Droplet digital PCR chip

Abstract

The present invention discloses a droplet digital PCR chip. The droplet digital PCR chip includes at least one chip unit, each chip unit includes a chip body formed by bonding a top piece and a bottom piece, the chip body is internally provided with an inlet chamber, a droplet storage chamber, and an injection hole. The injection hole connects with the inlet chamber, a plurality of droplet generating channels are disposed between the inlet chamber and the droplet storage chamber, a height of the droplet generating channel is smaller than a height of the droplet storage chamber, an injection fluid is injected into the inlet chamber through the injection hole, and the injection fluid is emulsified and enters the droplet storage chamber at a junction of the droplet generating channels and the droplet storage chamber.

Claims

1. A droplet digital PCR chip, comprising: at least one chip unit, wherein each chip unit comprises a chip body formed by bonding a top piece and a bottom piece and the chip body is internally provided with an inlet chamber, a droplet storage chamber, and an injection hole, wherein the injection hole connects with the inlet chamber, a plurality of droplet generating channels are disposed between the inlet chamber and the droplet storage chamber, a height of each of the droplet generating channels is smaller than a height of the droplet storage chamber, an injection fluid is injected into the inlet chamber through the injection hole, and the injection fluid is emulsified and enters the droplet storage chamber at a junction of the droplet generating channels and the droplet storage chamber; wherein the droplet storage chamber is in a U-shaped structure, the inlet chamber is in a linear structure, the droplet storage chamber is divided into a left arm and a right arm, the left arm and the right arm are disposed on two sides of the chip respectively and the inlet chamber is disposed between the two arms of the U-shaped structure; wherein a plurality of droplet generating channels are disposed along two sides of the inlet chamber to increase a speed of droplet generation; wherein the injection hole connecting to the inlet chamber is disposed on a surface of the chip, an end of the inlet chamber close to the injection hole is an injection end, an arrangement density of the droplet generating channels gradually increases from the injection end to another end of the inlet chamber.

2. The droplet digital PCR chip according to claim 1, wherein the chip further comprises an oil outlet chamber and an oil outlet hole, and the oil outlet chamber is connected to the droplet storage chamber through a plurality of oil outlet channels.

3. The droplet digital PCR chip according to claim 2, wherein the oil outlet chamber is disposed on an outer side of the droplet storage chamber.

4. The droplet digital PCR chip according to claim 1, wherein the chip is provided with a bubble storage groove, and the bubble storage groove is connected with the droplet storage chamber through at least one bubble channel.

5. The droplet digital PCR chip according to claim 1, wherein an injection flow channel is disposed between the injection hole and the inlet chamber, the injection flow channel extends a flowing time of injection fluid from the injection hole to the inlet chamber, and balances a pressure when the injection fluid enters into the droplet generating channel.

6. The droplet digital PCR chip according to claim 1, wherein a plurality of droplet generating channels are parallelly disposed between the inlet chamber and two arms.

7. The droplet digital PCR chip according to claim 6, wherein the droplet generating channels between the inlet chamber and the droplet storage chamber is disposed according to a pressure changing in the inlet channel, intervals between the droplet generating channels are small at a far end of the inlet chamber from the injection hole, and the intervals increases gradually to a near end of the inlet chamber from the injection hole, so that the pressure in each of the droplet generating channels is basically consistent.

8. The droplet digital PCR chip according to claim 1, wherein a bell mouth structure is formed by increasing a width of an end of the droplet generating channel close to the droplet storage chamber.

9. The droplet digital PCR chip according to claim 1, wherein a circular chamfer is disposed at an end of the droplet generating channel connecting with the droplet storage chamber.

10. The droplet digital PCR chip according to claim 1, wherein an upper surface of the droplet storage chamber is provided with at least one vent hole.

11. The droplet digital PCR chip according to claim 1, wherein a silicone plug is disposed at an inlet of the injection hole, and the silicone plug is provided with a through hole to connect with the inlet chamber.

