HK1098177B - Apparatus and methods for detecting nucleic acid in biological samples - Google Patents

Description

Apparatus and method for detecting nucleic acids in biological samples

Technical Field

The present invention relates to devices and methods for detecting nucleic acids in biological samples. The invention particularly relates to a novel device and method for detecting DNA sequences using field-assisted nucleic acid hybridization, and to a method for optimising the performance of such a device, and further extends to the use of field-assisted hybridization in any biological process involving charged entities.

Background

The advent of high density polynucleotide (e.g., DNA or RNA) array technology has changed the basic concepts of genomics and proteomics analysis. The transition from "dot blot" to "arrays on glass slides" and then to DNA microarrays (also known as DNA chips) has revolutionized the industry by making large-scale clinical diagnostic testing and screening processes practical. It is known that a typical microarray having reaction sites in a predetermined structure on a substrate shows a binding pattern when exposed to a sample of target nucleic acid fragments having a base sequence complementary to a capture fragment attached to the reaction sites. When a suitable detection mechanism is used, the binding mode and binding efficiency may be detected by optical or electronic means, which may include, for example, fluorescent labels, amperometric detection or impedance measurements.

The use of electrically-assisted nucleic acid hybridization is a technique known in the analysis of biological samples containing DNA, such as blood, plasma, urine, and the like. Conventionally, a chip for DNA detection is formed of one of various substances including glass, silica, and metal. On the chip surface, a number of electrical contacts are formed using known techniques. To detect a specific DNA sequence in a biological sample, capture probes consisting of complementary DNA fragments are attached to the chip surface by means of an adhesive layer, often an agarose gel. If a biological sample contains target DNA, the target DNA will bind to the complementary DNA fragment by hybridization, and such hybridization can be detected using various imaging techniques, thereby detecting the presence of the target DNA in the sample.

Prior Art

The nucleic acid fragments are charged and attracted to specific sites by electrostatic attraction using electrodes, so that by applying an appropriate current, the hybridisation process and hence the detection process can be accelerated. However, it is not possible to use electrodes in direct contact with the nucleic acid fragments due to the danger of electrochemical degradation or electrolysis of the sample. Conventionally, therefore, a permeation/adhesion layer is usually coated on the electrodes, as shown in US 5605662 and 6306348. The permeation/adhesion layer is typically composed of a porous substance, such as a sol-gel substance, a porous hydrogel substance, a porous oxide, and provides an adhesion surface that allows selective diffusion of small ions, also as a type of capture probe. Due to the pore size of the porous material, the porous material is generally too small to allow larger nucleic acid fragments to pass through, thereby reducing direct contact of the nucleic acid fragments with the electrode.

The prior art devices provide electrophoretic transport effects when a voltage is applied to the electrodes under the permeation/adhesion layer, without electrochemical degradation of the sample, and thus may improve hybridization. However, such prior art techniques capable of electrically induced hybridization are not without their disadvantages. For example, porous materials such as hydrogels and polymers are susceptible to degradation upon contact with aqueous solutions, various chemicals, and many ambient factors. The preparation of sol-gel materials is expensive and complicated, increasing production costs. Furthermore, porous materials are naturally fragile and tend to adsorb and trap unwanted foreign substances such as moisture hydrocarbons in the air, resulting in a shorter shelf life of the device.

Summary of The Invention

According to the present invention there is provided a device for detecting target nucleic acid in a sample comprising a substrate formed with at least one reaction unit, wherein said reaction unit comprises an attachment surface formed by a dielectric substance for attachment of a nucleic acid capture probe, wherein a metal electrode is provided in direct contact with said dielectric substance. The sample may comprise a biological substrate, and the sample may be a wastewater, a solution, or a reagent. The sample may also be a biological sample such as blood, plasma or urine.

Preferably, the electrode is provided below the adhesion surface, that is to say in contact with the side of the dielectric substance opposite the adhesion surface. However, it is conceivable that it may be applied in contact with one side of the dielectric substance or even in contact with the adhesive surface itself.

In a preferred embodiment of the invention, the dielectric substance is preferably an oxide, which may for example be selected from Al₂O₃、SiO₂And Ta₂O₅. The metal electrode may be formed of aluminum, for example.

