CN113447544A - Degradable transient NO sensor and preparation method thereof - Google Patents
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Abstract
The invention relates to a degradable transient NO sensor and a preparation method thereof. The invention discloses a degradable transient NO sensor, which comprises: a substrate formed from a levorotatory polylactic acid-polytrimethylene carbonate copolymer; a detection electrode comprising a working electrode, a counter electrode and a reference electrode, the detection electrode being formed on the substrate; a selectively permeable film at least partially coating the surface of the substrate, wherein the ratio of L-polylactic acid: the polytrimethylene carbonate ranges from 30:70 to 40: 60. The degradable transient NO sensor has the advantages of low detection limit, wide sensing range, high time resolution and excellent anti-interference performance. Meanwhile, the probe can be biologically absorbed, has flexibility and high stretching property, can be tightly attached to a detected part, reduces signal loss, obtains high current response and improves detection sensitivity.
Description
Technical Field
The invention relates to a degradable transient NO sensor and a preparation method thereof, belonging to the technical field of biological detection and medical detection.
Background
Accurate continuous measurement of key biomarkers in the human body is an important basis for health assessment, drug guidance, surgical protocols and post-operative monitoring. In particular, real-time monitoring of Nitric Oxide (NO) levels in a physiological environment plays a crucial role in neurotransmission, immune responses, the cardiovascular system, angiogenesis and microcirculation. Abnormal NO content is closely associated with inflammation, neurotoxicity and the development of cancer. For example, chondrocytes from osteoarthritis patients are associated with increased concentrations of NO and induce sustained nitric oxide synthase (iNOS) expression, thereby promoting inflammatory responses, chondrocyte apoptosis and cartilage degradation. As one of the leading causes of disability, osteoarthritis is expected to affect at least 1.3 billion people worldwide before 2050. Therefore, probing nitric oxide in the joint cavity is crucial for early intervention and treatment optimization in osteoarthritis patients. However, due to its short half-life (6-10s), low concentration (nM- μ M), high chemical activity and interference from other chemicals in biological systems (e.g. glucose, nitrite, uric acid, etc.). Several techniques have been proposed to detect the concentration of NO, including indirect methods such as measuring nitrite ions (NO) in solution2-) Griess analysis of concentration; and direct methods such as a fluorescence probe method, an electron spin resonance spectroscopy method, and a chemiluminescence method. Either these methods have insufficient detection limits or involve complicated sample preparation, thereby not enabling real-time measurement of Nitric Oxide (NO) levels in physiological environments.
In contrast, electrochemical sensors can provide rapid continuous NO detection with high sensitivity and low detection limits. However, most conventional electrochemical NO sensors are made of rigid materials, requiring surgical retrieval after implantation to eliminate unnecessary equipment load, which may cause severe irritation and expose the patient to the threat of infection complications.
In recent years, an emerging class of flexible and transient sensor devices has emerged that have mechanical properties that match biological tissues, can be absorbed or degraded, has the potential to eliminate the aforementioned drawbacks, can reduce potential foreign bodies and inflammation, and eliminate secondary surgery for device retrieval. For example, there are biodegradable and bioabsorbable sensor devices in the prior art that are capable of monitoring pressure and temperature in the brain, recording pressure and strain of tendon healing, etc.; in addition, there are bioabsorbable therapeutic devices for cardiovascular disease, peripheral nerve regeneration and infection mitigation.
Although various transient sensors having comparable performance to non-transient sensors have been available, the development of transient sensors having chemical sensing capabilities in physiological environments remains an unsolved problem in the art because it is difficult to satisfy both accurate sensing performance and degradability.
Disclosure of Invention
Problems to be solved by the invention
In order to solve the problems in the prior art, the invention provides a degradable transient NO sensor which has a low detection limit, a wide sensing range, a high time resolution and excellent anti-interference performance, is bioabsorbable, flexible and highly stretchable; meanwhile, the invention also provides a preparation method of the degradable transient NO sensor, and the preparation method is simple and feasible and is easy to realize industrial application.
