Zinc porphyrin-based nanofiber material, preparation method, visualized dual-modal fluorescence / colorimetric ratio biosensor and application

Abstract

The present invention provides a zinc porphyrin-based nanofiber material, a preparation method, a visual dual-modal fluorescence / colorimetric ratio biosensor and applications, belonging to the technical fields of electrochemiluminescence and colorimetry. The present invention synthesizes an Al-ZnTCPP@PAN nanofiber material by a solvothermal method. ZnTCPP has both fluorescence and colorimetric properties. After being compounded with PAN, the PO4 3‑ attacking the Al nodes can cause ZnTCPP to fall off, generating fluorescence / colorimetric signal changes for highly sensitive sensing of ALP. By calculating the ratio of the FL signal to the colorimetric signal, the detection limit of the material for ALP can be reduced and the sensing accuracy can be improved. At the same time, the visual dual-modal ratio sensing of ALP is realized by means of mobile phone photography and color recognition of the mobile phone APP.

Description

Technical Field

[0001] The present invention belongs to the technical field of colorimetry and fluorescence sensing, and in particular relates to a zinc porphyrin-based nanofiber material, a preparation method, a visualized dual-mode fluorescence / colorimetric ratio biosensor and applications. Background Art

[0002] Milk is one of the oldest natural dairy products and a common high-protein drink in our lives. It is popular for its rich nutrients and has become the best choice for calcium and protein supplementation for teenagers, middle-aged and elderly people. However, there are often some pathogens in milk that cause people to be infected with diseases, threatening human health. Currently, pasteurization is mostly used to sterilize dairy products. Alkaline phosphatase (ALP) is widely present in raw milk and biological tissues. It has relatively good heat resistance and is currently one of the most reliable indicators for evaluating the level of pasteurization. It plays an important role in dairy product safety testing.

[0003] At present, most of the reported ALP activity detection methods (colorimetry, fluorescence, electrochemistry, photoelectrochemistry and surface-enhanced Raman spectroscopy, etc.) need to rely on laboratory instruments, and achieving high-sensitivity and high-precision sensing is still a huge challenge. The demand for an easy-to-use and highly sensitive ALP biosensor is increasing to facilitate the immediate detection and timely assessment of related conditions. Compared with a single signal detection mode, a dual-modal sensor can provide more accurate and reliable results by reducing the interference of environmental factors through a self-calibration function. At the same time, the method of color recognition using mobile phone photography can realize the visual instant detection of the object to be tested. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the purpose of the present invention is to provide a zinc porphyrin-based nanofiber material, a preparation method, a visualized dual-modal fluorescence / colorimetric ratio biosensor and applications. The present invention first provides an Al-ZnTCPP@PAN nanofiber material having both fluorescence and colorimetric properties. The fluorescence / colorimetric ratio sensor prepared based on the Al-ZnTCPP@PAN nanofiber material can perform fluorescence / colorimetric (FL / Abs) ratio detection on alkaline phosphatase (ALP), and has the advantages of high sensitivity, high selectivity and simple preparation.

[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0006] The present invention first provides a method for preparing a zinc porphyrin-based nanofiber material, comprising the following steps:

[0007] Step 1: Prepare polyacrylonitrile membrane PAN by electrospinning;

[0008] Step 2: Using Atomic Layer Deposition of Al2 O 3 Al 2 O 3 @PAN;

[0009] Step 3: Add the Al obtained in step 2 2 O 3 @PAN is added to the mixed solution of ZnTCPP to react and obtain zinc porphyrin-based nanofiber material.

[0010] Preferably, the mixed solution of ZnTCPP comprises ZnTCPP, solvent DMF and deionized water.

[0011] Preferably, the reaction temperature in step 3 is 110-130° C., and the reaction time is 7-9 h.

[0012] The present invention also provides the zinc porphyrin-based nanofiber material prepared by the above preparation method.

[0013] The present invention also provides a visualized dual-mode fluorescence / colorimetric ratio biosensor, which is prepared from the zinc porphyrin-based nanofiber material.

[0014] The present invention also provides the above-mentioned visual dual-mode fluorescence / colorimetric ratio biosensor in detecting PO 4 3- Application in.

[0015] The present invention also provides the above-mentioned visual dual-mode fluorescence / colorimetric ratio biosensor for detecting PO 4 3- methods, including:

[0016] The visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in the sample to be tested, and fluorescence and colorimetric detection are performed after the reaction. The ratio of normalized fluorescence intensity to normalized electrochemiluminescence intensity is established to determine the PO 4 3- concentration, thereby detecting PO 4 3- concentration.

