CN116359299A - Electrochemical microneedle sensor for detecting glucose in fruits and vegetables and preparation method - Google Patents
Electrochemical microneedle sensor for detecting glucose in fruits and vegetables and preparation method Download PDFInfo
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- CN116359299A CN116359299A CN202310320470.5A CN202310320470A CN116359299A CN 116359299 A CN116359299 A CN 116359299A CN 202310320470 A CN202310320470 A CN 202310320470A CN 116359299 A CN116359299 A CN 116359299A
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Abstract
The invention discloses an electrochemical microneedle sensor for detecting glucose in fruits and vegetables and a preparation method thereof. The modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode pass through the hollow micro-needle and are electrically connected with the electrochemical workstation. The method comprises the following steps: adhering, insulating and packaging the micro platinum wire and the copper wire to obtain a micro platinum wire electrode; coating a working area of a micro platinum wire electrode to obtain a micro reference electrode; drying the working area after deposition, repeatedly immersing in a treatment solution and drying to obtain a modified micro platinum wire electrode; and enabling the micro platinum wire electrode, the micro reference electrode and the modified micro platinum wire electrode to pass through the hollow microneedle and packaging, and finally obtaining the electrochemical microneedle sensor. The preparation process of the microneedle sensor is simple and short in time consumption, interstitial fluid can be obtained under the condition that normal growth of fruits and vegetables is not affected, minimally invasive sensing of physiological growth information of the fruits and the vegetables is realized, an accurate, real-time and minimally invasive method for sensing glucose in the fruits and the vegetables is provided, and a reliable way is provided for obtaining the physiological growth information of the fruits and the vegetables.
Description
Technical Field
The invention relates to an electrochemical microneedle sensor, in particular to an electrochemical microneedle sensor for detecting glucose in fruits and vegetables and a preparation method thereof.
Background
Glucose has a high chemical activity and is more selective when detected in biological samples. With the development of enzyme engineering and nanoscience, more and more new technologies are being applied to the detection of glucose. According to the principle, the current methods for detecting glucose can be divided into a colorimetric method, a fluorescence method, a Raman spectroscopy method and an electrochemical method. The colorimetric detection of glucose is based on an enzyme and a color developer, glucose oxidase can oxidize glucose to produce hydrogen peroxide, which in the presence of peroxidase can cause color change of the color developer. The colorimetry is simple in operation, low in cost and high in speed, does not need a complex instrument, can read data by naked eyes, and is suitable for spot inspection. Raman scattering is a non-elastic process in which incident photons either lose energy or gain energy from the vibrational and rotational movements of the analyte molecules. The raman spectrum thus produced consists of bands specific to the molecular structure, thus producing a chemical fingerprint specific to the molecule. However, raman scattering signals are weak, which limits its application in chemical analysis. Since the fluorescence intensity interacts with glucose in a concentration-dependent manner, the fluorescence sensor detects glucose by using light of variable frequency, which has the advantage of being sensitive, fast, and non-destructive. Glucose concentration can be calculated by measuring the increase or decrease in fluorescence of a fluorescence donor associated with a glucose acceptor molecule. The electrochemical detection has the characteristics of high sensitivity, simplicity and rapidness in operation, good specificity and convenience in miniaturization and integration. Because glucose and oxidation products thereof have high electrochemical activity, electrochemical methods have been widely used in glucose detection. Glucose electrochemical sensors can be classified into a glucose electrochemical sensor and a non-enzyme electrochemical sensor according to the use or non-use of an enzyme. The enzyme sensor is based on glucose dehydrogenase or glucose oxidase, and has the advantages of high selectivity, high sensitivity, low detection limit and operability under physiological pH conditions. The non-enzyme sensor obtains signals by directly oxidizing glucose, and has the advantages of low cost, high stability and high response speed. Compared with the detection method, the method for detecting glucose has the advantages that the colorimetric method has poor anti-interference performance and low sensitivity; the fluorescence method is easy to be interfered by the background, is difficult to detect on site and has poor selectivity; raman spectroscopy is weak for glucose signals and is not conducive to detection. The electrochemical method has the characteristics of high sensitivity, simple and quick operation, easy microminiaturization and integration, and the like, and has great advantages in monitoring glucose signals in organisms. Besides intuitively displaying the glucose content, the electrochemical method can reflect the change of the glucose content in the organism in real time, and is favorable for realizing information real-time monitoring. With the development of medical means, products such as glucometers and the like are gradually marketed, animal blood sugar detection means are mature and developed, and the field of in-vivo glucose monitoring of fruits and vegetables is still lack of research. Therefore, development of the fruit and vegetable glucose monitoring device based on the electrochemical method can timely acquire fruit and vegetable physiological information and environmental information, and has important significance for developing agricultural productivity.