Means for solving the problems
The invention provides a degradable transient NO sensor, which comprises:
a substrate formed from a levorotatory polylactic acid-polytrimethylene carbonate copolymer;
a detection electrode comprising a working electrode, a counter electrode and a reference electrode, the detection electrode being formed on the substrate;
a selectively permeable membrane at least partially covering the surface of the substrate,
wherein, in the L-polylactic acid-poly trimethylene carbonate copolymer, the ratio of L-polylactic acid: the polytrimethylene carbonate ranges from 30:70 to 40: 60.
The degradable transient NO sensor according to the present invention, wherein the working electrode, the counter electrode and the reference electrode are formed of gold (Au).
The degradable transient NO sensor according to the present invention, wherein the permselective membrane is formed of polysugenol.
The degradable transient NO sensor is formed by dripping the L-polylactic acid-poly (trimethylene carbonate) copolymer to a glass template and peeling the glass template, wherein the surface roughness of the glass template is 1800-2200 meshes.
The degradable transient NO sensor provided by the invention is characterized in that the thickness of the substrate is 100-800 μm.
The degradable transient NO sensor is characterized in that the thickness of the detection electrode is 20-50 nm.
The degradable transient NO sensor according to the invention, wherein the thickness of the permselective membrane is 15-30 nm.
The invention also provides a preparation method of the degradable transient NO sensor, which comprises the following steps:
dripping the levorotatory polylactic acid-polytrimethylene carbonate copolymer to a glass template, and stripping to form a substrate;
forming a detection electrode on the substrate through magnetron sputtering;
and (3) immersing the substrate with the detection electrode into an electrochemical polymerization raw material solution, and forming a selective permeability film through electrochemical polymerization to obtain the degradable transient NO sensor.
According to the preparation method of the degradable transient NO sensor, the electrochemical polymerization raw material solution is a eugenol solution, and the concentration of the eugenol solution is 5-15 mM.
ADVANTAGEOUS EFFECTS OF INVENTION
The degradable transient NO sensor has the advantages of low detection limit, wide sensing range, high time resolution and excellent anti-interference performance. Meanwhile, the degradable transient NO sensor disclosed by the invention is bioabsorbable, flexible, highly stretchable and capable of being tightly attached to a detected part, so that the signal loss can be reduced, higher current response can be obtained, and the detection sensitivity can be improved. Meanwhile, the preparation method of the degradable transient NO sensor is simple and feasible, and is easy to realize industrial application, so that accurate and stable real-time NO monitoring can be provided under physiological conditions, and necessary diagnosis and treatment information can be provided for diagnosis and treatment of various diseases.
