CN110579527A - An electrophoresis microchip with an ion online enrichment device and its detection method - Google Patents

Abstract

the invention discloses an electrophoresis microchip with an ion online enrichment device, which comprises an electrophoresis microchip body and a sample injection pool arranged in the electrophoresis microchip body, wherein the sample injection pool is communicated with a sample enrichment pool through an ion enrichment flow channel, and the sample enrichment pool is communicated with a sample waste liquid pool through an ion sampling flow channel; the buffer solution injection pool and the buffer solution waste liquid pool are communicated through an ion separation flow channel; the ion sampling flow channel and the ion separation flow channel are vertically crossed and communicated with each other; the volume of the sample injection pool is larger than that of the sample enrichment pool, and the ion enrichment flow channel is positioned at the position where the distance between the sample injection pool and the sample enrichment pool is shortest. Also discloses a detection method using the electrophoresis microchip. The invention innovatively adopts a twice enrichment structure and a bell mouth shape, greatly improves the ion detection sensitivity of the electrophoresis microchip, and enables ions to be rapidly enriched, separated and detected on the electrophoresis microchip.

Description

An electrophoresis microchip with an ion online enrichment device and its detection method

technical field

The invention relates to the field of microfluidic chip electrophoresis, in particular to an electrophoresis microchip with an ion online enrichment device and a detection method.

technical background

Ion detection technology plays a vital role and significance in many fields such as clinical medicine, water quality monitoring, soil survey, food and drug quality management. For example, K ⁺ , Na ⁺ , Li ⁺ , NH ₄ ⁺ , Ca ²⁺ , Mg ²⁺ ions in soil are important indicators for soil survey; too much NH ₄ ⁺ , PO ₄ ⁺ ions in water will cause In eutrophication, the algae in the water body multiply rapidly, and other water body organisms die in large numbers, thus destroying the ecological balance; while heavy metal ions such as Cd ²⁺ and Pb ²⁺ in the water body and soil will gradually accumulate in the food chain, and eventually be brought into the human body to cause diseases . Due to the low concentration of ions in the environment, liquid chromatography, optical detection and mass spectrometry are mostly used for ion detection at present, but these three methods all rely on expensive equipment, and are time-consuming, difficult to integrate, and detection functions. Single and other shortcomings, which greatly limit the wide application of ion detection technology. At this time, microfluidic chip electrophoresis technology stands out in the field of ion detection due to its advantages of rapid detection, less reagent consumption, easy integration, and simultaneous detection of multiple ions. However, microfluidic electrophoresis chip technology has great limitations because of its low detection sensitivity. Therefore, there is an urgent need in this field for an electrophoretic microchip with the function of enriching ions on a chip.

At present, the commonly used enrichment methods are mainly divided into online enrichment and offline enrichment. Offline enrichment is mainly extraction technology, which has a large enrichment factor, but requires a long enrichment time. The online enrichment techniques are mainly field amplification enrichment, sweeping technique and isotachophoretic enrichment. Among them, isotachophoretic enrichment technology and sweeping technology, although the enrichment efficiency is high and the enrichment multiple is large, but the chip production is difficult, the cost is high, and the operation is cumbersome. Although the field amplification enrichment technology has the advantages of no need for other equipment, low cost and simple operation, it also has the disadvantages of low enrichment efficiency and small enrichment multiples. The electrophoresis microfluidic chip and detection method adopted in the present invention have the advantages of short enrichment time, high enrichment efficiency, large enrichment multiple, simple chip fabrication, low cost and simple operation.

Contents of the invention

The purpose of the present invention is to provide an electrophoresis microchip and detection method with an ion online enrichment device. The microelectrophoresis microchip and method solve the above-mentioned deficiencies in the prior art, and achieve ion enrichment, separation and detection in a short time. detection.

For reaching above-mentioned purpose, the technical solution step that the present invention takes is as follows:

An electrophoresis microchip with an ion online enrichment device, comprising an electrophoresis microchip body and a sample injection pool, a sample waste pool, a buffer injection pool, and a buffer waste pool arranged in the electrophoresis microchip body, the The sample injection pool communicates with the sample enrichment pool through the ion enrichment flow channel, and the sample enrichment pool communicates with the sample waste liquid pool through the ion sampling flow channel; the buffer injection pool and the buffer waste liquid pool are connected by ion The separation flow channel is connected; the ion sampling flow channel is vertically intersected with the ion separation flow channel and communicates internally; the volume of the sample injection pool is larger than the volume of the sample enrichment pool, and the ion enrichment flow channel is located in the sample The shortest distance between the injection pool and the sample enrichment pool.

