Cell Electrochemical Sensor Based on 3D Printing Technology and Application Thereof

Abstract

The disclosure relates to a cell electrochemical sensor based on a 3D printing technology and application thereof and belongs to the technical field of electrochemical sensors and toxin detection. The cell electrochemical sensor of the disclosure is constructed based on a 3D printing technology, and the construction method comprises the following steps: precisely depositing a cell/carbon nanofiber/GelMA composite hydrogel on a working electrode of a screen-printed carbon electrode through 3D printing, and carrying out curing to obtain the cell electrochemical sensor. The disclosure constructs a cell electrochemical sensor with a three-dimensional cell growth environment and rapid and sensitive response. The cell electrochemical sensor constructed by the disclosure can be used for quickly and effectively determining the combined effect type and effect degree of deoxynivalenol family toxins by combining an electrochemical impedance method and a combination index method.

Claims

1. A method for making a cell electrochemical sensor, wherein the method is based on a 3D printing technology comprising the following steps: (1) preparing a cell/carbon nanofiber/gelatin methacryloyl (GeMA) composite hydrogel: adding a carbon nanofiber solution into a GelMA solution for uniform mixing to obtain a carbon nanofiber/GelMA composite solution, and then uniformly mixing cells with the carbon nanofiber/GelMA composite solution to obtain the cell/carbon nanofiber/GeMA composite hydrogel; and (2) precisely depositing the cell/carbon nanofiber/GelMA composite hydrogel obtained in step (1) on a working electrode of a screen-printed carbon electrode through 3D printing, and carrying out curing to obtain the cell electrochemical sensor.

2. The method according to claim 1, wherein a final concentration of GeMA in the cell/carbon nanofiber/GelMA composite hydrogel is 5-15%.

3. The method according to claim 1, wherein a final concentration of cells in the cell/carbon nanofiber/GelMA composite hydrogel is 110.sup.6-110.sup.7/mL.

4. The method according to claim 1, wherein a final concentration of GeMA in the cell/carbon nanofiber/GelMA composite hydrogel is 5-7.5%.

5. The method according to claim 1, wherein a final concentration of carbon nanofibers in the cell/carbon nanofiber/GelMA composite hydrogel is 0.5-1 mg/mL.

6. The method according to claim 1, wherein the cells are lung adenocarcinoma epithelial cells A549.

7. The method according to claim 1, wherein before the step (1), the method further comprises: preparation of a GelMA solution: adding an LAP initiator into a DMEM cell culture medium for well mixing and dissolving to obtain a dissolved standard initiator solution, and then adding a GelMA material into the dissolved standard initiator solution for dissolving in a water bath in dark to obtain the GeMA solution.

8. The method according to claim 1, wherein the 3D printing comprises: pouring the cell/carbon nanofiber/GelMA composite hydrogel into a printing syringe, setting an initial needle position, a syringe temperature, a working platform temperature, an extrusion pressure, a graphic size, a graphic layer number and a nozzle walking speed, then precisely depositing the composite hydrogel on the working electrode of the screen-printed carbon electrode through 3D printing, and curing the composite hydrogel under a portable curing light source.

9. The cell electrochemical sensor made by the method according to claim 1, wherein the cell/carbon nanofiber/GelMA composite hydrogel is immobilized on a surface of a working electrode in the cell electrochemical sensor.

10. A method of using the cell electrochemical sensor according to claim 9 for evaluating toxicity of deoxynivalenol-family mycotoxins by, comprising: diluting one or more toxin standard substances with a DMEM cell culture medium into gradient concentration solutions; dropping the gradient concentration solutions on a working electrode printed with cells; placing the working electrode in an incubator for effect for 24 hours; and then carrying out electrochemical detection.

11. The method according to claim 10, wherein after the carrying out electrochemical detection, the method further comprises analyzing cytotoxicity and combined effect types of the toxins by using an electrochemical AC impedance method EIS and a combination index method (CI).

12. The method according to claim 10, wherein before diluting the one or more toxin standard substances with the DMEM cell culture medium into the gradient concentration solutions, the method further comprises: dropping the DMEM cell culture medium at the working electrode of the screen-printed carbon electrode to ensure that the culture medium can cover a cell/carbon nanofiber/GelMA complex on the working electrode to provide cells with nutrients needed for growth; placing the working electrode in an incubator with the carbon dioxide concentration of 5% and the humidity of 95% for incubation at 37 C. for 6-12 hours; and after incubation, removing the original culture medium on the working electrode.

