Patent Application
20250235865
The invention relates to an apparatus and a process for analyzing biomolecules through force spectroscopy. The invention applies in particular to the analysis of nucleic acid molecules such as DNA or RNA or proteins.
If biomolecules are attached to micro-scale beads, information about the structure of the biomolecules can be inferred by manipulating the beads and tracking their position with high resolution. For example, a nucleic acid molecule attached to a micro-scale bead can inform on the base sequence, the presence of biochemical modifications to the nucleic acid bases, and the interactions of the nucleic acid molecule with proteins such as polymerases, helicases, topoisomerase, etc.
Under particular experimental conditions, tracking the location of the bead in real time can be used to generate useful information about the structure of the DNA or RNA molecule to which it is attached. This can, in turn, be used to determine the molecule's gross organization, base sequence, the presence of biochemical modifications to the nucleic acid bases, and the interactions of the molecule with proteins such as polymerases, helicases, topoisomerases, etc.
For example, European patent EP3090803 discloses an apparatus for analyzing nucleic acid molecules, comprising: a bead on which one molecule is anchored at one end, a surface on which the molecule is anchored at the other end, an actuator adapted to cause the bead to move relative to said surface in one direction of motion, and a sensor adapted to measure a distance between the bead and the surface. The apparatus also comprises a well having an axis extending along the direction of motion of the bead and a bottom which is formed by said surface. The well is filled with an electrically conductive solution, and the bead being received in the well. The sensor is adapted to measure an impedance of the well, said impedance depending on a distance between the bead and the surface, in view of determining, from the measured impedance, the distance between the bead and the surface, and hence changes in the extension of the molecule with an accuracy up to the nanometer. From this information, the behavior of the molecule under various conditions can be inferred.
Typically, the control of the motion of the bead may rely on a magnetic force applied by the actuator on the bead. For example, the actuator comprises at least one magnet mounted to be displaceable along the direction of the axis of the well. The bead is made in a paramagnetic material, and is interposed between the bottom of the well and the magnet. The magnet allows a magnetic force to apply on the bead and consequently on the molecule to which it is anchored.
The sensor comprises a top electrode, a well and a bottom electrode. The top electrode is positioned over the top surface of the well, in contact with the electrically conductive solution. The top electrode is submitted to a known potential. The bottom electrode is positioned at the bottom surface of the well. An electronic circuit is provided to measure a current flowing between the electrodes. The current circulating between the electrodes is the measured signal.
The signal's strength depends on a ratio between the bead diameter and the microwell diameter at the bead's level. The cross-sectional area of the well perpendicular to the axis increases from the bottom surface to the top surface of the well. Thus, the strength of the signal indicates the depth of the bead within the well, which in turn indicates the molecule's extension.
However, the measured signal is weak, especially where the measurement precision is fundamentally limited by kT/C noise of the double layer capacitance on the bottom electrode.
Accordingly, there is a need for an apparatus for analyzing biomolecules using a biomolecule attached to a bead in a well, which allows a reduction of noise such as kT/C noise of the double layer capacitance and thermal noise of the well and thereby allows an increase in the measurement precision. Increasing precision reduces the number of measurement cycles required for statistical averaging, thereby allowing a reduced total measurement time, which helps achieve a cost-effective system with increased throughput.
The invention relates to an apparatus for biomolecule analysis comprising a plurality of cells, each cell comprising:
Other preferred, although non-limitative, aspects of the apparatus are as follows, isolated or in a technically feasible combination:
In a second aspect, the invention also relates to a process for manufacturing an apparatus as disclosed in the first aspect, said process comprising:
Step b) of depositing a first layer of a first material on the bottom electrode may be followed by a step b1) in which the first material of the first layer is removed except for a volume of first material above the bottom electrode, and wherein at step e) said volume of first material is etched to form the cavity. Dimensions of the cavity may be defined by the volume of first material left by the removal of the first material of the first layer in step b1). The cavity may be defined in the second layer of second material.
Other aspects, objects and advantages of the present invention will become better apparent upon reading the following detailed description of preferred embodiments thereof, given as non-limiting examples, and made with reference to the appended drawings wherein:
The apparatus includes a plurality of cells, each cell configured to carry out an analysis of a biomolecule. A biomolecule or biological molecule, is a molecule present in organisms that are essential to one or more typically biological processes. Typically, a biomolecule is a nucleic acid such as DNA or RNA, or a protein or complex of protein. Preferably, the biomolecule may be a double-stranded molecule of the hairpin type. Hairpin means a double helix wherein 5′ end of one strand is physically linked to the 3′ end of the other strand through an unpaired loop. This physical link can be either covalent or non-covalent, but is preferably a covalent bond. A hairpin thus consists of a double-stranded stem and an unpaired single-stranded loop. The biomolecule can also have other shapes, and especially may have some other reversible conformational change. For example, in the case of a protein, the biomolecule can be folded or unfolded.
