TWI731440B - Nucleic acid analysis apparatus and nucleic acid quantification method - Google Patents

Abstract

A nucleic acid analysis apparatus is used for quantifying the amount of a nucleic acid fragment in a sample reagent. The nucleic acid analysis apparatus includes a circuit board, a sensor chip, a microwell array, a temperature control element, and a microfluidic device. The sensor chip is disposed on the circuit board and includes a plurality of image sensors. The microwell array is disposed on the sensor chip and includes a plurality of microwells. Each microwell corresponds to one or more image sensors. The temperature control element is disposed on the circuit board. The microfluidic device is disposed on the microwell array and includes a microchannel. The microchannel is in communication with each of the microwells. The microfluidic device and the sensor chip are respectively disposed at two opposite sides of the microwell array. Furthermore, a nucleic acid quantification method using the nucleic acid analysis apparatus is provided.

Description

Nucleic acid analysis device and nucleic acid quantitative method

The present invention relates to an analysis device and a quantitative method, and particularly relates to a nucleic acid analysis device and a nucleic acid quantitative method.

In the current nucleic acid quantification technology, compared with the real-time quantitative polymerase chain reaction (real-time quantitative polymerase chain reaction) relative quantification method, the digital polymerase chain reaction (digital polymerase chain reaction) can provide more Approximately absolute quantitative approach.

However, since the quantitative method of digital polymerase chain reaction still needs to use the Poisson distribution principle to estimate the amount of nucleic acid, the quantitative method of digital polymerase chain reaction cannot be regarded as absolute quantification. In addition, the quantitative method of digital polymerase chain reaction has its disadvantages. For example, it usually takes 32 or more polymerase chain reaction (PCR) and takes about 2.5 to 4 hours.

The invention provides a nucleic acid analysis device and a nucleic acid quantification method, which can have higher sensitivity, less detection time and absolute quantification effects.

The nucleic acid analysis device of the present invention can be used to quantify the number of nucleic acid fragments in the sample reagent. The nucleic acid analysis device includes a circuit board, a sensing chip, a microwell array, a temperature control element, and a microfluidic element. The sensing chip is disposed on the circuit board and includes a plurality of image sensors. The micro-hole array is configured on the sensing chip and includes a plurality of micro-holes. Each microhole corresponds to one or more image sensors. The temperature control element is arranged on the circuit board. The microfluidic element is arranged on the micropore array and includes micro flow channels. The micro flow channel communicates with each micro hole. The microfluidic element and the sensing chip are respectively located on opposite sides of the microhole array.

In an embodiment of the present invention, the aforementioned nucleic acid analysis device further includes a filter and a wire. The filter is arranged on the sensing chip and located between the microhole array and the sensing chip. The wires are arranged on the circuit board. The wires can be used to electrically connect the sensing chip and the circuit board.

In an embodiment of the present invention, the above-mentioned temperature control element includes a plurality of heating elements. The heating element surrounds each micropore of the micropore array to regulate the temperature of each micropore.

In an embodiment of the present invention, the number of the aforementioned micropores is greater than the number of nucleic acid fragments in the sample reagent.

In an embodiment of the present invention, the number of nucleic acid fragments in each microwell is 0 to N, and N is an integer.

In an embodiment of the present invention, the distance between each of the aforementioned microholes and the corresponding image sensor is less than or equal to 10 microns.

The nucleic acid quantification method of the present invention can be used to quantify the number of nucleic acid fragments in the sample reagent. The nucleic acid quantification method includes the following steps. First, the above-mentioned nucleic acid analysis device is provided. Then, the sample reagent and the reaction reagent are dispensed into each microwell of the microwell array, and the reaction reagent includes a fluorescent label. Then, a predetermined number of polymerase chain reaction (PCR) is performed on the sample reagent to make the fluorescent label adhere to the nucleic acid fragment and release the fluorescent substance. Then, the intensity of the fluorescent signal of the fluorescent substance in each microwell is detected. Then, according to the intensity of the detected fluorescent signal, the number of nucleic acid fragments in each microwell is determined. Finally, the number of nucleic acid fragments in each microwell is added to obtain the number of nucleic acid fragments in the sample reagent.

