Molecularly imprinted material PtCo-MIP for kynurenine detection, preparation method, application and product

Abstract

The invention provides a molecularly imprinted material PtCo-MIP for kynurenine detection, a preparation method, application and a product, and belongs to the technical field of analytical chemistry. According to the invention, PtCo alloy is combined with a molecular imprinting technology; wherein the PtCo alloy has more excellent oxidase-like activity compared with Pt nanoparticles, and the molecular imprinting technology solves the problem of insufficient specific detection of a target factor by nano-enzyme by forming a cavity complementary with a kynurenine structure on the surface of the PtCo alloy, so that efficient and safe kynurenine specific recognition is realized, and the detection sensitivity is improved. And the blank of low-cost and high-specificity detection technology in objective diagnosis of depression is filled. The kynurenine detection kit has an extremely wide detection range on kynurenine, and the detection range and the sensitivity are far better than those of an existing commercial kit; meanwhile, the anti-interference capability is excellent, accurate detection can be performed even in a complex matrix, the detection performance is excellent, and an accurate biological basis is provided for early diagnosis of depression.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of analytical chemistry, and particularly relates to a molecular imprinting material PtCo@MIP for detecting kynurenine, a preparation method, application and product. BACKGROUND

[0002] Depression is a psychological disorder characterized by persistent low mood, loss of interest and pleasure, and is one of the major chronic and disabling mental illnesses in the world. Its "four high" characteristics-high prevalence, high recurrence rate, high disability rate, and high suicide rate, not only seriously damages the physical and mental health of patients, but also brings heavy burden to families and society. According to statistics from authoritative agencies such as the World Health Organization, the number of global depression patients has exceeded 350 million, and the number of people who die from depression each year is as high as 1 million. This series of shocking data reveals that early diagnosis of depression is the key to breaking the disease and reducing the risk of suicide.

[0003] However, the current diagnosis of depression mainly relies on clinical interviews and symptom assessment scales (such as the Hamilton Depression Scale and the Self-Rating Depression Scale), which are easily influenced by individual expression and subjective emotions, leading to high misdiagnosis rate and possibly exacerbating the patient's condition. Therefore, developing a convenient, accurate and low-cost detection method has become an urgent need for clinical practice and medical research.

[0004] In recent years, with the in-depth study of medical molecular biology, the pathophysiological mechanism of depression has been gradually revealed, and the correlation between inflammation and depression has become a research hotspot. Relevant reports and clinical studies have confirmed that the levels of various inflammatory factors (such as tumor necrosis factor-a and interleukin-6) in the central nervous system (such as brain tissue) and peripheral blood (such as serum and plasma) of patients with depression are significantly higher than those of healthy people. It is particularly worth noting that the levels of inflammatory factors in patients with depression with suicidal ideation or suicidal behavior are significantly higher, which provides an important biological clue for the objective diagnosis of depression. Further studies have shown that the abnormal increase in the level of inflammatory factors is closely related to the disorder of the "tryptophan-kynurenine metabolic pathway" in the body - under normal physiological conditions, tryptophan is mainly synthesized into neurotransmitters through the 5-hydroxytryptamine pathway to maintain emotional stability; when the body is in an inflammatory state, tryptophan will be more metabolized through the kynurenine pathway, leading to an abnormal increase in the generation of kynurenine in serum. Clinical detection data shows that the content of kynurenine in the serum of patients with depression is significantly higher than that of healthy people, and the change in its content has certain correlation with the severity and symptom of depression. This key finding confirms that by detecting the content of kynurenine in serum, early and objective diagnosis of depression can be achieved, providing a new direction for breaking through the limitations of traditional diagnosis methods.

[0005] However, the current clinical methods for detecting kynurenine, such as high performance liquid chromatography (HPLC), mass spectrometry (MS), high performance liquid chromatography-mass spectrometry (HPLC-MS) and capillary electrophoresis, although have high detection sensitivity and accuracy, are difficult to meet the needs of clinical popularization and early screening. These detection methods generally have two major problems: one is high detection cost - the required instrument equipment (such as mass spectrometer, high performance liquid chromatograph) is expensive, the maintenance cost is high, and the reagents and consumables used in the detection process are mostly imported or high-end products - resulting in high single detection cost, which is difficult to promote in primary medical institutions or large-scale screening; the second is complex and inconvenient operation - such methods have strict requirements on the experimental environment (such as requiring sterile and constant temperature laboratory), the detection process is complicated (such as complex sample pretreatment and long detection period), and professional technical personnel are required for operation and data analysis, which cannot realize rapid detection or on-site detection. Therefore, developing a kynurenine detection method with simple and convenient operation, low detection cost and high accuracy can not only fill the technical gap of current objective diagnosis of depression, but also provide a powerful tool for early screening, disease monitoring and efficacy evaluation of depression, which has important practical significance for improving the global prevention and treatment level of depression. SUMMARY

