comprises: At least one magnet is displaceably disposed on the top surface of the plate, the at least one magnet being configured to displace along an axis parallel to the axis of the hole; A pair of rods fixedly disposed on the top surface of the plate and on the opposite side of the hole, the pair of rods comprising a magnetic material, the pair of rods being configured to be magnetized under the influence of a magnetic field generated by at least one magnet.

3. The apparatus according to claim 1, wherein the sensor comprises: The main electrode is set on the top surface of the plate; The secondary electrode is disposed on the bottom surface of the plate; and An electronic circuit configured to measure the current between the main electrode and the secondary electrode.

4. The apparatus of claim 1, wherein the minimum cross-sectional area of ​​the hole along which the bead can move is greater than the diameter of the bead.

5. The apparatus of claim 1, wherein the cross-sectional area of ​​the hole, measured at the bottom of the hole, is greater than the diameter of the bead.

6. The apparatus of claim 1, wherein the cross-sectional area of ​​the pore portion along which the bead can move increases linearly with increasing distance from the bottom surface of the pore.

7. The apparatus of claim 1, wherein the beads comprise a paramagnetic material.

8. The apparatus of claim 1, wherein the plate includes a plurality of holes formed therein, each individual hole of the plurality of holes extending from the top surface into the plate along an axis perpendicular to the bottom surface; Nucleic acid molecules are disposed in each individual well of the plurality of wells and anchored at the first end to the bottom surface of the respective well; and The beads are disposed in each individual well of the plurality of wells, and the beads are anchored to the second end of the nucleic acid molecule in their respective wells.

9. The apparatus of claim 8, wherein the actuator comprises: At least one magnet is displaceably disposed on the top surface of the plate, the at least one magnet being configured to displace along an axis parallel to the axis of the hole; and A plurality of rods are fixedly disposed on the top surface of a plate, each rod extending between two adjacent holes of the plurality of holes, such that a pair of rods are associated with each hole and disposed on opposite sides of the respective hole, the plurality of rods comprising a magnetic material, the plurality of rods being configured to be magnetized under the influence of a magnetic field generated by a magnet.

10. An analytical method for nucleic acid molecules for non-diagnostic purposes, said method being carried out using the apparatus according to any one of claims 1-9, comprising: Nucleic acid molecules are provided in the pores such that the nucleic acid molecules are coupled to a bead at a second end and to the bottom surface of the pore at a first end, the second end being opposite to the first end, and the bead being movable along the axis of the pore; The impedance of the hole is measured as the bead moves along the axis of the hole; and The distance between the bead and the bottom surface of the hole is determined from the measured impedance.

11. The method of claim 10, wherein determining the distance between the bottom surface of the pore and the bead from the measured impedance includes determining the length of the nucleic acid molecule.

12. The method of claim 10, wherein providing the nucleic acid molecule in the well comprises: The beads are placed in a conductive solution within the pores; A first potential difference is applied between the main electrode and the secondary electrode to move the bead into contact with the bottom surface of the hole, wherein the main electrode is located near the top surface of the hole and the secondary electrode is located near the bottom surface of the hole; The beads are coupled to the nucleic acid molecules; and Reverse the potential difference between the main electrode and the secondary electrode to move the bead away from the bottom surface of the hole.

13. The method of claim 10, further comprising actuating the beads within the hole by applying a magnetic force to the beads to move the beads along the axis of the hole, thereby increasing the distance between the bottom surface of the hole and the beads.

14. The method of claim 10, wherein measuring the impedance of the orifice comprises measuring the resistance of the orifice, and wherein the resistance decreases linearly with increasing distance to the bottom surface of the orifice.

15. The method of claim 10, wherein the nucleic acid molecule comprises a double-stranded nucleic acid molecule, and the method further comprises applying tension to the double-stranded nucleic acid molecule to cause the strands of the double-stranded nucleic acid molecule to separate into single-stranded nucleic acid molecules.

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