13. The method according to claim 10, wherein the deoxynivalenol-family mycotoxins are one or two of deoxynivalenol (DON) and 15-acetyl-deoxynivalenol (15-ADON).

Description

BRIEF DESCRIPTION OF FIGURES

[0047] FIG. 1: A schematic diagram of the modification process of a screen-printed electrode.

[0048] FIG. 2: A schematic diagram of a 3D printing process for preparing a cell electrochemical sensor.

[0049] FIG. 3: Electrochemical characterization diagrams of a construction process of the cell electrochemical sensor, wherein FIG. 3A is an electrochemical signal obtained by cyclic voltammetry (CV); FIG. 3B is an electrochemical signal obtained by differential pulse voltammetry (DPV); FIG. 3C is an electrochemical signal obtained by electrochemical impedance spectroscopy (EIS); in FIGS. 3A-3C, a refers to a bare electrode (SPCE), b refers to an electrode modified with AuNPs (AuNP/SPCE), c refers to a carbon nanofiber/GeMA/AuNPs electrode (CN/GelMA/AuNP/SPCE), and d refers to a cell/carbon nanofiber/GeMA/AuNPs electrode (cells/CN/GelMA/AuNP/SPCE).

[0050] FIG. 4 is electrode-modified electron microscope characterization diagrams, wherein FIG. 4A refers to a bare electrode (SPCE); FIG. 4B refers to an electrode modificed with AuNPs (AuNP/SPCE-modified electrode); FIG. 4C refers to a cell/carbon nanofiber/GeMA/AuNPs electrode (cells/CN/GelMA/AuNP/SPCE-modified electrode); FIG. 4D refers to cells growing in clusters in a cell/carbon nanofiber/GeMA composite hydrogel.

[0051] FIG. 5: EIS detection result diagrams of the cell sensor when the cell electrochemical sensor is used for evaluating DON, 15-ADON and the combined effect of the two toxins, wherein FIG. 5A is an EIS detection result diagram of the cell sensor after 24 hours of DON stimulation, and a-f refer to the toxin concentrations of 0.1, 0.2, 0.5, 1, 2 and 5 g/mL respectively; FIG. 5B is an EIS detection result diagram of the cell sensor after 24 hours of 15-ADON stimulation, and a-f refer to the toxin concentrations of 0.1, 0.2, 0.5, 1, 2 and 5 g/mL respectively; FIG. 5C is an EIS detection result diagram of the cell sensor after 24 hours of DON and 15-ADON stimulation, and a-f refer to the toxin concentrations of 0.1+0.1, 0.2+0.2, 0.5+0.5, 1+1, 2+2 and 5+5 g/mL respectively; FIG. 5D is a correlation curve of different concentrations of DON and the electrochemical impedance value of the constructed sensor; FIG. 5E is a correlation curve of different concentrations of 15-ADON and the electrochemical impedance value of the constructed sensor; and FIG. 5F is a correlation curve of different concentrations of DON+15-ADON and the electrochemical impedance value of the constructed sensor.

[0052] FIG. 6: Experimental comparison results of the cell electrochemical sensor and a CCK8 method in evaluating the cytotoxicity, wherein FIG. 6A is DON; FIG. 6B is 15-ADON; and FIG. 6C is the combined effect of the two toxins DON and 15-ADON.

[0053] FIG. 7: An impedance spectrum diagram of electrochemical sensors prepared with different concentrations of the lung adenocarcinoma epithelial cells A549, wherein the cell concentration ranges of a-e are 110.sup.3/mL, 110.sup.4/mL, 110.sup.5/mL, 110.sup.6/mL and 110.sup.7/mL respectively.

[0054] FIG. 8: FIG. 8A refers to the survival rate of the lung adenocarcinoma epithelial cells A549 in different concentrations of GelMA hydrogels; FIG. 8B is a DPV detection result diagram of the electrode when different concentrations of GelMA hydrogels are printed on the screen-printed carbon electrode, wherein a-e refer to the GeMA concentrations of 5%, 7.5%, 10%, 12.5% and 15% respectively.

