Method for testing supercooling degree of nano-liter liquid drops by differential scanning calorimetry

Abstract

The invention discloses a method for testing the supercooling degree of nano-liter liquid drops by a differential scanning calorimetry. The method comprises the following steps: taking a liquid sample with the mass of more than 0.2 mg or the volume of more than 0.2 mu L, then placing the liquid sample in a crucible to form a plurality of sub-droplets, then carrying out DSC test to obtain the phase change enthalpy of the plurality of sub-droplets, and calculating to obtain the degree of supercooling of the plurality of sub-droplets; wherein the volume of at least one sub-droplet is less than 0.1 mu L. The method provided by the invention can accurately measure the freezing point temperature of the liquid drop below 50 nanoliters and even 5 nanoliters, widens the measurement range of the quantitative relationship between the liquid drop size and the supercooling behavior, and lays a technical foundation for researching the liquid coating dominated liquid drop phase change behavior.

Description

Technical Field

[0001] The present invention belongs to the technical field of supercooling degree testing, and particularly relates to a method for testing the supercooling degree of nanoliter droplets by differential scanning calorimetry. Background Art

[0002] Currently, the crystallization temperature (supercooling degree) of a liquid is mainly measured by differential scanning calorimetry (DSC method). However, when studying the dominant role of the liquid skin (a few molecular layers thick on the liquid surface) in the entire droplet, it is necessary to measure the crystallization temperature of very small volume (less than 50 nL) droplets to reveal the role of the skin in the droplet behavior. However, when the droplet is less than 0.1 mg (100 nL), the accuracy of the balance and the influence of liquid evaporation will both cause deviations in the sample mass (volume), making it difficult to accurately weigh and impossible to perform accurate DSC tests.

[0003] Currently, the crystallization process and crystallization temperature of 0.2 μL droplets can be measured by an optical microscope; the DSC method can also be used to test droplets above 0.1 μL. However, due to the influence of the weighing accuracy during the weighing process and the unstable mass of water evaporation, the measured sample mass (volume) has deviations, which in turn affects the construction of the quantitative relationship between droplet volume and supercooling behavior. Therefore, it is very difficult to measure the crystallization temperature (supercooling degree) of droplets below 0.1 μL or even below 50 nL.

[0004] The existing technology can use the DSC method to test droplets above 0.1 μL. However, the test accuracy has been affected by the weighing accuracy during the weighing process and the unstable mass of water evaporation, resulting in deviations in the measured sample mass (volume), which in turn affects the construction of the quantitative relationship between droplet volume and supercooling behavior, and it is difficult to reveal the role of the liquid skin in the droplet behavior. Summary of the Invention

[0005] The main purpose of the present invention is to provide a method for testing the supercooling degree of nanoliter droplets by differential scanning calorimetry to overcome the deficiencies of the prior art.

[0006] To achieve the foregoing invention purpose, the technical solutions adopted by the present invention include: An embodiment of the present invention provides a method for testing the supercooling degree of nanoliter droplets by differential scanning calorimetry, which includes: Providing a liquid sample; And taking a liquid sample with a mass above 0.2 mg or a volume above 0.2 μL, then placing the liquid sample in a crucible to form a plurality of sub-droplets, and then performing DSC tests to obtain the phase change enthalpies of the plurality of sub-droplets, and calculating to obtain the supercooling degrees of the plurality of sub-droplets; wherein, at least one sub-droplet has a volume less than 0.1 μL.

[0007] Compared with the prior art, the beneficial effects of the present invention are as follows: The method provided by the present invention can accurately measure the freezing point temperature of droplets below 50 nanoliters or even down to 5 nanoliters, broaden the measurement range of the quantitative relationship between droplet size and supercooling behavior, and lay a technical foundation for the research on the phase change behavior of droplet-dominated liquid skins. Description of the Drawings

[0008] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0009] Figures 1a - 1b They are respectively schematic diagrams of regular droplet tests and nanoliter-level droplet tests in the present invention; Figure 2 It is a conventional DSC test curve graph of the freezing point of 3 mg microliter-level water droplets in Embodiment 1 of the present invention; Figure 3 It is a conventional DSC test curve graph of the freezing point of 25 mg microliter-level water droplets in Embodiment 1 of the present invention; Figure 4 It is a DSC test phase change enthalpy curve graph of 5.2 nanoliter and 22.7 nanoliter independent water droplets in Embodiment 1 of the present invention; Figure 5 It is a DSC test initial crystallization temperature curve graph of 5.2 nanoliter and 22.7 nanoliter independent water droplets in Embodiment 1 of the present invention; Figure 6 It is a phase change enthalpy test curve graph of 5.8 nanoliter water droplets in Embodiment 2 of the present invention; Figure 7 It is an initial crystallization temperature test curve graph of 5.8 nanoliter water droplets in Embodiment 2 of the present invention; Figure 8 It is a phase change enthalpy test curve graph of 46.5 nanoliter water droplets in Embodiment 3 of the present invention; Figure 9 It is an initial crystallization temperature test curve graph of 46.5 nanoliter water droplets in Embodiment 3 of the present invention; Figure 10 It is a phase change enthalpy and initial crystallization temperature test curve graph of 43.6 nanoliter water droplets in Embodiment 4 of the present invention; Figures 11a - 11b It is a quantitative relationship graph between the crystallization temperature and the water droplet mass and volume in Embodiment 5 of the present invention. Detailed Embodiments

