Chiral ag nanofluorescent probe, preparation method thereof and application thereof in detection of feather keratin

Abstract

The application discloses a chiral Ag nano-fluorescent probe, a preparation method thereof and application of the chiral Ag nano-fluorescent probe in detection of feather keratin. Chiral cytosine nucleoside is used as a ligand to coordinate with Ag ions, and a chiral Ag nano-fluorescent probe with cytosine nucleoside as a ligand protection is prepared through reduction. The chiral Ag nano-fluorescent probe prepared by the method of the application is a multifunctional nano-probe with chirality and fluorescence, and can detect the hydrolysis degree of waste feather keratin from two different technical fields at the same time. The method for preparing the chiral Ag nano-fluorescent probe is relatively simple, the raw materials are cheap, easy to obtain and safe. The application provides a new thought for detecting the hydrolysis degree of waste feather keratin and rapidly screening effective waste feather degradation strains.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of keratin detection, and particularly relates to a chiral Ag nano-fluorescent probe, a preparation method thereof and application of the chiral Ag nano-fluorescent probe in detection of feather keratin. BACKGROUND

[0002] Feather is a derivative of epidermal cell keratinization of birds and poultry, accounts for about 10% of body weight, and is a main source of keratin, with a protein content of more than 90%. It is estimated that the feather by-products of poultry processing industry and down product production enterprises in China are more than one million tons per year. Because of high sulfur content in feather, the methods of landfill or combustion will cause serious environmental pollution. Therefore, the development and utilization of keratin can not only reduce environmental pollution, but also bring great economic benefits. However, because of the three-dimensional network structure formed in keratin molecules, there are a large number of disulfide bonds, which makes it difficult to extract keratin. In order to obtain keratin with good solubility, only methods such as selective opening of disulfide bonds can be used. At present, the methods for extracting keratin mainly include physical method, chemical method and biological method. Compared with chemical method and physical method, the biological enzymatic keratin treatment condition is relatively mild. However, the principle of feather degradation by keratinase producing bacteria is still unclear. We hope to detect the sulfur bond (thio group) opened by the enzymatic method to realize the purpose of rapid screening of feather hydrolysis strains.

[0003] At present, fluorescent labeling technology becomes a powerful tool for detecting thiol, which has high spatial and temporal resolution, high sensitivity, good selectivity, fast response and other advantages. However, the widely used fluorescent detection reagents, especially organic fluorescent reagents, are often complex in synthesis, poor in light stability, large in cytotoxicity, poor in biocompatibility, and there are a series of shortcomings such as the interference of background fluorescence. Therefore, the development of new biological inorganic materials and the development of new probes for detecting thiol under physiological conditions have become the research focus. In recent years, the application of coin metal (Au, Ag, Pt and Cu, etc.) fluorescent materials in biological systems has been deeply developed, and has shown an attractive application prospect. For example, in the fields of detecting gene fragments, proteins, biological small molecules and toxic metal ions using fluorescent method, coin metal nanomaterials show very high specificity. Among the numerous coin metal nanomaterials, there is also a strong affinity between the bases of DNA and metal cations. Therefore, the nanoclusters taking DNA (especially single-stranded DNA rich in cytosine bases in the sequence) as a template are expected to become a new type of inorganic biological fluorescent labeling probe to replace traditional organic fluorescent dyes (J.T. Petty, S.P. Story, J.C. Hsiang, R.M. Dickson. J. Phys. Chem. Lett., 2013, 4, 1148-1155; I. Diez, R.H.A. Ras. Nanoscale, 2011, 3, 1963-1970). Cytosine nucleoside (Cyt) as one of the pyrimidine nucleosides constituting nucleic acids is a kind of biological small molecule with biocompatibility. It has a simple molecular structure, low price and chirality. SUMMARY

[0004] The purpose of the present application is to use cheap chiral cytosine nucleoside as ligand, to successfully prepare chiral Ag nanofluorescent probe and its preparation method by sodium borohydride reduction method, namely chiral coin metal fluorescent probe, and then successfully apply it to the detection of feather keratin.

[0005] The chiral Ag nanofluorescent probe of the present application is prepared by the following method:

[0006] The chiral cytosine nucleoside as ligand is coordinated with Ag ion, and the chiral Ag nanofluorescent probe with cytosine nucleoside as ligand protection is prepared by reduction.

[0007] Preferably, the reduction is by sodium borohydride reduction method.

[0008] Preferably, the specific steps are:

[0009] Chiral cytosine nucleoside was dissolved in ethanol, then Ag salt was added and stirred until dissolved. Sodium borohydride was then added and stirred until an orange-red solution was formed. The supernatant was collected by centrifugation, and ether was added to the supernatant until the solution was saturated. The solution was allowed to stand until an orange-red solid precipitated. The orange-red solid was collected and dried to obtain the chiral Ag nanofluorescent probe.

