Inverted anti-evaporation device and method for trace sample

Abstract

The invention discloses an inverted anti-evaporation device and method for a trace sample, and the inverted anti-evaporation device comprises a liquid adding probe which is used for generating liquid drops; the reaction container is used for bearing sample liquid drops; the heating device is used for controlling the temperature in the reaction container, and a temperature gradient from hot to cold is formed in the heating device from bottom to top. Compared with a conventional coated phase, the method has the characteristics of simplicity in operation, rapidness and high efficiency, is not limited by an oil phase coated phase, and realizes flexible implementation of complex pretreatment operation through the steps of upright liquid adding and inverted reaction.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic analysis, and in particular to an inverted anti-evaporation device and method for trace samples. Background Art

[0002] Microfluidic technology refers to the science and technology involved in systems that use microchannels (with dimensions ranging from dozens to hundreds of micrometers) to process or manipulate tiny fluids (with volumes ranging from microliters to picoliters). Microfluidic systems have the advantages of low consumption of samples and reagents, strong ability to manipulate trace liquids, high analysis throughput, etc., and have become an effective technology for studying complex biological systems. With the emergence and rapid development of microfluidic technology in recent years, microfluidic technology has been widely applied in the fields of single-cell research, gene sequencing, medical diagnosis, environmental detection, and food safety.

[0003] In the microfluidic system for performing complex pretreatment operation steps, the problem of preventing the evaporation of trace liquids needs to be solved first. Currently, among the various microfluidic anti-evaporation methods that have been developed, most adopt covering with another phase medium that is immiscible with the sample liquid, such as mineral oil, etc., as the encapsulating phase to prevent the evaporation of microdroplets. For example, the Chinese patent document with the publication number CN115468684A discloses a microcalorimeter chip based on a flexible printed circuit process. Mineral oil is added to the surface of the substrate that has been subjected to oleophobic treatment to form a virtual reaction chamber, and a vacuum oil with a low saturated vapor pressure is used to encapsulate the sample, which can prevent its evaporation in a vacuum environment. However, the presence of the oil phase will greatly limit the operation process and application range of complex pretreatment of droplets.

[0004] Our laboratory has developed a microfluidic chip device for preventing droplet evaporation with a gas spacer (patent publication number CN106256436A), which can improve the problem of contact between the encapsulating phase and the sample droplet, but there are still certain limitations in the process of complex liquid addition operation.

[0005] Therefore, developing a device or method with low cost, flexible operation, simple and efficient for realizing anti-evaporation of trace liquids is still an urgent problem to be solved. Summary of the Invention

[0006] The present invention provides an inverted anti-evaporation device and method for trace samples, which can simply and efficiently achieve the anti-evaporation of trace liquids without being restricted by encapsulating phases such as oil phases.

[0007] An inverted anti-evaporation device includes:

[0008] A liquid addition probe for generating droplets;

[0009] A reaction container for holding sample droplets;

[0010] A heating device is used to control the temperature inside the reaction vessel after inversion, ensuring that a temperature gradient from hot to cold is formed inside the reaction vessel from bottom to top after inversion.

[0011] The present invention utilizes the basic law of gas convection in the changes of temperature gradient and density gradient, combined with an inverted device, to form an anti-evaporation device with the vapor formed by the heat vaporization of the surface of its own liquid droplets as the wrapping phase. Moreover, by ingeniously utilizing the surface tension and adsorption effect of the liquid droplets, when the reaction vessel is inverted, the liquid droplets will closely adhere to the bottom of the reactor. Compared with the conventional wrapping phase, the inverted anti-evaporation device of the present invention has the characteristics of simple, fast, and efficient operation, is not restricted by the wrapping phases such as oil phases, and through the steps of adding liquid in the upright position and reacting in the inverted position, the flexible realization of complex pretreatment operations can be achieved.

[0012] The present invention is applicable to occasions such as biochemical analysis of trace volume liquids, high-throughput drug screening, cell analysis, single-molecule analysis, etc., which require micro-liquid manipulation and heating reactions.

[0013] In the present invention, the sample liquid droplets can be any form and type of liquid, mainly determined according to actual needs. These liquid droplets include one or more of sample liquid, reaction liquid, buffer solution, dilution liquid, other functional solutions, including but not limited to aqueous phase, oil phase, etc.

