CN213434530U - Microfluidic device, microfluidic device and proteomics sample pretreatment platform - Google Patents
Microfluidic device, microfluidic device and proteomics sample pretreatment platform Download PDFInfo
- Publication number
- CN213434530U CN213434530U CN202020754619.2U CN202020754619U CN213434530U CN 213434530 U CN213434530 U CN 213434530U CN 202020754619 U CN202020754619 U CN 202020754619U CN 213434530 U CN213434530 U CN 213434530U
- Authority
- CN
- China
- Prior art keywords
- microfluidic device
- micro
- sample
- protein
- microfluidic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Abstract
The utility model provides a micro-fluidic device, a micro-fluidic device and a proteomics sample pretreatment platform, wherein the micro-fluidic device comprises a micro-fluidic chip and a reactor arranged in a micro-channel of the micro-fluidic chip, and the reactor comprises a protein sample pretreatment filler and a solid phase extraction membrane which are connected together; the micro-fluidic device integrates operations such as protein pre-enrichment, reduction, alkylation, enzymolysis, polypeptide desalting, elution, high pH value reverse phase fractionation and the like by taking air pressure as a driving force, and can be used for proteomic qualitative and quantitative analysis of a small amount of cell, tissue or body fluid (such as blood and urine) samples; the automatic proteomics sample pretreatment platform comprising the microfluidic device can realize automatic operation, and improve sample treatment flux and quantitative analysis accuracy.
Description
Technical Field
The utility model belongs to the technical field of proteomics, a micro-fluidic device, micro-fluidic device and proteomics sample pretreatment platform are related to.
Background
In proteomics research, after proteolysis of proteins in a sample into polypeptides, the polypeptides are used for liquid chromatography-mass spectrometry analysis to obtain protein information, and the separation and identification method is the most widely used proteomics research means at present.
Before proteomics analysis, complex sample pretreatment is required, which mainly comprises the following steps: cell lysis, protein component enrichment, protein disulfide bond opening and protection, protein enzymolysis, polypeptide component desalting and the like. In the current proteomics research strategy, the sample pretreatment steps are all manually completed, which greatly reduces the throughput of sample treatment, increases the loss of samples and reduces the reaction efficiency of related reactions. Therefore, a highly integrated and automated protein sample pretreatment technology is needed to reduce sample loss caused by manual operation to the maximum extent, improve system sensitivity, sample treatment flux and quantitative analysis accuracy, and simultaneously reduce workload of experimenters to the maximum extent and avoid experimental errors caused by human factors. To develop an automated proteomics analysis platform, new proteomics reactors need to be developed. The proteome reactor is a capillary filled with strong cation exchange resin, realizes the processes of enrichment, reduction, alkylation, enzymolysis and the like of proteins, and can identify 17 proteins from 300 cells (J.protein Res.2006,5, 2754-2759). The rare cell proteome reactor is based on a strong cation exchange resin capillary monolithic column, realizes the pre-enrichment, reduction, alkylation, enzymolysis and polypeptide fractionation of proteins, and identifies 409 and 2281 proteins from 5000 and 50000 cells respectively (mol. The two proteome reactors are both used for carrying out the protein treatment process in the capillary column, and the partial process of the protein sample pretreatment is integrated in the same device, so that the sample loss is effectively reduced. However, the packing is limited due to the influence of the inner diameter of the capillary column, so that the method is only suitable for processing a small amount of protein samples, and the processing process does not comprise processes such as polypeptide desalting and the like. An integrated proteomics technology is disclosed in anal. chem.2016,88,4864-4871, which realizes the whole process of pre-enrichment, reduction, alkylation, enzymolysis, polypeptide desalting, elution and high-pH reverse phase fractionation of proteins through a C18 membrane and an SCX filler filled in a pipette head, and can identify 1270 proteins from 2000 cells in a mass spectrum time of 1.4h and 7826 proteins from 100000 cells in a mass spectrum time of 22 h. Although this proteomics reactor works well, it requires the use of a centrifuge and is not suitable for a pneumatically driven automated proteomics sample pre-processing platform.
Therefore, the development of a novel microfluidic device which uses air pressure as a driving force and is applied to the pretreatment of proteomics samples is a problem to be solved at present.