[0010] The Ag salt can be silver tetrafluoroborate (AgBF4), silver trifluoromethanesulfonate (AgOTf), or silver trifluoroacetate (AgTFA).

[0011] Preferably, the mass ratio of Ag to cytosine is Ag:Cyt = 1:2.

[0012] Preferably, the mass ratio of NaBH4 to Ag is 1:12.

[0013] Preferably, the standing period until an orange-red solid precipitates is at 4-20°C.

[0014] The stirring continues until an orange-red solution is formed, and the reaction takes approximately 5–12 hours.

[0015] A second objective of this invention is to provide the application of the above-mentioned chiral Ag nanofluorescent probe in the detection of feather keratin.

[0016] A third objective of this invention is to provide a method for detecting feather keratin, comprising the following steps:

[0017] Chiral Ag nanofluorescent probes and keratin were mixed in a liquid environment for reaction, and then the emission wavelength λ was detected. em Fluorescence intensity around 620 nm, excitation wavelength λ ex =360nm, to determine the presence or concentration of keratin.

[0018] Preferably, the keratin can be waste keratin.

[0019] Preferably, the waste keratin is waste chicken feather keratin.

[0020] Preferably, the method also includes establishing a standard curve, specifically by mixing chiral Ag nanofluorescent probes and keratin standards of different concentrations in a liquid environment for reaction, and then detecting the emission wavelength λ. em Fluorescence intensity around 620 nm, excitation wavelength λ ex =360nm, thereby establishing a standard curve for keratin concentration and fluorescence intensity;

[0021] The chiral Ag nanofluorescent probe and the keratin sample to be tested are then mixed in a liquid environment for reaction, and the emission wavelength λ is then detected. em Fluorescence intensity = 620 nm, excitation wavelength λex = 360 nm, according to the measured fluorescence intensity and the standard curve, the concentration of the keratin to be detected is obtained.

[0022] The specific steps are as follows:

[0023] A, quantitatively take the chiral Ag nano fluorescent probe, and prepare a 5 mg / mL solution with deionized water for standby;

[0024] B, prepare a 1-100*10 -5 mol / L solution of keratin with deionized water for standby;

[0025] C, in a 3ml cuvette, sequentially add 20-50ul of chiral Ag nano fluorescent probe solution, 2.5ml of deionized water solution, 30-60ul of keratin solution with different concentrations, and deionized water to 3.0ml, shake well, and react at room temperature;

[0026] D, detect the fluorescence intensity of the emission wavelength λ em = 620 nm, and the excitation wavelength λ ex = 360 nm.

[0027] In order to solve the problem of fluorescence background interference, chiral cytosine nucleoside is used as a ligand to coordinate with Ag ions, and a biocompatible chiral Ag nano fluorescent probe with cytosine nucleoside as a ligand is successfully prepared by using sodium borohydride reduction method. The abandoned feather keratin is detected from the fields of chirality and fluorescence. In this experiment, a multifunctional chiral fluorescent probe is successfully synthesized, which can detect the abandoned feather keratin from two different fields at the same time, and has important significance in both theoretical research and practical application.

[0028] The principle of detecting abandoned feather keratin by the synthesized multifunctional chiral Ag nano fluorescent probe is as follows: (1) the circular dichroism spectrum (CD) signal of chiral cytosine nucleoside is in the near ultraviolet region (about 250-320 nm), and a new chiral center is formed after the orderly arrangement on the surface of Ag nano clusters, and the CD signal (about 350-600 nm) in the ultraviolet region or even visible region is generated, which can successfully avoid the interference of ligand background CD signal. Based on this condition, the CD signal in this wavelength range is fully utilized to detect the abandoned feather keratin. (2) the chiral silver nano fluorescent probe itself has strong orange-red fluorescence, and the fluorescence is quenched after the abandoned feather keratin is used, and the fluorescence intensity is inversely proportional to the concentration of the abandoned feather keratin, so as to indicate the existence or quantitative determination of the concentration of the abandoned feather keratin.

[0029] Effects and advantages of the present application:

[0030] The chiral Ag nano fluorescent probe prepared by the method has the advantages of chiral and fluorescent multifunctional nano probes, and can detect the hydrolysis degree of waste feather keratin from two different technical fields at the same time.

[0031] The method for preparing the chiral Ag nano fluorescent probe is relatively simple, the raw materials are cheap and easy to obtain, and safe.

[0032] The method for preparing the chiral Ag nano fluorescent probe is relatively simple, the raw materials are cheap and easy to obtain, and safe. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The excitation spectrum and emission spectrum diagram of the chiral Ag nano fluorescent probe prepared in Example 1 of the application.