Disclosure of Invention
In order to solve the problems in the background technology, the invention provides an electrochemical microneedle sensor for detecting fruit and vegetable glucose and a preparation method thereof. The invention integrates the nano composite interface with the hollow microneedle to prepare the minimally invasive electrochemical microneedle sensor, and can monitor glucose signals in the Pink-crown tomatoes and the aloe in real time.
With the development of micro-nano technology, as a very promising biosensing and drug delivery tool, micro-sized needles-microneedles have been widely used. For fruits and vegetables, the micro-needle can obtain interstitial fluid under the condition that normal growth of fruits and vegetables is not affected, and in-situ and real-time monitoring of physiological information of fruits and vegetables is realized. Electrochemical sensors provide an effective means for continuous monitoring of physiological information due to the inherent advantages of miniaturization and non-invasive sample collection. Therefore, electrochemical and micro-needle technologies are often combined to realize minimally invasive detection of fruit and vegetable physiological information. The combination of electrochemistry and micro-needles has great development prospect in the field of real-time perception of biological and physiological information.
The growth and development of fruits and vegetables are not separated from the production, transportation, storage and utilization of sugar, and the sugar is not only an important carbon source for producing energy and synthesizing biomacromolecules, but also serves as a signal molecule for regulating various physiological processes such as metabolism, adverse reaction and the like. Although various sugars play an important role in the embryo's process from development to senescence, glucose is the most basic unit, and forms a network with other signal molecules in the body of fruits and vegetables to regulate gene expression and protein synthesis, progression of the cell cycle, primary and secondary metabolism, and growth and development procedures to maintain normal growth of fruits and vegetables. Therefore, the detection of glucose in the fruit and vegetable is beneficial to knowing the transport mechanism of glucose in the fruit and vegetable, and the information of various stress factors can be indirectly obtained by monitoring glucose signals, so that the method has important significance for organ development and fruit and vegetable growth.
The technical scheme adopted by the invention is as follows:
1. an electrochemical microneedle sensor for detecting glucose in fruits and vegetables:
the electrochemical microneedle sensor comprises a hollow microneedle, a modified micro-platinum wire electrode, a micro-reference electrode and a micro-platinum wire electrode, wherein the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode penetrate through the hollow microneedle, and the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode are all electrically connected with an electrochemical workstation.
The hollow microneedle is rectangular, three quadrangular pyramid-shaped microneedle bodies are arranged on one side face of the hollow microneedle, grooves are formed in one side face of the hollow microneedle, which is far away from the microneedle bodies, a microneedle hole is formed in each of the three microneedle bodies along the length direction of the hollow microneedle bodies, the other ends of the modified micro platinum wire electrode, the micro reference electrode and the micro platinum wire electrode penetrate through the corresponding microneedle hole respectively and are electrically connected with an electrochemical workstation, and the middle parts of the modified micro platinum wire electrode, the micro reference electrode and the micro platinum wire electrode are solidified and packaged at the grooves of the hollow microneedle bodies through insulating glue.
One end of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode penetrate through the tip of one micro-needle body of each micro-needle through micro-pinholes, the distance from one end of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode to the tip of one micro-needle body of each micro-needle is 2mm, and the aperture of each micro-pinhole is the same, specifically 100-900 mu m.
2. A method for preparing an electrochemical microneedle sensor, which comprises the following steps:
the method comprises the following steps:
step 1) preparing hollow microneedles: hollow microneedles were obtained using transparent resin 3D printing with a print accuracy set to 20 μm.
Step 2) preparing a micro platinum wire electrode: and (3) adhering the other ends of the micro platinum wires and the copper wires, heating and curing, and then insulating and packaging the adhesion parts of the micro platinum wires and the copper wires to obtain the micro platinum wire electrode, wherein the micro platinum wire end of the micro platinum wire electrode is used as a working area.
Step 3) preparing a micro reference electrode: and (3) carrying out coating treatment on the working area of the micro-platinum wire electrode, and then heating and curing to obtain the micro-reference electrode.
Step 4) preparing a modified micro platinum wire electrode: and (3) adopting a timing current method, using a counter electrode, a reference electrode and a working electrode as a three-electrode system, carrying out deposition treatment on a working area of the micro-platinum wire electrode through electroplating solution, drying by nitrogen, repeatedly immersing the working area into a first treatment solution, drying, repeatedly immersing into a crosslinking solution, drying, immersing into a second treatment solution, and drying to obtain the modified micro-platinum wire electrode.
