Intelligent hydrogel patch for detecting penicilloic acid and preparation method thereof

Abstract

The invention relates to the technical field of biological detection, and provides an intelligent hydrogel patch for detecting penicilloic acid and a preparation method thereof. The patch is a three-layer intelligent hydrogel patch constructed by combining electrostatic spinning, coating and hydrogel technologies, and convenient and rapid detection of penicilloic acid is realized. The primary filtering layer is a heparinized cellulose nanofiber membrane and is used for intercepting blood cells and inhibiting blood coagulation, the secondary fine screening layer is a polydopamine coating charge selection interface and is used for allowing small molecule metabolites to pass through and reducing protein adsorption, the detection signal layer is penicilloic acid imprinted temperature-sensitive hydrogel, acrylic acid is used for providing hydrogen bond recognition sites, and the detection signal layer is used for detecting the content of the penicilloic acid imprinted temperature-sensitive hydrogel. The NIPAM is subjected to specific phase change under the trigger of the body temperature of 37 DEG C, that is, the penicilloic acid inhibits contraction and keeps transparent when being combined, and is turbid when not combined. The detection limit of the technology is as low as 0.1 mu g / mL, the specificity is high, the detection efficiency is high, the false positive rate is low, the biocompatibility is excellent, and an innovative solution is provided for home first aid instant penicillin allergy pre-detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to an intelligent hydrogel patch for detecting penicillin thiazolyl acid and a preparation method thereof. BACKGROUND

[0002] Penicillin, as a highly efficient, safe and economical β-lactam antibiotic, is one of the first choices for preventing and treating various bacterial infections (such as streptococcal infection, syphilis, pneumonia, etc.) after surgery. However, in clinical practice, as high as about 10% of patients report "penicillin allergy history", which constitutes a major obstacle to the use of the best first-line treatment drug. The key problem is that more than 90% of the "allergy history" labels are inaccurate, often due to non-specific skin rash in childhood, misdiagnosis, information transmission error or vague recall, etc. This widespread misjudgment seriously limits the application of penicillin, leading to serious clinical and public health consequences: patients are forced to use clinically selected broad-spectrum, less effective alternative drugs or more side effects (such as increased Clostridium difficile infection, risk of nephrotoxicity) and more expensive broad-spectrum alternative antibiotics (such as fluoroquinolones, clindamycin, vancomycin); more seriously, this significantly aggravates the overuse of broad-spectrum antibiotics, which is a key factor driving the emergence and spread of multi-drug resistant bacteria (such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), seriously threatening public health safety and increasing medical burden. In addition, the core mechanism of penicillin allergy is that penicillin metabolites such as penicillin thiazolyl acid (PTA), penicilloyl acid and penicillamine produced in the body, covalently bind to human proteins to form complete antigens (mainly penicilloyl-protein complex), triggering the body to produce specific IgE antibodies, leading to anaphylactic reactions when re-exposed. However, the current technology lacks specific recognition ability for the key metabolite PTA, which seriously limits the rational application of β-lactam antibiotics, further aggravating the crisis of bacterial drug resistance.

[0003] Currently, the penicillin allergy detection techniques used in China mainly include three types: 1) serum specific IgE detection, which mainly uses immunoassay (such as ImmunoCAP) to detect the anti-penicillin IgE antibody in serum. The detection process takes 2-4 hours and needs to rely on professional laboratory equipment. The key is that it cannot distinguish the antibodies against penicillin raw drugs and the main antigenic determinant (such as penicilloic acid), which significantly increases the risk of false positives. 2) Penicillin skin sensitivity test, which uses unstable penicillin G sodium salt solution as the antigen. The false positive rate is as high as 10-15%, and there is a risk of inducing severe allergic reactions, which needs to be operated under emergency conditions. 3) Graded drug challenge test, which is the gold standard for excluding allergies, but may cause fatal reactions such as anaphylactic shock. It is only suitable for low-risk patients and needs to be implemented under strict monitoring. In addition, among the main metabolic products of penicillin in the body, about 80% is PTA. Clinical serological studies have further confirmed that the core sensitizing antigen of penicillin allergy is the complex (PTA-protein) formed by the covalent combination of PTA and human proteins. However, the existing technology cannot directly detect such complexes simply and quickly, mainly because of the low structural stability, various forms (such as differences in binding sites and protein types), and dependence on complex serological analysis methods. Based on the above challenges, by directly capturing free PTA as a biomarker for penicillin allergy, the technical bottleneck of complex detection can be avoided, which is a more feasible alternative. SUMMARY

[0004] The purpose of the present application is to provide an intelligent hydrogel patch for detecting penicilloic acid to solve the above technical problems.

