Patent Application
20050142551
The present invention relates to methods of fabricating a device for the detection of target biological molecules from samples.
For the analysis and testing of nucleic acid molecules, amplification of a small amount of nucleic acid molecules, isolation of the amplified nucleic acid fragments, and other procedures are necessary. The polymerase chain reaction method is widely used for the amplification of nucleic acid molecules, in which an extremely small number of nucleic acid molecules or fragments can be multiplied by several orders of magnitude to provide detectable amounts of material. On the other hand, isolation and detection of particular nucleic acid molecules in a mixture requires a nucleic acid sequencer and fragment analyzer, in which gel electrophoresis and fluorescence detection are combined. However, electrophoresis becomes very labor-intensive as the number of samples or test items increases. For this reason, a simpler method of analysis using DNA oligonucleotide probes is becoming popular. In this method, many kinds of oligonucleotide probes are immobilized on the surface of a solid to make a probe array. When contacted with a sample, only nucleic acid molecules with specific sequences matching the oligonucleotide are trapped on the surface of the solid and detected.
This kind of isolation and detection method, in which biological probes are immobilized on the surface of a solid and hybridization proceeds between the probes and a sample, has long been known as a blotting method in which the presence of the target molecule is detected by a probe immobilized on a membrane using radioactive labeling. However, immobilization of a large number of probes on a small area has the advantage that only a small amount of sample is required, and a large number of probes can be used simultaneously.
There are several methods for production of such products. Probe molecules can be synthesized one base at a time by a photochemical reaction on small segments of a solid using the same photomasking techniques used in the semiconductor industry. In another method, a synthesized DNA, a PCR-amplified DNA, or a protein molecule is immobilized on a small segment of the surface of a solid for each probe. A third method is to use an inkjet droplet to deposit the biological probe onto the surface. After the biological probes are attached to the surface, the sample containing the target molecule to be analyzed is passed over the biological probes at a temperature conducive to rapid hybridization of the target molecule with the probes. A washing solution then removes all the unhybridized, unbound molecules.
This method requires the use of fluorescent or radioactive labels as additional materials. Such a system is expensive to use and is not amenable to being made portable for biological sample detection and identification. Furthermore, the hybridization reactions can take up to two hours, which for many uses, such as detecting biological warfare agents, is simply too long. Therefore, a need exists for a device and system which can rapidly detect target molecules from samples.
The present invention is directed to achieving these objectives.
The present invention relates to a method of manufacturing a detection device. The method first involves providing a substrate having a layer of aluminum between a first layer of photosensitive material and a base layer. Next, the substrate is subjected to a first level photolithography treatment to produce an aluminum electrical conductor containing conductive fingers with spaces between them. The spaces between the conductive fingers are covered with an electrical insulator material. Finally, biological probes are attached to the conductive fingers under conditions effective to form a gap between the biological probes on the spaced apart conductive fingers, where a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart conductive fingers. This bridges the gap between the biological probes, allowing detection of the target molecule.
Another aspect of the present invention relates to a method of manufacturing a detection device, which first involves providing a substrate having a base layer. Next, a first layer of electrical insulator material is deposited on one side of the base layer. A layer of aluminum is deposited on the first layer of electrical insulator material. Next, a first layer of photosensitive material is then coated onto the layer of aluminum. Certain portions of the first layer of photosensitive material are exposed to ultraviolet light through a first photomask, and the first layer of photosensitive material is developed and baked, leaving portions of the layer of aluminum uncovered. Then, the uncovered portions of the layer of aluminum are removed from the substrate, leaving portions of the first layer of electrical insulator material uncovered. Next, the photosensitive material remaining on the layer of aluminum is removed. Then, a diamond film is deposited on the substrate, and a second layer of photosensitive material is coated onto the diamond film. Next, the second layer of photosensitive material is exposed to ultraviolet light through a second photomask, and the second layer of photosensitive material is developed and baked, leaving portions of the diamond film uncovered. Then, the uncovered portions of the diamond film are removed from the layer of aluminum, where the exposing the second layer of photosensitive material, the developing and baking the second layer of photosensitive material, and the removing the uncovered portions of the diamond film are carried out such that only portions of the diamond film aligned with the conductive fingers will be removed, leaving portions of the second layer of photosensitive material on the substrate. Next, the second layer of photosensitive material remaining on the diamond film is removed. Finally, biological probes are attached to the conductive fingers under conditions effective to form a gap between the biological probes on the spaced apart conductive fingers, As a result, a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart conductive fingers to bridge the gap between the biological probes, allowing detection of the target molecule.
The present invention also relates to a method of manufacturing a detection device, which involves providing a substrate having an aluminum electrical conductor containing a plurality of coplanar conductive fingers with spaces between them, where the spaces are covered with an electrical insulator material. Biological probes are then attached to the conductive fingers under conditions effective to form a gap between probes on the spaced apart, coplanar, conductive fingers. As a result, a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart, coplanar, conductive fingers to bridge the gap between the biological probes, allowing detection of the target molecule.