[0006] The purpose of the present application is to provide a molecular imprinting material PtCo@MIP for kynurenine detection, a preparation method, an application and a product. The detection sensitivity of the molecular imprinting material PtCo@MIP for kynurenine detection is high, the specificity is good, the method is simple, the cost is low, and the method can provide accurate biological basis for early diagnosis of depression, meet the needs of on-site rapid detection, screening in primary medical institutions and other scenes, and solve the limitations of existing detection technologies which depend on professional laboratories and equipment.

[0007] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0008] The present application provides a molecular imprinting material PtCo@MIP for kynurenine detection. The preparation raw materials of the molecular imprinting material PtCo@MIP include the following components with the mass molar ratio:

[0009] Pt metal salt: cobalt metal salt: PVP, organic acid = 0.075-0.3 mmol: 0.075-0.3 mmol: 90-95 mg: 2-3 mmol.

[0010] Preferably, the Pt metal salt includes one of uranyl acetylacetone, platinum nitrate and potassium chloroplatinite, the cobalt metal salt includes one of cobalt acetylacetone, cobalt nitrate and cobalt chloride, and the organic acid includes one of benzoic acid, salicylic acid and sodium citrate.

[0011] The application also provides a preparation method of the molecular imprinting material PtCo@MIP for detecting kynurenine, comprising the following steps:

[0012] (1) Preparation of PtCo

[0013] The Pt-based metal salt, the Co-based metal salt, PVP, an organic acid and benzyl alcohol are mixed to obtain a mixture, and the mixture is sequentially subjected to ultrasonic treatment, stirring, heating, precipitation and washing to obtain PtCo.

[0014] (2) Synthesis of PtCo@MIP

[0015] The PtCo obtained in step (1) is dissolved to obtain a PtCo solution, the PtCo solution is mixed with a kynurenine solution, and then a dopamine hydrochloride solution is added, followed by ultrasonic treatment, stirring and self-polymerization, washing and removal of the template molecule to obtain the molecular imprinting material PtCo@MIP.

[0016] Preferably, in step (1), the ultrasonic treatment is performed for 20-40 min, the stirring is performed for 20-40 min, and the heating is performed at a temperature of 150-200℃ for 10-14 h.

[0017] Preferably, in step (2), the concentration of the PtCo solution is 1-1.5 mg / mL, the concentration of the kynurenine solution is 0.1-1 mg / mL, and the concentration of the dopamine hydrochloride solution is 0.1-1 mg / mL; the volume ratio of the PtCo solution to the kynurenine solution and the dopamine hydrochloride solution is 1-5:0.5-1.5:0.5-1.5.

[0018] Preferably, in step (2), the stirring is performed for 0.5-2 h, and the self-polymerization is performed for 0.5-20 h.

[0019] The application also provides the use of the molecular imprinting material PtCo@MIP for detecting kynurenine according to any one of claims 1-2 or the molecular imprinting material PtCo@MIP prepared by the preparation method according to claims 3-6 in the preparation of a product for detecting kynurenine.

[0020] The application also provides a paper-based colorimetric kit for detecting kynurenine, characterized in that the paper-based colorimetric kit for detecting kynurenine comprises the molecular imprinting material PtCo@MIP for detecting kynurenine according to any one of claims 1-2 or the molecular imprinting material PtCo@MIP prepared by the preparation method according to claims 3-6, test paper and TMB.

[0021] The application also provides a detection method of kynurenine molecules, characterized by comprising the following steps:

[0022] The PtCo@MIP and the sample to be detected are mixed for first incubation, TMB is added for second incubation, and then the absorbance value is detected to obtain the concentration of kynurenine molecules.

