An on-line double-gradient pre-concentration detection method for heavy metal ions in soil based on microchip electrophoresis non-contact conductivity detection

Abstract

The application discloses a kind of on-line double-gradient pre-concentration detection methods of soil heavy metal ions based on microchip electrophoresis non-contact conductivity detection, including preparation of the analyte sample to be measured, preparation of high concentration buffer as background buffer, preparation of microchip, microchip electrophoresis process and the like steps.When testing, the background buffer contains 2-(N-morpholinyl)ethanesulfonic acid, L-histidine and electroosmotic flow inhibitor;The analyte sample to be measured contains sample matrix liquid and sample extraction stock solution.Among them, the sample matrix liquid is low-concentration buffer, and glacial acetic acid is used to reduce pH value.In testing, due to the existence of concentration gradient and pH gradient between the analyte sample solution to be measured and the background buffer, the ions to be measured in the sample will slow down and accumulate at the contact surface of the two during migration, realizing on-line pre-concentration, and improving the detection sensitivity of the ions to be measured.The application greatly improves the separation degree of multiple heavy metal ions, and effectively improves the detection sensitivity of heavy metal ions.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of on-site rapid detection of soil heavy metals, and relates to an online double-gradient pre-concentration detection method for soil heavy metal ions based on microchip electrophoresis non-contact conductivity detection. The method is based on microchip electrophoresis, utilizes field-amplified sample stacking technology, and adjusts the pH value of the to-be-detected analyte to inhibit the convection dispersion generated in the field-amplified sample stacking process, so as to realize online pre-concentration of heavy metal ions. BACKGROUND

[0002] With the development of human industrial production and life activities, heavy metal pollution has become one of the main reasons for soil pollution. At present, effective detection methods for heavy metal ions mainly include atomic spectroscopy, mass spectrometry, chromatography and electrochemical analysis method. Among them, atomic spectroscopy, mass spectrometry and chromatography have defects such as complex detection equipment, high operation difficulty, high cost and large volume. The potential analysis method, polarography and capillary electrophoresis method in the electrochemical analysis method have defects such as long separation time, low sensitivity and difficulty in integration. For the detection of heavy metal ions, on-site rapid detection has become the main development trend.

[0003] Microchip electrophoresis technology is an extension of capillary electrophoresis technology. In the 1990s, Manz proposed the concept of micro total analyzer, which compressed the detection experiment on the chip, and opened the research boom of microchip electrophoresis. In 2001, Guijt proposed non-contact conductivity detection technology, which adopted the design of non-contact detection electrode to prolong the service life of the detection electrode. Although the microchip electrophoresis technology combined with the non-contact conductivity detection technology realizes the integration and on-site of heavy metal ion detection. However, its detection sensitivity still has the shortcoming of being low. Therefore, online pre-concentration of the sample becomes an important means to improve the detection sensitivity.

[0004] Online preconcentration techniques mainly include isoelectric focusing, sample sweeping, field-amplified sample stacking and dynamic pH junction. The isoelectric focusing technique is complicated in operation and the chip structure is complex, which is not suitable for on-site rapid detection. The sample sweeping technique has high requirements for the type and concentration of micelles, and the preconcentration time is long and the degree of specialization is high, which is also not suitable for on-site rapid detection. In the process of field-amplified sample stacking, the local non-uniform electroosmotic flow may cause the dispersion of analytes, which increases the width of sample zone and reduces the preconcentration effect. The dynamic pH junction technique changes the surface charge of the analyte to change the migration speed of the analyte, thereby improving the preconcentration effect. However, the dynamic pH junction technique cannot change the surface charge of heavy metal ions. Therefore, in the present application, based on the field-amplified sample stacking technique of microchip electrophoresis, by adding an electroosmotic flow inhibitor and adjusting the pH value of the to-be-tested analyte in the buffer, the size of the electroosmotic flow in the electrophoresis process and the convection dispersion generated in the field-amplified sample stacking process are inhibited, the concentration gradient and the pH gradient between the sample solution and the buffer are utilized to slow down the accumulation of the analyte in the migration process, and the migration time of the analyte is increased, so as to realize the online preconcentration of heavy metal ions. The present application is mainly based on the field-amplified sample stacking technique, by adjusting the buffer ratio and the pH value of the sample matrix liquid, an online double-gradient preconcentration detection method for soil heavy metal ions based on microchip electrophoresis non-contact conductivity detection is proposed. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application proposes an online double-gradient preconcentration detection method for soil heavy metal ions based on microchip electrophoresis non-contact conductivity detection, which utilizes the concentration gradient generated by field-amplified sample stacking and the pH gradient generated by adjusting the pH value of the to-be-tested analyte to inhibit the size of the electroosmotic flow and the convection dispersion in the field-amplified sample stacking process, and realizes the online preconcentration of the sample. The background buffer of the present application contains 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His) and cetyltrimethylammonium bromide (CTAB), wherein the cetyltrimethylammonium bromide (CTAB) controls the direction and size of the electroosmotic flow as an electroosmotic flow inhibitor. The sample matrix liquid contains low concentrations of 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His) and cetyltrimethylammonium bromide (CTAB), and uses glacial acetic acid to adjust the pH value, so as to form a concentration gradient and a pH gradient with the background buffer. By optimizing a series of important experimental parameters such as the CTAB concentration of the background buffer, the MES / His concentration and the pH value of the sample matrix liquid, the best conditions are applied to the detection of soil heavy metal ions, which is a simpler, more efficient and more sensitive technique. The present application can greatly improve the detection sensitivity of microchip electrophoresis for heavy metal ions, and compared with the previous preconcentration methods, it realizes simple and efficient operation while ensuring high enrichment effect.