[0005] The patch of the present application combines electrospinning, coating and hydrogel technology to construct a functional hydrogel patch, which is stacked from top to bottom with a primary filtration layer, a secondary fine screening layer and a detection signal layer.

[0006] Specifically, the primary filtration layer is a heparinized cellulose nanofiber membrane with a pore size of 3 μm and a surface zeta potential of ≤-30.65 mV for trapping blood cells and inhibiting blood clotting.

[0007] Specifically, the secondary fine screening layer is a polydopamine (PDA) coated charge selection interface with a pore size of 50 nm and a surface zeta potential of ≤-20.42 mV (pH 7.4) for reducing protein adsorption.

[0008] Specifically, the detection signal layer is a penicilloic acid imprinted temperature-sensitive hydrogel, which forms specific recognition cavities with penicilloic acid as the template molecule for capturing penicilloic acid; the temperature-sensitive hydrogel has a pore size of 20 nm and undergoes phase transition at 37°C body temperature to output a light transmittance change signal.

[0009] The mechanism of the smart hydrogel patch for detecting penicillin thiazole acid is as follows:

[0010] (1) In use, the blood is dropped into the patch, and the detection signal layer is tightly attached to the skin. At this time, the blood sample first enters from the center of the heparin cellulose fiber membrane of the primary filter layer, and after preliminary filtration, it flows through the PDA anti-fouling coating of the secondary fine screening layer, and is further reduced by the size screening and charge repulsion effect to reduce the influence of interfering substances, and finally reaches the core penicillin thiazole acid imprinted temperature-sensitive hydrogel detection signal layer.

[0011] (2) The molecularly imprinted hydrogel is constructed by copolymerization of acrylamide (AAm), acrylic acid (AAc), N-isopropyl acrylamide (NIPAM) and PTA, forming a penicillin thiazole acid imprinted temperature-sensitive hydrogel. Under the triggering action of body temperature (37℃), the penicillin thiazole acid molecular imprinting cavity can specifically capture the target penicillin thiazole acid through hydrogen bonding. Specifically, when the blood sample containing penicillin thiazole acid enters the hydrogel detection signal layer, the target is captured by the imprinting cavity, which inhibits the phase change shrinkage of the NIPAM component, so that the hydrogel remains soft and transparent; on the contrary, if there is no target binding, the body temperature will normally trigger the phase change shrinkage of NIPAM, causing the hydrogel to appear hard and turbid. The light transmittance of the captured penicillin thiazole acid is higher than that when the penicillin thiazole acid is not captured, and this significant difference in transparency realizes rapid and visual interpretation of the key sensitizing metabolite PTA of penicillin allergy.

[0012] Currently, there is no research report on the technology of detecting penicillin allergy by using molecularly imprinted hydrogel and triggering turbidity change by body temperature. The patch can directly analyze the whole blood sample without antibody labeling, and can complete the target capture and signal output of PTA within 10 minutes, providing an efficient and portable allergy detection solution for home emergency scenarios. Compared with traditional skin tests, the patch significantly reduces the false positive rate, and does not require skin puncture and professional interpretation, and has significant clinical transformation potential.

[0013] The application also provides a preparation method of the smart hydrogel patch for detecting penicillin thiazole acid, which specifically comprises the following steps:

[0014] (1) Heparinized cellulose nanofiber membrane preparation: 8wt% cellulose acetate (CA) is dissolved in a mixed solvent of acetone and N,N-dimethylacetamide, then heated and stirred at 50℃ water bath for 6h, and after cooling to 37℃, 2wt% heparin sodium is added and stirred for 2h to obtain spinning solution 1. The heparinized cellulose nanofiber membrane is prepared by using the spinning solution 1 as raw material for single-axis electrospinning. The electrospinning parameters are as follows: spinning speed is 0.003mm / s, spinning needle is 20G, voltage is 15kV, receiving distance is 13cm, temperature is 25℃, and relative humidity is 60%.

[0015] (2) Preparation of polydopamine coating charge selective interface: Dopamine is fully dissolved in 100 mM Tris buffer to obtain a deposition solution 1 with a concentration of 2 mg / mL, and the surface of the heparinized cellulose nanofiber membrane obtained in step (1) is subjected to plasma treatment for 1 min (100 W) by passing oxygen; and the activated heparinized cellulose nanofiber membrane is immersed in the deposition solution 1 at 25°C in the dark for 2 hours;