Drawings
FIG. 1 is a schematic diagram of a degradable transient NO sensor structure;
FIG. 2 is a schematic diagram of a process for preparing a degradable transient NO sensor;
FIG. 3 is a Fourier transform infrared spectroscopy test chart of sensor-I;
FIG. 4 is a graph of substrate response current measurements for sensor-I, sensor-II, and sensor-III;
FIG. 5 is a graph of tamper resistance measurements for sensor-I, sensor-IV, and sensor-V;
FIG. 6 Flexible display of sensor-I, (a) device bending, (b) device stretching;
FIG. 7 is a mechanical property test chart of sensor-I, (a) device bending, (b) device stretching;
FIG. 8 schematic diagram of an in vitro degradation experiment for sensor-I;
FIG. 9 Current response test plots of different concentrations of in vitro NO for sensor-I, (a) 0-100. mu.M, (b) 0-5. mu.M;
FIG. 10 is a linear plot of in vitro NO and response current for different concentrations of sensor-I, (a) 0-100. mu.M, (b) 0-5. mu.M;
FIG. 11 Current response test plot of in vitro NO in the presence of interfering substances for sensor-I: (a) response current curve, (b) quantitative analysis of anti-interference performance;
FIG. 12 stability test chart for sensor-I: (a) linear relation stability, (b) anti-interference performance stability;
FIG. 13 fluorescence photograph of cell proliferation of sensor-I;
FIG. 14 is a schematic representation of the results of the cell viability assay for sensor-I;
FIG. 15 schematic diagram of real-time monitoring of the release behavior of chondrocytes NO from knee joints of SD rats by sensor-I: (a) chondrocyte short-term NO-release behaviour test (b) chondrocyte 24 hour NO-release behaviour test;
FIG. 16 schematic diagram of real-time monitoring of release behavior of sensor-I on isolated liver of SD rat: (a) liver NO release assay (b) liver, kidney and heart NO release assay;
FIG. 17 schematic representation of the real-time monitoring of the release behaviour of the New Zealand white rabbit organ by sensor-I;
FIG. 18 schematic of real-time monitoring of NO concentration after sensor-I pericardium implantation: (a) a testing device, a response current curve (b), an electrocardiogram curve (c), a sensor and a NO concentration change curve measured by a Griess method;
FIG. 19 is a schematic illustration of the implantation of sensor-I in the joint cavity of a mammalian bone;
FIG. 20 schematic illustration of the implantation test of sensor-I in the joint cavity of a mammalian bone: (a) control group NO response current, (b) antibiotic group NO response current, (c) inflammatory factor group NO response current, (d) different group NO concentration changes.
Detailed Description
The invention provides a degradable transient NO sensor, as shown in figure 1, which comprises:
a substrate formed from a levorotatory polylactic acid-polytrimethylene carbonate copolymer (PLLA-PTMC);
a detection electrode comprising a working electrode, a counter electrode and a reference electrode, the detection electrode being formed on the substrate;
a selectively permeable membrane at least partially covering the surface of the substrate.
The substrate is formed by a left-handed polylactic acid-polytrimethylene carbonate copolymer (PLLA-PTMC), and the PLLA-PTMC is a flexible polymer and has high flexibility and stretchability, so that the degradable transient NO sensor can be endowed with good tensile and bending properties. The PLLA-PTMC copolymer may be biodegraded by hydrolysis.
Wherein, in the L-polylactic acid-poly trimethylene carbonate copolymer, the ratio of L-polylactic acid: the polytrimethylene carbonate ranges from 30:70 to 40: 60.
The degradable transient NO sensor according to the present invention, wherein the working electrode, the counter electrode and the reference electrode are formed of gold (Au). Because the bonding force between the poly (L-lactic acid) -poly (trimethylene carbonate) (PLLA-PTMC) and gold (Au) is strong, an intermediate bonding layer in the traditional process is not needed in the preparation process.
The detection mechanism of NO by the sensor is based on a standard three-electrode test system comprising a Working Electrode (WE), a Counter Electrode (CE) and a Reference Electrode (RE), as shown in fig. 1. When an NO oxidation potential is applied between WE and RE, NO is firstly oxidized to form nitrosyl cation on the surface of WE and then reacts with OH-in the solution to generate nitrite, so that an oxidation current is generated between WE and CE, the oxidation current intensity and the NO concentration are in a linear relation in a certain range, and therefore the NO concentration can be quantitatively analyzed by detecting the response current. The specific reaction process is as follows:
NO-e-→NO+ (1)
NO++OH-→HNO2 (2)
the sensing stability and the final electrode transient can be achieved by using ultra-thin gold nano-films, such as patterned ultra-thin gold nano-films, as a Working Electrode (WE), a Counter Electrode (CE) and a Reference Electrode (RE), wherein the degradable transient NO sensor according to the present invention, wherein the thickness of the detection electrode is 20-50 nm.
The degradable transient NO sensor according to the present invention, wherein the permselective membrane is formed of polysugenol.