[0014] Preferably, the volume of the liquid droplets generated by the liquid addition probe is from 1 picoliter to 1 milliliter.

[0015] Preferably, the temperature gradient should be greater than 0.1 °C, and the distance between the sample liquid droplets and the heat source should be greater than 100 μm.

[0016] More preferably, the temperature gradient should be greater than 1 °C, and the distance between the sample liquid droplets and the heat source should be greater than 0.5 cm.

[0017] Preferably, it may further include a temperature monitoring unit for real-time monitoring of the reaction temperature in the reaction vessel.

[0018] Optionally, the temperature monitoring unit includes a reaction monitoring container, a temperature sensor, and a temperature display; the structure of the reaction monitoring container is the same as that of the reaction container, and the reaction monitoring container is fixed on the upper part of the heating device in the same way after inversion; the temperature sensor is arranged at a position corresponding to the height of the sample liquid droplets in the reaction monitoring container for real-time monitoring of the reaction temperature in the reaction container and displaying it on the temperature display.

[0019] Since the inverted reaction container is placed in an environment with a temperature gradient, the temperature sensor for monitoring the reaction temperature should display the temperature at the bottom inside the reactor in real time for real-time monitoring of the reaction temperature; as a further preference, devices such as infrared imaging are used to achieve real-time monitoring of the temperature at the bottom of the target reaction container without reaching into the inside of the reaction container.

[0020] In the present invention, there are no special requirements for the inner wall material of the reaction vessel, including but not limited to inorganic materials, organic materials, composite materials, etc., such as glass, polyethylene, polypropylene, polytetrafluoroethylene, polyether ether ketone, etc. The specific material used is determined according to the experimental needs.

[0021] In the present invention, there are no special requirements for the bottom structure of the reaction vessel, as long as it can hold the sample droplets; the reaction vessel supports a structure with multiple container arrays arranged; the bottom structure of the reaction vessel can be the structure of the reactor itself or a structure processed externally.

[0022] Preferably, the bottom structure of the reactor is conical, so that after the reactor is inverted, the sample droplet is at the highest point inside the reactor, that is, the sample droplet is at the position with the lowest temperature in the reaction vessel.

[0023] In the present invention, the inversion angle of the reaction vessel is 120° to 180°.

[0024] In the present invention, the reaction vessel has two forms: closed and non-closed. Preferably, the closed form can better achieve the anti-evaporation effect, that is, the reaction vessel is provided with a sealing cover, the reaction vessel is inverted on the sealing cover, and is fixed to the upper part of the heating device.

[0025] Preferably, a protective droplet is added dropwise on the sealing cover to improve the anti-evaporation effect and move upward as a protection and supplement to the sample droplet; more preferably, a protective droplet with a volume smaller than the sample droplet is added dropwise on the sealing cover to further improve the anti-evaporation effect.

[0026] An inverted anti-evaporation method for trace samples, using the above inverted anti-evaporation device, specifically includes the following steps:

[0027] S1: Generate a sample droplet in the bottom structure of the reaction vessel using a liquid addition probe;

[0028] S2: Control the heating device to make its temperature reach the target set temperature;

[0029] S3: Invert the reaction vessel containing the sample droplet and fix it to the upper part of the heating device, so that a temperature gradient from hot to cold is formed from bottom to top inside the inverted reaction vessel;

[0030] Based on the inverted anti-evaporation device, there is a temperature gradient between the heating device and the sample droplet at the bottom of the inverted reaction vessel. The sample droplet is heated and vaporized around to form steam, and the hot air below convects upward due to the density gradient, inhibiting the downward diffusion of the steam. The hot steam preferentially condenses in the sample droplet at the lowest temperature in the reactor, protecting the remaining droplets from further vaporization, and achieving the effect of preventing evaporation of trace droplets.

[0031] S4: Heat and incubate the sample droplet according to the target temperature and target time required for the reaction; after the reaction ends, take out the sample for subsequent experiments.

[0032] Preferably, in step S2, a temperature monitoring unit is set up to monitor the reaction temperature in the reaction vessel in real time and display it on the temperature display.

[0033] Preferably, in step S3, the inversion angle of the reaction vessel is greater than 120°; as a further preference, the inversion angle of the reaction vessel is 180°.

