Patent Description:
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 <CIT> 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 the <NUM>' end of one strand is physically linked to the <NUM>' 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 (<NUM>,<NUM>,<NUM>), for instance greater than one hundred million (<NUM>,<NUM>,<NUM>). Since the cells share the same structure, only one cell will be described. With reference to <FIG>, each cell <NUM> includes a bottom surface <NUM> and a top surface <NUM> opposite the bottom surface <NUM>. An axis X extends between the bottom surface <NUM> and the top surface <NUM>, and is perpendicular to the bottom surface <NUM> and the top surface <NUM>.

The top surface <NUM> is the surface of a main layer <NUM> 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 <NUM> is formed on a substrate <NUM> also made of electrically insulating material. In this example, the interface between the layer <NUM> and the substrate <NUM> forms the bottom surface <NUM>.

A well <NUM> extends along the axis X from the top surface <NUM> towards the bottom surface <NUM>. The well <NUM> opens at the top surface <NUM> on a channel <NUM>. The well <NUM> forms a void in the main layer <NUM>. The well <NUM> is a so-called microwell, because the order of magnitude of their dimensions (depth, largest length of a cross-section) is about <NUM> or about <NUM>. For instance, a well <NUM> can have a depth along the axis X of a few micrometers, for instance comprised between <NUM> and <NUM> micrometers, for instance equal to <NUM>.

The well <NUM> 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 <NUM> decreases in a direction from the top surface <NUM> to the bottom surface <NUM>. The cross-sectional area of the well <NUM> may otherwise increase in a direction from the top surface <NUM> to the bottom surface <NUM>.

Preferably, the well <NUM> is rotationally symmetrical about the axis X. Viewed in a plane containing the axis X, the sidewall of the well <NUM> is not parallel with the axis X. The sidewall angle of the wall <NUM> is preferably comprised within <NUM>°-<NUM>° with respect to a plane perpendicular to the axis X. A diameter of a cross-section of the well <NUM> 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 <NUM> in a plane orthogonal to the axis X can range from a few hundred nanometers to a few micrometers, for instance about <NUM> or <NUM>. As in <FIG>, the well <NUM> may have a frustoconical shape (a truncated cone). Alternatively, the radius of the wall of the well <NUM> may be defined according to the distance z from the bottom surface <NUM> by an equation such as: <MAT> where I<NUM> is the height of the well <NUM> and r<NUM> is the bottom radius of the well <NUM>.

The well <NUM> and channel <NUM> are filled with an electrically conductive solution. The electrically conductive solution preferably has a conductivity of between <NUM>-<NUM> S/cm and <NUM>/cm, preferably between <NUM>-<NUM> S/cm and <NUM>-<NUM> S/cm, and more preferably between <NUM> to <NUM>/cm. For instance, the solution may be an aqueous solution of sodium chloride at a concentration of <NUM> mmol/m<NUM> (<NUM>). The solution may alternatively comprise a buffer compatible with the preservation of DNA molecules, such a buffered aqueous solution containing <NUM> Tris HCl and <NUM> EDTA and sodium Chloride at <NUM>. The buffer may also contain divalent cations compatible with enzymatic activities, such a <NUM> MgCl2. In some embodiments, the buffer should support electrophoresis (e.g. Tris Borate EDTA buffer).

A top electrode <NUM> is above the well <NUM>. In this example the top electrode <NUM> is separated from the top surface <NUM> by the channel <NUM>, but the top electrode <NUM> may also be arranged at the top surface <NUM>. The top electrode <NUM> is for example supported above the well <NUM> by a support plate <NUM>. The top electrode <NUM> is in contact with the electrically conductive solution. A bottom electrode <NUM> is arranged at the bottom surface <NUM>, typically on the surface of the substrate <NUM>. The bottom surface <NUM> is therefore formed by the bottom electrode <NUM> and by the surface of the substrate <NUM> where the bottom electrode <NUM> is absent. The top electrode <NUM> and the bottom electrode <NUM> are for example gold or platinum electrodes, but may be of any other suitable material.

A cavity <NUM> wider than the well <NUM> is arranged between the bottom electrode <NUM> and the well <NUM>. The cavity <NUM> is in fluid communication with the well <NUM> and therefore continues the void of the well <NUM> in the main layer <NUM> toward the bottom surface <NUM>, and more specifically to the bottom electrode <NUM> arranged at the bottom surface <NUM>. The cavity <NUM> is also filled with the electrically conductive solution. The bottom electrode <NUM> is larger than the cavity <NUM>, so that the bottom electrode <NUM> forms a bottom for the cavity <NUM>. The cavity <NUM> exposes the bottom electrode <NUM> at the bottom surface <NUM> to the electrically conductive solution filling the cavity <NUM>. A large area of the bottom electrode <NUM> is thus in contact with the electrically conductive solution.

