BIOASSAY DEVICE AND BIOASSAY SYSTEM COMPRISING THE SAME

A bioassay device includes a main body, a biomolecular image sensor and an electrical connection portion. The main body is formed with a sensor groove. The biomolecular image sensor is disposed in the sensor groove and includes an image sensor unit. The electrical connection portion is disposed at one side of the main body and electrically connected to the biomolecular image sensor. Such that, all of the units in bioassay are able to be disposed on the main body, and the bioassay device is a kit for bioassay.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to a bioassay device, and more particularly, to provide a bioassay device including all units used in bioassay and a bioassay system with the bioassay device with fully automated complex bioassay operation process.

2. The Prior Arts

Enzyme-Linked Immunosorbent Assay (ELISA) or Enzyme-linked immunoassay (EIA) is a specific antigen-antibody reaction test. The specific binding characteristics of the smear can detect the sample waiting to be tested, and in combination with the enzyme to carry out a color reaction, which can show the existence of a specific antigen or antibody, and can use the depth of the color for quantitative analysis, so as to achieve the purpose of detection and screening.

A biochip is a miniature device that utilizes biomaterials that can produce specific biochemical reactions with the biomolecules to be tested on a substrate, and can be quantified by a highly sensitive detection system. The biochip provides the advantages of low-cost bioanalytical testing capabilities. In molecular biology, biochips are basically miniaturized substrates that can perform hundreds or thousands of biochemical reactions simultaneously.

However, conventional ELISA is to use large and expensive equipment to receive optical or electronic signals to detect the status of biochemical molecular reactions after performing complex bioassay procedures, such as observation with a microscope and additional photography device to capture the screen for further analysis, so it takes a certain amount of time and manual operation. On the other hand, conventional biochips need to be additionally equipped with other expensive and large-scale image capture systems or equipment to detect and capture the luminescent images of the biochips after the bioassay process for subsequent analysis.

With the increasing popularity of the concept of point of care testing (POCT), that is, a personalized health test with a short analysis time and simple operation, in order to overcome the traditional biomolecular detection method, which requires large-scale equipment and complex bioassay process, it is imperative to develop more sensitive, simpler testing equipment and methods.

SUMMARY OF THE INVENTION

A primary objective of the present invention is to provide a bioassay device on which all units that will be used in bioassay are included.

Another objective of the present invention is to provide a bioassay system, which fully automates the complex bioassay operation process.

In order to achieve the aforementioned objectives, the present invention provides a bioassay device, which includes a main body, a biomolecular image sensor, and a first electrical connection portion. The main body defines a sensing groove. The biomolecular image sensor is disposed in the sensing groove and includes an image sensing unit. The first electrical connection portion is disposed at one side of the main body and is electrically connected to the biomolecular image sensor.

In a preferred embodiment, the diameter of the sensing groove tapers from the top to the bottom, and the biomolecular image sensor is disposed at the bottom of the sensing groove.

In a preferred embodiment, the bioassay device further includes a magnetic unit disposed in the main body and below the sensing groove.

In a preferred embodiment, the main body defines a reaction groove for accommodating a reactant.

In a preferred embodiment, the main body defines at least one reagent groove for accommodating a reagent.

In a preferred embodiment, the main body defines at least one first accommodating groove for accommodating a pipette tip.

In a preferred embodiment, the main body defines at least one second accommodating groove for accommodating a water absorbing unit.

In order to achieve the aforementioned objectives, the present invention provides a bioassay device, which includes a main body, a biomolecular image sensor, and a first electrical connection portion. The main body includes a cover body and a box body and defines a sensing groove, a reaction groove and at least one reagent groove, the cover body is disposed on the box body, and the sensing groove, the reaction groove and the at least one reagent groove all penetrate the top of the cover body and the top of the box body, the reaction groove is used for accommodating a reactant, and at least one reagent groove is used for accommodating a reagent. The biomolecular image sensor is disposed in the sensing groove and includes an image sensing unit. The first electrical connection portion is disposed at one side of the box body, and is electrically connected to the biomolecular image sensor.