Eugenol, the main chemical component of clove oil, has been used in the dental field for decades as an analgesic and has excellent biocompatibility. In the present invention, the use of polysugenol to form selectively permeable membranes facilitates the selectivity and specificity of NO sensing through hydrophobic repulsion, ionic interactions and molecular repulsion.
The degradable transient NO sensor is formed by dripping the L-polylactic acid-poly (trimethylene carbonate) copolymer to a glass template and peeling the glass template, wherein the surface roughness of the glass template is 1800-2200 meshes, and the optimal range is 2000 meshes.
In the above, the mesh number is defined as an example, and means the number of holes of a screen in an area of 1 square inch, and the higher the mesh number is, the more the screen holes are, the larger the surface roughness of the glass is.
The glass template with a certain surface roughness is adopted to form the substrate, so that the specific surface area of the substrate can be increased, larger response current can be obtained, and the detection sensitivity can be improved.
The degradable transient NO sensor according to the invention, wherein the thickness of the substrate is 100-500 μm.
The degradable transient NO sensor according to the invention, wherein the thickness of the permselective membrane is 15-30 nm.
Because the thickness of the detection electrode and the thickness of the selective permeability film are both in the nanometer level, the detection electrode and the selective permeability film are combined with the substrate, so that the whole sensor has good stretching and bending performance.
The invention also provides a preparation method of the degradable transient NO sensor, which comprises the following steps as shown in figure 2:
dripping the levorotatory polylactic acid-polytrimethylene carbonate copolymer to a glass template, and stripping to form a substrate;
forming a detection electrode on the substrate through magnetron sputtering; preferably, the sputtering voltage is 350-400V, the working current is 0.03-0.09A, and the sputtering rate is
And (3) immersing the substrate with the detection electrode into an electrochemical polymerization raw material solution, and forming a selective permeability film through electrochemical polymerization to obtain the degradable transient NO sensor.
Preferably, eugenol solutions of different concentrations are dissolved in NaOH solutionThe mixed solution is used as an electrolyte solution, Pt is used as a counter electrode, Ag/AgCl is used as a reference electrode, and the polymerization is carried out by using the CV function of an electrochemical workstation, wherein the scanning voltage range is 0-0.7V, and the scanning speed is 15-25 mV.s-1And 5-20 cycles of scanning. Wherein the concentration of the eugenol solution is 5-15 mM; the concentration of NaOH solution is 0.05-0.15M, and the volume is 40-80 mL.
According to the preparation method of the degradable transient NO sensor, the electrochemical polymerization raw material solution is a eugenol solution, and the concentration of the eugenol solution is 5-15 mM. When the concentration of the eugenol solution is within the range, the working electrode has better current response and anti-interference capability.
Polysugenol is deposited on the surface of the working electrode by means of electrochemical polymerization to minimize the interference of induction by related chemicals in biological systems (e.g., glucose, nitrite, uric acid, etc.).
Examples
Example 1
L-polylactic acid-polytrimethylene carbonate copolymer (30: 70): viscosity of 2.1mpa.s, commercially available from the bio-technology company of the large handle of the Jinan Dai.
Dripping the L-polylactic acid-poly (trimethylene carbonate) copolymer on the surface of the customized 2000-mesh rough glass, and stripping to form a substrate;
the detection electrode is formed by magnetron sputtering of a sputtering target material of gold (Au), and comprises a working electrode, a counter electrode and a reference electrode, wherein the sputtering voltage is 380V, the working current is 0.06A, and the sputtering rate is
The thickness of the detection electrode is about 32nm as shown by a step tester;
immersing a substrate with a detection electrode in an electrochemical polymerization raw material solution in which the concentration of eugenol is 10mM, and forming a selectively permeable film by electrochemical polymerization, wherein eugenol is dissolved in 60ml of 0.1M NaOH, the mixed solution is used as an electrolyte solution, Pt is used as a counter electrode, and Ag is used as an electrolyte solutionthe/AgCl is used as a reference electrode, polymerization is carried out by using the CV function of an electrochemical workstation, the scanning voltage range is 0-0.7V, and the scanning speed is 20 mV.s-1 Scan 10 cycles. The step tester test shows that the thickness of the selective permeability film is about 16nm, and the degradable transient NO sensor is obtained and recorded as sensor-I.