[0016] (3) Preparation of penicilloic acid imprinted temperature-sensitive hydrogel: 1.2 M N,N-methylene bisacrylamide (AAm), 0.4 M acrylic acid (AAc), 2.5 M N-isopropyl acrylamide (NIPAM), 0.06 M N,N'-methylene bisacrylamide (MBA), 0.2 mM PTA and a certain amount of photoinitiator Irgacure 2959 are mixed and stirred in the dark for 10 min to prepare a prepolymer solution, then the prepolymer solution is dropped on the surface of the polydopamine coating, a polyethylene terephthalate (PET) release film is used to control the thickness, and after 365 nm UV light treatment for 15 min, it is immersed in a mixture of methanol and acetic acid, oscillated at 30°C for 24 hours, completely removed PTA, washed with PBS at pH 7.4 to neutral, to obtain a smart hydrogel patch based on detection of penicillin metabolites;

[0017] Further, the volume ratio of acetone and N,N-dimethylacetamide in the mixed solvent in step (1) is 2:1.

[0018] Further, the pH of the Tris buffer in step (2) is 7.4.

[0019] Further, the concentration of the photoinitiator Irgacure 2959 in the prepolymer solution in step (3) is 0.15 wt%.

[0020] Further, the thickness of the hydrogel using the PET release film in step (3) is 200 μm.

[0021] Further, the volume ratio of methanol and acetic acid in the mixture in step (3) is 9:1.

[0022] Further, the patch is sealed and stored in the dark after freeze-drying, and is activated by immersion in 37°C physiological saline for 5 minutes before use.

[0023] The advantages of the present application are:

[0024] 1.The present application first combines heparinized cellulose fiber membrane and PDA coating for filtering PTA in blood, and constructs an intelligent hydrogel patch based on the detection of penicillin metabolites by combining PTA molecular imprinting temperature-sensitive hydrogel.The hydrogel patch of the present application has the performance of pre-detecting penicillin allergy, and directly detects penicillin metabolites PTA from whole blood without antibodies, which reduces the false positive rate and is fast compared with the existing technology of skin test, and is convenient for home first aid.

[0025] 2.The intelligent hydrogel patch of the present application uses heparinized cellulose fiber membrane and PDA coating two-layer material to filter whole blood, which is different from the serum detection and heparin / PDA mixed coating of traditional technology, and can quickly filter whole blood.

[0026] 3.The intelligent hydrogel patch of the present application uses body temperature as a detection energy source, and uses the temperature-sensitive phase change mechanism of NIPAM to specifically detect penicilloic acid, which is used for pre-detecting penicillin allergy. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a schematic diagram of the intelligent hydrogel patch for detecting penicilloic acid of the present application.

[0028] Figure 2 is the characterization of the lower critical solution temperature of the pre-polymer solution of the penicilloic acid imprinting temperature-sensitive hydrogel in the present application

[0029] Figure 3 (a) is the pore size characterization of the hydrogel patch of the present application; Figure 3 (b) is the Zeta potential characterization of the hydrogel patch of the present application; Figure 3 (c) is the protein adhesion characterization of the hydrogel patch of the present application.

[0030] Figure 4 (a) is the transmittance of the hydrogel patch of the present application after detection; Figure 4 (b) is the mechanical property of the hydrogel patch of the present application after detection.

[0031] Figure 5 is the detection performance of the hydrogel patch of the present application.

[0032] Figure 6 is the specificity performance of the hydrogel patch of the present application: Figure 6 (a) is the false positive rate; Figure 6 (b) is the cross-reactivity rate of different antibiotics.

[0033] Figure 7 is the biocompatibility of the hydrogel patch of the present application. DETAILED DESCRIPTION

[0034] The technical solutions described in the present application will be clearly and completely described below in combination with the drawings in the embodiments of the present application. Obviously, the embodiments described in the present specification are only a part of the feasible technical solutions of the present application, and other embodiments obtained by those skilled in the art on the basis of the embodiments of the present application without any creative labor should be regarded as belonging to the scope of protection of the present application.

[0035] Example 1: Preparation of an intelligent hydrogel patch for detecting penicilloic acid

[0036] (1) Preparation of heparinized cellulose nanofiber membrane: 8wt% CA was dissolved in a mixed solvent of acetone and N,N-dimethylacetamide (volume ratio 2:1), then heated and stirred in a 50℃ water bath for 6h, after cooling to 37℃, 2wt% heparin sodium was added and stirred for 2h to obtain spinning solution 1, using spinning solution 1 as raw material for uniaxial electrospinning, heparinized cellulose nanofiber membrane was obtained, the electrospinning parameters were set as follows: spinning speed was 0.003mm / s, spinning needle was 20G, voltage was 15kV, receiving distance was 13cm, temperature was 25℃, and relative humidity was 60%;