The depth or height H of the cavity <NUM> along the axis X is much smaller than the depth of the well <NUM> along the axis X. Preferably, the cavity <NUM> has a height H along the axis X comprised between <NUM> and <NUM>, preferably between <NUM> and <NUM> and more preferably comprised between <NUM> and <NUM>. The well <NUM> is at least twice as deep as the cavity <NUM> along the axis X, and preferably the well <NUM> is at least four to twenty times deeper than the cavity <NUM>. For example, the well has a height along the axis X greater than <NUM>, preferably greater than <NUM>, and more preferably greater than <NUM>.

The cavity <NUM> is however much wider than the well <NUM>. The cross-sectional area of the cavity <NUM>, perpendicular to the axis X, is at least twice a maximum cross-sectional area of the well <NUM>, and preferably at least four times a maximum cross-sectional area of the well <NUM>. For example, the cavity <NUM> has a largest cross-section perpendicularly to the axis X comprised between <NUM> and <NUM>. If the cross sections of the well <NUM> and the cavity <NUM> are circular, the diameter Dc of the cavity <NUM> is at least twice the maximal diameter DWmax of the well <NUM>. This is the case in the illustrated example of <FIG>, wherein the well <NUM> has a frustoconical shape and the cavity <NUM> has a cylindrical shape. As in this example, the cross-sectional area of the well <NUM> may decrease in a direction from the top surface <NUM> to the bottom surface <NUM> until a minimum is reached at a transition between the well <NUM> and the cavity <NUM>.

The cell <NUM> includes a bead <NUM> disposed within the well <NUM> at a position along the axis X. The bead <NUM> is of a spheroid shape, and is preferably spherical, even though it can be oval. The bead <NUM> has a diameter smaller than the diameter of the cross-section of the well <NUM>, but preferably close to the smaller diameter DWmin of the cross-section of the well <NUM>. Typically, the bead <NUM> has a diameter not greater than <NUM>. For instance, the bead <NUM> may have a diameter of about <NUM> or <NUM>. Preferably, the bead may be even smaller and have a diameter of less than <NUM>, for instance of <NUM>. The bead <NUM> 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 <NUM> is anchored to the bead <NUM> at a first end thereof, and to the bottom surface <NUM> on a second end thereof. Preferably, the second end of the biomolecule <NUM> is opposite the first end of the biomolecule <NUM>, i.e. the second end on the other side of the molecule with respect to the first end. Since the bottom surface <NUM> below the well <NUM> and cavity <NUM> is formed by the bottom electrode <NUM>, the second end of the biomolecule <NUM> is anchored to the bottom electrode <NUM>.

To achieve the anchoring of the biomolecule <NUM> on the bead <NUM> and on the bottom surface <NUM>, the bead <NUM> and the bottom surface <NUM> may be coated with a specific material adapted to bind with an end of the biomolecule <NUM>. 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 <NUM>, for example the exposed bottom electrode <NUM>, may be further coated with anti-Dig antibodies to bind a Dig-labelled end of the DNA / RNA molecule, see for instance <NPL>.

The bead <NUM> is attached to the bottom surface <NUM> via the biomolecule <NUM>, but the bead <NUM> is moveable relative to the bottom surface <NUM>. In particular, the bead <NUM> can move in the well <NUM> along the direction of the axis X. The depth of the position of the bead <NUM> within the well <NUM> can thus change, depending on the forces applying on the bead <NUM>.

In order to apply a force on the bead <NUM> along this axis X, the cell <NUM> may further include an actuator adapted to cause the bead <NUM> to move in translation along said axis. The control of the motion of the bead <NUM> may rely on a magnetic force applied by the actuator on the bead <NUM>. In that case, the bead <NUM> is made of a paramagnetic material, such as a superparamagnetic material. For instance, the bead <NUM> may be made in latex with incorporated ferrites, and coated with streptavidin for anchoring the biomolecule <NUM>. 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 <NUM> is then positioned between the bottom surface <NUM> and the magnets. These magnets allow one to apply a force on the bead <NUM> and consequently on the biomolecule <NUM> to which it is anchored. By moving the magnets closer to or further from the bead <NUM> along the axis X, one changes the magnetic field and thus controls the magnitude of the force applied to the bead <NUM>, thus controlling the stretching of the biomolecule <NUM> in the direction of the axis X.