[0034] Preferably, in step S3, invert the reaction vessel on the sealing cover and then fix it to the upper part of the heating device; preferably, drop a protective droplet on the sealing cover, and the protective droplet forms a thermal vapor when heated and moves upward to protect and supplement the sample droplet; as a further preference, the volume of the protective droplet is smaller than that of the sample droplet.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The present invention utilizes the gas convection law generated by temperature gradient and density gradient to achieve the anti-evaporation effect of trace droplets, enabling the thermal vapor generated by the droplets to form a wrapping phase for the droplets themselves, without the need for other phase media to directly contact the sample, thereby avoiding the pollution problem of the phase media and eliminating the limitations of the phase media;

[0037] 2. In the present invention, the volume of the trace droplet can be reduced to the nanoliter level, which can effectively reduce the consumption of the sample and reagent, enable the reaction to proceed in a microscale system, improve the mass transfer efficiency of the sample, and accelerate the progress of the reaction;

[0038] 3. The inverted anti-evaporation device adopted by the present invention is applicable to any form and type of droplets, any form and type of reaction vessels, as well as any form of liquid addition method and the pre-treatment process that requires heating, and is simple, flexible, convenient, and reliable to operate;

[0039] 4. The present invention is applicable to array-type liquid addition devices and array-type reaction vessels, with multiple samples; at the same time, it has the characteristics of automation and high throughput;

[0040] 5. The present invention is widely applicable to the heating reaction of trace liquids. Description of the Drawings

[0041] Figure 1 It is the schematic diagram of the principle of the inverted anti-evaporation device in the embodiment of the present invention;

[0042] Figure 2 It is the schematic diagram of the inverted anti-evaporation device and the operation process in the embodiment of the present invention;

[0043] Figure 3Schematic diagram of the inverted capping anti-evaporation device and operation process in the embodiment of the present invention;

[0044] Figure 4 Schematic diagram of temperature control of the inverted and inverted capping anti-evaporation devices in the embodiment of the present invention;

[0045] Figure 5 Schematic diagram of the shape of the reaction vessel in the embodiment of the present invention;

[0046] Figure 6 Schematic diagram of the inverted grooved anti-evaporation device and operation process in the embodiment of the present invention;

[0047] Figure 7 Anti-evaporation experimental result diagram of the inverted capping anti-evaporation device in the embodiment of the present invention.

[0048] In the figure: 1 - liquid addition probe, 2 - sample liquid droplet, 3 - reaction vessel, 4 - sealing cap, 5 - heating device, 6 - placement rack, 7 - temperature sensor, 8 - temperature display, 9 - reaction monitoring vessel, 10 - droplet vapor, 11 - thermal cap, 12 - protective droplet. Detailed implementation manners

[0049] The following further describes the present invention in detail with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not impose any limitations on it.

[0050] Referring to the accompanying drawings, the preferred embodiments according to the present invention will be described in detail below.

[0051] Embodiment 1

[0052] Compare the device and working principle of conventional heating cap anti-evaporation with the inverted anti-evaporation of the present invention. For the specific schematic diagram, see Figure 1 .

[0053] For the conventional heating cap anti-evaporation device, as shown in (a) of Figure 1 , by heating the cap 11, it is possible to prevent the steam formed during the heating process and the cooling process after the heating ends from condensing into droplets at the upper sealing cap, so that the steam formed by vaporization will preferentially condense and recover at the bottom of the reaction vessel tube. However, the method of conventional heating cap anti-evaporation inevitably has the sample steam spreading throughout the reaction vessel. Therefore, there are requirements for the initial volume of the reaction droplet. When the droplet volume is reduced to an ultra-trace volume and the droplets are all vaporized to form steam during the heating process but still do not reach the saturated vapor pressure, the situation of droplet evaporation to dryness will occur.

[0054] Compared with the anti-evaporation method of the conventional heating cap, the inverted anti-evaporation device adopted by the present invention, as shown in Figure 1As shown in Fig. (b), by cleverly utilizing the law of gas convection generated by the changes in temperature gradient and density gradient, the traditional temperature difference form of hot at the top and cold at the bottom is transformed into a temperature gradient form of cold at the top and hot at the bottom. And with the inverted placement of the reaction vessel 3, the droplet vapor 10 formed by the vaporization of the surrounding heated trace sample droplet 2 will be resisted by the upward convection of the hot air below, so it is restricted from diffusing downward and accumulates around the sample droplet 2, making the vapor molecules generated by the droplet serve as a wrapping phase to protect the remaining droplet from further vaporization, achieving the effect of preventing the evaporation of ultra-trace droplets.