[0017] The present invention also provides the use of the above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor in detecting ALP concentration.

[0018] The present invention also provides a method for detecting ALP concentration using the above-mentioned visualized dual-mode fluorescence / colorimetric ratio biosensor, comprising:

[0019] The above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in a sample to be tested containing ALP and phosphate monoester, and fluorescence and colorimetric detection are performed after the reaction, and the relationship between the ratio of the normalized fluorescence intensity and the normalized colorimetric intensity and the ALP concentration is established to detect the ALP concentration.

[0020] The present invention also provides a method for visualizing the detection of ALP concentration, comprising the following steps:

[0021] The above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in a sample to be tested containing ALP and phosphate monoester. After the reaction, fluorescence and colorimetric detection are performed, followed by taking a photo with a mobile phone. Color recognition is performed using a mobile phone APP, and the relationship between the ratio of the normalized visualized fluorescence intensity and the normalized visualized colorimetric intensity and the ALP concentration is established, thereby visually detecting the ALP concentration.

[0022] The beneficial effects of the present invention are as follows:

[0023] (1) The zinc porphyrin-based nanofiber material Al-ZnTCPP@PAN provided by the present invention has a simple synthesis process and takes less time. ZnTCPP has good FL and colorimetric properties, and FL / Abs ratio detection can be used to detect PO 4 3- and ALP concentration with high sensitivity and reliability.

[0024] (2) The Al-ZnTCPP@PAN of the present invention is a convenient and sensitive tool suitable for detecting ALP, which can realize visual detection of ALP concentration. The method has the advantage of on-site instant detection, with a linear range of 0.1-10 mU / mL and a detection limit as low as 0.039 mU / mL. It has broad application prospects in the field of fluorescence and colorimetric sensing detection.

[0025] (3) The detection method of the present invention is simple to operate, has universal applicability, and is easy to mass produce. In addition, the Al-ZnTCPP@PAN fiber membrane is easy to store and use, and the fluorescence / colorimetric dual-mode ratio sensor can be prepared at any time as needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is the preparation process of Al-ZnTCPP@PAN in Example 1 of the present invention;

[0028] Figure 2Characterization diagrams of Al-ZnTCPP@PAN of Example 1 of the present invention, wherein (1) is the X-ray diffraction (XRD) spectrum of Al-ZnTCPP@PAN, (2) is the infrared spectrum of Al-ZnTCPP@PAN, and (3) is the X-ray photoelectron spectroscopy (XPS) of Al-ZnTCPP@PAN;

[0029] Figure 3 The stimulation responses of Al-ZnTCPP@PAN in Example 3 of the present invention at different ALP concentrations are (1) fluorescence spectra, (2) colorimetric spectra, and (3) ratio linear relationship diagrams;

[0030] Figure 4 This is the visualized dual-modal ratiometric sensing of Al-ZnTCPP@PAN in different ALP concentrations in Example 3 of the present invention;

[0031] Figure 5 Schematic diagram of the mechanism of detecting ALP concentration by the fluorescence / colorimetric dual-modal ratio sensor based on Al-ZnTCPP@PAN in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] As introduced in the background technology, dual-modal ratio sensing has better selectivity and higher sensitivity than single signal sensing. After introducing mobile phone photography for color recognition, visual on-site instant detection can be achieved.

[0033] The present invention first provides a method for preparing a zinc porphyrin-based nanofiber material, comprising the following steps:

[0034] Step 1: preparing polyacrylonitrile membrane PAN by electrospinning; the electrospinning method is preferably as follows: mixing polyacrylonitrile powder with a solvent to obtain a spinning solution, wherein the solvent is preferably N,N-dimethylformamide (DMF), the mass fraction of the spinning solution is preferably 10%, and the mass g of the polyacrylonitrile powder: the volume mL of the solvent is preferably 0.1:1;

[0035] The spinning solution is then subjected to electrospinning, and the electrospinning parameters are: DC voltage 18kV, spinning temperature 22°C, spinning humidity 25%, injection speed 0.8mL / h, and receiving plate distance 18cm. Spinning is stopped after 3 hours, and the fiber membrane is removed and dried. The drying condition is preferably placed in a 40°C oven for 4 hours to obtain polyacrylonitrile membrane PAN.

[0036] Step 2: Using Atomic Layer Deposition of Al 2 O 3 Al2 O 3 @PAN;

[0037] Step 3: Add the Al obtained in step 2 2 O 3 @PAN is added to the mixed solution of ZnTCPP for reaction, and the product is washed and dried to obtain zinc porphyrin-based nanofiber material. The mixed solution of ZnTCPP includes ZnTCPP, solvent DMF and deionized water, and the mass mg of ZnTCPP: volume mL of DMF: volume mL of deionized water is preferably 7:1:3; the reaction temperature is preferably 110-130°C, more preferably 120°C, and the reaction time is preferably 7-9h, more preferably 8h.

