Trypsin detection method based on hydrogel-filled microchannels

Abstract

The application discloses a method for detecting trypsin in a hydrogel microchannel filled with glutaraldehyde cross-linked with human serum albumin. Trypsin has specific hydrolysis on HSA, and the hydrogel will expose carboxyl after hydrolysis. Therefore, when the hydrogel is hydrolyzed by trypsin, the change of the charge density of the hydrogel microchannel will be triggered, leading to the change of the ion current, so that the detection of trypsin is realized. The performance of the trypsin sensor is adjusted by adjusting experimental influencing factors (such as HSA concentration, electrolyte concentration, pH value and reaction time). Good feedback is obtained in actual sample measurement. The present study successfully performs microscale ICR experiments in the microchannel, thereby successfully constructing the trypsin sensor. The hydrogel-filled microchannel not only retains the advantages of the original nanochannel, but also solves the defects of nanochannel blockage, high operation difficulty and the like. It can also provide some references for the measurement of macromolecular targets in nanochannels / microchannels.

Description

Technical Field

[0001] The invention belongs to the field of biological detection, and particularly relates to filling a microchannel with hydrogel to generate rectification and using the rectification for electrochemical detection of trypsin. Background Art

[0002] Ionic current rectification (ICR) has attracted much attention due to its importance in fabricating nanofluidic transistors, mimicking biological gated ion channels, and building sensing devices. Micrometer-scale ICR occurs when the asymmetry within a micropore increases or the charge density is large enough.

[0003] Hydrogel is a type of extremely hydrophilic gel with a three-dimensional network structure. Its structural controllability and charge tunability make it an effective material for filling nano / micro channels, and on this basis, new nano / micro channel sensors can be developed. Currently, there are literature reports on several applications of hydrogels filling micro-nano channels. Studies have shown that the use of hydrogels in nanopores has great potential. However, no one has used micron-scale channels to detect large molecular targets. Trypsin is present in the digestive system and can hydrolyze protein molecules into small peptides to promote their absorption. Abnormal trypsin levels are highly correlated with several serious diseases, such as pancreatic cancer, pancreatitis, cystic fibrosis and biliary cirrhosis. Therefore, trypsin detection is of great significance for clinical diagnosis. This application combines the advantages of hydrogels and microchannel electrochemical sensors for the first time to construct a biological detection method and apply it to the analysis of trypsin. Summary of the Invention

[0004] To achieve the above objectives, the present invention discloses a method for detecting trypsin based on hydrogel-filled microchannels, using the following technical scheme: First, quartz glass capillaries are immersed in piranha solution for 8 hours, thoroughly rinsed with water, and then air-dried. Subsequently, these glass tubes are converted into 3.0μm microchannels using a laser drawing instrument, and the pore size uniformity of each microchannel is verified using an optical microscope. Next, the microchannels are immersed in a mixture of glutaraldehyde (50%) and human serum albumin (180mg / mL) (the volume ratio of 50% glutaraldehyde and 180mg / mL human serum albumin is 50:8) for 10 seconds. Due to the siphon effect, the mixed solution forms a hydrogel inside the microchannel. After the mixed solution forms a hydrogel in the microchannel, it is filled with 100mM potassium chloride solution to obtain a hydrogel microchannel. The hydrogel microchannel is immersed in trypsin solutions of different concentrations for 1 hour and then rinsed with 100mM potassium chloride solution to remove residual trypsin. Finally, the hydrogel microchannel is electrochemically tested to determine whether micron-scale ICR is generated.

[0005] Furthermore, a trypsin sensor was constructed using the aforementioned method. The feasibility of the constructed trypsin detection sensor was then investigated. When trypsin solutions of varying concentrations were inserted into the hydrogel microchannel, the rectification ratio was observed to vary significantly with the concentration of trypsin. To improve sensor performance, multiple parameters were optimized. Because HSA concentration affects the hardness of the hydrogel and, consequently, its filling efficiency within the microchannel, experiments were conducted using a gradient HSA concentration range of 125 mg / mL to 250 mg / mL. The optimal concentration was found to be 180 mg / mL. Furthermore, the strength of the electrolyte affects the strength of the double layer, so optimization was also required. The optimal KCl concentration was found to be 100 mM within the range of 1–1500 mM. Furthermore, the reaction time between trypsin and the hydrogel also had a certain influence on rectification. Experiments were conducted using different reaction times, and the optimal detection reaction time was found to be 60 minutes within the range of 10–120 minutes, optimizing the sensor's performance. Finally, a comprehensive linear model was established.

[0006] The beneficial effects of the present invention are as follows:

[0007] (1) Through the optimization of conditions, hydrogels can be quickly formed in microchannels within 10 s.

[0008] (2) Electrochemical testing of hydrogel microchannels revealed that they can achieve micron-scale ICR. This approach not only maintains the high spatiotemporal resolution and sensitivity of nanopores, but also addresses the issues of fragile nanopore tips, difficult operation, and inconvenience in detecting large molecular targets.

[0009] (3) A practical trypsin detection method was designed using this hydrogel microchannel. Compared with other trypsin sensors, it has a wider linear range and higher sensitivity. At the same time, this also provides some reference for the measurement of large molecular targets in nano / microchannels. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Optical microscope (A) and transmission electron microscope (B) images of the hydrogel-filled microtubes in Example 1 of the present invention.

