CN114366092A - Micro-needle sensor based on electro-codeposition electron mediator and preparation method thereof - Google Patents
Micro-needle sensor based on electro-codeposition electron mediator and preparation method thereof Download PDFInfo
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- CN114366092A CN114366092A CN202111560889.5A CN202111560889A CN114366092A CN 114366092 A CN114366092 A CN 114366092A CN 202111560889 A CN202111560889 A CN 202111560889A CN 114366092 A CN114366092 A CN 114366092A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
- A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase
- A61B5/14865—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue using enzyme electrodes, e.g. with immobilised oxidase invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors
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- A—HUMAN NECESSITIES
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- A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
- A61B5/00—Measuring for diagnostic purposes; Identification of persons
- A61B5/68—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient
- A61B5/6846—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive
- A61B5/6847—Arrangements of detecting, measuring or recording means, e.g. sensors, in relation to patient specially adapted to be brought in contact with an internal body part, i.e. invasive mounted on an invasive device
- A61B5/685—Microneedles
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- A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
- A61B2562/02—Details of sensors specially adapted for in-vivo measurements
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- A—HUMAN NECESSITIES
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- A61B2562/12—Manufacturing methods specially adapted for producing sensors for in-vivo measurements
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- A—HUMAN NECESSITIES
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- A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
- A61B2562/12—Manufacturing methods specially adapted for producing sensors for in-vivo measurements
- A61B2562/125—Manufacturing methods specially adapted for producing sensors for in-vivo measurements characterised by the manufacture of electrodes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61B2562/00—Details of sensors; Constructional details of sensor housings or probes; Accessories for sensors
- A61B2562/16—Details of sensor housings or probes; Details of structural supports for sensors
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- Health & Medical Sciences (AREA)
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Abstract
The invention discloses a micro-needle sensor based on an electro-codeposition electronic mediator and a preparation method thereof, the micro-needle sensor comprises a substrate and a micro-needle array positioned on the substrate, the surface of the micro-needle array is covered with a conducting layer and a composite material electronic mediator layer, the composite material electronic mediator layer is composed of carbon nano tubes, a hydrogel electronic mediator and enzyme, and the composite material electronic mediator layer is fixed on the surface of the conducting layer of the micro-needle array through electro-codeposition. The electronic modification material of the microneedle sensor in the embodiment of the invention is not easy to fall off, has good stability and high sensitivity, and can be widely applied to the technical field of sensors.
Description
Technical Field
The invention relates to the technical field of sensors, in particular to a micro-needle sensor based on an electro-codeposition electron mediator and a preparation method thereof.
Background
The microneedle sensor is widely used for detecting various physiological indexes such as blood glucose value, cholesterol value or uric acid value, but in the related art, the microneedle sensor has the following problems: 1. the device is easily influenced by a test environment, and has poor anti-interference capability and low sensitivity; 2. the electronic modification material on the surface of the microneedle is not firmly adhered, and the microneedle is easy to fall off in the process of inserting into the skin, so that the electronic modification is diffused and lost in the tissues in vivo.
Disclosure of Invention
In view of this, an object of the embodiments of the present invention is to provide a micro-needle sensor based on an electrodeposited electron mediator and a method for manufacturing the same, in which an electron modification material is not easy to fall off, and has good stability and high sensitivity.
In a first aspect, an embodiment of the present invention provides a microneedle sensor based on an electrodeposited electron mediator, including a substrate and a microneedle array located on the substrate, where a conductive layer and a composite electron mediator layer are covered on a surface of the microneedle array, the composite electron mediator is composed of a carbon nanotube, a hydrogel electron mediator and an enzyme, and the composite electron mediator layer is fixed on a surface of the conductive layer of the microneedle array through electrodeposition.
Optionally, the microneedle array comprises a hollow microneedle array and a sheet microneedle array.
Optionally, the material of the microneedle array comprises stainless steel.
Optionally, the hydrogel electron mediator comprises a reducing macromolecule.
Optionally, the reducing macromolecule comprises a metallic osmium complex.
Optionally, the material of the conductive layer comprises any one of gold, chromium or platinum.
In a second aspect, embodiments of the present invention provide a method for preparing a microneedle sensor based on an electrodeposited electron mediator, including:
preparing a microneedle electrode, and preparing a conductive layer on the surface of the microneedle electrode;
preparing a composite material electron mediator solution, wherein the composite material electron mediator solution comprises carbon nano tubes, a hydrogel electron mediator and an enzyme;
and fixing the composite material electronic mediator on the surface of the conductive layer of the microneedle electrode by adopting electrodeposition.
Optionally, the preparing of the conductive layer on the surface of the microneedle electrode specifically includes:
cleaning and drying the microneedle electrode;
treating the dried microneedle electrode by using soldering flux;
the microneedle electrode treated with the flux is placed in a solution containing a conductive metal, and a conductive layer is prepared by electrochemical deposition.
Optionally, the preparing of the composite material electron mediator solution specifically includes:
respectively preparing an electron mediator solution and a carbon nano tube solution;
centrifuging the electron mediator solution and the carbon nanotube solution according to a first preset proportion to prepare a doping solution;
providing an enzyme solution;
and centrifuging the doping solution and the enzyme solution according to a second preset proportion to prepare the enzyme doping solution.
Optionally, the fixing the composite electronic mediator on the surface of the conductive layer of the microneedle electrode by using electrodeposition specifically includes:
immersing a microneedle electrode comprising a conductive layer in the composite electron mediator solution;
fixing the composite material electronic mediator on the surface of the conductive layer of the microneedle electrode by adopting electrodeposition;
and immersing the microneedle electrode covered with the conductive layer and the composite material electronic medium layer into a buffer solution for a preset time.