The apparatus may contain several hundreds or thousands of wells, up to several tens of millions or hundreds of millions, arranged in a planar pattern. The number of cells may preferably be greater than several thousands or millions, preferably greater than ten million (10,000,000), for instance greater than one hundred million (100,000,000). Since the cells share the same structure, only one cell will be described. With reference to
The top surface 4 is the surface of a main layer 6 of electrically insulating material, for example a semiconductor such as silicon, glass, a non-conducting polymer such as polyimide, an organic dielectric, or a resin. The layer 6 is formed on a substrate 8 also made of electrically insulating material. In this example, the interface between the layer 6 and the substrate 8 forms the bottom surface 2.
A well 10 extends along the axis X from the top surface 4 towards the bottom surface 2. The well 10 opens at the top surface 4 on a channel 12. The well 10 forms a void in the main layer 6. The well 10 is a so-called microwell, because the order of magnitude of their dimensions (depth, largest length of a cross-section) is about 1 μm or about 0.1 μm. For instance, a well 10 can have a depth along the axis X of a few micrometers, for instance comprised between 1 and 10 micrometers, for instance equal to 8 μm.
The well 10 has a cross-sectional area perpendicular to the axis X, which monotonically varies along the axis X between a minimum and a maximum. Typically, the cross-sectional area of the well 10 decreases in a direction from the top surface 4 to the bottom surface 2. The cross-sectional area of the well 10 may otherwise increase in a direction from the top surface 4 to the bottom surface 2.
Preferably, the well 10 is rotationally symmetrical about the axis X. Viewed in a plane containing the axis X, the sidewall of the well 10 is not parallel with the axis X. The sidewall angle of the wall 10 is preferably comprised within 70°-89° with respect to a plane perpendicular to the axis X. A diameter of a cross-section of the well 10 in a plane orthogonal to the axis X varies between a minimum diameter DWmin and a maximum diameter DWmax. A largest length or diameter of a cross-section of the well 10 in a plane orthogonal to the axis X can range from a few hundred nanometers to a few micrometers, for instance about 4 or 5 μm. As in
z=I
0 tan h2(r−r0)
where I0 is the height of the well 10 and r0 is the bottom radius of the well 10.
The well 10 and channel 12 are filled with an electrically conductive solution. The electrically conductive solution preferably has a conductivity of between 10−7 S/cm and 10 S/cm, preferably between 10−4 S/cm and 10−1 S/cm, and more preferably between 1 to 20 mS/cm. For instance, the solution may be an aqueous solution of sodium chloride at a concentration of 100 mmol/m3 (100 mM). The solution may alternatively comprise a buffer compatible with the preservation of DNA molecules, such a buffered aqueous solution containing 10 mM Tris HCl and 0.1 mM EDTA and sodium Chloride at 100 mM. The buffer may also contain divalent cations compatible with enzymatic activities, such a 10 mM MgCl2. In some embodiments, the buffer should support electrophoresis (e.g. Tris Borate EDTA buffer).
A top electrode 14 is above the well 10. In this example the top electrode 14 is separated from the top surface 4 by the channel 12, but the top electrode 14 may also be arranged at the top surface 4. The top electrode 14 is for example supported above the well 10 by a support plate 16. The top electrode 14 is in contact with the electrically conductive solution. A bottom electrode 18 is arranged at the bottom surface 2, typically on the surface of the substrate 8. The bottom surface 2 is therefore formed by the bottom electrode 18 and by the surface of the substrate 8 where the bottom electrode 18 is absent. The top electrode 14 and the bottom electrode 18 are for example gold or platinum electrodes, but may be of any other suitable material.
A cavity 20 wider than the well 10 is arranged between the bottom electrode 18 and the well 10. The cavity 20 is in fluid communication with the well 10 and therefore continues the void of the well 10 in the main layer 6 toward the bottom surface 2, and more specifically to the bottom electrode 18 arranged at the bottom surface 2. The cavity 20 is also filled with the electrically conductive solution. The bottom electrode 18 is larger than the cavity 20, so that the bottom electrode 18 forms a bottom for the cavity 20. The cavity 20 exposes the bottom electrode 18 at the bottom surface 2 to the electrically conductive solution filling the cavity 20. A large area of the bottom electrode 18 is thus in contact with the electrically conductive solution.