In a further solution, the volume ratio of the sample injection pool to the sample enrichment pool is not greater than 12:1; the sample injection pool is a semicircular arc structure, and there are three ion enrichment flow channels, and the three ion enrichment channels The enrichment flow channel and the ion sampling flow channel are arranged in a cross.

The number of ion-enrichment flow channels is at least one, preferably three, and the sample injection pool can be in other shapes, such as elliptical, special-shaped structures, etc., as long as the length of the ion-enrichment flow channels is within the allowable range of processing conditions. the better. In this device, the sample injection pool is designed as a semi-circular arc structure, the purpose is to make the lengths of the three enrichment flow channels equal, and the short enrichment flow channels will increase the enrichment efficiency.

The volume of the sample injection pool is larger than the volume of the sample enrichment pool, and the ions in the sample solution migrate from the large-volume sample injection pool to the small-volume sample enrichment pool to form the first enrichment of ions. In order to obtain a better ion enrichment effect, the volume ratio of the two should not be greater than 12:1. If it is greater than 12:1, a saturation effect will appear.

In a further solution, the connection between the sample injection pool and the ion enrichment flow channel is in the shape of an outwardly protruding bell mouth, and the connection between the sample enrichment pool and the ion enrichment flow channel is in the shape of an inverted bell mouth protruding inward.

The design of this bell mouth shape is to ensure that the flow resistance of ions from the sample injection cell to the sample enrichment cell is small, and the flow resistance of the opposite direction is relatively large, so that a lower enrichment voltage can obtain a higher The speed of ion enrichment also reduces the diffusion of ions from high concentration to low concentration.

In a further scheme, the concentration of the low-concentration buffer A in the sample injection pool, the sample enrichment pool and the ion enrichment flow channel is less than that in the sample waste liquid pool, buffer injection pool, buffer waste liquid pool, ion sampling flow channel and The concentration of high-concentration buffer B in the ion separation flow channel forms a concentration gradient to enrich ions.

In a further scheme, the concentration ratio of the high-concentration buffer B to the low-concentration buffer A is 2:1-10:1.

The concentration ratio is generally 2:1-10:1. If it is less than 2:1, there will be no obvious enrichment effect. If it is greater than 10:1, it will cause electroosmotic flow disturbance, cause laminar flow effect, and reduce the enrichment effect.

In a further solution, the body of the electrophoresis microchip consists of a micropipe layer, an insulating layer, and an electrode layer from top to bottom; the ion enrichment flow channel, ion sampling flow channel and ion separation flow channel are arranged in the micropipe layer, The electrode layer is provided with an ion detection electrode, and the ion detection electrode is composed of a transmitting electrode and a receiving electrode arranged at the tail of the ion separation flow channel, and the transmitting electrode and the receiving electrode are arranged in parallel.

In a further scheme, each of the sample injection pool, the sample waste pool, the buffer injection pool and the buffer waste pool is provided with a high-voltage electrode; the sample enrichment pool is provided with at least one high-voltage electrode; wherein the sample enrichment The high-voltage electrodes in the collection pool and the sample injection pool are connected to form a high voltage enrichment, the high-voltage electrodes in the sample enrichment pool and the sample waste pool are connected to form a high-voltage sample injection, and the high-voltage electrodes in the buffer injection pool and the buffer waste pool The connection creates a separate high voltage.

Another object of the present invention is to provide a detection method using the above-mentioned electrophoresis microchip, comprising the following steps:

(1) Prepare two buffer solutions with different concentrations, that is, low concentration buffer A and high concentration buffer B, and dissolve the sample to be tested in low concentration buffer A to make a sample solution;

(2) Add high-concentration buffer B into the sample waste pool, buffer injection pool, and buffer waste pool, and fill the ion injection channel and ion separation channel; add low-concentration buffer A into the sample rich pool, and fill the ion enrichment flow channel; add the sample solution into the sample injection pool; fill it all up;

(3) Enrichment high pressure is applied between the sample enrichment pool and the sample injection pool, so that the ions in the sample solution in the sample injection pool migrate to the sample enrichment pool to achieve the first enrichment of ions;

(4) Apply high injection pressure between the sample enrichment pool and the sample waste pool, and the ions in the sample solution migrate from the sample enrichment pool to the sample waste pool through the ion injection flow channel. The step surface of high concentration buffer B forms the second enrichment of ions;

(5) When the ions reach the intersection of the ion injection flow channel and the ion separation flow channel, turn on the separation high pressure, that is, apply the separation high pressure between the buffer injection pool and the buffer waste pool, and the ions in the sample solution will detect the ion The electrode migrates, is detected by the ion detection electrode, and enters the buffer waste reservoir.

The ions flow from the low-concentration sample enrichment pool to the high-concentration ion sampling flow channel, and are enriched for the second time on the solution concentration step surface, and the enriched ions continue to flow to the sample waste pool under the influence of the injection voltage , the separation voltage is turned on when the ions reach the intersection, and the ions move to the detection electrode, and finally enter the buffer waste pool after passing through the detection electrode.