SUMMERY OF THE UTILITY MODEL
Aiming at the defects of the prior art, the utility model aims to provide a micro-fluidic device, a micro-fluidic device and an automatic proteomics sample pretreatment platform, wherein the sample treatment process is driven by air pressure, and can realize the pretreatment of protein samples, including the whole processes of protein pre-enrichment, reduction, alkylation, enzymolysis, polypeptide desalting, elution and high pH value reverse phase classification, and can be used for proteomics qualitative and quantitative analysis of a small amount of cells, tissues or body fluid (such as blood and urine) samples; furthermore, the utility model discloses a micro-fluidic device can realize the automation mechanized operation, improves sample treatment flux and quantitative analysis accuracy.
In order to achieve the purpose of the utility model, the utility model adopts the following technical proposal:
an object of the utility model is to provide a micro-fluidic device, micro-fluidic device includes micro-fluidic chip and sets up the reactor in micro-fluidic chip's microchannel, the reactor is including the protein sample pretreatment filler and the solid phase extraction membrane that link together.
The utility model discloses in, the number of microchannel is 1-100, and when the number of microchannel be 2-100, a plurality of microchannels are independent each other separately, can include the reactor, also can not include the reactor, and the concrete selection of reactor can be the same, also can be different, and the technical staff in the art can adjust according to actual need.
The utility model discloses in, the material of micro-fluidic chip is resistant organic solvent's material, preferred polystyrene.
The utility model discloses in, micro-fluidic chip's shape is trapezoidal tetragonal body, the arbitrary one or at least two kinds's in cuboid or the cylinder combination, preferred trapezoidal tetragonal body.
In the present invention, the shape of the microchannel includes any one of a right circular truncated cone, a cylinder, or a rectangular parallelepiped, or a combination of at least two of them.
The utility model discloses in, the microchannel is including connecting positive round platform body section and the cylinder section that sets up, positive round platform body section includes cross-section end and bottom surface end, the cross-section end of positive round platform body section is connected with the cylinder section.
In the present invention, the reactor is disposed in the positive section of the microchannel.
The utility model discloses in, along bottom surface end to cross-section end direction, protein sample pretreatment filler and solid-phase extraction membrane have set gradually in the positive round body section.
In the present invention, the protein sample pretreatment packing comprises a strong cation exchange resin packing and/or a strong anion exchange resin packing, preferably a combination of a strong cation exchange resin packing and a strong anion exchange resin packing, the strong cation exchange resin packing being exemplarily selected from a sulfonic group strong cation exchange resin packing. The strong anion exchange resin filler is illustratively selected from quaternary ammonium based strong anion exchange resin fillers.
In the utility model discloses, the solid phase extraction membrane is C18 membrane.
A second object of the present invention is to provide a microfluidic device, comprising:
the fixer comprises a shell and an accommodating chamber positioned in the shell;
a microfluidic device for one of the purposes disposed in the holder-receiving chamber;
connecting pieces embedded at two ends of the fixer shell;
and the capillary tube penetrates through the connecting piece and is communicated with the micro-channel of the microfluidic device.
The utility model discloses a micro-fluidic device can realize the automation mechanized operation: for example, the microfluidic device can be used to automatically process samples at high throughput on an agilent capillary electrophoresis apparatus.
In the present invention, the housing is formed by a first housing and a second housing which are connected to each other in a butt joint manner, that is, the first housing and the second housing are detachably connected to each other.
The utility model discloses in, the surface of connecting piece is provided with the locating part, the locating part is used for restricting the fixer at the ascending relative movement of axial.
The utility model discloses in, the locating part is the boss, set up the recess with boss engaged with on the contact surface of fixer both ends and connecting piece, through the meshing effect restriction fixer of boss and recess is at the ascending relative movement of axial.
The utility model discloses a third of purpose provides an automatic change proteomics sample pretreatment platform, automatic change proteomics sample pretreatment platform includes closed system, the second of purpose micro-fluidic device and collecting system, closed system and collecting system all link to each other with the capillary at micro-fluidic device both ends.
In the utility model discloses in, the number of micro-fluidic device is 1-100, the number of micro-fluidic device is 2-100, parallelly connected setting between the different micro-fluidic devices.
The utility model discloses in, the closed system is including the airtight container that is provided with gas passage and the sample placer who is arranged in the airtight container, sample placer is used for placing the sample.
In the present invention, the collection system includes a collection device for collecting the sample after the reaction system.