The implementation of the embodiment of the invention has the following beneficial effects: the surface of the microneedle array in the embodiment of the invention is modified with a composite material electronic medium layer, the composite material electronic medium layer consists of a carbon nano tube, a hydrogel electronic medium and an enzyme, and the composite material electronic medium layer is fixed on the surface of a conducting layer of the microneedle array through electrodeposition; by modifying the composite material electron mediator comprising the carbon nano tube, the hydrogel electron mediator and the enzyme, the influence of a testing environment is reduced, the strength of a detection signal is increased, and the stability and the sensitivity are improved; the electronic mediator of the composite material is more firmly fixed by an electrodeposition technology and is not easy to fall off, so that the stability is further improved.
Drawings
FIG. 1 is a schematic structural diagram of a micro-needle sensor based on an electrodeposited electron mediator according to an embodiment of the present invention;
FIG. 2 is a schematic flow chart illustrating steps of a method for fabricating a microneedle sensor based on an electrodeposited electron mediator, according to an embodiment of the present invention;
fig. 3 is an SEM image of a sheet metal microneedle according to an embodiment of the present invention;
FIG. 4 is a SEM comparison of an electron dielectric layer of a hydrogel prepared by electrodeposition and physical dip coating according to an embodiment of the present invention;
FIG. 5 is a SEM comparison of various dielectric layer electrodes provided in accordance with an embodiment of the present invention;
FIG. 6 is a graph comparing i-t curves of electrode response to glucose for different electron mediator layers according to an embodiment of the present invention;
FIG. 7 is a graph comparing i-t curves of an electrode of an electronic mediator layer of a carbon nanotube composite hydrogel and an electrode of an electronic mediator layer of osmium according to an embodiment of the present invention;
fig. 8 is a comparison graph of cyclic voltammetry tests of different microneedle sensing electrodes provided by an embodiment of the present invention;
fig. 9 is a comparison graph of a microneedle blood glucose sensor used in subcutaneous blood glucose testing of an animal according to an embodiment of the present invention.
Detailed Description
The invention is described in further detail below with reference to the figures and the specific embodiments. The step numbers in the following embodiments are provided only for convenience of illustration, the order between the steps is not limited at all, and the execution order of each step in the embodiments can be adapted according to the understanding of those skilled in the art.
As shown in fig. 1, an embodiment of the present invention provides a microneedle sensor based on an electrodeposited electron mediator, including a substrate 1-1 and a microneedle array 1-2 located on the substrate 1-1, wherein a conductive layer 1-4 and a composite electron mediator layer 1-5 are covered on a surface of a microneedle electrode 1-3 in the microneedle array 1-2, the composite electron mediator is composed of a carbon nanotube, a hydrogel electron mediator and an enzyme, and the composite electron mediator layer 1-5 is fixed on a surface of the conductive layer 1-4 of the microneedle array by electrodeposition. Wherein, 1-6 represents the circuit structure schematic diagram of the sensing layer composed of the conducting layer 1-4 and the composite material electronic medium layer 1-5.
It should be noted that the enzyme includes glucose oxidase, lactase, cholesterol enzyme, catalase or uricase, etc., and the enzyme is specifically determined according to the application example, and the embodiment of the present invention is not particularly limited.
As can be understood by those skilled in the art, in the carbon nanotube, hydrogel electron mediator and enzyme composite system, the carbon nanotube solution comprises isopropanol solution of multi-walled carbon nanotubes, acetic acid solution of multi-walled carbon nanotubes, aqueous solution of single-walled carbon nanotubes, acetic acid solution of single-walled carbon nanotubes and the like, so as to improve the response effect and improve the sensing performance.
Optionally, the microneedle array comprises a hollow microneedle array and a sheet microneedle array.
Optionally, the material of the microneedle array comprises stainless steel.
As will be appreciated by those skilled in the art, the shape of the microneedles of the microneedle array includes, but is not limited to, hollow microneedles or sheet-like microneedles, such as solid microneedles; materials for microneedle arrays include, but are not limited to, stainless steel, such as formed by 3d printing a polymer followed by sputtering of chromium or platinum; the specific shape or material of the microneedles of the microneedle array is determined according to the actual application, and the embodiments of the present invention are not particularly limited.
Optionally, the hydrogel electron mediator comprises a reducing macromolecule.
Optionally, the reducing macromolecule comprises a metallic osmium complex.
It should be noted that specific materials of the hydrogel electron mediator include, but are not limited to, reducing polymers and metal osmium complexes, and the specific materials of the hydrogel electron mediator need to be determined according to practical applications, and the embodiment of the present invention is not particularly limited.
Optionally, the material of the conductive layer comprises any one of gold, chromium or platinum.
As will be appreciated by those skilled in the art, gold, chromium or platinum are good conductive materials, are conductive well and stable. The material of the conductive layer includes, but is not limited to, gold, chromium, or platinum, and the embodiment of the present invention is not particularly limited.
The implementation of the embodiment of the invention has the following beneficial effects: the surface of the microneedle array in the embodiment of the invention is modified with a composite material electronic medium layer, the composite material electronic medium layer consists of a carbon nano tube, a hydrogel electronic medium and an enzyme, and the composite material electronic medium layer is fixed on the surface of a conducting layer of the microneedle array through electrodeposition; by modifying the composite material electron mediator comprising the carbon nano tube, the hydrogel electron mediator and the enzyme, the influence of a testing environment is reduced, the strength of a detection signal is increased, and the stability and the sensitivity are improved; the electronic mediator of the composite material is more firmly fixed by an electrodeposition technology and is not easy to fall off, so that the stability is further improved.