[0034] Figure 2 The CD diagram and UV diagram of the chiral Ag nano fluorescent probe prepared in Example 1 (in the diagram, 1-6 are chiral silver nano fluorescent probes with decreasing concentrations in the test process).

[0035] Figure 3 The fluorescence spectrum diagram of the waste feather keratin with different concentrations added into the probe solution prepared in Example 1.

[0036] Figure 4 The visible light diagram of the probe solution (left) and the solution after adding waste feather keratin (right) in Example 1. DETAILED DESCRIPTION

[0037] The following examples are further illustrations of the application, but not limitations of the application.

[0038] Example 1

[0039] 1. At room temperature, 48 mg of Cyt was ultrasonically dissolved in 3 mL of ethanol.

[0040] 2. 19.5 mg of AgBF4 was added to the solution of step 1, and continuously stirred until dissolved.

[0041] 3. At room temperature, 0.05 mg of NaBH4 was added to the solution of step 2, and continuously stirred until an orange-red solution was formed, and the reaction was about 6 h.

[0042] 4. The solution obtained in step 3 was transferred to a centrifuge for 3 min (8000 r / min), and the supernatant was collected.

[0043] 5. In the supernatant of step 4, 2-3 mL of ether solvent was added to saturate the solution, and placed at (4-20 DEG C) for 12 h, and orange-red solid was precipitated at the bottom.

[0044] 6. The orange-red solid obtained in step 5 was centrifuged for 3 min (8000 r / min) to obtain an orange-red solid for standby use.

[0045] 7. The product obtained in step 6 was dissolved in deionized water to obtain a 5 mg / mL solution for standby use at room temperature.

[0046] 8. The waste chicken feather keratin was dissolved in deionized water to obtain a solution with different concentrations (1-100 x 10 -5 mol / L) for standby use.

[0047] 9. In a 3.0 mL cuvette, 50 μL of the solution prepared in step 7, 2.5 mL of deionized water and 50 μL of the waste keratin solution with different concentrations (10, 20, 30, 40, 50, 60, 80 and 100 x 10 -5 mol / L) were sequentially added, and deionized water was added to 3.0 mL to shake and react for 5 min at room temperature.

[0048] 10. The fluorescence intensity of the probe solution with an emission wavelength λ em = 620 nm (excitation wavelength λ ex = 360 nm) was detected by using a Duetta fluorescence and absorption spectrometer.

[0049] The orange-red solid obtained in step 6 is a chiral Ag nanofluorescent probe (AgBF4+Cyt), Figure 2 is the UV absorption spectrum and CD diagram of the chiral Ag nanofluorescent probe prepared in Example 1. Figure 3 is the fluorescence spectrum diagram of the probe solution of Example 1 to which the waste feather keratin with different concentrations is added. Figure 4 is the visible light diagram of the probe solution of Example 1 (left, concentration is 5 mg / mL) and the solution after the addition of the waste feather keratin (right; concentration is 100 x 10 -5 mol / L).

[0050] From Figure 1 , 2 , 3 and 4, it can be seen that the synthesized chiral Ag nanofluorescent probe forms a new chiral center, successfully escapes the interference of the CD signal of the ligand Cyt between 400-500 nm. With the increase of the concentration of the waste feather keratin, the fluorescence enhancement of the probe is continuously quenched, and the keratin can be successfully detected, and the fluorescence intensity is inversely proportional to the concentration of the waste keratin, thereby indicating the presence or quantitative determination of the concentration of the waste keratin.

[0051] Example 2:

[0052] 1. At room temperature, 48 mg of Cyt was ultrasonically dissolved in 3 mL of ethanol.

[0053] 2. 25.7 mg of AgOTf was added to the solution of step 1 and stirred until dissolved.

[0054] 3. 0.05 mg of NaBH4 was added to the solution of step 2 at room temperature and stirred until an orange-red solution was formed. The reaction was allowed to proceed for about 6 h.

[0055] 4. The solution of step 3 was transferred to a centrifuge tube and centrifuged for 3 min at 8000 rpm. The supernatant was collected.

[0056] 5. To the supernatant of step 4, 2-3 mL of diethyl ether was added to saturate the solution. The solution was allowed to stand at 4-20 °C for 12 h. An orange-red solid was precipitated at the bottom.

[0057] 6. The suspension of step 5 was centrifuged for 3 min at 8000 rpm to obtain an orange-red solid, which was ready for use.

[0058] Example 3:

[0059] 1. 48 mg of Cyt was dissolved in 3 mL of ethanol at room temperature.

[0060] 2. 22.1 mg of AgTFA was added to the solution of step 1 and stirred until dissolved.