In a preferred embodiment of the invention, the device may comprise a multilayer structure comprising firstly a substrate layer and secondly an insulating layer formed on said substrate layer, a third layer formed on said insulating layer and comprising a patterned conductive region defining at least one metal electrode, and a fourth layer comprising a region of at least one dielectric substance, wherein each of said metal electrodes in said third layer is covered by a region of dielectric substance in said fourth layer. Preferably, the mode conductive regions of the third layer are separated by regions formed of a dielectric substance. More preferably, the dielectric substance regions in the fourth layer are separated by a region of passivating substance that may extend to an edge of the dielectric substance region to define the reaction cell.

Viewed from another broad aspect, the present invention provides a method of performing field assisted hybridization in the detection of a nucleic acid target in a biological sample, comprising the steps of: providing a reaction cell having an attachment surface formed by a dielectric substance, providing a metal electrode below in direct contact with said dielectric substance, attaching a nucleic acid capture probe to said attachment surface, adding a sample to said reaction cell, and applying a voltage to said electrode. The sample may comprise a biological substrate and the sample may be a wastewater, a solution or a reagent. The sample may also be a biological sample such as plasma or urine.

The voltage may be adapted as a continuous voltage, or may be a smoothly varying or pulsed voltage.

Viewed from another broad aspect, the invention also provides a method of attracting or repelling charged entities to or from a surface of a reaction unit when performing a biological reaction, comprising the steps of: a dielectric substance is provided as said surface, an electric field being generated by inducing charge separation in said dielectric substance. The charged entity may be a nucleic acid molecule.

Viewed from a further aspect, the invention also extends to a method of forming an array of reaction units for performing a biological assay, comprising the steps of: the method comprises the steps of preparing a metal electrode pattern on an insulating substance, depositing a dielectric substance area on the metal electrode, and forming a frame around the edge of the upper surface of the dielectric substance area to limit the reaction unit.

Preferably, for example, the method may comprise: depositing a layer of metal on an insulating surface, covering a desired pattern of the metal layer with photoresist, removing the remaining metal layer by an etching process, depositing a layer of the dielectric substance on top of the pattern metal, whereby the dielectric substance covers the pattern metal and occupies the area between the pattern electrodes, depositing a passivation layer on top of the layer of dielectric substance, patterning the passivation layer with photoresist, removing the passivation layer to reveal the dielectric substance covering the metal electrodes to define the reaction cell.

Brief Description of Drawings

Some embodiments of the invention will now be described by way of example and with reference to the accompanying drawings, in which:

figure 1 is a cross-sectional view through a chip according to one embodiment of the invention,

figure 2 is a view similar to figure 1 but showing the chip in use,

figure 3 is a schematic diagram showing the underlying principle constituting a preferred embodiment of the invention,

figure 4 illustrates steps in a possible fabrication process,

figures 5(a) and (b) show the results of the first test,

FIGS. 6(a) and (b) show the results of the second test, and

fig. 7(a) and (b) show the results of the third test.

Detailed description of the preferred embodiments

FIG. 1 illustrates in cross-section a portion of an embodiment of the invention comprising three units for receiving a sample buffer, but it will be appreciated that any number of units may be provided and that they are typically formed in a matrix array.

An apparatus according to one embodiment of the invention is fabricated by sequential deposition on a silicon substrate using conventional deposition techniques. First, (FIG. 4(a)) a layer of SiO having a thickness of between about 200nm and 500nm is formed by any suitable technique including thermal oxidation or by any suitable deposition technique such as sputtering, e-beam evaporation, and the like₂The formed insulating layer is formed on a silicon substrate. On top of the insulating layer, an aluminum layer is again formed with a thickness between about 500nm and 1000nm using any conventional deposition technique (fig. 4 (b)).

Once the aluminum layer has been formed, it is patterned using a layer of photoresist (fig. 4(c)), the exposed areas are removed by etching (fig. 4(d)) and the photoresist is removed (fig. 4 (e)). Then with Al₂O₃Coating the chip (FIG. 4(f)) to a thickness of between 50-500nm to form Al between the Al regions formed on the silica substrate₂O₃And (4) a region. Then Plasma Enhanced Chemical Vapor Deposition (PECVD) or the like is performed on Al₂O₃Upper precipitate of, for example, Si₃N₄(iii) the passivation layer of (1) (fig. 4 (g)). The passivation layer is then patterned with photoresist (fig. 4(h)), and then etched (fig. 4(i)) to develop Al₂O₃A region which becomes a reaction unit adhesion surface. Finally, the photoresist is removed (fig. 4 (j)).