According to the embodiment, the conductive stainless steel microneedle array is prepared by laser micro-cutting or micro-processing and other modes, the microneedles have painless transdermal performance, and the risks of pain or tissue damage and the like caused by implanting the electrodes in a body can be reduced. The electrode takes a metal microneedle array (a stainless steel substrate and a surface gold/platinum conducting layer) as a base material, the surface of the metal microneedle array is composed of an electronic mediator sensing layer of a carbon nanotube/hydrogel electronic mediator/enzyme composite material system, the carbon nanotube/hydrogel electronic mediator/enzyme composite material system is treated by adopting a blended carbon nanotube, hydrogel electronic mediator and enzyme electrodeposition technology, wherein the electronic mediator layer is the carbon nanotube/hydrogel electronic mediator/enzyme composite material system, the hydrogel electronic mediator mainly comprises metal complex macromolecules such as metal osmium (or ruthenium) complex-polyvinylpyrrolidone (or bipyridyl or derivatives thereof) and the like, the enzyme can be various types of oxidase such as glucose oxidase, lactase, cholesterol enzyme, uricase, catalase and the like, and the metal microneedle array is prepared on the surface of the microneedle by means of the co-electrodeposition technology to form a uniform coating. By adopting the carbon nano tube/hydrogel electronic mediator/enzyme composite material system as the electronic mediator layer, the micro-needle array can be based on the second generation blood sugar sensing principle of the electronic mediator, the electrochemical test process can use low voltage as bias voltage (for example-0.1V), and the interference of other redox substances in vivo such as vitamins can be effectively avoided. Meanwhile, the detection and sensing of the electrode based on the second generation blood sugar sensing principle depends on the direct transfer of electrons from an enzyme activity center to the electrode without using hydrogen peroxide as an intermediate state electron carrier, so that the electrode has no dependence on oxygen concentration, and the prepared microneedle blood sugar electrode can overcome the problem of oxygen shortage in tissues during in vivo sensing. Compared with the electrode coating prepared by the conventional ways of dip coating, dropping coating and the like, the blending electrodeposition preparation method of the sensing material layer of the sensing electrode has excellent uniformity and mechanical stability, greatly improves the linear detection range of the microneedle blood glucose electrode, and enables the living body to detect blood glucose fluctuation.
As shown in fig. 2, an embodiment of the present invention provides a method for preparing a microneedle sensor based on an electrodeposited electron mediator, including:
s100, preparing a microneedle electrode, and preparing a conducting layer on the surface of the microneedle electrode.
Specifically, the microneedle electrode was prepared as follows: 5-20 syringe needle tubes of GB 23g, 26g, 28g and 30g with the length of 10/20/30mm are taken, a marking machine is used for carrying out laser etching on the microneedles with the power of 50-90 w, the advancing speed of 20-50 mm/s and the advancing times of 100-400 times, and the hollow microneedles are cut into the uniform length of 4/6/8/10 mm.
Optionally, the preparing of the conductive layer on the surface of the microneedle electrode specifically includes:
s110, cleaning and drying the microneedle electrode;
s120, treating the dried microneedle electrode by using soldering flux;
and S130, putting the micro-needle electrode treated by the soldering flux into a solution containing conductive metal, and preparing a conductive layer through electrochemical deposition.
Specifically, the preparation process of the conductive layer is as follows: 1. and soaking the cut microneedles in absolute ethyl alcohol, cleaning for 20-40 min in ultrasound, taking out, putting into a drying oven at 40-80 ℃, and drying for later use. 2. Preparing the stainless steel soldering flux containing 24% of ZnO, 30% of NH4Cl, 6% of HCl, 30% of CH3COOH, 12% of H2O and 3% of surfactant by volume. 3. And (3) immersing the microneedle into stainless steel soldering flux for ultrasonic treatment, taking out after 200s/300s/400s, and slightly sucking away the redundant solution on the surface of the microneedle by using dust-free paper. 4. And taking out the micro-needle, connecting the micro-needle with an electrochemical workstation, taking the micro-needle as a working electrode, taking a gold sheet electrode as a counter electrode, and taking a silver-silver chloride electrode as a reference electrode. Adding 8mL of aqueous solution of gold sodium sulfite with the concentration of 2/4/6mmol/L into a 10mL beaker, immersing three electrodes into the solution, and carrying out electrochemical deposition of gold on the microneedles by using a multi-step constant current method in the gold sodium sulfite electroplating solution, wherein the parameters are as follows: the first stage is as follows: 0.00006A, 15 s; and a second stage: -0.00006A, 15 s; and a third stage: 0.02A, 200s/300s/400 s. After deposition, the mixture is put into a deionized water solution for gentle rinsing and then is naturally dried after being taken out.
The counter electrode was prepared as follows: and connecting the micro-needle with the gold electrochemically deposited to an electrochemical workstation, wherein the micro-needle is used as a working electrode, a platinum sheet electrode is used as a counter electrode, and a silver-silver chloride electrode is used as a reference electrode. Adding 8mL of aqueous solution of sodium platinum sulfite with the concentration of 2/4/6mmol/L into a 10mL beaker, immersing three electrodes into the solution, and performing electrochemical platinum deposition on the microneedles by using a multi-step constant current method to electroplate the platinum solution in the sodium platinum sulfite, wherein the parameters are as follows: the first stage is as follows: 0.00006A, 15 s; and a second stage: -0.00006A, 15 s; and a third stage: 0.02A, 200s/300s/400 s. After deposition, the mixture is put into a deionized water solution for gentle rinsing and then is naturally dried after being taken out.
The reference electrode was prepared as follows: taking the micro-needle with the electrochemical deposition of gold, coating the micro-needle with silver/silver chloride ink, and drying the micro-needle in an oven at 40 ℃/60 ℃/80 ℃. And taking out after drying, performing immersion coating by using a methanol solution (a methanol solution of PVB) of polyvinyl butyral with the mass fraction of 0.5%/1%/1.5%/2%, and airing at room temperature for more than 12/14/16 hours.
S200, preparing a composite material electron mediator solution, wherein the composite material electron mediator solution comprises carbon nano tubes, a hydrogel electron mediator and an enzyme.
Optionally, the preparing of the composite material electron mediator solution specifically includes:
s210, preparing an electron mediator solution and a carbon nano tube solution respectively;
s220, centrifuging the electron mediator solution and the carbon nanotube solution according to a first preset proportion to prepare a doping solution;
s230, providing an enzyme solution;
s240, centrifuging the doping solution and the enzyme solution according to a second preset proportion to prepare the enzyme doping solution.