12. The droplet digital PCR chip according to claim 1, wherein droplets are laid in a single, a double, or a multi-layer array in the droplet storage chamber of the chip, and the droplets remain stable after a reaction or a detection.

13. The droplet digital PCR chip according to claim 1, wherein the chip comprises a plurality of chip units, and the chip units are parallelly arranged.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a schematic cross-sectional view of a droplet digital PCR chip in embodiment 1;

(2) FIG. 2 illustrates a schematic structural view of a top piece on an inner side of the chip;

(3) FIG. 3 illustrates a schematic cross-sectional view of the droplet digital PCR chip of FIG. 1 along the A-A direction;

(4) FIG. 4 illustrates a partial enlarged view of area B in FIG. 2;

(5) FIG. 5 illustrates a schematic partial structural view of a droplet generating channel and a droplet storage chamber;

(6) FIG. 6 illustrates a schematic structural view of a droplet digital PCR chip in embodiment 2;

(7) FIG. 7 illustrates a schematic structural view of a droplet digital PCR chip in embodiment 3;

(8) FIG. 8 illustrates a view of generated droplets in embodiment 4;

(9) FIG. 9 illustrates droplet fluorescent signals in embodiment 4;

(10) FIG. 10 illustrates a schematic structural view of a droplet digital PCR chip in embodiment 5;

(11) FIG. 11 illustrates a schematic structural view of a chip unit in FIG. 10.

DETAILED DESCRIPTION OF THE INVENTION

(12) The following examples, including the experiments conducted and results achieved are provided for illustrative purpose only and are not to be construed as limiting upon the present invention.

Embodiment 1

(13) As shown in FIGS. 1 to 5, a droplet digital PCR chip includes a chip unit, and the chip unit includes a chip body formed by bonding a top piece 1 and a bottom piece 2. The chip body is internally provided with an inlet chamber 3, a droplet storage chamber 4, and an oil outlet chamber 5.

(14) In the present invention, the internal structures of the chip body include, but is not limited to, an inlet chamber, a droplet storage chamber, and an oil outlet chamber. The internal structures may be disposed on an inner wall of the top piece, or on an inner wall of the bottom piece of the chip, or formed after a bonding of the top and bottom pieces, as long as the top and bottom pieces are bonded together to form a desired microstructure.

(15) In this embodiment, the microstructure is located on the inner wall of the top piece, that is, a side of the top piece facing the inner wall of the chip.

(16) Specifically, as shown in FIG. 2, the droplet storage chamber 4 is in a U-shaped structure. The inlet chamber 3 is in a linear structure, and is disposed between two arms of the U-shaped structure. A plurality of droplet generating channels 6 are parallelly disposed between the inlet chamber and the two arms. The oil outlet chamber 5 is located on an outer side of the U-shaped structure. A plurality of oil outlet channels 7 are provided between the droplet storage chamber 4 and the oil outlet chamber 5. Heights of the droplet generating channel 6 and the oil outlet channel 7 both are smaller than a height of the droplet storage chamber 4, so that a specific terrace with a function of droplets generation is formed at a junction between the droplet generating channel 6 and the droplet storage chamber 4.

(17) The droplet storage chamber 4 is in a U-shaped structure, so that the elongated inlet chamber 3 extends into an interior of the droplet storage chamber 4. A plurality of droplet generating channels 6 can be disposed on a top of and along two sides of the inlet chamber 3 to increase a speed of droplet generation. Meanwhile, it also makes all boundaries of the droplet storage chamber 4 not too far from the droplet generating channels, avoiding generated droplets migrate over long distances to cause damage and loss.

(18) An injection hole 8 connecting to the inlet chamber 3 and an oil drain hole 9 connecting to the oil outlet chamber 5 both are disposed on a surface of the chip.

