TWI236532B - Multichannel polynucleotide molecule sequencing method - Google Patents

Abstract

This invention relates to a multichannel polynucleotide molecule sequencing method. It comprises the following procedures: firstly, it fabricates a thin film on a chip by microfabrication technology. Secondly, it sets a plurality of nano-scaled apertures on the thin film as ion channel. Furthermore, for each aperture, it installs a set of sub-pico ampere ion current measuring circuit, an independent ion channel electric field, and a set of controllers. The controller measures the ion current of each base of the polynucleotide jammed by the aperture. Moreover, it transforms the current into base sequence. Finally, it records and compares the sequence in a polynucleotide molecule database.

Description

1236532 V. Description of the invention (1) Polynucleic acid molecules include DNA (Rna) and RiboNucleic Acid (RNA), etc. The following description of the present invention is only described by DNA. To perform the analysis, the DNA is first quenched from the cell nucleus and purified. The procedure is: 1. Soak in a detergent to destroy the cell membrane. 2. Centrifuge to remove the debr is protein enzymes, leaving only the DNA in the solution. . The DNA analysis is generally divided into two parts: one is diagnosis, which detects only the existence of a specific test base pair; the other is sequencing, which generates a true base pair sequence. There are five basic chemical procedures for general analysis, such as those described in CH Mast rang el 〇, MA Burns, and DT Burke Microfabricated Devices for Genetic Diagnostics, " Proceedings of the IEEE, Vol. 86, No. 80, 1998, Aug. : 1. Chemical amplification. The double-stranded fragment is first heated to separate into two single-stranded strands, and the single-stranded strands are synthesized into double strands using an enzyme polymerase. This procedure is called poly I per chain reaction PCR (ploymerase chain). reaction) A typical amplification procedure uses high-temperature resistant Taq polymerase enzyme quenched from the heat-resistant microorganism The r mu s aquat f eus, and with an unknown DNA template (t emp 1 ate), a sufficient amount of ribose The nucleotides (d NTP 's) are mixed with primers (pr i mer s) that determine the origin of replication. 2. Add fluorescent dye to DNA fragments. 3. Use restriction enzymes for fragmentation or digestion ° 4. Separation (s e p a r a t i ο n), currently more commonly used methods, such as electrophoresis

Page 4 1236532 V. Description of the invention (2) (electrophoretic), such as capillary electrophoresis (CE), mainly separates DNA fragments of different lengths. Five, there are two common methods of sequence reading. A. Sanger sequencing scheme (Sanger sequencing scheme): mainly put a small amount of ddNTP's (ddA, ddC, ddG, or ddT), making the aggregation action incomplete, and Stop at ddNTP's, then use capillary electrophoresis, and then read optically with a fluorescent microscope. B. Another method: a procedure using a fixed DNA probe array to achieve hybridization. The above-mentioned conventional methods are time-consuming and expensive. The enzymes and sample dosages used are large and expensive. The production of poly I per chain reaction (P C R), DNA probes, etc. is complicated and expensive. The use of ion channels for the sequencing of gene sequences. There is US patent 1186095357 which mainly uses biochemical films, such as lipid bi layer membrane, and channel proteins such as ma 11 ο ρ ori η (L am B ) p 〇re or bacteria 1 ρ 〇 re-forming eggs

The main disadvantages of white matter, etc., are that it cannot be made into multiple channels and read the gene sequence at the same time, and it is not easy to combine the reading circuit with the control circuit; in addition, the ion channel does not use semiconductor processes, and it is not compatible with micro-electromechanical processes. Completed, so it is not easy to make, and there is little flexibility in changing the size, length, and location of the channel. Furthermore, there is a lack of a simpler, cheaper, and truly effective method that allows DNA to be effectively straightened before entering the channel, so that one base can pass through the channel, making the DNA sequence clear and legible. In addition, this