Compared with the prior art, the utility model discloses following beneficial effect has:
(1) the utility model discloses a be applied to micro-fluidic device of proteomics sample pretreatment can accomplish operations such as the pre-enrichment of protein, reduction, alkylation, enzymolysis, polypeptide's desalination, elution and high pH value reverse phase are hierarchical through atmospheric pressure drive, realize the proteomics of a small amount of cells, tissue or body fluid (such as blood, urine) sample qualitative and quantitative analysis; furthermore, the utility model discloses a micro-fluidic device can realize the automation mechanized operation, improves sample treatment flux and quantitative analysis accuracy.
(2) The utility model discloses a micro-fluidic device can realize mass production, low cost.
(3) The utility model discloses a be applied to micro-fluidic device of proteomics sample pretreatment can be through parallelly connected multichannel reactors such as two passageways, four-channel or eight passageways of formation, or parallelly connected a plurality of microchannels and the reactor of setting up in a micro-fluidic chip, handles a plurality of protein samples simultaneously, improves the sample treatment flux.
Drawings
Fig. 1 is a schematic structural diagram of a microfluidic device according to an embodiment of the present invention;
fig. 2 is a schematic structural diagram of a microfluidic device according to an embodiment of the present invention;
figure 3 is a schematic structural diagram of an automated proteomics sample pre-processing platform in an embodiment of the present invention;
wherein 100 is a protein sample pretreatment filler, 101 is a solid phase extraction membrane, 102 is a microfluidic chip, 10 is a fixer, 11 is a microfluidic device, 12 is a connecting piece, 13 is a capillary tube, 14 is a limiting piece, 1 is a microfluidic device, 2 is a closed system, 3 is a collection system, 21 is a gas channel, 22 is a closed container, and 23 is a sample placing device;
FIG. 4 is a graph of the flow rate of the microfluidic device at different pressures in an automated reaction system of water through protein in example 1;
FIG. 5 is a flow rate chart of the microfluidic device in example 1 at a pressure of 3bar in an automated reaction system in which different solvents were passed through the protein;
FIG. 6 is a chromatogram of a sample of HEK 293T cells processed by the microfluidic device of example 1;
FIG. 7 is a graph of the enzymatic and alkylation efficiencies of the microfluidic device of example 1 for three consecutive treatments of a protein sample;
figure 8 is a statistical plot of the amount of proteins and polypeptides identified in plasma samples treated with the microfluidic device at unfractionated and high pH levels in example 1.
Detailed Description
The technical solution of the present invention will be further explained by the following embodiments. It should be understood by those skilled in the art that the described embodiments are merely provided to assist in understanding the present invention and should not be construed as specifically limiting the present invention.
The present embodiment provides a microfluidic device, as shown in fig. 1, as can be seen from fig. 1, the microfluidic device includes a microfluidic chip 102 and a reactor disposed in a microchannel of the microfluidic chip 102, the reactor includes a protein sample pretreatment packing 100 and a solid phase extraction membrane 101 connected together; the microchannel comprises a right circular truncated cone section and a cylinder section which are connected from left to right, the right circular truncated cone section comprises a section end and a bottom end, the section end of the circular truncated cone section is connected with the cylinder section, and the protein sample pretreatment filler and the solid-phase extraction membrane are positioned on the right circular truncated cone section of the microchannel.
The embodiment provides a microfluidic device, as shown in fig. 2, including a holder 10 (including a housing and an accommodating chamber), a microfluidic device 11 (where the microfluidic device 11 is the microfluidic device provided above) disposed in the accommodating chamber inside the holder, a connecting part 12 embedded at two ends of the holder, and a capillary 13 penetrating through the connecting part 12, where the capillary 13 is communicated with a microchannel of the microfluidic device 11, the housing is formed by butting a first housing and a second housing, an outer surface of the connecting part 12 is provided with a limiting part 14 for limiting relative movement of the holder 10 in an axial direction, where the limiting part is a boss, a contact surface between two ends of the holder 10 and the connecting part is provided with a groove engaged with the boss, and the relative movement of the holder 10 in the axial direction is limited by the engagement between the boss and the groove.
The embodiment also provides an automatic proteomics sample pretreatment platform, as shown in fig. 3, comprising a closed system 2, a microfluidic device 1 (wherein the microfluidic device is the microfluidic device provided above) and a collection system 3, wherein the closed system 2 and the collection system 3 are both connected with capillaries at two ends of the microfluidic device 1; the closed system 2 comprises a closed container 22 provided with a gas passage 21, and a sample placement device 23 located in the closed container 22.
