CN1267565C - Microdrop oscillatory silicone groove polymeraxe chair reaction biological chip - Google Patents

Abstract

The invention relates to a silicon tank type polymerase chain reaction biological chip of micro-droplet oscillation type. It includes a microgroove, a constant temperature zone separated by a thermal insulation groove on the back of the microgroove, a resistive micro temperature sensor on the back of the microgroove, and micro heaters placed symmetrically on both sides of the temperature sensor. It is widely used in the fields of biological genome research, forensic science, genetic analysis, treatment effect evaluation and medical diagnosis. Samples and reactants oscillate in the form of micro-droplets in a tank with three constant temperature zones to realize the denaturation, annealing and extension processes of polymerase chain reaction (PCR), which reduces the driving pressure required for biochip work , to avoid the impact of possible air bubbles on the function of the chip; the constant temperature zone integrated on the chip is conducive to the rapid heating and cooling process of the PCR reaction mixture, and it is convenient to change the temperature of the working area to optimize the PCR process, or to achieve some special requirements; Manufactured by silicon micromachining technology, it is convenient for mass production, small in size and low in cost.

Description

Micro droplet oscillating silicon tank polymerase chain reaction biochip

technical field

The invention belongs to the field of medical instruments, in particular to the design of a nucleic acid PCR clinical diagnosis biochip suitable for high throughput, rapidity and automation.

Background technique

Polymerase chain reaction (PCR, Polymerase Chain Reaction) is one of the important means of modern nucleic acid analysis. It can amplify the number of target nucleic acid molecules nearly doubled in one cycle, and after 20-30 cycles, the limited nucleic acid The molecule was amplified ¹⁰⁹ times, so as to realize the enrichment of the target molecule and prepare for further analysis. The birth of the PCR process has greatly accelerated the process of research on the genome structure of various organisms, and has a wide range of applications in forensic science, genetic analysis, treatment effect evaluation, and medical diagnosis.

In recent years, with the development of micro-processing technology, microchips used in clinical laboratory research have been widely valued. Micro Total Analysis Systems (Micro Total Analysis Systems) based on microchips have become a research hotspot in analytical chemistry, clinical medicine, and biology, and micro PCR chips, an important part of micro total analysis systems, have attracted many researchers Notice. Since the work of the micro-PCR chip was reported for the first time in 1993, many research groups have successively invested in the research work of the micro-PCR chip. Because the characteristic scale of the chip is on the order of microns, the micro-PCR chip can achieve a very high heating and cooling rate (generally on the order of 10°C/s or higher), thus greatly improving the time efficiency of the PCR process, making it usually takes several The detection process of hours or tens of minutes is accelerated to a dozen or even minutes; at the same time, the sample volume required by the micro-PCR chip is very small (microliter or even nanoliter), which can greatly reduce the cost of the PCR process. And reduce the possibility of pollution. In 1999, my country's patent 99114416.3 proposed a PCR microarray probe cycle detection biochip, which was realized by conventional methods. The schematic diagram is shown in Figure 1. In the figure, 1 is a microchannel, 2, 3, 4, and 5 are microreactors, 6 is a PCR reaction system, 7 is a small iron block, and 8 is a magnet. Its working principle is: the PCR reaction solution 6 is injected between the two small iron blocks 7 in the microchannel 1 in advance, and then covered and sealed with a transparent film, and the sample is injected into the PCR reaction solution through the film by a syringe or other sample injectors , and then pressed and fixed with a glass piece; after shaking and mixing, the PCR reaction can be started. Firstly, the denaturation is carried out in the 2nd area, and then the small iron block 7 in the microgroove is driven by the magnet 8 and the reaction system solution 1 is quickly moved to the Anneal in zone 3, then move to zone 4 for extension, then quickly move to zone 2 to start the second cycle after extension, or move to zone 5 for hybridization reaction and then move to zone 2 to start the second cycle; continue the cycle process like this , until the desired result is obtained. The above-mentioned chip uses droplet circulation to complete the PCR process through three externally realized constant temperature regions, which has many advantages. The disadvantages of this micro-PCR chip are: 1) it is not based on silicon micro-processing technology, and is not compatible with IC technology, so that the chip needs relatively complex and huge external equipment to provide control and communication, etc., which is difficult to produce in large quantities at low cost; 2) the structure The size is large, and it takes a long time to complete the PCR process; 3) the PCR operation in each channel of the same array needs to have the same denaturation, annealing and extension temperature; 4) it is difficult to realize automatic control, and the efficiency is low. In 2001, my country's patent 01103687.7 proposed a DNA-PCR biochip composed of a micro-well reaction chamber made on a silicon substrate by MEMS technology, an integrated platinum resistance heater and a temperature sensor, and packaged on a PCB board. It is realized by silicon micromachining technology, and its schematic diagram is shown in Figure 2. In the figure, 9 is a microwell type reaction chamber, 10 is a microgroove, 11 is a glass cover slip, 12 is a sample injection hole, 13 is a silicon nitride film, and 14 is a platinum resistance heating element and a temperature sensor integrated in the chip. Its working principle is: the PCR reaction solution enters the micro-well reaction chamber 9 through the micro-groove 10 through the injection hole 12, and realizes the heating and cooling requirements of the PCR process under the action of the platinum resistance heating element, the temperature sensor 14 and the cooling of the external fluid. . The disadvantages of this micro-PCR chip are: 1) the chip is close to the airtight microcavity, and during the PCR denaturation process, since the temperature is close to the boiling point of the working fluid, bubbles are easily generated and the chip structure is destroyed; 2) it is not based on microfluidic technology, so it is difficult Integration with other micro-devices constitutes a real lab-on-chip (Lab-on-Chip); 3) The chip uses an external fluid (air) to realize the cooling process, and the cooling rate is relatively low.