In a further scheme, the buffer solution is prepared from histidine/2-morphineethanesulfonic acid, 18-crown ether-6 and deionized water, wherein the concentration ratio of high-concentration buffer B and low-concentration buffer A is 2:1-10:1;

In step (3), the enrichment high voltage is 200-800V and the time is 30-90s,

In step (4), the sampling voltage is 300-600V, and the time is 10-20s.

The separation high voltage in step (5) is 500-2000V.

In the above steps, the applied voltage is inversely proportional to the time, the time required for voltage application decreases when the voltage increases, and the time increases when the voltage decreases.

In a further scheme, the low-concentration buffer A is a pH=6.0 solution formed by mixing 5mM/L histidine/2-morphineethanesulfonic acid, 0.5mM/L18-crown-6 and deionized water; High-concentration buffer B is a pH=6.0 solution formed by mixing 20 mM/L histidine/2-morphineethanesulfonic acid, 0.5 mM/L 18-crown-6 and deionized water.

In the invention, a sample enrichment pool is arranged between the sample injection pool and the sample waste liquid pool, and the sample injection pool and the sample enrichment pool are connected through an ion enrichment flow channel. The shorter the length of the ion-enrichment flow channel within the allowable range of the processing conditions, the better, that is, it is set at the shortest distance between the sample injection pool and the sample enrichment pool. This is because too long ion enrichment channel will greatly reduce the ion enrichment efficiency. The number of enrichment channels is at least 1, such as 1, 2, 3, 4, etc. can realize the present application.

Multiple high-voltage electrodes can be set in the sample injection cell, and the number of high-voltage electrodes corresponds to the number of ion-enrichment flow channels, that is, several high-voltage electrodes are provided for several ion-enrichment flow channels. A high-voltage electrode is respectively arranged in the sample enrichment pool, the sample waste liquid pool, the buffer solution injection pool and the buffer solution waste liquid pool. Electrophoresis is formed between the two after electrification.

This application has the function of ion enrichment twice, the first enrichment is realized by the volume difference between the sample injection pool and the sample enrichment pool, that is, the volume of the sample injection pool is larger than the volume of the sample enrichment pool, and the two The volume ratio is not greater than 12:1. Because there is a saturation effect in the volume ratio of the two, when the volume ratio is greater than 12:1, a saturation effect occurs. When the enrichment high voltage is turned on, the ions to be measured in the sample solution migrate from the large-volume sample injection pool to the small-volume sample enrichment pool under the action of electric field force and electroosmotic flow force to complete the first enrichment.

Two buffers with different concentrations are injected into different regions of the electrophoresis microchip, and the concentration ratio is 2:1-10:1. Too high a concentration ratio will disturb the electroosmotic flow in the channel and cause laminar flow, thereby reducing the richness. In order to improve the collection effect, the sample injection pool, the ion enrichment flow channel, and the sample enrichment pool are injected with a low-concentration buffer, and other areas are injected with a high-concentration buffer. When the injection high pressure is turned on, different buffer concentrations will lead to different field strengths in buffers with different concentrations. When the buffer concentration is low, the field strength increases, and when the buffer concentration is high, the field strength decreases. Different concentrations will complete the second enrichment on the concentration gradient surface.

Because the ions in the sample solution diffuse from high concentration to low concentration, after ion enrichment, the ion concentration in the sample injection pool is much smaller than the ion concentration in the sample enrichment pool, and the ions flow from the sample enrichment pool to The sample injects into the cell to diffuse, and the concentration of ions in the enriched sample enrichment cell increases, so the diffusion phenomenon is also enhanced. Therefore, in the present invention, the connection between one end of the ion enrichment channel and the sample injection pool is in the shape of a bell mouth, and the connection between the other end and the sample enrichment pool is in the shape of an inverted bell mouth, so as to ensure that ions flow from the sample injection pool to the sample enrichment The flow resistance of the cell is small, while the flow resistance of the reverse flow is relatively large, so that a lower enrichment voltage can obtain a higher ion enrichment speed, and reduce the concentration of ions from the sample after the first enrichment. Diffusion from the collecting pool to the sample injection pool.