[0023] Preferably, the first incubation time is 20-40 min, and the second incubation time is 5-20 min.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] (1) The application combines nano-enzyme (PtCo alloy) with molecular imprinting technology to assist in diagnosing depression through colorimetric detection of kynurenine; the PtCo alloy has more excellent oxidase-like activity than Pt nanoparticles, and the molecular imprinting technology forms a cavity complementary to the structure of kynurenine on the surface of the PtCo alloy, thereby solving the problem of insufficient specific detection of target factors by nano-enzyme, realizing efficient and safe specific recognition of kynurenine, and filling the gap of low-cost and high-specificity detection technology in objective diagnosis of depression. The colorimetric detection method based on PtCo@MIP has a very wide detection range for kynurenine (L-kyn), the low-concentration end can cover 0.48 μM, the high-concentration end can reach 960 μM, and the detection limit is as low as 0.0282 μM, which is far superior to the existing high-performance liquid chromatography (HPLC) detection method and commercial reagent kit in terms of detection range and sensitivity. At the same time, it has excellent anti-interference ability and can accurately detect in complex matrix, and has excellent detection performance, providing accurate biological basis for early diagnosis of depression.

[0026] (2) The application is convenient and efficient to operate, and does not require complex pretreatment. The colorimetric analysis platform based on PtCo@MIP can directly analyze kynurenine serum samples. The detection result is intuitive, and the specific detection of kynurenine can be realized by observing the color change of the color developing substrate TMB (blue without kynurenine, and lighter color with kynurenine) or detecting ultraviolet absorbance, without complex operation of professional personnel.

[0027] (3) The application has strong practicability and is suitable for multiple scene detection requirements. The paper-based colorimetric sensor constructed by combining the detection method with paper also has a wide detection range (4.8 μM-960 μM), and takes into account the detection performance and convenience, which can meet the needs of on-site rapid detection, screening in primary medical institutions and other multiple scene requirements, and solves the limitations of existing detection technologies which rely on professional laboratories and equipment. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained based on these drawings without creative labor.

[0029] Figure 1 The morphology of PtCo in Embodiment 1 of the present application;

[0030] Figure 2 The XRD spectrum of PtCo in Embodiment 1 of the present application;

[0031] Figure 3 The absorbance comparison chart of Pt and PtCo in Embodiment 1 of the present application;

[0032] Figure 4 The enzyme kinetics test result chart in Embodiment 1 of the present application, wherein a is the oxygenase-like enzyme activity kinetics test result of Pt and PtCo; b is the Lineweaver-Burk chart of the oxygenase-like enzyme activity of Pt and PtCo;

[0033] Figure 5 The XPS comparison chart of PtCo and Pt in the present application;

[0034] Figure 6 The PtCo@MIP characterization result chart in the present application, wherein a is the TEM analysis result, b is the element mapping image, c is the FTIR spectrum, d is the TGA result, and e is the zeta potential test result of PtCo and PtCo@MIP;

[0035] Figure 7 The detection result of kynurenine in Embodiment 1 of the present application;

[0036] Figure 8 The relationship between kynurenine and absorbance in Embodiment 1 of the present application;

[0037] Figure 9 The relationship between the color intensity (R value) on the paper-based colorimetric sensor and the kynurenine concentration in Embodiment 6 of the present application;

[0038] Figure 10 The anti-interference experiment result chart in the present application. DETAILED DESCRIPTION

[0039] The various exemplary embodiments of the present application will now be described in detail, which should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.

[0040] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, for example, concentrations, amounts, and the like, every concentration between the upper and lower limit of that range is also specifically included within the scope of the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0041] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference for the disclosure and

[0042] Many modifications and variations of this application of the present application can be made without departing from its spirit or scope, which will be apparent to those skilled in the art. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only. It is to be understood that the application is not limited in scope by the specific embodiments described herein. Rather, the intent is to embrace all changes and modifications that are within the spirit and scope of the application.

[0043] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0044] Example 1

[0045] Example 1 of the present application provides a preparation method of molecularly imprinted material PtCo@MIP, the specific steps are as follows:

[0046] (1) Synthesis of PtCo

[0047] 1) Mix 0.15 mmol of platinum (II) acetylacetone, 0.3 mmol of cobalt (II) acetylacetone, 3 mmol of benzoic acid, 93.33 mg of PVP (molecular weight is 58000) and 35 mL of benzyl alcohol, ultrasonic for 30 minutes, then magnetic stirring at 500 r / min for 30 minutes, after the mixed solution is completely dispersed, transfer the dispersed solution to a polytetrafluoroethylene lined high-pressure reaction kettle, heat at 180℃ for 12 hours, after heating, add acetone to precipitate, separate the precipitate, wash 6 times with ethanol-acetone 1:1 mixture (same below), obtain PtCo, characterize PtCo, the results are shown in Figures 1-2 The PtCo is dispersed into distilled water to prepare a PtCo solution for standby use.