[0011] Figure 2 This is an IV test diagram of the microchannel modified with hydrogel in Example 2 of the present invention.

[0012] Figure 3 This is a feasibility diagram for detecting trypsin in Example 3 of the present invention.

[0013] Figure 4 This is the working curve for detecting trypsin in Example 4 of the present invention. DETAILED DESCRIPTION

[0014] The application will be further described in detail below with reference to the examples.

[0015] Example 1

[0016] First, the quartz glass capillary was soaked in piranha solution for 8 hours, rinsed thoroughly with water, and then air dried. Subsequently, the capillary was converted into a microchannel using a carbon dioxide laser pipette (P-2000). The pipette (P-2000) converted the capillary into a microchannel with a uniform 3.0 micrometer diameter at the microcapillary orifice. The uniformity of the microchannel orifice was verified using an optical microscope. Next, the microchannel was treated by immersing it in a mixed solution of glutaraldehyde (50%) and human serum albumin (180 mg / mL) (volume ratio of 50:8) for 10 seconds. After 10 seconds, the mixed solution formed a hydrogel within the microchannel, and a 100 mM KCl solution was injected into the hydrogel microchannel to obtain a hydrogel microchannel. The hydrogel filling was confirmed using an optical microscope and a transmission electron microscope. The optical microscope and transmission electron microscope images of the hydrogel-filled microcapillary are shown in FIGS. 1 and 2, respectively. Figure 1

[0017] Example 2

[0018] I-V curve testing of the hydrogel microchannel was performed using the CV mode of the electrochemical workstation with a scan voltage range of -1 V to 1 V. The scan rate was 0.05 V / s. An Ag / AgCl electrode was inserted into the microchannel as the working electrode, and another Ag / AgCl electrode was placed in the reaction cell as the reference electrode and auxiliary electrode. The electrolyte solution used was 100 mM KCl. In all cases, the electrolyte solution in the microchannel was matched with that in the reaction cell. It was found that the I-V curve exhibited an asymmetric shape, indicating the success of the microscale ICR. The I-V testing graph of the hydrogel-modified microchannel is shown in FIG. 3. Figure 2

[0019] Example 3

[0020] The trypsin was dissolved in a PBS buffer with pH = 7.4, and the hydrogel microchannel was soaked in a trypsin solution with concentrations of 1 and 100 μg / mL for one hour, and then rinsed with a 100 mM KCl solution to remove the residual trypsin. Finally, I-V testing was performed on the hydrogel microchannel. The scan voltage range was -1 V to 1 V. The scan rate was 0.05 V / s. It was found that different concentrations of trypsin could change the ionic current of the hydrogel (I). Figure 3

[0021] Example 4

[0022] ​​​The hydrogel microchannels were soaked in 0.01, 0.1, 1, 10, 100 μg / mL trypsin solution for one hour, and then rinsed with 100 mM KCl solution to remove residual trypsin. The hydrogel microchannels were tested for I-V. The scan voltage range was -1 V to 1 V. The scan rate was 0.05 V / s. A working curve for detecting trypsin was made. Figure 4

[0023] It is to be understood that the application is not limited to the examples described above, which can be modified or adapted in various ways by those skilled in the art without thereby departing from the scope of the application as defined in the appended claims.​

Claims

1. A trypsin detection method based on hydrogel-filled microchannels, characterized by: The hydrogel of human serum albumin cross-linked with glutaraldehyde was filled into the microchannel, and electrochemical testing was performed using an electrochemical workstation. Trypsin is used to hydrolyze serum albumin. The carboxyl groups of the hydrogel are exposed after being hydrolyzed by trypsin, which leads to changes in ion current. The detection of trypsin is achieved by detecting the changes in current. The hydrogel was filled into the microchannels by: (1) immersing quartz glass capillaries in piranha solution for 8 hours, rinsing thoroughly with water, and then air-drying; then, using a carbon dioxide laser drawer, these capillaries were converted into microchannels with a diameter of 3.0 μm; (2) the formed microchannels were immersed in a mixture of 50% glutaraldehyde and 180 mg / mL human serum albumin for 10 seconds; The electrochemical test using the electrochemical workstation includes: performing an IV test on the prepared hydrogel microchannel using the electrochemical workstation; The IV curve showed an asymmetric diode-like shape, with negative current exceeding positive current. Specifically, the IV curve was captured using an electrochemical workstation with a sweep voltage range of -1 V to 1 V. An Ag / AgCl electrode was inserted into the microchannel as the working electrode, while another Ag / AgCl electrode was placed in the reaction cell and served as both the reference electrode and the auxiliary electrode. The electrolyte solution used was 100 mM KCl. The trypsin detection method is as follows: the prepared hydrogel microchannel is immersed in trypsin solutions of different concentrations for one hour, and then rinsed with 100mM KCl solution to remove residual trypsin; then, an IV curve test is performed using an electrochemical workstation; and a working curve is plotted using the ratio of the current value at a negative potential to the current value at a positive potential as the vertical axis and the trypsin concentration as the horizontal axis.

2. The trypsin detection method based on hydrogel-filled microchannel according to claim 1, characterized in that The volume ratio of the 50% glutaraldehyde to 180 mg / mL human serum albumin is 50:

8.

3. The trypsin detection method based on hydrogel-filled microchannel according to claim 1, characterized in that: The concentration of the trypsin solution is 0.01-100 μg / mL.

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