Step 5) preparing an electrochemical microneedle sensor: the method comprises the steps of respectively penetrating a micro-platinum wire electrode, a micro-reference electrode and a modified micro-platinum wire electrode through a micro-needle body of a hollow micro-needle body, placing the working area ends of the micro-platinum wire electrode, the micro-reference electrode and the modified micro-platinum wire electrode outside the tip end of the micro-needle body of the hollow micro-needle body, respectively penetrating the copper wire ends of the micro-platinum wire electrode, the micro-reference electrode and the modified micro-platinum wire electrode through a micro-needle hole, and then electrically connecting the copper wire ends with an electrochemical workstation, wherein the middle parts of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode are solidified and packaged through insulating glue at the groove of the hollow micro-needle body, and finally obtaining the electrochemical micro-needle sensor.
In the step 2), the other ends of the micro platinum wires and the copper wires are adhered by using epoxy conductive adhesive and then heated and cured for more than 2 hours in a baking oven at 70 ℃, and then the adhesion part of the micro platinum wires and the copper wires is insulated and packaged by using insulating adhesive to obtain a micro platinum wire electrode, wherein a 2mm length area of one end of the micro platinum wires of the micro platinum wire electrode is used as a working area.
In the step 3), the working area of the micro-platinum wire electrode is subjected to uniform coating treatment by using Ag/Agcl slurry, and then is heated and cured in a baking oven at 70 ℃ for 2 hours to obtain the micro-reference electrode.
In the step 5), the middle parts of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode are packaged at the groove of the hollow micro-needle body through insulating glue and dried and cured for 2 hours at normal temperature.
In the step 4), the electroplating solution is specifically trichlorogold acid HAucl with the concentration of 10mg/mL 4 And (3) using a timing current method, taking a micro-platinum wire electrode as a working electrode, taking an Ag/Agcl electrode as a reference electrode, taking a platinum electrode as a counter electrode, maintaining the potential of-0.66V for less than 180s through electroplating solution, depositing gold nano-particles AuNPs on a working area of the micro-platinum wire electrode, and drying by nitrogen to finish the deposition treatment.
In the step 4), the first treatment solution is specifically 50mg/ml Nafion, the crosslinking agent is specifically glucose oxidase crosslinking solution, and the second treatment solution is specifically 15mg/ml PU; repeatedly immersing the working area for 4 times into 50mg/ml Nafion for 10s, repeatedly taking out and drying at normal temperature until Nafion is completely volatilized, repeatedly immersing the working area for 5 times into glucose oxidase cross-linking liquid for 1min, repeatedly taking out and drying at normal temperature, finally immersing the working area into 15mg/ml PU for 3s, and drying at normal temperature to obtain the modified micro platinum wire electrode;
in the step 4), the glucose oxidase cross-linking liquid is specifically prepared by taking phosphate buffer solution as a solvent, adopting glucose oxidase GOX with the concentration of 20-60 mg/ml, and glutaraldehyde GA with the mass ratio of glucose oxidase to bovine serum albumin BSA of 1:1 and the volume fraction of 0.2-1%.
3. A minimally invasive real-time sensing method of an electrochemical microneedle sensor comprises the following steps:
the modified micro-platinum wire electrode, the micro-reference electrode and the working area of the micro-platinum wire electrode of the electrochemical microneedle sensor are inserted into stems or leaves of fruits and vegetables containing glucose, and the change of signals transmitted by the electrochemical microneedle sensor is monitored in real time through an electrochemical workstation, so that the real-time monitoring and sensing of the change of the glucose concentration of the fruits and vegetables are realized.
The beneficial effects of the invention are as follows:
1. the invention constructs the microneedle sensor integrating the nano composite interface and the hollow microneedle, and the microneedle sensor is used for monitoring glucose in real time, so that the operation process is simple and the time consumption is short.
2. The nanomaterial can be used for shortening the electron tunneling distance between the enzyme and the electrode surface through partial expansion of the enzyme in the adsorption process, so that the accessibility of a redox center is increased, the direct transfer of electrons is promoted, the nanomaterial can be used for increasing the surface roughness of the electrode material, so that more enzyme is adsorbed on the electrode surface, the enzyme loading capacity is increased, and the efficiency and the sensitivity of the sensor are improved. The nano composite micro interface constructed in the working area of the microneedle sensor has high selectivity, anti-interference performance and good biocompatibility by combining the design of the anti-interference layer, the identification element and the diffusion limiting layer.
3. The hollow microneedle prepared by the invention can obtain interstitial fluid under the condition of not affecting the normal growth of fruits and vegetables, and realize minimally invasive perception of physiological growth information. The combination of the electrochemical sensing interface and the microneedle technology provides a new idea for the real-time sensing of the physiological information of fruits and vegetables.