The principle of the second enrichment is to create different field strengths according to different buffer concentrations, so that ions can be enriched at the sudden change in field strength. When the ion concentration in the sample enrichment pool is too high, the low concentration buffer is The ions dissolved in it increase the concentration, so the concentration gradient of the two buffer solutions with different concentrations decreases, and thus the gradient of the field strength change also decreases, thus reducing the efficiency of the second ion enrichment. Therefore, high-voltage electrodes are respectively set in the sample injection pool, sample enrichment pool, sample waste pool, buffer injection pool, and buffer waste pool; the high-voltage electrodes in the sample enrichment pool and sample injection pool are connected to form a rich The high voltage is collected to promote ion migration to form the first enrichment; the high voltage electrode in the sample enrichment pool and the sample waste liquid pool is connected to form a sample injection high pressure to promote the second enrichment of ions. In addition, the high-voltage electrodes in the buffer injection pool and the buffer waste pool are connected to form a separation high voltage, which promotes the migration of ions in the sample solution to the ion detection electrode for detection.

The ion detection electrode is composed of a transmitting electrode and a receiving electrode arranged at the tail of the ion separation flow channel. The two electrodes are placed in parallel and an insulating film is arranged on the upper surface to ensure that it does not contact the fluid in the ion separation flow channel above.

The body of the electrophoretic microchip has a layered design from top to bottom, and it has a micropipe layer, an insulating layer and an electrode layer from top to bottom. The micropipe layer and insulating layer are made of plastic materials; the micropipe is processed by precision numerical control metal punch, and then the concave micropipe is made on the plastic plate by hot pressing; the electrode layer can be directly used on the PCB board or on the glass Manufactured by magnetron sputtering metal electrodes on the substrate; the insulating layer can be composed of a plastic film with a thickness of less than 0.2 mm. The micropipe layer and the plastic film acting as the insulating layer are firmly combined through a thermocompression bonding process to form a complete micropipe and ensure that the fluid in the pipe does not come into contact with the electrode layer.

The steps of the thermocompression bonding process are as follows: firstly, ultrasonic cleaning is performed on the pipe layer of the microchip. The cleaning water is a 9:1 mixture of deionized water and ethanol, and the temperature is set at 30°C. After cleaning for 3 minutes, replace the cleaning water and repeat 3 times; then use plasma to clean the pipe layer of the microchip and the plastic film serving as the insulating layer Machine for surface activation treatment, the treatment time is set for 3 minutes; finally, the micropipe layer and the plastic film of the insulating layer are bonded by thermocompression, the bonding temperature is 110°C, the time is 40min, and the pressure is 0.55MPa. The combination of the micropipe layer and the insulating layer and the electrode layer are assembled and fixed by U-shaped clips, which are convenient for disassembly and reuse.

Therefore, the present invention utilizes electric field force and electroosmotic flow force to make ions migrate from the large-volume sample injection pool to the small-volume sample enrichment pool to form the first ion enrichment; the sample injection pool is added before the sample enrichment pool, The sample injection pool and the sample enrichment pool connected to the two ends of the ion enrichment flow channel are respectively designed in the shape of a bell mouth and an inverted bell mouth shape. Through the change of flow resistance, the flow of ions from high concentration buffer to low concentration buffer is reduced. The phenomenon of diffusion; then use the solution concentration gradient to form an electric field gradient, so that the ions are enriched on the concentration gradient surface, and the second enrichment is completed; finally, the ion injection, separation and detection are completed.

Therefore, the electrophoretic microfluidic chip and detection method adopted in the present invention have the advantages of short enrichment time, high enrichment efficiency, large enrichment multiple, simple chip fabrication, low cost, and simple operation. The invention can be applied to soil survey, water quality monitoring, clinical medical treatment and other industries.

Description of drawings

Fig. 1 is a structural representation of the present invention;

Fig. 2 is the top view of Fig. 1;

Fig. 3 is an enlarged schematic diagram of part A in Fig. 2;

Fig. 4 is the connection schematic diagram when there is one ion separation channel in the present invention;

Fig. 5 is the connection schematic diagram when there are five ion separation channels in the present invention;

Fig. 6 is B-B sectional view among Fig. 2;

Fig. 7 is the electrophoresis process diagram of detection process;

Fig. 8 is a comparative electrophoresis spectrum using the detection method of the present invention and the traditional detection method.

Explanation of reference signs:

1-micropipe layer, 2-insulating layer, 3-electrode layer, 4-ion enrichment flow channel, 5-ion sampling flow channel, 6-ion separation flow channel, 7-sample injection pool, 8-sample enrichment pool, 9-sample waste pool, 10-buffer injection pool, 11-buffer waste pool, 12-transmitting electrode, 13-receiving electrode.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing:

Example 1:

As shown in Figure 1-6, an electrophoresis microchip with an ion online enrichment device includes an electrophoresis microchip body and a sample injection pool 7, a sample waste liquid pool 9, and a buffer injection pool arranged in the electrophoresis microchip body. Pool 10, buffer solution waste liquid pool 11, the sample injection pool 7 communicates with the sample enrichment pool 8 through the ion enrichment flow channel 4, and the sample enrichment pool 8 communicates with the sample waste liquid pool through the ion sampling flow channel 5 9 are connected; the buffer injection pool 10 and the buffer waste pool 11 are communicated through the ion separation flow channel 6; the ion sampling flow channel 5 is vertically intersected with the ion separation flow channel 6 and communicated internally; the The volume of the sample injection pool 7 is greater than that of the sample enrichment pool 8 , and the ion enrichment channel 4 is located at the shortest distance between the sample injection pool 7 and the sample enrichment pool 8 .