Example 2
The same procedure as in example 1 was followed except that 1000 mesh customized rough glass was used in place of the rough glass in example 1, to obtain a degradable transient NO sensor, denoted sensor-II.
Example 3
The same procedure as in example 1 was repeated except that the glass having a smooth surface was used in place of the rough glass in example 1, to obtain a degradable transient NO sensor, denoted as sensor-III.
Example 4
The concentration of eugenol in the electrochemical polymerization raw material solution is changed to 5mM, and the rest is the same as that in the example 1, so that the degradable transient NO sensor is obtained and is marked as a sensor-IV.
Example 5
The concentration of eugenol in the electrochemical polymerization raw material solution is changed to 15mM, and the rest is the same as that in the example 1, so that the degradable transient NO sensor is obtained and is marked as a sensor-V.
Performance testing
Fourier transform infrared spectroscopy testing
The semi-finished product without the formation of the selectively permeable film and the sensor-I were subjected to Fourier transform infrared spectroscopy using a German Nax corporation tester (X70), and the results are shown in FIG. 3.
As can be seen from fig. 3, the electrode surface after plating the selectively permeable film exhibits aliphatic and aromatic functional groups containing different oxygen-containing groups, indicating that eugenol has been successfully polymerized on the electrode surface.
Response current testing
A traditional three-electrode electrochemical system is adopted, and a gold nano-film is used as a working electrode, a counter electrode and a reference electrode. The test was performed in a PBS solution at 37 ℃ by a chronoamperometry (chronoamperometry) method in an electrochemical workstation, and different NO concentration solutions and response currents corresponding thereto were obtained by continuously dropping different amounts of the NO standard solution in the PBS solution, and the results are shown in fig. 4. The substrates in examples 1 and 2 had higher corresponding currents compared to substrates made using smooth-surfaced glass.
Anti-interference test
Glucose (GLU), sodium Nitrite (Nitrite), sodium Nitrate (Nitrate), Ascorbic Acid (AA) and Uric Acid (UA) are used as comparison substances, anti-interference tests are carried out on a sensor-I, a sensor-IV and a sensor-V, the selectivity and the specificity of the NO sensor are researched by using common interference substances in a biological system, such as Glucose (Gluose), sodium Nitrite (Nitrite), sodium Nitrate (Nitrate), sodium Nitrate (NaNO3), Ascorbic Acid (AA) and Uric Acid (UA), a traditional three-electrode electrochemical system is adopted, and a gold NaNO film is used as a working electrode, a counter electrode and a reference electrode. The test was performed in a PBS solution at 37 ℃ by a chronoamperometry (chronoamperometry) method in an electrochemical workstation. In the selectivity test, NO and interferents (glucose, sodium nitrite, sodium nitrate, ascorbic acid and uric acid) were added in sequence to PBS and the response current was recorded. The concentrations of NO and the interfering agent were 0.1mM and 0.5mM, respectively, and the interference resistance of the sensor was evaluated by determining the ratio of the response current to the interfering chemical to the response current to NO, and the results are shown in fig. 5.
As shown in FIG. 5, the results show that the working electrode has better current response and anti-interference capability when the concentration of eugenol in the solution is 5-15 mM, especially when the concentration of the plating solution is 10 mM.
Mechanical Property test
As shown in FIG. 6, sensor-I has good bending and stretching capabilities.
