Microdroplet reactor for ultra-low volume sample processing and methods of use

Abstract

This invention discloses a microdroplet reactor for ultra-micro sample processing, comprising: a reaction vessel for holding sample microdroplets; reagents placed inside the reaction vessel to maintain humidity; a sealing sheet for sealing the reaction vessel; a retainer for ensuring a tight seal between the sealing sheet and the reaction vessel; and a temperature controller placed at the bottom of the reaction vessel for regulating the temperature inside the reaction vessel. This invention also discloses a method for performing multi-step biochemical reactions of ultra-micro samples based on the microdroplet reactor. The microdroplet reactor of this invention features simple structure, low cost, high-throughput scalability, and good instrument compatibility. The method of use offers advantages such as low sample and reagent consumption, high reaction efficiency, minimal reaction loss, and no reagent contamination. It is suitable for fields such as micro-sample analysis, single-cell analysis, protein analysis, and nucleic acid analysis, and is particularly suitable for single-cell proteomics analysis.

Description

Technical Field

[0001] This invention relates to the field of microfluidic biochemical analysis technology, specifically to a microdroplet reactor for ultra-micro sample processing and its usage method. Background Technology

[0002] The processing and characterization of ultra-micro (microgram-level) biochemical samples has always been a significant challenge in the field of biochemical analysis. With the development of high-precision biochemical analysis techniques such as single-cell analysis, the amount of sample being analyzed is becoming increasingly smaller, rendering conventional reaction vessels such as centrifuge tubes and test tubes inadequate for handling ultra-micro samples. In contrast, droplet microfluidics technology utilizes two immiscible liquid phases to generate dispersed microdroplets for experimental operations. Droplet volumes are typically in the nanoliter to picoliter range, making it particularly suitable for the analysis and detection of valuable samples. Microdroplet reactions offer numerous advantages, including low sample consumption, minimal cross-contamination, rapid reagent mixing, high reaction rates, good operational accuracy, and high reproducibility, and are currently widely used in various biochemical analysis fields such as single-cell analysis, enzyme analysis, protein analysis, and nucleic acid analysis.

[0003] Current droplet microfluidics mainly generate microdroplets by applying a certain external force to disrupt the tension balance between the water and oil phases. These methods include T-channel method, flow focusing method, co-flow focusing method, electric field driving method, optical control method, and microvalve driving method.

[0004] For example, the patent specification with publication number CN105363503B discloses a multi-component microdroplet microfluidic chip, which consists of a capping layer, a hydrophobic chip layer, a hydrophilic chip layer, and a substrate layer stacked sequentially. The capping layer includes five reagent inlets: one aqueous phase A inlet, two aqueous phase B inlets from the capping layer itself, and two oil phase inlets. The hydrophobic chip layer is a focused flow microchannel structure, including one aqueous phase A inlet channel, two oil phase inlet channels, and one water-in-oil microchannel. The droplet generation chambers are connected by a cross-shaped focused flow microdroplet generation channel; they also include two aqueous phase B inlets of the hydrophobic chip layer, which cooperate with the capping layer; the hydrophilic chip layer is a focused flow microchannel structure, including one microdroplet introduction channel, two aqueous phase B introduction channels, and one water-in-oil multi-component microdroplet generation chamber, which are connected by a cross-shaped focused flow water-in-oil multi-component microdroplet generation channel; the substrate layer includes one water-in-oil multi-component microdroplet collection outlet.

[0005] The methods described above can flexibly and conveniently generate a large number of stable microdroplets, and can also achieve various biochemical reaction operations such as sample extraction and reagent addition through droplet splitting, fusion, internal mixing, and capture. However, since most of these methods require the use of oil-phase solutions such as mineral oil or fluorinated oil to disperse the droplets, some lipophilic samples may dissolve in the oil, resulting in sample loss. Furthermore, when extracting reactants from microdroplets, a small amount of oil-phase solution is inevitably introduced, which not only interferes with the separation of analytical instruments such as liquid chromatography, but also reduces the electrospray efficiency of high-resolution mass spectrometry systems, thus affecting sample identification and analysis.