Example 1
In this example, parameters of the microfluidic device were set: the micro-fluidic chip is a trapezoidal cuboid, the height is 8.57mm, the width is 7.00mm, the side length of the upper bottom surface is 5.35mm, and the side length of the lower bottom surface is 6.32 mm; the diameter of the bottom surface of the positive round body section is 1.46mm, the diameter of the cross section is 0.8mm, and the height is 7.27 mm; the diameter of the bottom surface of the cylinder section is 1.46mm, and the height is 1.3 mm; in the embodiment, the pretreatment filler of the protein sample is the combination of sulfonic strong cation exchange resin filler and quaternary ammonium strong anion exchange resin filler, and the dosage is 1 mg; the solid phase extraction membrane is a C18 membrane, and the number of layers is 5; the capillary had an inner diameter of 200 μm, an outer diameter of 360 μm and a length of 15 cm.
This embodiment utilizes this micro-fluidic device to test the volume of the water that flows through the protein reactor under the same time (2min) different pressures (2bar, 4bar, 6bar, 8bar, 10bar), and figure 4 is the velocity of flow curve graph of water when pressure and passing through the protein reactor in this embodiment, and can know from figure 4, the utility model discloses a micro-fluidic device's pressure and velocity of flow have fine linear relation.
In the embodiment, the microfluidic device is used for testing the flow rates of different solvents in the proteome reactor when the pressure is 3bar, and as can be seen from fig. 5, the flow rate of the solution with high contents of methanol, acetonitrile and acetonitrile is relatively high, and the flow rate of water is relatively low.
As can be seen from fig. 4 and 5, the flow rate of the solvent in the microfluidic device can be controlled by the pressure in this embodiment.
The embodiment also tests the enzymolysis efficiency of the protein sample (HEK 293T cell sample) processed by the microfluidic device, fig. 6 is a chromatogram peak diagram of the HEK 293T cell sample processed by the microfluidic device, and as can be seen from fig. 6, the chromatogram peak has narrow width and uniform distribution, which indicates that the enzymolysis efficiency of the sample is high.
The enzymolysis efficiency and the alkylation efficiency of the HEK 293T cell sample processed by the microfluidic device are tested for three times continuously, and as can be seen from fig. 7, the alkylation efficiency of three repeated experiments is close to 1, and the enzymolysis efficiency exceeds 97%.
As can be seen from fig. 6 and 7, the microfluidic device of the present embodiment has no influence on the enzymolysis effect when processing the protein sample, and has a good sample processing effect.
The microfluidic device of this embodiment and the existing SISPROT proteome reactor were used to process the same 10. mu.g plasma sample, and then the detection was performed by LC-MS, and the detection results are shown in Table 1:
TABLE 1
| Technique of | Identification of the amount of polypeptide | Identification of protein amounts |
| Proteome reactor of the present embodiment | 2550 | 215 |
| SISPROT proteome reactor | 2703 | 233 |
As can be seen from table 1, when 10 μ g of plasma samples were treated in the same way (without high pH reverse fractionation of the polypeptides), 2550 polypeptides and 215 proteins could be identified by the microfluidic device of this embodiment, which has a similar effect to the SISPROT proteome reactor, indicating that the microfluidic device of this embodiment has a better sensitivity.
The micro-fluidic device of the embodiment is used for processing the trace bovine serum albumin, and the detection result is as follows through liquid chromatography-mass spectrometry detection:
TABLE 2
As can be seen from table 2, the microfluidic device of the present embodiment can identify 5 polypeptides and a coverage of 7.58% when handling bovine serum albumin with a protein amount as small as 2ng, and can achieve a coverage of 80.81% when the protein amount reaches 2 μ g; the results show that the microfluidic device of the present embodiment also has a good effect on processing a trace amount of protein samples.
The microfluidic device in this embodiment also integrates high pH reverse phase fractionation of polypeptides, thereby increasing the amount of polypeptide and protein identification, as shown in fig. 3:
TABLE 3
| Grading | Identification of the amount of polypeptide | Identification of protein amounts |
| 3％ACN | 898 | 166 |
| 6％ACN | 1516 | 207 |
| 9％ACN | 1514 | 204 |
| 15％ACN | 2170 | 220 |
| 80％ACN | 1921 | 207 |
| Merging |
4154 | 317 |
As shown in Table 3, 20. mu.g of plasma samples were treated with the proteome reactor of the present embodiment, and the polypeptides were sequentially eluted using 5mmol/L ammonium formate solutions containing 3%, 6%, 9%, 15%, 80% acetonitrile at pH 10, and 5 fractions were performed, and finally 4154 polypeptides and 317 proteins were identified in total.