Tensile tests were performed 1000 times at 90 degree angle using an electronic universal materials tester (model WDW3020 available from new company, vinpockets) at 20% and 50% strain. As shown in fig. 7, the sensor electrode resistance was substantially unchanged after 1000 tensile/bending tests.
In vitro degradation test
In vitro degradation testing of sensor-I was performed in phosphate buffered saline (PBS solution) at 65 ℃ and the results are shown in FIG. 8.
In the accelerated degradation process of the sensor-I under the condition of in vitro temperature rise, the PLLA-PTMC substrate material and the polysugenol film are gradually hydrolyzed along with the extension of the soaking time, the Au electrode attached to the PLLA-PTMC substrate material and the polysugenol film are gradually cracked into nano particles along with the degradation of the substrate, and the sensor-I is completely degraded after 15 weeks.
In vitro NO assay
The in vitro NO test is the same as the response current test described previously.
FIG. 9 shows the relationship between NO concentration and response current for the in vitro NO test of sensor-I. As can be seen from fig. 9, the response current gradually increases with the increase of the NO concentration, the response time is less than 350ms, and the fast response is important for the real-time detection of NO. After each concentration tested in the graph, the current gradually decreased due to the slower diffusion rate of NO in PBS solution.
FIG. 10 shows the relationship between NO concentration and response current of sensor-I, and it can be seen from FIG. 10 that the relationship between NO concentration and response current is divided into two linear intervals of 0-5 μ M and 5-100 μ M, in which the detection sensitivities of the sensor to NO are 5.29nA/μ M and 4.17nA/μ M, respectively, and further tests show that the detection limit of the sensor is 3.97nM when the signal-to-noise ratio is 3.
In vitro NO assay in the Presence of interfering substances
The procedure is the same as that described for the in vitro NO test, except that interfering substances are added.
During in vivo testing, NO often coexists with other substances, and therefore, the selective specific response of the sensor to NO pairs is particularly important. The response current of the electrode of the sensor-I is tested when some common interfering substances exist in the body together with NO based on the sensor-I, the test result is shown in FIG. 11a, and the quantitative calculation result is shown in FIG. 11 b. As can be seen from fig. 11, in an environment where the concentration of the interfering substance is 5 times that of NO, the sensor has good anti-interference effects on Glucose (GLU), sodium Nitrite (Nitrite), sodium Nitrate (Nitrite), Ascorbic Acid (AA), and Uric Acid (UA), and the maximum interference current is less than 15% of the NO response current value.
Stability test
And the stability test is that the response current test and the anti-interference test are repeated every other day within 14 days. The specific test method is the same as the method described above.
In addition to the linear relationship and anti-interference characteristics, operational stability is another important property of the NO sensor, and is directly related to the in vitro and in vivo operational life of the NO sensor. FIG. 12 shows the stability test curves of sensor-I linearity (FIG. 12a) and interference rejection characteristics (FIG. 12b) over a two-week period. As can be seen from fig. 12a, in 7 bodies, the slope of the linear relationship curve increased slightly, but the change was small, the slope increased significantly after 7 days, and the current value reached about 1.5 times that of day 1 on day 14; as can be seen from FIG. 12b, the anti-interference effect of the sensor is kept better within 7 days, and after 7 days, the response current of all interfering substances except glucose rises obviously. The increase in the slope of the linear relationship and the progressive deterioration of the interference resistance is mainly due to the gradual thinning and dissolution in the permselective film poly-eugenol PBS solution.
Biocompatibility testing
To investigate the biocompatibility of the sensor, the whole device was co-cultured with human aortic vascular smooth muscle cells (HA-VSMCs) for 5 days, and cell proliferation fluorescence images and corresponding optical microscope images were observed. The experimental group is human aortic vascular smooth muscle cells cultured together with the device, and the control group is human aortic vascular smooth muscle cells without the device and with the rest culture conditions consistent with those of the experimental group.