As a further solution, as shown in Figure 3, the volume ratio of the sample injection pool 7 to the sample enrichment pool 8 is not greater than 12:1; the sample injection pool 7 is a semicircular arc structure, and the ion enrichment flow channel 4 There are three, and the three ion enrichment flow channels 4 and the ion sampling flow channels 5 are arranged in a cross. The connection between the sample injection pool 7 and the ion enrichment flow channel 4 is in the shape of a trumpet protruding outward, and the connection between the sample enrichment pool 8 and the ion enrichment flow channel 4 is in the shape of an inverted bell mouth protruding inward.

The design of this bell mouth shape is to ensure that the flow resistance of ions from the sample injection cell to the sample enrichment cell is small, and the flow resistance of the opposite direction is relatively large, so that a lower enrichment voltage can obtain a higher The speed of ion enrichment also reduces the diffusion of ions from high concentration to low concentration.

The number of ion-enriching flow channels 4 is at least one, and there is only one ion-enriching flow channel 4 as shown in FIG. 4 , and there are five ion-enriching flow channels 4 as shown in FIG. 5 . The shape of the sample injection pool 7 can also be various, as long as the length of the ion enrichment channel 4 is as short as possible.

Preferably there are three ion enrichment flow channels 4, and the sample injection pool 7 is a semicircular arc structure, the purpose is to make the lengths of the three enrichment flow channels equal, and the short enrichment flow channels will increase the enrichment efficiency.

The volume of the sample injection pool 7 is larger than that of the sample enrichment pool 8, and the ions in the sample solution migrate from the large volume sample injection pool to the small volume sample enrichment pool to form the first enrichment of ions. In order to obtain a better ion enrichment effect, the volume ratio of the two should not be greater than 12:1. If it is greater than 12:1, a saturation effect will appear.

In a further scheme, the sample injection pool 7, the sample enrichment pool 8 and the ion enrichment flow channel 4 are all filled with low-concentration buffer A, the sample waste pool 9, the buffer injection pool 10, and the buffer waste pool 11 , the ion sampling channel 5 and the ion separation channel 6 are filled with high-concentration buffer B, and the concentration of low-concentration buffer A is lower than that of high-concentration buffer B, so as to form a concentration gradient and enrich ions. The sample solution to be tested is compatible with the low-concentration buffer A and injected into the sample injection pool 7 .

In a further scheme, the concentration ratio of the high-concentration buffer B to the low-concentration buffer A is 2:1-10:1.

The concentration ratio is generally 2:1-10:1. If it is less than 2:1, there will be no obvious enrichment effect. If it is greater than 10:1, it will cause electroosmotic flow disturbance, cause laminar flow effect, and reduce the enrichment effect.

The length of the ion-enrichment flow channel is as short as possible within the allowable range of processing, and the length of the preferred ion-enrichment flow channel 4 of the present invention is 500 μm; the length of the ion sampling flow channel 5 is 16 mm, and the ion separation flow channel 6 The length of the ion sampling flow channel 5 and the ion separation flow channel 6 is 53mm, the distance from the sub-detection electrode to the intersection of the ion sampling flow channel 5 and the ion separation flow channel 6 is 35mm, and the distance from the buffer solution waste liquid pool 11 is 45mm; the ion enrichment flow channel 4, the ion inlet The width and depth of the sample channel 5 and the ion separation channel 6 are both 100 μm.

In a further solution, the body of the electrophoresis microchip is sequentially composed of a micropipe layer 1, an insulating layer 2 and an electrode layer 3; the ion enrichment flow channel 4, the ion sampling flow channel 5 and the ion separation flow channel 6 are set In the micropipe layer 1, the electrode layer 3 is provided with an ion detection electrode, and the ion detection electrode is composed of a transmitting electrode 12 and a receiving electrode 13 arranged at the tail of the ion separation channel 6, and the transmitting electrode 12, The receiving electrodes 13 are arranged in parallel (as shown in FIG. 6 ).