In order to achieve the aforementioned objectives, the present invention provides a bioassay system, including a casing, a control device, a mounting seat, a robotic arm, a pipette nozzle, and a bioassay device. The control device is disposed inside the casing. The mounting seat is disposed inside the casing, and has a second electrical connection portion. The second electrical connection portion is electrically connected to the control device. The robotic arm is disposed inside the casing and is electrically connected to the control device. The pipette nozzle is disposed on the robotic arm and is electrically connected to the control device. The bioassay device includes a main body, a biomolecular image sensor and a first electrical connection portion. The main body defines a sensing groove. The biomolecular image sensor is disposed in the sensing groove and includes an image sensing unit. The first electrical connection portion is disposed at one side of the main body, and is electrically connected to the biomolecular image sensor and the second electrical connection portion.

In a preferred embodiment, the bioassay system further includes a first magnet, and the first magnet is disposed on the mounting seat.

The effect of the present invention is that the bioassay device of the present invention can dispose all the units that will be used in bioassay on the main body, so as to form a special set for bioassay.

Furthermore, the bioassay system of the present invention can fully automate the complex bioassay operation process, so as to achieve the following advantages: first, it reduces manual operation steps, avoids human interference, and can shorten the detection time; second, reduce the volume and weight of the system and reduce costs; third, improve the sensitivity to achieve the detection effect of a single molecule; fourth, expand the scope of application; fifth, avoid the waste of specimens and minimize the amount of specimens used; sixth, achieve the goal of using the same small amount of sample to perform ultra-trace detection of multiple target molecules in a single detection.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As shown inFIG.1toFIG.6, the present invention provides a bioassay system, including a casing1, a control device2, a mounting seat3, a robotic arm4, a pipette nozzle5, a bioassay device6, a light source7, a vibration device8, and a first magnet9. The outer surface of the casing1is provided with a display screen101, and the display screen101can display relevant information of the progress of the bioassay operation; the casing1is provided with a door panel102. The control device2is disposed inside the casing1. The mounting seat3is disposed inside the casing1and has a second electrical connection portion301, and the second electrical connecting portion301is electrically connected to the control device2; the inner side of the mounting seat3has a plurality of positioning grooves302. The robotic arm4is disposed inside the casing1and is electrically connected to the control device2. The pipette nozzle5is disposed on the robotic arm4and is electrically connected to the control device2. The light source7is disposed inside the casing1and is electrically connected to the control device2. The vibration device8and the first magnet9are both disposed on the mounting seat3.

As shown inFIGS.7to10, the bioassay device6includes a main body10, a biomolecular image sensor20, a first electrical connection portion30, and a magnetic unit40.

As shown inFIGS.7to10, the main body10includes a cover body110and a box body120and defines a sensing groove11, a reaction groove12, a plurality of reagent grooves131-133, and a plurality of first accommodating grooves141-148. The cover body110is disposed on the box body120, and the sensing groove11, the reaction groove12, the reagent grooves131-133, and the first accommodating grooves141-148all penetrate through the top of the cover body110and the top of the box body120. The cover body110defines the second accommodating grooves16, and the second accommodating grooves16penetrate through the top of the cover body110. The reaction groove12is used for accommodating a reactant, the reagent grooves131-133are respectively used to accommodate different reagents, and the first accommodating grooves141-148are respectively used to accommodate a plurality of pipette tips151-158and a plurality of first water absorbing units171, which are located below the pipette tips151-158, and the second accommodating grooves16are respectively used for accommodating a plurality of second water absorbing units172.

Specifically, the box body120has a first side120, a second side1202, a third side1203, and a fourth side1204. The top part of the first side1201protrudes to form a protruding platform1201A, a concave portion1201B is formed inward below the protruding platform1201A, and the surface of the concave portion1201B is further recessed to form a bonding groove1201C. The sensing groove11is close to the center of the main body10, and the diameter of the sensing groove11tapers from the top to the bottom; more specifically, the sensing groove11forms an inverted tapered portion111in the box body120, and an opening112is defined at the bottom of the inverted tapered portion111. The reaction groove12and the reagent grooves131-133penetrate through the top of the protruding platform1201A. The first accommodating grooves141-148are respectively located on both sides of the sensing groove11and are disposed in two rows, wherein one row of the first accommodating grooves141-144is close to the second side1202of the box body120, and the other row of the first accommodating grooves145-148are located close to the fourth side1204of the box body120. The second accommodating grooves16are respectively located between the sensing groove11and the reagent grooves131-133and between the sensing groove11and the reaction groove12. The second side1202and the fourth side1204of the box body120respectively have a plurality of anti-slip bars18and a plurality of positioning blocks19.