The force applied to the bead <NUM> 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 <NUM>, 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 <NUM> can be varied. The actuator may include the top electrode <NUM> and bottom electrode <NUM>. Other ways of controlling the force applied to the bead <NUM> can be used, such as optical or acoustic tweezers, the latter implying application of acoustic waves on the bead, see for instance <NPL> , or <NPL>.

The apparatus allows determining the position of the bead <NUM> within the well <NUM>, for example the depth of the bead <NUM>, or the distance between the bead <NUM> and the bottom surface <NUM> corresponding to the length of the biomolecule <NUM> anchored to the bead <NUM> and the bottom surface <NUM>, by monitoring the impedance, in particular the resistance (or conductance) between the bottom electrode <NUM> at the bottom surface <NUM> and the top electrode <NUM>.

Since the well <NUM> has a cross-section that varies with the depth or the distance along the axis X from the bottom surface <NUM>, and as the bead <NUM> is of constant size, the bead <NUM> occupies a varying proportion of the internal volume of the well <NUM>. As an example, in reference to <FIG> in which the area of a cross-section of the well <NUM> perpendicular to the axis X strictly increases with the distance from the bottom surface <NUM>, if the bead <NUM> is very close to the bottom of the well <NUM>, it occupies a major proportion of the portion of the well <NUM> extending around the bead <NUM>. The resistance of this portion is therefore increased dramatically because there is very little space left for the conductive solution. As the overall resistance of the cell <NUM> is the integral of the resistance along the whole depth of the well <NUM> and cavity <NUM> along the axis X, the overall resistance is also increased dramatically. Conversely, if the bead <NUM> is close to the top surface <NUM>, it occupies a minor proportion of the portion of the well <NUM> extending around the bead <NUM>. The resistance of this portion is thus less increased than previously, and the overall resistance of the well <NUM> is smaller than previously.

The resistance of the well <NUM> thus depends on the distance between the bead <NUM> and the bottom surface <NUM>, which depends on the length of the biomolecule <NUM> attached to the bead <NUM>. By measuring the impedance between the top electrode <NUM> and the bottom electrode <NUM>, it is possible to derive the length of the biomolecule <NUM>.

The top electrode <NUM> and the bottom electrode <NUM> are in contact with the electrically conductive solution. One of the electrodes <NUM>, <NUM> is connected to a voltage source (not shown), preferably an AC voltage source, and is set to reference peak voltage V<NUM> at least intermittently. The reference peak voltage V<NUM> is preferably below <NUM> V, and more preferably below <NUM> V to avoid electrolysis phenomenon within the well <NUM>. For example, the reference peak voltage V<NUM> is comprised between <NUM> and <NUM> mV, preferably comprised between <NUM> and <NUM> mV. This electrode is used as a reference electrode (preferably the top electrode <NUM>) and may be common to several cells <NUM>, whereas the other electrode used as a measurement electrode (preferably the bottom electrode <NUM>) is specific to each cell <NUM> or well <NUM>.

An electronic circuit measures the impedance between the two electrodes <NUM>, <NUM> 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 <NUM> 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. <CIT> ; <CIT> ; <CIT> ; <CIT>. Details can be found in patent <CIT>.

As a non-limiting example, the process can for instance be carried out according to the sequence disclosed in document <CIT>, to which one can refer for more details about the implementation of the sequence. In this example, the biomolecule <NUM> is a hairpin nucleic acid molecule.

First, the bead is actuated to separate the two strands of the hairpin biomolecule <NUM>, by applying a tension about <NUM> pN or more on the molecule, for instance equal to <NUM> pN. The distance between the bead <NUM> and the bottom surface <NUM> is derived from the impedance measurement, which corresponds to the total length of the opened hairpin biomolecule <NUM>. A piece of single-stranded nucleic acid molecule is then hybridized with one of the strands of the biomolecule <NUM>.

The bead <NUM> is then actuated to release the tension applied to the biomolecule <NUM>. The nucleic acid biomolecule <NUM> 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 <NUM> comprises a sequence which is complementary to a part of the hairpin biomolecule <NUM>. Moreover the continuing measurement of the length of the biomolecule <NUM> during the re-hybridization of the hairpin, including the measurement of the length of the biomolecule <NUM> during the pause when the hairpin biomolecule <NUM> is partly re-hybridized, allows determining the position of the said sequence in the biomolecule <NUM>. Indeed, the comparison between the length of the biomolecule <NUM> at the moment of the pause and the total length of the biomolecule <NUM> allows inferring the exact position of the hybridized nucleic acid biomolecule <NUM>, from which the sequence of the biomolecule <NUM> at said position can be deduced.