In a further scheme, each of the sample injection pool 7, the sample waste pool 9, the buffer injection pool 10 and the buffer waste pool 11 is provided with a high-voltage electrode; the sample enrichment pool 8 is provided with at least one high-voltage electrode. Electrodes; wherein the high-voltage electrodes in the sample enrichment pool 8 and the sample injection pool 7 are connected to form a high-voltage enrichment, the high-voltage electrodes in the sample enrichment pool 8 and the sample waste liquid pool 9 are connected to form a sample injection high voltage, and the buffer solution is injected into the pool 10 is connected to the high-voltage electrode in the buffer solution waste pool 11 to form a high voltage separation.

Example 2:

A detection method using the above-mentioned electrophoresis microchip, comprising the following steps:

(1) Mix histidine/2-morphineethanesulfonic acid, 18-crown-6 and deionized water to form high-concentration buffer B and low-concentration buffer A with a concentration ratio of 2:1;

Mix the sample to be tested with low-concentration buffer A at a volume ratio of 1:1 to make a sample solution;

(2) Add high-concentration buffer B into the sample waste liquid pool 9, buffer injection pool 10 and buffer waste liquid pool 11, and fill the ion sampling channel 5 and ion separation channel 6; Liquid A is added to the sample enrichment pool 8, and filled with the ion enrichment channel 4; the sample solution is added to the sample injection pool 7; it is completely filled; wherein the volume ratio of the sample injection pool 7 to the sample enrichment pool 8 is 12 :1;

(3) Apply an enrichment high voltage of 700V for 30s between the sample enrichment pool 8 and the sample injection pool 7, so that the ions in the sample solution in the sample injection pool 7 migrate to the sample enrichment pool 8 to realize the first ion separation. one enrichment;

(4) Apply a 400V sample injection high pressure between the sample enrichment pool 8 and the sample waste liquid pool 9 for 20s, and the ions in the sample solution flow from the sample enrichment pool 8 to the sample waste liquid through the ion injection channel 5 Pool 9 migrates in the direction, forming the second enrichment of ions on the step surface of low-concentration buffer A and high-concentration buffer B;

(5) When the ions arrive at the intersection of the ion sampling channel 5 and the ion separation channel 6, turn on the separation high pressure, that is, apply a 1500V separation high pressure between the buffer injection pool 10 and the buffer waste pool 11, and the sample The ions in the solution migrate to the ion detection electrode, the ion detection electrode detects them, and then enter the buffer solution waste pool 11 .

A detection signal of 5Vpp and a frequency of 900kHz is applied to the transmitting electrode 12. When the ions pass through the ion detection electrode, they will be captured by the receiving electrode 13, so as to enrich, separate and detect ions in the experimental sample solution. The detection method mainly adopts capacitive coupling non-contact conductometric detection, but it is not limited to this detection method, and other methods that can detect the ion concentration in the solution are also feasible.

Example 3:

A detection method using the above-mentioned electrophoresis microchip, comprising the following steps:

(1) Low-concentration buffer A is prepared by mixing 5mM/L histidine/2-morphineethanesulfonic acid, 0.5mM/L 18-crown-6 and deionized water; it is composed of 20mM/L histidine/ 2-Morphineethanesulfonic acid, 0.5mM/L 18-crown-6 and deionized water are mixed to form high-concentration buffer B, and the pH of low-concentration buffer A and high-concentration buffer B is 6.0;

The aqueous solution of KCl, NaCl, and LiCl (both analytical reagents) is used as the sample to be tested, and then the sample to be tested is mixed with low concentration buffer A at a volume ratio of 1:1 to make the sample solution with an ion concentration;

(2) Add high-concentration buffer B into the sample waste liquid pool 9, buffer injection pool 10 and buffer waste liquid pool 11, and fill the ion sampling channel 5 and ion separation channel 6; Liquid A is added to the sample enrichment pool 8, and filled with the ion enrichment flow channel 4; the sample solution is added to the sample injection pool 7; fully filled (as shown in Figure 7 (I)), wherein the sample injection pool 7 and The volume ratio of the sample enrichment pool 8 is 5:1;

(3) Apply a 400V enrichment high voltage between the sample enrichment pool 8 and the sample injection pool 7 for 40 s, so that the ions in the sample solution in the sample injection pool 7 migrate to the sample enrichment pool 8 to realize the first ion separation. One enrichment (as shown in Figure 7 ((II)));

(4) Apply a 500V sample injection high pressure between the sample enrichment pool 8 and the sample waste liquid pool 9 for 14s, and the ions in the sample solution flow from the sample enrichment pool 8 to the sample waste liquid through the ion injection channel 5 Pool 9 migrates in the direction, forming the second enrichment of ions on the step surface of low-concentration buffer A and high-concentration buffer B (as shown in Figure 7 (III));

The ions flow from the low-concentration sample enrichment pool to the high-concentration ion sampling flow channel, and are enriched for the second time on the solution concentration step surface, and the enriched ions continue to flow to the sample waste pool under the influence of the injection voltage , the separation voltage is turned on when the ions reach the intersection, and the ions move to the detection electrode, and finally enter the buffer waste pool after passing through the detection electrode.