[0038] The present invention also provides the zinc porphyrin-based nanofiber material prepared by the above preparation method.

[0039] The present invention also provides a visual dual-mode fluorescence / colorimetric ratio biosensor, which is prepared from the above zinc porphyrin-based nanofiber material. The specific preparation preferably includes:

[0040] The prepared Al-ZnTCPP@PAN was placed in 300 μL of different PO 4 3- The fiber membrane was taken out and placed in 200 μL of HAC-NaAC buffer (pH=3.5) containing 0.6 mM TMB for 2 minutes, and measured using an enzyme reader. Subsequently, the solution in the centrifuge tube was transferred to a cuvette to obtain the FL spectrum, and a visual dual-modal fluorescence / colorimetric ratio biosensor was obtained.

[0041] The present invention also provides the above-mentioned visual dual-mode fluorescence / colorimetric ratio biosensor in detecting PO 4 3- Application in.

[0042] The present invention also provides the above-mentioned visual dual-mode fluorescence / colorimetric ratio biosensor for detecting PO 4 3- methods, including:

[0043] The visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in the sample to be tested, and fluorescence and colorimetric detection are performed after the reaction. The ratio of normalized fluorescence intensity to normalized electrochemiluminescence intensity is established to determine the PO 4 3- concentration, thereby detecting PO 4 3- concentration.

[0044] The present invention also provides the use of the above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor in detecting ALP concentration.

[0045] The present invention also provides a method for detecting ALP concentration using the above-mentioned visualized dual-mode fluorescence / colorimetric ratio biosensor, comprising:

[0046] The above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in a sample to be tested containing ALP and phosphate monoester, and fluorescence and colorimetric detection are performed after the reaction, and the relationship between the ratio of the normalized fluorescence intensity and the normalized colorimetric intensity and the ALP concentration is established to detect the ALP concentration.

[0047] Due to the hydrolysis of AA2P by Al-ZnTCPP@PAN and ALP, PO 4 3- The interaction leads to the enhancement of fluorescence signal and the weakening of colorimetric signal, realizing FL / Abs dual-modal ratio biosensing of ALP. This detection method is simple and practical, with a linear range of 0.1-10mU / mL and a detection limit as low as 0.024mU / mL.

[0048] The present invention synthesizes Al-ZnTCPP@PAN nanofiber material by solvent thermal method. ZnTCPP has both fluorescence and colorimetric properties. 2 O 3 @After PAN compounding, ALP can be used to hydrolyze AA2P to produce PO 4 3- Attacking Al nodes causes ZnTCPP to fall off, resulting in FL / Abs signal changes, which is used for high-sensitivity sensing of ALP.

[0049] The present invention grows ZnTCPP in situ on Al 2 O 3 @PAN membrane surface, so that the obtained Al-ZnTCPP@PAN nanofiber material can catalyze O 2 The reactive oxygen species are generated, and TMB is oxidized to produce a colorimetric signal. It also has fluorescent properties, and a dual-modal ratiometric sensing platform is successfully built. 4 3- When attacking the Al node on the surface of Al-ZnTCPP@PAN, ZnTCPP falls off and the FL signal is enhanced. At the same time, the amount of ZnTCPP on the surface of Al-ZnTCPP@PAN decreases, resulting in a decrease in the colorimetric intensity, thus realizing FL / Abs dual-modal ratio signal detection. The post-reaction solution is then photographed with a mobile phone, and color recognition software is used to realize visual dual-modal ratio detection of ALP.

[0050] A method for visualizing ALP concentration detection of the present invention comprises the following steps:

[0051] The fluorescence / colorimetric dual-mode ratio biosensor of the Al-ZnTCPP@PAN nanofiber material is immersed in the sample to be tested containing ALP and phosphate monoester, and the fluorescence and colorimetric detection are performed after the reaction, followed by mobile phone photography, and color recognition using the mobile phone APP to establish the relationship between the ratio of the normalized visualized fluorescence intensity and the normalized visualized colorimetric intensity and the ALP concentration, thereby visually detecting the ALP concentration. This method has the advantage of on-site instant detection, with a linear range of 0.1 to 10 mU / mL and a detection limit as low as 0.039 mU / mL.

[0052] The phosphate monoester includes L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P). The phosphate monoester is catalyzed by ALP to produce PO 4 3- substrate.