[0037] (2) Preparation of polydopamine coating charge selection interface: dopamine was fully dissolved in Tris buffer (pH 7.4, 100mM) to obtain a deposition solution 1 with a concentration of 2mg / mL, the surface of the heparinized cellulose nanofiber membrane obtained in step (1) was subjected to plasma treatment for 1min (100W) by passing oxygen, and the activated heparinized cellulose nanofiber membrane was immersed in the deposition solution 1 and treated at 25℃ in the dark for 2h;

[0038] (3) Preparation of penicilloic acid imprinted temperature-sensitive hydrogel: 1.2M AAm, 0.4M AAc, 2.5M NIPAM, 0.06M MBA, 0.15wt% photoinitiator Irgacure 2959 and 0.2mM PTA were mixed under light shielding to prepare a prepolymer solution, the prepolymer solution was dropped on the surface of the PDA coating, a PET release film was covered to control the thickness to 200μm, 365nm UV light was used for treatment for 15min, then the sample was immersed in a mixture of methanol and acetic acid (volume ratio 9:1), oscillated at 30℃ for 24h, PTA was completely removed, the sample was washed with PBS at pH 7.4 until neutral, an intelligent hydrogel patch for detecting penicillin metabolites was obtained, and after freeze-drying, the sample was sealed and stored in the dark, and before use, the sample needed to be rehydrated and activated by immersing it in 37℃ physiological saline for 5min.

[0039] An intelligent hydrogel patch for detecting penicilloic acid is prepared in this embodiment, as shown in Figure 1 Fig. 1, which is sequentially stacked from top to bottom with a primary filter layer, a secondary fine screening layer and a detection signal layer, and a blood sample is added at the center position of the primary filter layer.

[0040] Preparation of the smart hydrogel patch without heparinization treatment

[0041] This comparative example is compared with Example 1, except that the cellulose nanofiber membrane is not subjected to heparinization treatment.

[0042] Preparation of the smart hydrogel patch without PDA coating

[0043] This comparative example is compared with Example 1, except that the heparinized cellulose nanofiber membrane is not subjected to PDA coating treatment.

[0044] Preparation of the smart hydrogel patch without molecular imprinting

[0045] This comparative example is compared with Example 1, except that the pre-polymer solution for hydrogel synthesis does not add PTA, and the smart hydrogel patch without molecular imprinting is obtained after 15 minutes of 365 nm UV light treatment.

[0046] Preparation of the smart hydrogel patch without heparin membrane / PDA coating

[0047] This comparative example is compared with Example 1, except that there is no heparinized cellulose nanofiber membrane layer and PDA coating treatment, and only a penicilloyl acid imprinted temperature-sensitive hydrogel layer.

[0048] Preparation of the smart hydrogel patch with PDA / heparin mixed coating

[0049] This comparative example is compared with Example 1, except that the heparinized cellulose fiber membrane and PDA coating are replaced by one-step heparin / PDA mixed coating treatment on the cellulose fiber membrane.

[0050] (1) Preparation of cellulose nanofiber membrane: 8wt% CA was dissolved in a mixed solvent of acetone and N,N-dimethylacetamide with a volume ratio of 2:1, heated and stirred in a 50°C water bath for 6h to obtain spinning solution 1. Cellulose nanofiber membrane was prepared using spinning solution 1 as raw material by uniaxial electrospinning. The electrospinning parameters were set as follows: spinning speed was 0.003mm / s, spinning needle was 20G, voltage was 15kV, receiving distance was 13cm, temperature was 25°C, and relative humidity was 60%;

[0051] (2) Preparation of heparin / PDA coating: Dopamine and heparin (mass ratio of 1:1) were dissolved in Tris buffer (pH 7.4, 100 mM) to obtain a deposition solution 1 with a concentration of 2 mg / mL. The cellulose nanofiber membrane obtained in step (1) was subjected to plasma treatment for 1 min (100 W) by passing oxygen through the surface of the membrane. The activated cellulose nanofiber membrane was immersed in the deposition solution 1 at 25°C for 2 hours in the dark.

[0052] Test Example 1: Characterization of the minimum critical solution temperature of the pre-polymer solution of the penicilloyl acid imprinted temperature-sensitive hydrogel prepared in Example 1

[0053] The pre-polymer solution at different temperatures (20-50°C) was placed in a sample cell for measurement by UV spectrophotometry, and the absorbance at a wavelength of 500 nm was measured. As shown in Figure 2 , the absorbance of the pre-polymer solution increased significantly at 33°C as the inflection point. In addition, the absorbance reached 1.58 a.u. at 37°C, and the physical state of the pre-polymer solution was also significantly turbid compared to that at 25°C, because this was the lower critical solution temperature of the temperature-sensitive polymer NIPAM. Therefore, the minimum critical solution temperature of the pre-polymer solution of the penicilloyl acid imprinted temperature-sensitive hydrogel prepared in Example 1 was 33°C, i.e., the hydrogel patch prepared in Example 1 could undergo phase transition at a body temperature of 37°C.