For the implanted device in vivo, the good biocompatibility can effectively reduce rejection reaction and inflammatory reaction, reduce pain of patients and promote wound healing, and is an important guarantee for normal work and safe implantation of the device. Human primary vascular smooth muscle cells and the device are co-cultured, and the biocompatibility of the implanted device can be effectively and scientifically judged through cell proliferation conditions and cell activity tests. FIG. 13 shows fluorescence pictures of cell co-culture of sensor-I, and it can be seen that cell proliferation is significant with the number of days of culture, and the proliferation is not significantly different from that of the control group. FIG. 14 shows the results of cell viability assays for sensor-I, which were similar in cell co-cultured cells to the control group. The two groups of test results show that the NO sensor has good biocompatibility.
In vitro cell and tissue NO real-time testing
Real-time monitoring of SD rat knee joint chondrocyte NO release behavior
Chondrocytes were seeded with interleukin (IL-1. beta.) in Phosphate Buffered Saline (PBS) at 37 deg.C, the NO sensor was immersed around the chondrocytes, and the response current of the sensor was observed and recorded by adding the NO reaction substrates L-arginine (L-Arg) and NO enzyme blocker (L-NAME). FIG. 15a shows the real-time monitoring of the NO release behavior of knee chondrocytes from SD rats by sensor-I. The increase in current due to cellular NO release was further demonstrated by an increase in response current following the addition of the NO reaction substrate L-arginine (L-Arg) at 60 seconds in the presence of inflammatory factor (IL-1. beta.) stimulation, followed by the addition of NO enzyme blocker (L-NAME) at 180 seconds, with the current then decreasing to the background. On this basis, a long-term real-time monitoring of the behavior of chondrocyte NO release was continued, as shown in fig. 15 b. As can be seen from FIG. 15b, the response current rises significantly after the addition of L-Arg. Meanwhile, the NO release amount of the chondrocytes is synchronously monitored by utilizing a clinically traditional Griess method. It can be seen that the variation trend of the test result of the Griess method is similar to that of the test result of the sensor, and the effectiveness and the accuracy of the sensor for NO detection are proved. But NO is the oxidation product of NO as tested by Griess method2-It is the cumulative dose of NO released and therefore its absolute value of NO measurement is higher than the sensor-I measurement.
Real-time monitoring of release behavior of isolated liver of SD rat
Isolated liver from Sprague-Dawley (SD) rats was placed in PBS solution, NO sensor was placed near the liver, and sensor response current was observed and recorded by addition of the NO reaction substrates L-arginine (L-Arg) and NO enzyme blocker (L-NAME). Figure 16a shows the application of sensor-I to real-time monitoring of the release behavior of SD rat ex vivo liver. As can be seen from FIG. 16a, the response current gradually increased after the addition of L-Arg and then gradually decreased with the increase in the concentration of added L-NAME, indicating that the sensor can effectively detect the liver NO release signal. Subsequently, the sensor is also successfully applied to the detection of NO release from heart and kidney of SD rat and brain, heart, liver and kidney of New Zealand white rabbit, as shown in FIG. 16b, and the test result shows that the sensor-I has practical effectiveness and universality for the release of NO from organs in vivo.
As shown in fig. 17, sensor-I was applied to real-time monitoring of the release behavior of new zealand white rabbit organs, showing substantial effectiveness and universality.
