Patent Application
20070240985
The embodiments will be described with reference to the accompanying drawing.
As shown in
The flow channel 110 is for carrying a solution containing at least one of target nucleic acids and nucleic acid detection bodies. The probe electrodes are arranged at intervals to form a row along the flow channel 110. Although not shown, the nucleic acid detection device 100 has, in addition to the probe electrodes, a counter electrode to measure an electrochemical reaction, and preferably a counter electrode and a reference electrode, in the flow channel 110. The counter electrode and reference electrode are respectively electrically connected to the signal input/output pads 106 through wiring lines formed in or on the surface of the nucleic acid detection device 100.
The nucleic acid detection device 100 is used as it is mounted on a known nucleic acid detection apparatus. The nucleic acid detection apparatus supplies the solution to the flow channel 110 and measures the current flowing through the electrodes through the signal input/output pads 106.
As shown in
Referring to
The smaller the area of the probe electrode E, the higher for sensitivity. In other words, the smaller the maximal width of the probe electrode E, i.e., a diameter R for a circular probe electrode, the better. When, however, the diameter R of the probe electrode E is almost equal to a thickness d of the passivation film 124, in the stable flow described above, the wall of the passivation film 124 existing around the probe electrode E hinders the solution from smoothly entering the opening, so that the solution dose not reach the surface of the probe electrode E easily. Accordingly, difficulty in detection increases. Such a phenomenon appears when the diameter R of the nucleic acid detection probe immobilization electrode E is smaller than about 100 times the thickness d of the passivation film 124.
This embodiment is directed to a structure that allows the solution to easily reach even a probe electrode E with a small area.
As shown in
As shown in
The nucleic acid detection device according to this embodiment is fabricated in the following manner.
A silicon substrate is made with the anisotropic etching to form structures on the substrate surface such that an area around the electrode forms an inclined surface. The material of the substrate is not limited to silicon, but semiconductor, glass, resin, ceramics, metal, or the like may be employed. The inclined surface may alternatively be formed by etching, sputtering, deposition, cutting, molding, printing, or the like. The inclined surface is formed so that the angle θ of the inclined surface with respect to the flat bottom surface satisfies tan θ>d/R with respect to the designed supplying direction of the solution of the flow channel. After that, a metal thin film is formed by sputtering. Alternatively, the metal thin film may be formed by deposition, printing, or the like. After that, wiring portions, pad portions, and the like are patterned by etching where necessary. After that, a passivation film is formed by coating. Alternatively, the passivation film may be formed by sputtering, deposition, printing, or the like. After that, the passivation film is selectively etched to form openings, so the metal thin film is partly exposed through the openings to serve as electrodes. The technique to form the openings is not particularly specified.
Subsequently, by dropping a solution containing nucleic acid probes onto the electrodes, the nucleic acid probes are immobilized on the electrodes to form probe electrodes. Alternatively, the nucleic acid probes may be immobilized by adsorption. By dropping different types of solutions containing different types of nucleic acid probes onto different electrodes, a device for detecting different nucleic acids can be formed.
A component having a preformed flow channel structure is adhered or contact-bonded to the structure formed in this manner to finish a nucleic acid detection device. The electrode forming step and nucleic acid probe immobilization step described above may be applied to a structure that is etched or cut in advance into a flow channel shape.
The nucleic acid detection device according to this embodiment is used in the following manner.
A solution containing target nucleic acids is supplied to the flow channel to cause a hybridization reaction. As the nucleic acid detection device according to this embodiment has an inclined portion in an area around each electrode and the angle θ of the inclined surface with respect to the flat bottom surface satisfies tan θ>d/R, the target nucleic acids contained in the solution are supplied to the probe electrodes efficiently. Thus, even a small amount of nucleic acids sufficiently allows a hybridization reaction. In contrast to this, if tan θ>d/R is not satisfied, the flowing solution cannot directly collide against the probe electrodes. Hence, the target nucleic acids contained in the solution are not supplied to the probe electrodes efficiently.
A washing buffer is supplied to the flow channel to clean the flow channel. As tan θ>d/R is satisfied, the washing buffer is supplied to the probe electrodes efficiently, to clean the probe electrodes reliably.
A solution containing nucleic acid recognition bodies is supplied to the flow channel to cause the hybridized nucleic acids to react with the nucleic acid recognition bodies. As tan θ>d/R is satisfied, the nucleic acid recognition bodies are supplied to the probe electrodes efficiently, to cause the reaction reliably.
The electrochemical response of the nucleic acid recognition bodies is measured to detect the nucleic acids.
The basic structure of a nucleic acid detection device according to the second embodiment is the same as that of the first embodiment. This embodiment is directed to another structure that allows a solution to easily reach even a probe electrode E with a small area as well.
As shown in
The nucleic acid detection device according to this embodiment is fabricated in the following manner.