[0054] Test Example 2: Characterization of the pore size, Zeta potential, and protein adhesion of the hydrogel patch prepared in Example 1

[0055] The microstructure of the hydrogel patch was characterized by field emission scanning electron microscopy (SEM). After the material was freeze-dried for 24 h, the sample was fixed with conductive glue, and the surface and cross-section were observed, and the pore size and thickness were counted. As shown in Figure 3 (a), the pore sizes of the heparinized cellulose nanofiber membrane, PDA coating, and penicilloyl acid imprinted temperature-sensitive hydrogel were 3000 nm, 50 nm, and 20 nm, respectively.

[0056] The Zeta potential of the fiber membrane and PDA coating in the hydrogel patch was evaluated by a Zeta potential instrument. The coating of the hydrogel patch was fixed outward on the electrode holder of a flat sample cell, and the instrument detected the movement of ions in the electrolyte under the action of an electric field by laser irradiation of the coating surface. As shown in Figure 3 (b), the Zeta potentials of the fiber membrane and PDA coating were -30.65 mV and -20.42 mV, respectively, because the carboxyl groups in PDA dominated the negative charge, making the surface negatively charged, which could repel negatively charged proteins and thus achieve anti-protein contamination.

[0057] Protein adhesion evaluation was performed on the fiber membrane and PDA coating surface of the hydrogel patch. The hydrogel patch was placed in 250 μL of fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) solution (2 mg / mL) and incubated in the dark for 2 hours, and the sample surface was repeatedly washed with PBS solution. The sample was observed by laser confocal microscopy and the fluorescence image calculation was recorded. The results are shown in Figure 3 As shown in (c), the fluorescence intensity of the fiber membrane surface reached 74.3 a.u., and the fluorescence intensity of the PDA coating surface was 14.8 a.u. This is because the fiber membrane has a certain hydrophilicity and can adsorb the BSA-FITC solution, while the PDA coating is hydrophobic and has a negative charge on the surface, making it difficult to adsorb the BSA-FITC solution. Therefore, the PDA coating in the hydrogel patch prepared in Example 1 has the ability to reduce protein adsorption.

[0058] Test Example 3: Transmittance and mechanical property characterization of the hydrogel patches prepared in Example 1 and Comparative Examples 1-5

[0059] After adding 50 μL of blood samples containing / without PTA to the center of the primary filter layer of the hydrogel patch, the patch was immediately turned over to make the hydrogel surface of the detection signal layer adhere to the skin, and the body temperature triggered the temperature-sensitive phase change. After the signal was stable, the naked eye could determine whether the hydrogel was transparent or turbid.

[0060] (1) Transmittance characterization: After the detection was completed, the transmittance of the hydrogel patch was tested by ultraviolet spectrophotometry. The hydrogel patch was placed in a sample cell for measurement, and the measurement wavelength range was 380-800 nm. The results are shown in Figure 4 As shown in (a), in the PTA-containing blood sample (positive) group, the transmittance of Example 1 reached 92%, because the blood was filtered and PTA was specifically screened into the detection signal layer to inhibit the NIPAM phase change, and the sample was in a transparent state. The transmittance of Comparative Example 1 and Comparative Example 2 was 33% and 47%, respectively, because the lack of heparin for anticoagulation or the lack of PDA for small molecule screening resulted in the partial absence of blood filtration, causing impurities to enter the detection signal layer and affect the NIPAM phase change, resulting in a slightly turbid state. The transmittance of Comparative Example 3 reached 61%, because the hydrogel in the detection signal layer could not specifically recognize PTA, and the sample entered the region and reacted to form a transparent state. The transmittance of Comparative Example 4 reached 51%, because the patch did not have a primary filter layer and a secondary fine screening layer for blood pretreatment, and the whole blood entered the detection signal layer, making it difficult to screen PTA for reaction, and the unfiltered whole blood reduced the transparency of the hydrogel. The transmittance of Comparative Example 5 was 60%, because although the patch pretreated the blood, it was only a layer of heparin / PDA mixed coating, which was difficult to gradually filter the blood to screen out PTA, resulting in a moderate transparency.