In an embodiment of the present invention, the aforementioned microfluidic element further includes an opening communicating with the microfluidic channel. The step of distributing sample reagents and reaction reagents into each microwell of the microwell array includes the following steps. The sample reagent and the reaction reagent are respectively injected into the opening. Sample reagents and reaction reagents flow into each microwell of the microwell array along the microfluidic channel of the microfluidic element.

In an embodiment of the present invention, the above step of performing polymerase chain reaction on the sample reagent a predetermined number of times includes the following steps. A temperature control element is used to regulate the temperature of the micropores so that the temperature of the micropores circulates between 45°C and 95°C or the temperature of the micropores is maintained at a fixed temperature to perform the polymerase chain reaction.

In an embodiment of the present invention, the step of detecting the intensity of the fluorescent signal of the fluorescent substance in each microwell includes the following steps. One or more image sensors corresponding to each micro-hole are used to independently read the fluorescent signal intensity of the fluorescent substance in the micro-hole.

In an embodiment of the present invention, the step of judging the number of nucleic acid fragments in each microwell based on the intensity of the detected fluorescent signal includes the following steps. First, sort the detected fluorescent signal intensity from low to high. Next, the number of nucleic acid fragments in the microwell with the lowest fluorescence signal intensity is represented as one, and as the ranking is higher, the number of nucleic acid fragments in the corresponding microwell is sequentially increased by one. Finally, the number of nucleic acid fragments in the microwell where the intensity of the fluorescent signal cannot be detected is expressed as zero.

Based on the foregoing, in the nucleic acid analysis device and nucleic acid quantification method of this embodiment, the nucleic acid analysis device includes a sensing wafer, a microwell array, a temperature control element, and a microfluidic element. With the configuration of the microfluidic element, the sample reagents can be evenly distributed in the microwells of the microwell array. With the configuration of the sensing chip, the sensitivity of sample detection can be improved, and the detection time can be reduced. In addition, since the number of nucleic acid fragments in each microwell can be judged according to the intensity of the detected fluorescent signal, the nucleic acid quantification method provided in this embodiment has an effect of absolute quantification.

FIG. 1A is a three-dimensional schematic diagram of a nucleic acid analysis device according to an embodiment of the present invention. For clarity of the drawings and convenience of description, FIG. 1A omits several elements, such as the microfluidic element 150 and so on. FIG. 1B is a schematic cross-sectional view of the nucleic acid analysis device of FIG. 1A along the section line A-A'. Referring to FIGS. 1A and 1B at the same time, the nucleic acid analysis device 10 of this embodiment includes a circuit board 110, a sensing chip 120, a microwell array 130, a temperature control element 140 and a microfluidic element 150. In this embodiment, the circuit board 110 may include bonding pads 112. The bonding pad 112 can be electrically connected to an external electronic component, and output the signal detected by the nucleic acid analysis device 10 to an external electronic component (not shown).

In addition, in this embodiment, the nucleic acid analysis device 10 can be used to quantify the number of nucleic acid fragments 162 in the sample reagent 160.

The sensing chip 120 is disposed on the circuit board 110, and the sensing chip 120 includes a pixel array and a plurality of bonding pads 124. In this embodiment, the pixel array includes, for example, 368×184, 1024×1024, or 4096×3072 pixels, but it is not limited thereto. Each pixel includes an image sensor 122, and the image sensor 122 can be, for example, a photodiode as a component for sensing light signals, and can convert the light signals into electrical signals and output them to be readable Pattern. In some embodiments, the image sensor 122 may be, for example, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor or a CCD (Charge Coupled Device) image sensor, but it is not limited thereto. In this embodiment, the material of the sensing chip 120 may include silicon, but it is not limited to this. Multiple image sensors 122