A metal thin film is formed on a substrate by sputtering. Alternatively, the metal thin film may be formed by deposition, printing, or the like. After that, wiring portions, pad portions, and the like are patterned by etching where necessary. After that, a passivation film is formed by coating. Alternatively, the passivation film may be formed by sputtering, deposition, printing, or the like. After that, the passivation film is selectively etched to form openings, so the metal thin film is partly exposed through the openings to serve as electrodes. The technique to form the openings is not particularly specified. At this time, the passivation film is formed to satisfy d2>d1 with respect to the supplying direction of the solution of the flow channel which is designed in advance.
After that, nucleic acid probes are immobilized and a flow channel is formed in the same manner as in the first embodiment.
The nucleic acid detection device according to this embodiment is used in the following manner.
A solution containing target nucleic acids is supplied to the flow channel to cause a hybridization reaction. As the passivation film of the nucleic acid detection device according to this embodiment satisfies d2>d1, the target nucleic acids contained in the solution are supplied to the probe electrodes efficiently. Thus, even a small amount of nucleic acids sufficiently allows a hybridization reaction. In contrast to this, if the passivation film does not satisfy d2>d1, the flowing solution is not sufficiently supplied to the probe electrodes. Hence, the target nucleic acids contained in the solution are not supplied to the probe electrodes efficiently.
After that, washing, reaction with the nucleic acid recognition bodies, and measurement of an electrochemical reaction are performed in the same manner as in the first embodiment to detect nucleic acids.
The basic structure of a nucleic acid detection device according to the third embodiment is the same as that of the first embodiment. This embodiment is directed to still another structure that allows a solution to easily reach even a probe electrode E with a small area as well.
As shown in
As shown in
In the nucleic acid detection device according to this embodiment, the flow of the solution is a laminar flow until reaching the projection 110p. When colliding against the projection 110p, the laminar flow changes to a turbulent flow, and thereafter is restored to the laminar flow. The right-hand side of the above inequality is known to express a distance through which the flow of a fluid changes from a turbulent flow to a laminar flow. Accordingly, as far as the distance L satisfies the above inequality, the flow of the solution that has collided against the upstream end of the projection 110p and transformed into a turbulent flow reaches each probe electrode E in the form of the turbulent flow. This supplies at least one of the target nucleic acid and nucleic acid recognition body contained in the solution to the probe electrode E.
The upper limit of the distance L was determined on the basis of the following consideration.
When the solution flows in from a tube inlet at a uniform velocity, a boundary layer develops along the tube wall. When the boundary layer increases its thickness and reaches the center, the velocity distribution in the tube becomes constant (e.g., a parabolic velocity distribution). The region until reaching the fully developed flow is called an entrance region, and its length is called an inlet length X.
For a laminar flow, X/D is a function of Re. According to Boussinesq, X/D≧0.065×Re.
The magnification of a dimensionless number Re which is defined by the following inequality and named Reynolds number serves to discriminate whether an in-tube flow is a laminar flow or a turbulent flow.
Re=ρul/μ
where l is the typical length of the flow, u is the typical flow velocity, ρ is the density of the fluid, and u is the viscosity of the fluid. As the number Re is a dimensionless number, its value is the same regardless of the employed system of units as far as the state of the flow is the same. For a flow in the tube, Re=ρuD/μ is employed by using an inner diameter D of the tube to substitute the typical length l of the flow.
When the section of the pipeline is not circular, turbulent flow transition, pressure loss, and the like can be dealt with in the same manner as a circular cylindrical tube if employing an equivalent diameter De which is defined by the following inequality as the typical length of Re:
De=4S/lp
where S is the sectional area of the flow, and lp is the perimeter of the periphery of the solid wall with which the flow is in contact, i.e., a wetted perimeter.
In the nucleic acid detection device according to this embodiment, if the distance L is smaller than the entrance distance X, i.e., if L<X, the turbulent flow generated at the upstream end of the projection 110p reaches the probe electrode E before being restored to a laminar flow. Substitution of X=0.065×Re×D for L<X yields the relationship described above. In this case, a wall peripheral perimeter Lp of the flow channel substitutes for the wetted perimeter lp in accordance with the structure of the nucleic acid detection device.
The nucleic acid detection device according to this embodiment is fabricated in the following manner.
A substrate is etched or cut to form structures on the substrate surface such that an area around each electrode projects from the substrate surface. The material of the substrate is not limited to silicon, but semiconductor, glass, resin, ceramics, metal, or the like may be employed. After that, a metal thin film is formed on the substrate by sputtering. Alternatively, the metal thin film may be formed by deposition, printing, or the like. After that, wiring portions, pad portions, and the like are patterned by etching where necessary. After that, a passivation film is formed by coating. Alternatively, the passivation film may be formed by sputtering, deposition, printing, or the like. After that, the passivation film is selectively etched to form openings, so the metal thin film is partly exposed through the openings to serve as electrodes. The technique to form the openings is not particularly specified. At this time, the openings are formed to satisfy L<0.065×Re×D with respect to the designed supplying direction of the solution of the flow channel.