[0061] The transmittance of the blood sample (negative) group without PTA of Example 1, Comparative Example 1 and Comparative Example 2 was 20%, 17% and 26%, respectively, because the blood was filtered and no PTA in the blood reacted with the detection signal layer to form turbidity; the transmittance of Comparative Example 3 was 70%, because the detection signal layer could not specifically recognize PTA and the sample entered the hydrogel to form transparency; the transmittance of Comparative Examples 4 and 5 was 46% and 55%, respectively, because the blood sample lacked a blood pretreatment zone or was not sufficiently filtered, which reduced the transparency of the hydrogel. Therefore, the above results show that the hydrogel patch of Example 1 can accurately recognize PTA and visually express it in the form of transparency (transparent / turbid) after filtering the blood sample with / without PTA.

[0062] (2) Mechanical property characterization: The hydrogel patch after detection was tested for mechanical properties using a rotational rheometer at an oscillation frequency of 50 rad / s at 25°C. The results are shown in FIG. 8. Figure 4 As shown in FIG. 8(b), the storage modulus of Example 1 in the positive group was 1.2 kPa, while that in the negative group was 25 kPa, because PTA was filtered into the hydrogel to make it soft, and PTA did not enter the hydrogel to react in a stiff state; the storage modulus of Comparative Example 3 in the positive group was 5.5 kPa, while that in the negative group was 5.8 kPa, because the detection signal layer of the hydrogel could not specifically recognize PTA, and the sample reacted in the detection signal layer to form a medium soft state; the storage modulus of Comparative Example 4 in the positive group was 7.8 kPa, while that in the negative group was 9.5 kPa, because the patch had no blood pretreatment zone, and whole blood entered the detection signal layer, making it difficult to filter PTA to react, and the hydrogel normally underwent phase transition to form a slightly stiff state; the storage modulus of Comparative Example 5 in the positive group was 8.2 kPa, while that in the negative group was 8.8 kPa, because the patch had only a heparin / PDA mixed coating, which was difficult to gradually filter blood to filter PTA into the hydrogel to undergo phase transition, and the hydrogel was in a slightly stiff state.

[0063] In summary, the hydrogel patch of Example 1 can accurately recognize PTA and visually express it in the form of different mechanical properties (soft / stiff) after filtering the blood sample with / without PTA.

[0064] Test Example 4: Detection performance characterization of the hydrogel patches prepared in Example 1 and Comparative Examples 1-5

[0065] 50 μL of blood samples containing PTA at different concentrations (0.1, 0.5, 1, 10, 100 ng / mL) were added to the center of the primary filter layer of the hydrogel patch, and the samples entered the patch and were detected by penicillin thiazole acid in the detection signal layer, and their detection rates were evaluated. The results are shown in FIG. 9. Figure 5As shown in (a), the sample detection rate of Example 1 reached 98.5% for 0.1 ng / mL, and reached 100% for other concentrations, because it effectively filtered the blood and screened PTA into the detection signal layer for detection. The sample detection rates of Comparative Examples 1-5 were all less than 50% for 0.1 ng / mL and 0.5 ng / mL, because they could not well filter and screen PTA in the blood for detection. The sample detection rates of Comparative Examples 1-5 all reached 100% for 100 ng / mL, because they had a certain effect of detecting PTA, but the precision was limited. In summary, the hydrogel patch of Example 1 can filter the blood samples of different concentrations of PTA, accurately identify PTA, and the detection limit reaches 0.1-100 ng / mL.

[0066] Test Example 5: Specificity characterization of the hydrogel patches prepared in Example 1 and Comparative Examples 1-5

[0067] (1) False positive rate characterization: 100 groups of blood samples without PTA were used to detect penicillin thiazole acid for the hydrogel patches prepared in Example 1 and Comparative Examples 1-5, and the false positive rate was evaluated according to the detection results. The results are shown in Figure 6 As shown in (a), the false positive rate of Example 1 for blood samples without PTA was only 2%, because it could specifically identify PTA for detection. The false positive rates of Comparative Examples 1-5 were all greater than 20%, because it was difficult to specifically screen PTA into the detection signal layer for detection, and it was easy to identify other impurities for reaction.

[0068] (2) Cross-reaction rate characterization: In order to compare the detection effect of the hydrogel patch on other antibiotics, 100 groups of blood samples containing penicillin G (10 μg / mL), ceftriaxone (150 μg / mL), and vancomycin (30 μg / mL) were used to detect the hydrogel patches prepared in Example 1, Comparative Example 3, and Comparative Example 5, and the cross-reaction rate was evaluated according to the detection results. The results are shown in Figure 6 As shown in (b), the cross-reaction rates of Example 1 for the three drugs were all less than 5%, because the hydrogel patch could specifically identify PTA. The cross-reaction rate of Comparative Example 3 for penicillin G reached 52%, and the cross-reaction rates for other drugs were less than 20%, because penicillin G was a drug very similar to PTA, and the hydrogel with PTA as a template had difficulty in identifying PTA for detection, while other drugs were screened by the blood pretreatment area, and the cross-reaction rate was slightly lower. The cross-reaction rate of Comparative Example 3 for penicillin G reached 40%, because the heparin / PDA mixed coating had difficulty in screening PTA for detection.