The micro-hole array 130 is configured on the sensing wafer 120, and the micro-hole array 130 includes a plurality of micro-holes 132. In this embodiment, although FIG. 1A schematically shows that the micropores 132 in the micropore array 130 are arranged in a 4×4 matrix and the number of the micropores 132 is 16, the present invention does not apply to the micropores 132. The arrangement and the number are restricted. That is to say, in some embodiments, the microwells in the microwell array can also be arranged in a 12×8, 24×16 or other matrix manner, and the number of the microwells can also be, for example, 96, 384. Or other quantities. In this embodiment, each microhole 132 corresponds to one pixel (ie, the image sensor 122), but it is not limited to this. That is to say, in other embodiments, each microhole 132 may also correspond to two pixels (ie, the image sensor 122), as shown in FIG. 2. In some embodiments, each microhole can also correspond to more than two pixels (ie, image sensors) (not shown). For example, when the sensor chip 120 has 368×184 pixels, there must be at least 67712 corresponding microholes 132. In this embodiment, the image sensor 122 can be used to sense the fluorescent signal generated in the microhole 132. In addition, in this embodiment, the material of the microwell array 130 is, for example, silicon or aluminum, but it is not limited to this, as long as it is a material that is opaque and does not react with biomolecules and reagents. The biomolecules and reagents include, for example, nucleic acid, enzyme, nucleoside triphosphate and other reagents involved in the reaction, but not limited to this. In some embodiments, the microholes 132 of the microhole array 130 are, for example, holes dug into the silicon substrate by a deep reactive ion etching (DRIE) process.

1A and 1B again, in this embodiment, the number of micropores 132 is, for example, greater than the number of nucleic acid fragments 162 in the sample reagent 160, so that it can be avoided when the sample reagent 160 is evenly distributed in the micropores. After the holes 132, there is more than one nucleic acid fragment 162 in each microwell 132. However, in some embodiments, although the number of micropores 132 is greater than the number of nucleic acid fragments 162 in the sample reagent 160, according to the Poisson distribution principle, when the sample reagent 160 is evenly distributed in each micropore 132 Thereafter, the number of nucleic acid fragments 162 in each microwell 132 may also be 0 to N, and N is an integer.

In addition, in this embodiment, since the distance D between each micro-hole 132 and the corresponding image sensor 122 is, for example, less than or equal to 10 microns, the image sensor 122 has a better response to the fluorescent signal in the micro-hole 132. Sensitive. Specifically, since the number of photons that the image sensor 122 can receive is inversely proportional to the square of the distance D, the distance D is also the distance between the fluorescent signal in the microhole 132 and the corresponding image sensor 122. Therefore, compared with the conventional micro-hole and the image sensor with a distance of 10 cm or more, the number of photons that the image sensor 122 of this embodiment can receive under the same sensing area It is more than ¹⁰⁸ times the number of photons that the conventional image sensor can receive. In other words, compared with the conventional nucleic acid analysis device, the nucleic acid analysis device 10 of this embodiment can obtain a lower fluorescence signal intensity, that is, the detection limit of the nucleic acid analysis device 10 of this embodiment is lower. And the sensitivity is high.

In this embodiment, the temperature control element 140 is disposed on the circuit board 110. The temperature control element 140 may include a plurality of heating elements 142. The heating element 142 surrounds each micro-hole 132 of the micro-hole array 130 to regulate the temperature of each micro-hole 132. In some embodiments, the heating elements 142 of the temperature control element 140 may be distributed around the micropores 132 in a staggered arrangement, so as to uniformly and quickly control the temperature of each micropore 132, thereby making the temperature of each micropore 132 The sample reagent 160 can reach the reaction temperature uniformly and quickly.

In this embodiment, the microfluidic element 150 includes a microfluidic channel 152, a first opening 154, a second opening 156, and a cover 158. The microfluidic element 150 is arranged on the microwell array 130. The microfluidic element 150 and the sensing chip 120 are respectively located on opposite sides of the microhole array 130. In detail, in this embodiment, after the cover 158, the encapsulant 159, and the microhole array 130 are assembled, an accommodating space (ie, the micro flow channel 152) can be defined. In other words, the micro flow channel 152 is located between the cover 158, the encapsulant 159, and the microhole array 130. The first opening 154 and the second opening 156 are openings on the cover 158, and can be used as an inlet and an outlet for injecting the sample reagent 160, respectively. In addition, since the first opening 154 and the second opening 156 are respectively connected to the micro flow channel 152, and the micro flow channel 152 is connected to each micro hole 132 of the micro hole array 130, the sample reagent injected from the first opening 154 160 can directly flow into the micro flow channel 152, and then the sample reagent 160 can be evenly distributed in the micro wells 132 along the micro flow channel 152.