In vivo implanted NO real time testing
Real-time monitoring of NO concentration after pericardium implantation
Nitroglycerin (hereinafter referred to as NTG) can be decomposed to release NO in the living body, and thus is a commonly used clinical drug for relieving and treating chronic heart failure and angina pectoris. However, the dosage of nitroglycerin suitable for biological individuals is very different, and excessive nitroglycerin may cause reflex tachycardia, so that the real-time monitoring of the concentration of NO released by the organism in the heart after NTG input is very important. The test rabbits were continuously fed NTG at a constant rate through the ear vein, and the NO concentration was monitored in real time by inserting sensor-I between the pericardium and heart of New Zealand white rabbits, in the manner shown in FIG. 18 a. As can be seen from the graph of fig. 18a, NO increases in the pericardial fluid in response to the NTG input, and the response current continues to rise as the amount of drug is increased, indicating that NO is continuously generated and the concentration continues to increase in the heart. Meanwhile, since the sensor-I has a very thin thickness and good flexibility and can be closely attached to the surface of the heart, the sensor-I can simultaneously record an electrocardiogram curve in addition to detecting NO signals, as shown in FIG. 18b, which can provide an important basis for timely adjusting the clinical dosage of NTG. In addition, similar to the cellular assay, the NO detection was performed with sensor-I, along with the Griess method, and the results are shown in FIG. 18 c. As can be seen from FIG. 18c, the changes in NO concentration measured by the two methods are consistent, indicating that sensor-I can effectively detect the change in NO concentration in the pericardium after drug infusion.
Implantation test of a mammalian osteoarticular cavity
As shown in fig. 19, a sensor-I is implanted in the bone joint cavity of a mammal rabbit, wireless transmission of data is realized through a wireless control circuit, and the experimental rabbit is divided into a control group, an antibiotic group and an inflammatory factor group. The results of continuous detection of NO concentrations in joint cavities of different groups of experimental rabbits are shown in fig. 20, and it can be seen from fig. 20 that NO concentrations in joint cavities of control group and antibiotic group rabbits are very low and have NO obvious difference, while NO response current in joint cavities of inflammation factor group rabbits is obviously increased, and the corresponding NO concentrations are also obviously increased. The sensor-I can effectively detect and discriminate NO concentration in the joint cavity under the condition of complete implantation, and shows good clinical application prospect.
Claims (9)
1. A degradable transient NO sensor, comprising:
a substrate formed from a levorotatory polylactic acid-polytrimethylene carbonate copolymer;
a detection electrode comprising a working electrode, a counter electrode and a reference electrode, the detection electrode being formed on the substrate;
a selectively permeable membrane at least partially covering the surface of the substrate,
wherein, in the L-polylactic acid-poly trimethylene carbonate copolymer, the ratio of L-polylactic acid: the polytrimethylene carbonate ranges from 30:70 to 40: 60.
2. The degradable transient NO sensor of claim 1, wherein the working, counter and reference electrodes are formed of gold (Au).
3. The degradable transient NO sensor of claim 1 or 2, wherein the permselective membrane is formed of polysugenol.
4. The degradable transient NO sensor of any one of claims 1 to 3, wherein the substrate is formed by dispensing the L-PLA-PTMC copolymer onto a glass template and peeling off the glass template, wherein the glass template has a surface roughness of 1800-2200 mesh.
5. The degradable transient NO sensor of any of claims 1 to 4, wherein the thickness of the substrate is 100-800 μm.
6. The degradable transient NO sensor of any one of claims 1 to 5, wherein the thickness of the detection electrode is 20-50 nm.
7. The degradable transient NO sensor of any of claims 1 to 6, wherein the selectively permeable membrane has a thickness of 15-30 nm.
8. A method of making a degradable transient NO sensor according to any of claims 1-7, comprising the steps of:
dripping the levorotatory polylactic acid-polytrimethylene carbonate copolymer to a glass template, and stripping to form a substrate;
forming a detection electrode on the substrate through magnetron sputtering;
and (3) immersing the substrate with the detection electrode into an electrochemical polymerization raw material solution, and forming a selective permeability film through electrochemical polymerization to obtain the degradable transient NO sensor.
9. The method for preparing the degradable transient NO sensor, according to claim 8, wherein the electrochemical polymerization raw material solution is a eugenol solution, and the concentration of the eugenol solution is 5-15 mM.
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| CN115248241B (en) * | 2022-07-28 | 2024-08-20 | 清华大学 | Flexible NO sensor and preparation method thereof |
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