[0006] Therefore, developing an oil-free, flexible microdroplet reactor and performing continuous multi-step reaction processing in situ within the microdroplets is of great significance for the precise biochemical analysis of ultra-micro samples. Summary of the Invention

[0007] One objective of this invention is to provide a microdroplet reactor for ultra-micro sample processing. This reactor achieves a multi-step reaction process of microdroplets based on the flexible opening and closing of a sealing sheet, without introducing contaminants such as mineral oil, and is compatible with common analytical instruments such as liquid chromatography and mass spectrometry.

[0008] A microdroplet reactor for ultra-micro sample processing, comprising:

[0009] Reaction vessel, used to hold sample microdroplets;

[0010] The reagents are placed inside the reaction vessel to maintain humidity;

[0011] A sealing strip, used to seal the reaction vessel;

[0012] A retainer is used to ensure that the sealing sheet is sealed and fitted tightly to the reaction vessel;

[0013] A temperature controller is placed at the bottom of the reaction vessel to regulate the temperature inside the reaction vessel.

[0014] Preferably, the reaction vessel is a centrifuge tube, sample vial cap, multi-well plate, or microchip array material, preferably one that is simple, readily available, inexpensive, and does not interact with the sample and reagents. More preferably, when the sample quantity is small, the cap of a low-protein-adsorption centrifuge tube is used as the reaction vessel; when the sample quantity is large, a multi-well plate is used as the reaction vessel.

[0015] Preferably, the reagent is deionized water, ammonium bicarbonate solution, or phosphate buffer solution. It typically has a lower ion concentration than the sample solution and is primarily used for preferential evaporation during heating, maintaining the saturated vapor pressure within the reaction vessel, preserving humidity, and inhibiting excessively rapid evaporation of the sample droplets.

[0016] Preferably, the sealing sheet is made of transparent tape, silicone gasket, sealing film, or aluminum foil. More preferably, transparent tape is used as the sealing sheet, and a roller structure is used to automatically seal and open the sealing sheet.

[0017] Preferably, the retainer is a metal weight, a plastic clip, or a clamp. More preferably, a metal weight is used to press the sealing sheet down by gravity, preventing the sealing sheet from detaching due to increased pressure inside the reaction vessel during heating.

[0018] Preferably, the temperature controller uses a heating element, a cooling element, a constant temperature chamber, or an oven. More preferably, a combination of a heating element and a cooling element is used to construct the temperature controller.

[0019] One object of the present invention is to provide a method for multi-step biochemical reactions of ultra-micro samples based on the above-mentioned microdroplet reactor, the method comprising the following steps:

[0020] (1) Use a liquid manipulation probe to drop a nano-sized sample and reagent into the center of the reaction vessel, or to aspirate a portion of the reaction solution from it, and carry out one or more steps of reaction in situ.

[0021] (2) Add a moisturizing agent to the inner edge of the reaction vessel away from the sample reaction droplets, and replenish the moisturizing agent as needed during the multi-step reaction process;

[0022] (3) Before and after each reaction step, open the sealing sheet to add reagents or remove the reaction solution, and close the sealing sheet to seal the reaction container.

[0023] (4) Fix the sealing sheet to control the temperature of the sample reaction environment.

[0024] This method has the advantages of low sample and reagent consumption, high reaction efficiency, low reaction loss, and no reagent contamination. It is suitable for fields such as trace sample analysis, single-cell analysis, protein analysis, and nucleic acid analysis, and is especially suitable for single-cell proteomics analysis.