1236532 V. Explanation of the invention (3) The patent can not only read every base by the action of electric field and integrated circuit. [Technical objective] One of the objectives of the present invention is to provide an effective method for reading a gene sequence, which mainly uses a plurality of ion channel mechanisms to accelerate the speed of sequence reading. Another object of the present invention is to reduce the cost by using polymerase chain reaction PCR or using electrophoresis and other procedures that require the use of biochemical materials. A third object of the present invention is to provide a method for sequentially reading a longer sequence, and it is not necessary to use a short MA probe like a hybridization method. The fourth objective of the present invention is to provide a simple, cheap and truly effective method to make DN A straighten effectively before entering the channel, so that one test base and one base can pass through the channel to make the DNA sequence clear and distinguishable. . A fifth object of the present invention is to provide a method for reading a plurality of types of DNA at a time without having to singularize DNA. A sixth object of the present invention is to provide a reading device that can be integrated into a wafer form, which is completed using a semiconductor process and a micro-electro-mechanical process. In summary, the main purpose of the present invention is to provide an efficient method for gene sequencing, which uses multiple ion channel mechanisms, uses a semiconductor process, and combines it with a microcomputer and electrical process to increase the speed of sequencing. In order to clearly describe the 4 methods and the device of the present invention, the inventors have provided illustrations, figure numbers, and detailed descriptions as follows: [Illustration]

1236532 5. Description of the invention (4) Figure 1 is a schematic diagram of a device for sequencing genes of a polyion channel used in the present invention. Figure 2 is a flow chart of a complete gene sequencing. 3 is a schematic diagram of DNA straightening in the method of the present invention. Figure 4 is The electronic circuit of the inventive method. FIG. 5 is a current-voltage converter circuit used in the present invention. FIG. 6 is an embodiment of the ion channel used in the present invention. FIG. 7 is a top view of the embodiment configuration of the ion channel used in the invention. Schematic diagram for reading the DNA sequence using the ion channel of the present invention Figure No. 1 Device for sequencing genes in a polyion channel 3 Nano channel 5 Ion current 7 Second cavity 1 1 process 1 1 3 process three 1 5 process five 2 0 electrode System 2 2 substrate &

2 4 First electrode 26 Straightened DNA 2 Single-stranded DNA 4 Each test base 6 First pocket 1 2 Process 2 1 4 Process 4 19 Thin film 21 Second electrode

2 3 DC electrode / signal input line 25 Unstraightened DNA 3 0 P ic ampere current measurement electricity 1236532 V. Description of the invention (5) Sub-circuit 3 2 High-speed analog digital converter 34 Display (LCD) 3 6 Memory 3 8 DC electric field voltage 10 0 First substrate 1 0 2 5 4. 7 Shallow groove with 4 degree oblique angle 10 4 Upper layer of first substrate 1 0 6 Bowl-shaped nano channel 201—Layered gold (Au) runner Layer 3 0 3 Twenty nano channels 311 First-stage amplifier circuit 3 1 3 Third-stage amplifier circuit 31 Current / voltage converter 3 3 Microcontroller 3 5 External computer 37 RS-2 3 2 Communication protocol 3 9 AC electric field voltage 10 1 Silicon nitride (Si 3N 4) thin film 1 0 3—Layer of gold (Au) 105 Lower layer of the first substrate 2 0 0 Di-based material or silicon 3 0 1 Glass material or polymer material 3 0 2Injection port 301 Field control and current detection circuit 3 1 2 First stage amplification circuit [Mechanism of gene reading] F 3, Ersuo # 'is a multi-ion channel reading gene II'V used in the present invention … Using the spot MA formed on the negative side of multiple nanochannels 3 will be inserted into the second cavity 7 formed on the solution side of a gasified salt such as potassium gas (KC1) or sodium chloride. t;

1236532 V. Description of the invention (6) Different, and the length of the nano channel is about 0.5-1 nm, and its pore diameter is less than about 4 nm, so each base 4 can block the channel to different degrees according to its size, resulting in The frequency of potassium ions flowing through the channel, that is, ion current 5, is different, so the DNA sequence can be read correspondingly. The object read by the present invention can be DNA or message RNA, because DNA includes genes and intergenic regions, and messenger RNA belongs to the gene code, so reading them is more direct. From the above overview, we know that the basic concept of the D N A sequence passing through the channel is to achieve: 1. Four bases, A, G, C, and T. The degree of blocking the channel is significantly different and identifiable, and it is not affected by the channel size variation. 2. D N A passes through the channel, it should be ensured that the 5 'lead leads through the channel, not blocking. 3. The speed at which DN A passes through the channel should not be affected by the length of the DNA or its sequence combination, that is, the time for each base to pass through the hole is the same, and the number of bases passing through the channel per unit time is fixed, and at least five to six consecutive Identical bases can be clearly identified. 4. Multiple channels can simultaneously read DNA sequences, regardless of the length and type of DNA, to increase the speed of reading DNA sequences. [Agreed countermeasures] The entire process of gene reading, as shown in Figure 2, is explained as follows: Process 1 1 1 Use conventional methods to extract single or multiple types of DNA from which the sequence is to be read. An appropriate concentration of a solution such as a chloride salt.