4. The invention realizes the minimally invasive real-time sensing technology of glucose in the Pink-crown tomatoes and the aloe, explores a precise, real-time and minimally invasive method for sensing glucose in fruits and vegetables, and provides a reliable way for acquiring the physiological information of fruit and vegetable growth.
Drawings
FIG. 1 (a) is an SEM image of the working area MPt-Au interfacial electron microscope at 300 times magnification;
FIG. 1 (b) is an SEM image of the working area MPt-Au interfacial electron microscope at 5000 magnification;
FIG. 1 (c) is an SEM image of the working area MPt-Au interfacial electron microscope at 30000 magnification;
FIG. 1 (d) is an SEM image of the working area MPt-Au interfacial electron microscope at 50000 magnification;
FIG. 2 (a) is an SEM image of the MPt-Au interface of the working area;
FIG. 2 (b) is an SEM image of the working area MPt-Au-Nafion interface;
FIG. 2 (c) is an SEM image of the working area MPt-Au-Nafion-GOX interface;
FIG. 2 (d) is an SEM image of the working area MPt-Au-Nafion-GOX-PU interface;
FIG. 3 (a) is a top view of a hollow microneedle body;
fig. 3 (b) is a bottom view of the hollow microneedle body;
FIG. 3 (c) is a front view of the hollow microneedle body;
FIG. 3 (d) is a side view of the hollow microneedle body;
FIG. 4 (a) is a graph of the current response of the microneedle sensor versus glucose concentration for a glucose concentration range of 0-10 mM;
FIG. 4 (b) is a graph of the current response of the microneedle sensor versus glucose concentration (operating potential 0.7V, supporting electrolyte 1 XPBS) for a glucose concentration range of 5-100 mM;
FIG. 5 is a graph showing the amperometric response of a microneedle sensor to glucose, ascorbic acid, fructose, sucrose, lactose, maltose, raffinose (operating potential 0.7V);
FIG. 6 (a) is a repetitive normalized current histogram of a microneedle sensor;
FIG. 6 (b) is a normalized current histogram of stability of the microneedle sensor;
fig. 7 (a) is a graph of a microneedle sensor monitoring glucose signals in tomato body over 12h in real time;
FIG. 7 (b) is a graph of the microneedle sensor monitoring glucose signals in aloe body over 12 hours in real time;
FIG. 8 is a graph of a microneedle sensor monitoring glucose signals in tomato body for 12h in real time under salt stress;
fig. 9 (a) is a schematic diagram showing healing of tomato stem wound sites within days 1, 2, 3, 4, 5, 6, 14, and 15;
FIG. 9 (b) is a schematic illustration of aloe vera leaf wound healing within days 1, 2, 3, 4, 5, 6, 14, 15.
Detailed Description
The invention will be described in further detail with reference to the accompanying drawings and specific examples.
The embodiment of the invention specifically comprises the following steps:
example 1:
the preparation method of the electrochemical microneedle sensor comprises the following steps:
step 1) preparing hollow microneedles: hollow microneedles were obtained using transparent resin 3D printing with a print accuracy set to 20 μm.
Step 2) preparing a micro platinum wire electrode: and (3) using epoxy conductive adhesive to adhere the other ends of the micro platinum wire and the copper wire, heating and curing the mixture in an oven at 70 ℃ for more than 2 hours, and then insulating and packaging the adhesion part of the micro platinum wire and the copper wire by using insulating adhesive to obtain a micro platinum wire electrode, wherein a 2mm length area of one end of the micro platinum wire electrode is used as a working area.
Step 3) preparing a micro reference electrode: and carrying out uniform coating treatment on the working area of the micro-platinum wire electrode by using Ag/Agcl slurry, and then heating and curing the mixture in a baking oven at 70 ℃ for 2 hours to obtain the micro-reference electrode.
Step 4) preparing a modified micro platinum wire electrode: adopting a timing current method, using a counter electrode, a reference electrode and a working electrode as a three-electrode system, taking a micro platinum wire electrode as the working electrode, taking an Ag/Agcl electrode as the reference electrode, taking a platinum electrode as the counter electrode, and carrying out deposition treatment, namely, maintaining for 150s at a potential of-0.66V, passing through 10mg/mL of tetrachloroauric acid HAucl 4 Depositing gold nano-particles AuNPs on a working area of a micro platinum wire electrode, drying by nitrogen, repeatedly immersing the working area for 4 times into 50mg/ml naphthol solution Nafion for 10s and repeatedly taking out and drying at normal temperature until the Nafion is completely volatilized, repeatedly immersing the working area for 5 times into glucose oxidase crosslinking solution for 1min and repeatedly taking out and drying at normal temperature, finally immersing the working area into 15mg/ml polyurethane solution PU for 3s and drying at normal temperatureFinally, the modified micro platinum wire electrode is obtained.