As shown in fig. 8, the amount of the polypeptide and the amount of the protein identified by the high pH reverse phase fractionation of the microfluidic device in this embodiment are 1.7 times and 1.5 times, respectively, as compared with those identified by the non-fractionation.
Example 2
The embodiment provides an automated proteomics sample pretreatment platform, and the microfluidic device in the automated proteomics sample pretreatment platform is the same as that in embodiment 1.
The embodiment also provides an application of the automated proteomics sample pretreatment platform in qualitative and quantitative analysis of protein samples, which comprises the following steps:
(1) diluting the plasma sample protein concentration of about 60 μ g/. mu.L to 1 μ g/. mu.L with 20mmol/L aqueous solution of N- (2-hydroxyethyl) piperazine-N-2 sulfonic acid (HEPES) having pH 7.4;
(2) transferring various required reagents into a reagent tube and placing the reagent tube into a closed container, opening a two-way valve, introducing nitrogen, pressurizing by using the nitrogen to enable a sample or the reagent in the reagent tube to flow through a microfluidic device, and respectively activating the microfluidic device by 80 mu L of methanol and 40 mu L of 10mmol/L HEPES (pH 7.4) aqueous solution; after activation, putting the protein sample with the concentration of 1 mug/muL into a reagent tube, pressurizing by nitrogen to enable the protein sample to flow through a microfluidic device, and enabling the protein sample to be enriched on the SCX/SAX mixed filler;
(3) the microfluidic device was washed with 10mmol/L HEPES aqueous solution containing 20% acetonitrile and pure acetonitrile, and 50mmol/L Dithiothreitol (DTT) in 20mmol/L HEPES (pH 8.0) aqueous solution was added to react at room temperature for 30 minutes to complete the reduction of the protein. Then, 20. mu.L of 20mmol/L HEPES (pH 8.0) is added to wash off DTT, 10mmol/L of iodoacetyl ammonium solution containing trypsin is added to react for 60 minutes at room temperature in the dark environment, and the alkylation and enzymolysis of protein are completed;
(4) the resulting polypeptide was transferred from the SCX/SAX mixed packing to a C18 membrane using 60. mu.L of 500mmol/L aqueous ammonium formate solution and 60. mu.L of 500mmol/L aqueous sodium chloride solution;
(5) adding 60 mu L of 1% formic acid aqueous solution for desalting, eluting the polypeptide from the C18 membrane by using 0.5% acetic acid aqueous solution containing 80% acetonitrile, collecting the eluted polypeptide in a collecting tube, placing the collecting tube in a freeze dryer for freeze drying, and redissolving the polypeptide in 0.1% formic acid aqueous solution, namely detecting by using liquid chromatography-mass spectrometry.
The amount of polypeptides and proteins detected in 10. mu.g plasma samples using liquid chromatography-mass spectrometry is shown in Table 4:
TABLE 4
| Number of experiments | Identification of the amount of polypeptide | Identification of protein amounts |
| |
2442 | 214 |
| |
2657 | 215 |
As can be seen from table 4, more than 2400 polypeptides and 210 proteins can be identified in two repeated experiments, which indicates that the microfluidic device applied to the proteomic sample pretreatment has good stability in the protein sample pretreatment by air pressure driving.
Example 3
The embodiment provides an automated proteomics sample pretreatment platform, and the microfluidic device in the automated proteomics sample pretreatment platform is the same as that in embodiment 1.