The result of this fabrication process is the multi-layer structure of fig. 1. Aluminum regions are formed on the silicon dioxide insulating layer, and these aluminum regions are coated with Al₂O₃And (5) separating. In the presence of aluminum and Al₂O₃On top of the layer is formed a layer comprising a passivated substance Si on top of the aluminium region₃N₄Separated from each other Al₂O₃Region, passivationSubstance Si₃N₄Subsequently covered with Al₂O₃Region, separating the aluminum region on the lower layer. The passivating material also extends over the top Al₂O₃To define a surface on which a biological sample is placed for analysis.

It will thus be appreciated that in the embodiment shown in figure 1, a chip is formed from three units 1-3, each of which is made of Al underlaid with aluminium₂O₃And (4) forming. When the aluminum region is formed by etching, although not shown in fig. 1, electrical connections may also be formed to allow application of a voltage to the aluminum region.

Once the chip of fig. 1 has been fabricated, it can be used as the basis for many different biological tests and analyses. In particular, each of units 1-3 in FIG. 1 may be provided with a suitable capture probe as shown in FIG. 2. Depending on the test to be performed, each unit may be provided with the same capture probe or with a different capture probe having a nucleic acid fragment complementary to the fragment in the sample to be searched for in the test or assay. In the embodiment of fig. 2, units 1-3 are all identical and a drop of sample containing buffer is added to the units to cover all three units.

It will be appreciated by those skilled in the art that if the sample contains nucleic acid fragments complementary to the capture probes, they will bind to the capture probes by a hybridisation process, which can be detected by known techniques. Since the nucleic acid fragments are charged, the hybridization can be enhanced by providing an electric field that will attract the desired nucleic acid fragments towards the adhesion surface and the capture probes. The mechanism by which this embodiment can be implemented is shown in figure 3.

In particular, if a voltage is applied to the aluminum electrode under the cell, as shown in FIG. 3, due to Al₂O₃Is a dielectric substance, the charge separation will be in Al₂O₃Internal generation of which polarity will depend on the application to Al₂O₃Polarity of voltage of the lower aluminum electrode. As shown in the left side of FIG. 3, if a positive voltage is applied to the aluminum electrode, Al₂O₃Will also have a positive voltage, which attracts negatively charged fragments and repels positively charged fragments. Conversely, if a negative voltage is applied to the aluminum electrode, Al₂O₃Will also have a negative voltage, which attracts positively charged fragments and repels negatively charged fragments, as shown on the right hand side of fig. 3. Thus directly underlying and directly contacting Al₂O₃The aluminum electrodes in contact with the adhesion surface selectively apply a voltage that selectively attracts/repels the nucleic acid fragments, thereby electrically inducing hybridization. It should be understood that the voltage can be applied in many different ways. The voltage may be, for example, a constant continuous voltage, may be a smoothly varying voltage, or may be pulsed, with regular pulses or in any desired pattern.

A particular advantage of the present invention, at least in its preferred form, is that unwanted electrochemical reactions and/or electrolysis can be completely avoided compared to the prior art, since there is no electron transfer between the sample solution and the surface of the dielectric layer. Thus, the nucleic acid fragments can be electrically pulled to the adhesion surface without electrochemical degradation. A further important advantage of the field assisted hybridization method and apparatus of the present invention, at least in preferred forms, is that the salt concentration and pH of the sample do not change. These parameters are key factors that affect the efficiency of hybridization and the stability of the hybridized nucleic acid fragments. The prior art electrically assisted hybridization techniques result in significant changes in salt concentration and pH due to electrochemical reactions, and other techniques such as specific buffers are required to compensate for these effects. A further advantage of the present invention is that no electrochemical reactions occur, which in turn indicates that the solutions/reagents involved in the detection process are not disturbed. No bubble formation and/or precipitation occurs during the detection process, which is important in improving the quality of the detection signal.