A measurement signal reflecting accurately the changes of the impedance of the well <NUM> is therefore of paramount importance for the analysis of the biomolecule <NUM>. Due to the cavity <NUM>, the conductive solution is in contact with the bottom electrode <NUM> over a large area, larger than the largest cross-section of the well <NUM>. 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 <NUM> in comparison with a cell <NUM> without a cavity <NUM>, without having too small a measurement signal. More cells <NUM> 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 <NUM> makes the manufacturing of the apparatus easier. The shape of the well <NUM> must be carefully designed, and the manufacturing process must respect the determined shape of the well <NUM>. The shape of the well <NUM> dictates the relationship between the measurement signal and the position of the bead <NUM>. Any inaccuracy in the dimensions of the well <NUM> 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 <NUM> between the bottom electrode <NUM> and the well <NUM> allows relaxing the strict design parameters of the well <NUM> near the bottom electrode <NUM> since the dimensions of the cavity <NUM> 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 <NUM> is indeed to provide a large contact area between the bottom electrode <NUM> and the conductive solution, and this function involves few design constraints.

Adding the cavity <NUM> between the well <NUM> and the bottom electrode <NUM> can be made in any way known by the person skilled in the art. Two ways of manufacturing a cell <NUM> with a cavity <NUM> between the well <NUM> and the bottom electrode <NUM> 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 <NUM> is chosen to correspond to the desired height (or depth) of the cavity <NUM>.

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 <FIG>, a first process for manufacturing an apparatus first involves the step a) of forming a bottom electrode <NUM> on a dielectric substrate <NUM>. The dielectric substrate <NUM> can be for example glass, or a silicon wafer with an insulating coating such as silicon dioxide or silicon nitride or combination thereof. As mentioned above, the bottom electrode <NUM> may be made of gold or platinum deposited on the substrate through conventional deposition techniques such as electron beam evaporation or sputtering. The bottom surface <NUM> is thereby formed by the upper surface of the dielectric substrate <NUM> and the bottom electrode <NUM> thereon.

In step b) illustrated in <FIG>, a first layer <NUM> of a first material is deposited onto the bottom electrode <NUM> and the surface of the substrate <NUM> other than the bottom electrode <NUM>. In step c) illustrated in <FIG>, a second layer <NUM> of a second material is deposited to cover the first layer <NUM>.

In step d) illustrated in <FIG>, the well <NUM> is formed in the second layer <NUM> of second material, thereby exposing an area <NUM> of first material at the bottom of the well <NUM>. The well <NUM> is formed for example by transfer of reflow resist pattern, or dry etching with control of sidewall angle, or nano imprint lithography.

In step e) illustrated in <FIG>, the cavity <NUM> is formed by etching first material through the well <NUM>. The etching starts by removing the first material from the area <NUM> of first material exposed by the well <NUM>. The first material is removed on all the thickness of the first layer <NUM>. A pre-cavity is formed in the first layer <NUM>, with a cross-section that increases as the etching goes on. When the cavity <NUM> of desired dimensions is obtained, the etching is stopped. The dimensions of the cavity <NUM> can therefore be controlled by the duration of the etching. The remaining of the first layer <NUM> is kept. The cavity <NUM> is defined in the first layer <NUM> of first material. The first layer <NUM> and the second layer <NUM> form together the main layer <NUM> of electrically insulating material.

Since the first material is etched through the well <NUM>, the cavity <NUM> thus formed is automatically aligned with the well <NUM>. 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 <FIG>, the top electrode <NUM> is arranged, for example by depositing metal such as gold on a support plate <NUM> and bringing the support plate <NUM> above the well <NUM>, at a distance of the top surface <NUM> to leave room for the channel <NUM>. In a further step g) the well <NUM> and cavity <NUM> are filled with the electrically conductive solution, the biomolecule <NUM> is anchored at a first end to the bottom surface <NUM> and at a second end to the bead <NUM>, to obtain a cell <NUM> as illustrated in <FIG>.

