CN115980370A - Based on PLA/NiFe 2 O 4 Flexible magnetoelastic biological test paper and preparation method thereof - Google Patents
Based on PLA/NiFe 2 O 4 Flexible magnetoelastic biological test paper and preparation method thereof Download PDFInfo
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Abstract
The invention provides a method based on PLA/NiFe 2 O 4 The magnetoelastic biological test paper and the preparation method thereof belong to the technical field of biosensors; the invention adopts NiFe 2 O 4 The material and PLA are mixed to prepare a wire, and then 3D printing is carried out to prepare a substrate layer, so that the test paper has excellent electromagnetic properties, and stress signals on the surface of the test paper can be sensitively converted into electromagnetic signals; the carbon dot surface amino coupling antibody is utilized to enable the antibody to have the characteristic of fluorescence, biological functional modification of the test paper surface is achieved, and the problems that complex operation of modifying the antibody by using a reduction gold layer and the gold layer are easy to fall are solved; based on good biocompatibility and flexibility of the NC membrane, the membrane can be applied to wearable health monitoring equipment or the field of biological and medical detection; the biological detection test paper has the advantages of low cost, simple operation, high sensitivity and the like.
Description
Technical Field
The invention relates to the technical field of biosensors, in particular to a biosensor based on PLA/NiFe 2 O 4 The flexible magnetoelastic biological test paper and the preparation method thereof.
Background
At present, rigid metal alloy materials such as iron-nickel alloy, nickel-cobalt alloy and the like are used as substrates for preparing the magnetoelastic biosensor, the hardness of the substrates is high, a plurality of expensive large-sized instruments and complicated manufacturing processes are needed in cutting, plasticity and processing, the operation is time-consuming and labor-consuming, and further the application is limited. The biosensor substrate printed by the 3D printing technology does not need later cutting, processing and the like, can be directly printed into an ideal model, simplifies the manufacturing process of the substrate, and ensures that the operation process becomes simple and time-saving. However, most of the wires used in the fused deposition 3D printer are made of common single materials, and only the required structure can be obtained, but the electromagnetic properties are lost, and it is difficult to modify the printed structure in the later period.
The surface biological modification of the magnetoelastic biosensor is generally to evaporate AuNPs particles with the thickness of 30-50nm on the surface of a substrate, then to aminate the surface of the substrate by utilizing a reducing gold layer, and then to modify an antibody. The operation of modifying the antibody by using the gold reducing layer is complex, and the gold layer is easy to drop after the antibody is modified by using the gold reducing layer, so that the detection performance and sensitivity are reduced.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a PLA/NiFe-based alloy 2 O 4 The flexible magnetoelastic biological test paper and the preparation method thereof.
In order to achieve the above object, the present invention is achieved by the following technical solutions.
Based on PLA/NiFe 2 O 4 The flexible magnetoelastic biological detection test paper comprises a substrate layer with a three-dimensional grid-shaped structure, wherein an NC film is arranged on the upper surface of the substrate layer, and the surface of the NC film is modified with antibodies-CDs; the base layer is made of PLA and NiFe 2 O 4 The substrate layer has electrical conductivity and magnetism; the antibody-CDs are produced by reacting an antibody withCarbon sites are coupled.
Preferably, the base layer is printed by a 3D printer.
Preferably, the substrate layer is doped with MWCNTs to obtain conductivity.
Based on PLA/NiFe 2 O 4 The preparation method of the flexible magnetoelastic biological test paper comprises the following steps:
1) Preparing a base layer: mixing NiFe 2 O 4 Adding the mixture into melted PLA, uniformly mixing, extruding wires, and obtaining a three-dimensional latticed substrate layer in a 3D printing mode;
2) Soaking the substrate layer in MWCNTs solution to enable the MWCNTs to be attached to the pores and the surface of the substrate layer, and enabling the substrate layer to obtain conductivity and magnetism;
3) Adhering an NC film on the surface of the substrate layer to prepare a double-layer composite structure;
4) Antibody modification: and soaking the double-layer composite structure in an antibody-CDs solution to ensure that the antibody-CDs are fully fixed and modified on the surface of the NC membrane.
Preferably, niFe 2 O 4 The weight percentage of the PLA is 20wt%.
Preferably, the double-layer composite structure is soaked in absolute ethyl alcohol to ensure that the NC membrane is completely transparent, and is dried and then subjected to antibody modification.
Preferably, the preparation method of the antibody-CDs comprises mixing and shaking the diluted antibody solution and a solution containing EDC and NHS for activation, so that carboxyl in the antibody is activated into NHS ester; and then, dripping the CDs solution into the activated antibody solution, and mixing and shaking to obtain the antibody solution coupled with the carbon dots.