Page 9 1236532 V. Inventing ft. (7) Process 2 12 Warm to 7 0-9 5 ° C, so that the DNA or messenger RNA maintains a single strand state, there will be no secondary structure. Without increasing the pH of the solution, maintain about 7-8, make the single strand of D N A negatively charged, and the adjacent test groups will repel each other, so that the single strand of DNA or messenger RNA can maintain a state close to a straight line. If the messenger RNA is read, because the maximum length of the messenger RNA will not exceed 10 K b p ’, it must be maintained at South temperature (9 5 C)’ so as not to be destroyed by restriction enzymes. The heating requirements required in this process can be provided by a heater of an external device, or a micro-heater can be provided in the cavity to provide temperature control by micro-processing technology. Process III 1 3 As shown in FIG. 3, the electrode system 20 is vacuum-deposited the first (gold) electrode 2 4 on the substrate 22 forming the first cavity 6, and the second ( Gold) electrode 21, put the solution containing DNA into the first cavity 6 between the two electrodes, at this time the DNA is randomly rolled into a ball 201, when an electric field of 1 MV / m, 1 MHz is applied between the two electrodes, DNA will be straightened 202 (refer to S. Suzuki, e t. A 1., " Quantitative Ana lysis of DNA Orientation in Stationary AC Electric Fields Using Fluorescence Anisotropy, n IEEE Trans. Industry Applications Vol. 34, NO. 1, Jan / Feb, 1998, PP7 5-8 3), in the present invention, a DC electrode / signal input line 23 is additionally provided at the second electrode terminal 21, and a DC bias is added to attract negatively charged DNA Toward the second electrode 21, the straightened DNA passes through the channel 3 provided on the thin film 19.

Page 10 1236532 5. Description of the invention (8) The distance between the two electrodes is preferably more than twice the length of the DNA. The position of the electrode below the channel is larger than that of the first electrode 24, which is covered with the first cavity ( The bottom surface is set as the negative end; the second electrode 21 is above the thin film 19, and its area is small, and it can be properly designed to restrict the area of the straightened DNA. Since the second electrode 21 is passed through the thin film 19, (Non-conductor) isolation, so that the straightened DNA will not be adsorbed and fixed on the second electrode 2 process 4 1 4 blocking the reading of the ionic current Only one straight DNA passes through the nano-hole, each alkali The base stays in the nanopore for a controlled period of time, and potassium ions or nano ions will pass through the nanopore when the base is blocked, and the ion current can be read by the current / voltage conversion circuit. Process 5 1 5 Interpretation of DNA sequence by gene sequence conversion Generally speaking, the ion current after base blocking is larger than that before non-blocking in GATC order, and there are obvious class differences. Refer to Figure 8, so you can use three Bit Analogy A bit converter is used to convert the sequence into an AGCT sequence and store the sequence in the memory of the microcontroller for other subsequent uses. 〇 [Electronic circuit of the ion channel] The current flowing through the channel of the ion, the current is about twice pic 〇 to hundreds of Pic 0 (10-12) amps. The conventional technology is to use large-scale instruments or electronic circuits. In order to reduce and integrate them on the chip, the present invention redesigns them.