Specifically, the preparation process of the composite material electron mediator solution is as follows: 1. using thiobetaine methyl acrylate (SBMA) with osmium complex NH2-(CH2)6(m-bim)Os(bpy)2·2PF6Copolymerization was performed to form a solution of osmium electron mediator and an equal volume of one part of a solution of multi-walled Carbon Nanotubes (CNTs) dissolved in Isopropanol (IPA) was prepared. In a 15mL centrifuge tube, osmium electron mediator and carbon nanotube doping solutions (hereinafter referred to as doping solutions) having carbon nanotube concentrations of 0%, 20%, 40%, 60%, 80%, and 100% (v/v) were prepared at volume ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5, respectively. 2. 500mg of glucose oxidase is accurately weighed and dissolved in 10mL of PBS buffer solution to prepare a glucose oxidase solution with the concentration of 50 mg/mL. 3. In a 15mL centrifuge tube, a doping solution and a glucose solution are respectively added according to the volume ratio of 1:1, 1:3 and 1:4 to prepare a doping solution (hereinafter referred to as enzyme doping solution) of an osmium electron mediator, a carbon nanotube and glucose oxidase with the volume fractions of 50%, 25% and 20% (v/v).
S300, fixing the composite material electronic mediator on the surface of the conductive layer of the microneedle electrode by adopting electrodeposition.
Optionally, the fixing the composite electronic mediator on the surface of the conductive layer of the microneedle electrode by using electrodeposition specifically includes:
s310, immersing the microneedle electrode comprising the conductive layer into the composite material electronic mediator solution;
s320, fixing the composite material electronic mediator on the surface of the conductive layer of the microneedle electrode by adopting electrodeposition;
and S330, immersing the microneedle electrode covered with the conductive layer and the composite material electronic dielectric layer into a buffer solution for a preset time.
Specifically, the method for the electrodeposition of the composite electron mediator is as follows: and connecting the micro-needle with the gold electrochemically deposited to an electrochemical workstation, wherein the micro-needle is used as a working electrode, and a platinum sheet electrode is used as a counter electrode and a reference electrode. Adding 8mL of enzyme doping solution with volume fraction of 50%/25%/20% into a 10mL beaker, immersing three electrodes into the solution, controlling the needle point of the microneedle to be immersed into the enzyme doping solution with different concentrations by controlling the length of about 0.5/1/2mm, and carrying out electrochemical codeposition on the microneedle in the enzyme doping solution by using a multi-step constant pressure method, wherein the parameters are as follows: the first stage is as follows: 1A, 200s/300s/400 s. And (3) taking out the electrode after deposition, airing the electrode at room temperature for more than 12/14/16 hours, putting the prepared microneedle detection electrode into PBS buffer solution for standing, taking out the microneedle detection electrode after 8/10/12 hours, naturally airing the microneedle detection electrode at room temperature for 4/6/8 hours, and then putting the microneedle detection electrode in a constant temperature box at 25 ℃ for storage.
It is noted that the composite electron mediator by electrodeposition can be characterized by the following method: and (3) taking the prepared metal microneedle electrode, sputtering for 100s in a gold spraying sputtering instrument at 80% power to uniformly plate a layer of gold material on the surface of the electrode, and observing the appearance under a scanning electron microscope.
It can be understood by those skilled in the art that the microneedle electrode prepared by the above method can be characterized by potential scanning, and the specific method is as follows: taking one 10mL beaker, taking the modified working electrode as a working electrode, taking a commercial platinum electrode as a counter electrode, taking an Ag/AgCl electrode as a reference electrode, putting the counter electrode into the beaker, adding 10mL of PBS buffer solution into the beaker, and immersing the electrode into the solution. The electrochemical workstation software was turned on and the Cyclic-volt measurement option was selected. Setting parameters: init E: -0.2V, High E: 0.8V, Low E: -0.2V, Final E: -0.2V, Initial Scan policy: negative, Scan Rate:0.1V/s, sweet Segments:10, Sample Interval: 0.001V. The measured peak was observed.
It should be noted that the performance characterization process of the microneedle electrode to the glucose response is as follows: taking one 10mL beaker, taking the modified working electrode as a working electrode, taking a commercial platinum electrode as a counter electrode, taking an Ag/AgCl electrode as a reference electrode, putting the counter electrode into the beaker, adding 10mL of PBS buffer solution into the beaker, and immersing the electrode into the solution. The electrochemical workstation software is opened, and the Amperometric i-t Curve option is selected. Setting parameters: init E: 0.5V, Sample Interval:0.1s, Run Time: 300s, Sensitivity: 0.00001A/V. Glucose solutions of different concentrations were added to the solution, so that the concentration of the solution varied: glucose response was measured at different concentrations at 2mM/4mM/6mM/8mM/10mM/12mM/14mM/16mM/18mM/20 mM.