[0055] On the basis of the above inverted anti-evaporation device, the inverted anti-evaporation device is further optimized, and a protective droplet 12 with a volume smaller than that of the sample droplet 2 is generated on the sealing cover 4, as Figure 1 shown in Fig. (c). When the device is placed in a heating environment, the protective droplet 12 is heated to form hot vapor and moves upward to protect and supplement the sample droplet 2.

[0056] Example 2

[0057] The device and operation process of inverted anti-evaporation are as Figure 2 shown. The steps include:

[0058] Step 1: Use the liquid addition probe 1 to generate an ultra-trace sample droplet 2 at the bottom of the reaction vessel 3; the volume of the sample droplet 2 is from picoliter to microliter; the bottom shape of the reaction vessel 3 has different structures for different usage scenarios, and the structural examples are as Figure 5 shown. At the same time, multiple reaction vessels 3 can be arranged to form a reaction vessel array for the inverted anti-evaporation process of high-throughput sample heating reaction, as Figure 5 shown in Fig. (f).

[0059] Step 2: After turning the reaction vessel 3 containing the sample droplet 2 by 180 degrees, make the opening of the reaction vessel 3 face downward and place it on the placement rack 6 above the heating device 5; the placement rack 6 can be selected according to the shape of the reaction vessel 3, and keep a sufficient distance between the sample droplet 2 and the heating device 5 to form a temperature difference greater than 1 °C.

[0060] Step 3: Control the heating device 5 and observe the temperature display 8 to make the temperature at the bottom of the reaction vessel 3 reach the target temperature; the temperature display 8 has a corresponding temperature sensor 7, as Figure 4 shown, placed at the bottom of the reaction monitoring vessel 9 at the same height as the reaction vessel 3 for real-time monitoring of the actual temperature of the reaction vessel 3 to achieve precise control of the reaction temperature.

[0061] Example 3

[0062] The device and operation process of inverted sealed anti-evaporation are as Figure 3 shown. The steps include:

[0063] Step 1: Use the liquid addition probe 1 to generate ultra-trace sample droplets 2 at the bottom of the reaction vessel 3; the volume of the sample droplets 2 is from picoliters to microliters; the bottom shape of the reaction vessel 3 has different structures for different usage scenarios, and the structure examples are as Figure 5 shown, including (a) round bottom, (b) flat bottom, (c) convex bottom, (d) conical bottom, (f) square bottom, (g) grooved conical bottom, (h) grooved microstructured bottom, (f) multi-reaction vessel array. At the same time, multiple sealed reaction vessels 3 can be arranged to form a reaction vessel array for the inverted anti-evaporation process of high-throughput sample heating reaction, such as Figure 5 shown in (f).

[0064] Step 2: Cover the reaction vessel 3 containing the sample droplets 2 with a matching sealing cap 4 to make the sample droplets 2 in a sealed environment; after turning it 180 degrees, make the opening of the reaction vessel 3 face down and place it on the placement rack 6 above the heating device 5; the placement rack 6 can be selected according to the shape of the reaction vessel to keep a sufficient distance between the sample droplets 2 and the heating device 5 to form a temperature difference greater than 0.1 °C.

[0065] Step 3: Control the heating device 5 and observe the temperature display 8 to make the temperature at the bottom of the reaction vessel 3 reach the target temperature; the temperature display 8 has a corresponding temperature sensor 7, as Figure 4 shown, placed at the bottom of the reaction monitoring vessel 9 at the same height as the reaction vessel 3 for real-time monitoring of the actual temperature of the reaction vessel 3 to achieve precise control of the reaction temperature.

[0066] Example 4

[0067] The device and operation process for anti-evaporation with an inverted grooved structure, as Figure 6 shown. The steps include:

[0068] Step 1: Use the liquid addition probe 1 to directly generate trace sample droplets 2 at the grooved opening; the volume of the sample droplets 2 is from picoliters to microliters; the bottom of the reaction vessel is a grooved structure for different usage scenarios, and the structure examples are as Figure 6 shown.