[0010] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and a large number of practices, have been able to propose the technical solution of the present invention. Mainly, when preparing the sample, a number of sub-droplets of different sizes and independent of each other are placed in a crucible, and then DSC testing is carried out. Through the quantitative calculation of the phase change enthalpy of each sub-droplet, the droplet mass (or volume) corresponding to each independent sub-droplet can be obtained, and then the crystallization temperature (supercooling degree) of droplets with different masses / volumes can be obtained, realizing the supercooling degree test of nanoliter-level (less than 50 nL) droplets.

[0011] The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0012] The present invention provides a method of placing a number of sub-droplets of different sizes and independent of each other in a crucible during sample preparation, with the total droplet mass maintained above 0.2 mg (0.2 microliters). Utilizing the characteristic that the droplet volume affects the supercooling degree, it ensures that sub-droplets of different sizes crystallize separately at different temperatures, generating independent sub-droplet crystallization peaks, and thus realizing the supercooling degree test of nanoliter-level (less than 50 nL) droplets.

[0013] One aspect of the embodiments of the present invention provides a method for testing the supercooling degree of nanoliter droplets by differential scanning calorimetry, including: Providing a liquid sample; And, taking a liquid sample with a mass above 0.2 mg or a volume above 0.2 μL, then placing the liquid sample in a crucible to form a plurality of sub-droplets, and then performing DSC testing to obtain the phase change enthalpy of the plurality of sub-droplets, and obtaining the supercooling degree of the plurality of sub-droplets through calculation; wherein, at least one sub-droplet has a volume less than 0.1 μL.

[0014] In some more specific implementation schemes, the plurality of sub-droplets can be obtained by installing a pipette tip on a pipette gun to suck the liquid and then discharging a plurality of small droplets on the crucible.

[0015] In some more specific implementation schemes, the method specifically includes: modifying the surface of the crucible to be hydrophilic or hydrophobic, at least ensuring that when the liquid sample is placed on the surface of the crucible, it does not spread and forms a plurality of independent spherical crown-shaped droplets.

[0016] In some more specific implementation schemes, the liquid sample includes any one of water, glycerol, and salt solution, and is not limited thereto.

[0017] In some more specific embodiments, the method specifically includes: taking a liquid sample with a mass of more than 0.2 mg or a volume of more than 0.2 μL, then placing the liquid sample in a crucible to form a plurality of sub-droplets that are independent of each other, and then performing DSC testing to obtain the phase change enthalpy of each sub-droplet.

[0018] In some more specific embodiments, the method specifically includes: calculating the mass or volume of each sub-droplet based on the total mass or total volume of the liquid sample and the phase change enthalpy of each sub-droplet, and then obtaining the crystallization temperature of each sub-droplet.

[0019] In some more specific embodiments, the method specifically includes: calculating the supercooling degree of each sub-droplet based on the crystallization temperature of each sub-droplet and the theoretical phase change temperature of the liquid sample, so as to realize the testing of the supercooling degree of nanoliter droplets.

[0020] Furthermore, establish the relationship between the volume of each sub-droplet and the supercooling degree, so as to obtain the law of droplet phase change behavior dominated by the liquid skin.

[0021] In some more specific embodiments, at least one of the plurality of sub-droplets has a volume of less than 50 nL.

[0022] In some even more specific embodiments, the method for testing the supercooling degree of nanoliter droplets by differential scanning calorimetry includes: 1) Select a liquid, take a sample of the liquid, and the mass should be kept above 0.2 mg (200 nL), then perform DSC testing on the conventional liquid sample (such as Figure 1a ), to obtain the crystallization temperature of the liquid sample, and the difference between the crystallization temperature and the theoretical phase change temperature (melting temperature) is the supercooling degree; 2) Test the supercooling degree of droplets below 0.1 μL or even below 50 nL. Since it is not easy to accurately weigh, place a number of sub-droplets of different sizes and independent of each other in the crucible during sample preparation, and the total mass of the liquid sample is above 0.2 mg (0.2 μL), and perform DSC testing on multiple sub-droplets (such as Figure 1b ), the test curve will show multiple exothermic peaks, and record the phase change enthalpy of each exothermic peak in turn; 3) Calculate the mass (or volume) of each droplet based on the total mass of the liquid sample, the total phase change enthalpy (the sum of the phase change enthalpies of each sub-droplet) and the phase change enthalpy of each sub-droplet, and the crystallization temperature of the small-volume droplets can be obtained, and then the supercooling degree (the difference between the crystallization temperature and the theoretical phase change temperature (melting temperature)) can be obtained; 4) By testing the methods in steps 2) and 3) multiple times, quantitative relationship data points of the volume and supercooling degree of multiple droplets can be obtained, so as to reveal the role of the liquid skin in the supercooling behavior of droplets.