In this embodiment, the nucleic acid analysis device 10 may further include a filter 170. The filter 170 is disposed on the sensing chip 120, and the filter 170 is located between the microhole array 130 and the sensing chip 120. Since the image sensor 122 of the sensor chip 120 can receive any light signals from the microholes 132, the filter 170 can be configured to filter out the light wavebands (such as excitation light) that are not to be sensed. And the specific light waveband of the side to be detected passes through the filter 170 and is sensed by the image sensor 122. Therefore, in this embodiment, the setting of the filter 170 can be used to improve the S/N ratio. In some embodiments, if it is desired to detect all the optical signals from the micro-holes 132 without other noise interference, the filter 170 may not be required. In some embodiments, the filter 170 may include an absorption filter 172 and an interference filter 174. The absorption filter 172 is, for example, a lens with a filter effect, and has the characteristics of an absorption spectrum. The interference filter 174 can produce an interference effect on the light, so as to pass the light of the wavelength range to be detected.

In this embodiment, the nucleic acid analysis device 10 may further include a wire 180. The wire 180 is configured on the circuit board 110. The wires 180 can be used to electrically connect the bonding pads 124 of the sensing chip 120 and the bonding pads 112 of the circuit board 110, so as to output signals detected by the sensing chip 120 to external electronic components through the bonding pads 112 of the circuit board 110 .

In short, in the nucleic acid analysis device 10 of this embodiment, the microfluidic element 150 can be used to evenly dispense the sample reagent 160 into the micropores 132 of the micropore array 130, so that there are differences in the micropores 132. There will be more than one nucleic acid fragment 162. By arranging the sensor chip 120 corresponding to each micro-hole 132, the detection sensitivity of the sample reagent 160 can be improved, and the detection time can be reduced.

FIG. 3 shows a flowchart of a nucleic acid quantification method according to an embodiment of the present invention. Please refer to FIG. 1A, FIG. 1B and FIG. 3 at the same time. The nucleic acid quantification method of this embodiment can be used to quantify the number of nucleic acid fragments 162 in the sample reagent 160. In the nucleic acid quantification method of this embodiment, first, step S210 is performed to provide the nucleic acid analysis device 10. The nucleic acid analysis device 10 includes a circuit board 110, a sensing wafer 120, a microwell array 130, a temperature control element 140 and a microfluidic element 150.

Next, step S220 is performed, and the sample reagent 160 and the reaction reagent 164 are equally divided into the microwells 132 of the microwell array 130, respectively. In detail, in this embodiment, first, a uniformly mixed sample reagent 160 (or a uniformly mixed reaction reagent 164) is injected into the first opening 154, so that the sample reagent 160 (or reaction reagent 164) is removed from the first opening 154. The opening 154 flows into the micro channel 152 communicating with the first opening 154. Next, the sample reagent 160 (or the reaction reagent 164) is flowed into each of the micropores 132 communicating with the micro flow channel 152 along the micro flow channel 152, so that the sample reagent 160 (or the reaction reagent 164) can be evenly distributed to Inside each micropore 132. In some embodiments, after the sample reagent 160 and the reaction reagent 164 are dispensed into the micropores 132, mineral oil can be added to the first opening 154 so that the mineral oil covers the micropores in the microchannel 152 The array 130 avoids the mutual interference and evaporation of reagents between the microwells 132. In this embodiment, the reaction reagent 164 includes a fluorescent label 166, a polymerase, a primer, a buffer, and the like. The fluorescent label 166 includes a fluorescent substance. In this embodiment, the fluorescent label 166 can be adhered to the adhesive portion of the nucleic acid fragment 162, and the fluorescent label 166 does not fluoresce. Only when the newly synthesized DNA is copied to the adhesion site, the fluorescent substance in the fluorescent marker 166 is released, and the fluorescent substance can be excited to emit a fluorescent signal.

