CN212610526U - Device for storing micro-droplets and reading system - Google Patents

Abstract

The utility model provides a device for storing micro-droplets and a reading system, and discloses a micro-droplet storage device, which comprises a plurality of cavities for accommodating micro-droplets, wherein each cavity accommodates one droplet; the storage device comprises a substrate and a cover plate, wherein the substrate is provided with a plurality of micro-liquid-drop cavities, each cavity can contain a liquid drop, and the cover plate seals the cavities; in some approaches, each cavity is connected by a microchannel. Such a device may be arranged on a rotating carrier to enable reading of the detection result.

Description

Device for storing micro-droplets and reading system

Technical Field

The present invention relates to a device for storing micro-droplets and a reading system, and more particularly, to a system and method for reading micro-droplet reaction results.

Background

"fluid biopsy" is the fastest growing Technology in the field of precision medicine, and is rated by MIT Technology Review as a ten-year-old breakthrough Technology in 2015. The "liquid biopsy" is a subject to be examined for cell-free dna (cfdna). The detection of cfDNA mutation has profound significance in the aspects of early cancer diagnosis, cancer staging, drug target, real-time monitoring of curative effect and the like, and is helpful for accurate cancer treatment of patients. In addition, non-invasive prenatal screening is another emerging screening method to diagnose whether an infant has a genetic problem by analyzing the circulating ultra-low amount of cell-free fetal DNA in the maternal blood stream. Therefore, being able to sensitively detect and absolutely quantify cfDNA is of great importance for the diagnosis, prognosis and treatment of cancer and genetic diseases. However, because the cfDNA content is very low, the traditional real-time fluorescent PCR technology cannot identify the ultra-low frequency mutation in the high wild-type background due to the self-sensitivity limitation, and is not suitable for cfDNA detection.

In recent years, the advent of digital PCR technology is expected to successfully address the challenges facing cfDNA detection. The work flow of the digital PCR technology is as follows: the diluted sample is first distributed to a large number of microreactors such that each microreactor contains one or zero target DNA molecules for single-molecule template PCR amplification. The copy number of the target DNA molecule is obtained by counting the number of positive reaction units (containing the target DNA) and negative reaction units (not containing the target DNA) and by statistical methods. Because the reaction units are independent from each other, the target genes with low abundance are not interfered by other DNA of the sample in the independent reaction units. Therefore, compared with real-time fluorescent PCR, the digital PCR technology has more excellent sensitivity, specificity and accuracy, and is a highly effective method for detecting low-abundance DNA. The digital PCR technology can successfully quantify the type and the amount of cfDNA existing in the plasma of a patient, and provides possibility for early diagnosis of cancer, drug response monitoring and prognosis decision.

Current digital PCR systems can be divided into two categories, including microcavity and droplet-based digital PCR systems. Microcavity-based digital PCR systems (e.g., Fluidigm, QuantStudio) integrate control valves or hydrophilic and hydrophobic modification of the surface to enable sample loading into pre-fabricated reaction wells on the chip. However, microwell structures inevitably suffer from several drawbacks, including complex design and manufacturing processes, high sample solution flow and seal failure rates on the chip, and relatively high cost (e.g., Fluidigm chips $ 500 per chip). Droplet-based digital PCR systems (e.g., Bio-Rad or RainDance) utilize droplet microfluidics to generate water-in-oil emulsions for sample discretization. These highly uniform droplets can serve as isolated reactors, well suited for high-throughput chemical and biological analysis. Both Bio-Rad and RainDance systems use microfluidic chips to form droplets at high frequency (e.g., several kHz), then collect the droplets in a PCR tube for PCR amplification, then aspirate and flow the droplet emulsion in a microchannel, and inject a spacer fluid to separate and align the droplets, identify and count the microchannels. The identification is performed by fluorescence reading or photographing, but the result reading of droplets flowing in a microchannel (flow cytometer) requires precise instrument and equipment, and each droplet needs to be read for result testing and then the result of the test is recorded. The droplets are generally read by a flow method using a photomultiplier, and although the droplets are sampled at a kHz rate, the whole reading and analysis still requires several hours, and there is a problem that the droplet reading time is long. Is not favorable for the wide application of the digital PCR.