[0025] Adding a humidifying agent droplets to the inner edge of the reaction vessel primarily aims to increase the humidity within the reaction space, preventing the nanoliter-sized reaction droplets from evaporating too quickly. Preferably, 2.5–10 μL of deionized water can be added. According to Raoult's law, at the same temperature, the saturated vapor pressure of the humidifying agent is higher than that of the sample reaction solution containing a higher solute concentration. Therefore, after the vapor pressure inside the reaction vessel reaches the saturated vapor pressure of the sample reaction solution, the humidifying agent will continue to evaporate because it has not yet reached its saturated vapor pressure. This protects the sample droplets from drying out under extreme conditions such as high temperatures, ensuring that the reactants in the sample droplets remain in a high-concentration ionic state, resulting in high reaction efficiency and low adsorption and phase transition losses. Practice has shown that low-concentration deionized water droplets can effectively compensate for the evaporation of the sample droplets.

[0026] Methods for opening and closing the sealing sheet include, but are not limited to, tape sealing and compression sealing. Preferably, transparent tape with a width slightly larger than the diameter of the reaction vessel opening can be used for sealing. Rollers are used to press the transparent tape to promote a complete seal; the rollers can be driven by a motor for automated operation. Using transparent tape sealing is not only cost-effective and easy to operate, but also allows for real-time observation of the sample's reaction status and assessment of the reactor's sealing effect based on the amount of water mist.

[0027] The methods for securing the sealing sheet include, but are not limited to, using gravity to press it down or clamping it, with the main purpose of preventing air leakage from the sealing sheet due to increased gas pressure inside the reaction vessel during heating, reaction, or other processes. Preferably, the sealing sheet can be secured with a heavy object.

[0028] The beneficial effects of this invention are:

[0029] (1) The microdroplet reactor in this invention can maintain the droplet shape without the intervention of the oil phase. It can maintain the micro-volume required for efficient reaction of micro-samples, and will not cause loss of lipophilic solutes. Moreover, it will not introduce oil phase contamination and can be compatible with commonly used analytical instruments such as liquid chromatography and mass spectrometry.

[0030] (2) The microdroplet reactor in this invention can be opened and closed flexibly and conveniently. Reagents can be added freely according to actual needs, or reaction solutions can be drawn. It is very suitable for long-chain complex reaction processes with small sample amounts, many reaction steps, and real-time monitoring of the reaction process. It is especially suitable for biochemical analysis of ultra-micro samples.

[0031] (3) The droplet moisturizing method in this invention can effectively reduce the evaporation of the sample solution. Even under high temperature conditions, it can ensure that the sample droplets do not evaporate, so that the sample solution always maintains a highly active and highly concentrated ion solution reaction state. Compared with conventional reactors, it not only greatly improves the sample reaction efficiency, but also reduces the surface adsorption and phase transition losses of the sample.

[0032] (4) Because conventional reaction vessels produced in batches are used, or even centrifuge tube caps discarded in routine operations are used to build reactors, the cost is low. It is easy to select appropriate materials to build matching microdroplet reactors according to actual application scenarios. It is also easy to use arrayed structures and automated systems to improve the throughput and automation level of sample analysis. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the microdroplet reactor structure based on a centrifuge tube cap in Example 1;

[0034] Figure 2Images of the sample state after heating the microdroplet reactor based on centrifuge tube cap in Example 1 at 95°C for 30 minutes and 37°C for 2 hours, respectively.

[0035] Figure 3 This is a schematic diagram of the automated microdroplet reactor structure based on a porous plate in Example 2;

[0036] In the diagram: 1. Sample reaction droplet; 2. Deionized water droplet; 3. Tube cap; 4. Transparent tape; 5. Silicone gasket; 6. Metal block; 7. Temperature controller; 8. Porous plate; 9. Liquid manipulation probe; 10. Sealing film; 11. Roller; 12. Mechanical rod. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] like Figure 1 As shown, a microdroplet reactor based on a centrifuge tube cap includes a tube cap 3 for carrying a low protein adsorption centrifuge tube for holding the sample, a transparent tape 4 for sealing the tube cap 3, a silicone gasket 5 for ensuring the transparent tape 4 adheres tightly, a metal block 6 for fixing the transparent tape 4 by gravity, and a temperature controller 7 located at the bottom of the tube cap 3; sample reaction droplets 1 and deionized water droplets 2 for maintaining humidity inside the tube cap 3 are added to the tube cap 3.