It will also be appreciated that preferred embodiments of the invention are described in the context of detecting accelerated hybridisation in nucleic acid fragments, and that the invention is more generally applicable to any biological process involving charged entities where it is desirable to control the movement of such charged entities by attracting the entities to a surface or repelling the entities from a surface.

Fig. 5 to 7 show the results of a number of experiments using the structures of fig. 1 to 3 with and without a voltage applied to the aluminium electrode underneath the cell. It will of course be appreciated that in all of these examples, the reaction times, applied voltages and other parameters are fully exemplary and may be varied as desired.

FIGS. 5(a) and (b) show controls in which no voltage was applied to the aluminum electrode in both cases, and therefore hybridization was carried out without electrical assistance. In this example, the target oligomer in the sample was synthetic β -actin (91 bases, pure) and the hybridization time was 90 minutes. The target oligomer is present in the sample of fig. 5(a) and absent in the sample of fig. 5 (b). In neither fig. 5(a) or 5(b) a voltage was applied to the aluminum electrode, but the cell in fig. 5(a) was significantly darker than in 5(b) due to the presence of the target oligomer in the sample of fig. 5 (a).

The situation in fig. 6(a) and (b) is the same as in fig. 5(a) and (b) because the same target oligomer is provided in the sample of fig. 6(a) and not in the sample of fig. 6 (b). However, in this example, a voltage of +10V was applied to the aluminum electrode and the hybridization time was reduced to 10 minutes. A comparison of FIGS. 5(a) and 6(a) shows that even though the hybridization time has been sufficiently reduced, the cells are significantly darker in FIG. 6(a), demonstrating the effectiveness of the applied voltage in accelerating hybridization. There is a similarity between fig. 5(b) and 6(b), where the target oligomer is absent, showing that the applied voltage does not result in any false positive results.

Fig. 7(a) - (c) illustrate a third example, wherein the oligomer of interest in the sample is an Avarian Influenza Virus (AIV). Subtype H5 (250 bases mixed with other non-specific oligomers). In all three cases (a) - (c), the hybridization time was 10 minutes. The differences between fig. 7(a) - (c) are as follows: in fig. 7(a) the target oligomer is present in the sample and a +10V voltage is applied to the aluminum electrode below the cell; in fig. 7(b) no target oligomer was present in the sample and a +10V voltage was applied to the aluminum electrode below the cell; in fig. 7(c) the target oligomer was present in the sample but no voltage was applied to the aluminum electrode below the cell. This example again shows that applying a +10V voltage to the electrodes results in an accelerated and intense signal of hybridization (very dark area in the cell of fig. 7 (a)) at a hybridization time of only 10 minutes. In comparison, the similarity of the characterization between FIG. 7(b) (no target and no voltage applied) and FIG. 7(c) (target and no voltage applied) shows that efficient hybridization cannot be obtained within the same time period (10 minutes) without electrically assisted hybridization.

The present invention, at least in its form of application, provides a simple, low cost device which allows field assisted hybridisation and/or other biological processes to be carried out at a faster rate with high performance, which can be applied to many possible applications. The present invention uses the principle of charge separation in a dielectric substance that is in contact with an electrode to which a voltage is applied. In the above embodiment, the electrode is aluminum and the dielectric substance is Al₂O₃But other combinations of metal electrodes and dielectric adhesion surfaces are possible. For example, SiO2, Ta2O5 may be used as adhesion surfaces for oxide-based dielectric substances.

In contrast to prior art devices using an osmotic layer, the oxide-based dielectric layer in direct contact with the electrodes provides a robust, compact structure that is chemically inert to most acids, alkali metals, and other reagents commonly used in biological reactions. The structure is also stable to ambient factors such as temperature and humidity and is less subject to physical damage. The production costs will be lower and the device can be easily manufactured using standard deposition techniques and other microelectronics manufacturing techniques. Indeed the use of microelectronic deposition and fabrication techniques in the fabrication of the devices of the present invention also has the advantage that the devices can be readily incorporated into other devices produced using the same or similar techniques.

Claims (27)

1. A device for detecting target nucleic acids in a sample, comprising a matrix formed by at least one reaction unit, wherein said reaction unit comprises an attachment surface defined by a dielectric layer for attaching nucleic acid capture probes and for attracting or repelling charged entities, wherein a metal electrode is provided in direct contact with said dielectric layer, and wherein said device is adapted to generate a charge separation in said dielectric layer, but without current flow or electron transfer between said dielectric layer and the sample.