Contents of the invention

The purpose of the present invention is to provide a micro-droplet oscillation type silicon tank polymerase chain reaction biochip.

The present invention is characterized in that it is a biochip made of silicon as a base material and made by silicon micromachining technology. It contains a straight microgroove, which makes the PCR mixture oscillate in its groove in the form of droplets to achieve Denaturation, annealing and extension process of PCR; constant temperature zone, it has at least two, it is located at the back of the above-mentioned straight microgroove, between each constant temperature zone is separated from each other by heat-insulated microgroove; resistive micro temperature sensor, it is located in the above-mentioned constant temperature zone Center; micro-electric heater: it is symmetrically placed on both sides of the above-mentioned resistive micro-temperature sensor.

Tests have proved that it is suitable for silicon micromachining, compatible with IC technology, and has the advantages of low cost, small size, high speed, and good performance.

Description of drawings

Fig. 1 is the structural representation of existing PCR microarray probe cycle detection type biochip;

Fig. 2 is the structural representation of existing DNA-PCR biochip and the miniature thermal cycler using this chip;

Fig. 3 is the front view of embodiment structure of the present invention;

Fig. 4 is the cross-sectional view of A-A plane of embodiment structure of the present invention;

Fig. 5 is the sectional view of B-B plane of embodiment structure of the present invention;

Fig. 6 is the embodiment structure C-C plane sectional view of the present invention;

Fig. 7 is a schematic diagram of the silicon micromachining process of the present invention.

Detailed ways

A kind of droplet oscillation type silicon tank type polymerase chain reaction (PCR) biological chip embodiment of the present invention design, in conjunction with accompanying drawing, describe in detail as follows:

The structure of the present embodiment is shown in Fig. 3, 4, 5, 6, mainly by etching on the silicon base 21, and by the microchannel 24 (the liquid droplet 22 of sample and reactant in it) that is closed by glass cover 23 motion), three constant temperature working zones 16, inlet holes 15 and outlet holes 17; the three constant temperature zones are separated by heat insulation grooves 20, and two heaters 18 and a temperature sensor 19 are arranged symmetrically on each constant temperature zone. The thickness of the silicon base 21 is 300 μm; the microchannel 24 is 32,000 μm long, with a trapezoidal cross-section (formed according to the <110> crystal orientation in bulk silicon processing), with an upper bottom width of 600 μm, a lower bottom width of 1000 μm, and a height of 280 μm; three constant temperature zones 16 Both are 5000 μm long and 1600 μm wide; the heater 18 has a width of 200 μm, a length of 4900 μm, and a height of 0.6 μm; the temperature sensor 19 has a width of 50 μm, a length of 5000 μm, and a height of 0.6 μm. The heaters are symmetrically arranged on both sides of the temperature sensor, and the distance between them is 1100 μm.