Specifically, the animal experiment test process of the microneedle sensing electrode glucose electrode is as follows: 1. preparation of experimental rats: STZ-Type 1DR rats which are 10 weeks old are purchased, no obvious abnormality is detected, after adaptive breeding is carried out for 7 days, 3 rats with the blood sugar level exceeding 250mg/dL for two consecutive days and 3 rats with the blood sugar level not exceeding 160mg/dL for two consecutive days are selected for carrying out experiments. 2. The anesthesia method comprises the following steps: rats were sedated and then general anesthesia was performed by intraperitoneal injection of 2% sodium pentobarbital solution (0.2mL/100 g). The body temperature of the rats was kept constant using a thermostatic electric heating plate. After the experimental device (microneedle sensor) is arranged, the experimental device is continuously placed on the constant-temperature electric heating plate until the experimental device is revived. Intramuscular injection of a revival solution reduces the chance of accidental death from anesthesia. 3. After the rats were completely anesthetized, the skin of the back was prepared in an area of about 3.5X 3.5cm2. Corresponding experiments were performed according to experimental groups (see below for details). 4. After the skin preparation of group 1 rats (blood glucose levels exceeding 250mg/dL for two consecutive days) was completed, microneedle sensors were applied to the dorsal skin sites and fixed using a mouse vest. Subcutaneous interstitial fluid glucose signals were measured every 15 minutes for the sensing channel. The microneedle sensor was worn continuously for 72 hours, during which time the rat's normal diet was filled with water and the rat was injected with glucose solution at specific time intervals to induce blood glucose excursions. The rat tail was sampled every first 24 hours at intervals of 30min and every second 48 hours at intervals of 1 hour, and the actual blood glucose values were measured using a commercial blood glucose meter as a control. After removal of the vest and microneedle sensors and incubation for an additional 24 hours, the rats were sacrificed by intraperitoneal injection of excess anesthetic (2% sodium pentobarbital solution). Fixing, embedding and slicing local skin at microneedle application position, and staining with hematoxylin-eosin (H) staining method&E) The sections were stained and observed for changes in skin structure, inflammatory cells and possible puncture wounds. 5. Group 2 rats (blood glucose level not exceeding 160mg/dL on two consecutive days) will have microneedlesThe sensor was applied to the dorsal skin site and fixed using a mouse vest. Subcutaneous interstitial fluid glucose signals were measured every 15 minutes for the sensing channel. The microneedle sensor was worn continuously for 72 hours, during which time the rat's normal diet was filled with water and the rat was injected with glucose solution at specific time intervals to induce blood glucose excursions. The rat tail was sampled every first 24 hours at intervals of 30min and every second 48 hours at intervals of 1 hour, and the actual blood glucose values were measured using a commercial blood glucose meter as a control. After removal of the vest and microneedle sensors and incubation for an additional 24 hours, the rats were sacrificed by intraperitoneal injection of excess anesthetic (2% sodium pentobarbital solution). Fixing, embedding and slicing local skin at microneedle application position, and staining with hematoxylin-eosin (H) staining method&E) The sections were stained and observed for changes in skin structure, inflammatory cells and possible puncture wounds. 6. And evaluating performance indexes such as accuracy, sensitivity and the like of the corrected blood glucose monitoring. After removal of the vest and microneedle sensors and incubation for another 24 hours, the rats were sacrificed by intraperitoneal injection of excess anesthetic.
The preparation, testing, etc. of the microneedle sensor will be described below with reference to several examples.
Example one
The microneedle blood glucose sensing electrode based on the electro-codeposition carbon nanotube composite hydrogel electronic mediator is prepared by the following steps:
A. preparation of microneedle electrode (pretreatment of working electrode microneedle)
SEM images of the prepared sheet-like microneedles are shown in fig. 3. Soaking the cut microneedle by using absolute ethyl alcohol, cleaning for 40min in ultrasonic waves, taking out, putting into an oven with the temperature of 80 ℃, and drying for later use. Preparing the stainless steel soldering flux containing 24% of ZnO, 30% of NH4Cl, 6% of HCl, 30% of CH3COOH, 12% of H2O and 3% of surfactant by volume. And (3) immersing the microneedle into the stainless steel soldering flux for ultrasonic treatment, taking out after 200s, and slightly sucking away the redundant solution on the surface of the microneedle by using dust-free paper. And taking out the micro-needle, connecting the micro-needle with an electrochemical workstation, taking the micro-needle as a working electrode, taking a gold sheet electrode as a counter electrode, and taking a silver-silver chloride electrode as a reference electrode. Adding 8mL of 2mmol/L aqueous solution of gold sodium sulfite into a 10mL beaker, immersing three electrodes into the solution, and performing electrochemical gold deposition on the microneedles by using a multi-step constant current method to obtain gold electroplated solution of gold sodium sulfite, wherein the parameters are as follows: the first stage is as follows: 0.00006A, 15 s; and a second stage: -0.00006A, 15 s; and a third stage: 0.02A, 400 s. After deposition, the mixture is put into a deionized water solution for gentle rinsing and then is naturally dried after being taken out.
B. Preparation of carbon nano tube composite hydrogel electron mediator solution
Using thiobetaine methyl acrylate (SBMA) with osmium complex NH2-(CH2)6(m-bim)Os(bpy)2·2PF6Copolymerization was carried out to form an osmium electron mediator solution, and equal volumes of each solution of multi-walled Carbon Nanotubes (CNTs) dissolved in isopropyl alcohol (IPA) were prepared. In a 15mL centrifuge tube, an osmium electron mediator with a carbon nanotube concentration of 20% (v/v) and a carbon nanotube doping solution (hereinafter referred to as a doping solution) were prepared at a volume ratio of 4: 1. 500mg of glucose oxidase is accurately weighed and dissolved in 10mL of PBS buffer solution to prepare a glucose oxidase solution with the concentration of 50 mg/mL. In a 15mL centrifuge tube, a doping solution and a glucose solution are respectively added according to the volume ratio of 1:1, and a doping solution (hereinafter referred to as an enzyme doping solution) of an osmium electron mediator, a carbon nanotube and glucose oxidase with the volume fraction of 50% (v/v) is prepared.
C. Preparation of electron dielectric layer modified enzyme electrode by using electrodeposition method
And connecting the micro-needle with the gold electrochemically deposited to an electrochemical workstation, wherein the micro-needle is used as a working electrode, and a platinum sheet electrode is used as a counter electrode and a reference electrode. Adding 8mL of enzyme doping solution with volume fraction of 50% into a 10mL beaker, immersing three electrodes into the solution, controlling the length of the needle point of the microneedle to be about 2mm, immersing the microneedle into the enzyme doping solution, and performing electrochemical codeposition on the microneedle in the enzyme doping solution by using a multi-step constant voltage method, wherein the parameters are as follows: the first stage is as follows: 1A, 300 s. And taking out the electrode after deposition, airing at room temperature for more than 16 hours, putting the prepared microneedle detection electrode into PBS buffer solution for standing, taking out after 8 hours, naturally airing at room temperature for 8 hours, and then placing the electrode in a constant temperature box at 25 ℃ for storage.
