CN112858408B - Flexible electrochemical biosensor and construction method thereof - Google Patents
Flexible electrochemical biosensor and construction method thereof Download PDFInfo
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses a flexible electrochemical biosensor and a construction method thereof. Belongs to the technical field of biochemical analysis. The method comprises the following steps: in an organic phase system, electrochemically polymerizing to synthesize a PEE-PPy (pentaerythritol ethoxylate-polypyrrole) membrane; preparing a PPy composite film in a water phase system by taking the PEE-PPy film as a working electrode; and (3) adding the PPy composite film into a chloroauric acid solution for treatment, then placing the PPy composite film into a CEA (carcinoembryonic antigen) aptamer solution for treatment, and sealing to obtain the flexible electrochemical biosensor. The flexible electrochemical biosensor prepared by the invention can quantitatively detect CEA. The device has flexibility and self-supporting property, and the signal of the sensor can be kept stable under the condition of mechanical deformation.
Description
Technical Field
The invention relates to the technical field of biochemical analysis, in particular to a flexible electrochemical biosensor and a construction method thereof.
Background
Flexible electronic devices have the characteristics of good flexibility, lightness, low cost and the like, and are therefore widely studied for constructing an ideal platform for personalization. Flexible electronic devices have been used in a wide range of applications in information, energy, medical, defense, etc. fields, such as flexible electronic displays, organic light emitting diodes, thin film solar cells, supercapacitors, flexible sensors, etc. The flexible sensor can effectively monitor the health condition of people in real time, provide health-related information for users, and further strengthen management on chronic diseases.
The flexible electrochemical biosensors reported today are made flexible and wearable mainly by combining the sensor electrodes and a flexible substrate. Flexible substrates include flexible polymer substrates (PDMS, polyurethane, polyimide, etc.) or wearable materials (gloves, glasses and clothing), etc. The flexible electrochemical biosensor can detect molecular markers in biological tissue fluid and blood samples.
However, such flexible electrochemical sensors require the incorporation of additional flexible substrates and current collectors, and the integration and design process of the devices is complicated. Moreover, the modulus difference between each component of such electrochemical sensors (including the sensor electrode material, the flexible substrate and the current collector) is large, and the repeated mechanical deformation and frequent use can cause peeling and delamination between the components, which affects the performance of the electrochemical sensor. Therefore, such electrochemical sensors are weak in stability, durability and flexibility. In order to improve the flexibility and stability of the device, it is important to construct a flexible electrochemical sensor based on a self-supporting electrode design.
Therefore, how to provide a flexible electrochemical biosensor is a problem that needs to be solved by those skilled in the art.
Disclosure of Invention
In view of the above, the present invention provides a flexible electrochemical biosensor and a method for constructing the same. The self-supporting and flexible properties of the sensor electrode are utilized and used for detecting CEA (carcinoembryonic antigen). The constraint of a flexible substrate and a current collector of the traditional flexible sensor is broken, and the flexible sensor is simple in construction and has self-supporting property and flexibility. Wherein, after the gold nanoparticles (AuNPs) are introduced, the aptamer can be modified on the surface of the electrode through self-assembly, and MCH is used as a blocking agent to keep good recognition capability of the biosensor. High sensitivity, good selectivity, and excellent long-term stability and mechanical deformation stability. Various mechanical deformations can be tolerated without affecting the performance of the sensor. Provides a new method and thought for designing the flexible electrochemical biosensor, and has great development potential in the fields of flexibility and wearable electronics.
In order to achieve the purpose, the invention adopts the following technical scheme:
a construction method of a flexible electrochemical biosensor comprises the following steps:
(1) Mixing a pure isopropanol solution and a boron trifluoride diethyl etherate solution, and sequentially adding PEE and pyrrole monomers to prepare an organic phase electrolyte; electrochemically polymerizing a three-electrode system in the prepared organic phase electrolyte under the conditions of constant current and ice-water bath to synthesize a PEE-PPy film, wherein the working electrode is a glass sheet sputtered with titanium and platinum;
(2) Sequentially adding pyrrole monomer, 2-sodium naphthalene sulfonate and surfactant into the aqueous solution, adjusting the pH of the electrolyte by using 2-naphthalenesulfonic acid, and preparing aqueous electrolyte; selecting a three-electrode system, taking a PEE-PPy film as a working electrode, and electrochemically polymerizing under the conditions of constant polymerization voltage and ice-water bath to obtain a PPy composite film;
(3) And adding the PPy composite film into a chloroauric acid solution for treatment, then placing the PPy composite film into a CEA aptamer solution for treatment, and sealing to obtain the flexible electrochemical biosensor.