[0006] To achieve the above object, the technical scheme adopted by the present application is:

[0007] A soil heavy metal ion online double-gradient pre-concentration detection method based on microchip electrophoresis non-contact conductivity detection, comprising the following steps:

[0008] (I) Preparation of the sample to be detected:

[0009] Grind the sample soil through a sieve and heat it in a dryer for 30-45 min. Then, mix the heated sample soil with the extraction agent and ultrasonically treat for 30-60 min. After standing, take the supernatant, filter it with filter paper, and filter it with a 0.22 μm water filter to obtain the sample extraction stock solution; preferably, the extraction agent is ammonium acetate solution.

[0010] Prepare a low-concentration buffer as the sample matrix liquid, and the low-concentration buffer comprises 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His), and an electroosmotic flow inhibitor; preferably, the concentration of 2-(N-morpholino) ethanesulfonic acid in the low-concentration buffer is 1.25-10 mM / L, the concentration of L-histidine is 1.25-10 mM / L, and the concentration of the electroosmotic flow inhibitor is 0.0025-0.1 mM / L; further preferably, the concentration of MES is 2.5 mM / L, the concentration of His is 2.5 mM / L, the electroosmotic flow inhibitor is cetyltrimethylammonium bromide (CTAB), and the concentration of CTAB is 0.01 mM / L.

[0011] Mix the sample extraction stock solution and the sample matrix liquid at a volume ratio of 1:1, adjust the pH value of the solution to 3-6 with 4% (v / v) glacial acetic acid to obtain the sample to be detected.

[0012] (II) Preparation of the background buffer:

[0013] Prepare a mixed solution of 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His), and cetyltrimethylammonium bromide (CTAB) as the background buffer; preferably, the concentration of MES is 20 mM / L, the concentration of His is 20 mM / L, and the concentration of CTAB is 0.01 mM / L.

[0014] (III) Provide a microchip:

[0015] The microchip comprises a cross microchannel, the cross microchannel comprises a first channel and a second channel which are in communication with each other, two ends of the first channel are respectively formed with a sample cell and a first reservoir, two ends of the second channel are respectively formed with a second reservoir and a third reservoir; the second channel is arranged with a detection area at one end close to the third reservoir, the detection area is composed of a transmitting electrode and a receiving electrode. The transmitting electrode transmits a high-frequency alternating current signal, due to polarization, the receiving electrode receives an alternating current signal of the same frequency, the amplitude of the alternating current signal is affected by the conductivity in the solution, when the analyte migrates to the detection area, the conductivity of the detection area changes, thereby the amplitude of the current signal received by the receiving electrode changes, an electrophoretic peak is obtained, and the information of the type and concentration of the analyte is contained in the electrophoretic peak.