Another aspect of the present invention relates to a method of manufacturing a detection device. The method first involves providing a substrate having a layer of aluminum and a first layer of photosensitive material. Then, the substrate is subjected to a first level photolithography treatment to produce an aluminum electrical conductor having conductive fingers with spaces between them. Finally, biological probes are attached to the conductive fingers under conditions effective to form a gap between the biological probes on the spaced apart conductive fingers. As a result, a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart conductive fingers to bridge the gap between the biological probes, allowing detection of the target molecule.
The present invention provides methods of fabricating a device for rapidly detecting the presence of biological material. The target molecule either itself or as a support is used to complete an electrical circuit. The presence of the target molecule is indicated by the ability to conduct an electrical signal through the circuit. In the case where the target molecule is not present, the circuit will not be completed. Thus, the target molecule acts as a switch. The presence of the target molecule provides an “on” signal for an electrical circuit, whereas the lack of the target molecule is interpreted as an “off” signal. Due to the direct detection of the target molecule, the device allows for extremely sensitive detection of target molecules connecting two electrical conductors.
FIGS. 1A-P illustrate the sequence of steps necessary for fabricating a device for detecting the presence of a target molecule.
The present invention relates to a method of manufacturing a detection device. The method first involves providing a substrate having a layer of aluminum between a first layer of photosensitive material and a base layer. Next, the substrate is subjected to a first level photolithography treatment to produce an aluminum electrical conductor containing conductive fingers with spaces between them. The spaces between the conductive fingers are covered with an electrical insulator material. Finally, biological probes are attached to the conductive fingers under conditions effective to form a gap between the biological probes on the spaced apart conductive fingers, where a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart conductive fingers. This bridges the gap between the biological probes, allowing detection of the target molecule.
Another aspect of the present invention relates to a method of manufacturing a detection device, which first involves providing a substrate having a base layer. Next, a first layer of electrical insulator material is deposited on one side of the base layer. A layer of aluminum is deposited on the first layer of electrical insulator material. Next, a first layer of photosensitive material is then coated onto the layer of aluminum. Certain portions of the first layer of photosensitive material are exposed to ultraviolet light through a first photomask, and the first layer of photosensitive material is developed and baked, leaving portions of the layer of aluminum uncovered. Then, the uncovered portions of the layer of aluminum are removed from the substrate, leaving portions of the first layer of electrical insulator material uncovered. Next, the photosensitive material remaining on the layer of aluminum is removed. Then, a diamond film is deposited on the substrate, and a second layer of photosensitive material is coated onto the diamond film. Next, the second layer of photosensitive material is exposed to ultraviolet light through a second photomask, and the second layer of photosensitive material is developed and baked, leaving portions of the diamond film uncovered. Then, the uncovered portions of the diamond film are removed from the layer of aluminum, where the exposing the second layer of photosensitive material, the developing and baking the second layer of photosensitive material, and the removing the uncovered portions of the diamond film are carried out such that only portions of the diamond film aligned with the conductive fingers will be removed, leaving portions of the second layer of photosensitive material on the substrate. Next, the second layer of photosensitive material remaining on the diamond film is removed. Finally, biological probes are attached to the conductive fingers under conditions effective to form a gap between the biological probes on the spaced apart conductive fingers. As a result, a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart conductive fingers to bridge the gap between the biological probes, allowing detection of the target molecule.
The present invention also relates to a method of manufacturing a detection device, which involves providing a substrate having an aluminum electrical conductor containing a plurality of coplanar conductive fingers with spaces between them, where the spaces are covered with an electrical insulator material. Biological probes are then attached to the conductive fingers under conditions effective to form a gap between probes on the spaced apart, coplanar, conductive fingers. As a result, a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart, coplanar, conductive fingers to bridge the gap between the biological probes, allowing detection of the target molecule.
Another aspect of the present invention relates to a method of manufacturing a detection device. The method first involves providing a substrate having a layer of aluminum and a first layer of photosensitive material. Then, the substrate is subjected to a first level photolithography treatment to produce an aluminum electrical conductor having conductive fingers with spaces between them. Finally, biological probes are attached to the conductive fingers under conditions effective to form a gap between the biological probes on the spaced apart conductive fingers. As a result, a target molecule, if present in a sample, can bind to a pair of the biological probes on the spaced apart conductive fingers to bridge the gap between the biological probes, allowing detection of the target molecule.
One embodiment of the method of the present invention is set forth in FIGS. 1A-P which depict the sequence of steps necessary for fabricating a device for detecting the presence of a target molecule.