[0069] Step 2: Cover the upper end of the grooved opening with a sealing cap 4 that can be selected according to the shape of the reaction vessel 3, and place the reaction vessel on the placement rack 6 above the heating device 5; the placement rack 6 can be selected according to the shape of the reaction vessel 3 to keep a sufficient distance between the sample droplets 2 and the heating device 5 to form a temperature difference greater than 1 °C.

[0070] Step 3: Control the heating device 5 and observe the temperature display 8 to make the temperature at the bottom of the reaction vessel 3 reach the target temperature; the temperature display 8 has a corresponding temperature sensor 7, as Figure 4As shown, it is placed at the bottom of the reaction monitoring container 9 at the same height as the reaction container 3, used to monitor the actual temperature of the reaction container 3 in real time, and achieve precise control of the reaction temperature.

[0071] Example 5

[0072] Anti-evaporation experiment of the inverted sealed anti-evaporation device. Using a liquid addition probe to form a droplet with a volume of 500 nL and distilled water as the solution at the bottom of the reaction container (as shown in (c) below), perform the inverted sealed anti-evaporation operation according to Example 3 above, and let it carry out heating reactions under the conditions of 37 °C for 2 h, 60 °C for 30 min, and 95 °C for 10 min respectively, and evaluate the evaporation of the micro-droplets under different heating conditions. The results are as Figure 5 shown below. Figure 7 as

[0073] The above embodiments have described the technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modification, supplement, and equivalent replacement made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An inverted anti-evaporation device, characterized in that, Comprising: A liquid addition probe for generating liquid droplets; A reaction vessel for holding sample liquid droplets; A heating device for controlling the temperature inside the inverted reaction vessel to ensure that a temperature gradient from hot to cold is formed from bottom to top inside the inverted reaction vessel.

2. The inverted anti-evaporation device according to claim 1, characterized in that, The volume of the liquid droplets generated by the liquid addition probe is from 1 picoliter to 1 milliliter.

3. The inverted anti-evaporation device according to claim 1, characterized in that, The conditions for forming the temperature gradient are: the temperature gradient should be greater than 0.1 °C, and the distance between the sample liquid droplet and the heat source should be greater than 100 μm.

4. The inverted anti-evaporation device according to claim 1, characterized in that, It further includes a temperature monitoring unit for real-time monitoring of the reaction temperature in the reaction vessel.

5. The inverted anti-evaporation device according to claim 1, wherein, The bottom structure of the reaction vessel is for holding sample liquid droplets; the reaction vessel supports a structure arranged in multiple container arrays.

6. The inverted anti-evaporation device according to claim 1, characterized in that, The inverted angle of the reaction vessel is 120° to 180°.

7. The inverted anti-evaporation device according to claim 1, characterized in that, The reaction vessel is provided with a sealing cover, the reaction vessel is inverted on the sealing cover and fixed to the upper part of the heating device.

8. The inverted anti-evaporation device according to claim 7, wherein, A protective liquid droplet is added dropwise on the sealing cover.

9. An inverted anti-evaporation method for trace samples, characterized in that, Using the inverted anti-evaporation device according to any one of claims 1 to 8, specifically including the following steps: S1: Using the liquid addition probe to generate a sample liquid droplet in the bottom structure of the reaction vessel; S2: Controlling the heating device to make its temperature reach the target set temperature; S3: Inverting the reaction vessel containing the sample liquid droplet and fixing it to the upper part of the heating device, so that a temperature gradient from hot to cold is formed from bottom to top inside the inverted reaction vessel; Based on the inverted anti-evaporation device, there is a temperature gradient between the heating device and the sample liquid droplet at the bottom of the inverted reaction vessel. The sample liquid droplet is heated and vaporized to form steam around it, and the hot air below convects upward due to the density gradient, inhibiting the diffusion of the steam; at the same time, the hot steam preferentially condenses in the sample liquid droplet at the lowest temperature point in the reaction vessel, protecting the remaining liquid droplets from further vaporization, achieving the effect of preventing evaporation of micro liquid droplets; S4: Heating and incubating the sample liquid droplet according to the target temperature and target time required for the reaction; after the reaction ends, taking out the sample for subsequent experiments.

10. The inverted anti-evaporation method for trace samples according to claim 9, characterized in that, In step S3, the reaction vessel is inverted on the sealing cover and then fixed to the upper part of the heating device; a protective liquid droplet is added dropwise on the sealing cover, and the protective liquid droplet is heated to form hot steam and moves upward to protect and supplement the sample liquid droplet.

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