The depth or height H of the cavity 20 along the axis X is much smaller than the depth of the well 10 along the axis X. Preferably, the cavity 20 has a height H along the axis X comprised between 0.05 μm and 1 μm, preferably between 0.1 μm and 0.8 μm and more preferably comprised between 0.2 μm and 0.5 μm. The well 10 is at least twice as deep as the cavity 20 along the axis X, and preferably the well 10 is at least four to twenty times deeper than the cavity 20. For example, the well has a height along the axis X greater than 1 μm, preferably greater than 2 μm, and more preferably greater than 5 μm.
The cavity 20 is however much wider than the well 10. The cross-sectional area of the cavity 20, perpendicular to the axis X, is at least twice a maximum cross-sectional area of the well 10, and preferably at least four times a maximum cross-sectional area of the well 10. For example, the cavity 20 has a largest cross-section perpendicularly to the axis X comprised between 2 μm and 7 μm. If the cross sections of the well 10 and the cavity 20 are circular, the diameter Dc of the cavity 20 is at least twice the maximal diameter DWmax of the well 10. This is the case in the illustrated example of
The cell 1 includes a bead 22 disposed within the well 10 at a position along the axis X. The bead 22 is of a spheroid shape, and is preferably spherical, even though it can be oval. The bead 22 has a diameter smaller than the diameter of the cross-section of the well 10, but preferably close to the smaller diameter DWmin of the cross-section of the well 10. Typically, the bead 22 has a diameter not greater than 5 μm. For instance, the bead 22 may have a diameter of about 1.5 μm or 1 μm. Preferably, the bead may be even smaller and have a diameter of less than 1 μm, for instance of 0.3 μm. The bead 20 must have a different conductivity than the conductivity of the solution. The bead is preferably electrically insulating.
To perform an analysis of a biomolecule, a biomolecule 24 is anchored to the bead 22 at a first end thereof, and to the bottom surface 2 on a second end thereof. Preferably, the second end of the biomolecule 24 is opposite the first end of the biomolecule 24, i.e. the second end on the other side of the molecule with respect to the first end. Since the bottom surface 2 below the well 10 and cavity 20 is formed by the bottom electrode 18, the second end of the biomolecule 24 is anchored to the bottom electrode 18.
To achieve the anchoring of the biomolecule 24 on the bead 22 and on the bottom surface 2, the bead 22 and the bottom surface 2 may be coated with a specific material adapted to bind with an end of the biomolecule 24. For instance, the DNA or RNA molecules may be labelled with biotin at one end, digoxigenin (Dig) at another end, and the beads may be coated with streptavidin to bind with a labelled (for example biotin) end of a DNA/RNA hairpin molecule, and the bottom surface 2, for example the exposed bottom electrode 18, may be further coated with anti-Dig antibodies to bind a Dig-labelled end of the DNA/RNA molecule, see for instance Hunter M M, Margolies M N, Ju A, Haber E, “High-affinity monoclonal antibodies to the cardiac glycoside, digoxin”, Journal of Immunology, 1982 September; 129(3): 1165-1172.
The bead 22 is attached to the bottom surface 2 via the biomolecule 24, but the bead 22 is moveable relative to the bottom surface 2. In particular, the bead 22 can move in the well 10 along the direction of the axis X. The depth of the position of the bead 22 within the well 10 can thus change, depending on the forces applying on the bead 22.
In order to apply a force on the bead 22 along this axis X, the cell 1 may further include an actuator adapted to cause the bead 22 to move in translation along said axis. The control of the motion of the bead 22 may rely on a magnetic force applied by the actuator on the bead 22. In that case, the bead 22 is made of a paramagnetic material, such as a superparamagnetic material. For instance, the bead 22 may be made in latex with incorporated ferrites, and coated with streptavidin for anchoring the biomolecule 24. The actuator may include at least one permanent magnet, which can be controlled to move in translation along the axis X. The actuator may include two permanent magnets, positioned at equal distance of the axis X and having their magnetic poles aligned perpendicular to the axis X, the North pole of a magnet facing the South pole of the other. The bead 22 is then positioned between the bottom surface 2 and the magnets. These magnets allow one to apply a force on the bead 22 and consequently on the biomolecule 24 to which it is anchored. By moving the magnets closer to or further from the bead 22 along the axis X, one changes the magnetic field and thus controls the magnitude of the force applied to the bead 22, thus controlling the stretching of the biomolecule 24 in the direction of the axis X.