[0048] Figure 1 The morphology of PtCo and Figure 2 The XRD spectrum shows that the PtCo with a size of about 3nm is successfully prepared.

[0049] 2) Evaluation of oxidase-like activity

[0050] The concentration of PtCo solution was adjusted to 100μg / mL, 20μL PtCo solution and 20μL TMB solution (50mM) were added to 960μL Hac-NaAc buffer (0.2M, pH4.0), and the absorbance at 652nm was measured after 10min reaction at 25℃ using UV-Vis spectrophotometer. The results are shown in Figure 3

[0051] The results show that the maximum absorbance of PtCo is greater than that of Pt. Figure 3

[0052] 3) Enzyme kinetics analysis

[0053] Enzyme kinetics analysis: 100μg / mL Pt or PtCo was added to Hac-NaAc buffer solution (pH=4, 0.2M) respectively, and different concentrations of TMB were added as catalysts. Then the absorbance of each concentration of TMB at 652nm was measured. The kinetic constants K m and V max were calculated according to Michaelis-Menten equation. The results are shown in Figure 4

[0054] V=V max [S] / (K m +[S])

[0055] Where V is the initial reaction rate, [S] is the substrate concentration, V max is the maximum reaction rate, K m is the Michaelis constant, which is the substrate concentration when the reaction rate reaches half of V max . V max and K m are calculated according to Lineweaver-Burk double-reciprocal plot (1 / V and 1 / [S]). The reaction rate (V) can be calculated according to the amount of TMB oxidation (oxTMB) per unit time. The concentration of oxTMB is determined using the Beer-Lambert law, expressed as A=εbc, where A is the absorbance at 652nm, ε is the molar absorption coefficient (ε 652nm =39,000M -1 cm -1 ), b is the path length of light through the sample, and c is the molar concentration of oxTMB. ​​

[0056] Figure 4 The K content of PtCo nanoparticles is shown to be... m (0.2) smaller than K of Pt nanoparticles m (0.25), V of PtCo max (15.57) and K cat (14.18) are all higher than the V of Pt. max (3.76) and Kcat (3.67), meaning that PtCo has better oxidase-like activity than Pt.

[0057] 4) Binding energy

[0058] The binding energy between PtCo and Pt was measured, and the results are as follows: Figure 5 As shown.

[0059] Figure 5 The results show that the binding energy of Pt 4f in PtCo nanoparticles is lower than that of Pt 4f in pure Pt nanoparticles, while the binding energy of Co0 2p (779.5 eV) and Co2 in PtCo nanoparticles are higher. + The binding energy of the 2p orbital (781.4 eV) is greater than that of the standard binding energy of the 2p orbital in elemental Co (778.2 eV) and that of Co in CoO. 2+ The high standard binding energy of the 2P orbital (779.7 eV) and the change in the binding energy of Pt 4f and Co 2p in PtCo nanoparticles indicate that there is a strong electronic interaction between Pt and Co. This interaction can optimize the electron redistribution on the catalyst surface, optimize the adsorption and desorption of oxygen and oxygen intermediates, thereby improving the activity of oxidases and proving the formation of PtCo alloys.

[0060] (2) Synthesis of PtCo@MIP

[0061] 5 mg of PtCo was dispersed in 4 mL of Tris-HCl buffer (10 mM, pH 8.5), and 1 mL of template molecule L-kyn (1 mg / mL, solvent: ultrapure water: acetonitrile = 9:1) was added. The mixture was stirred at room temperature for 2 h, then 1 mL of dopamine hydrochloride (0.25 mg / mL) was added, and the mixture was sonicated for 10 min. After stirring at room temperature for 1 h, the mixture was self-polymerized. After the polymerization was completed, the sample was washed three times with water, and then repeatedly washed with a methanol: acetic acid = 9:1 mixed solution to remove the template molecule, thus obtaining PtCo@MIP. The PtCo@MIP was characterized, and the results are as follows: Figure 6 As shown.

[0062] Figure 6 TEM analysis revealed a 2.2 nm thick imprinted layer on the PtCo@MIP surface, and elemental mapping also showed some nitrogen (N) elements aggregated on the PtCo surface. FTIR spectroscopy indicated that PtCo@MIP had a 1614 cm⁻¹ core.-1 The PtCo@MIP showed a clear N-H bending vibration peak, similar to polydopamine (PDA), while the PtCo alloy did not have this peak. The TGA results showed that the weight loss rate of PtCo@MIP was 19.1%, which was significantly higher than that of PtCo, which was 7.3%, indicating that in addition to the evaporation of some water vapor, the imprint layer also decomposed significantly.