With reference to <FIG>, a second process for manufacturing an apparatus first involves the step a) of forming a bottom electrode <NUM> on a dielectric substrate <NUM>. The dielectric substrate <NUM> can be for example glass, a silicon wafer with an insulating coating such as silicon dioxide or silicon nitride or combination thereof. As mentioned above, the bottom electrode <NUM> may be made of gold or platinum deposited on the substrate through conventional deposition techniques such as electron beam evaporation or sputtering. The bottom surface <NUM> is formed by the upper surface of the dielectric substrate <NUM> and the bottom electrode <NUM> thereon.

In step b) illustrated in <FIG>, a first layer <NUM> of a first material is deposited on the bottom electrode <NUM>. The first layer <NUM> is also deposited on parts of the bottom surface <NUM> of the substrate <NUM> other than the bottom electrode <NUM>. The first layer <NUM> thus covers the entire bottom surface <NUM>.

In a step b1) following the step b) of depositing a first layer of a first material onto the bottom electrode <NUM> and illustrated by <FIG>, the first material of the first layer <NUM> is removed except for a volume <NUM> of first material above the bottom electrode <NUM>. The volume <NUM> of first material which is left correspond to the desired volume of the cavity <NUM>. The first layer <NUM> of first material now consists in the remaining volume <NUM> of first material. The removal of the first material of the first layer <NUM> is for example performed by wet etching or dry etching using standard techniques with appropriate selectivity well known to those skilled in the art.

In step c) illustrated by <FIG>, a second layer <NUM> of a second material is deposited to cover the first layer <NUM>, which is now restricted to the remaining volume <NUM> of first material in this embodiment. The second layer <NUM> also covers the bottom surface <NUM> where the first material was previously removed.

In step d) illustrated by <FIG>, the well <NUM> is formed in the second layer <NUM> of second material, thereby exposing an area <NUM> of first material of the volume <NUM> of first material above the bottom electrode <NUM>. The well <NUM> is formed for example by transfer of reflow resist pattern, or dry etching with control of sidewall angle, or nano imprint lithography.

In step e) illustrated in <FIG>, the cavity <NUM> is formed by etching first material through the well <NUM>. The etching starts by removing the first material from the area <NUM> of first material exposed by the well <NUM>, and is carried out until the volume <NUM> of first material above the bottom electrode <NUM> is entirely removed, thus leaving the cavity <NUM>. Dimensions of the cavity are defined by the volume <NUM> of first material left by the removal of the first material of the first layer <NUM> in step b1). Consequently, the size and shape of the cavity <NUM> is defined before the etching, which lead to an etching process extremely tolerant to process variations, especially regarding duration variations. In this embodiment, the entirety of the first material is removed either during step b1) or step e). The main layer <NUM> of electrically insulating material is therefore constituted only of the second layer <NUM> of second material.

In this approach, the cavity <NUM> is defined in the second layer <NUM> of second material, since the first layer <NUM> no longer exists. The dimensions of the cavity <NUM> 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 <NUM> and the volume <NUM> of first material.

Claim 1:
An apparatus for biomolecule analysis, comprising a plurality of cells (<NUM>), each cell (<NUM>) comprising:
- a bottom surface (<NUM>) and a top surface (<NUM>) opposite the bottom surface (<NUM>), an axis (X) extending between the bottom surface (<NUM>) and the top surface (<NUM>);
- a well (<NUM>) extending along the axis (X) from the top surface (<NUM>) towards the bottom surface (<NUM>), wherein the well (<NUM>) has a cross-sectional area perpendicular to the axis (X), which monotonically varies along the axis of the well (<NUM>) between a minimum and a maximum;
- a molecule (<NUM>) anchored at a first end to the bottom surface (<NUM>);
- a bead (<NUM>) to which a second end of the biomolecule is anchored, the bead (<NUM>) disposed within the well (<NUM>) at a position;
- a electrically conductive solution filling the well (<NUM>);
- a top electrode (<NUM>) above the well, and a bottom electrode (<NUM>) at the bottom surface (<NUM>), both the top electrode (<NUM>) and the bottom electrode (<NUM>) in contact with the electrically conductive solution,
wherein the apparatus comprises a sensor configured to measure an impedance between the top electrode (<NUM>) and the bottom electrode (<NUM>), the impedance depending on the depth of the bead (<NUM>) within the well (<NUM>),
characterized in that a cavity (<NUM>) wider than the well (<NUM>) is arranged between the bottom electrode (<NUM>) and the well (<NUM>), said cavity (<NUM>) in fluid communication with the well (<NUM>) and exposing the bottom electrode (<NUM>) at the bottom surface to the electrically conductive solution filling the cavity (<NUM>).