Preferably, the length of the cover body110is 100 mm, the width of the cover body110is 60 mm, the length of the box body120is 68 mm, the width of the box body120is 60 mm, and the total of the height of the cover body110and the height of the box body120is 93 mm.

Preferably, the first water-absorbing units171and the second water-absorbing units172can be made of absorbent cotton, cloth, paper, sponge or water-absorbing resin and other materials with water-absorbing capacity. However, it is not limited to thereto, and any material with water absorbing ability can be the first water absorbing units171and the second water absorbing units172of the present invention.

As shown inFIG.6andFIG.8, the biomolecular image sensor20is disposed in the opening112of the sensing groove11and includes an image sensing unit21. The structures of the two biomolecular image sensors20will be described below, however, the present invention is not limited thereto, and any type of biomolecular image sensors20can be disposed in the sensing groove11.

As shown inFIG.11, the first biomolecular image sensor20A includes the image sensing unit21, a microstructure layer22, and a readout circuit23. The image sensing unit21includes a plurality of unit pixels211disposed in an array on a substrate212. The unit pixel211generates an electron after receiving an incident light. The surface of the image sensing unit21receiving the incident light is defined as a light receiving surface. The microstructure layer22is disposed on the light receiving surface of the image sensing unit21and has a plurality of microstructures221disposed in a specific shape repeatedly, wherein each of the microstructures221corresponds to a single unit pixel211respectively. The readout circuit23is coupled to the unit pixels211, and the readout circuit23generates a voltage signal according to the electrons, which is used as a signal reading value. Each microstructure221can accommodate a carrier25carrying a biomolecule24to be tested.

Specifically, the carrier25can be a microparticle, and through the EDC/NHS reaction, the biomolecule24to be tested, such as an antibody, is linked with the microparticle by forming an amide bond; the antibody, the microparticle and the sample to be tested are then mixed, so as to grab the antigen to be tested from the sample to be tested; then the secondary antibody of the modified biotin is combined with the antigen; the biotin is combined with streptavidin to bring a plurality of horseradish peroxidase (HRP) molecule to form a complex of microparticle-antibody-antigen-antibody-biotin-streptavidin-polyHRP. The complex is the reactant of the first biomolecular image sensor20A.

Preferably, the microparticles are preferably magnetic beads of 1-3 μm, and the magnetic beads may use magnetic elements, such as iron (Fe), nickel (Ni), cobalt (Co), etc., neodymium iron boron (Nb—Fe—B) and other ferromagnetic alloys, or magnetic materials of Fe3O4, Fe2O3, FeO. Alternatively, non-magnetic materials such as Au, sepharose, polystyrene, and SiO2can also be used for the microparticles.

Preferably, only a single carrier25needs to be accommodated in a single microstructure221as much as possible, so the size and height of the microstructures221need to match the size and height of the carrier25. More specifically, the diameter of the microstructure221is preferably 1.3-1.8 times larger than the diameter of the microparticles, the depth is preferably 1.2-1.3 times larger than the diameter of the microparticles, and the aspect ratio is preferably 1-1.2 times. With 2 μm beads, the micropores are about 2.5-3 μm in diameter and about 2.5 μm deep. The distance between the micropores is about 2-3 pm. For example, if the carrier25is a microparticle with a diameter of 2 μm, and the microstructures21can be grooves with a diameter of 2.5-3 μm and a depth of 2.5 μm, and the spacing is 2-3 μm.

As shown inFIG.12, the second type of biomolecular image sensor20B includes the image sensing unit21, a plurality of detection molecules26, and a readout circuit23. The image sensing unit21includes a plurality of unit pixels211disposed in an array on a substrate. The unit pixel211generates an electron after receiving an incident light. The surface of the image sensing unit21receiving the incident light is defined as a light receiving surface. The detection molecules26are disposed on the light-receiving surface of the image sensing unit21and used to bind a biomolecule to be tested (not shown). A plurality of detection molecules261form a combination of detection molecules, and each of the combinations of detection molecules corresponds to a single unit pixel211respectively. The readout circuit23is coupled to the unit pixels211, and the readout circuit23generates a voltage signal according to the electrons, which is used as a signal reading value.