Step 5) preparing an electrochemical microneedle sensor: the method comprises the steps of respectively penetrating a micro-platinum wire electrode, a micro-reference electrode and a modified micro-platinum wire electrode through a micro-needle body of a hollow micro-needle body, placing the working area ends of the micro-platinum wire electrode, the micro-reference electrode and the modified micro-platinum wire electrode outside the tip end of the micro-needle body of the hollow micro-needle body, respectively penetrating the copper wire ends of the micro-platinum wire electrode, the micro-reference electrode and the modified micro-platinum wire electrode through a micro-needle hole, electrically connecting the copper wire ends with an electrochemical workstation, and finally drying and curing the middle parts of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode at the groove of the hollow micro-needle body through insulating glue for 2 hours at normal temperature to package, thereby finally obtaining the electrochemical micro-needle sensor. The glucose oxidase cross-linking solution is prepared by using 1mL of phosphate buffer solution as a solvent, and adopting 30mg of glucose oxidase GOX, 30mg of bovine serum albumin BSA and 6 mu L of glutaraldehyde GA.
The invention relates to a minimally invasive real-time sensing method of an electrochemical microneedle sensor, which comprises the following steps:
the modified micro-platinum wire electrode, the micro-reference electrode and the working area of the micro-platinum wire electrode of the electrochemical microneedle sensor are inserted into stems or leaves of fruits and vegetables containing glucose, and the change of signals transmitted by the electrochemical microneedle sensor is monitored in real time through an electrochemical workstation, so that the real-time monitoring and sensing of the change of the glucose concentration of the fruits and vegetables are realized.
The morphology analysis of the nanocomposite micro interface at the working area of the microneedle sensor of embodiment 1 of the present invention is specifically as follows:
microscopic morphology of MPt-Au surface gold nanoparticle AuNPs at a reduction time of 150s was observed by scanning electron microscope SEM, and the results are shown in fig. 1 (a), fig. 1 (b), fig. 1 (c) and fig. 1 (d). Compared with a bare micro-platinum wire, the deposition of AuNPs enables compact particles to appear on the surface of the micro-platinum wire, besides improving the electron transfer efficiency, the nano structure also enables the surface roughness of the micro-platinum wire to be improved, and the subsequent crosslinking adsorption of glucose oxidase is facilitated. The micro platinum wires were surface analyzed by X-ray spectroscopy. After electrochemical reduction treatment, gold elements appear on the surface of the micro-platinum wire, and the construction of the gold nano micro-interface is successfully realized by the method provided by the verification again.
As shown in fig. 2 (a), fig. 2 (b), fig. 2 (c) and fig. 2 (d), the SEM images of MPt-Au, MPt-Au-Nafion-GOX-PU interfaces were sequentially obtained, and the interface morphology of the micro platinum wire after layer-by-layer modification was analyzed by SEM characterization. The dense granular structure demonstrates successful reduction of AuNPs, followed by blurring of the nanointerface, which is caused by successful coating of Nafion membranes. The cross-linked adsorption of glucose oxidase allows the bottommost nanostructure to be further covered and the nanostructure on the view to disappear completely. Finally, modification of the polyurethane resulted in the formation of a rounded small raised structure at the interface, demonstrating successful formation of the polyurethane structure.
The hollow microneedle body of the microneedle sensor of the embodiment of the present invention is shown in fig. 3 (a), fig. 3 (b), fig. 3 (c) and fig. 3 (d). The hollow microneedle substrate was square 6mm long and 6mm wide, and had a thickness of 2mm. In order to facilitate the fixation of the copper wire at the tail end of the microelectrode, a hollow structure is designed for the penetration of the copper wire, the thickness of the hollow part is 1.4mm, the microneedle body is in a quadrangular pyramid shape, the pyramid base is a square with the length of 1.1mm and the width of 1.1mm, the pyramid height is 2mm, and the aperture of the hollow microneedle is 300 mu m. Three microelectrodes are integrated into a hollow microneedle and encapsulated with insulating glue and connected to an electrochemical workstation.