This method first involves providing a substrate having base layer 2, as shown in
First layer of electrical insulator material 4 is deposited on one side of base layer 2, as shown in
Layer of aluminum 6, an electrically conductive material, is deposited on first layer of electrical insulator material 4, as shown in
First layer of photosensitive material 8 is coated onto layer of aluminum 6, as shown in
Certain portions of first layer of photosensitive material 8 are exposed to ultraviolet light 10 through first photomask 12, as shown in
First layer of photosensitive material 8 is developed and baked, leaving portions of layer of aluminum 6 uncovered, as shown in
Uncovered portions of layer of aluminum 6 are removed from the substrate, leaving portions of first layer of electrical insulator material 4 uncovered, as shown in
Photosensitive material 8 remaining on layer of aluminum 6 is removed, as shown in
Next, diamond film 14, an electrical insulator material, is deposited on the substrate, as shown in
Second layer of photosensitive material 16 is coated onto the diamond film 14, as shown in
Second layer of photosensitive material 16 is exposed to ultraviolet light 10 through a second photomask 18, as shown in
Exposed second layer of photosensitive material 16 is developed and baked, leaving portions of diamond film 14 uncovered, as shown in
As shown in
Second layer of photosensitive material remaining 16 on diamond film 14 is removed, as shown in
The top view of the final fabricated device with two contact cuts 20 and an active area with spaced apart conductive fingers 22 exposed is shown in
Finally, biological probes 24 are attached to conductive fingers 22 under conditions effective to form a gap between biological probes 24 on spaced apart conductive fingers 22, as shown in
In one embodiment of the present invention, the biological probes are proteins or antibodies.
The fabricated device of the present invention is used to detect target molecules from samples. As shown in
Optionally, after hybridization of the target molecules to sets of biological probes, the target molecule can be coated with a conductor, such as a metal, as described in U.S. patent application Ser. Nos. 60/095,096 or 60/099,506, which are hereby incorporated by reference in their entirety. The coated target molecule can then conduct electricity across the gap between the pair of probes, thus producing a detectable signal indicative of the presence of a target molecule.
The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
Fabrication began with a bare 6″ silicon wafer (n-type or p-type) with (100) orientation, a resistivity of 5-15 ohm·cm, and a thickness of 675+50 μm.
A layer of silicon hard coat, as a first electrical insulator layer, was spin-coated on one side of the silicon wafer. A puddle of silicon hard coat (methylsilsesquioxane solution; SHC1200, GE Silicones, Waterford, N.Y.), about 3 inches in diameter, was dispensed onto the wafer, which was then spun at 3000 rpm for 180 sec.
A layer of aluminum was either sputtered or evaporated to the desired thickness onto the layer of silicon hard coat. When sputtered, power was set at 2000 Watts, argon flow at 63 sccm, presputter time at 5 min, chamber pressure at 106 Torr, deposition pressure at 5 mTorr, and deposition rate at 240 A/min. Alternatively, an aluminum pellet was evaporated using the evaporator (CVC, Port Townsend, Wash.) set at an appropriate chamber pressure (low 10−6 Torr) to give an approximate thickness of 2000 Å.
A first layer of photoresist was coated onto the aluminum layer. The wafer was coated using coat trac or was hand-coated using Oir 620, a positive photoresist containing novalac resin, diazonaphthoquinone, casting solvent, additives, and surfactants (Olin, Norwalk, Conn.). When hand-coating, the substrate must be dehydration baked at 200° C. for 2 min on a hot plate, the photoresist must be dispensed at 4500 rpm for 1 min, and the substrate must be soft baked at 90° C. for 1 min on a hot plate.
The substrate was subjected to a first level lithography treatment, using an i-line stepper (Canon, Japan). The stepper job was loaded using the command, “ST DNA3_level 1.” The exposure dose was set at 130 mj/cm2.
The exposed wafer was developed in a base developer (Shipley CD26, Shipley, Marboro, Mass.) for 1 min and rinsed with water. The wafer was dried and hard baked on a hot plate at 120° C. for 2 min.
The exposed aluminum was removed by etching the wafer with aluminum etchant at 50° C., and rinsing well in DI water. The etching time depended on the thickness of aluminum, and the etch rate was 2000 Å/min.
The photoresist remaining on the aluminum layer was removed with acetone and rinsed with DI water.
A diamond film, as a second electrical insulator layer, was spin-coated onto the substrate.
The substrate was then coated with photoresist as previously described.
The substrate was subjected to a second level lithography treatment to open active areas and contact cuts, using an i-line stepper (Canon, Japan). The stepper job was loaded using the command, “ST DNA3_level 1.” The substrate was exposed and a post-exposure bake was performed as described for the first level lithography. An exposure dose of 130 mj/cm2 was used.
The exposed substrate was developed in the base developer for 1 min and rinsed with water. Then, the substrate was dried and hard baked on a hot plate at 120° C. for 2 min.
Next, the exposed diamond film was removed by plasma etching in O2. This step is not necessary if photoresist is used as the second electrical insulator layer.
Next, the photoresist remaining on the aluminum layer was removed with acetone. This step is not necessary if photoresist is used as the second electrical insulator layer.
Finally, biological probe molecules are attached to the active area of the fabricated device with the aluminum electrical conductors exposed.
Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/332,282, filed Nov. 21, 2001, which is hereby incorporated by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US02/37257 | 11/19/2002 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 60332282 | Nov 2001 | US |