SUMMERY OF THE UTILITY MODEL

In order to solve these conventional problems, the present invention provides an apparatus for storing micro-droplets and a reading system, and discloses a novel micro-droplet structure that can perform reading of a droplet reaction result in a photographing manner. Also, results for multiple droplets, e.g., 10, 20, 30, 50, or 100 test results, can be read at a time.

An aspect of the utility model is to provide a storage device of well micro-droplet, the device is including the cavity that holds the micro-droplet, and the cavity holds and to hold a plurality of liquid drops. The storage device comprises a base plate and a cover plate, wherein a plurality of micro-liquid drop cavities are formed in the base plate, each cavity can contain a plurality of liquid drops, and the cover plate seals the cavities. In some forms, the device further comprises a chamber for storing excess oily substance, the chamber being in fluid communication with the chamber for storing the droplets. In some embodiments, the cavity for storing the oily substance is in communication with the cavity for storing the liquid droplet via a microchannel. In some embodiments, the cavity for storing the droplets is a thin layer structure, so that the droplets can be distributed in a single layer in the cavity.

The cavity is a single-layer cavity structure, and a micropore structure for storing single liquid drops is not included in the cavity. In fact, the cavity is a microcavity, and a plurality of droplets can be stored in a single-layer structure in the cavity, and the droplets are spread flat in the cavity.

When the reaction of the liquid drop is completed, the reaction result of the liquid drop can be obtained by photographing the device, for example, if fluorescence is generated, a fluorescent bright spot (positive) is formed in the photographed image, and a negative result is formed if no bright spot appears.

In some embodiments, the storage device is in the form of a curved surface, or the cavity itself is in the form of a curved surface, and the storage device with the curved surface is arranged on a rotating carrier, and along with the rotation of the carrier, the storage device with the curved surface is driven to rotate, so as to read the droplet result on the storage device, and the reading mode is a photographing mode, for example, a CCD camera is used for photographing.

In some embodiments, the cavity is a circular arc, and the carrier is circular. The storage device may be arranged on a circular carrier and the result of the plurality of droplets in the storage chamber is read as the carrier rotates.

In some embodiments, the carrier is a disk structure and the cavity of the storage device is shaped to match the curvature of the disk.

In some embodiments, the width of the cavity is consistent with or smaller than the imaging area of the photograph. Therefore, the result of all the droplets in the imaging area can be shot by each shooting, then the repeated shooting sites can be fitted after all the shots are shot, the result of all the droplets in the whole storage area is formed, then statistical analysis is carried out, and finally the number of the analyzed substances in a certain sample is given.

The samples of these can be any samples, such as samples of blood, saliva, sweat, spinal fluid.

The analyte may be any analyte, such as an antibody, an antigen, or a nucleic acid.

The storage device herein may be a place where the droplets are stored or a place where each droplet reacts. When a heating reaction is required, a heating module can be arranged on the carrier, so that the cavity for storing the liquid drops is directly heated on the carrier to carry out the necessary reaction, such as an amplification reaction and the like.

The labels may be fluorescent labels or any labels that can be identified so that the results of each reaction of the tiled microdroplets can be read by taking a picture. In some approaches, a plurality of microdroplet storage devices are carried on a carrier.

In order to facilitate the storage and reading of the storage device on the carrier, the device comprises an upper layer and a lower layer, wherein the lower layer is a base layer, a cavity structure for storing liquid drops is arranged on the base layer, and then a cover is covered to seal the cavity, so that a liquid drop storage cavity is formed. The cover is typically transparent or light transmissive. In some embodiments, the substrate layer is configured with structures that mate with the micro-droplet storage device to hold the droplet storage device in place.

In some embodiments, the device is a disk structure comprising a plurality of curved cavities, each cavity being capable of storing a plurality of droplets, and no micro-porous structure in the cavity for storing droplets. The cavity is a planar structure, and a layer of liquid drops is laid in the planar thin-layer cavity and can be photographed by a photographic device.