[0016] (iv) The microchip electrophoresis process comprises a preparation stage, a sample injection stage, a separation stage and a detection stage, wherein:

[0017] The preparation stage process is as follows: after the cross microchannel is flushed with a background buffer solution, the sample cell, the first reservoir, the second reservoir and the third reservoir are filled with the background buffer solution; for the first use, the microchannel is first flushed with 1mM NaOH solution for 15min and then distilled water for 15min; for non-first use, the microchannel is flushed with 1mM NaOH solution for 5min and then distilled water for 10min.

[0018] The sample injection stage process is as follows: the background buffer solution in the sample cell is replaced with the analyte sample to be tested, a high-voltage electrode is placed in each of the sample cell, the first reservoir, the second reservoir and the third reservoir, and the high-voltage electrode is preferably a platinum metal electrode; a 500-2000V voltage is applied to the sample cell for 14s, the first reservoir is grounded for 14s, and the second reservoir and the third reservoir are suspended for 14s; during the process, because there are concentration gradient and pH gradient on both sides of the interface between the analyte to be tested and the background buffer solution, the heavy metal ions to be tested are slowed down and accumulated at the interface, thereby realizing pre-concentration in the sample injection stage.

[0019] The separation stage process is as follows: the voltage is switched, a 1000-2000V voltage is applied to the second reservoir for 80s, the third reservoir is grounded for 80s, and the sample cell and the first reservoir are suspended for 80s. During the process, part of the sample reaching the cross intersection is pushed into the second channel, different types of heavy metal ions to be tested are separated into different sample bands due to different migration speeds, and different heavy metal ion bands to be tested are in contact with the background buffer solution at two interfaces, so that the heavy metal ion bands to be tested are continuously shortened during migration and are slowed down and accumulated at the front interface, thereby realizing pre-concentration in the separation stage.

[0020] The detection stage process is that when the sample zone reaches the detection area, an electropherogram is drawn by collecting the amplitude change caused by the change of electric conductance.

[0021] The present application has the advantages of:

[0022] The present application is based on field-amplified sample stacking technology, adjusts the pH value of the analyte to be detected, suppresses the convection dispersion in the pre-concentration process, and uses the concentration gradient and pH value gradient of the analyte to be detected and the background buffer to establish an online double-gradient pre-concentration detection method based on microchip electrophoresis for heavy metal ions in soil, and the method is used for pre-concentration of heavy metal ion samples; the method is successfully applied to pre-concentration and detection of heavy metal ions in real soil extract, and the sensitivity of heavy metal lead and cadmium is improved by 26.40 times and 43.73 times respectively; the resolution of heavy metal lead and cadmium is improved from 0.41 to 1.38. The method has the advantages of simplicity, high efficiency and low cost, and is suitable for portable microchip electrophoresis systems. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a structural schematic diagram of a microchip.

[0024] Figure 2 It is a pre-concentration mechanism diagram.

[0025] Figure 3 It is an electropherogram obtained under different conditions of an electroosmotic flow inhibitor (CTAB) concentration.

[0026] Figure 4 It is the influence of different sample matrix liquid concentrations on the pre-concentration effect.

[0027] Figure 5 It is an electropherogram of the sample matrix liquid under different pH conditions.

[0028] Figure 6 It is the influence of the pH value of the sample matrix liquid on the heavy metal ions to be detected.

[0029] Figure 7 It is a comparison of electropherograms of a traditional microchip electrophoresis method and the pre-concentration method proposed in the present application.

[0030] The drawings show that: 1-cross microchannel, 101-injection channel, 102-separation channel, 2-sample cell, 3-first liquid pool, 4-second liquid pool, 5-third liquid pool, 6-emission electrode, 7-receiving electrode. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with examples, so that those skilled in the art can better understand the present application and implement it, but the examples are not limiting to the present application.

[0032] In addition, the preparation process in the following examples is a conventional means in the existing technology in the art, and thus will not be described in detail.

[0033] A soil heavy metal ion online double-gradient pre-concentration detection method based on microchip electrophoresis non-contact conductivity detection, comprising the following steps:

[0034] (I) Preparation of the sample of the analyte to be detected:

[0035] Grind the sample soil through a sieve and heat it in a dryer for 30-45 min. Then, mix the dried sample soil with the extraction agent and ultrasonically treat for 30-60 min. After standing, take the supernatant, filter it with filter paper, and filter it with a 0.22 μm water filter to obtain the sample extraction stock solution; preferably, the extraction agent is ammonium acetate solution.