(5) When the ions reach the intersection of the ion sampling flow channel 5 and the ion separation flow channel 6, turn on the separation high pressure, that is, apply a separation high pressure of 1000V between the buffer injection pool 10 and the buffer waste liquid pool 11, and the sample The ions in the solution migrate to the ion detection electrode (as shown in FIG. 7 (IV) ), the ion detection electrode detects them, and then enter the buffer solution waste pool 11 .

A detection signal of 5Vpp and a frequency of 900kHz is applied to the transmitting electrode 12. When the ions pass through the ion detection electrode, they will be captured by the receiving electrode 13, so as to enrich, separate and detect ions in the experimental sample solution. The detection method mainly adopts capacitive coupling non-contact conductometric detection, but it is not limited to this detection method, and other methods that can detect the ion concentration in the solution are also feasible.

In the invention, a sample enrichment pool is arranged between the sample injection pool and the sample waste liquid pool, and the sample injection pool and the sample enrichment pool are connected through an ion enrichment flow channel. The shorter the length of the ion-enrichment flow channel within the allowable range of the processing conditions, the better, that is, it is set at the shortest distance between the sample injection pool and the sample enrichment pool. This is because too long ion enrichment channel will greatly reduce the ion enrichment efficiency.

As shown in FIG. 8 , using a mixed solution of K ⁺ , Na ⁺ , and Li ⁺ ions as the sample solution, the electrophoresis spectra were compared using the detection method of this embodiment and the traditional cross electrophoresis microchip detection. Among them, A is the electrophoresis spectrum obtained by using the electrophoresis microchip and detection method of the present invention, and the concentration of each ion (K ⁺ , Na ⁺ , Li ⁺ ) in the sample solution is 100 μm/L; B is the traditional detection method. In the obtained electrophoresis spectrum, the concentration of each ion (K ⁺ , Na ⁺ , Li ⁺ ions) in the sample solution is 500 μm/L. It can be seen from the figure that the enrichment ^and enhancement of K ⁺ , Na ^{+ and} Li ⁺ three ^ions obtained by the detection method of this method are 37, 43, 46 times. In addition, the enrichment, separation and detection of ions are completed within 100 seconds by using the electrophoresis microchip and detection method described in the present invention. Therefore, experiments have proved that the present invention can complete the enrichment and separation detection of ions with a very short experimental time, a large enrichment multiple, a simple and low-cost microfluidic chip, and simple operations.

The examples described above are only descriptions of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention. Any person familiar with the art, without departing from the design spirit and scope of the present invention, can make a complete understanding of the present invention. All improvements and modifications made should fall within the scope of protection determined by the claims of the present invention.

Claims (10)

1. The utility model provides an electrophoresis microchip with online enrichment facility of ion which characterized in that: the electrophoresis microchip comprises an electrophoresis microchip body, and a sample injection pool (7), a sample waste liquid pool (9), a buffer liquid injection pool (10) and a buffer liquid waste liquid pool (11) which are arranged in the electrophoresis microchip body, wherein the sample injection pool (7) is communicated with a sample enrichment pool (8) through an ion enrichment flow channel (4), and the sample enrichment pool (8) is communicated with the sample waste liquid pool (9) through an ion sampling flow channel (5); the buffer solution injection pool (10) and the buffer solution waste liquid pool (11) are communicated through an ion separation flow channel (6); the ion sampling flow channel (5) and the ion separation flow channel (6) are vertically crossed and communicated with each other; the volume of the sample injection pool (7) is larger than that of the sample enrichment pool (8), and the ion enrichment flow channel (4) is positioned at the shortest distance between the sample injection pool (7) and the sample enrichment pool (8).

2. electrophoresis microchip according to claim 1, characterized in that: the volume ratio of the sample injection pool (7) to the sample enrichment pool (8) is not more than 12: 1; the sample injection pool (7) is of a semicircular arc structure, the number of the ion enrichment flow channels (4) is three, and the ion enrichment flow channels (4) and the ion sampling flow channel (5) are arranged in a cross manner.

3. Electrophoresis microchip according to claim 1, characterized in that: the sample injection pool (7) and the ion enrichment flow channel (4) are communicated to form a horn mouth shape protruding outwards, and the sample enrichment pool (8) and the ion enrichment flow channel (4) are communicated to form an inverted horn mouth shape protruding inwards.

4. Electrophoresis microchip according to claim 1, characterized in that: the concentration of the low-concentration buffer solution A in the sample injection pool (7), the sample enrichment pool (8) and the ion enrichment flow channel (4) is less than that of the high-concentration buffer solution B in the sample waste liquid pool (9), the buffer solution injection pool (10), the buffer solution waste liquid pool (11), the ion sample injection flow channel (5) and the ion separation flow channel (6), and a concentration gradient is formed to enrich ions.