(19) An end of the inlet chamber close to the injection hole 8 is defined as an injection end. An elongated connecting channel 10 is disposed between the injection end and the injection hole 8. An arrangement density of the droplet generating channels 6 gradually increases from the injection end to another end of the inlet chamber 3. This arrangement with a gradient density allows the droplets to be generated at a similar rate in different locations. If the droplet generating channels are evenly disposed, a pressure on the droplet generating channels 6 close to the injection hole is larger than those at far side, driving the aqueous phase flowing faster and generating more droplets.

(20) An end of the droplet generating channel 6 connecting with the droplet storage chamber 4 is provided with a circular chamfer 11. A length of the circular chamfer 11 in a droplet formation direction is ranging from 1 μm to 500 μm. The length of the circular chamfer 11 in the droplet formation direction affects a size of the generated droplets. Within a certain range, the size of the generated droplets increases as the length increases.

(21) A cross section of the droplet generating channel 6 is rectangular with a width ranging from 10 μm to 500 μm and a height ranging from 1 μm to 400 μm. A depth of droplet storage chamber is ranging from 2 μm to 1000 μm. A width of the droplet generating channel 6 close to the end connecting with the droplet storage chamber 4 becomes larger to form a bell mouth structure. The size of the droplets is directly related to a size of the droplet generating channel 6, which means the larger the inlet channels 6, the larger the droplets. Thus, the size of droplet generating channel 6 needs to be within a reasonable range. A bell mouth structure facilitates the generation and movement of droplets.

(22) The injection hole 8 is sealed by a silicone plug which has a through hole. Due to good elasticity of silicone material, a pipette can be tightly matched with the silicone plug during a loading of oil phase or aqueous phase, which ensures the airtightness of the chip channels to make a stable flow rate and pressure and homogeneous droplets generated.

(23) The chip is also engraved with a series of circles of known size in the inlet chamber 3 as a scale, which can be conveniently used to measure the size of the droplets when inspected under a microscope.

(24) When using, the oil phase is firstly injected into inner chambers of the chip through the injection hole 8. After the chip is filled with the oil phase and all bubbles are expelled from the inner chambers, the aqueous phase is injected into the inlet chamber 3 through the injection hole 8 under a certain pressure. When the aqueous phase enters the droplet storage chamber 4 through the droplet generating channels 6, a terrace is formed at the junction of the droplet generating channel 6 and the droplet storage chamber 4 as the height of droplet generating channel 6 is smaller than the droplet storage chamber 4. When the aqueous phase enters a relatively wide droplet storage chamber 4 from the relatively narrow droplet generating channel 6, the aqueous phase partially enters the droplet storage chamber 4 breaks with the aqueous phase in the droplet generating channel 6 as a flow rate is accelerated by a surface tension, forming droplets. After the generated droplets enter the droplet storage chamber 4, the corresponding volume of the oil phase is drained to the oil outlet chamber 5 through the oil outlet channels 7. The oil phase is eventually drained through the oil outlet hole 9 that connects with the oil outlet chamber 5. A height of the oil outlet channels 7 is smaller than a height of the droplet storage chamber 4, so that the droplets are not easily drained from the oil outlet channel 7. Large number of droplets are finally laid in the droplet storage chamber 4. The water-in-oil droplets exist independently, while outer oil phases are mixed with each other, which is equivalent to the presence of droplets of mutually independent aqueous phases in the oil phase. After the droplets are generated, the chip can be directly put into a thermal cycler for a PCR amplification. After the amplification, the chip can be placed into an analysis instrument for imaging and reading fluorescent signals.

Embodiment 2

(25) A droplet digital PCR chip includes a chip unit 100. The chip unit 100 also includes a chip body formed by bonding a top piece and a bottom piece, as illustrated in Embodiment 1.

(26) The main difference is that the chip body is provided with an inlet chamber and a droplet storage chamber. However, there is no oil outlet chamber.

(27) Moreover, the chip may further be provided with a bubble storage groove 12. The bubble storage groove 12 is connected to the droplet storage chamber 4 through a plurality of bubble channels 13.