Example two
The second embodiment is different from the first embodiment in that the step of preparing the electron mediator solution of the carbon nanotube composite hydrogel in the step B is replaced by the following steps:
using thiobetaine methyl acrylate (SBMA) with osmium complex NH2-(CH2)6(m-bim)Os(bpy)2·2PF6Copolymerization was carried out to form an osmium electron mediator solution, and equal volumes of each solution of multi-walled Carbon Nanotubes (CNTs) dissolved in isopropyl alcohol (IPA) were prepared. In a 15mL centrifuge tube, an osmium electron mediator with a carbon nanotube concentration of 50% (v/v) and a carbon nanotube doping solution (hereinafter referred to as a doping solution) were prepared at a volume ratio of 1: 1.
Accurately weighing 500mg of glucose oxidase, dissolving the glucose oxidase in 10mL of PBS buffer solution, and preparing glucose oxidase solution with the concentration of 50mg/mL
In a 15mL centrifuge tube, a doping solution and a glucose solution are respectively added according to the volume ratio of 1:1, and a doping solution (hereinafter referred to as an enzyme doping solution) of an osmium electron mediator, a carbon nanotube and glucose oxidase with the volume fraction of 50% (v/v) is prepared.
EXAMPLE III
In-vivo animal experimental tests were performed using the microneedle blood glucose sensing electrode based on the electrodeposited electron mediator prepared in example one, and the determination steps were as follows:
1. preparation of experimental rats: STZ-Type 1DR rats which are 10 weeks old are purchased, no obvious abnormality is detected, after adaptive breeding is carried out for 7 days, rats with the blood sugar level exceeding 250mg/dL for two consecutive days and 1 rat with the blood sugar level not exceeding 160mg/dL for two consecutive days are selected for carrying out experiments.
2. The anesthesia method comprises the following steps: rats were sedated and then general anesthesia was performed by intraperitoneal injection of 2% sodium pentobarbital solution (0.2mL/100 g). The body temperature of the rats was kept constant using a thermostatic electric heating plate. After the experimental device (microneedle sensor) is arranged, the experimental device is continuously placed on the constant-temperature electric heating plate until the experimental device is revived. Intramuscular injection of a revival solution reduces the chance of accidental death from anesthesia.
3. After the rats were completely anesthetized, the rats were placed on their backsSkin preparation is performed on part of the skin, and the area of the skin preparation is about 3.5 x 3.5cm2And carrying out experiments.
4. After the skin preparation of rats (blood glucose levels exceeding 250mg/dL for two consecutive days) was completed, microneedle sensors were applied to the dorsal skin sites and fixed using a mouse vest. Subcutaneous interstitial fluid glucose signals were measured every 15 minutes for the sensing channel. The microneedle sensor was worn continuously for 72 hours, during which time the rat's normal diet was filled with water and the rat was injected with glucose solution at specific time intervals to induce blood glucose excursions. The rat tail was sampled every first 24 hours at intervals of 30min and every second 48 hours at intervals of 1 hour, and the actual blood glucose values were measured using a commercial blood glucose meter as a control. After removal of the vest and microneedle sensors and incubation for an additional 24 hours, the rats were sacrificed by intraperitoneal injection of excess anesthetic (2% sodium pentobarbital solution). Local skin at the position where the microneedle is applied is taken, fixed, embedded and sliced, the section is stained by hematoxylin-eosin (H & E) staining method, and the change of skin structure, inflammatory cells and possible puncture wound is observed.
5. And evaluating performance indexes such as accuracy, sensitivity and the like of the corrected blood glucose monitoring. After removal of the vest and microneedle sensors and incubation for another 24 hours, the rats were sacrificed by intraperitoneal injection of excess anesthetic.
Example four
The difference from the first embodiment is that: the physical dip coating method is adopted to replace the electrodeposition process.
EXAMPLE five
The difference from the first embodiment is that: replacing the carbon nano tube composite hydrogel electron mediator with a Prussian blue electron mediator, and replacing the steps B and C with the following steps:
connecting an electrochemical workstation, taking the gold-plated micro-needle as a working electrode, taking a platinum sheet electrode as a counter electrode, and taking a silver-silver chloride electrode as a reference electrode. Adding 8mL of 2mmol/L aqueous solution of platinum sodium sulfite into a 10mL beaker, immersing three electrodes into the solution, and performing electrochemical gold deposition on the microneedles by using a multi-step constant current method to obtain gold-plated gold solution of the gold sodium sulfite, wherein the parameters are as follows: the first stage is as follows: 0.00006A, 15 s; and a second stage: -0.00006A, 15 s; and a third stage: 0.02A, 100 s. After deposition, the mixture is put into a deionized water solution for gentle rinsing and then is naturally dried after being taken out.
Connecting an electrochemical workstation, taking the micro-needle plated with platinum as a working electrode, taking a platinum sheet electrode as a counter electrode, and taking a silver-silver chloride electrode as a reference electrode. 2.5mM FeCl was added to a 10mL beaker3、100mM KCl、2.5mM K3Fe(CN)6And 100mM HCl, and Prussian blue was deposited using a voltammetric sweep at a rate of 20mV/S and a range of 0-0.5V for 8 cycles.
Preparation of enzyme adhesive: solution 1: dissolving glucose oxidase in PBS to prepare a glucose oxidase solution; solution 2: bovine Serum Albumin (BSA) is dissolved in PBS to prepare BSA solution (BSA @ PBS, the concentration is 80 mg/mL); solution 3: 100uL of glutaraldehyde (glutanic dialdehydee, GA, 2.5%). The solution 1-2-3 was uniformly mixed in a unit of uL of 50-250-.
Slowly extending the microneedle electrode plated with the Prussian blue into the adhesive solution, and slowly pulling for 2s to form a sensing layer.