[0040] The cap 3 can be cut from a 1.5 ml centrifuge tube with low protein adsorption and is used to hold the sample reaction droplet 1; the transparent tape 4 is slightly wider than the opening diameter of the cap 3 so as to seal tightly; the temperature controller 7 can be composed of heating or cooling plates, or temperature control instruments such as constant temperature chambers or ovens can be used to control the internal temperature of the microreactor.

[0041] Taking single-cell proteomics sample processing as an example, the method is as follows:

[0042] like Figure 1The microdroplet reactor based on a centrifuge tube cap was constructed as shown. Using a liquid manipulation probe, three drops of 2.5 μL deionized water (2) were added to the inner edge of the cap (3). Next, a 100 nm sample droplet (1) containing a single test cell was added to the center of the cap (3), followed by the addition of the first reaction reagent—a cell lysis agent. Transparent tape (4) was affixed to the top of the cap (3), and then a silicone gasket (5) and a metal block (6) were sequentially covered, ensuring a tight seal and preventing the tape from easily falling off. The sealed cap (3) was then placed on a temperature controller (7) for a controlled reaction time. Remove the metal block 6, silicone pad 5, and transparent tape 4. Repeat the above operation, adding reducing reagent, alkylating reagent, enzymatic digestion reagent, isotope labeling reagent, and reaction termination reagent in the same manner to complete the multi-step sample pretreatment operation, including cell lysis, reduction, enzymatic digestion, isotope labeling, and reaction termination. The volume of reagent added in each step is 100 nanoliters. The reaction temperature varies from 15°C to 95°C, and the reaction time varies from 20 minutes to 2 hours. After each reaction, deionized water is added as needed based on the evaporation of deionized water droplets 2.

[0043] like Figure 2 As shown, after heating at 95°C for 30 minutes and maintaining a constant temperature at 37°C for 2 hours, the deionized water droplet 2 partially evaporated and condensed on the inner surface of the transparent tape 4, but the sample reaction droplet 1 was never completely evaporated.

[0044] After sample pretreatment, the sample reaction droplet 1 is aspirated using a liquid manipulation probe, and its contact surface is repeatedly rinsed. The solution is then transferred to a commercially available end-tube or vial. Chromatographic separation and mass spectrometric detection of the reaction solution are performed using a liquid chromatography-mass spectrometry (LC-MS) system. Using this microdroplet reactor for single-cell sample pretreatment, over 1000 proteins can be quantitatively identified from a single HeLa cell in a label-free manner.

[0045] Example 2

[0046] like Figure 3 As shown, an automated microdroplet reactor based on a porous plate includes a porous plate 8, a liquid manipulation probe 9 for adding and extracting samples and reagents, a sealing membrane 10 for sealing the pores of the porous plate 8, a roller 11 for unfolding and flattening the sealing membrane 10, a mechanical rod 12 for pushing the roller forward and backward, and a temperature controller 7 located at the bottom of the porous plate 8; each pore of the porous plate 8 contains a sample reaction droplet 1 and a deionized water droplet 2 for maintaining the humidity inside the pores of the porous plate 8.

[0047] The size and number of pores in the multi-well plate 8 can be selected or customized according to actual usage requirements; the liquid manipulation probe 9 is a capillary probe, or a droplet ejector or a commercial pipette can also be used, and it can also be arranged in an array structure for batch pipetting operations; the mechanical rod 12 pushes the roller 11 under the drive of the motor to control the sealing film 10 to seal and open each reaction pore of the multi-well plate 8.