Then, step S230 is performed to perform polymerase chain reaction (PCR) on the sample reagent 160 a predetermined number of times, so that the fluorescent label 166 is bonded to the nucleic acid fragment 162 and the fluorescent substance is released. In detail, in this embodiment, the temperature control element 140 can be used to control the temperature of the micropores 132. For example, the heating element 142 is used to heat the micropores 132 so that the temperature of the micropores 132 is between 45°C and 95°C. Cycle time, or maintain the temperature of the microwell 132 at a fixed temperature (for example, 60° C., but not limited to this) to perform the polymerase chain reaction. In addition, the temperature control element 140 may also include a cooling chip (not shown) to achieve a rapid cooling effect. In this embodiment, the predetermined number of times is, for example, n times, where n is an integer, n is greater than or equal to 1, and n is less than 32. In this example, when the first polymerase chain reaction is performed, the fluorescent substance can be released; and every time the polymerase chain reaction is increased, the released amount can be increased in multiples. Fluorescent substance.

Then, step S240 is performed to detect the fluorescent signal intensity of the fluorescent substance in each micro-hole 132. In detail, in this embodiment, after each polymerase chain reaction, one image sensor 122 (or multiple image sensors 122) corresponding to each microhole 132 can be used to independently The intensity of the fluorescent signal of the fluorescent substance in the microhole 132 is read. In other words, the image sensor 122 corresponding to each micro-hole 132 can independently detect the fluorescent signal intensity of the fluorescent substance in the micro-hole 132. In addition, in some embodiments, when there are more than two image sensors 122 corresponding to each micro-hole 132, each image sensor 122 corresponding to the micro-hole 132 can also independently detect different Fluorescent signal.

Then, step S250 is performed to determine the number of nucleic acid fragments 162 in each microwell 132 according to the intensity of the detected fluorescent signal. In detail, in this embodiment, after performing a predetermined number of polymerase chain reactions, it should be possible to detect fluorescent signals of different intensities in different micropores 132, and the fluorescent signals of different intensities have multiples of relationship. Then, when sorting the intensity of the detected fluorescent signals from low to high, they can be classified as 1, 2, 3, or 4 times the fluorescent signal intensities. In addition, because the more nucleic acid fragments 162 in the microwells 132, the higher the intensity of the fluorescent signal obtained after the polymerase chain reaction. Therefore, the number of nucleic acid fragments 162 in the microwell 132 with the lowest fluorescence signal intensity (that is, 1 times the fluorescence signal intensity) is represented as one, and the higher the sequence is (that is, 2 times, 3 times, 4 times) Fluorescence signal intensity), sequentially increase the number of nucleic acid fragments 162 in the corresponding microwells 132 by one. For example, there should be 1 nucleic acid fragment 162 in the microwell 132 where the fluorescence signal intensity of 1 time is detected; there should be 2 nucleic acid fragments in the microwell 132 where the fluorescence signal intensity is 2 times the intensity of detection. 162; There should be 3 nucleic acid fragments 162 in the microwell 132 where the fluorescence signal intensity of 3 times is detected; there should be 4 nucleic acid fragments 162 in the microwell 132 where the fluorescence signal intensity of 4 times is detected; However, there should be 0 nucleic acid fragments 162 in the microwell 132 where the intensity of the fluorescent signal cannot be detected. In other words, in this embodiment, the number of nucleic acid fragments 162 in each micropore 132 can be determined according to the relative multiple relationship of the fluorescence signal intensity between different micropores 132.

Finally, step S260 is performed to add the number of nucleic acid fragments 162 in each microwell 132 to obtain the number of nucleic acid fragments 162 in the sample reagent 160. At this time, the number of nucleic acid fragments 162 in the sample reagent 160 has been quantified, and the number should be the absolute number of nucleic acid fragments 162 in the sample reagent 160.

In short, in the nucleic acid quantification method of this embodiment, the number of nucleic acid fragments 162 in each micropore 132 can be determined according to the relative multiple relationship of the fluorescence signal intensity between different micropores 132, so this embodiment The nucleic acid quantification method provided in the example has the effect of absolute quantification.

[ Experimental example 1]

Figure 4 illustrates the distribution of nucleic acid fragments in the microwell array in an experimental example of the present invention. For clarity of the drawings and convenience of description, FIG. 4 omits several elements of the nucleic acid analysis device 10a.

Referring to FIG. 4, the nucleic acid analysis device 10a of this embodiment is similar to the nucleic acid analysis device 10 of FIG. 1B, and the main difference between the two is that the microwells 132a in the microwell array 130a of the nucleic acid analysis device 10a are It is arranged in an 8×4 matrix, and the number of micropores 132 is 32, as shown in FIG. 4.