Preferably, the following components: in the three-electrode system in the step (1), the counter electrode is a stainless steel electrode; the reference electrode is a silver wire.
Preferably: in the step (1), the mass fraction of boron trifluoride diethyl etherate solution is 47.0-47.7% by weight; the volume ratio of the pure isopropanol solution to the boron trifluoride diethyl etherate solution is 7:3; the volume ratio of PEE is 5%; the concentration of pyrrole monomer is 0.05M; the current density of the constant current is 0.8-1.0 mA/cm 2 (ii) a The temperature of the ice water bath is 0-4 ℃; the time of electrochemical polymerization is 80-90 min; the thickness of the PEE-PPy film is 14-15 μm.
Preferably: and (3) in the three-electrode system in the step (2), taking Ag/AgCl as a reference electrode and taking a stainless steel electrode as a counter electrode.
Preferably: adjusting the pH value to 3 in the step (2); the concentrations of pyrrole monomer, sodium 2-naphthalenesulfonate and surfactant were 0.05M, 0.1M and 0.025M, respectively; the surface active agent is triton X-100; the polymerization voltage is 0.80-0.90V, and the time of electrochemical polymerization is 15-20 min.
Preferably: the step (3) is specifically as follows:
1) Placing the PPy composite film in a chloroauric acid solution and KNO 3 Depositing for 400s under the voltage of-0.4V in the mixed solution to obtain a thin film electrode;
2) The membrane electrode was soaked in the CEA aptamer solution for 12-14 hours, and finally the remaining activation sites were blocked with MCH.
Has the advantages that: a layer of gold nanoparticles is uniformly covered on the surface of the thin film electrode through electrochemical deposition;
connecting the CEA aptamer and the gold nanoparticles through the self-assembly effect, and fixing the CEA aptamer and the gold nanoparticles on the surface of the membrane electrode;
the remaining activation sites are blocked in order to ensure the recognition ability of the constructed flexible electrochemical biosensor.
The deposition voltage of the gold nanoparticles is optimized, and the impedance response of the constructed thin film electrode is maximum when the voltage is-0.4V. above-0.4V or below-0.4V, the impedance response is reduced;
the deposition time of the gold nanoparticles is optimized, and when the deposition time is 400s, the impedance response of the thin film electrode is the maximum;
when the membrane electrode is soaked in the CEA aptamer solution for 12-14 hours, the CEA aptamer can be successfully connected to the surface of the membrane. If the time is too short, the connection of the aptamer is not sufficient, and the aptamer can be saturated within about 12 to 14 hours;
preferably: chloroauric acid solution and KNO 3 The concentration of (A) was 0.5mM, the aptamer concentration was 2.0. Mu.M, and the CEA incubation time was 60min.
Has the advantages that: the concentration of the CEA aptamer is optimized, and the impedance response of the thin film electrode is close to saturation at 2.0 mu M;
the incubation time of the CEA is optimized, the impedance response of the sensor continuously increases along with the increase of the incubation time of the CEA, and the impedance response is close to the balance when the incubation time is 60min.
The invention also provides the flexible electrochemical biosensor prepared by the construction method.
The invention also provides application of the flexible electrochemical biosensor in detecting CEA preparations, instruments or wearable electronic products.
According to the technical scheme, compared with the prior art, the invention discloses and provides the flexible electrochemical biosensor and the construction method thereof, and the technical effect is that the flexible electrochemical biosensor prepared by the invention can quantitatively detect CEA. The device has flexibility and self-supporting nature, and the signal of sensor can remain stable under mechanical deformation condition, and is specific:
1. and preparing a flexible self-supporting electrochemical biosensor based on the prepared flexible conductive PPy composite film, wherein the flexible self-supporting electrochemical biosensor is used for detecting CEA.