The force applied to the bead 22 can be created by other configurations, in alternative or complement. The actuator may include a permanent magnet and a strip covered with a magnetizable material positioned at a fixed distance relative to the well 10, of about a few micrometers. By bringing the permanent magnet closer or further from the strip covered with magnetizable material the field applied by said strip on the bead 22 can be varied. The actuator may include the top electrode 14 and bottom electrode 18. Other ways of controlling the force applied to the bead 22 can be used, such as optical or acoustic tweezers, the latter implying application of acoustic waves on the bead, see for instance G. Sitters, D. Kamsma, G. Thalhammer, M. Ritsch-Marte, E. J. G. Peterman and G. L. J. Wuite, “Acoustic Force spectroscopy”, in Nature Methods, Vol. 12, No 1, January 2015, or X. Ding, Z. S. Lin, B. Kiraly, H. Yue, S. Li, I. Chiang, J. Shi, S. J. Benkovic and T. J. Huang, “On-Chip Manipulation of single microparticles, cells, and organisms using surface acoustic waves”, PNAS, Jul. 10, 2012, vol. 109, no. 28, 11105-11109.
The apparatus allows determining the position of the bead 22 within the well 10, for example the depth of the bead 22, or the distance between the bead 22 and the bottom surface 2 corresponding to the length of the biomolecule 24 anchored to the bead 22 and the bottom surface 2, by monitoring the impedance, in particular the resistance (or conductance) between the bottom electrode 18 at the bottom surface 2 and the top electrode 14.
Since the well 10 has a cross-section that varies with the depth or the distance along the axis X from the bottom surface 2, and as the bead 22 is of constant size, the bead 22 occupies a varying proportion of the internal volume of the well 10. As an example, in reference to
The resistance of the well 10 thus depends on the distance between the bead 22 and the bottom surface 2, which depends on the length of the biomolecule 24 attached to the bead 22. By measuring the impedance between the top electrode 14 and the bottom electrode 18, it is possible to derive the length of the biomolecule 24.
The top electrode 14 and the bottom electrode 18 are in contact with the electrically conductive solution. One of the electrodes 14, 18 is connected to a voltage source (not shown), preferably an AC voltage source, and is set to reference peak voltage V0 at least intermittently. The reference peak voltage V0 is preferably below 1.2 V, and more preferably below 0.8 V to avoid electrolysis phenomenon within the well 10. For example, the reference peak voltage V0 is comprised between 10 and 500 mV, preferably comprised between 50 and 300 mV. This electrode is used as a reference electrode (preferably the top electrode 14) and may be common to several cells 1, whereas the other electrode used as a measurement electrode (preferably the bottom electrode 18) is specific to each cell 1 or well 10.
An electronic circuit measures the impedance between the two electrodes 14, 18 by measuring the current flowing between them. The electronic circuit may comprise a known resistance connected in series with the measurement electrode. A potential difference at the poles of the resistance may be measured to infer the current flowing through the resistance. Amplification circuitry may be provided to amplify the measurement signal. For sake of simplicity, the electronic circuit and the various electric connections are not represented.
The analysis of the biomolecule 24 is carried out on the basis of the measurement signal. For example, the analysis may comprise the determination of a nucleic acid sequence, i.e. the deciphering of the actual succession of bases in a nucleic acid, but also the determination of other pieces of information on the nucleic acid sequence, such as the detection of a particular sequence in a nucleic acid molecule, the detection of a difference between the sequences of two different nucleic acid molecules, or the binding of a protein to a specific sequence, see e.g. WO 2011/147931; WO 2011/147929; WO 2013/093005; WO 2014/114687. Details can be found in patent EP3090803.
As a non-limiting example, the process can for instance be carried out according to the sequence disclosed in document EP2390351, to which one can refer for more details about the implementation of the sequence. In this example, the biomolecule 24 is a hairpin nucleic acid molecule.
First, the bead is actuated to separate the two strands of the hairpin biomolecule 24, by applying a tension about 15 pN or more on the molecule, for instance equal to 18 pN. The distance between the bead 22 and the bottom surface 2 is derived from the impedance measurement, which corresponds to the total length of the opened hairpin biomolecule 24. A piece of single-stranded nucleic acid molecule is then hybridized with one of the strands of the biomolecule 24.