[0048] Taking the screening of drug combinations using trace cells as an example, the method is as follows:

[0049] like Figure 3 The diagram illustrates an automated microdroplet reactor based on a multi-well plate. Using a droplet manipulation probe 9, 10 μL of deionized water droplets 2 are added to one edge of each well in the multi-well plate 8. Then, using the probe 9, appropriate amounts of sample cell suspension and corresponding drug reagents are sequentially added to each reaction well, generating sample reaction droplets 1. A mechanical rod 12 drives the movement of rollers 11 on both sides, ensuring that all wells except the one to be processed are always sealed by a sealing membrane 10. At regular intervals, appropriate amounts of deionized water, drug reagents, and culture medium are added. Throughout the reaction, a temperature controller 7 maintains the reaction environment at a suitable temperature. By comparing the effects of different drug types, combinations, and dosages on cell viability, suitable drug formulations can be rapidly screened for specific cell types, showing particularly good application results for precious clinical cell samples.

[0050] This invention provides a low-cost, high-efficiency, pollution-free, low-operation-barrier, and highly compatible method for fabricating and using a microdroplet reactor. Compared to microdroplet reactors involving an oil phase, this microdroplet reactor exhibits less sample loss, no oil phase contamination, and does not affect processes such as liquid chromatography separation and mass spectrometry spraying. It also boasts good instrument compatibility and can withstand harsh temperature fluctuations. Compared to customized microfluidic chips, this microdroplet reactor is simpler to fabricate, lower in cost, and more compatible with instruments. It can also be flexibly opened and closed, allowing for reagent addition and reaction monitoring, making it suitable for continuous, multi-step, long-chain, complex reaction processes. Therefore, the flexibly openable and closable microdroplet reactor and its usage method in this invention are highly suitable for long-chain biochemical processing of ultra-micro samples, and can be applied in fields such as single-cell analysis, microbial research, and multi-omics analysis, especially in the field of single-cell proteomics analysis.

[0051] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A microdroplet reactor for ultra-micro sample processing, characterized in that, include: The reaction vessel is used to hold sample microdroplets, the volume of which is nanoliters. A humidifying agent is placed inside the reaction vessel to maintain humidity, and the ion concentration of the humidifying agent is lower than that of the sample microdroplets; A sealing strip, used to seal the reaction vessel; A retainer is used to ensure that the sealing sheet is sealed and fitted tightly to the reaction vessel; A temperature controller is placed at the bottom of the reaction vessel to regulate the temperature inside the reaction vessel.

2. The microdroplet reactor according to claim 1, characterized in that, The reaction vessel is a centrifuge tube, a sample vial cap, a multi-well plate, or a microporous chip.

3. The microdroplet reactor according to claim 1, characterized in that, The moisturizing agent is deionized water, ammonium bicarbonate solution, or phosphate buffer solution.

4. The microdroplet reactor according to claim 1, characterized in that, The sealing sheet can be made of transparent tape, silicone gasket, sealing film or aluminum foil.

5. The microdroplet reactor according to claim 1, characterized in that, The fastener uses a metal weight, plastic buckle, or clip.

6. The microdroplet reactor according to claim 1, characterized in that, The temperature controller uses a heating element, a cooling element, a constant temperature chamber, or an oven.

7. A method for performing multi-step biochemical reactions of ultra-micro samples, characterized in that, The method using the microdroplet reactor for ultra-micro sample processing according to any one of claims 1 to 6 includes the following steps: (1) Use a liquid manipulation probe to drop a nano-sized amount of sample and reagent into the center of the reaction vessel, or to aspirate a portion of the reaction solution to carry out one or more steps of reaction in situ. (2) Add a moisturizing agent to the inner edge of the reaction vessel away from the sample reaction droplets, and replenish the moisturizing agent as needed during the multi-step reaction process; (3) Before and after each reaction step, open the sealing sheet to add reagents or aspirate the reaction solution, and close the sealing sheet to seal the reaction container. (4) Fix the sealing sheet to control the temperature of the sample reaction environment.

8. The method according to claim 7, characterized in that, Add 2.5 to 10 microliters of deionized water in step (2).

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