2. The flexible conductive PPy composite film has a sandwich structure, and is constructed by continuously synthesizing 2-naphthalenesulfonate (2-NS) doped PPy on the basis of a PEE doped PPy film (PEE-PPy film).
3. And performing electrochemical modification on the surface of the film electrode based on the flexible conductive PPy composite film to construct the self-supporting flexible aptamer sensor. Gold nanoparticles are firstly fixed on the surface of a film electrode by an electrochemical deposition method, and a CEA aptamer is connected to the surface of the electrode through self-assembly, so that the flexible and self-supporting aptamer sensor is finally and successfully constructed.
4. The aptamer sensor is simple to construct, does not need an additional elastic substrate and a current collector, and has self-supporting property. The flexibility is good, the stability is strong, and the performance of the sensor can still be kept stable when various mechanical deformations occur. Such flexible self-supporting electrochemical biosensors have great potential in applications of flexible and wearable electronics.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic diagram of the preparation process of the flexible self-supporting electrochemical biosensor for CEA detection provided by the invention.
FIG. 2 is a schematic diagram of the impedance response of the flexible electrochemical biosensor provided by the present invention to CEA.
FIG. 3 is a schematic diagram of the conditions for constructing the electrochemical biosensor and the optimization of CEA incubation time provided by the present invention, wherein (a) the voltage for depositing gold nanoparticles is optimized, (b) the time for depositing gold nanoparticles is optimized, (c) the CEA aptamer concentration is optimized, and (d) the CEA incubation time is optimized.
FIG. 4 is a schematic diagram of the impedance response of the electrochemical biosensor to CEA of different concentrations, wherein (a) the impedance response of the electrochemical biosensor to CEA is illustrated in a partial enlarged view with Log f being 1; (b) a linear calibration curve of the rate of change of impedance and CEA concentration.
FIG. 5 is a schematic diagram of the long-term stability of an electrochemical biosensor according to the present invention.
FIG. 6 is a schematic diagram of a deformation test of an electrochemical biosensor provided by the present invention, wherein (a) the impedance change of the electrochemical biosensor before and after deformation, (b) the impedance change of the electrochemical biosensor after incubation of CEA before and after deformation, (c) the stress-strain curve of the PPy composite thin film, and (d) the response of the electrochemical biosensor to impedance under different strains (twist; fold; knot; knotting).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The embodiment of the invention discloses a flexible electrochemical biosensor and a construction method thereof.
In the examples, the required starting materials are all commercially available, for example PEE (pentaerythritol ethoxylate), CAS number: 30599-15-6; PPy (polypyrrole), MCH (6-mercapto-1-hexanol), etc., the required equipment is all available from commercial sources, such as chenhua electrochemical workstation 660E; CEA (carcinoembryonic antigen) aptamer, manufactured by Venetian corporation, is not specifically listed here.
Example 1
Preparation of a (Sandwich-structured) PPy composite film
The principle is as follows: and (3) continuously synthesizing 2-naphthalenesulfonate (2-NS) doped PPy on the basis of the PEE doped PPy thin film, and finally obtaining the PPy composite thin film with a sandwich structure (see figure 1).
The synthesis steps are as follows:
1) In an organic phase system, a PEE-doped PPy film, namely a PEE-PPy film for short, is synthesized by electrochemical polymerization.
The method specifically comprises the following steps: isopropanol (pure isopropanol solution) and boron trifluoride diethyl etherate (mass fraction in terms of boron trifluoride is 47.0-47.7%) are mixed according to the volume ratio of 7:3, then PEE with the volume ratio of 5% is added, and finally 0.05M pyrrole monomer is added to prepare the organic phase electrolyte.