According to the Poisson distribution principle, when the sample reagent is evenly distributed in the micropores 132a, the distribution situation is shown in FIG. 4. For example, the number of nucleic acid fragments in each microwell 132a may be 0 to 4, but there may be 5, 6, 7, or more than 7, but only 5, 6, 7, or 7. The above probability is almost close to 0, so it is not listed.

[ Experimental example 2]

The example shows that sample reagents A, B, C, D, and E containing different numbers of nucleic acid fragments are respectively loaded into a wafer containing 20,000 microwell arrays. The micropore array has 20,000 micropores, and the total number of nucleic acid fragments of the sample reagents A, B, C, D, and E is less than the number of micropores of the micropore array. According to the Baisong distribution, the distribution of the number of nucleic acid fragments that can be filled with different concentrations of sample reagents to the microwells of the wafer is shown. The results are shown in Table 1. This table only lists the distribution of the highest probability before, that is, the number of nucleic acid fragments is 0, The distribution of 1, 2, 3, and 4, and since the probability of the number of more than 5 has been close to zero, it is not listed.

Table 1 Before packaging The number of micropores corresponding to the number of each nucleic acid fragment Sample reagent Number of micropores Total number of nucleic acid fragments λ 1 nucleic acid fragment 2 nucleic acid fragments 3 nucleic acid fragments 4 nucleic acid fragments A 20000 100 0.005 99.5 0.2 0.0 0.0 B 20000 200 0.01 198.0 1.0 0.0 0.0 C 20000 500 0.025 487.7 6.1 0.1 0.0 D 20000 1000 0.05 951.2 23.8 0.4 0.0 E 20000 2000 0.1 1809.7 90.5 3.0 0.1 *λ can be regarded as a concentration, or a mathematical expectation value, that is, the probability of a nucleic acid fragment in each microwell.

From the results in Table 1, it can be seen that without considering the distribution of 0 nucleic acid fragments, when the sample reagents A, B, C, D, and E are aliquoted, most of the microwells will only have 1 nucleic acid fragment, and there is no Or only a few micropores will have 2 or more nucleic acid fragments.

In summary, in the nucleic acid analysis device and nucleic acid quantification method of this embodiment, the nucleic acid analysis device includes a sensing chip, a microwell array, a temperature control element, and a microfluidic element. With the configuration of the microfluidic element, the sample reagents can be evenly distributed in the microwells of the microwell array. With the configuration of the sensing chip, the sensitivity of sample detection can be improved, and the detection time can be reduced. In addition, since the number of nucleic acid fragments in each microwell can be judged according to the number of polymerase chain reactions corresponding to the intensity of the fluorescent signal reaching the threshold, the nucleic acid quantification method provided in this embodiment is capable of absolute quantification. effect.

10: Nucleic acid analysis device 110: Circuit board 112: Bonding pad 120: Sensing chip 122: Image sensor 124: Bonding pad 130: Microwell Array 132: Microporous 140: Temperature control element 142: Heating element 150: Microfluidic components 152: Micro channel 154: The first opening 156: Second opening 158: Cap 159: Encapsulation colloid 160: sample reagent 162: Nucleic Acid Fragment 164: Reagents 166: Fluorescent marker 170: Filter 172: Absorption filter 174: Interference filter 180: Wire D: distance S210, S220, S230, S240, S250, S260: steps

FIG. 1A is a schematic top view of a nucleic acid analysis device according to an embodiment of the present invention. FIG. 1B is a schematic cross-sectional view of the nucleic acid analysis device of FIG. 1A along the section line A-A'. FIG. 2 is a schematic cross-sectional view of a nucleic acid analysis device according to another embodiment of the present invention. FIG. 3 shows a flowchart of a nucleic acid quantification method according to an embodiment of the present invention. Figure 4 illustrates the distribution of nucleic acid fragments in the microwell array in an experimental example of the present invention.