(28) The bubble storage groove 12 is mainly used for storing the bubbles in the droplet storage chamber 4. There is no special requirement for a position and a structure thereof. Considering a layout and rational utilization of the whole chip, the bubble storage groove 12 can be disposed at a corner space of the chip to utilize the corner space as much as possible without occupying the main space of the chip.

(29) In this embodiment, the bubble storage groove 12 and corresponding bubble channels 13 are provided at an end of the inlet channel.

(30) Further comparison with embodiment 1, an injection flow channel 14 is disposed between the injection hole 8 and the inlet chamber 3. The injection flow channel 14 can extend a time of injection fluid flow from the injection hole 8 to the inlet chamber 3, and balance a pressure when the injection fluid enters into the droplet generating channel 6.

(31) The injection flow channel 14 can be in various forms. In order to increase the pressure equilibrium of injection fluid enter into the droplet inlet channel and the stability of the droplets, the injection flow channel 14 can be designed as long and tortuous as possible.

Embodiment 3

(32) As shown in FIG. 7, a plurality of chip units 100 are disposed on one chip body. The structure of each chip unit 100 is the same as that in Embodiment 2.

Embodiment 4

(33) A plurality of chip units are disposed on one chip body, and the structure of each chip unit is the same as that in Embodiment 1.

Embodiment 5

(34) By using the droplet digital PCR chip in Embodiment 1, and 70% mineral oil+30% Tetradecane+3% EM90+3% Triton X-100 as the oil phase (the ratio of mineral oil and Tetradecane is mass/mass ratio to form the main component of the oil phase, the ratio of EM90 and TritonX-100 is mass/mass ratio to be added additionally to the main component), the droplet generation and PCR amplification are carried out. The process is described in detail as follows:

(35) (1) Preparing the oil phase.

(36) (2) Preparing the aqueous phase, i.e. the PCR reaction mixture.

(37) The template was derived from non-small cell lung cancer (NSCLC) cell line H1975, with both T790M and L858R mutations.

(38) The primer sequences are:

(39) TABLE-US-00001 (SEQ ID NO: 1) F: 5′-GCCTGCTGGGCATCTG-3′; (SEQ ID NO: 2) R: 5′-TCTTTGTGTTCCCGGACATAGAC-3′;

(40) The probe sequence is:

(41) 5′- FAM- ATGAGCTGCATGATGAG -MGB-NFQ -3′ (SEQ ID NO: 3), wherein FAM is a fluorescent reporter and NFQ is a quencher.

(42) PCR reaction mixture:

(43) TABLE-US-00002 Components Volume (μl) 2 × PCR buffer(Taq polymerase, 7.5 dNTP {grave over ( )} Mg.sup.2+ includes) BSA (1%) 1.5 Primer F (10 μM) 0.3 Primer R (10 μM) 0.3 Probe (5 μM) 0.3 Template (5 ng/μl) 1.0 Water, nuclease-free 4.1 Total Volume 15
(3) Filling the droplet digital PCR chip with oil phase.
(4) Generating droplets with the step emulsification method while adding the aqueous phase to the oil phase using a syringe pump.
(5) Performing PCR amplification according to the following procedure:
96° C. for 10 min, and 40 cycles of 30 s at 98° C. and 60 s at 62° C., followed by 62° C. for 60 s, incubate at 25° C.
(6) After amplification, observing the morphology of the droplets under a microscope. If the droplets are homogenous and stable, detect the fluorescent signal with a chip scanner.

(44) Results: After the PCR amplification, the droplets were homogenous, and the diameter was about 80 μm. There was hardly any broken and fused droplet (FIG. 8), and strong fluorescent signals can be detected by chip scanner (FIG. 9).