The embodiment also provides an application of the automated proteomics sample pretreatment platform in qualitative and quantitative analysis of protein samples, which comprises the following steps:
(1) a sample of HEK 293T cells was lysed using a compatible lysis buffer (the components of the lysis buffer included 10mmol/L HEPES, pH 7.4, 600mmol/L guanidine hydrochloride, 1% DDM, 1mmol/L Na)3VO4And protease inhibitor), adding 0.1% formic acid to acidify the sample solution to pH 2 after the cleavage is completed;
(2) and transferring various required reagents into the reagent tube and placing the reagents into a closed container, opening the two-way valve, introducing nitrogen, and pressurizing by the nitrogen to enable the sample or the reagent in the reagent tube to flow through the microfluidic device. The micro-fluidic device is firstly activated by 80 mu L of methanol, 20 mu L of 100mmol/L potassium citrate aqueous solution and 20 mu L of 10mmol/L potassium citrate aqueous solution respectively; after activation, putting a protein sample into a reagent tube, pressurizing by nitrogen to enable the sample to slowly flow through a microfluidic device, and enabling the protein to be enriched on the SCX/SAX combined filler;
(3) washing off the surfactant DDM bound to the C18 membrane with an 8mmol/L aqueous solution of potassium citrate containing 20% acetonitrile; then, adding a solution of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) of 10mmol/L, and reacting at room temperature for 15 minutes to complete the reduction of the protein; then 20 mu L of 20mmol/L HEPES is added, the pH value is 8.0, TCEP is washed off, 10mmol/L of iodoacetyl ammonium solution containing trypsin is added, and the reaction is carried out for 60 minutes at room temperature in a dark environment, so that the alkylation and the enzymolysis of the protein are completed;
(4) 60 mu L of 500mmol/L ammonium formate aqueous solution and 60 mu L of 500mmol/L sodium chloride aqueous solution are added to transfer the generated polypeptide from the SCX/SAX mixed filler to a C18 film;
(5) 60. mu.L of 1% aqueous formic acid was added to remove the salt. Finally, the polypeptide was eluted from C18 membrane 3 using 0.5% aqueous acetic acid containing 80% acetonitrile; the eluted polypeptide is collected in a collecting pipe, then is placed in a freeze dryer for freeze drying and then is redissolved in 0.1% formic acid aqueous solution, and then the detection can be carried out by liquid chromatography-mass spectrometry.
The amounts of polypeptide and protein detected by LC-MS are shown in Table 5:
TABLE 5
| Number of experiments | Identification of the amount of polypeptide | Identification of protein amounts |
| |
31244 | 4553 |
| |
31837 | 4426 |
| |
31855 | 4489 |
As can be seen from table 5, 30000 polypeptides and 4500 proteins can be identified in 3 repeated experiments, which indicates that the protein sample processed by the automated proteomic sample pretreatment platform of this example has good reproducibility.
Example 4
The embodiment provides an automated proteomics sample pretreatment platform, and the microfluidic device in the automated proteomics sample pretreatment platform is the same as that in embodiment 1.
The embodiment also provides an application of the automated proteomics sample pretreatment platform in qualitative and quantitative analysis of protein samples, which comprises the following steps:
(1) a sample of HEK 293T cells was lysed using a compatible lysis buffer (lysis buffer composition 10mmol/L HEPES, pH 7.4, 600mmol/L guanidine hydrochloride, 1% DDM, 1mmol/L Na)3VO4And protease inhibitor), adding 0.1% formic acid to acidify the sample solution to pH 2 after the cleavage is completed;
(2) and transferring various required reagents into the reagent tube and placing the reagents into a closed container, opening the two-way valve, introducing nitrogen, and pressurizing by the nitrogen to enable the sample or the reagents to flow through the microfluidic device. The micro-fluidic device is firstly activated by 80 mu L of methanol, 20 mu L of 100mmol/L potassium citrate aqueous solution and 20 mu L of 10mmol/L potassium citrate aqueous solution respectively; after activation, putting a protein sample into a reagent tube, pressurizing by nitrogen to enable the sample to slowly flow through a microfluidic device, and enabling the protein to be enriched on the strong cation/strong anion exchange resin mixed filler;
(3) washing off the bound C with an 8mmol/L aqueous potassium citrate solution containing 20% acetonitrile18Surfactant DDM on film 3; then, adding a solution of tris (2-carboxyethyl) phosphine hydrochloride (TCEP) of 10mmol/L, and reacting at room temperature for 15 minutes to complete the reduction of the protein; then 20 mu L of 20mmol/L HEPES is added, the pH value is 8.0, TCEP is washed off, 10mmol/L of iodoacetyl ammonium solution containing trypsin is added, and the reaction is carried out for 60 minutes at room temperature in a dark environment, so that the alkylation and the enzymolysis of the protein are completed;
(4) 60 mu L of 500mmol/L ammonium formate aqueous solution and 60 mu L of 500mmol/L sodium chloride aqueous solution are added to transfer the generated polypeptide from the SCX/SAX mixed filler to a C18 film;
(5) 60. mu.L of 5mmol/L ammonium formate aqueous solution was added for desalting. Finally, the polypeptide is eluted by using 5mmol/L ammonium formate solution with pH value of 10 and containing 3%, 6%, 9%, 15% and 80% acetonitrile respectively, namely high pH value reverse phase grading is carried out; the eluted polypeptide is collected in a collecting pipe, then is placed in a freeze dryer for freeze drying and then is redissolved in 0.1% formic acid aqueous solution, and then the detection can be carried out by liquid chromatography-mass spectrometry.
