Patent ID: 12240752

DETAILED DESCRIPTION

In the following description of favorable exemplary embodiments of the present disclosure, identical or similar reference signs are used for the elements which are depicted in the various figures and act in a similar manner, in order to dispense with a repeated description of said elements.

FIG.1shows a schematic representation of a sequencing unit100according to one exemplary embodiment. The sequencing unit100comprises a graphene layer102having a sequencing pore104for sequencing a biochemical material, in this case for determination of DNA fragment length by way of example. The graphene layer102is a layer which was generated by thermal conversion of a self-assembled monolayer prestructured with the sequencing pore104in a thermal lithography method. A conversion temperature for the thermal conversion was chosen such that the sequencing pore104was reduced in size, as a result of closing up, to a diameter suitable for sequencing the biochemical material. A suitable conversion temperature can therefore be selected depending on the desired diameter of the sequencing pore104.

According to this exemplary embodiment, the graphene layer102forms a base of a cavern element106having a cavern108for prestorage of the biochemical material. The cavern108is, by way of example, formed as a channel crossing the cavern element106in a substantially straight manner and having an entry opening110and exit opening112lying opposite the entry opening. The sequencing pore104lies opposite the exit opening112, meaning that an exit cross-section of the cavern108is defined by the sequencing pore104.

According to the exemplary embodiment shown inFIG.1, the graphene layer102has a plurality of further sequencing pores114for sequencing the biochemical material or further biochemical materials. The further sequencing pores114are generated analogously to the sequencing pore104. The cavern element106is accordingly formed with multiple further caverns116for accommodating the biochemical materials, the respective exit openings of the further caverns116each lying opposite one further sequencing pore114.

The sequencing unit100is, for example, integrable in a lab-on-a-chip environment, indicated by a dashed frame. By way of example, the sequencing unit100according toFIG.1is realized as a bonded component composed of a cavern wafer having nanopores at the base and a cap-shaped counter electrode118. The graphene layer102is covered by the counter electrode118. The sequencing unit100is connected to a measurement device122for current measurement via the counter electrode118and multiple further electrodes120which are each arranged at an entry opening of the caverns108,116. In particular, the sequencing unit100is realized as a LATE-qPCR Sanger sequencing array in silicon with a downstream graphene-based nanopore sequencer via determination of DNA fragment length.

Self-assembled monolayers, called monolayers for short hereinafter, are absolutely ideal as precursor layer for creating the sequencing pores in the thermal lithography method, especially by thermal scanning probe lithography, t-SPL for short, since precision and reproducibility of this structuring method are particularly good owing to the very low and extremely homogeneous, here monomolecular, layer thickness. Furthermore, at an AFM tip temperature of 900° C. to 950° C., the t-SPL method generates temperatures of 300° C. to 400° C. on the layer surface in a localized and instantaneous manner, this being distinctly above the decomposition temperature of the monolayer in an oxygenous environment, for example in air or in an oxygen-enriched atmosphere or in pure oxygen. The decomposition temperature is, for example, approx. 250° C. As a result, the erosion of the monolayer in the region concerned commences instantly, with the result that the “burning” of a nanopore, also called sequencing pore above, into the monolayer can take place extremely rapidly. Since only comparatively few nanopores are generated across the wafer surface, for example not more than two nanopores per qPCR array cell, and respectively only one or not more than two points are initiated and “burnt” as points per qPCR array cell to this end, the process of producing the sequencing unit100is also very economical, since the problems which can occur in connection with dense writing of large surface areas with a multiplicity of differently dimensioned and extended structures are avoided. On the contrary, what is sufficient here per qPCR array cell is the initiation of respectively one or not more than two positions and the “burning” of respectively a single nanopoint or nanodot at each position. Advantageously, points and no complex structural geometries are thus written in the nanometer and micrometer range.

The resolution limit of the t-SPL method is typically between 10 and 15 nm. In the case of nanopores for DNA sequencing via determination of DNA fragment length, this would be somewhat too large and would distinctly reduce the electric signals or the electric signal shift. The mechanism of converting the monolayer into the graphene layer102improves the situation significantly because there is usually present in the monolayer for graphene formation an excess of carbon that leads to the formation of graphene bilayers or trilayers, i.e., of multiple layers of graphene instead of a single layer of graphene, also called a monolayer. Owing to the excess of carbon, what occurs in the region of the nanopores is a “closing-up” proceeding from the edges of the nanopores, with the result that the nanopores in the graphene become distinctly smaller than were primarily generated in the monolayer. With optimal process control of the thermal annealing, a nanopore size of 1 to 2 nm can thus be set, the character of the graphene layer102changing from a bilayer or trilayer to a monolayer with increasing proximity to a nanopore.