Preferably, the image sensing unit can be a CMOS image sensor manufactured by using a semiconductor process, and after the packaging is completed, its surface is oxidized to form a light-penetrating flat surface composed of SiO2. The flat surface is the protective layer at the light-receiving surface for receiving incident light. Next, a plurality of detection molecules21is directly fixed on the light-receiving surface by means of chemical modification, for example, by using oxygen atoms in SiO2on the light-receiving surface, firstly with 3-aminopropyl, APTES silane compounds for surface-modifying to have amine groups (NH2) on the surface, so as to interact with detection molecules21such as antibodies, receptor proteins, DNA, aptamers, or other chemical molecules to create bonds to directly immobilize the detection molecules21on the light-receiving surface of the image sensing unit. More specifically, the EDC/NHS reaction can be used to make the carboxyl group on the antibody as the detection molecule21bond with the amine group on the light-receiving surface, so as to be immobilized on the light-receiving surface; or, glutaraldehyde can be used to connect protein G and the amine group on the light-receiving surface. Since protein G can bind to the Fc region of most antibodies, it can flexibly change the specific antibody as detection molecule21according to different biomolecules to be tested. The antibody is mixed with the sample to be tested to capture the antigen to be tested from the sample to be tested; then the secondary antibody of the modified biotin is combined with the antigen; the biotin is combined with streptavidin to bring in multiple horseradish peroxidase (HRP) molecules; to form antibody-antigen-antibody-biotin-streptavidin-polyHRP complex. The complex is the reactant of the second biomolecular image sensor20B.

In a preferred embodiment, the biomolecular image sensor20is used for biological or chemical analysis, such as detecting the presence and/or concentration of a biomolecule24in a sample to be tested; that is, the incident light can be a light emitted by the fluorescent label, reporter molecule label, or chemiluminescent label of the biomolecule to be tested, and more specifically, the biomolecule24to be tested by the biomolecular image sensor20, in the process of a biological or chemical analysis, can react with other molecules and emit incident light such as chemiluminescence or fluorescence. Moreover, the biomolecules24to be tested can be, for example, proteins, peptides, antibodies, nucleic acids, etc. According to the present invention, the fluorescent label can be, but not limited to, FITC, HEX, FAM, TAMRA, Cy3, Cy5, quantum dot, etc., and can be used with a quencher dye.

When the biomolecule24to be tested binds to the chemiluminescent label, the reactant at least includes the biomolecule24to be tested in the sample to be tested, the reaction groove12accommodates the reactant, and the three reagent grooves131-133accommodate phosphate buffered saline (PBS), Luminol, also known as photosensitizer, and peroxide respectively.

When the biomolecule24to be tested binds to the fluorescent label, the reactant at least includes the biomolecule24to be tested in the sample to be tested, the reaction groove12accommodates the reactant, the reagent groove131accommodates reagents such as phosphate buffered saline; the remaining reagent grooves132-133are not injected with any reagents and can even be omitted.

In a preferred embodiment, the image sensing unit21may be a backside illuminated Complementary Metal-Oxide-Semiconductor (CMOS) image sensor or a front illuminated CMOS image sensor, but the invention is not limited thereto.

In a preferred embodiment, the unit pixel211includes a photoelectric conversion unit (not shown), and the photoelectric conversion unit may be a unit that generates and accumulates electrons corresponding to incident light. For example, the unit pixel211may be a photodiode, a photo transistor, a photo gate, a pinned photo diode (PPD), an avalanche photodiode (APD), single-photon avalanche diode (SPAD), photomultiplier tube (PMT), or any combination thereof.

In some embodiments, the biomolecular image sensor20includes a glass substrate (not shown) and the image sensing unit21, and the image sensing unit21is disposed on the glass substrate.

As shown inFIG.6,FIG.7,FIG.8, andFIG.10, the first electrical connection portion30is disposed in the bonding groove1201C of the first side1201of the box body120, and is electrically connected to the readout circuit23.

As shown inFIG.10, the magnetic unit40includes a support base41and a second magnet42. The support base41is disposed in the box body120and is located below the sensing groove11, and the second magnet42is disposed on the support base41and aligned with the opening112at the bottom of the reverse tapered portion111. In other words, the second magnet42is located directly below the biomolecular image sensor20.