After that, nucleic acid probes are immobilized, and a flow channel is formed, in the same manner as in the first embodiment.
The nucleic acid detection device according to this embodiment is used in the following manner.
A solution containing target nucleic acids is supplied to the flow channel to cause a hybridization reaction. The nucleic acid detection device according to this embodiment has a projection in an area around each electrode, and the distance L from the upstream end of the projection to the probe electrode satisfies L<0.065×Re×D. Thus, the flow of the solution collides against the upstream end of the projection to form a turbulent flow, and reaches the probe electrode before being restored to a laminar flow. Thus, the target nucleic acids contained in the solution are supplied to the probe electrodes efficiently. Thus, even a small amount of nucleic acids sufficiently allows a hybridization reaction. If the flow of the solution forms a laminar flow, the velocity decreases in the vicinity of the wall surfaces of the flow channel, i.e., on the probe electrodes, and the target nucleic acids contained in the solution are not supplied to the probe electrodes efficiently.
After that, washing, reaction with the nucleic acid recognition bodies, and measurement of an electrochemical reaction are performed in the same manner as in the first embodiment to detect nucleic acids.
As shown in
The flow channel 210 is for carrying a solution containing at least one of target nucleic acids and nucleic acid detection bodies. The probe electrodes E are arranged at intervals to form a row along the flow channel 210.
The nucleic acid detection device 200 incorporates a circuit which measures an electrochemical response of the nucleic acid recognition body for nucleic acid detection. The circuit is formed in a circuit area CA located around an electrode area EA where a metal film patterned to form a probe electrode is arranged.
As shown in
The metal film 222 electrically is connected to a circuit C built in the circuit area CA. In a structure 220 before forming the passivation film 224, usually, the surface of the electrode area EA is desirably flat, but the surface of the circuit area CA may have many steps.
To improve the measurement sensitivity of the electrochemical response, the probe electrode E is preferably small. To supply at least one of the target nucleic acid and nucleic acid recognition body in the solution to the probe electrode E efficiently, the thinner the passivation film 224, the better. As the surface of the circuit area CA has the many steps, if the passivation film 224 is thin, the corners of the steps tend to be exposed readily, and it is difficult to reliably cover the circuit area CA. Accordingly, the passivation film 224 must be thick to reliably cover the circuit area CA. The thick passivation film 224, however, interrupts at least one of the target nucleic acid and nucleic acid recognition body in the solution from reaching the probe electrode E.
This embodiment is directed to a structure that allows the solution to readily reach even a probe electrode E with a small area while reliably covering the steps.
As shown in
The thick portion 234b of the passivation film 234 reliably covers the steps on the surface of the circuit area CA of the structure 220. In addition, as the portion 234a present around the probe electrode E is thin, the solution can reach the probe electrode E readily.
The nucleic acid detection device according to this embodiment is fabricated in the following manner.
A metal thin film is formed on a semiconductor substrate including a circuit, or a substrate having steps and made of glass, resin, ceramics, metal, or the like by sputtering. Alternatively, the metal thin film may be formed by deposition, printing, or the like. After that, wiring portions, pad portions, and the like are patterned by etching where necessary. After that, a passivation film is formed by coating. Alternatively, the passivation film may be formed by sputtering, deposition, printing, or the like. After that, the passivation film is selectively etched to form openings, so the metal thin film is partly exposed through the openings to serve as electrodes. The technique to form the openings is not particularly specified. At this time, the passivation film is formed such that a portion of it which covers each electrode area EA becomes thinner than a portion of it which covers the circuit area CA. The procedure to form the passivation film is not particularly specified. For example, the passivation film can be obtained by forming a comparatively thin first passivation film, forming openings that define electrodes in the comparatively thin first passivation film, forming a comparatively thick second passivation film, and forming openings larger than those in the first passivation film in the second passivation film. According to another procedure, the passivation film can be obtained by forming both first and second passivation films, forming openings larger than electrodes in the second passivation film, and thereafter forming openings that define the electrodes of the first passivation film. According to still another procedure, the passivation film can be obtained by forming a second passivation film first, forming openings larger than the electrodes in the second passivation film, thereafter forming a first passivation film, and forming openings that define the electrodes in the first passivation film. The first and second passivation films can be of the same type or different types. Three or more types of passivation films can be used. This structure can decrease the thickness of the first passivation film that defines the electrodes. At this time, to cover portions having large steps, a passivation film having a large thickness may be formed.
After that, the nucleic acid probes are immobilized to form the probe electrodes in the same manner as in the first embodiment.