[0055] FIG. 9: FIG. 9A is an EIS detection result diagram of the cell sensor when the DON toxin stimulates the cell electrochemical sensor prepared by a dropping method, wherein a-f refer to the DON toxin concentrations of 0.1, 0.5, 2, 0.2, 1 and 5 g/mL respectively; and FIG. 9B is a correlation curve graph of the DON toxin and the sensor impedance value.

DETAILED DESCRIPTION

[0056] The preferred examples of the disclosure will be described below. It should be understood that the examples are used for better explaining the disclosure and are not intended to limit the disclosure. The carbon nanofibers used in the following examples and comparative example are purchased from Xianfeng Nano, the model is XFM60, the diameter is 200-600 nm, and the length is 5-50 m.

Example 1 Preparation of a Cell Electrochemical Sensor

[0057] A method for constructing a cell electrochemical sensor based on a 3D printing technology (shown as FIG. 2) comprises the following steps:

[0058] (1) Preparation of a gelatin methacryloyl (GelMA) solution: An LAP initiator is added into a DMEM cell culture medium to make the final concentration reach 0.5%, and then dissolving is carried out in a water bath at 60 C. for 30 min in the dark to obtain a dissolved standard initiator solution. A GelMA material is added into the dissolved standard initiator solution for dissolving in a water bath at 60 C. for 30 min in the dark, and shaking is carried out 3 times during the period to obtain a GelMA solution (the mass concentration is 7.5%); and then the obtained GeMA solution is filtered with a 0.22 m sterile filter membrane into a clean container for use.

[0059] (2) Preparation of a cell/carbon nanofiber/GelMA composite hydrogel: A certain amount of carbon nanofibers are added into a phosphate buffer solution (PBS) to make the concentration of carbon nanofibers reach 1 mg/mL, ultrasonic treatment is carried out for 2 hours, and then a prepared carbon nanofiber solution is placed under an ultraviolet light for irradiation overnight. A certain amount of the carbon nanofiber solution is added into the GeMA solution obtained by filtration in step (1), the mixed solution is thoroughly mixed to make the GeMA concentration reach 7.5% and the carbon nanofiber solid filling amount reach 0.05%, and a carbon nanofiber/GelMA composite solution is obtained. The human lung adenocarcinoma epithelial cells A549 are uniformly mixed with the carbon nanofiber/GelMA composite solution, and the cell concentration is adjusted to be 110.sup.6/mL to obtain the cell/carbon nanofiber/GeMA composite hydrogel.

[0060] (3) Modification and electroplating of a screen-printed carbon electrode with gold nanoparticles (shown as FIG. 1): The screen-printed carbon electrode needs to be activated in a 0.5 mmol/L sulfuric acid solution first and then scanned by cyclic voltammetry, the voltage is 0.2 V-0.6 V, the scanning rate is 100 mV/s, and the scanning circle number is 8. The activated screen-printed carbon electrode is immersed in a 10 mL of a gold electroplating solution containing 1% chloroauric acid and 1 mmol/L sulfuric acid solution, a time-current method is adopted, the electroplating voltage is 0.3 V, the electroplating time is 120 seconds, and then the electrode is rinsed with ultrapure water, dried with nitrogen and placed under an ultraviolet light overnight for irradiation and sterilization to obtain the screen-printed carbon electrode modified and electroplated with gold nanoparticles.

[0061] (4) 3D printing: The cell/carbon nanofiber/GelMA composite hydrogel prepared in step (2) is poured into a printing syringe, the syringe temperature is set to be 26 C., the working platform temperature is 3 C., the extrusion pressure is 0.1 MPa, the graphic length is 3 mm, the graphic width is 3 mm, the printing layer number is 1, the nozzle walking speed is 300 mm/min, the screen-printed carbon electrode modified and electroplated with gold nanoparticles prepared in step (3) is placed at a specific position on the working platform, a printing procedure is run to precisely deposit the composite hydrogel on the working electrode of the screen-printed carbon electrode, and the composite hydrogel is irradiated with a portable curing light source with the wavelength of 405 nm for 10-20 seconds for curing to obtain the cell electrochemical sensor.