A 20 μ g sample of HEK 293T cell protein was treated and subjected to high pH reverse phase fractionation using the procedure described in this example, and the amounts of polypeptides and proteins identified are shown in table 6:
TABLE 6
| Grading | Identification of the amount of polypeptide | Identification of protein amounts |
| 3％ACN | 15501 | 4869 |
| 6％ACN | 24283 | 5694 |
| 9％ACN | 23068 | 5511 |
| 15％ACN | 30923 | 5860 |
| 80％ACN | 33707 | 5797 |
| Merging results | 92412 | 8010 |
As can be seen from Table 6, the amounts of the identified polypeptides and proteins in each fraction are relatively average, and finally, 92412 polypeptides and 8010 proteins are identified by combining the results, which indicates that the protein automated reaction system of the present embodiment has good sensitivity in processing protein samples.
Comparative example 1
The scheme of the comparative example is CN106770814A, and compared with the scheme of the example 1, in the comparative example 1, the sample enters the proteome reactor under the action of centrifugal force in a centrifugal mode, but the starting and the ending of the reaction cannot be controlled in time in the mode, and the centrifugal force has the function of throwing the sample to the inner wall surface of the pipette tip, so that the sample cannot easily enter the exchange resin filler, and the continuous reaction is not facilitated; in the embodiment 1, the microfluidic device can ensure the continuous reaction under the pushing of the air pressure, and in addition, the generation and the interruption of the reaction can be adjusted at any time by controlling the air pressure. In addition, comparative example 1 used only a strong cation exchange resin filler which could not effectively bind proteins having an isoelectric point below 2, resulting in sample loss; while the strong cation/strong anion exchange resin mixed filler is used in the embodiment 1, the two fillers have complementary action and can effectively bind protein, and the sample loss is hardly caused.
The applicant states that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and those skilled in the art should understand that any changes or substitutions easily conceivable by those skilled in the art within the technical scope of the present invention are within the protection scope and the disclosure scope of the present invention.
Claims (10)
1. The microfluidic device is characterized by comprising a microfluidic chip and a reactor arranged in a microchannel of the microfluidic chip, wherein the reactor comprises a protein sample pretreatment filler and a solid-phase extraction membrane which are connected together.
2. The microfluidic device according to claim 1, wherein the microfluidic chip is in the shape of any one of a trapezoid, a square, a cuboid or a cylinder, or a combination of at least two of the foregoing;
the shape of the microchannel comprises any one or combination of at least two of a right circular truncated cone, a cylinder or a cuboid;
the number of the micro-channels is 1-100.
3. The microfluidic device according to claim 2, wherein the microchannel comprises a right circular truncated cone section and a cylinder section arranged in connection, the right circular truncated cone section comprising a cross-sectional end and a bottom surface end, the cross-sectional end of the right circular truncated cone section being connected to the cylinder section;
the reactor is arranged on the right round platform section of the microchannel;
along the direction from the bottom surface end to the cross section end, a protein sample pretreatment filler and a solid phase extraction membrane are sequentially arranged in the positive round platform section.
4. The microfluidic device according to claim 1, wherein the protein sample pretreatment packing comprises a strong cation exchange resin packing and/or a strong anion exchange resin packing;
the solid phase extraction membrane is a C18 membrane.
5. A microfluidic device, characterized in that it comprises:
the fixer comprises a shell and an accommodating chamber positioned in the shell;
a microfluidic device according to any one of claims 1 to 4 disposed in the holder receiving chamber;
connecting pieces embedded at two ends of the shell of the fixer;
and the capillary tube penetrates through the connecting piece and is communicated with the micro-channel of the microfluidic device.
6. The microfluidic device according to claim 5, wherein the housing is formed by a first housing and a second housing in butt joint;
the outer surface of the connecting piece is provided with a limiting piece, and the limiting piece is used for limiting the relative movement of the fixator in the axial direction.