[0036] Prepare a low-concentration buffer as the sample matrix liquid, and the low-concentration buffer comprises 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His), and an electroosmotic flow inhibitor; preferably, the concentration of 2-(N-morpholino) ethanesulfonic acid in the low-concentration buffer is 1.25-10 mM / L, the concentration of L-histidine is 1.25-10 mM / L, and the concentration of the electroosmotic flow inhibitor is 0.0025-0.1 M / L; further preferably, the concentration of MES is 2.5 mM / L, the concentration of His is 2.5 mM / L, and the concentration of CTAB is 0.01 mM / L.

[0037] Mix the sample extraction stock solution and the sample matrix liquid in a volume ratio of 1:1, adjust the pH value of the solution to 3-6 with 4% (v / v) glacial acetic acid to obtain the sample of the analyte to be detected.

[0038] (II) Preparation of the background buffer:

[0039] Prepare a mixed solution of 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His), and cetyltrimethylammonium bromide (CTAB) as the background buffer; preferably, the concentration of MES is 20 mM / L, the concentration of His is 20 mM / L, and the concentration of CTAB is 0.01 mM / L.

[0040] (III) Preparation of the microchip:

[0041] Cut a 5 mm thick polymethyl methacrylate (PMMA) bottom plate and a 0.2 mm thick polymethyl methacrylate (PMMA) top plate into a 30×70 rectangle using a laser cutting machine to obtain a microchip base and a microchip bonding cover plate.

[0042] The microchip substrate is clamped on a precision micro-milling machine, and a 0.1 mm milling cutter is used to process the cross micro-channel 1 at a high speed of 10000 rpm, the cross micro-channel 1 comprising a first channel 101 and a second channel 102 in communication with each other; the whole process is opened to cutting fluid, and the cross section of the micro-channel is 100 μm wide and 100 μm deep.

[0043] The precision micro-milling machine tool is replaced, and a 2 mm milling cutter is selected to process four liquid reservoirs at the four end points of the cross micro-channel at a speed of 4500 rpm, which are a sample pool 2, a first liquid reservoir 3, a second liquid reservoir 4 and a third liquid reservoir 5, to obtain an open microchip;

[0044] The open microchip and the microchip bonding cover plate are taken, and ultrasonic cleaning is used for 15 min, followed by plasma treatment for 5 min, and the treated surfaces are bonded, and the hot stamping technology is used in a hot press at a pressure of 63 kg and a temperature of 101 ℃ for 15 min to obtain a microchip; the second channel in the microchip is arranged with a detection area at one end close to the third liquid reservoir, and the detection area is composed of a transmitting electrode 6 and a receiving electrode 7. The transmitting electrode 6 transmits a high-frequency alternating current signal, and due to polarization, the receiving electrode 7 receives an alternating current signal of the same frequency. The amplitude of the alternating current signal is affected by the conductivity of the solution. When the analyte migrates to the detection area, the conductivity of the detection area changes, thereby changing the amplitude of the current signal received by the receiving electrode, obtaining an electrophoretic peak, and the information of the analyte species, concentration, etc. is contained in the electrophoretic peak.

[0045] (Four) Microchip electrophoresis process, including preparation stage, sample injection stage, separation stage and detection stage, Figure 1 The principle of pre-concentration of the method is mainly explained, which includes:

[0046] The preparation stage is as follows Figure 2 A: After flushing the cross micro-channel 1 with background buffer, the sample pool 2, the first liquid reservoir 3, the second liquid reservoir 4 and the third liquid reservoir 5 are filled with background buffer;