The nucleic acid detection device according to this embodiment is used in the following manner.
A solution containing target nucleic acids is supplied to the flow channel to cause a hybridization reaction. As the passivation film which defines the probe electrodes is thin, the target nucleic acids contained in the solution are supplied to the probe electrodes efficiently. Thus, even a small amount of nucleic acids sufficiently allows a hybridization reaction.
After that, washing, reaction with the nucleic acid recognition bodies, and measurement of an electrochemical reaction are performed in the same manner as in the first embodiment to detect nucleic acids.
The basic structure of a nucleic acid detection device according to the fifth embodiment is the same as that of the fourth embodiment. This embodiment is directed to still another structure that allows a solution to easily reach even a probe electrode E with a small area as well while reliably covering steps.
As shown in
The planarization layer 242 reliably covers steps on the surface of the circuit area CA of the structure 240. The metal film 222 is stacked on the flat upper surface of the planarization layer 242. After patterning the metal film 222, the passivation film 244 is stacked on the metal film 222. Thus, the passivation film 244 only need cover the metal film 222 reliably. Accordingly, the passivation film 244 may be a thin film. This allows the solution to reach the probe electrode E easily.
The nucleic acid detection device according to this embodiment is fabricated in the following manner.
A semiconductor substrate including a circuit, or a substrate made of glass, resin, ceramics, metal, or the like and having steps is subjected to a planarization process to decrease the steps. The planarization technique is not particularly specified, and may be etching using a solution or a gas, or mechanical polishing such as the CMP process (Chemical Mechanical Planarization). After the planarization, a metal thin film is formed on the planarization layer by sputtering. Alternatively, the metal thin film may be formed by deposition, printing, or the like. After that, wiring portions, pad portions, and the like are patterned by etching where necessary. After that, a passivation film is formed by coating. Alternatively, the passivation film may be formed by sputtering, deposition, printing, or the like. After that, the passivation film is selectively etched to form openings, so the metal thin film is partly exposed through the openings to serve as electrodes. The technique to form the openings is not particularly specified. The planarization process allows use of a thinner passivation film.
After that, nucleic acid probes are immobilized to form probe electrodes in the same manner as in the first embodiment.
The nucleic acid detection device according to this embodiment is used in the following manner.
A solution containing target nucleic acids is supplied to the flow channel to cause a hybridization reaction. As the passivation film which defines the probe electrodes is thin, the target nucleic acids contained in the solution are supplied to the probe electrodes efficiently. Thus, even a small amount of nucleic acids sufficiently allows a hybridization reaction.
After that, washing, reaction with the nucleic acid recognition bodies, and measurement of an electrochemical reaction are performed in the same manner as in the first embodiment to detect nucleic acids.
An example that uses the nucleic acid detection device according to the third embodiment will be described in detail.
1. Preparation of Nucleic Acid Detection Device
A nucleic acid detection device 1 with a structure that satisfied the requirements described in the third embodiment and a nucleic acid detection device 2 with a structure that did not satisfy them were fabricated. In each of the nucleic acid detection devices 1 and 2, electrodes having different areas are formed two for each area.
Nucleic acid probes having the following sequences were immobilized to the respective electrodes of each area of the nucleic acid detection devices 1 and 2.
Probe A: gtttctgcac ccgga
Probe B: gtttctgcgc ccgga
Flow channels were similarly formed in the nucleic acid detection devices 1 and 2 having the immobilized nucleic acid probes.
2. Respective Types of Nucleic Acid Reactions
A sample nucleic acid having the following sequence including that complementary with the probe A was used.
Sample Nucleic Acid: ggcctccgct ctcgcttcgc ctctttcacc ccgcgcccag ccccgccccg cgccgcgaag aaatgaaact cacagaccct gtgctgaggg cggctccggg cgcagaaacg aaacctagct
A 2×SSC solution containing the sample nucleic acid was supplied to the flow channels of the respective nucleic acid detection devices 1 and 2 to fill the electrodes. The devices 1 and 2 were then set still at 35° C. for 60 min to cause a hybridization reaction of the sample nucleic acid with the nucleic acid probes. Subsequently, a 0.2×SSC solution was supplied to the respective flow channels to fill the electrodes. The devices 1 and 2 were then set still at 35° C. or 60 min to cause a washing reaction. Subsequently, a phosphoric acid solution containing 50 μM of Hoechst 33258 as a nucleic acid detection body was supplied to the respective flow channels to fill the electrodes, to cause the nucleic acid and Hoechst 33258 to interact with each other.
3. Electrochemical Measurement
The embodiments of the present invention are described above with reference to the accompanying drawings. Note that the present invention is not limited to the above embodiments. Various modifications and changes may be made without departing from the spirit and scope of the present invention.
Additional advantages and modifications will readily occur to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein. Accordingly, various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-111178 | Apr 2006 | JP | national |