10: Nucleic acid analysis device 110: Circuit board 112: Bonding pad 120: Sensing chip 122: Image sensor 124: Bonding pad 130: Microwell Array 132: Microporous 140: Temperature control element 142: Heating element 150: Microfluidic components 152: Micro channel 154: The first opening 156: Second opening 158: Cap 159: Encapsulation colloid 160: sample reagent 162: Nucleic Acid Fragment 164: Reagents 166: Fluorescent marker 170: Filter 172: Absorption filter 174: Interference filter 180: Wire D: distance

Claims (10)

A nucleic acid analysis device for quantifying the quantity of a nucleic acid fragment in a sample reagent, comprising: a circuit board; a sensing chip arranged on the circuit board, including a plurality of image sensors; and a microwell array, The sensor chip is arranged on the sensor chip and includes a plurality of micro holes, wherein each of the micro holes corresponds to one or more of the image sensor; a temperature control element arranged on the circuit board; and a microfluidic element arranged The microwell array includes a microfluidic channel, wherein the microfluidic channel communicates with each of the microwells, and the microfluidic element and the sensing chip are respectively located on opposite sides of the microwell array, wherein the The number of micropores is greater than the number of nucleic acid fragments in the sample reagent. The nucleic acid analysis device described in claim 1 further includes: a filter arranged on the sensing chip and located between the microhole array and the sensing chip; and a wire arranged on the sensor chip. The circuit board is used to electrically connect the sensing chip and the circuit board. According to the nucleic acid analysis device described in claim 1, wherein the temperature control element includes a plurality of heating elements, and the heating elements surround each of the micropores of the micropore array to regulate the temperature of each of the micropores. According to the nucleic acid analysis device described in item 1 of the scope of patent application, the number of the nucleic acid fragments in each microwell is 0 to N, and N is an integer. According to the nucleic acid analysis device described in item 1 of the scope of patent application, the distance between each of the micropores and the corresponding image sensor is less than or equal to 10 micrometers. A nucleic acid quantification method for quantifying the quantity of a nucleic acid fragment in a sample reagent, comprising: providing a nucleic acid analysis device as described in item 1 of the scope of patent application; distributing the sample reagent and a reaction reagent to the microwell In each of the microwells of the array, the reaction reagent includes a fluorescent label; a predetermined number of polymerase chain reactions are performed on the sample reagent to make the fluorescent label adhere to the nucleic acid fragment and release a fluorescent label Optical substance; detecting the intensity of a fluorescent signal of the fluorescent substance in each of the microwells; and judging the number of the nucleic acid fragments in each of the microwells based on the detected intensity of the fluorescent signal; and adding up The number of the nucleic acid fragment in each microwell is used to obtain the number of the nucleic acid fragment in the sample reagent. According to the nucleic acid quantification method described in item 6 of the scope of patent application, the microfluidic element further includes an opening communicating with the microchannel, and the sample reagent and the reaction reagent are dispensed into each of the microwells of the microwell array. The steps in the well include: respectively injecting the sample reagent and the reaction reagent into the opening; and the sample reagent and the reaction reagent flow into each of the microchannels of the microfluidic element along the microchannel of the microfluidic element.孔内。 In the hole. The nucleic acid quantification method according to item 6 of the scope of patent application, wherein the step of performing the polymerase chain reaction of the predetermined number of times on the sample reagent includes: using the temperature control element to adjust the temperature of the micropores to make The temperature of the micropores is cycled between 45°C and 95°C or the temperature of the micropores is maintained at a fixed temperature to perform the polymerase chain reaction. The nucleic acid quantification method according to claim 6, wherein the step of detecting the fluorescence signal intensity of the fluorescent substance in each of the microwells includes: using one or more of the images corresponding to each of the microwells The sensor independently reads the intensity of the fluorescent signal of the fluorescent substance in the microhole. The nucleic acid quantification method according to claim 6 of the patent application, wherein the step of judging the quantity of the nucleic acid fragment in each microwell according to the intensity of the detected fluorescent signal includes: comparing the detected fluorescent signal The intensity is sorted from low to high; the number of the nucleic acid fragment in the microwell with the lowest intensity of the fluorescent signal is expressed as 1, and as the ranking is higher, the corresponding nucleic acid fragments in the microwell are sequentially made Increase the number of the nucleic acid fragments by 1; and denote the number of the nucleic acid fragments in the microwell where the intensity of the fluorescent signal cannot be detected as 0.

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