[0023] In view of the problem that it is very difficult to measure the crystallization temperature (supercooling degree) of droplets below 0.1 μL or even below 50 nL, the present invention provides a method for DSC measurement, in which the total mass is above 0.2 mg (0.2 μL), but during sample preparation, a number of sub-droplets of different sizes and independent of each other are placed in a crucible. By quantitatively calculating the phase change enthalpy, the droplet mass (or volume) corresponding to each phase change enthalpy is obtained, and the crystallization temperature and supercooling degree of droplets with different masses / volumes are studied, thus realizing the measurement of the supercooling degree of very small volumes (less than 50 nL).

[0024] The technical solution of the present invention will be further described in detail below in conjunction with several preferred embodiments and accompanying drawings. These embodiments are implemented on the premise of the technical solution of the invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0025] In the following embodiments, the experimental materials used can be obtained from conventional biochemical reagent companies without special instructions.

[0026] Example 1: Measurement of supercooling degree of 5.2 nL and 22.7 nL water droplets (1) Sampling deionized water, with a water droplet mass of 3 mg, a micro-liter droplet, and performing DSC measurement on conventional droplets (such as Figure 1a Figure 2 ). It is found that the freezing point (icing) temperature of the water droplet is -18.8 °C, and the supercooling degree is 18.8 °C (0 °C - (-18.8 °C)); (2) Continuing to sample deionized water, increasing the water droplet mass to about 25 mg, and performing DSC measurement on conventional water droplets ( Figure 3 ). It is found that when the water droplet is greater than 20 mg, the freezing point is still near -20 °C, which is close to the test result of 3 mg; (3) Measuring the supercooling degree of droplets below 0.1 μL or even below 50 nL. Since it is not easy to accurately weigh, during sample preparation, a number of sub-droplets of different sizes and independent of each other are placed in a crucible, and DSC measurement on nano-liter droplets is performed (such as Figure 1b Figure 4 ). The test curve shows three exothermic peaks, and the phase change enthalpies from right to left are 203.3 J / g, 17.0 J / g, and 3.9 J / g respectively.

[0027] (4) The total mass of the droplets is 0.3 mg. Calculating, the minimum water droplet mass is 0.0052 mg, and the volume is 0.0052 μL ≈ 5.2 nL. The freezing point (icing) temperature of the 5.2 nL water droplet is -35.7 °C, and the supercooling degree is 35.7 °C.

[0028] ​​(5). Similarly, the calculated mass of the middle water droplet is 0.0227 mg, and the volume is 0.0227 μL ≈ 22.7 nL. The freezing (icing) temperature of the 22.7-nanoliter water droplet is -34.0 ºC, and the supercooling degree is 34.0 ºC (as Figure 5 ).

[0029] Example 2: Supercooling degree test of 5.8-nanoliter water droplets (1). Take a sample of deionized water. When making the sample, place several sub-droplets of different sizes and independent of each other in the crucible, and conduct a DSC test on nanoliter droplets (as Figure 1b , Figure 6 ). The test curve shows 4 exothermic peaks. Among them, the 1st to 3rd peaks from right to left are connected and it is difficult to independently measure the phase change enthalpy. Therefore, the overall phase change enthalpy is measured to be 235.7 J / g, and the phase change enthalpy measured on the far left is 4.4 J / g.

[0030] (2). The total mass of the droplets is 0.320 mg. The calculated minimum water droplet mass is 0.0058 mg, and the volume is 0.0058 μL ≈ 5.8 nL. The freezing (icing) temperature of the 5.8-nanoliter water droplet is -34.7 ºC, and the supercooling degree is 34.7 ºC (0 ºC - (-34.7 ºC)) (as Figure 7 ).

[0031] (3). Comparing with the test of the 5.2-nanoliter water droplet in Example 1, it can be seen that their volumes are close and the supercooling degrees are also close, indicating that this test method has stability.

[0032] Example 3: Supercooling degree test of 46.7-nanoliter water droplets (1). Take a sample of deionized water. When making the sample, place several sub-droplets of different sizes and independent of each other in the crucible, and conduct a DSC test on nanoliter droplets (as Figure 1b , Figure 8 ). The test curve shows 5 exothermic peaks. Among them, the 3rd and 4th peaks from right to left are connected and it is difficult to independently measure the phase change enthalpy. Therefore, calculate the overall phase change enthalpy. Thus, the phase change enthalpies from right to left are 124.9 J / g, 27.3 J / g, 110.3 J / g, and 2.4 J / g in turn.