[0053] The present invention provides a reaction solution of ALP (0-15 mU / mL) and AA2P of different concentrations as the test sample. The ALP and AA2P reaction solution is prepared by mixing 50 μL of ALP of different concentrations (0-15 mU / mL) with 50 μL of 4 mM AA2P (containing 2.5 mM MgCl 2 ) was mixed with a Tris-HCl buffer solution (pH=10, 50 mM) and incubated at 37°C for 50 minutes. Subsequently, the pH was adjusted to 6.8 to prepare an ALP and AA2P reaction solution.

[0054] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments and comparative examples.

[0055] Zinc porphyrin (ZnTCPP) was purchased from J&K Reagent Company.

[0056] Example 1

[0057] Preparation method of Al-ZnTCPP@PAN nanofiber material, such as Figure 1 As shown, the steps are as follows:

[0058] (1) 0.5 g of polyacrylonitrile (PAN) powder was mixed with 5 mL of N,N-dimethylformamide (DMF), stirred at room temperature for 4 h, and a uniform spinning solution with a mass fraction of 10% was prepared. After cooling, the spinning solution was injected into a syringe for electrospinning. The electrospinning parameters were: DC voltage 18 kV, spinning temperature 22 °C, spinning humidity 25%, injection speed 0.8 mL / h, and receiving plate distance 18 cm. Spinning was stopped after 3 h, the fiber membrane was removed, and placed in a 40 °C oven to dry for 4 h. Subsequently, the Al 2 O3 Al 2 O 3 @PAN;

[0059] (2) Mix 7 g ZnTCPP with 1 mL DMF and 3 mL deionized water and add 0.3 × 0.3 cm 2 Al 2 O 3 @PAN fiber membrane was reacted at 120℃ for 8h.

[0060] (3) Wash with deionized water and ethanol three times respectively.

[0061] (4) After drying, Al-ZnTCPP@PAN nanofiber material is obtained.

[0062] The Al-ZnTCPP@PAN nanofiber material prepared in Example 1 was characterized by XRD. Figure 2 As shown in (1), the XRD pattern shows that the PAN film and Al 2 O 3 @PAN has no obvious characteristic peak at 2θ = 5-10°, while Al-ZnTCPP MOF powder and Al-ZnTCPP@PAN have a main characteristic peak at 2θ = 7.6°, which is consistent with the simulation image and corresponds to the (201) crystal plane, indicating that a MOF structure is formed on the fiber surface. 2 O 3 @PAN comparison, Al-ZnTCPP MOF powder and Al-ZnTCPP@PAN at 996 cm -1 There is an obvious characteristic peak at , which is derived from the zinc porphyrin in the MOF structure. It also shows that Zn did not fall off from the porphyrin ring during the preparation of the functionalized fiber membrane. Figure 2 As shown in (2) in the figure. X-ray photoelectron spectroscopy (XPS) is used to identify the valence state of the element. For O1s peak separation, three characteristic peaks were observed at 529.5, 531.1 and 533.3 eV, belonging to Al 2 O 3 The Al-O bond between ZnTCPP and the CO and C=O bonds of the carboxyl group in ZnTCPP, that is, the carboxyl group of ZnTCPP and Al 2 O 3 @PAN forms covalent bonds between Al nodes, such as Figure 2 As shown in (3) in .

[0063] Example 2

[0064] ZnTCPP / Ti obtained in Example 1 3 C 2 Tx The composite nanomaterial is a fluorescence / colorimetric dual-modal ratio biosensor based on ZnTCPP.

[0065] The prepared Al-ZnTCPP@PAN was placed in 300 μL of different PO 4 3- The fiber membrane was taken out and placed in 200 μL of HAC-NaAC buffer (pH=3.5) containing 0.6 mM TMB for 2 minutes, and measured using an enzyme reader. Subsequently, the solution in the centrifuge tube was transferred to a cuvette to obtain the FL spectrum, and a visual dual-modal fluorescence / colorimetric ratio biosensor was obtained.

[0066] Example 3

[0067] Take 300 μL of ALP and AA2P reaction solutions of different concentrations in a centrifuge tube, immerse the Al-ZnTCPP@PAN nanofiber material in the test solution, react for 8 minutes, then put the taken out fiber membrane into HAC-NaAC buffer (200 μL, pH = 3.5) containing TMB (0.6 mM), irradiate with a 15 W flashlight for 2 minutes, measure with an enzyme reader, and determine the FL of the solution in the centrifuge tube at the same time.