[0047] The sample injection stage is as follows Figure 2B: In this stage, we first use the syringe to remove the background buffer in the sample cell, and then use the pipette to add 50 μL of the analyte to be tested into the sample cell. Then, four high-voltage platinum electrodes are placed in the sample cell and three reservoirs, and the voltage conditions are set as follows: 500 V voltage is applied to the sample cell, the first reservoir is grounded, the second reservoir and the third reservoir are suspended, and the duration is 14 s. In this stage, due to the generation of the sample injection high voltage, the heavy metal cations in the analyte to be tested will move towards the first reservoir. In addition, due to the concentration gradient and pH gradient on both sides of the contact surface between the analyte to be tested and the background buffer, the heavy metal cations in the analyte to be tested will slow down and accumulate at the contact surface during migration, realizing the pre-concentration in the injection stage. The principle of the heavy metal cations slowing down and accumulating at the contact surface is as follows: first, we add a certain amount of electroosmotic flow inhibitor to the background buffer and the sample matrix solution, so that the electroosmotic flow changes direction, but its size cannot change the direction of the heavy metal cations moving towards the first reservoir. Secondly, there is a concentration gradient between the background buffer and the analyte to be tested, so their conductivities are different, and the migration rate of the heavy metal cations in them is also different, which is faster in the analyte to be tested than in the background buffer. Therefore, when the heavy metal cations pass through the contact surface, they slow down and accumulate due to the change in migration speed. In addition, there is a pH gradient between the background buffer and the analyte to be tested, and pH value is an important factor affecting electroosmotic flow, which is positively correlated. At this time, due to the presence of the electroosmotic flow inhibitor, the direction of the electroosmotic flow is from the first reservoir to the sample cell, and the pH value of the analyte to be tested is lower than that of the background buffer, so the electroosmotic flow in the analyte to be tested is smaller than that in the background buffer. Therefore, when the heavy metal cations pass through the contact surface, the migration rate of the heavy metal cations will decrease due to the increase of the electroosmotic flow, and will further slow down and accumulate at the contact surface.

[0048] The separation stage is as follows: Figure 2 C: Switch the voltage, apply 1000 V voltage to the second reservoir, while grounding the third reservoir, suspending the sample cell and the first reservoir, and the duration is 80 s. In this stage, the analyte to be tested will be pushed into the second channel at the cross intersection, and different types of heavy metal cations will gradually separate to form different zones in the second channel to pass through the detection area. In this process, the sample zone will form two contact surfaces with the background buffer before and after the sample zone, and the contact surface in the migration direction will continue to slow down and accumulate as in the injection stage. The heavy metal cations in the rear contact surface will quickly migrate to the contact surface in the migration direction due to the concentration gradient and pH gradient, so the sample zone will continuously narrow, further improving the pre-concentration effect of the heavy metal cations.

[0049] The detection stage is as follows: Figure 2D: The sample zone is pre-concentrated before it reaches the detection region. Different kinds of heavy metal cations are pre-concentrated and separated when they reach the detection region. The detection region draws electropherogram by collecting the amplitude change caused by the change of conductance.

[0050] Example 1

[0051] This example studies the effect of different concentrations of the electroosmotic flow inhibitor (CTAB) in the background buffer (0.0025, 0.01, 0.025, 0.05, 0.1 mM / L) on the electroosmotic flow and heavy metal cations. The cadmium chloride is dissolved in distilled water and mixed with 20 mM sample matrix solution 1:1 to prepare the standard sample for analysis.

[0052] The sample matrix solution contains 20 mM / L 2-(N-morpholino) ethanesulfonic acid (MES), 20 mM / L L-histidine (His) and the corresponding concentration of the electroosmotic flow inhibitor, with a pH value of 6.1. The background buffer contains 20 mM 2-(N-morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (His) and the corresponding concentration of the electroosmotic flow inhibitor, with a pH value of 6.1.

[0053] The specific steps for analyzing the concentration of the electroosmotic flow inhibitor are as follows:

[0054] 1) Flush the microchannel

[0055] When the microchannel is used for the first time, flush the microchannel with 1 mM NaOH solution for 15 min and distilled water for 15 min. When the microchannel is not used for the first time, flush the microchannel with 1 mM NaOH solution for 5 min and distilled water for 10 min.

[0056] 2) Flush the microchannel with the background buffer for 10-15 min before use.

[0057] 3) Inject 50 μL of the sample into the sample cell and insert the high-voltage platinum electrode.

[0058] 4) During the injection stage, apply a voltage of 500 V to the sample cell, ground the first reservoir, and leave the second and third reservoirs floating, with a duration of 14 s.

[0059] 5) During the separation stage, apply a voltage of 1000 V to the second reservoir, ground the third reservoir, and leave the sample cell and the first reservoir floating, with a duration of 80 s.