[0063] The zeta potential test was carried out by dispersing PtCo and PtCo@MIP in aqueous solution, and the results are shown in FIG. 8e. Figure 6

[0064] Figure 6 As shown in FIG. 8e, the zeta potential of the PtCo alloy was -11.65 mV before imprinting, and the zeta potential increased to -7.69 mV after imprinting, which may be due to the influence of PDA-derived ammonium ions.

[0065] Figure 6 It is shown that the PtCo@MIP molecularly imprinted material was successfully constructed.

[0066] Figure 7 It is shown that after the addition of kynurenine, the color of the system becomes lighter, and the absorbance decreases.

[0067] Take 100 μL of PtCo@MIP (concentration 0.1 mg / ml) into a centrifuge tube, continue to add 100 μL of kynurenine (concentration 0.5 mg / ml) and mix. Incubate the mixture in a 37°C water bath for 30 min to promote the interaction between PtCo@MIP and kynurenine. After incubation, transfer 20 μL of the incubation solution to a small centrifuge tube. Then, add 960 μL of Hac-NaAc buffer solution and 20 μL of TMB to the test tube, and react at 25°C for 10 min. Finally, measure the absorbance at 652 nm using a UV-visible spectrophotometer. The results are shown in FIG. 7.

[0068] Figure 7 It is shown that after the addition of kynurenine, the color of the system becomes lighter, and the absorbance decreases.

[0069] (3) Detection by standard addition method

[0070] 1) Establishment of kynurenine-absorbance curve

[0071] Add 100 μL of kynurenine (concentration 0.1-0.5 mg / mL) to a 1.5 mL centrifuge tube, respectively, then add 100 μL of PtCo@MIP (concentration 0.5 mg / mL) to each test tube and mix. Incubate the mixture in a 37°C water bath for 30 min to promote the interaction between PtCo@MIP and kynurenine.

[0072] ​After incubation, 20 μL of the incubation solution was transferred to a small centrifuge tube, and 960 μL of Hac-NaAc buffer solution and 20 μL of TMB were continuously added, and the reaction was carried out at 25°C for 10 minutes. Finally, the absorbance at 652 nm was measured using a UV-Vis spectrophotometer, and the results are shown in Figure 8

[0073] Figure 8 It is shown that with the increase of the content of kynurenine, the color of the solution system gradually lightens, and the absorbance gradually decreases, and presents a linear relationship in the range of 0.48 μM to 960 μM of kynurenine concentration.

[0074] 2) A 5 μM kynurenine serum solution was prepared using human serum, 100 μL of the kynurenine serum solution was mixed with 100 μL of PtCo@MIP with a concentration of 0.15 mg / mL in a separate test tube, and incubated at 37°C for 30 minutes. After incubation, 40 μL of the mixed system was transferred to a separate small centrifuge tube, 940 μL of Hac-NaAc buffer solution (pH = 4, 0.2 M) and 20 μL of TMB solution with a concentration of 50 mM were added. After incubation at 25°C for another 10 minutes, the absorbance of the resulting solution was measured using a UV-Vis spectrophotometer. The concentration of kynurenine was obtained by the kynurenine-absorbance curve, and finally the recovery of added kynurenine was calculated.

[0075] The results show that the kynurenine concentration detected by the present application is 5.3 μM. The recovery rate is 106%, indicating that the analysis system has good accuracy.

[0076] Example 2

[0077] The present application provides a preparation method of the molecular imprinting material PtCo@MIP, and the specific steps are as follows:

[0078] (1) Synthesis of PtCo

[0079] 0.3 mmol of platinum (II) acetylacetone, 0.3 mmol of cobalt (II) acetylacetone, 3 mmol of benzoic acid, 93.33 mg of PVP (molecular weight 40000) and 35 mL of benzyl alcohol were mixed, ultrasonic for 30 minutes, and then magnetically stirred at 500 r / min for 30 minutes. After the mixed solution was completely dispersed, the dispersed solution was transferred to a polytetrafluoroethylene lined high-pressure reaction kettle, heated at 160°C for 12 hours, and then precipitated with acetone after heating. The precipitate was separated and washed with an ethanol-acetone mixture for 6 times to obtain PtCo. The PtCo was dispersed in distilled water to prepare a PtCo solution for standby.