[0068] As the ALP concentration increases, the FL intensity increases and the colorimetric intensity gradually decreases. The data changes are as follows: Figure 3 (1) and (2). By fitting the results of FL and colorimetry, the linear range of FL and colorimetric dual-modal detection is 0.1-10mU / mL, the FL detection LOD is 0.035mU / mL, and the colorimetric LOD is 0.030mU / mL. After normalizing FL and colorimetric intensity, the linear relationship between ALP concentration and FL / Abs is obtained, as shown in 3(3). The ratio LOD is 0.024mU / mL, and the LOD of the ratio mode is significantly lower than that of single mode detection. This example illustrates that the dual-modal ratio sensing based on Al-ZnTCPP@PAN nanofiber material has a low detection limit, a wide detection range, and good application value.

[0069] Example 4

[0070] The 96-well plate and the cuvette under 375 nm ultraviolet light in Example 3 were photographed by mobile phone, and the color was recognized by mobile phone software to record the different PO 4 3- All data were normalized using FL and the maximum value of the colorimetric response as references to bring the data into a unified range of 0 to 1. The visualized normalized data were then ratio-processed to determine the PO concentration.4 3- / Quantitative analysis of ALP.

[0071] The colorimetric samples and fluorescent samples of ALP with different concentrations were photographed, and the obtained photos were imported into the mobile phone color recognition software. The scatter plot and linear relationship diagram were obtained according to the RGB values. Figure 4 (1) and (2) show that the linear range is 0.1 to 10 mU / mL. The relationship between the normalized ratio of the fluorescence R value and the colorimetric G value and the ALP concentration is as follows: Figure 4 (3) shows that after taking the logarithm, a linear fit was performed, and the linear range was 0.1-10 mU / mL, and the detection limit was 0.039 mU / mL, which was lower than the single-mode fluorescence (0.051 mU / mL) and colorimetric (0.046 mU / mL). The dual-mode ratio sensing platform showed high sensitivity, indicating that it is suitable for the high-sensitivity detection of ALP in actual dairy products. The schematic diagram of the fluorescence / colorimetric dual-mode ratio sensing is shown in Figure 5 shown.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a zinc porphyrin-based nanofiber material, characterized in that: The following steps are involved: Step 1: Prepare polyacrylonitrile membrane PAN by electrospinning; Step 2: Prepare Al2O3@PAN by atomic layer deposition of Al2O3; Step 3: adding the Al2O3@PAN obtained in step 2 to the mixed solution of ZnTCPP to react and obtain zinc porphyrin-based nanofiber material; The mixed solution of ZnTCPP comprises ZnTCPP, solvent DMF and deionized water; The reaction temperature in step 3 is 110-130° C., and the reaction time is 7-9 hours.

2. The zinc porphyrin-based nanofiber material prepared by the preparation method according to claim 1.

3. A visual dual-modal fluorescence / colorimetric ratio biosensor, characterized in that: It is prepared from the zinc porphyrin-based nanofiber material described in claim 2.

4. The visual dual-modal fluorescence / colorimetric ratio biosensor of claim 3 is used to detect PO4 3- Application in.

5. The use according to claim 4, characterized in that: The described visual dual-modal fluorescence / colorimetric ratiometric biosensor detects PO4 3- methods, including: The visualized dual-mode fluorescence / colorimetric ratio biosensor is immersed in the sample to be tested, and fluorescence and colorimetric detection are performed after the reaction. The ratio of normalized fluorescence intensity to normalized electrochemiluminescence intensity is established to determine the ratio of PO4 3- The relationship between the concentration and the detection of PO4 3- concentration.

6. Use of the visualized dual-modal fluorescence / colorimetric ratio biosensor according to claim 3 in detecting ALP concentration.

7. The use according to claim 6, characterized in that: The method for detecting ALP concentration using a visualized dual-modal fluorescence / colorimetric ratio biosensor comprises: The above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in a sample to be tested containing ALP and phosphate monoester, and fluorescence and colorimetric detection are performed after the reaction, and the relationship between the ratio of the normalized fluorescence intensity and the normalized colorimetric intensity and the ALP concentration is established to detect the ALP concentration.

8. The use according to claim 6, characterized in that: The method for visualizing the ALP concentration comprises the following steps: The above-mentioned visualized dual-modal fluorescence / colorimetric ratio biosensor is immersed in a sample to be tested containing ALP and phosphate monoester. After the reaction, fluorescence and colorimetric detection are performed, followed by taking a photo with a mobile phone. Color recognition is performed using a mobile phone APP, and the relationship between the ratio of the normalized visualized fluorescence intensity and the normalized visualized colorimetric intensity and the ALP concentration is established, thereby visually detecting the ALP concentration.

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