Based on the optimization result, combining with a timing current method, the ampere response of the microneedle sensor to glucose with different concentrations is examined, and a corresponding detection model is established: i=0.00012c+0.0109 (c=0 to 10 mM), i= 0.9126log C-2.568 (c=5 to 100 mM), I represents current, and C represents glucose concentration. The glucose concentrations were 100. Mu.M, 500. Mu.M, 1mM, 5mM, 10mM, 25mM, 50mM, and 100mM, respectively, and the results were shown in FIG. 4 (a) and FIG. 4 (b). As the glucose concentration increases, the current response of the microneedle sensor increases significantly. And drawing a scatter diagram by taking the glucose concentration or the logarithmic value of the glucose concentration as the X axis and the current value as the Y axis, and performing linear fitting. From the graph, when the glucose concentration ranges from 0mM to 10mM, the ampere response of the microneedle sensor is positively correlated with the glucose concentration, and the linear regression equation is I=0.00012C+0.0109, and the linear correlation coefficient is 0.9891; when the glucose concentration ranges from 5mM to 100mM, the current response of the sensor is positively correlated with the logarithm of the glucose concentration, and the linear regression equation is I= 0.9126log C-2.5680, and the linear correlation coefficient is 0.8453.
The anti-interference, repeatability and stability tests are specifically as follows:
the amperometric response of the microneedle sensor to six interfering substances, ascorbic acid, fructose, sucrose, lactose, maltose and raffinose, when detecting glucose was tested. Firstly, a buffer solution is dripped on a microneedle sensor, a pipette is used for removing the liquid after a signal is stable, a glucose solution is added again, other interferents are added in the same mode after the signal is stable, and the pipette gun head is not contacted with a working area of the microneedle sensor in the operation process, so that the damage to devices is avoided. The concentration of each substance is selected to be the same as the previous section according to the relative content of the seven components in the fruit and vegetable body, and the result is shown in figure 5. The current response of the microneedle sensor to the interfering substance is close to zero, and glucose with equal concentration is added again after the interfering substance is added, the ampere response of the microneedle sensor is not changed obviously compared with the response of glucose added for the first time, so that the microneedle sensor has good anti-interference performance and repeatability, and has great application potential in actual detection.
In the actual application scene of the sensing device, the repeatability and the stability of the sensor have practical significance. To investigate the reproducibility of the proposed microneedle sensor, the same microneedle sensor was used here in cycles 6 times, and based on chronoamperometry, the normalized currents for 5mM test object in 6 replicates were compared. Stability was assessed by testing the normalized current of 3 different batches of fabricated microneedle sensors for an isoconcentration analyte. As shown in fig. 6 (a) and fig. 6 (b), for the measured object with the same concentration, the normalized current RSD obtained by recycling 1 microneedle sensor for 6 times is 6.36%, and the normalized current RSD of 3 microneedle sensors with different manufacturing batches is 4.79%, which indicates that the current response difference between the microneedle sensors in recycling and between the microneedle sensors with different batches is not large, so as to meet the requirement of actual detection.
The feasibility analysis is specifically as follows:
in view of the complex matrix effect in the fruit and vegetable tissue, the application feasibility of the constructed microneedle sensor and the quantitative detection model in a fruit and vegetable system is examined. The feasibility verification was performed using a labeled recovery method, and the test was performed in tomato stem grinding fluid using a standard addition method using a microneedle sensor. The stem extracts from three normally grown tomato plants were diluted in a ratio of 1:9 with the grinding fluid and PBS buffer, glucose was added at low concentration (100. Mu.M) and high concentration (5 mM) respectively as supporting electrolytes, and the mixture was detected using a microneedle sensor, and the current response was substituted into the detection model to obtain a glucose concentration value, which was compared with the actual addition amount, and the recovery rate, which was the ratio of the theoretical concentration to the actual addition concentration obtained by the detection model, was calculated according to this, and the results were shown in Table 1. The results show that the recovery rate of the 6 groups of samples is between 92% and 118%, and the positive and negative errors are less than 20%. The results prove that the manufactured microneedle sensor has good biocompatibility in the tomato in-vivo environment, and the interface constructed by the experiment and the established detection model have high feasibility in the application scene in the fruit and vegetable.