5. Electrophoresis microchip according to claim 4, characterized in that: the concentration ratio of the high-concentration buffer solution B to the low-concentration buffer solution A is 2:1-10: 1.

6. Electrophoresis microchip according to claim 1, characterized in that: the electrophoresis microchip body is sequentially provided with a microchannel layer (1), an insulating layer (2) and an electrode layer (3) from top to bottom; the ion enrichment flow channel (4), the ion sampling flow channel (5) and the ion separation flow channel (6) are arranged in the micro-pipeline layer (1), an ion detection electrode is arranged in the electrode layer (3), the ion detection electrode is composed of an emitting electrode (12) and a receiving electrode (13) which are arranged at the tail part of the ion separation flow channel (6), and the emitting electrode (12) and the receiving electrode (13) are arranged in parallel.

7. Electrophoresis microchip according to claim 1, characterized in that: the sample injection pool (7), the sample waste liquid pool (9), the buffer injection pool (10) and the buffer waste liquid pool (11) are respectively provided with a high-voltage electrode; the sample enrichment pool (8) is provided with at least one high-voltage electrode; wherein the high voltage electrodes in the sample enrichment pool (8) and the sample injection pool (7) are connected to form an enrichment high voltage, the high voltage electrodes in the sample enrichment pool (8) and the sample waste liquid pool (9) are connected to form a sample injection high voltage, and the high voltage electrodes in the buffer injection pool (10) and the buffer waste liquid pool (11) are connected to form a separation high voltage.

8. an assay method using the electrophoresis microchip according to any one of claims 1 to 7, characterized in that: the method comprises the following steps:

(1) preparing two buffers with different concentrations, namely a low-concentration buffer A and a high-concentration buffer B, and dissolving a sample to be detected in the low-concentration buffer A to prepare a sample solution;

(2) Adding a high-concentration buffer solution B into a sample waste liquid pool (9), a buffer solution injection pool (10) and a buffer solution waste liquid pool (11), and filling an ion sample injection flow channel (5) and an ion separation flow channel (6); adding a low-concentration buffer solution A into a sample enrichment pool (8), and filling an ion enrichment flow channel (4); adding the sample solution into a sample injection pool (7); filling the mixture completely;

(3) Applying an enrichment high pressure between the sample enrichment pool (8) and the sample injection pool (7) to enable ions in the sample solution in the sample injection pool (7) to migrate into the sample enrichment pool (8) to realize the first enrichment of the ions;

(4) applying sample injection high pressure between the sample enrichment pool (8) and the sample waste liquid pool (9), wherein ions in the sample solution migrate from the sample enrichment pool (8) to the direction of the sample waste liquid pool (9) through the ion sample injection flow channel (5), and form secondary enrichment of the ions on the step surfaces of the low-concentration buffer solution A and the high-concentration buffer solution B;

ions flow from the low-concentration sample enrichment pool to the high-concentration ion sample injection flow channel, are enriched for the second time on the solution concentration step surface, the enriched ions continue to flow to the sample waste liquid pool under the influence of the sample injection voltage, the separation voltage is started when the ions reach the crossroad, the ions move to the detection electrode, and finally enter the buffer liquid waste liquid pool after passing through the detection electrode;

(5) when the ions reach the intersection of the ion sample introduction flow channel (5) and the ion separation flow channel (6), the separation high pressure is started, namely the separation high pressure is applied between the buffer solution injection pool (10) and the buffer solution waste liquid pool (11), the ions in the sample solution migrate to the ion detection electrode, the ion detection electrode detects the ions, and then the ions enter the buffer solution waste liquid pool (11).

9. the detection method according to claim 8, characterized in that: the buffer solution is prepared from histidine/2-morphine ethane sulfonic acid, 18-crown ether-6 and deionized water, wherein the concentration ratio of the high-concentration buffer solution B to the low-concentration buffer solution A is 2:1-10: 1;

The enrichment high pressure in the step (3) is 200-800V, the time is 30-90s,

In the step (4), the injection voltage is 300-,

The separation pressure in the step (5) is 500-2000V.

10. the detection method according to claim 9, characterized in that: the low-concentration buffer solution A is a solution with the pH value of 6.0 formed by mixing 5mM/L histidine/2-morphine ethanesulfonic acid, 0.5 mM/L18-crown ether-6 and deionized water; the high concentration buffer B was a solution having a pH of 6.0 prepared by mixing 20mM/L histidine/2-morphine ethane sulfonic acid, 0.5 mM/L18-crown-6 and deionized water.