[0062] The cell electrochemical sensor obtained in step (4) needs to undergo cell incubation before application, and specific steps comprise: dropping a 150 L of the DMEM cell culture medium at the working electrode of the screen-printed carbon electrode to ensure that the culture medium can cover a cell/carbon nanofiber/GelMA complex on the working electrode to provide cells with nutrients needed for growth, and then placing the working electrode in an incubator with the carbon dioxide concentration of 5% and the humidity of 95% for incubation at 37 C. for 6 hours to obtain the incubated cell electrochemical sensor.

[0063] Cyclic voltammetry, differential pulse voltammetry, an AC impedance method and scanning electron microscope characterization are performed on the cell electrochemical sensor after incubation. The conditions of the voltammetry method are: the voltage of 0.2 V-0.6 V and the scanning rate of 100 mV/s; the condition of the AC impedance method is: the frequency range of 1 Hz-10.sup.5 Hz. In the scanning electron microscope characterization, samples containing the cells A549 are tested after the cells are cultured in the CN/GeMA composite hydrogel for 48 hours.

[0064] As shown in FIG. 3, compared with a bare screen-printed carbon electrode, the electrical signal of the gold-plated screen-printed carbon electrode is significantly enhanced, and it is indicated that the gold nanoparticles have excellent conductivity. When the carbon nanofiber/GelMA hydrogel is deposited on the working electrode, the electrical signal is reduced to some extent. When the cell/carbon nanofiber/GelMA hydrogel is deposited on the working electrode, the electrical signal is further reduced due to the insulation of cell membranes, it is indicated that the cells are successfully modified on the electrode surface, and preparation of the cell electrochemical sensor is completed.

[0065] The electrode-modified electron microscope characterization diagram in FIG. 4 shows that AuNPs are evenly distributed on the electrode surface (FIG. 4B). FIG. 4C shows uniform immobilization of carbon nanofibers in the GeMA hydrogel, which increases catalytically active sites for achieving better electrochemical performance. After the cells A549 are introduced into the screen-printed carbon electrode, the cells are firmly immobilized and evenly distributed in the CN/GelMA composite hydrogel. In addition, the cells are also aggregated in the composite hydrogel, which maintain complete cell morphologies (FIG. 4D), the good physical state of the cells in the hydrogel is confirmed, and redox probes are prevented from entering the electrode surface, so that the electron transfer resistance is increased.

Example 2 Application of a Cell Electrochemical Sensor Based on a 3D Printing Technology

[0066] The incubated electrochemical sensor obtained in example 1 is used for evaluating the cytotoxicity of deoxynivalenol-family mycotoxins, and specific operations are as follows:

[0067] (1) Drug stimulation: An original culture medium on a working electrode is removed, toxin standard substances are diluted with a DMEM cell culture medium into gradient concentration solutions, and then 150 L of DON toxin solutions in the concentration range of 0.1, 0.2, 0.5, 1, 2 and 5 g/mL, 150 L of 15-ADON toxin solutions in the concentration range of 0.1, 0.2, 0.5, 1, 2 and 5 g/mL and 150 L of DON+15-ADON toxin solutions in the concentration range of 0.1+0.1, 0.2+0.2, 0.5+0.5, 1+1, 2+2 and 5+5 g/mL are taken and dropped on the working electrode printed with cells respectively, the working electrode is placed in an incubator for effect for 24 hours, and corresponding impedance values are measured.

[0068] (2) Detection of electrochemical signal values: A 150 L of 2.5 mM Fe(CN).sub.6.sup.3/4 PBS solution is used as an electrode reaction system, the frequency range of an electrochemical AC impedance method (EIS) is 1 Hz-10.sup.5 Hz, and the impedance value is fitted by Zview software and calculated through the best equivalent circuit. The toxic effects on cells are generated after different doses of toxins stimulate the cells, EIS is used for analyzing the toxicity of the toxins on the lung adenocarcinoma epithelial cells A549, and a calculation method is as follows:

[00004]$\mathrm{Inhibition}.Math..Math.\mathrm{rate}.Math..Math.\left(\%\right)=\left(1-\frac{R_{\mathrm{dosing}}-R_{\mathrm{carbon}.Math..Math.\mathrm{nanofiber}/\mathrm{GeIMA}}}{R_{0.Math.\mathrm{dosing}}-R_{\mathrm{carbon}.Math..Math.\mathrm{nanofiber}/\mathrm{GeIMA}}}\right)100$

[0069] wherein R.sub.dosing refers to the impedance value of the screen-printed carbon electrode (SPE) with toxin stimulation and bioprinting modification, R.sub.0dosing refers to the impedance value of the screen-printed carbon electrode (SPE) with bioprinting modification and without toxin stimulation, and R.sub.carbon nanofiber/GelMA refers to the impedance value of the SPE with modification of carbon nanofibers and GelMA hydrogel and without cells.