2. A device as claimed in claim 1, wherein said electrode is in contact with an edge of said dielectric layer opposite said adhesion surface.

3. A device as claimed in claim 1, wherein said dielectric is an oxide.

4. A device as claimed in claim 3, wherein said dielectric is selected from Al₂O₃、SiO₂Or Ta₂O₅。

5. An apparatus as claimed in claim 1, wherein said electrodes are formed of aluminium.

6. A device as claimed in claim 1, wherein said dielectric comprises Al₂O₃And the electrodes are formed of aluminum.

7. The device as claimed in claim 1 comprising a plurality of layers: comprising a first substrate layer, a second insulating layer formed on said substrate layer, a third layer formed on said insulating layer and comprising patterned conductive regions defining at least one metal electrode, and a fourth layer comprising at least one region of dielectric material, wherein each of said metal electrodes in said third layer is covered by a region of dielectric material in said fourth layer.

8. A device as claimed in claim 7, wherein the patterned conductive regions of the third layer are separated by regions formed of a dielectric substance.

9. A device as claimed in claim 7, wherein said regions of dielectric substance in said fourth layer are separated by regions of a passivating substance.

10. A device as claimed in claim 9, wherein said passivation substance region extends to the edge of said dielectric substance region to define said reaction cell.

11. A device as claimed in claim 1 wherein said sample comprises a biological substrate.

12. An apparatus for detecting a target biological substance of a charged entity in a sample, comprising: a matrix formed of at least one reaction cell, wherein said reaction cell comprises a surface defined by a dielectric layer for attracting or repelling charged entities, wherein a metal electrode is provided in direct contact with said dielectric layer, and wherein said device is adapted to generate charge separation in said dielectric layer, but no current flow or electron transfer between said dielectric layer and a sample.

13. A method for field assisted hybridization in detecting a target nucleic acid in a sample, comprising the steps of: providing a reaction cell having an adhesion surface formed by a layer of a dielectric substance defining an adhesion surface, providing a metal electrode in direct contact with the dielectric adhesion layer, adhering a nucleic acid capture probe to the dielectric adhesion layer, adding a sample to the reaction cell, and applying a voltage to the metal electrode for creating charge separation in the dielectric adhesion layer, but not creating a current flow or electron transfer between the dielectric adhesion layer and the sample.

14. A method as claimed in claim 13, wherein said electrode is provided in contact with a side of said dielectric adhesion layer opposite said adhesion surface.

15. A method as claimed in claim 13, wherein said voltage is a continuously applied voltage.

16. A method as claimed in claim 13, wherein the voltage is applied as a series of pulses.

17. A method as claimed in claim 13 wherein the sample comprises a biological substrate.

18. A method of attracting or repelling charged entities to or from a surface of a reaction unit when performing a biological reaction, comprising the steps of: providing a dielectric layer defining said surface, an electric field being generated by inducing charge separation in said dielectric layer, but no current flow or electron transfer being generated between said dielectric layer and the sample.

19. A method as claimed in claim 18, wherein charge separation in said dielectric layer is induced by placing electrodes in direct contact with said dielectric layer and applying a voltage to said electrodes.

20. A method as claimed in claim 19, wherein a continuous voltage is applied to said electrodes.

21. A method as claimed in claim 19, wherein a pulsed voltage is applied to said electrodes.

22. A method as claimed in claim 19, wherein the electrodes are placed in contact with a surface of the dielectric layer opposite to the surface to which the charged entities are attracted or repelled.

23. A method as claimed in claim 13, wherein said electrode is in contact with an edge of said dielectric layer opposite said adhesion surface.

24. A method as claimed in claim 13, wherein said dielectric substance is an oxide.

25. A method as claimed in claim 13, wherein said electrodes are formed from aluminium.

26. A method as claimed in claim 13, wherein said dielectric substance comprises Al₂O₃And the electrodes are formed of aluminum.

27. A method as claimed in claim 13, wherein said dielectric substance is selected from Al₂O₃、SiO₂Or Ta₂O₅。