Page 11 1236532 V. Description of the invention (9) ---- Integrated and integrated circuits, described below: Daddy test 'P 1 co Ampere current measurement electronic circuit 3 〇 It can be roughly divided into five independent sub-circuits' Including 丨 current converter (IV converter) 9 night current electric field voltage ^ _ • • parent current electric field voltage 4. three-bit high-speed analog digital converter 5 · microcontroller and so on. The first-class, low-noise operational amplifier used in voltage converters, referring to Figure 5 ', is composed of two #differential amplifiers. The input bias current is equal to 0.03pA, the input voltage is equal to 2pF, and the voltage noise density is It is 15xl0-17 VVHz at 0.1MHz, and the gain-bandwidth product is 100MHz. Because the current / voltage converter 31 is used, the purpose is to convert the current of 1 PA to 1 OmV. Therefore, the required resistance of the conventional circuit needs to be 1 ohm. lysilic〇n) N-well, may cause too much circuit area. Therefore, it is completed in sections. The first stage of operational amplification 3 1 1 uses a feedback resistor 丨 〇〇ohm ohms to convert 1 pa current to 0 · 1 mVt ^ voltage. The second stage 3 丨 2 uses a voltage follower As a buffer, the third stage 3 1 3 is a differential amplifier with an amplification factor of 丨 〇〇, so the combined amplification gain of the three stages is 1 〇! QV 〇1 t / A. The three-bit high-speed analog digital converter 32 aims to set the four voltage levels corresponding to the four test bases of dnA and a reference level. There are six voltage levels when unblocked, so it needs to be converted into three. Bit, the basic structure is a flash circuit (refer to D. Α · Johns and K. Martin, 1997, Analog Integrated Circuit Design, pp.507-513), the resistance between the levels, corresponding to four It can be adjusted according to the difference of the base level and the voltage level when it is not blocked. Its conversion speed is 100MHz-500MHz, which can make

1236532 V. Description of the Invention (ίο) Multiple channels are used to read multiple channels. The purpose of the AC electric field voltage 3 9 ′ is to apply an electric field of 1 μV / m, 1 M Η z between the two electrodes, to straighten the D N A. The DC electric field voltage is 38, the purpose is to match the straightening effect of the AC electric field voltage, and apply a DC bias voltage to one of the electrodes to make the straight j) NA pass through the channel provided on the electrode, in order to reduce noise Impact (refer to Hami J i et. Al., 1981, Pfluegers Arch. Eur. J. Physiol 3 9 1: 8 5-1 0 0), provide a way to reduce the speed of DNA through the nanopore, so that each base can Suspended in the nanopore, without the influence of diffusion, there is time for the ions to pass through, basically controlling the sampling time to 0.5 ~ 2 microseconds, and its DC bias level is about zero to negative pressure, and The turn-on DC pause bias level is approximately 60-150mV. This DC electric field bias voltage can be applied through the current-to-voltage converter 3 丨 shown in Figure 5, that is, input V cmd at the positive terminal of the first-stage amplifier, transfer to the negative terminal through the virtual zero potential difference, and go through the current signal terminal I. The local electric field voltage is generated, and the amplification at the first stage is subtracted by the differential amplifier, and only the amplified value of the ion current I is taken out. The functions of the microcontroller 33 include: adjusting the voltage level and frequency of the AC electric field voltage, the level of the DC electric field voltage, the on-time and pause time of the driving DNA signal, and the communication with the three-bit high-speed analog digital converter. Conversion and storage of gene sequence code, or comparison with the gene sequence of memory 3 6 for known diseases, and display on the display (LCD) 34, or temporary gene sequence code, through RS- 2 3 2 communication protocol 3 7. Transfer to external computer 3 5. The microcontroller can also use commercially available products, and the built-in memory