Embodiment 6

(45) As shown in FIG. 10, a plurality of chip units 100 are disposed on one chip, and the structure of each chip unit 100 is basically identical (FIG. 11). The design principle of the main structure of the chip unit is the same as that in Embodiments 1 and 2. The inlet chamber, the droplet storage chamber and the oil outlet chamber are also provided inside the chip unit.

(46) Different from embodiment 1, an injection flow channel 14 is disposed between the injection hole 8 and the inlet chamber 3 as that in Embodiment 2. The injection flow channel 14 can extend a flowing time of injection fluid from the injection hole 8 to the inlet chamber 3, and balance a pressure when the injection fluid enters into the droplet generating channel 6.

(47) In the present embodiment, the injection flow channel 14 is disposed in a ring shape. An upper half of the annular flow channel stores part of the aqueous phase, so that the lower half, which connects with the droplet generating channel, always maintains a certain amount of pressure from aqueous phase. With such design, the annular flow channel is used to disperse and equilibrate the flow pressure, so that the pressure is evenly released. Meanwhile, the intervals between the droplet generating channels can be adjusted according to the change of the flow pressure, so that the pressure of each droplet generating channel is equilibrated at maximum degree and the droplets are homogenous. Thus, the force on droplets is unidirectional and the droplets move in one direction, which reduces the risk of droplet fusion.

(48) In addition, the oil outlet chamber is disposed in a regular U shape, surrounding the three sides of the droplet storage chamber. The oil outlet channel 7 is evenly disposed between the droplet storage chamber and the oil outlet chamber. This kind of oil outlet channel 7 reduces the oil drain pressure, facilitating the quick drain of the oil phase.

Embodiment 7

(49) Using the droplet digital PCR chip in Embodiment 6, and 70% mineral oil+30% Tetradecane+3% EM90+3% Triton X-100 as the oil phase (the ratio of mineral oil and Tetradecane is mass/mass ratio to form the main component of the oil phase, the ratio of EM90 and TritonX-100 is mass/mass ratio to be added additionally to the main component), the droplet generation and PCR amplification are carried out. The process is described in detail as follows:

(50) (1) Preparing the oil phase.

(51) (2) Preparing the aqueous phase, i.e. the PCR reaction mixture.

(52) The template was derived from non-small cell lung cancer (NSCLC) cell line H1975, with both T790M and L858R mutations.

(53) The primer sequences are:

(54) TABLE-US-00003 (SEQ ID NO: 1) F: 5′-GCCTGCTGGGCATCTG-3′; (SEQ ID NO: 2) R: 5′-TCTTTGTGTTCCCGGACATAGAC-3′;

(55) The probe sequence is:

(56) 5′- FAM- ATGAGCTGCATGATGAG -MGB-NFQ -3′ (SEQ ID NO: 3), wherein FAM is a fluorescent reporter and NFQ is a quencher.

(57) PCR Mixture:

(58) TABLE-US-00004 Components Volume (μl) 2 × PCR buffer(Taq polymerase, 7.5 dNTP {grave over ( )} Mg.sup.2+ includes) BSA (1%) 1.5 Primer F (10 μM) 0.3 Primer R (10 μM) 0.3 Probe (5 μM) 0.3 Template (5 ng/μl) 1.0 Water, nuclease-free 4.1 Total Volume 15
(3) Filling the droplet digital PCR chip with oil phase.
(4) Generating droplets with the step emulsification method while adding the aqueous phase to the oil phase using a syringe pump.
(5) Performing PCR amplification according to the following procedure:
96° C. for 10 min and 40 cycles of 30 s at 98° C. and 60 s at 62° C., followed by 62° C. for 60 s, incubate at 25° C.
(6) After amplification, observing the morphology of the droplets under a microscope. If the droplets are homogenous and stable, detect the fluorescent signal with a chip scanner.

(59) Results: After the PCR amplification, the droplets were homogenous, and the diameter was about 80-120 μm. There was hardly any broken and fused droplet (FIG. 8), and strong fluorescent signals can be detected by chip scanner.