In some modes, an identifiable mark is arranged on the carrier or the micro-droplet storage device, the mark is used for starting reading of the device, for example, a light-transmitting hole or some color label, when the micro-droplet storage device is installed on the carrier, the position identification is carried out through the identification module, so that the initial position of the micro-droplet is judged, and then the shooting or reading of the reaction result of the droplet is carried out from the initial position. In fact, in some alternative ways, the initial position of the motor is the same and is restored after each test. The chip is provided with a clamping groove, and the chip is placed in the same position. When the camera is used for taking a picture, the position of the chip can be known only by knowing the angle of rotation of the motor. This is achieved by means of control and programming.

In some embodiments, the device further comprises a cavity for storing the oily substance, wherein the cavity is in fluid communication with the cavity for storing the liquid droplets. In some embodiments, the liquid drop storage device further comprises a micro-hole, wherein the micro-hole is communicated with the cavity for storing the liquid drop. After the liquid drops enter the cavity for storing the liquid drops from the micropores, the liquid drops enter the cavity for storing the oil phase through the channel, and the rest liquid drops are left in the cavity for storing the oil liquid drops. Thus, the cavity storing the droplets is located upstream of the cavity storing the oil phase.

In another aspect, the present invention also provides a method for reading a reaction result stored in a micro-droplet, the method comprising:

providing a storage device for storing droplets, the device comprising a cavity for storing droplets, the droplets being tiled in the cavity,

moving the storage device;

and the reading equipment is used for photographing the cavity of the storage equipment to acquire an image.

In a preferred embodiment, the movement of the storage device is performed in a rotating or swiveling manner.

In a preferred manner, the storage device is arranged on a carrier, the movement of which drives the movement of the storage device.

In a preferred form, the storage means is in the form of a disc and the cavity is also in the form of a curved surface. In some embodiments, the disk is rotated to rotate the storage device.

In some embodiments, the reading device reads in the form of a photomicrograph. In some modes, the mode of photographing is performed in a CCD mode.

In some approaches, the width of the droplet storage device is the same or the same as the area from which the photograph is read.

Advantageous effects

The utility model discloses a mode of disc carries out reading of little liquid drop chip result, and is different with the mode of traditional loss cell instrument. The efficiency of reading results is increased and in addition the cost is reduced.

Drawings

Fig. 1 is a schematic structural diagram of the reading system of the present invention.

Fig. 2 is a top view of a structure of a storage device on a carrier.

Fig. 3 is a schematic view of different forms of storage devices on a carrier.

Fig. 4 is a schematic perspective view of a carrier, in which fig. 4A is a pattern of a disk structure including 4 structures for storing liquid droplets in one embodiment. Fig. 4B is a schematic perspective view of the back surface of the disk structure, fig. 4C is a schematic perspective view of the base body, and fig. 4D is a schematic sectional view of the first liquid and oil phase-storing chamber.

Detailed Description

The structures referred to by the invention or the technical terms used therein are further described below, if not otherwise indicated, in accordance with the common general term of the art.

In some embodiments of the present invention, as shown in fig. 1, a system for reading microdroplet chips is provided, which includes a rotating base 30, a rotating plate or carrier 10 (also referred to herein as a storage device) disposed on the base, and a microdroplet chip disposed on the carrier for reading the result. The rotating base is connected with the stepping motor, so that the rotation of the stepping motor drives the rotating base to rotate, and the connection relation can be realized by the known prior technical scheme.

The carrier 10 disposed on the rotating base 30 can be disposed on the base in a fixed manner or a detachable manner. Typically the carrier or storage device is a unitary structure that is freely removable from the base. In some modes, the carrier comprises a base body and a cover, and the cavity structure is arranged on the base body. The chamber structure is in the form of a chamber 105 shown in fig. 2, and includes a chamber in which a plurality of micro-droplets are stored, the droplets being in a tiled chamber. When the detection is needed, the device with the disc structure is placed on the base, and the stepping motor is started to enable the optical camera to shoot the test result of part of micro-droplets in the cavity. Therefore, a camera device 20, such as a CCD camera, is positioned directly above the storage device, directly opposite the underlying device 10. Of course, in some feasible ways, the carrier and the camera are arranged in the same vertical direction, if not in the vertical direction, at least in one straight direction, for example, the carrier has an angle, for example, an angle with the horizontal line, and the camera is parallel to the carrier and also at the same angle with the horizontal.