EXAMPLE six
The difference from the first embodiment is that: replacing the carbon nanotube composite hydrogel electron mediator with a carbon nanotube electron mediator, and replacing the solution preparation in the step B with the following steps:
one part of a solution of multi-walled Carbon Nanotubes (CNTs) dissolved in Isopropanol (IPA) was prepared. 500mg of glucose oxidase is accurately weighed and dissolved in 10mL of PBS buffer solution to prepare a glucose oxidase solution with the concentration of 50 mg/mL. In a 15mL centrifuge tube, a carbon nanotube solution and a glucose solution were added respectively according to a volume ratio of 1:1, respectively, to prepare a doping solution of carbon nanotubes and glucose oxidase (hereinafter referred to as enzyme doping solution) with a volume fraction of 50% (v/v).
EXAMPLE seven
The difference from the first embodiment is that: and (3) replacing the carbon nano tube composite hydrogel electron mediator with an osmium electron mediator, and replacing (solution preparation) in the step B with the following steps:
using thiobetaine methyl acrylate (SBMA) with osmium complex NH2-(CH2)6(m-bim)Os(bpy)2·2PF6Copolymerization is carried out to form an osmium electron mediator solution. 500mg of glucose oxidase is accurately weighed and dissolved in 10mL of PBS buffer solution to prepare a glucose oxidase solution with the concentration of 50 mg/mL. In a 15mL centrifuge tube, an osmium electron mediator solution and a glucose solution were added at a volume ratio of 1:1, respectively, to prepare a doped solution of an osmium electron mediator and glucose oxidase (hereinafter referred to as an enzyme doped solution) having a volume fraction of 50% (v/v).
The analysis of the sensing layer in the above embodiments is as follows:
referring to fig. 4, fig. 4(a) is an SEM image of the modified carbon nanotube composite hydrogel electronic dielectric layer prepared by the co-electrodeposition process, and fig. 4(b) is an SEM image of the modified carbon nanotube composite hydrogel electronic dielectric layer prepared by the physical dip coating process. As can be seen from fig. 4, the adhesion between the carbon nanotube material of the sensing layer prepared by physical dip coating and the electrode substrate is not firm, the modification effect is not uniform, the film forming property is poor, the mechanical wear resistance is weak, and the carbon nanotube material is easy to fall off, so that the response range of the electrode is limited, and the sensing performance is weak. Compared with the method for modifying the carbon nano tube by a physical dip coating method, the carbon nano tube composite hydrogel electronic mediator glucose sensing electrode modified by the electro-codeposition process is tightly adhered to the electrode substrate, is uniformly modified, and has good film forming property and strong mechanical wear resistance.
Referring to fig. 5, (a) in fig. 5 is an SEM image of the electrode with carbon nanotube composite hydrogel as the electron mediator layer, fig. 5(b) is an SEM image of the electrode with prussian blue as the electron mediator layer, fig. 5(c) is an SEM image of the electrode with carbon nanotube electrochemically plated platinum as the electron mediator layer, and fig. 5(d) is an SEM image of the electrode with osmium electron mediator as the electron mediator layer. As can be seen from fig. 5, the sensing layer modified by prussian blue as the electronic dielectric layer is affected by surface tension, is easy to crack, is not firmly adhered to the electrode substrate, has poor film-forming property, has a limited response range at low voltage, is low in sensitivity, and has weak sensing performance; the sensing layer modified by the osmium electron mediator only has poor adhesion with a metal substrate layer, uneven material distribution, poor film forming property, low sensitivity and weaker sensing performance; compared with the Prussian blue electronic mediator and the osmium electronic mediator, the composite hydrogel electronic mediator glucose sensing electrode obtained by modifying the hydrogel electronic mediator layer is tightly adhered to the electrode substrate, is uniformly modified, has good film forming property and strong mechanical wear resistance, and is not easy to fall off.
The results of the performance tests on glucose response in the above examples are as follows:
taking one 10mL beaker, taking the modified working electrode as a working electrode, taking a commercial platinum electrode as a counter electrode, taking an Ag/AgCl electrode as a reference electrode, putting the counter electrode into the beaker, adding 10mL of PBS buffer solution into the beaker, and immersing the electrode into the solution. The electrochemical workstation software is opened, and the Amperometric i-t Curve option is selected. Setting parameters: init E:0.2V, Sample Interval:0.1s, Run Time: 300s, Sensitivity: 0.00001A/V.
Glucose response was measured at different concentrations in 20mL solutions by adding glucose solutions at concentrations that varied from 0mM/2mM/4mM/6mM/8mM/10mM/12mM/14mM/16mM/18mM/20mM every 50 seconds.
Referring to fig. 6, fig. 7 and table one, fig. 6(a) is an i-t curve of the carbon nanotube composite hydrogel as the electrode of the electronic medium layer, fig. 6(b) is an i-t curve of the carbon nanotube composite hydrogel with different ratios as the electrode of the electronic medium layer, fig. 6(c) is an i-t curve of the prussian blue as the electrode of the electronic medium layer, fig. 6(d) is an i-t curve of the carbon nanotube electrochemically platinized carbon nanotube as the electrode of the electronic medium layer, fig. 7 is an i-t curve of the electrode of the electronic medium layer and the electrode of the osmium electronic medium layer, and table one is glucose response test data of different embodiments in the PBS buffer solution.
Watch 1
| Performance of | Example one | EXAMPLE five | EXAMPLE six | EXAMPLE seven |
| Detection interval | 0-20mM | 0-1mM | 1-10mM | 0-16mM |
| Detection limit | 200uM | 200uM | 200uM | 200uM |
| Sensitivity of the probe | 120nA/mM | 20nA/mM | 7nA/mM | 30nA/mM |
As can be seen from fig. 6 and 7 and table one: compared with the fifth embodiment and the sixth embodiment, the first embodiment can perform electrochemical sensing at a low voltage of less than 0.2V, broadens the linear interval of glucose, has obvious steps, and greatly improves the sensitivity of glucose detection; compared with the seventh embodiment, the first embodiment has obvious steps, and the sensitivity of the detection of glucose is greatly improved.