[0080] (2) Synthesis of PtCo@MIP

[0081] ​Take 5mg of PtCo dispersion into 4mL of Tris-HCl buffer (10 mM, PH = 8.5), add 1mL of template molecule L-kyn (2mg / mL, solvent is ultrapure water: acetonitrile = 9:1), stir at room temperature for 0.5h, then add 1mI of dopamine hydrochloride (0.5mg / mL), ultrasonic oscillation for 10min, stir at room temperature for 1h, after the end, wash with water three times, and then use the mixed solution of methanol:acetic acid = 9:1 to wash repeatedly, wash away the template molecule, and get PtCo@MIP.

[0082] (3) The method of Example 1 is used to detect and calculate the performance of the PtCo@MIP prepared in this example, and the results show that the kynurenine concentration of the detection system in this example is 5.4μM. The recovery rate is 108%, indicating that the analysis system has good accuracy.

[0083] Example 3

[0084] Example 3 of the present application provides a preparation method of molecularly imprinted material PtCo@MIP, and the specific steps are as follows:

[0085] (1) Synthesis of PtCo

[0086] Mix 0.3mmol of platinum (II) acetylacetone, 0.15mmol of cobalt (II) acetylacetone, 3mmol of salicylic acid, 93.33mg of PVP (molecular weight 8000) and 35mL of benzyl alcohol, ultrasonic for 30 minutes, then magnetic stirring at 500r / min for 30 minutes, make the mixed solution completely dispersed, then transfer the dispersed solution to a polytetrafluoroethylene lined high-pressure reaction kettle, heat at 180℃ for 12 hours, after heating, add acetone to precipitate, separate the precipitate, wash with ethanol-acetone mixture for 6 times, and get PtCo. Disperse PtCo into distilled water to prepare PtCo solution for standby.

[0087] (2) Synthesis of PtCo@MIP

[0088] Take 5mg of PtCo dispersion into 4mL of Tris-HCl buffer (10 mM, PH = 8.5), add 1mI of template molecule L-kyn (1mg / mI, solvent is ultrapure water: acetonitrile = 9:1), stir at room temperature for 2h, then add 1mI of dopamine hydrochloride (2mg / m1), ultrasonic oscillation for 10min, stir at room temperature for 8h, after the end, wash with water three times, and then use the mixed solution of methanol:acetic acid = 9:1 to wash repeatedly, wash away the template molecule, and get PtCo@MIP.

[0089] (3) The human serum was used to prepare a 10 mM kynurenine serum solution, and the performance of the PtCo@MIP prepared in this example was detected by the method of Example 1 and calculated, and the results showed that the kynurenine concentration obtained by the detection system in this example was 10.4 mM, and the recovery rate was 104%, indicating that the analysis system had good accuracy.

[0090] Example 4

[0091] Example 4 of the present application provides a preparation method of the molecular imprinting material PtCo@MIP, and the specific steps are as follows:

[0092] (1) Synthesis of PtCo

[0093] 0.15 mmol of platinum (II) nitrate, 0.3 mmol of cobalt (II) nitrate, 3 mmol of sodium citrate, 93.33 mg of PVP (molecular weight 8000) and 35 mI of benzyl alcohol were mixed, ultrasonic for 30 minutes, then magnetic stirring at 500 r / min for 30 minutes, the mixed solution was completely dispersed, then the dispersed solution was transferred to a polytetrafluoroethylene lined high-pressure reaction kettle, heated at 160℃ for 12 hours, after heating, acetone was added for precipitation, the precipitate was separated and washed with ethanol-acetone mixture for 6 times to obtain PtCo. The PtCo was dispersed in distilled water to prepare a PtCo solution for standby.

[0094] (2) Synthesis of PtCo@MIP

[0095] 5 mg of PtCo was dispersed in 4 mL of Tris-HCl buffer (10 mM, pH 8.5), 1 mL of template molecule L-kyn (0.5 mg / mL, solvent: ultrapure water: acetonitrile = 9:1) was added, stirred at room temperature for 2 h, then 1 mI of dopamine hydrochloride (0.25 mg / m1) was added, ultrasonic oscillation for 10 min, and then stirred at room temperature for 4 h. After the end, it was washed with water for three times, and then washed with a mixture of methanol:acetic acid = 9:1 repeatedly to wash off the template molecule to obtain PtCo@MIP.

[0096] (3) The human serum was used to prepare a 15 mM kynurenine serum solution, and the performance of the PtCo@MIP prepared in this example was detected by the method of Example 1 and calculated, and the results showed that the kynurenine concentration obtained by the detection system in this example was 15.7 mM. The recovery rate was 105%, indicating that the analysis system had good accuracy.