[0070] The combined effect type can be determined by substituting the inhibition rate obtained after the sensor is stimulated by toxins into a combination index formula, and a combined effect formula in the combination index method is:

[00005]$\frac{f_a}{f_u}={\left(\frac{D}{D_m}\right)}^m$

[0071] wherein f.sub.a refers to a cell damage effect rate, f.sub.u refers to a cell undamaged effect rate, D refers to a toxin concentration, D.sub.m refers to a toxin concentration when the cell damage effect rate reaches 50%, and m refers to a dose-effect curve coefficient.

[0072] A calculation formula of the CI index is:

[00006]${\mathrm{CI}}_x={.Math.}_{j=1}^n.Math.\frac{{\left(D\right)}_j}{{\left(D_x\right)}_j}$

[0073] wherein (D).sub.j refers to the required concentration when x % damage effect is caused by the combined effect of toxins, (D.sub.x).sub.j refers to the concentration when x % damage effect is caused by a single toxin, and if CI>0.9, the combined effect type of toxins is considered to be antagonism; if CI=0.9-1, the combined effect type of toxins is considered to be an additive effect; and if CI<0.9, the combined effect type of toxins is considered to be synergism.

[0074] (3) Result judgment

TABLE-US-00001 TABLE 1 CI index values obtained after analysis of the combined effect of mycotoxins by the cell electrochemical sensor Concentration of Inhibition mycotoxins/(g/mL) rate/100 Combined DON 15-ADON f.sub.a CI effect type 0.1 0.1 0.0877 1.23033 Antagonism 0.2 0.2 0.2768 0.80967 Synergism 0.5 0.5 0.4027 1.28417 Antagonism 1 1 0.5122 1.79919 Antagonism 2 2 0.6426 2.33635 Antagonism 5 5 0.6778 5.14921 Antagonism

[0075] As shown in FIG. 5, the impedance value of the cell sensor prepared in example 1 is gradually reduced with the increasing dose of DON in the concentration range of 0.1-5 g/mL and the increasing dose of 15-ADON in the concentration range of 0.1-5 g/mL, the reason is that after different doses of toxins stimulate the cells A549, the cells can undergo different degrees of apoptosis, lysis, and morphological changes, and thus changes of impedance electrochemical signals are caused. The IC.sub.50 value of DON measured by the EIS method is 0.9281 g/mL, the IC.sub.50 value of 15-ADON is 1.2560 g/mL, the IC.sub.50 value of DON and 15-ADON during combined effect is 2.279 g/mL, and specific results are shown in Table 1. It is found by using the cell electrochemical sensor constructed in example 1 to analyze the combined effect type of DON and an acetylated derivative 15-ADON thereof that the two toxins generally show an antagonistic effect.

Example 3 a Verification Experiment

[0076] The CCK8 method is used for detecting the cytotoxicity of DON, 15-ADON and DON+15-ADON alone and in combination. The lung adenocarcinoma epithelial cells A549 with the density of 510.sup.4/mL are adhered to the wall of a 96-well plate for inoculation, a culture medium is removed after culture for 24 hours, and 100 L of the toxin solution same as that in example 2 is added. After 24 hours of toxin stimulation, the supernatant is sucked out, a 100 L of culture medium containing 10% CCK8 is added into each well for incubation at 37 C. for 2 hours, then the absorbance value is measured with a microplate reader at 450 nm, the cell activity inhibition rate is calculated, and the calculation method is as follows:

[00007]$\mathrm{Inhibition}.Math..Math.\mathrm{rate}.Math..Math.\left(\%\right)=\left(1-\frac{{\mathrm{OD}}_{\mathrm{dosing}}-{\mathrm{OD}}_{\mathrm{blank}}}{{\mathrm{OD}}_{0.Math.\mathrm{dosing}}-{\mathrm{OD}}_{\mathrm{blank}}}\right)100$

[0077] wherein OD.sub.dosing refers to the absorbance value after 24 hours of toxin stimulation, OD.sub.0dosing refers to the absorbance value after 24 hours of toxin-free stimulation, and OD.sub.blank refers to the absorbance value of the pure cell culture medium.