Before starting the bioassay, first, as shown inFIG.1, the door panel102is opened. Then, the hand abuts on the anti-slip bars18to prevent the hand from slipping. Then, as shown inFIGS.2to7, the bioassay device6is moved into the casing1, and the positioning blocks19are fixed in the positioning grooves302, so that the bioassay device6is quickly positioned on the mounting seat3. At this point, the first electrical connection portion30is electrically connected to the second electrical connection portion301, and the first magnet9is located below the protruding platform1201A.

As shown inFIG.13AtoFIG.13C, the present invention utilizes chemiluminescence for the first biomolecular image sensor20A to detect biomolecules, including the following steps:

In step S100, the reactants are uniformly mixed in phosphate buffered saline. More specifically, the control device2controls the movement of the robotic arm4, and the robotic arm4controls the movement of the pipette nozzle5. Step S100further includes: the pipette nozzle5takes out the pipette tips151-154from the first accommodating grooves141-144; the pipette nozzle5uses the pipette tips151-154to suck phosphate buffered saline from the reagent groove131; the pipette nozzle5uses the pipette tips151-154to add phosphate buffered saline into the reaction groove12to form a reaction solution; the first magnet9moves to the bottom of the protruding platform1201A, and the first magnet9provides a magnetic force to adsorb the reactant onto the side wall of the reaction groove12, and the pipette tips151-154are utilized by the pipette nozzle5to draw phosphate buffered saline from the reaction groove12; the pipette tips151-154are placed by the pipette nozzle5in the first accommodating grooves141-144; the first magnet9is removed, so that the reactants fall off from the side walls of the reaction groove12.

In step S101, the reactant and phosphate buffered saline are mixed into a reaction solution and injected into the sensing groove11. Step S101further includes: the pipette nozzle5takes out the pipette tip155from the first accommodating groove145and uses the pipette tip155to suck phosphate buffered saline from the reagent groove131; the phosphate buffered saline is injected into the reaction groove12, and the reactants are suspended in phosphate buffered saline to form a reaction solution; the pipette nozzle5uses the pipette tip155to suck the reaction solution and inject the reaction solution into the sensing groove11, and the reactants are easy to follow the sidewall of the inverted tapered portion111to flow onto the surface of the biomolecular image sensor20.

In step S102, the reactants fall into the microstructures221. More specifically, the means for the carrier25to fall into the microstructures221include the following two: (1) the control device2controls the vibration device8to generate vibration, and the vibration provided by the vibration device8is transmitted to the bioassay device6through the mounting seat3, and the carrier25is affected by the vibration and falls into the microstructures221; (2) the second magnet42is located directly under the sensing groove11, the second magnet42provides a magnetic force, and the carrier25is subjected to the magnetic force and falls into the microstructures221under the influence. In other words, if the bioassay system is equipped with the vibration device8, the bioassay device6may not be equipped with the magnetic unit40; if the bioassay system is not equipped with the vibration device8, the bioassay device must be equipped with the magnetic unit40; only one of the two options is needed so as to save costs.

Step S103, removing the phosphate buffered saline in the sensing groove11. Step S103further includes: the pipette nozzle5uses the pipette tip155to suck phosphate buffered saline from the sensing groove11;

Step S104, preparing a chemiluminescent solution (ECL solution). Step S104further includes: the pipette nozzle5takes out the pipette tip156from the first accommodating groove146; the pipette nozzle5uses the pipette tip156to suck luminol from the reagent groove132; luminol is injected into reagent groove133and forms a chemiluminescent solution with peroxide. It is worth noting that the pipette nozzle5can also be moved to the reagent groove133to absorb the peroxide, and then the peroxide is injected into the reagent groove132to form a chemiluminescent solution with luminol.

In step S105, the chemiluminescent solution is injected into the sensing groove11. Step S105further includes: the pipette nozzle5uses the pipette tip156to suck the chemiluminescent solution; the pipette nozzle5uses the pipette tip156to inject the chemiluminescent solution into the sensing groove11, and the chemiluminescent solution easily follows the sidewall of the inverted tapered portion111to flow onto the surface of the biomolecular image sensor20. The chemiluminescent solution can make the chemiluminescent label of the biomolecule24to be tested emit light.