Ideal conditions are thus achieved for the application of nanopore sequencing by means of a precisest possible determination of DNA fragment length via current modulation during passage of fragments through the nanopore: the membrane thickness immediately at the pore opening approximately corresponds to a graphene monolayer with a pore diameter of 1 to 2 nm.

Provided below by way of example is a detailed description of a total process for integrating graphene production with nanopore generation by introduction of self-assembled monolayers and t-SPL into the production of a sequencing qPCR array. The specified dimensions and layer thicknesses or layer materials used in the process flow are merely to be understood as exemplary data.

FIGS.2ato2hshow schematically a total process for producing a sequencing unit fromFIG.1. At the start of the process, a sequence of layers composed of, for example, 250 nm silicon dioxide204resulting from thermal oxidation and, thereabove, 280 nm silicon nitride206resulting from low-pressure vapor deposition is grown onto a front side and rear side of a silicon wafer208over the entire surface. This is shown inFIG.2a. The compressive stress of the thermal silicon dioxide is compensated or overcompensated for by the tensile stress of the deposited silicon nitride. The specified layer thicknesses are only intended to illustrate how a tensile stress in the layer structure can be generated altogether through the interplay of compressively stressed and tensilely stressed layers through appropriate dimensioning of the layer thicknesses. The resulting tensile stress is important later in the process in order to avoid breaking of freed layer membranes by “arching” and to ensure sufficient stability.

In a further process step, the geometries of qPCR array cell elements are defined on the front side of the wafer by photolithography using a sufficiently thick photoresist mask210, as shown inFIG.2b. In a self-adjusting process, the mask windows are first opened in the silicon nitride layer206and the silicon dioxide layer204by etching from the front side of the wafer. This is shown inFIG.2c. Thereafter, using the same masking and by means of a DRIE process (DRIE=deep reactive ion etching), the caverns108,116are etched through the silicon wafer208from the front side of the wafer as far as the stop on the silicon dioxide204of the rear side of the wafer. The result is shown inFIG.2d. The caverns108,116represent the later qPCR array cell elements. The thickness of the silicon dioxide layer204tolerates a certain overetching to compensate for deviations in etching-rate uniformity across the wafer surface. By way of example, it is assumed that, in the case of said overetching, up to 100 nm silicon dioxide can be lost in the cavern region. The silicon dioxide layer204thus has, in line with the exemplarily chosen initial layer thickness of 250 nm, additionally a residual thickness of 150 nm. The silicon dioxide layer204should not break through as far as the silicon nitride layer206during overetching, because silicon nitride does not have sufficient stability in the DRIE process. For a person skilled in the art, it is obvious what measures with respect to oxide thickness can allow an adjustment to a higher or lower overetching to be tolerated. After the silicon has been etched through, the membranes of the cavern bases are each self-supporting, i.e., should be tensilely stressed as mentioned above in order not to break and to have a sufficient stability for the follow-up processes.

Thereafter, as shown inFIG.2e, the photoresist mask210is removed from the front side of the wafer and the silicon wafer208is thermally oxidized, with, for example, 2.5 μm silicon dioxide being grown on the side walls of the caverns108,116in the qPCR array cell elements. As a consequence of this thermal oxidation process, a portion of the silicon nitride layer206is likewise oxidized, but very much more slowly than is the case on free silicon surfaces. For 2.5 μm silicon dioxide grown thermally on silicon, what can be typically assumed is 30 nm silicon nitride which is converted into an approx. 40 nm thick Re oxide212, i.e., silicon dioxide, as is evident fromFIG.2f.

The Re oxide212has hydrophilic surface properties. If, on the front side in the follow-up process, it is removed from the front side of the wafer by selective etching, for example using buffered hydrofluoric acid, what arises is a comparatively hydrophobic front side of the wafer composed of silicon nitride, as shown inFIG.2g. In the case of this etching process, approx. 50 to 100 nm silicon dioxide are likewise lost in the caverns108,116on the side walls and on the cavern base, with the result that not less than 50 nm silicon dioxide204together with the overlying layer structure of approx. 250 nm silicon nitride and 40 nm Re oxide212remain on the cavern base, wherein the Re oxide212should not be removed from the rear side of the wafer. Since the rear side of the wafer remains hydrophilic owing to the Re oxide212, what can then be carried out is the coating process to coat the rear side of the wafer with a precursor layer214, in this case a self-assembled monolayer, for generating the graphene layer. After deposition, the monolayer214is provided with nanopores by means of t-SPL. Thereafter, a metallic layer is vapor-deposited onto the rear side of the wafer, for example 300 nm copper or nickel. The underlying monolayer214is thermally converted to graphene. At the same time, the annealing process also determines the size of the nanopores, since the carbon has a certain mobility during the temperature treatment and the 10 to 15 nm pores close up slowly from the edges proceeding from the t-SPL structuring of the monolayer214.