[0033] (2). The total mass of the droplets is 5.160 mg. The calculated minimum water droplet mass is 0.0467 mg, and the volume is 0.0467 μL ≈ 46.7 nL. The freezing (icing) temperature of the 46.7-nanoliter water droplet is -27.1 ºC, and the supercooling degree is 27.1 ºC (0 ºC - (-27.1 ºC)) (as Figure 9 ).

[0034] Example 4: Supercooling Degree Test of 3.6-Nanoliter Water Drops (1). Take a sample of deionized water. When preparing the sample, place several sub-droplets of different sizes and independent of each other in the crucible, and conduct DSC tests on nanoliter droplets (such as Figure 1b , Figure 10 ). The test curve shows 5 exothermic peaks. The phase change enthalpies from right to left are 53.8 J / g, 112.4 J / g, 32.1 J / g, 28.0 J / g, and 4.0 J / g in sequence.

[0035] (2). The total mass of the droplets is 0.205 mg. It is calculated that the mass of the smallest water droplet (the sub-droplet corresponding to 4.0 J / g) is 0.0036 mg, and the volume is 0.0036 μL ≈ 3.6 nL. The freezing (icing) temperature of the 3.6-nanoliter water droplet is -36.5 ºC, and the supercooling degree is 36.5 ºC (0 ºC - (-36.5 ºC)) (such as Figure 10 ).

[0036] Example 5: Influence of Water Droplet Volume on Supercooling Degree

[0037] (1). By comparing the crystallization temperatures of water droplets with different volumes in Examples 1 - 4, it can be found that there is a direct relationship between the water droplet volume and the crystallization temperature, as shown in Figures 11a - 11b ; It can be seen from Figures 11a - 11b that as the volume or mass of the water droplet decreases, the crystallization temperature also decreases (the supercooling degree increases). There is a mutation near 20 nanoliters (0.02 mg), and the supercooling degree drops sharply, indicating that the influence of the water skin (the role played by the water skin in the whole water droplet) is getting larger and gradually dominates the supercooling behavior of the droplet.

[0038] In addition, the inventors of this case also referred to the foregoing examples and conducted tests with other raw materials, process operations, and process conditions described in this specification, and all obtained relatively ideal results.

[0039] It should be understood that the technical solutions of the present invention are not limited to the limitations of the above specific implementation cases. Any technical deformation made according to the technical solutions of the present invention without departing from the purpose of the present invention and the scope protected by the claims falls within the protection scope of the present invention.

Claims

1. A method for measuring the supercooling degree of a nanoliter droplet by differential scanning calorimetry, characterized in that, Including: Providing a liquid sample; And taking a liquid sample with a mass of more than 0.2 mg or a volume of more than 0.2 μL, then placing the liquid sample in a crucible to form a plurality of sub-droplets, and then performing DSC testing to obtain the phase change enthalpy of the plurality of sub-droplets, and calculating to further obtain the supercooling degree of the plurality of sub-droplets; wherein, the volume of at least one sub-droplet is less than 0.1 μL.

2. The method according to claim 1, wherein Specifically including: Modifying the surface of the crucible to be hydrophilic or hydrophobic, at least such that when the liquid sample is placed on the surface of the crucible, it does not spread and forms a plurality of independent spherical crown-shaped droplets.

3. The method according to claim 1, characterized in that: The liquid sample includes any one of water, glycerol, and salt solution.

4. The method according to claim 1, wherein Specifically including: Taking a liquid sample with a mass of more than 0.2 mg or a volume of more than 0.2 μL, then placing the liquid sample in a crucible to form a plurality of sub-droplets and each sub-droplet is independent of each other, and then performing DSC testing to obtain the phase change enthalpy of each sub-droplet.

5. The method according to claim 4, wherein Specifically including: Calculating the mass or volume of each sub-droplet based on the total mass or total volume of the liquid sample and the phase change enthalpy of each sub-droplet, and further obtaining the crystallization temperature of each sub-droplet.

6. The method according to claim 5, wherein Specifically including: Calculating the supercooling degree of each sub-droplet based on the crystallization temperature of each sub-droplet and the theoretical phase change temperature of the liquid sample, so as to realize the testing of the supercooling degree of nanoliter droplets.

7. The method according to claim 6, wherein: Establishing the relationship between the volume of each sub-droplet and the supercooling degree, so as to obtain the droplet phase change behavior law dominated by the liquid skin.

8. The method according to claim 1, characterized in that: The volume of at least one of the plurality of sub-droplets is less than 50 nL.

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