Preferably, the dilution is performed by diluting the antibody solution with a PBS solution.
Preferably, the 3D printing is printing using a fused deposition 3D printer.
Preferably, the antibody-modified test strips are incubated in a BSA solution to block non-specific binding sites.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adopts PLA/NiFe 2 O 4 The + NC/CDs flexible magnetoelastic composite structure precisely controls the shape structure of the test paper by using a 3D printing technology, has good electromagnetic property and excellent mechanical property, and can well realize the conversion from biological signals to electromagnetic signals; meanwhile, based on the three-dimensional latticed multilayer composite structure, the sensitivity of the detection test paper is effectively improved.
2. The invention utilizes the coupling of CDs surface group-amino and antibody molecules as biological recognition elements, and utilizes the luminescence condition of the fluorescence characteristic of CDs under ultraviolet light, so that the distribution of CDs + antibodies on the surface of the test paper can be visually observed, and the multi-mode recognition of fluorescence signals and electromagnetic signals is realized. Amino groups on the surface of CDs can be well coupled with carboxyl groups of the antibody, so that the fixation of the antibody is realized, and the detection of various substances can be realized based on antigen-antibody specific recognition.
3. The invention expands the functions of the traditional magnetoelastic biosensor, can detect different antigens by changing the types of the modified antibodies, increases the utilization rate of the detection test paper and enlarges the application range. The preparation method of the flexible magnetoelastic biological test paper is simple and easy to implement and can be produced in batches.
Drawings
FIG. 1 shows a PLA/NiFe-based composition according to the present invention 2 O 4 The structural schematic diagram of the flexible magnetoelastic biological test paper is shown;
FIG. 2 shows that the present invention is based on PLA/NiFe 2 O 4 The flow schematic diagram of the preparation method of the flexible magnetoelastic biological test paper;
in the figure: reference numeral 1 denotes a basal layer, 2 denotes an NC membrane, 3 denotes CDs, 4 denotes an antibody, and 5 denotes an antigen.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The technical solution of the present invention is described in detail below with reference to the embodiments and the drawings, but the scope of protection is not limited thereto.
Example 1
Referring to FIG. 1, this example proposes a PLA/NiFe-based alloy 2 O 4 The bottom of the flexible magnetoelastic biological test paper is a basal layer 1 with a cubic grid structure, and the size of the basal layer 1 is 4.5mm x 1.5mm. The NC membrane 2 is adhered to the upper surface of the substrate layer 1, and the surface of the NC membrane 2 is modified with antibodies-CDs; the material of the basal layer 1 is PLA and NiFe 2 O 4 The composite wire is printed and prepared into a required cubic grid structure with gaps as a substrate layer 1 by using a fused deposition type 3D printer; and the substrate layer 1 obtains conductivity by doping MWCNTs; antibody-CDs are formed by coupling antibody 4 to carbon-dotted CDs 3.
Example 2
Referring to FIG. 2, and by way of example, there is provided a PLA/NiFe-based alloy as described in example 1 2 O 4 The preparation method of the magnetoelastic biological test paper comprises the following steps:
selecting basic materials: PLA/NiFe 2 O 4 Composite wires, NC films, CDs (raw materials are m-phenylenediamine and aspartic acid) and MWCNTs.
Step 1: PLA/NiFe 2 O 4 Preparing a composite wire:
1) Heating PLA to 210 ℃ to melt the PLA;
2) Mixing NiFe 2 O 4 The powder is added to the melted PLA (NiFe) 2 O 4 20 wt%) was uniformly mixed, and then a wire having a diameter of 1.75mm was extruded using an extruder and cooled, to prepare a wire suitable for 3D printing.
Step 2: the prepared wire rod was printed with a cubic lattice structure having voids therein as a base layer 1 of the test paper using a fused deposition 3D printer, and the size of the base layer 1 was 4.5mm by 1.5mm.
And step 3: and (2) soaking the substrate layer 1 in MWCNTs solution, standing for 20min, attaching the MWCNTs to the pores and the surface of the cubic grid structure to enable the cubic grid structure to obtain conductivity, taking out the cubic grid structure, and drying for later use.
And 4, step 4: preparing a double-layer composite structure by using the substrate layer 1 and the NC film 2:
1) NC film 2 was cut into 4.5mm by 4.5mm squares for use.
2) The NC film 2 is adhered to the surface of the base layer 1 to form a two-layer composite structure.