1236532 V. Description of the invention (11) 36 can also be expanded by additional memory, so that the number of channels readable by the present invention and the length of DNA read by each channel can be longer. [Ion channel fabrication] The fabrication of ion channels is completed by the following micromachining techniques: Step one, refer to Figure 6 (a) 'Firstly select P-Type (100) single crystal silicon as the first substrate 100, Then, on the first substrate 100, a low-pressure chemical vapor deposition (LPCVD) Fang Wu was deposited on both sides of the first substrate 100 to form a 50nm-10 0nm silicon nitride (Si core) film 101 on both sides. Step two, referring to FIG. 6 (b), using a photolithographic surname engraving technique to pattern the lower half of the substrate by dry etching, and then placing the first substrate 100 at 85. Etching while stirring in potassium hydroxide (K0H) of C, and using the characteristics of the single crystal silicon substrate to etch a shallow groove 1 0 2 of 5 4 · 7 4 degree oblique angle; Step 3, refer to FIG. 6 (c). Works on light lithography and uses a sputter to compete for a layer of chromium (Cr) on the substrate and a layer of gold (Au) 1 〇3, and uses peeling (L 丨 f 〇 ff) The required pattern is left in the process for the second electrode 21 and the DC electrode / signal input line 2 3. The chromium metal layer mainly functions to increase the adhesion between the gold and the silicon substrate f, so It is not necessary to be too thick; step four, referring to FIG. 6 (d), and then using the photolithography technique to react the ion etching machine to the upper layer 104 of the first substrate and the lower layer 105 of the first substrate

Page 14 1236532 V. Description of the invention (12) (R I E) 'Carve a nitride stone I insect out of the window, and place the substrate at 8 5. etch in potassium hydroxide (K 0 Η) of c while stirring for a period of time until the first substrate at 104 and 105 penetrates into the injection hole;

Step 5 'Referring to FIG. 6 (e), a photoresist coater is used to coat a layer of about 1000 nm photoresist (PMMA) on the back of the upper silicon nitride film, and then exposed to an appropriate dose of electron beam. The silicon nitride thin film 1 01 was then dry-etched with a reactive ion etcher by controlling the etching time or using a feedback mechanism (Jiali Li et. Al. M Ion-beam sculpting at nanometer length scales M, Nature, Vol. 412, July 2001, pp. 166-169) can obtain a bowl-shaped communication

Channel 10, where the minimum aperture of this channel can be controlled at 0.5 to 4 nm as required. If it is a tiny channel that allows only a single strand of DNA to flow, its pore diameter can be set to 0.5-2 nm; in addition, it can also be controlled by steaming or splashing key oxides such as Si 0 The size and thickness of a channel aperture, and then the first substrate was etched while being stirred in potassium hydroxide (K0H) at 85 ° C., and stopped until it contacted the silicon nitride (S i A 〇 film 1 01); related The equipment and conditions required for making nano-scale aperture masks are as follows: Electron beam system Elionix ELS-3300, maximum acceleration voltage 30kV, electron beam diameter 6nm-lum depends on current, such as 7nm at 7pA; maximum exposure area field size 500um or 200um; SAL601 negative photoresistor, the photoresist thickness should be minimized to reduce the sca 11 ering effect. For example, SAL601-SR7 is diluted with MICROPOS IT thinner at a ratio of 2: 3 6000rijm to a thickness of 100nm. .

Page 15 1236532 V. Description of the invention (13) Step 6, refer to FIG. 6 (f), then take a piece of P-Type (100) single crystal silicon as the second substrate 2 0 0, and use photolithography to etch Technology to etch silicon nitride; Step 7, refer to Figure 6 (g), and then use potassium hydroxide (K Η) at 85 ° C to etch the desired shape; Step 8, refer to Figure 6 (h), Then use a sputtering machine to sputter a layer of chromium (Cr) and then a layer of gold (Au) 201 on the upper half plane of the second substrate as a second electrode 2 that provides an electric field and a measurement signal 2 Step 9: Referring to FIG. 6 (i), the first substrate 100 and the second substrate 2000 are heated to 390 ° C to perform the bonding of the silicon / gold / stone evening eutectic alloy or Adhesive or co-fusion gold bonding, in which the distance between the two substrates and the size of the channel can be controlled by designing a silicon nitride pattern; Step 10, refer to Figure 6 (j), and finally a layer of glass or polymer material Or the flow channel layer 3 0 1 of silicon is bonded to the two-layer substrate of FIG. 6 (i), thus completing the structural diagram of the DNA sequence reading element; φ In addition It is also possible to design some micropumps in the glass layer to assist D N A and buffer solution (B u f f r r) in the flow channel. Referring to FIG. 7, it is a configuration top view of one embodiment of the present invention. Contains