[0060] The results obtained are as follows: Figure 3The results are shown in the figure. It can be seen from the figure that as the concentration of the electroosmotic flow inhibitor increases, the peak of the heavy metal cadmium becomes smaller and the migration time increases. This is related to the inhibition of electroosmotic flow. When the concentration of the electroosmotic flow inhibitor reaches 0.05 mM / L, the electrophoretic peak of the heavy metal cadmium disappears completely, indicating that the electroosmotic flow is reversed and the ion migration direction is changed. When the concentration of the electroosmotic flow inhibitor is between 0.0025-0.025 mM / L, the electrophoretic peak of the heavy metal cadmium becomes smaller and the migration time increases, indicating that the electroosmotic flow is reversed but the ion migration direction does not change. In summary, the present application selects 0.01 mM / L electroosmotic flow inhibitor for subsequent research.

[0061] Example 2

[0062] This example studies the influence of different concentrations of sample matrix liquid (1.25, 2.5, 10, 15, 20 mM / L) on the preconcentration effect. The cadmium chloride is dissolved in distilled water and mixed with different concentrations of sample matrix liquid 1:1 to prepare standard samples for analysis.

[0063] The sample matrix liquid contains different concentrations (1.25, 2.5, 10, 15, 20 mM / L) of 2-(N-morpholino) ethanesulfonic acid (MES), L-histidine (His) and 0.01 mM / L electroosmotic flow inhibitor, and the pH value is 6.1. The background buffer contains 20 mM 2-(N-morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (His) and 0.01 mM / L electroosmotic flow inhibitor, and the pH value is 6.1; the rest of the conditions are the same as in Example 1.

[0064] The specific steps for analyzing the sample matrix liquid are as follows:

[0065] 1) Flush the microchannel

[0066] When the microchannel is used for the first time, flush the microchannel with 1 mM NaOH solution for 15 min and distilled water for 15 min; for non-first use, flush the microchannel with 1 mM NaOH solution for 5 min and distilled water for 10 min.

[0067] 2) Flush the microchannel with background buffer for 10-15 min before use.

[0068] 3) Inject 50 μL of sample into the sample pool and insert the high-voltage platinum electrode.

[0069] 4) In the injection stage, apply a voltage of 500 V to the sample pool, the first reservoir is grounded, and the second and third reservoirs are suspended, with a duration of 14 s.

[0070] 5) In the separation stage, apply a voltage of 1000 V to the second reservoir, ground the third reservoir, and suspend the sample pool and the first reservoir, with a duration of 80 s.

[0071] The results are as follows Figure 4 As shown in the figure, the electrophoresis peak height and sensitivity increase as the sample matrix concentration decreases. However, when the sample matrix concentration decreases from 2.5 mM / L to 1.25 mM / L, the electrophoresis peak height and sensitivity decrease from 24.10 mV and 48.20 mV / mM to 20.63 mV and 42.26 mV / mM, respectively. This is because when the sample matrix concentration is too low, the concentration difference between it and the background buffer (BGE) becomes too large, causing turbulence in the electroosmotic flow at the interface and resulting in poor concentration. Therefore, this invention selects 2.5 mM / L as the optimal concentration of the sample matrix for subsequent research.

[0072] Example 3

[0073] This example investigated the effect of sample matrix solution pH (3, 3.5, 4, 4.5, 6) on the pre-concentration effect. Cadmium chloride was dissolved in distilled water and mixed 1:1 with 2.5 mM / L sample matrix solution, and the pH was adjusted using 4% (v / v) glacial acetic acid as a standard sample for analysis.

[0074] The sample matrix solution contained 2.5 mM / L 2-(N-morpholino)ethanesulfonic acid (MES), 2.5 mM / L L-histidine (His), and 0.01 mM / L electroosmotic inhibitor. The background buffer contained 20 mM / L 2-(N-morpholino)ethanesulfonic acid (MES), 20 mM / L L-histidine (His), and 0.01 mM / L electroosmotic inhibitor, with a pH of 6.1.

[0075] The specific steps for analyzing the sample matrix solution are as follows:

[0076] 1) Flushing the microchannels

[0077] When using the microchannel for the first time, rinse the microchannel with 1mM NaOH solution for 15 min and then with distilled water for 15 min. For subsequent uses, rinse the microchannel with 1mM NaOH solution for 5 min and then with distilled water for 10 min.

[0078] 2) Rinse the microchannel with background buffer for 10-15 minutes before use.

[0079] 3) Inject 50 μL of sample into the sample cell and insert the high-voltage platinum electrode.