[0097] Example 5

[0098] Example 5 of the present application provides a preparation method of the molecular imprinting material PtCo@MIP, and the specific steps are as follows:

[0099] (1) Synthesis of PtCo

[0100] 0.15 mmol of potassium chloroplatinite, 0.3 mmol of cobalt (II) chloride, 3 mmol of sodium citrate, 93.33 mg of PVP (molecular weight 8000) and 35 mL of benzyl alcohol were mixed, and after ultrasonic treatment for 30 minutes, 500 r / min magnetic stirring was carried out for 30 minutes, and after the mixed solution was completely dispersed, the dispersed solution was transferred to a polytetrafluoroethylene lined high-pressure reaction kettle, heated at 160 DEG C for 12 hours, and after heating was completed, acetone was added for precipitation, the precipitate was separated, and ethanol-acetone mixture was used for washing 6 times to obtain PtCo. The PtCo was dispersed in distilled water to prepare a PtCo solution for standby.

[0101] (2) Synthesis of PtCo@MIP

[0102] 5 mg of PtCo was taken and dispersed in 4 mL of Tris-HCl buffer (10 mM, pH = 8.5), 1 mI of template molecule L-kyn (0.5 mg / mL, solvent: ultrapure water: acetonitrile = 9:1) was added, and stirred at room temperature for 2 h, then 1 mL of dopamine hydrochloride (0.5 mg / m1) was added, ultrasonic oscillation was carried out for 10 min, and stirring was carried out at room temperature for 4 h. After the end, it was washed with water for three times, and then a mixed solution of methanol: acetic acid = 9:1 was repeatedly washed to wash off the template molecule to obtain PtCo@MIP.

[0103] (3) The human serum was used to prepare 10 mM of kynurenine serum solution, and the method of example 1 was used to carry out the standard addition method detection and calculate the performance of the PtCo@MIP prepared in this example. The results show that the kynurenine concentration of the detection system in this example is 10.7 mM. The recovery rate is 107%, which shows that the analysis system has good accuracy.

[0104] Example 6

[0105] Example 6 of the present application is based on the preparation of paper-based colorimetric sensor in example 1, and the effect is detected, and the specific steps are as follows:

[0106] (1) Prepare a solution of kynurenine with a concentration of 1-1000 μM, cut Whatman No. 1 filter paper into circular paper pieces with a diameter of 0.8 cm. According to the method of Example 1, incubate the PtCo@MIP prepared in Example 1 with kynurenine for 30 minutes, take 10 μL of the incubated solution, drop it on the circular paper piece, dry at 37°C to form a paper-based biochip, then drop 10 μL of a mixture of sodium acetate-acetic acid buffer and TMB (2 mM) onto the dried paper-based biochip, react at room temperature for 10 minutes. Then take a picture with a smart phone, identify the RGB value with Image J, detect the relationship between the color intensity (R value) on the paper-based colorimetric sensor and the concentration of kynurenine, and the results are shown in Figure 9 .

[0107] Figure 9 It is shown that as the content of kynurenine added increases, the color of the paper-based colorimetric sensor gradually lightens, and by identifying the RGB value with Image J, it is found that the R value increases with the increase of the content of kynurenine, and has a linear relationship in the range of 4.8 μM to 960 μM.

[0108] (2) Prepare kynurenine solutions with concentrations of 5, 10 and 15 μM respectively, and use the paper-based colorimetric sensor and the purchased commercial kit for detection, and the results are shown in Table 1

[0109] Table 1 Detection effect of paper-based colorimetric sensor

[0110]

[0111] Table 1 shows that the recovery rate of the paper-based colorimetric method of the application in the serum spiking experiment is better than that of the commercial kit, and the relative standard deviation is lower than that of the commercial kit, indicating that the paper-based colorimetric method has excellent detection performance.

[0112] Test Example 1

[0113] In Test Example 1 of the application, the kynurenine-absorbance curve of Example 1 is used to calculate the detection limit and the detection range, and the specific steps are as follows:

[0114] Kynurenine is detected by using conventional quantitative fluorescence determination, HPLC-MS / MS, molecular imprinting solid-phase extraction device, molecular imprinting 2D photonic crystal hydrogel sensor, HPLC combined with ultraviolet and fluorescence detection, high-throughput HPLC, red fluorescent gold nanoclusters (AuNCs), and terbium nanoclusters, and the detection limit and the detection range are calculated, and the results are shown in Table 2.