The bead 22 is then actuated to release the tension applied to the biomolecule 24. The nucleic acid biomolecule 24 then rezips to reform a hairpin. However, the presence of a single-stranded nucleic acid molecule hybridized to one of the nucleic acid strands leads to a pause in re-hybridization (or rezipping) of the hairpin. The detection of such a pause indicates that the single-stranded nucleic acid biomolecule 24 comprises a sequence which is complementary to a part of the hairpin biomolecule 24. Moreover the continuing measurement of the length of the biomolecule 24 during the re-hybridization of the hairpin, including the measurement of the length of the biomolecule 24 during the pause when the hairpin biomolecule 24 is partly re-hybridized, allows determining the position of the said sequence in the biomolecule 24. Indeed, the comparison between the length of the biomolecule 24 at the moment of the pause and the total length of the biomolecule 24 allows inferring the exact position of the hybridized nucleic acid biomolecule 24, from which the sequence of the biomolecule 24 at said position can be deduced.
A measurement signal reflecting accurately the changes of the impedance of the well 10 is therefore of paramount importance for the analysis of the biomolecule 24. Due to the cavity 20, the conductive solution is in contact with the bottom electrode 18 over a large area, larger than the largest cross-section of the well 10. The increased electrode area in contact with the conductive solution leads to a significant increase in the measurement signal's strength, improving the signal to noise ratio. It is then possible to reduce the size of the cell 1 in comparison with a cell 1 without a cavity 20, without having too small a measurement signal. More cells 1 can thus be accommodated in the same volume, greatly improving the throughput of the apparatus without increasing the cost in the same proportion. It is also possible to accelerate the measure since the measurement signal is more stable and accurate values are more easily obtained, thereby increasing the throughput of the apparatus without increasing the cost.
The presence of the cavity 20 makes the manufacturing of the apparatus easier. The shape of the well 10 must be carefully designed, and the manufacturing process must respect the determined shape of the well 10. The shape of the well 10 dictates the relationship between the measurement signal and the position of the bead 22. Any inaccuracy in the dimensions of the well 10 may lead to measurement errors. With known manufacturing techniques, it may be difficult to ensure accurate dimensions of a bottom portion of a well in contact with a bottom electrode. Having the cavity 20 between the bottom electrode 18 and the well 10 allows relaxing the strict design parameters of the well 10 near the bottom electrode 18 since the dimensions of the cavity 20 do not have to be strictly controlled, or at least only global parameters such as cavity height H and diameter are to be considered. Constraining parameters such as the slope of the cavity wall no longer need to be considered. The main function of the cavity 20 is indeed to provide a large contact area between the bottom electrode 18 and the conductive solution, and this function involves few design constraints.
Adding the cavity 20 between the well 10 and the bottom electrode 18 can be made in any way known by the person skilled in the art. Two ways of manufacturing a cell 1 with a cavity 20 between the well 10 and the bottom electrode 18 will now be described. The two examples share many aspects, which can be summarized in the following steps:
The first material is an electrically insulating material, for example a semiconductor such as silicon, or insulator such as silicon dioxide, silicon nitride, glass, a non-conducting polymer such as polyimide, an organic dielectric, or a resin. The thickness (or height) of the first layer 30 is chosen to correspond to the desired height (or depth) of the cavity 20.
The second material is also an electrically insulating material, and can be a semiconductor such as silicon, or insulator such as silicon dioxide, silicon nitride, glass, a non-conducting polymer such as polyimide, an organic dielectric, or a resin. The first material and the second material are chosen so as to be able to selectively etch the first material without altering the second material, one example being a first material of silicon dioxide and a second material of polyimide, where the first material can be etched selectively from the second material using hydrofluoric acid solution.
With reference to
In step b) illustrated in
In step d) illustrated in
In step e) illustrated in
Since the first material is etched through the well 10, the cavity 20 thus formed is automatically aligned with the well 10. This approach allows a shorter process, with fewer steps, in particular few alignment steps. The duration of the etching must however be precisely controlled. Variations between cavity sizes of different cells may also appear.
In step f) illustrated by
With reference to
In step b) illustrated in
In a step b1) following the step b) of depositing a first layer of a first material onto the bottom electrode 18 and illustrated by
In step c) illustrated by
In step d) illustrated by
In step e) illustrated in
In this approach, the cavity 20 is defined in the second layer 32 of second material, since the first layer 30 no longer exists. The dimensions of the cavity 20 are more precisely defined, which improves cavity size uniformity between different cells. Also, the etching is much easily controlled since the cavity size is not defined by the etching duration. This approach however requires an additional alignment step between the well 10 and the volume 36 of first material.
In step f) illustrated by
While the present invention has been described with respect to certain preferred embodiments, it is obvious that it is in no way limited thereto and it comprises all the technical equivalents of the means described and their combinations. In particular, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the appended claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/077363 | 9/30/2022 | WO |