[0069] The above results all show that the false positive rate of the hydrogel patch of Example 1 is only 2%, and the probability of identifying negative samples as positive is extremely low; the cross-reaction rate of detection of different antibiotics is less than 5%, and the probability of identifying other antibiotics as positive is very low, so the hydrogel patch of Example 1 can accurately and specifically identify PTA.

[0070] Test Example 6: Biocompatibility characterization of the hydrogel patches prepared in Example 1 and Comparative Examples 1-5

[0071] (1) Anticoagulant performance characterization: the hydrogel patch and 4% red blood cell suspension were co-incubated for 2 hours, and the absorbance of the sample supernatant at 540 nm was further determined to evaluate the hemolysis rate. As shown in Figure 7 , the hemolysis rates of Example 1 and Comparative Examples 1-5 were all less than 5%, which showed that the materials used in the system all had good biological safety, but the relative hemolysis rates of Comparative Example 1 and Comparative Example 4 were slightly higher, because they both did not have heparin component, which affected the anticoagulant effect of the hydrogel patch.

[0072] (2) Cell activity characterization: after co-incubation of the hydrogel patch and cells, cck-8 was used for cell activity detection to evaluate the cell survival rate. As shown in Figure 7 , the cell survival rates of Example 1 and Comparative Examples 1-5 were all greater than 80%, which showed that the materials used in the system all had biological safety and could be in contact with the human body.

[0073] The above tests show that the materials used in Example 1 all have good biocompatibility.

[0074] Test Example 7: Accuracy characterization of penicillin allergy pre-detection of the hydrogel patch prepared in Example 1

[0075] In order to compare the accuracy of the hydrogel patch prepared in Example 1 and the skin test (intradermal injection of penicillin) of the prior art in the pre-detection of penicillin allergy, first, 50 μL of fingertip blood samples of 20 groups of penicillin allergic patients and healthy people were taken, the blood samples were dropped onto the center of the primary filter layer of the hydrogel patch prepared in Example 1, then the hydrogel surface of the detection signal layer was tightly attached to the surface of the human skin, the blood samples were detected, and after the detection signal was stable, the average detection time, the detection result and the positive rate were recorded. Then, 0.1 mL of 500 U / mL penicillin G sodium salt was intradermally injected into the penicillin allergic patients and healthy people, and after the local skin reaction was stable, the positive / negative was judged, and the average detection time was recorded. The detection results are shown in Table 1, the positive rate of the blood samples of the penicillin allergic patients in Example 1 reaches 85%, the positive rate of the healthy people is only 5%, and the detection time is within 10 minutes, because the hydrogel patch of Example 1 directly and specifically detects the penicillin metabolite PTA, the accuracy and detection efficiency are higher; the positive rate of the penicillin allergic patients in the prior art skin test is 70%, the positive rate of the healthy people is 10%, and the detection time is about 30 minutes, because the skin test method is based on the skin state and detects the penicillin drug itself, the performance of the human body will be different, resulting in a high false positive rate. In summary, compared with the prior art skin test, the hydrogel patch prepared in Example 1 has higher accuracy, shorter detection time and convenient operation.

[0076] Table 1 Positive rate of pre-detection of penicillin allergy of the hydrogel patch prepared in Example 1 and the skin test

[0077]

[0078] In summary, the intelligent hydrogel patch for detecting penicillin metabolites has the ability to pre-detect penicillin allergy, and experiments have proved that it has the effects of high specificity, convenient operation, high detection efficiency, detection limit of 0.1-100 ng / mL, low false positive rate and visual interpretation of results, and is simple and convenient and is expected to be widely used in the pre-detection of penicillin allergy in home first aid scenes.

[0079] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and supplements without departing from the principles of the present application, and these improvements and supplements should also be considered within the protection scope of the present application.

Claims

1. A smart hydrogel patch for detecting penicillin, characterized in that: The patch is stacked from top to bottom with a primary filtration layer, a secondary fine screening layer and a detection signal layer: The primary filtration layer is a heparinized cellulose nanofiber membrane with a pore size of 3 μm. The surface of the heparinized cellulose nanofiber membrane is loaded with heparin, and the surface zeta potential is ≤-30.65 mV, which is used to intercept blood cells and inhibit coagulation; The secondary fine sieve layer is a polydopamine coating with a pore size of 50 nm; the surface of the polydopamine coating is enriched with negative charges, and the zeta potential of the polydopamine coating is ≤-20.42 mV, which is used to reduce protein adsorption; The detection signal layer is a penicillin-thiazolyl acid-imprinted temperature-sensitive hydrogel, which uses penicillin-thiazolyl acid as a template molecule to form specific recognition holes for capturing penicillin-thiazolyl acid; the temperature-sensitive hydrogel has a pore size of 20nm, undergoes a phase change at body temperature of 37°C, and outputs a transmittance change signal.