[0080] 4) During the sample injection stage, a voltage of 500V is applied to the sample cell, the first liquid storage cell is grounded, and the second and third liquid storage cells are suspended for 14 seconds.

[0081] 5) Separation phase, 1000V applied to the second reservoir, the third reservoir grounded, the sample reservoir and the first reservoir left floating, duration 80s.

[0082] The results are shown in Figures 1-3. Figure 5 and Figure 6 As shown in Figures 1-3, as the pH value decreases, the corresponding ion peak of the electropherogram is higher and the peak width is narrower, and the electropherogram peak is more acute. The peak height reaches 202 mV at pH = 3, and the relative standard deviation (RSD) of the peak height under the same experimental conditions is 8.71%. In addition, the migration time increases as the pH value decreases. The increase in migration time can be due to the decrease in pH value, which reduces the electroosmotic flow and reduces the mobility of the ions to be tested. At the same time, the increase in migration time also increases the time for the sample zone to accumulate at the interface. The decrease in peak width is because as the pH value decreases, the concentration effect of the ions to be tested improves, the ions are more concentrated, the time through the detection region is shorter, and the peak width is smaller. In summary, the sample matrix solution with a pH value of 3 is selected as the optimal pH value for subsequent research.

[0083] Example 4

[0084] The actual pre-concentration effect of the present application was observed. The conventional microchip electrophoresis method was compared with the pre-concentration method proposed in the present application. The lead and cadmium contaminated soil extract was mixed with 2.5 mM / L sample matrix solution at a ratio of 1:1, and 4% (v / v) glacial acetic acid was used to adjust the pH value to 3 as the sample for analysis.

[0085] The sample matrix solution contains 2.5 mM / L 2-(N-morpholino) ethanesulfonic acid (MES), 2.5 mM / L L-histidine (His), and 0.01 mM / L electroosmotic flow inhibitor, and the pH value is 3.0. The background buffer contains 20 mM 2-(N-morpholino) ethanesulfonic acid (MES), 20 mM L-histidine (His), and 0.01 mM / L electroosmotic flow inhibitor, and the pH value is 6.1.

[0086] The specific steps for analyzing the sample matrix solution are as follows:

[0087] 1) Flush the microchannel

[0088] When the microchannel is used for the first time, flush the microchannel with 1 mM NaOH solution for 15 min, and then flush with distilled water for 15 min. For non-first use, flush the microchannel with 1 mM NaOH solution for 5 min, and then flush with distilled water for 10 min.

[0089] 2) Flush the microchannel with background buffer for 10-15 min before use.

[0090] 3) Inject 50 μL of sample into the sample reservoir and insert the high-voltage platinum electrode.

[0091] 4) Injection phase, 500V voltage applied to sample reservoir, first reservoir grounded, second and third reservoirs left floating, duration 14s.

[0092] 5) Separation phase, 1000V applied to second reservoir, third reservoir grounded, sample and first reservoirs left floating, duration 80s.

[0093] The results are shown in Figs. 4(a) and 4(b). Figure 7 As shown in Figs. 4(a) and 4(b), in the microchip electrophoresis non-contact conductivity detection method without using the double-gradient pre-concentration method proposed by the present application, the separated lead and cadmium electrophoresis peaks were observed, but the signal intensity was weak and the resolution was poor, as shown in Fig. 4(a). Figure 7 After using the soil heavy metal ion online double-gradient pre-concentration detection method based on microchip electrophoresis non-contact conductivity detection proposed by the present application, the lead and cadmium were well concentrated, and strong enhanced peaks appeared at 42s and 55s, respectively, as shown in Fig. 4(b). Figure 7 In addition, the pre-concentration method of the present application can visibly improve the resolution (R) of lead and cadmium. The improvement of the resolution is related to the aggregation effect of the online double-gradient pre-concentration method on the ions in the sample zone. Compared with the traditional microchip electrophoresis method, the pre-concentration method proposed by the present application can improve the sensitivity of heavy metals lead and cadmium by 26.4 and 43.73 times, respectively. In addition, the pre-concentration and separation detection of heavy metal ions are completed within 90s by using the pre-concentration method proposed by the present application. Therefore, the experiment proves that the present application can obtain a larger pre-concentration multiple in a very short experimental time, and the microchip is simple and low in cost, and the operation for pre-concentration and separation detection of heavy metal ions is simple.