In step S106, the chemiluminescent solution in the sensing groove11is removed. Step S106further includes: the pipette nozzle5draws the chemiluminescent solution from the sensing groove11by using the pipette tip156;

In step S107, each of the unit pixels211respectively detects an incident light in a single microstructure221, the incident light including the light emitted by the chemiluminescent label of the biomolecule24to be tested.

In step S108, the incident light received by each of the unit pixels211is transmitted through the unit pixels211to generate an electron.

In step S109, a voltage signal is generated according to the electrons through the readout circuit23, and the voltage signal is transmitted to the control device2through the first electrical connection portion30and the second electrical connection portion301.

In step S110, the control device2analyzes whether the biomolecule24to be tested exists or not according to the voltage signal.

In step S111, if the biomolecule24to be tested exists, the control device2further compares it with the standard concentration curve to obtain the concentration of the biomolecule24to be tested.

In step S112, the control device2analyzes whether the signal reading value exceeds the threshold value according to the voltage signal.

In step S113, the control device2defines the unit pixel whose measured signal reading value exceeds the threshold value as1.

In step S114, the control device2defines the unit pixel whose measured signal reading value does not exceed the threshold value as0.

In step S115, the control device2calculates the total number of unit pixels as1and compares it with the standard concentration curve to obtain the concentration of the biomolecule24to be tested.

In some embodiments, the bioassay system can be not configured with the vibration device8and the first magnet9, the bioassay device may not be configured with the magnetic unit40, and the reagent groove contains a surfactant. Step S102in these embodiments includes: the pipette nozzle5takes out the pipette tip157from the first accommodating groove147; the pipette nozzle5uses the pipette tip157to suck the surfactant from the reagent groove; the surfactant is injected into the sensing groove11, and the surfactant easily flows along the sidewall of the inverted tapered portion111onto the surface of the biomolecular image sensor20; the pipette nozzle5places the pipette tip157into the first accommodating groove147. Thereby, the surfactant can significantly reduce the surface tension of the reaction solution, so that the carrier25can fall into the microstructures221smoothly. Compared with the vibration force or the magnetic force, reducing the surface tension of the reaction solution can increase the ratio of the carrier25falling into the microstructures221. Furthermore, the bioassay system omits the vibrating device8and the first magnet9and the bioassay device6omits the magnetic unit40, so that the manufacturing cost can be further reduced, and the volume and weight can also be reduced.

It is worth noting that, during the movement of the pipette nozzle5, the pipette tips151-158can first contact the second water absorbing units172in the second accommodating grooves16, and the second water absorbing units172in the second accommodating grooves16can absorb the liquid attached to the outer walls or tips of the pipette tips151-158, so as to prevent the liquid from dripping into the reagent grooves131-133and contaminating the reagents. After the pipette tips151-158return to the first accommodating grooves141-148, the first water absorbing units171in the first accommodating grooves141-148can absorb the pipette tips151-158to prevent the residual liquid from volatilizing into the casing1and contaminating the reagent.

As shown inFIG.13AtoFIG.13D, the difference between the method of the present invention for detecting biomolecules for the first biomolecular image sensor20A using photoluminescence and the aforementioned method is that step S116replaces S104-S106, and step S107is slightly different. In other words, step S116is further included between step S103and step S107, wherein the control device2controls the light source7to aim at the inside of the sensing groove11to emit light, and the light excites the fluorescent label to emit light. Preferably, the light source7is a laser diode, the light emitted by the laser diode is a laser light, and the fluorescent label is excited by the laser light to emit light. In step S107, the incident light includes the light emitted by the fluorescent label of the biomolecule24to be tested.

The present invention utilizes chemiluminescence or photoluminescence for the second biomolecular image sensor20B to detect biomolecules. The difference from the foregoing method is that steps S100and S102are omitted.

In summary, the bioassay device of the present invention can set all the units that will be used in bioassay on the main body, so as to form a bioassay-specific set.

Furthermore, the bioassay system of the present invention can fully automate the complex bioassay operation process, so as to achieve the following advantages: first, it reduces manual operation steps, avoids human interference, and can shorten the detection time; second, reduce the volume and weight of the system and reduce costs; third, improve the sensitivity to achieve the detection effect of a single molecule; fourth, expand the scope of application; fifth, avoid the waste of specimens and minimize the amount of specimens used; sixth, achieve the goal of using the same small amount of sample to perform ultra-trace detection of multiple target molecules in a single detection.