7. The microfluidic device according to claim 6, wherein the position-limiting member is a boss, a groove engaged with the boss is formed on a contact surface between the two ends of the holder and the connecting member, and the relative movement of the holder in the axial direction is limited by the engagement between the boss and the groove.
8. An automated proteomics sample pretreatment platform, comprising a closed system, the microfluidic device according to claim 5 and a collection system, wherein the closed system and the collection system are connected to capillaries at two ends of the microfluidic device.
9. The automated proteomic sample pre-processing platform of claim 8, wherein the number of the microfluidic devices is 1-100, the number of the microfluidic devices is 2-100, and different microfluidic devices are connected in parallel.
10. The automated proteomic sample pre-processing platform of claim 8, wherein the closed system comprises a closed container with a gas channel and a sample placement device in the closed container for placing a sample;
the collection system includes a collection device for collecting the reacted sample.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020754619.2U CN213434530U (en) | 2020-05-09 | 2020-05-09 | Microfluidic device, microfluidic device and proteomics sample pretreatment platform |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202020754619.2U CN213434530U (en) | 2020-05-09 | 2020-05-09 | Microfluidic device, microfluidic device and proteomics sample pretreatment platform |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN213434530U true CN213434530U (en) | 2021-06-15 |
Family
ID=76288913
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202020754619.2U Active CN213434530U (en) | 2020-05-09 | 2020-05-09 | Microfluidic device, microfluidic device and proteomics sample pretreatment platform |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN213434530U (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115184526A (en) * | 2022-05-31 | 2022-10-14 | 南方科技大学 | Mixed-mode-filler-based small-amount cell proteomics reactor and application thereof |
-
2020
- 2020-05-09 CN CN202020754619.2U patent/CN213434530U/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115184526A (en) * | 2022-05-31 | 2022-10-14 | 南方科技大学 | Mixed-mode-filler-based small-amount cell proteomics reactor and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Freire et al. | Proteome-on-a-chip: mirage, or on the horizon? | |
| Lion et al. | Why the move to microfluidics for protein analysis? | |
| Massolini et al. | Immobilized trypsin systems coupled on‐line to separation methods: Recent developments and analytical applications | |
| WO2006080186A1 (en) | Detection device using cartridge | |
| Ekström et al. | On‐chip microextraction for proteomic sample preparation of in‐gel digests | |
| EP1688739A1 (en) | Devices, systems and methods for separation | |
| US20060219637A1 (en) | Devices, systems and methods for liquid chromatography | |
| US11198118B2 (en) | Integrated modular unit containing one or more analyte concentrator-microreactor devices to be coupled to a cartridge-cassette and methods of operation | |
| JP2902111B2 (en) | Disposable reaction vessel for solid-phase immunoassay and method for measuring components detectable by immunoreaction | |
| EP2972331A1 (en) | Microfluidic distributing device | |
| WO2015072265A1 (en) | Capillary device for separation and analysis, microfluidic chip for separation and analysis, protein or peptide analysis method, electrophoresis device, and microfluidic chip electrophoresis device for separation and analysis | |
| Hušek et al. | Advances in amino acid analysis | |
| Wouters et al. | A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots | |
| US6475799B1 (en) | Electrochemical analysis system | |
| CN111437896A (en) | Microfluidic device and application thereof | |
| CN213434530U (en) | Microfluidic device, microfluidic device and proteomics sample pretreatment platform | |
| Tůma | Progress in on-line, at-line, and in-line coupling of sample treatment with capillary and microchip electrophoresis over the past 10 years: A review | |
| Roberg-Larsen et al. | Recent advances in on-line upfront devices for sensitive bioanalytical nano LC methods | |
| CN101685051A (en) | Open-tubular capillary column enriching phosphoeptide or phosphorylated protein and method | |
| US20190317059A1 (en) | High throughput body fluid protein sample preparation device and applications thereof | |
| Lee et al. | Development of an efficient on-chip digestion system for protein analysis using MALDI-TOF MS | |
| JP2007163252A (en) | Measuring cartridge for liquid chromatograph, and liquid chromatograph device | |
| WO2017166897A1 (en) | Proteomic reactor, protein chromatographic separation platform and use thereof | |
| Wallman et al. | Autonomous protein sample processing on‐chip using solid‐phase microextraction, capillary force pumping, and microdispensing | |
| Chen et al. | The combination of flow injection with electrophoresis using capillaries and chips |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| GR01 | Patent grant | ||
| GR01 | Patent grant |