Page 16! 236532 V. Description of the invention (14) The injection port 3 0 2 provides a solution containing D N A, and 20 nano-channels 3 0 3 are actuated by four sets of electric field control and current detection circuits 3 0 4. The electric field control and current detection circuit 304 can be completed on the first substrate 100 by the integrated circuit manufacturing process, and the fabrication of the nano-ion channel 3 03 is based on the above micro-addition as the post-processing of the integrated circuit manufacturing process. (Post processing).实施 例 _ Implementation examples _

The resistivity of 100 nm nitride stone deposited by LPCVD is 1016 ~ 4x10 16 ohm-cm. After packaging, as shown in Figure 6 (j), the upper cover forms a cavity, which can be equivalent to a drop. Pipette (refer to et * al., 1981, Pfluegers Arch. Eur. J. Physiol 3 9 1: 8 5-1 〇〇), so the overall effect of the present invention, not only has the film suction (Patch cl amp), because the micro-fabricated nitride nitride film has better resistivity, and the read circuit is directly fabricated to etch the north and south, and the noise of the moon is smaller. .

Basically, the "conductance of the nanopore" at any given time is determined by the resistance of the ions through the nanopore, and the ions leaving or entering the nanopore. Under the conditions of 1M NaC1, 10mM Tris PH7.4 l20mV, 2m, 1nm nanometer pore length, the unblocked current is about two PA, and the time for each test group to pass through the nanopore is about 5.3. us, in order to reduce the response speed and low noise of the amplifier and analog digital converter (ADC), it is necessary to slow down the speed of each base through the nanopore, which can be achieved by the following methods, such as increasing Solution viscosity, or lower DC bias

Page 17 1236532 V. Description of the invention (15) Small, or make the DC bias voltage have a synchronization signal, about 200 KHz or lower, the time ratio of applying positive and negative electricity is about 1: 1 to 1:10 , The ratio of the voltage magnitude is 1 0: 1 to 1: 1. Fig. 8 is a schematic diagram for reading a DNA sequence using the ion channel of the present invention. In order to avoid having multiple bases in the nanopore at the same time, and therefore cannot recognize a single base, the best way is to make the length of the nanopore close to the length of a single test base (pitch) is about 0.4 nm . Just as mentioned before, the nano-perforation is manufactured by using ion reactive etching to make the perforation into a bowl shape, and the minimum thickness of the bottom can reach 0.4 nm. · Due to the symmetry of the electrical structure of the double-stranded DNA, there are two possible directions for generating g after the application of static electricity, and the two orders are exactly opposite. If a single-stranded DNA is used, the symmetry of the electrical structure no longer exists. The 5 'head has a large electric charge, and it can be achieved by applying an alternating electric field or not, and a DC bias voltage. The 5' head passes through the nanopore first. In addition, it can also be read by a reliable sequence and compared with the data to get the correct result. the sequence of. In summary, the present invention combines the semiconductor manufacturing process with the design and fabrication of multiple ion channel mechanisms of the micro-electro-mechanical manufacturing process, which can indeed make the sequence reading speed faster and more effective. Therefore, an invention patent application has been filed according to law. According to the content described above, other related changes should be included in the scope of applying for an invention patent as long as they do not depart from the spirit of the present invention.

Page 1236532 Brief description of the diagram [Figure not] Figure 1 is a schematic diagram of a device for sequencing genes of a multi-ion channel used in the present invention. The entire sequence of gene sequencing is a schematic diagram of DNA straightening in the method of the present invention. The electronic circuit is a current-voltage converter circuit used in the present invention. An example of the ion channel used in the present invention is made of the embodiment of the present invention. The above configuration is shown in FIG. Figure 6 View 7 Figure 8 is a schematic diagram of the DNA sequence read using the ion channel of the present invention Figure No. 2 Single strand DNA 4 Each base 6 First pocket

1 2 process two 1 4 process four 19 thin film 21 second electrode 2 3 DC electrode / signal input line 2 5 unstraightened DNA 1 polyion channel sequencing gene device 3 nano channel 5 ion flow 7 second cavity 1 1 process 1 3 process 3 1 5 process 5 2 0 electrode system 2 2 substrate 24 first electrode