2. The smart hydrogel patch for detecting penicillin according to claim 1, characterized in that: The detection signal layer is in close contact with the skin when in use, and the blood sample enters from the center of the primary filter layer of the patch and enters the secondary fine screening layer and the detection signal layer in sequence.

3. The smart hydrogel patch for detecting penicillin according to claim 1, characterized in that: When penicillin is captured by the penicillin molecular imprinting holes at 37° C., the phase change of the temperature-sensitive hydrogel is inhibited, and the transmittance of the temperature-sensitive hydrogel is higher than that when penicillin is not captured.

4. A method for preparing the smart hydrogel patch for detecting penicillin as claimed in claim 1, characterized in that: The preparation method comprises the following steps: (1) Preparation of heparinized cellulose nanofiber membrane: 8 wt% cellulose acetate was dissolved in a mixed solvent of acetone and N,N-dimethylacetamide, then heated in a water bath at 50°C with stirring for 6 h. After cooling to 37°C, 2 wt% sodium heparin was added and stirred for 2 h to obtain a spinning solution 1. The spinning solution 1 was used as a raw material for uniaxial electrospinning to obtain a heparinized cellulose nanofiber membrane. The electrospinning parameters were set as follows: spinning speed of 0.003 mm / s, spinning needle of 20 G, voltage of 15 kV, receiving distance of 13 cm, temperature of 25°C, and relative humidity of 60%. (2) Preparation of the charge selective interface of the polydopamine coating: Dopamine was fully dissolved in 100 mM Tris buffer (pH 7.4) to obtain a deposition solution 1 with a concentration of 2 mg / mL. The surface of the heparinized cellulose nanofiber membrane obtained in step (1) was subjected to a 100 W plasma treatment for 1 min by passing oxygen. The activated heparinized cellulose nanofiber membrane was immersed in the deposition solution 1 at 25°C in the dark for 2 hours. (3) Preparation of penicillin thiazole acid-imprinted thermosensitive hydrogel: 1.2 M N,N-methylenebisacrylamide, 0.4 M acrylic acid, 2.5 M N-isopropylacrylamide, 0.06 M N,N'-methylenebisacrylamide, 0.2 mM penicillin thiazole acid and a certain amount of photoinitiator Irgacure 2959 were mixed and stirred in the dark for 10 min to prepare a prepolymer solution. The prepolymer solution was then dropped onto the surface of the polydopamine coating and covered with a polyethylene terephthalate release film to control the thickness. After irradiation with 365 nm UV light for 15 min, the prepolymer was immersed in a mixture of methanol and acetic acid and shaken at 30 °C for 24 h to completely remove penicillin thiazole acid. The prepolymer solution was then rinsed with PBS at pH 7.4 until neutral, thereby obtaining a smart hydrogel patch based on the detection of penicillin metabolites.

5. The method for preparing a smart hydrogel patch for detecting penicillin according to claim 3, characterized in that: The volume ratio of acetone to N,N-dimethylacetamide in the mixed solvent of step (1) is 2:

1.

6. The method for preparing a smart hydrogel patch for detecting penicillin according to claim 3, characterized in that: The concentration of the photoinitiator Irgacure 2959 in the prepolymer solution in step (3) is 0.15 wt %.

7. The method for preparing a smart hydrogel patch for detecting penicillin according to claim 3, characterized in that: The hydrogel in step (3) uses a PET release film to control the thickness to 200 μm.

8. The method for preparing a smart hydrogel patch for detecting penicillin according to claim 3, characterized in that: The volume ratio of the methanol and acetic acid mixture in step (3) is 9:

1.

9. The method for preparing a smart hydrogel patch for detecting penicillin according to claim 3, characterized in that: The patch in step (3) is sealed and stored away from light after freeze-drying, and is rehydrated and activated before use by immersing it in 37°C saline for 5 minutes.

10. Use of the smart hydrogel patch according to any one of claims 1 to 9 in a penicillin allergy detection kit, characterized in that: The test sample of the kit is blood. After the blood is applied to the patch, the detection signal layer of the patch needs to be placed close to the skin to activate the detection signal layer for penicillin at a body temperature of 37°C.

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