Table 1 recovery of glucose from tomato shoot extract by microneedle sensor
The in-situ monitoring is specifically as follows:
in view of the excellent performance of the microneedle sensor based on the MPt-Au-Nafion-GOX-PU composite nano micro interface on glucose detection, the microneedle sensor is applied to in-situ minimally invasive sensing of glucose signals in fruits and vegetables. Here, the tomato powder crown and aloe are taken as experimental objects, and the feasibility of in-situ monitoring of fruit and vegetable physiological information by the microneedle sensor is explored. The micro-needle sensor is inserted into the stems of tomatoes and the leaves of aloe, the fixation of the micro-electrode is realized by means of the rigidity of the micro-needle substrate, meanwhile, the damage and the influence of the sensing process on fruits and vegetables are reduced by utilizing the characteristics of the minimally invasive device, and the copper wire on the back of the micro-needle sensor is connected with the electrochemical workstation for electrochemical detection. During the growth of fruits and vegetables, the glucose content can change rhythmically as a photosynthesis product and a respiration substrate. When fruits and vegetables are subjected to environmental stress, the contents of glucose in the fruits and vegetables are also changed under the influence of multiple signal paths as an important class in bioactive molecules. Based on this principle, tomato and aloe plants were monitored in real time for a period of 12h, and the change in the current response of the device was recorded, and the results are shown in fig. 7 (a) and fig. 7 (b). The results show that the current signals of the microneedle sensors in tomato and aloe show similar change rules in the 12h day and night detection process. After entering the night, photosynthesis of plants is weakened, and respiration consumes glucose, so that current signals are reduced; in contrast, after the day, photosynthesis is enhanced, glucose gradually synthesizes and accumulates, and signals rise.
In addition, in order to simulate osmotic stress which may occur in the environment, a 200mM NaCl solution was added to the soil where tomato plants were grown, and the trend of the current signal was observed, and the result is shown in FIG. 8. The signal did not increase when salt stressed tomato plants entered the day compared to normally grown tomato plants, possibly due to the inhibitory effect of salt on photosynthesis. Experimental results show that the microneedle sensor has good feasibility in real-time sensing of glucose in fruits and vegetables, and has good application prospects in acquisition of stress information.
The wound assessment is specifically as follows:
to evaluate the wound healing of the fruits and vegetables during the detection process by the microneedle sensor and the possible effect of the insertion of the microneedle sensor on the normal growth of the fruits and vegetables, the wound morphology of the tomato and aloe body surface by the microneedle sensor was continuously monitored for 15 days, and the recovery thereof was observed and recorded by an electron microscope, as shown in fig. 9 (a) and 9 (b). The results showed that with the prolongation of the healing time, the wound sites of both tomato and aloe formed callus, and no adverse conditions such as tissue necrosis occurred. And simultaneously comparing growth conditions of 4 plants on the 1 st day and the 15 th day after the microneedle sensor is inserted, and recording by single-shot back shooting. On day 15 of microneedle sensor insertion, both tomato and aloe showed normal growth, and the results indicate that penetration of the microneedle sensor did not affect normal growth of the plants.
Claims (10)
1. An electrochemical microneedle sensor for fruit and vegetable glucose detection, which is characterized in that: the micro-electrode comprises a hollow micro-needle, a modified micro-platinum wire electrode, a micro-reference electrode and a micro-platinum wire electrode, wherein the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode penetrate through the hollow micro-needle, and the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode are all electrically connected with an electrochemical workstation.
2. An electrochemical microneedle sensor for fruit and vegetable glucose detection according to claim 1, wherein: the hollow microneedle is rectangular, three quadrangular pyramid-shaped microneedle bodies are arranged on one side face of the hollow microneedle, grooves are formed in one side face of the hollow microneedle, which is far away from the microneedle bodies, a microneedle hole is formed in each of the three microneedle bodies along the length direction of the hollow microneedle bodies, the other ends of the modified micro platinum wire electrode, the micro reference electrode and the micro platinum wire electrode penetrate through the corresponding microneedle hole respectively and are electrically connected with an electrochemical workstation, and the middle parts of the modified micro platinum wire electrode, the micro reference electrode and the micro platinum wire electrode are solidified and packaged at the grooves of the hollow microneedle bodies through insulating glue.
3. An electrochemical microneedle sensor for detecting glucose in fruits and vegetables according to claim 2, wherein: one end of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode penetrate through the tip of one micro-needle body of each micro-needle through micro-pinholes, the distance from one end of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode to the tip of one micro-needle body of each micro-needle is 2mm, and the aperture of each micro-pinhole is the same, specifically 100-900 mu m.