Page 12 1236532

Schematic description 26 Straightened DNA 31 Current / voltage converter 3 3 Microcontroller 3 5 External computer 37 RS- 2 3 2 Communication protocol 3 9 AC electric field voltage 10 1 Silicon nitride (Si 3N4) film 3 0 Pic 〇 Amp current measurement electronic circuit 3 2 High-speed analog digital converter 34 Display (LCD) 3 6 Memory 3 8 DC electric field voltage 10 0 First substrate 1 0 2 5 4. 7 4 degree shallow groove Upper layer 1 0 3—Layer of gold (A u) 10 5 First substrate lower layer 10 4 First substrate 106 Bowl-shaped nano channel 201—Layer channel layer of gold (Au) 3 0 3 Twenty nanometers Channel 3 1 1 First-stage amplifier circuit 3 1 3 Third-stage amplifier circuit 2 0 0 Second substrate 3 0 1 Glass material or polymer material or silicon 3 0 2 Injection port 3 0 4 Electric field control and current detection circuit 3 12 Second stage amplifier circuit

Page 20

Claims (1)

023 ^ 532 ^ 公. Temporal engraving 『Beauty Carvings si 1. A multi-channel polynuclear sequencing method, which uses micro-machined technical rice-level perforations, as a single or sub-solution between the acupoints. The first cavity-for each perforation, an alternating electric field is made so that the first and second films are perpendicular to each other, and the acid molecule is passed through the film group p A (1 0 _1 amps). Each base of the molecule is passed through-a set of controllers and Direction, the measured value of the ion channel sub-current is further or further compared with polynucleotide molecules (polynuleotide molecules method includes the fabrication of a thin film on a wafer, and the creation of a plurality of nano-channels on the thin film, so that the thin film is between the first Dimples and dimples of various polynucleotide molecules are placed in a cavity that has been placed properly. Use an integrated circuit process to set a separate ion through the pocket. Straighten the polynucleotide molecules in the direction and thinly place an ion. The DC bias voltage of the channel makes the perforation provided on the straightened nuclei reach the second cavity, and an ion current measurement circuit is set to measure the ion current blocking the perforation when the polynucleotide hole is used to control the ions. Large DC bias of the channel The magnitude and frequency of the AC electric field, and the processing of the ion signal, conversion into a base sequence, and recording, the acid molecular database alignment. 2. The sequence of the polynucleotide molecules as described in the first patent application scope The method, the so-called AC electric field of the ion channel, mainly consists of appropriately disposing the first electrode on the side where the first cavity is not in contact with the film, and the second electrode on the side where the second cavity is in contact with the film, and defining its scope of action. When the solution containing the polynucleotide molecule is placed in the cavity between the two electrodes, the polynucleotide molecules that were randomly rolled into a group will be subject to the appropriate voltage and frequency, such as lMV / m, 1MHz. The electric field is between the two electrodes and it is straightened without two Page 21 1236532 6. Scope of Patent Application Sub-structure. 3. The method for sequencing polynucleotide molecules according to item 1 in the scope of the patent application, wherein the so-called DC bias of the ion channel is mainly provided by using a second electrode terminal on the thin film at the second cavity. A DC electrode / signal input line is near the perforation, and a DC bias voltage is applied to attract negatively charged polynucleotide molecules to move toward the perforation direction, so that the straightened polynucleotide molecules pass through the perforations provided on the film, or intermittently. Change the direction of the DC bias, so that the polyglycolic acid molecules stay in the perforation intermittently. 4. The method for sequencing polynucleotide molecules according to item 1 of the scope of patent application, wherein the so-called multiple nano-scale perforations refer to the pore size and shape, the number of pores, and the thickness at the minimum pore diameter, Can be completed by micro-processing technology as required. 5. The method for sequencing a polynucleotide molecule as described in item 1 of the scope of the patent application, wherein the solution in the cavity can be provided with temperature control by an external heater as required, or a micro-processing technology can be used to set a micro- The heater provides temperature control in the recess. 6. The method for sequencing polynucleotide molecules according to item 1 of the scope of the patent application, wherein the so-called multi-channel sequencing refers to that multiple nano-scale perforations with corresponding control electric fields and read circuits can each be simultaneously Sequencing a string of polynucleotide molecules. Page 12 1253632 Page 23