[0078] It can be seen from FIG. 6 that the measurement structure of the cell electrochemical sensor constructed in example 1 for evaluating the cytotoxicity of vomitoxin is in good consistency with the results measured by a traditional cytotoxicology method, and the cell electrochemical sensor can be used for effectively determining the cytotoxicity of toxins.

Example 4 Optimization of Preparation Parameters of the Cell/Carbon Nanofiber/GeMA Composite Hydrogel

[0079] (1) Final Concentration of Cells

[0080] According to example 1, a series of gradient cell concentrations of cell/carbon nanofiber/GelMA hydrogels are prepared to make the final concentrations of the lung adenocarcinoma epithelial cells A549 reach 110.sup.3/mL, 110.sup.4/mL, 110.sup.5/mL, 110.sup.6/mL and 110.sup.7/mL respectively, other parameters remain unchanged, then the cell/carbon nanofiber/GeMA hydrogels are deposited on the gold-plated screen-printed carbon electrode through 3D printing to obtain different cell concentrations of electrochemical sensors, and electrochemical signals are tested by an AC impedance method.

[0081] As shown in FIG. 7, as the cell concentration is increased, the blocking effect of cell membranes on the current is improved, and the impedance value of the cell electrochemical sensor is increased. When the cell concentration reaches 110.sup.6/mL-110.sup.7/mL, the impedance value is no longer increased significantly, it is indicated that the cells on the electrode surface are saturated at this time, and the formed cell electrochemical sensor stays in a relatively stable state. Therefore, the cell concentration range of 110.sup.6/mL-110.sup.7/mL is adopted.

[0082] (2) Final Concentration of GeMA

[0083] According to example 1, the final GeMA concentrations are adjusted to 5%, 7.5%, 10%, 12.5% and 15% respectively to prepare cell/GelMA hydrogels, then the cell/GelMA hydrogels are deposited on the gold-plated screen-printed carbon electrode through 3D printing, a calcein-AM/PI cell live and dead double staining kit is used for detecting the cell viability of cells incubated in different concentrations of GelMA hydrogels for 72 hours, and differential pulse voltammetry is used for testing the electrochemical signals.

[0084] A cell live and dead staining experiment is carried out according to instructions of the kit: the cell/GelMA hydrogel is observed under a laser confocal microscope after staining, and yellow-green fluorescent live cells and red fluorescent dead cells are observed at the same time at an excitation wavelength of 49010 nm. In addition, the dead cells are observed separately at an excitation wavelength of 545 nm, and then cell viability statistics is carried out through a counting function of an instrument.

[0085] As shown in FIG. 8A, the cells A549 have a high survival rate in the GeMA concentration range of 5-7.5%. It can be seen from FIG. 8B that as the concentration of GeMA is increased, the current value of the screen-printed carbon electrode is reduced, the impedance value is increased, and in order to ensure the activity of the cells in the sensor and the sensitivity of the sensor, the GelMA concentration range of 5-7.5% is adopted. If the GeMA concentration is too low, the requirements of 3D printing cannot be met.

Comparative Example 1

[0086] The 3D printing method of example 1 is adjusted to a dropping method, that is to say, a 10 L of cell/carbon nanofiber/GelMA hydrogel is taken and dropped on the screen-printed working electrode through a pipette, other parameters remained unchanged, and the cell electrochemical sensor is prepared.

[0087] The prepared cell electrochemical sensor is used for electrochemical detection of DON cell toxicity, and results of EIS detection and impedance value changing with toxin concentrations are shown as FIG. 9. It can be seen that there is no regular linear relationship between the dose of the DON toxin and the impedance value of the cell electrochemical sensor, and there are large errors between parallel dose groups. It is indicated that the deposition of the cells, carbon nanofibers and hydrogel on the screen-printed carbon electrode in a dropping method can cause the problem of uneven distribution of cells and materials due to human errors, thereby reducing the detection precision.

[0088] Therefore, the 3D printing method can be used for more precisely positioning biological materials and living cells, human errors are reduced to a certain extent, mass production is allowed, and the method has the advantages of ensuring the precision and being convenient in operation.