Using a Chenghua electrochemical workstation 660E, synthesizing a PEE-PPy membrane by adopting a three-electrode system in an electrochemical polymerization mode, wherein the working electrode is a glass sheet sputtered with titanium and platinum, the counter electrode is a stainless steel electrode (model 304), and the reference electrode is a silver wire; adopting a constant current method to ensure that the current density is 0.8-1.0 mA/cm 2 And (3) performing lower polymerization, performing electropolymerization in an ice water bath (at 0-4 ℃) for 80-90 min, stripping a PEE-PPy film formed by polymerization from the upper surface of the working electrode, and measuring the final thickness of the PEE-PPy film by a micrometer screw to be 14-15 mu m.
2) The prepared PEE-PPy film is used as a working electrode to continuously synthesize 2-naphthalenesulfonate doped PPy in a water phase system, and finally the PPy composite film with a sandwich structure is obtained.
The method specifically comprises the following steps:
pyrrole monomer (0.05M) and 2-sodium naphthalenesulfonate (0.1M) are sequentially added into the aqueous solution, 0.025M triton X-100 is selected as a surfactant, and 2-naphthalenesulfonic acid is used for adjusting the pH value of the electrolyte to 3 to prepare the aqueous electrolyte.
A three-electrode system is selected, a PEE-PPy film is used as a working electrode, ag/AgCl is used as a reference electrode, a stainless steel electrode (type 304) is used as a counter electrode, and polymerization is carried out for 15-20 min in an ice-water bath under the condition that the polymerization voltage is constant to be 0.80-0.90V, so that the PPy composite film with a sandwich structure is finally formed.
Example 2
Construction of self-supporting electrochemical biosensors
(1) The synthesized PPy composite film was placed in a 0.5mM chloroauric acid solution (containing 0.5M KNO) 3 ) Depositing for 400s under the voltage of-0.4V, and uniformly covering a layer of gold nanoparticles on the surface of the thin film electrode.
(2) Soaking the membrane electrode deposited with the gold nanoparticles in a CEA aptamer (aptamer sequence: 5'-SH-ATA CCA GCT TAT TCA ATT-3') solution overnight (12-14 hours), connecting the CEA aptamer and the gold nanoparticles through the self-assembly effect, and fixing the membrane electrode on the surface of the membrane electrode.
Finally, MCH was used to block the remaining activation sites to ensure the recognition ability of the constructed biosensor.
Example 3
Experimental condition optimization of flexible electrochemical biosensor
Detecting CEA by using non-Faraday impedance, wherein the detection method comprises the following steps:
directly using the constructed biological aptamer film electrode as a working electrode to be connected with an electrochemical workstation, using a saturated calomel electrode and a platinum wire as a reference electrode and a counter electrode respectively, and detecting CEA (10 CEA) in a PBS (0.2M, pH 7.4) solution - 6 g/mL)。
The results show that:
the response of the CEA to the change in impedance is evident, as shown in FIG. 2CEA/MCH/Aptamer/Au/PPy, and the remaining controls are: PPy, au/PPy-PPy composite film covered with a layer of gold nanoparticles, aptamer/Au/PPy combined film electrode, MCH/Aptamer/Au/PPy-sealed Aptamer sensor.
Respectively optimizing experimental conditions (including deposition voltage, deposition time and CEA aptamer concentration of gold nanoparticles) for constructing the biological aptamer sensor and incubation time of CEA, and finally obtaining optimized experimental conditions: the deposition voltage was-0.4V, the deposition time was 400s, the aptamer concentration was 2.0. Mu.M and the CEA incubation time was 60min, as shown in FIG. 3.
Example 4
Performance testing of flexible electrochemical biosensors
(1) High sensitivity
The flexible electrochemical biosensor based on the all-polymer self-supporting electrode shows different impedance responses to CEA with different concentrations and high sensitivity, as shown in FIG. 4.
A detection step: the constructed flexible electrochemical biosensor is directly used as a working electrode and is connected with an electrochemical workstation, and the saturated calomel electrode and the platinum wire are respectively used as a reference electrode and a counter electrode. Linear range of the sensor is 10 -10 g/mL-10 -6 g/mL，R 2 =0.998, with a minimum detection limit of 0.033ng/mL.
(2) Long term stability
The flexible electrochemical biosensor exhibits excellent long-term stability. As shown in fig. 5, after being stored in PBS solution for 15 days, the impedance response retention rate can still be maintained at 97.06%, and good long-term stability is shown.