In some embodiments, the carrier is detachable from the base 30, and the test results are read when the micro droplet chip is placed on the base. In some embodiments, the shape of the cavity of the core of the microdroplet is in the form of a curved surface, such as shown in fig. 2, and the microdroplet chip 102 is in the form of a curved surface, including spaces with stored droplets, which constitute curved surfaces. In some embodiments, the micro-droplet chip has opposite chair corners 105,106, where small holes may be provided, in communication with the external atmosphere, for the evacuation of excess air. Of course, some holes may be provided for the input of droplets. It is also convenient to provide a groove structure or bayonet structure 111 on the carrier or device, and to design the same similar structure on the base, so as to allow the disc 10 to be secured to the base 30. In some embodiments, when the carrier 10 is in the form of a disk, the curved surface of the cavity for storing the micro-droplets overlaps with the center of the disk or is concentric with the center of the disk. It can also be understood that the arc of the curved surface of the cavity of the droplet chip is the same as the arc of the disk, or one end of the arc of the disk overlaps the curved surface of the droplet chip. In some embodiments, a through hole 113 is also formed in the middle of the disk, and is also used to fix the disk chip to the base 30. The shape of the through hole is arbitrary, and the through hole can be assembled or separated through the structure which is matched with the base conveniently.

In some embodiments, the width of the area imaged or photographed by the camera is the same as the width of the cavity in the micro-droplet chip, or the width of the cavity of the micro-droplet chip is smaller than the area to be imaged, where the cavity is used to accommodate the micro-droplets. Therefore, the area or the area of the micro-droplet shot each time is covered by all the imaging areas in the transverse direction or is positioned in the imaging area, so that as long as the disk chip on the base is driven to rotate along with the rotation of the base 30, the rotating speed and the shooting frequency are set, and each time the disk chip rotates by a distance which is equal to or less than the imaging area, the micro-droplet images in different imaging areas are shot longitudinally along with the rotation. Thus, with rotation, the results of micro-droplets in different areas can be continuously photographed. Since each microdroplet represents a test result, 10,100,200,300,500 or more drops can be obtained simultaneously in a region. This is highly efficient in obtaining multiple test results at different times. Different from the detection method of the traditional flow cytometry instrument, the flow cytometry can only detect a single liquid drop at a time, so that the efficiency is low and the cost is high, and the utility model discloses can accomplish reading of a plurality of liquid drop results in very short time.

In some embodiments, the shape of the cavity of the droplet storage device is a curved shape, such as the different curved shape shown in fig. 3. For example, a four-part curved pattern 102, a two-half-circle curved pattern 103, or a pattern similar to a full circular ring 104 may be possible. The rotating base is circular, and may be in other shapes, such as square, oval, or diamond, but all can rotate to rotate the micro-droplet storage device.

In some aspects, the disk-shaped chip includes a plurality of cavity regions. For example, as shown in fig. 4, the disc-shaped chip comprises a two-layer structure, a base 110 and a cover 109, wherein a cavity structure 114 for storing micro-droplets is disposed on the base, and the cavity is a planar recessed region. The depth of the recessed region is comparable to the diameter of the droplet or slightly greater than the depth of the droplet size. For example, the depth of the cavity is typically 80um, the length can be any length, and the diameter of the droplets is typically 70-80 um. Of course, the depth may be any depth, and may vary with the diameter of the droplet, but it is preferred that the droplet is spread in a monolayer within the cavity. The cavity does not have a plurality of wells, each of which holds one droplet, and this is a conventional chip structure, i.e. a conventional structure with wells, where each well is used to store one droplet. The present invention finds that such a microporous structure is not required, but only a cavity structure, which is a planar structure, resembling the cavities of some thin layers. When the chamber is manufactured, the cover 109 is closed, thereby forming a completely sealed chamber. In some modes, the thin-layer cavity is only used for accommodating a layer of liquid drops, and the liquid drops are flatly laid in the cavity, so that the test result can be read in a photographing mode. In some embodiments, the device further comprises another chamber, which is substantially parallel to the droplet storage chamber 114, but has a width less than or equal to the width of the droplet storage chamber, and is primarily used to store excess oil. The droplet generally comprises a sample liquid and necessary reagents, and further comprises a layer of oily substance outside the droplet, so that the droplets can exist independently, and when the liquid is formed or stored in the cavity 114, the excessive oily substance can be discharged or stored in the cavity 115.