[0115] Table 2 Detection limit and detection range of different methods

[0116]

[0117] As shown in Table 2, the present invention has a wider (lower and higher) detection range and a lower detection limit.

[0118] Experimental Example 2

[0119] Experimental Example 2 of this invention tested the anti-interference effect of the PtCo@MIP prepared in Example 1. The specific steps are as follows:

[0120] Referring to step (3) of Example 1, part 2), Na is added to a 5 μM kynurenine serum solution. + K + Ca 2+ Potential interfering substances such as Glu and BSA were detected, and the results were as follows: Figure 10 As shown.

[0121] Figure 10 The display shows that Na + K + Ca 2+ Potential interfering substances such as Glu and BSA have little impact on the detection method of this invention and can be ignored, so this invention has high specificity.

[0122] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A molecularly imprinted material PtCo@MIP for the detection of kynurenine, characterized in that, The raw materials for preparing the molecularly imprinted material PtCo@MIP include the following components in the following mass molar ratio: Pt-based metal salt: cobalt-based metal salt: PVP: organic acid = 0.075-0.3 mmol: 0.075-0.3 mmol: 90-95 mg: 2-3 mmol.

2. The molecularly imprinted material PtCo@MIP for kynurenine detection according to claim 1, characterized in that, The Pt-based metal salt includes one of platinum acetylacetonate, platinum nitrate, and potassium chloroplatinate; the cobalt-based metal salt includes one of cobalt acetylacetonate, cobalt nitrate, and cobalt chloride; and the organic acid includes one of benzoic acid, salicylic acid, and sodium citrate.

3. A method for preparing the molecularly imprinted material PtCo@MIP for kynurenine detection as described in any one of claims 1 to 2, characterized in that, Includes the following steps: (1) Preparation of PtCo A mixture of Pt-based metal salt, cobalt-based metal salt, PVP, organic acid, and benzyl alcohol was obtained. The mixture was then subjected to ultrasonication, stirring, heating, precipitation, and washing to obtain PtCo. (2) Synthesis of PtCo@MIP PtCo from step (1) was dissolved to obtain a PtCo solution. The PtCo solution was mixed with a kynurenine template molecule solution and stirred. Then, dopamine hydrochloride solution was added, and the mixture was ultrasonically vibrated and stirred to self-polymerize. After washing, the template molecules were removed to obtain the molecularly imprinted material PtCo@MIP.

4. The method for preparing the molecularly imprinted material PtCo@MIP for kynurenine detection according to claim 3, characterized in that, In step (1), the ultrasound time is 20-40 min, the stirring time is 20-40 min, the heating temperature is 150-200℃, and the heating time is 10-14 h.

5. The method for preparing the molecularly imprinted material PtCo@MIP for kynurenine detection according to claim 3, characterized in that, In step (2), the concentration of the PtCo solution is 1-1.5 mg / mL, the concentration of the kynurenine solution is 0.1-1 mg / mL, the concentration of the dopamine hydrochloride solution is 0.1-1 mg / mL, and the volume ratio of the PtCo solution to the kynurenine solution and the dopamine hydrochloride solution is 1-5:0.5-1.5:0.5-1.

5.

6. The method for preparing the molecularly imprinted material PtCo@MIP for kynurenine detection according to claim 3, characterized in that, In step (2), the stirring time is 0.5 to 2 hours, and the stirring self-polymerization time is 0.5 to 20 hours.

7. The use of the molecularly imprinted material PtCo@MIP for kynurenine detection as described in any one of claims 1 to 2, or the molecularly imprinted material PtCo@MIP for kynurenine detection prepared by the preparation method described in claims 3 to 6, in the preparation of kynurenine detection products or depression detection products.

8. A paper-based colorimetric reagent kit for detecting kynurenine or depression, characterized in that, The paper-based colorimetric kit for kynurenine detection comprises the molecularly imprinted material PtCo@MIP for kynurenine detection as described in any one of claims 1 to 2, or the molecularly imprinted material PtCo@MIP for kynurenine detection prepared by the preparation method described in claims 3 to 6, test paper, and TMB.

9. A method for detecting kynurenine molecules, characterized in that, Includes the following steps: PtCo@MIP and the sample to be tested were mixed and incubated for the first time. TMB was added and incubated again. The absorbance value was then measured to obtain the concentration of kynurenine molecules.

10. The method for detecting kynurenine molecules according to claim 9, characterized in that, The first incubation period is 20 to 40 minutes, and the second incubation period is 5 to 20 minutes.

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