(3) Resistance to mechanical deformation
The flexible electrochemical biosensor prepared by the embodiment has good mechanical property and high flexibility. Various mechanical deformations are possible, and the substrate impedance response of the constructed sensor hardly changes when various mechanical deformations such as twisting, folding and knotting occur, as shown in fig. 6 a. And the sensor was subjected to various deformations after incubation of CEA with an impedance response signal rate of change within 4%, as shown in fig. 6 b.
In combination with the stress-strain curve of the PPy composite film, the thin film electrode has very good elongation at break, as shown in fig. 6 c. At 25% mechanical deformation, the impedance signal response of the sensor is substantially unchanged, as shown in fig. 6 d. The experimental results prove that the flexible electrochemical biosensor prepared by the embodiment has good mechanical deformation stability, and can completely bear deformation motion of a human body when being applied to wearable detection.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A construction method of a flexible electrochemical biosensor is characterized by comprising the following steps:
(1) Mixing a pure isopropanol solution and a boron trifluoride diethyl etherate solution, and sequentially adding PEE and a pyrrole monomer to prepare an organic phase electrolyte; electrochemically polymerizing a three-electrode system in the prepared organic phase electrolyte under the conditions of constant current and ice-water bath to synthesize a PEE-PPy film, wherein the working electrode is a glass sheet sputtered with titanium and platinum;
(2) Sequentially adding pyrrole monomer, 2-sodium naphthalene sulfonate and surfactant into the aqueous solution, adjusting the pH of the electrolyte by using 2-naphthalenesulfonic acid, and preparing aqueous electrolyte; selecting a three-electrode system, taking a PEE-PPy film as a working electrode, and electrochemically polymerizing under the conditions of constant polymerization voltage and ice-water bath to obtain a PPy composite film;
(3) Adding a PPy composite film into a chloroauric acid solution for treatment, then placing the PPy composite film in a CEA aptamer solution for treatment, and sealing to obtain a flexible electrochemical biosensor;
the PEE: pentaerythritol ethoxylate, CAS number: 30599-15-6.
2. The method of claim 1, wherein in the three-electrode system of step (1), the counter electrode is a stainless steel electrode; the reference electrode is a silver wire.
3. The method of claim 1 or 2, wherein step (1) is performed byThe mass fraction of boron trifluoride ether solution is 47.0-47.7% by boron trifluoride; the volume ratio of the pure isopropanol solution to the boron trifluoride diethyl etherate solution is 7:3; the volume ratio of the PEE is 5 percent; the concentration of pyrrole monomer is 0.05M; the current density of the constant current is 0.8-1.0 mA/cm 2 (ii) a The temperature of the ice-water bath is 0-4 ℃; the time of the electrochemical polymerization is 80-90 min; the thickness of the PEE-PPy film is 14-15 mu m.
4. The method of claim 1, wherein step (2) comprises using Ag/AgCl as a reference electrode and a stainless steel electrode as a counter electrode.
5. The method of claim 1 or 4, wherein the pH of step (2) is adjusted to 3; the concentrations of the pyrrole monomer, the sodium 2-naphthalenesulfonate and the surfactant are 0.05M, 0.1M and 0.025M respectively; the surfactant is triton X-100; the polymerization voltage is 0.80-0.90V, and the time of electrochemical polymerization is 15-20 min.
6. The construction method according to claim 1, wherein the step (3) is specifically:
1) Placing the PPy composite film in a chloroauric acid solution and KNO 3 Depositing for 400s under the voltage of-0.4V in the mixed solution to obtain a thin film electrode;
2) The membrane electrode was soaked in the CEA aptamer solution for 12-14 hours, and finally the remaining activated sites were blocked with MCH.
7. The method of claim 6, wherein said chloroauric acid solution and KNO 3 The concentration of (A) was 0.5mM, the aptamer concentration was 2.0. Mu.M, and the CEA incubation time was 60min.
8. A flexible electrochemical biosensor prepared according to any one of the construction methods of claims 1 to 7.
9. Use of the flexible electrochemical biosensor of claim 8 in the detection of a CEA formulation, an instrument, or a wearable electronic product.
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