The results of cyclic voltammetry tests performed on the microneedle blood glucose sensing electrodes prepared in the above examples were as follows:
referring to fig. 8, fig. 8(a) is a CV chart of the electrode using the carbon nanotube composite hydrogel as the electron mediator layer, fig. 8(b) is a CV chart of the electrode using prussian blue as the electron mediator layer, and fig. 8(c) is a CV chart of the electrode using the carbon nanotube electrochemical platinum plating as the electron mediator layer. Compared with the fifth example and the sixth example, the peak value of the CV is obviously not shown, and the peak value of the CV is obviously shown in the first example 1 and is about 0.2V; as can be seen from CV and It test results, compared with Prussian blue electron mediators, carbon nanotube electron mediators and osmium electron mediators glucose sensing electrodes, when the carbon nanotube composite hydrogel electron mediator glucose sensing electrode is used as a working electrode for glucose detection, the detection limit is obviously improved, the sensing performance is good, the sensitivity is high, and the linear range is improved to 20 mM. When the working electrode is used for detecting glucose, the stability of the working electrode is greatly improved.
Blood glucose test of living animals was performed using the micro-needle blood glucose sensing electrode based on the electrodeposited electron mediator prepared in example 1. Referring to fig. 9, fig. 9(a) shows the first day of the in vivo animal experimental test of the microneedle blood glucose sensing electrode, fig. 9(b) shows the second day of the in vivo animal experimental test of the microneedle blood glucose sensing electrode, and fig. 9(c) shows the third day of the in vivo animal experimental test of the microneedle blood glucose sensing electrode. From fig. 9, it can be seen that the microneedle electrode based on the composite hydrogel electron mediator shows better performance in the in vivo experiment with the rat as the object, different from other structures, can perform detection even under the voltage of <0.2V, conforms to the trend of blood glucose change measured by commercial detection equipment, can continuously and stably detect the blood glucose fluctuation in the living body for 3 days, and has a detection range of 0-25mM in vivo, high accuracy and good sensitivity.
From the above test results, it can be seen that: according to the embodiment of the invention, the carbon nano tube/hydrogel electronic mediator/enzyme composite material system composite material electronic mediator layer is fixed on the surface of the microneedle by a co-electrodeposition technology to form a uniform coating, and the microneedle blood glucose sensing electrode is prepared. The electronic mediator layer of the composite material greatly improves the linear detection range of the microneedle blood glucose electrode, so that the electrode has excellent uniformity and mechanical stability; the common electronic mediator is not firmly adhered, is easy to fall off, is not uniform and the like in the surface treatment process of the microneedle electrode. The sensing performance of the microneedle blood glucose electrode is remarkably improved, a cyclic voltammetry test (CV) curve is uniform and symmetrical, an obvious redox peak is achieved, an ampere-time test (It) curve is stable and has large gradient, and electrochemical sensing can be performed at low voltage of less than 0.2V. Compared with other sensing materials, the linear range of glucose sensing is obviously improved, the linear range of the microneedle blood glucose sensor in vitro can reach 0-20mM, blood glucose fluctuation can be continuously and stably detected in a living body at lower voltage, and the detection range in the living body reaches 0-25 mM. Compared with the traditional BG method, the method has the advantages of reduced error, widened linear range, improved response range, more obvious discrimination, improved sensitivity and improved stability.
While the preferred embodiments of the present invention have been illustrated and described, 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. A micro-needle sensor based on an electro-codeposition electron mediator is characterized by comprising a substrate and a micro-needle array positioned on the substrate, wherein the surface of the micro-needle array is covered with a conducting layer and a composite material electron mediator layer, the composite material electron mediator is composed of carbon nano tubes, a hydrogel electron mediator and enzymes, and the composite material electron mediator layer is fixed on the surface of the conducting layer of the micro-needle array through electro-codeposition.
2. A microneedle sensor according to claim 1, wherein the microneedle array comprises a hollow microneedle array and a sheet-like microneedle array.
3. A microneedle sensor according to claim 1, wherein the material of the microneedle array comprises stainless steel.
4. A microneedle sensor according to claim 1, wherein the hydrogel electron mediator comprises a reducing macromolecule.
5. A microneedle sensor as claimed in claim 4, wherein the reducing polymer comprises a metal osmium complex.
6. A microneedle sensor according to claim 1, wherein the material of the conductive layer comprises any one of gold, chromium or platinum.
7. A preparation method of a micro-needle sensor based on an electro-codeposition electron mediator is characterized by comprising the following steps:
preparing a microneedle electrode, and preparing a conductive layer on the surface of the microneedle electrode;
preparing a composite material electron mediator solution, wherein the composite material electron mediator solution comprises carbon nano tubes, a hydrogel electron mediator and an enzyme;
and fixing the composite material electronic mediator on the surface of the conductive layer of the microneedle electrode by adopting electrodeposition.
8. The preparation method according to claim 7, wherein the preparing of the conductive layer on the surface of the microneedle electrode specifically comprises:
cleaning and drying the microneedle electrode;
treating the dried microneedle electrode by using soldering flux;
the microneedle electrode treated with the flux is placed in a solution containing a conductive metal, and a conductive layer is prepared by electrochemical deposition.
9. The preparation method according to claim 7, wherein the preparation of the composite material electron mediator solution specifically comprises:
respectively preparing an electron mediator solution and a carbon nano tube solution;
centrifuging the electron mediator solution and the carbon nanotube solution according to a first preset proportion to prepare a doping solution;
providing an enzyme solution;
and centrifuging the doping solution and the enzyme solution according to a second preset proportion to prepare the enzyme doping solution.
10. The preparation method according to claim 7, wherein the fixing of the composite electronic mediator on the surface of the conductive layer of the microneedle electrode by using the electro-codeposition specifically comprises:
immersing a microneedle electrode comprising a conductive layer in the composite electron mediator solution;
fixing the composite material electronic mediator on the surface of the conductive layer of the microneedle electrode by adopting electrodeposition;
and immersing the microneedle electrode covered with the conductive layer and the composite material electronic medium layer into a buffer solution for a preset time.
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