4. A method of manufacturing an electrochemical microneedle sensor according to any one of claims 1-3, characterized in that: the method comprises the following steps:
step 1) preparing hollow microneedles: 3D printing by using transparent resin to obtain hollow microneedles;
step 2) preparing a micro platinum wire electrode: the other end of the micro platinum wire is adhered to the other end of the copper wire and then heated and solidified, then the adhesion part of the micro platinum wire and the copper wire is insulated and packaged to obtain a micro platinum wire electrode, and the micro platinum wire end of the micro platinum wire electrode is used as a working area;
step 3) preparing a micro reference electrode: coating the working area of the micro platinum wire electrode, and then heating and curing to obtain a micro reference electrode;
step 4) preparing a modified micro platinum wire electrode: adopting a timing current method, using a counter electrode, a reference electrode and a working electrode as a three-electrode system, carrying out deposition treatment on a working area of a micro platinum wire electrode through electroplating solution, drying by nitrogen, repeatedly immersing the working area into a first treatment solution, drying, repeatedly immersing into a crosslinking solution, drying, finally immersing into a second treatment solution, and drying to obtain the modified micro platinum wire electrode;
step 5) preparing an electrochemical microneedle sensor: the method comprises the steps of respectively penetrating a micro-platinum wire electrode, a micro-reference electrode and a modified micro-platinum wire electrode through a micro-needle body of a hollow micro-needle body, placing the working area ends of the micro-platinum wire electrode, the micro-reference electrode and the modified micro-platinum wire electrode outside the tip end of the micro-needle body of the hollow micro-needle body, respectively penetrating the copper wire ends of the micro-platinum wire electrode, the micro-reference electrode and the modified micro-platinum wire electrode through a micro-needle hole, and then electrically connecting the copper wire ends with an electrochemical workstation, wherein the middle parts of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode are solidified and packaged through insulating glue at the groove of the hollow micro-needle body, and finally obtaining the electrochemical micro-needle sensor.
5. The method for manufacturing an electrochemical microarray sensor according to claim 4, wherein:
in the step 2), the other ends of the micro platinum wires and the copper wires are adhered by using epoxy conductive adhesive and then heated and cured for more than 2 hours in a baking oven at 70 ℃, and then the adhesion part of the micro platinum wires and the copper wires is insulated and packaged by using insulating adhesive to obtain a micro platinum wire electrode, wherein a 2mm length area of one end of the micro platinum wires of the micro platinum wire electrode is used as a working area.
6. The method for manufacturing an electrochemical microneedle sensor according to claim 4, wherein:
in the step 3), the working area of the micro platinum wire electrode is uniformly coated with Ag/Agcl slurry, and then heated and cured in a baking oven at 70 ℃ for 2 hours to obtain a micro reference electrode;
in the step 5), the middle parts of the modified micro-platinum wire electrode, the micro-reference electrode and the micro-platinum wire electrode are packaged at the groove of the hollow micro-needle body through insulating glue and dried and cured for 2 hours at normal temperature.
7. The method for manufacturing an electrochemical microneedle sensor according to claim 4, wherein: in the step 4), the electroplating solution is specifically trichlorogold acid HAucl with the concentration of 10mg/mL 4 And (3) using a timing current method, taking a micro-platinum wire electrode as a working electrode, taking an Ag/Agcl electrode as a reference electrode, taking a platinum electrode as a counter electrode, maintaining the potential of-0.66V for less than 180s through electroplating solution, depositing gold nano-particles AuNPs on a working area of the micro-platinum wire electrode, and drying by nitrogen to finish the deposition treatment.
8. The method for manufacturing an electrochemical microneedle sensor according to claim 4, wherein: in the step 4), the first treatment solution is specifically 50mg/ml Nafion, the crosslinking agent is specifically glucose oxidase crosslinking solution, and the second treatment solution is specifically 15mg/ml PU; repeatedly immersing the working area for 4 times into 50mg/ml naphthol solution Nafion for 10s, repeatedly taking out and drying at normal temperature until Nafion is completely volatilized, repeatedly immersing the working area for 5 times into glucose oxidase cross-linking liquid for 1min, repeatedly taking out and drying at normal temperature, finally immersing into 15mg/ml polyurethane solution PU for 3s, and drying at normal temperature to obtain the modified micro platinum wire electrode.
9. The method for manufacturing an electrochemical microneedle sensor according to claim 4, wherein: in the step 4), the glucose oxidase cross-linking liquid is prepared by taking phosphate buffer solution as a solvent and adopting glucose oxidase GOX with the concentration of 20-60 mg/ml, bovine serum albumin BSA and glutaraldehyde GA with the volume fraction of 0.2% -1%, wherein the mass ratio of the glucose oxidase to the bovine serum albumin BSA is 1:1.
10. The electrochemical microneedle sensor according to any one of claims 1 to 3 or the method for minimally invasive real-time sensing of an electrochemical microneedle sensor prepared by the method for preparing any one of claims 4 to 9, wherein: the modified micro-platinum wire electrode, the micro-reference electrode and the working area of the micro-platinum wire electrode of the electrochemical microneedle sensor are inserted into stems or leaves of fruits and vegetables containing glucose, and the change of signals transmitted by the electrochemical microneedle sensor is monitored in real time through an electrochemical workstation, so that the real-time monitoring and sensing of the change of the glucose concentration of the fruits and vegetables are realized.
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