The droplets are generally mixed with an oil phase that surrounds the droplets, and generally, the droplets float on the surface of the oil phase, and the oil phase is located under the droplets, and when the droplets are driven into the micropores, the oil phase first enters the cavity 114, and then flows into the cavity 115 through the channel 117 along with the injection of the oil phase. Continuing with the injection of the oil phase, the droplets also enter cavity 114 and the oil phase continues to flow into cavity 115. The volume of cavity 115 is comparable to the volume of the oil phase, so that the oil phase fills 115 and the droplets are spread flat in cavity 114. Or alternatively. It is also to be construed that the droplet is driven into the cavity from the 116 hole by the pipette tip. Because the density of the oil phase is higher than that of the water phase in the oil-water system, the liquid drops float above and the oil phase sinks at the bottom, when the liquid drops are injected into the cavity, the redundant oil flows into the cavity firstly, passes through the cavity 114 and finally flows into the cavity 115. The volume of the cavity 115 is approximately equal to the volume of the excess oil. The floating droplet is just left in the chamber 114 when it is hit. Certainly, in order to realize communication, the cavity for containing the oil phase is provided with an air hole communicated with the outside, so that air is conveniently discharged, and the liquid flow is realized.

Therefore, the chamber for containing or storing the liquid droplets is located upstream of the chamber for containing or storing the oil phase, and the chamber for containing or storing the oil phase is located downstream of the chamber for containing or storing the liquid droplets, divided in the direction of flow of the liquid. The liquid inlet orifice 116 is located upstream of the chamber for receiving or storing the liquid droplets.

It will be appreciated that such a dual chamber chip for storing droplets may be one or more, for example in a different pattern on a disc as shown in figure 3, such that the reaction results of the droplets are read by rotating the disc.

It will also be appreciated that these droplets are prepared by making a liquid structure, and that the droplets can be prepared using known techniques. Of course, the prepared droplets can be put on the chip of the present invention for reaction, for example, amplification of nucleic acid, and the result can be read by photographing at the same time as the amplification or after the completion of the amplification. Of course, these droplets may be reacted and then transferred to the chip structure of the present invention, and the result may be read.

The above description is merely illustrative of specific embodiments of the present invention and should not be construed as limiting the scope of the invention.

Claims (15)

1. A device for storing micro-droplets, which comprises a cavity for storing the micro-droplets, and is characterized in that the cavity in the device is of a thin-layer structure, so that the micro-droplets are distributed in the cavity in a single layer, and the shape of the cavity is a curved surface.

2. The device of claim 1, wherein the device is in the shape of a ring.

3. The device of claim 2, wherein the device is shaped as a ring.

4. The device of claim 1, wherein the width of the chamber is equal to or less than the width of the imaged area to be photographed.

5. The device of claim 1, wherein the chamber of the device is in the shape of an arc of a circle.

6. A device for storing microdroplets as claimed in claim 1 wherein the height of the cavity is greater than or equal to the diameter of the stored droplet.

7. A device for storing micro-droplets according to claim 1, wherein the chamber does not comprise a micro-porous structure for storing droplets.

8. The device of claim 1, further comprising a chamber for storing an oily substance, wherein the chamber for storing an oily substance is in fluid communication with the chamber for storing droplets.

9. The device of claim 8, wherein the droplet storage chamber is located upstream of the oil phase storage chamber.

10. A device for storing microdroplets as claimed in claim 9 wherein the height of the droplet storage chamber is between 5-300 um.

11. Reading system comprising a device for taking pictures, a rotating carrier, characterized in that the carrier is provided with means for storing microdroplets as claimed in one of claims 1 to 5.

12. A reading system according to claim 11, wherein the rotatable carrier is connected to a stepper motor, the rotation of the carrier being effected by the stepper motor.

13. A reading system according to claim 12, wherein said carrier is disc shaped.

14. A reading system according to claim 11, wherein said means for taking pictures is a CCD camera.

15. A reading system according to claim 11, wherein said means for taking a picture is located above the means for storing microdroplets.

Images