[0061] 3. 0.05 mg of NaBH4 was added to the solution of step 2 at room temperature and stirred until an orange-red solution was formed. The reaction was allowed to proceed for about 6 h.

[0062] 4. The solution of step 3 was transferred to a centrifuge tube and centrifuged for 3 min at 8000 rpm. The supernatant was collected.

[0063] 5. To the supernatant of step 4, 2-3 mL of diethyl ether was added to saturate the solution. The solution was allowed to stand at 4-20 °C for 12 h. An orange-red solid was precipitated at the bottom.

[0064] 6. The suspension of step 5 was centrifuged for 3 min at 8000 rpm to obtain an orange-red solid, which was ready for use.

[0065] The above description of the specific embodiments of the present application in conjunction with the accompanying drawings is not intended to limit the scope of the present application. Those skilled in the art should understand that various modifications or variations can be made to the technical solutions of the present application without departing from the scope of the present application.

Claims

1. The application of chiral Ag fluorescent nanoprobes in the detection of feather keratin, wherein the preparation method of the chiral Ag fluorescent nanoprobes includes the following steps: Chiral cytosine nucleoside was dissolved in ethanol, then Ag salt was added and stirred to dissolve. Sodium borohydride was then added and stirred until an orange-red solution was formed. The supernatant was collected by centrifugation, and ether was added to the supernatant until the solution was saturated. The solution was allowed to stand until an orange-red solid precipitated. The orange-red solid was collected and dried to obtain the chiral Ag nanofluorescent probe. The Ag salt is silver tetrafluoroborate, silver trifluoromethanesulfonate, or silver trifluoroacetate; The mass ratio of Ag to cytosine nucleoside is Ag:Cyt = 1:2; the mass ratio of NaBH4 to Ag is 1:

12. The step of letting the mixture stand until an orange-red solid precipitates is performed at 4–20°C; the step of stirring until an orange-red solution is formed takes 5–12 hours.

2. A method for detecting feather keratin, characterized in that, Includes the following steps: Chiral Ag nanofluorescent probes and keratin were mixed in a liquid environment to carry out the reaction, and then the emission wavelength was detected. λ em Fluorescence intensity around 620 nm, excitation wavelength λ ex = 360 nm, to determine the presence or concentration of keratin; The preparation method of the chiral Ag nanofluorescent probe includes the following steps: Chiral cytosine nucleoside was dissolved in ethanol, then Ag salt was added and stirred to dissolve. Sodium borohydride was then added and stirred until an orange-red solution was formed. The supernatant was collected by centrifugation, and ether was added to the supernatant until the solution was saturated. The solution was allowed to stand until an orange-red solid precipitated. The orange-red solid was collected and dried to obtain the chiral Ag nanofluorescent probe. The Ag salt is silver tetrafluoroborate, silver trifluoromethanesulfonate, or silver trifluoroacetate; The mass ratio of Ag to cytosine nucleoside is Ag:Cyt = 1:2; the mass ratio of NaBH4 to Ag is 1:

12. The step of letting the mixture stand until an orange-red solid precipitates is performed at 4–20°C; the step of stirring until an orange-red solution is formed takes 5–12 hours.

3. The method according to claim 2, characterized in that, This also includes establishing a standard curve, specifically involving mixing chiral Ag nanofluorescent probes and keratin standards of different concentrations in a liquid environment for reaction, followed by detecting the emission wavelength. λ em Fluorescence intensity around 620 nm, excitation wavelength λ ex = 360 nm, thereby establishing a standard curve for keratin concentration and fluorescence intensity; The chiral Ag nanofluorescent probe and the keratin sample to be tested are then mixed in a liquid environment to react, and the emission wavelength is then detected. λ em Fluorescence intensity at 620 nm, excitation wavelength λ ex = 360 nm. The concentration of the keratin to be tested is obtained by comparing the measured fluorescence intensity with the standard curve. The keratin is waste keratin.

4. The method according to claim 3, characterized in that, The waste keratin mentioned is waste chicken feather keratin.

5. The method according to claim 2, characterized in that, The specific steps are as follows: A. Weigh out the chiral Ag nanofluorescent probe quantitatively and prepare a 5 mg / mL solution with deionized water for later use; B. Prepare keratin to a concentration of 1×100 ×10 using deionized water. ‒5 Solutions of different mol / L concentrations are available for use; C. Add 20-50 µL of chiral Ag nanofluorescent probe solution, 2.5 mL of deionized water solution, and 30-60 µL of keratin solutions of different concentrations to a 3 mL colorimetric tube in sequence. Make up the volume with deionized water to 3.0 mL, shake well, and react at room temperature. D. Detecting emission wavelength using a fluorescence spectrometer λ em Fluorescence intensity at 620 nm, excitation wavelength λ ex = 360 nm.

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