[0094] The above examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Any skilled person in the art can make various improvements and modifications to the present application without departing from the design spirit and scope of the present application, and the improvements and modifications shall fall within the protection scope of the present application.

Claims

1. A method for online dual-gradient pre-concentration detection of heavy metal ions in soil based on microchip electrophoresis non-contact conductivity detection, characterized in that: The method comprises the following steps: Preparation of a sample to be tested: soil sample is soaked in a leaching agent to obtain a sample leaching stock solution; The sample leaching stock solution is mixed with a low-concentration buffer, and then an acid is added to reduce the pH value to obtain a sample to be tested; A high-concentration buffer is prepared as a background buffer, and the concentration of the high-concentration buffer is higher than that of the low-concentration buffer; A microchip is provided: the microchip comprises a cross-shaped microchannel, the cross-shaped microchannel comprises a first channel and a second channel which are in communication with each other, two ends of the first channel are respectively formed with a sample pool and a first reservoir, and two ends of the second channel are respectively formed with a second reservoir and a third reservoir; a detection area is arranged at one end of the second channel close to the third reservoir, and the detection area is composed of an emitting electrode and a receiving electrode; A microchip electrophoresis process comprises a preparation stage, a sample injection stage, a separation stage and a detection stage, wherein: In the preparation stage, the cross-shaped microchannel is flushed with the background buffer, and then the sample pool, the first reservoir, the second reservoir and the third reservoir are filled with the background buffer; In the sample injection stage, the background buffer in the sample pool is replaced by the sample to be tested, and a high-voltage electrode is arranged in each of the sample pool, the first reservoir, the second reservoir and the third reservoir; a voltage is applied to the sample pool, the first reservoir is grounded, and the second reservoir and the third reservoir are suspended; at this time, the sample to be tested is pre-concentrated and moves to the intersection of the cross-shaped microchannel; In the separation stage, a voltage is applied to the second reservoir, the third reservoir is grounded, and the sample pool and the first reservoir are suspended; at this time, the sample to be tested is pushed into the second channel, and different types of heavy metal cations are gradually separated in the second channel to form different sample bands and pass through the detection area; In the detection stage, when the sample band reaches the detection area, an electropherogram is drawn by collecting the amplitude change caused by the change in conductance; The low-concentration buffer and the high-concentration buffer have the same components, including 2-(N-morpholino) ethanesulfonic acid, L-histidine and an electroosmotic flow inhibitor; the concentration of 2-(N-morpholino) ethanesulfonic acid in the low-concentration buffer is 1.25-10 mM / L, the concentration of L-histidine is 1.25-10 mM / L, and the concentration of the electroosmotic flow inhibitor is 0.0025-0.1 M / L; the concentration of 2-(N-morpholino) ethanesulfonic acid in the high-concentration buffer is 20 mM / L, the concentration of L-histidine is 20 mM / L, and the concentration of the electroosmotic flow inhibitor is 0.0025-0.1 M / L.

2. The method according to claim 1, wherein the method is characterized by: The leaching agent is an ammonium acetate solution.

3. The method according to claim 1, wherein the method is characterized by: The concentration of the electroosmotic flow inhibitor in the low-concentration buffer and the high-concentration buffer is 0.01 mM / L.

4. The method according to claim 1 or 3, wherein the method is characterized by: The electroosmotic flow inhibitor is hexadecyl trimethyl ammonium bromide.

5. The method according to claim 1, wherein the method is characterized by: The concentration of 2-(N-morpholino) ethanesulfonic acid in the low-concentration buffer is 2.5 mM / L, and the concentration of L-histidine is 2.5 mM / L.

6. The method according to claim 1, wherein the method is characterized by: The acid is glacial acetic acid, hydrochloric acid or nitric acid; and the pH value of the sample to be tested is 3.0-6.

0.

7. The method for online dual-gradient pre-concentration detection of heavy metal ions in soil based on microchip electrophoresis non-contact conductivity detection according to claim 1, characterized in that: The voltage applied to the sample pool is 500-2000V, and the voltage applied to the second liquid pool is 1000-2000V.

8. The method for online dual-gradient pre-concentration detection of heavy metal ions in soil based on microchip electrophoresis non-contact conductivity detection according to claim 1, characterized in that: The high-voltage electrode is a platinum metal electrode.

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