After removal of the metal layer, the graphene layer102with the generated nanopores is exposed on the layer structure of the cavern base. This is shown inFIG.2h. Now, the layer structure of the cavern base is etched through in steps as far as the caverns108,114, specifically preferably by means of isotropically etching selective wet-etching chemistries, with the graphene layer102serving as masking for the etching process. Proceeding from the nanopores, the silicon dioxide layers are successively etched through from the rear side of the wafer, for example by means of highly dilute or buffered hydrofluoric acid, whereas phosphoric acid is used, for example, to etch through the silicon nitride interlayer. For the self-adjusting etching process with the aid of the nanopore-provided graphene layer102as masking, what are thus used in three steps are dilute (buffered) hydrofluoric acid for the Re oxide212, phosphoric acid for the silicon nitride206and dilute (buffered) hydrofluoric acid for the underlying silicon dioxide204. In the case of this procedure with use of the graphene nanopores as masking layer for the etching of the underlying dielectrics, a particular advantage achieved is that said dielectrics are substantially maintained outside the nanopores, mechanically support the graphene layer102and thus ensure additional stability.

After generation of a metallized layer216on the front side of the wafer and the bonding of a counter wafer to connection surfaces218on the rear side of the wafer, an array cell structure is available for connection to a measurement device, as schematically represented inFIG.1.

A further advantage of this process sequence is that the front side of the wafer is relatively hydrophobic owing to the silicon nitride layer206, whereas the cavern walls in the qPCR array cells are hydrophilic owing to the silicon dioxide layer204on the side walls and on the cavern base. This means that the qPCR array cells can be easily filled with aqueous media, since they are virtually sucked into the array cells and held therein owing to the hydrophilic environment. Furthermore, the closure of the array cells on the front side can be achieved particularly effectively, reliably and reproducibly by means of oils, preferably by means of fluorine oils or fluorocarbons of high dielectric strength and insulation such as perfluoropolyether or FC40 or FC77 (manufacturer: 3M), since the hydrophobic front side of the wafer repels a water film or a water film can be very easily and completely displaced therefrom by the advancing fluorine oils or fluorocarbons. As a result, both electrical and biochemical crosstalk between the array cell elements is suppressed in an effective manner, since a water film connecting the array cell elements is suppressed in an effective manner by the hydrophobic surface properties. The hydrophobic effect of the surface can, if needed, be additionally further enhanced, for example by printing with perfluoroalkyltrichlorosilanes such as, for example, perfluorooctyltrichlorosilane or perfluorodecyltrichlorosilane.

FIG.3shows a flow chart of a method300according to one exemplary embodiment. The method300can, for example, be carried out to produce a sequencing unit as described above on the basis ofFIGS.1to2h. This involves generating, in a step310, a prestructured layer in a thermal lithography method by creating at least one sequencing pore in a precursor layer, especially a self-assembled monolayer. In a further step320, it is converted into a graphene layer by heating to a particular conversion temperature. At the same time, the sequencing pore is reduced in size by a certain extent depending on the conversion temperature chosen.

By means of such a method, it is, for example, possible for nanopores of greater than 10 nm to be written into a self-assembled monolayer in a particularly economical manner by means of commercially available machines and commercially available processes and to be converted to a graphene layer having appropriate nanopores in a subsequent thermal process using a temporarily applied metal layer. Owing to carbon migration at high temperatures, the nanopores in the graphene can be reduced in size distinctly below the resolution limit of 10 to 15 nm of the thermal lithography process. Furthermore, a reduction of the layer thickness of the generated graphene layer immediately around the nanopores is advantageously achieved, typically from a bilayer or trilayer structure toward a monolayer structure, this increasing the accuracy of sequencing or the accuracy of determination of DNA fragment length. Since only comparatively few nanopores are written as points on the wafers, for example not more than two nanopores per qPCR array cell, the process of nanopore generation by means of t-SPL is extremely economical and rapidly performable.

If an exemplary embodiment comprises an “and/or” link between a first feature and a second feature, this is to be interpreted as meaning that the exemplary embodiment comprises both the first feature and the second feature according to one embodiment and either only the first feature or only the second feature according to a further embodiment.