In this embodiment, the silicon micromachining method is used to process the sensor structure. The basic processing process is shown in Figure 7. Since the central cross section can fully explain the processing process, only the processing process of the central cross section of the chip is completely drawn in Figure 7. .

First, a silicon dioxide layer 26 with a thickness of 6000 Å is formed on the upper and lower surfaces of the <100> silicon wafer 25 by wet thermal oxidation, as a mask for wet etching of silicon (Fig. 6-a); then through the photoresist 27 Protection, single-sided photolithography to form windows for manufacturing heating wires, temperature sensors and electrical contacts (Figure 6-b); metallization and stripping and growing resistance materials (Cr/Pt, 28) to form heating wires, temperature sensors and electrical contacts sheet (Fig. 6-c); the silicon dioxide on the other side of the silicon base is photolithographically removed to form wet etching openings (Fig. 6-d); wet etching (EPW etching solution) forms micro-channels (Fig. 6-e); Float away the residual silicon dioxide on the surface of the silicon substrate on the opening side of the microchannel (Figure 6-f), and perform silicon-glass bonding (the channel surface is bonded to glass 29), forming a glass carrier and a complete microchannel (Figure 6- g); the silicon dioxide on the silicon base surface on the other side is photolithographically removed to form wet etching openings (Figure 6-h); wet etching (EPW etching solution) forms heat insulation grooves (Figure 6-i).

The working principle of the present invention is based on the oscillating movement of droplets in a microgroove with three constant temperature zones to realize the PCR process, but the difference from the domestic patent 99114416.3 is that the present invention adopts a solution suitable for silicon micromachining, which can achieve low cost It is mass-produced and compatible with the IC process, and the control circuit can be integrated; the chip structure is small, and the time to complete the PCR process is short; the constant temperature area and the microgroove are integrated on the same chip, and the same array can be adjusted according to the needs of PCR. Control temperature; can realize automatic control. The difference from the domestic patent 01103687.7 is that the present invention adopts a new droplet microfluidic technology, which can not only avoid damage to the chip by bubbles that may occur during the denaturation stage, but also can be integrated with other microfluidic devices to form a real lab-on-a-chip (Lab-on-Chip); the present invention realizes the requirement of the temperature change of the PCR process through the PCR reaction solution continuously passing through three constant temperature zones, thereby being able to realize a heating and cooling rate far greater than that of the microwell type PCR chip.

Features and scope of application of the present invention:

1. Manufactured by silicon micro-processing technology, it has the characteristics of small size, low cost and good performance;

2. Using droplet microfluidic technology, the droplet oscillates in the microgroove with three constant temperature zones to realize the PCR process, with fast heating and cooling speed and small consumption of samples and reactants;

3. The constant temperature zone and the microchannel are integrated on the same chip, and the control temperature of different channels of the same array can be adjusted according to the needs of PCR;

4. The present invention can be applied in different occasions, and is especially suitable for clinical detection under the requirements of high automation, rapidity and high throughput.

Claims (1)

1, microdrop oscillatory type silicone groove type polymerase chain reaction (PCR) biochip, this chip contains microreactor and flat-temperature zone, it is characterized in that, and it is a kind of biochip of making base material and making of bulk silicon process with silicon, and it is made up of following unit:

A straight microflute: it make the PCR mixed solution with the drop form in its groove vibratory movement to realize sex change, annealing and the extension process of PCR;

The flat-temperature zone: it has two or three, and it is positioned at the above-mentioned straight microflute back side, opens with adiabatic microflute is apart between each flat-temperature zone;

Resistance-type microtemperature transmitter: it is positioned at center, above-mentioned flat-temperature zone;

Little electric heater: it places above-mentioned resistance-type microtemperature transmitter both sides symmetrically.

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