3) Soaking the double-layer composite structure in absolute ethyl alcohol, and standing for 10min to ensure that the NC film 2 on the surface of the double-layer composite structure is completely transparent;
4) After the NC film 2 completely appeared transparent, it was carefully picked up with tweezers, placed in a dry container, allowed to stand for 1 hour, and taken out after it was completely dried.
And 5: coupling carbon dots to antibodies to prepare antibody-CDs:
1) Activating the antibody: the antibody solution was diluted with a PBS solution to obtain an appropriate concentration of antibody 4. The antibody solution is mixed and shaken for 30min with a solution containing 4 mg/mL EDC and 4 mg/mL NHS at 37 ℃, so that carboxyl in the antibody 4 is activated into NHS ester, and the NHS ester is more efficiently combined with amino on the surface of the carbon spots CDs 3.
2) And (3) dripping CDs solution with the same volume into the activated antibody solution under stirring, and mixing and shaking at 37 ℃ for 30min to obtain the antibody solution coupled with carbon dots, namely antibody-CDs.
And 6: antibody modification: by utilizing the characteristic that the antibody-CDs can be attached to tiny pores of the NC membrane 2, the surface of the test paper is modified with the required antibody 4:
1) And (4) soaking the double-layer composite structure obtained in the step (4) in an antibody-CDs solution, and standing for 1h at 37 ℃ so that antibody molecules are fully fixed and modified on the surface of the test paper.
2) The antibody-modified strip was removed from the solution and washed three times with PBS solution to remove antibody molecules that were physically adsorbed on the surface. And soaking the test paper in BSA solution, standing for 0.5h in an environment at 37 ℃, and blocking the locus, thus finishing the preparation of the flexible magnetoelastic biological detection test paper. The biological test paper is adopted to detect the antigen 5.
While the invention has been described in further detail with reference to specific preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. Based on PLA/NiFe 2 O 4 The flexible magnetoelastic biological detection test paper is characterized by comprising a substrate layer with a three-dimensional latticed structure, wherein an NC film is arranged on the upper surface of the substrate layer, and antibodies-CDs are modified on the surface of the NC film; the base layer is made of PLA and NiFe 2 O 4 The substrate layer has electrical conductivity and magnetism; the antibody-CDs are formed by coupling the antibody to a carbon site.
2. A PLA/NiFe based alloy according to claim 1 2 O 4 The flexible magnetoelastic biological test paper is characterized in that the basal layer is printed by a 3D printer.
3. A PLA/NiFe based on according to claim 1 2 O 4 The flexible magnetoelastic biological detection test paper is characterized in that the substrate layer obtains electric conductivity by doping conductive materials MWCNTs.
4. A PLA/NiFe based alloy as claimed in any one of claims 1 to 3 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized by comprising the following steps:
1) Preparing a base layer: mixing NiFe 2 O 4 Adding the mixture into melted PLA, uniformly mixing, extruding wires, and obtaining a substrate layer with a three-dimensional grid structure in a 3D printing mode;
2) Soaking the substrate layer in MWCNTs solution to enable the MWCNTs to be attached to the pores and the surface of the substrate layer, and enabling the substrate layer to obtain conductivity and magnetism;
3) Adhering an NC film on the surface of the substrate layer to prepare a double-layer composite structure;
4) Antibody modification: and (3) incubating the double-layer composite structure in an antibody-CDs solution, so that the antibody-CDs are fully fixed and modified on the surface of the NC membrane.
5. A PLA/NiFe based alloy according to claim 4 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized in that the method comprises the steps of 2 O 4 The weight percentage of the PLA is 20wt%.
6. A PLA/NiFe based alloy according to claim 4 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized in that the double-layer composite structure is soaked in absolute ethyl alcohol to ensure that an NC membrane is completely transparent, and then antibody modification is carried out after drying.
7. A PLA/NiFe based alloy according to claim 4 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized in that the preparation method of the antibody-CDs is that diluted antibody solution and solution containing EDC and NHS are mixed and vibrated for activation, so that carboxyl in the antibody is activated into NHS ester; and then dripping the CDs solution into the activated antibody solution, and mixing and oscillating to obtain the antibody solution coupled with the carbon dots.
8. A PLA/NiFe based alloy according to claim 7 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized in that the dilution is to dilute the antibody solution by adopting PBS solution.
9. A PLA/NiFe based alloy according to claim 4 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized in that the 3D printing is performed by using a fused deposition type 3D printer.
10. Root of herbaceous plantsPLA/NiFe-based, according to claim 4 2 O 4 The preparation method of the flexible magnetoelastic biological test paper is characterized in that the test paper modified by the antibody is incubated in BSA solution to seal the nonspecific binding site.
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