CN115728281A - Fluorescent probe for detecting L-tryptophan and preparation thereof - Google Patents
Fluorescent probe for detecting L-tryptophan and preparation thereof Download PDFInfo
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Abstract
Description
技术领域technical field
本发明属于荧光分析技术领域，具体涉及一种用于L-色氨酸检测的荧光探针及其制备。The invention belongs to the technical field of fluorescence analysis, and in particular relates to a fluorescent probe for detecting L-tryptophan and its preparation.
背景技术Background technique
发光量子点具有独特的尺寸依赖性、磁性、光学和电化学等特性，与其他含有重金属的量子点带有毒性以及潜在的环境危害性相比，目前主要致力于寻找无毒的非重金属的量子点，其中，硫量子点由于其具有良好的化学稳定性、水分散性以及生物相容性而备受关注。Luminescent quantum dots have unique size-dependent, magnetic, optical, and electrochemical properties. Compared with other quantum dots containing heavy metals, which are toxic and potentially harmful to the environment, the current focus is on finding non-toxic, non-heavy metal quantum dots. Among them, sulfur quantum dots have attracted much attention due to their good chemical stability, water dispersibility and biocompatibility.
L-色氨酸是蛋白质合成的必需成分，是动物和人类生长和代谢所需的一种芳香氨基酸。L-色氨酸的缺乏会导致白内障、糖尿病、抑郁症等疾病，且L-色氨酸不能在体内合成，它需要通过饮食或补充来获得。因此，L-色氨酸的分析检测在制药、环境、临床和工业研究中具有重要意义。而荧光探针是一种新型的检测试剂，由于其具有较高灵敏度、较低检测成本、样品处理简单、操作方便、测定快速以及实时检测的优点而备受人们的青睐。但是，到目前为止，利用硫量子点作为荧光探针用于L-色氨酸检测尚未见报道。L-Tryptophan is an essential component of protein synthesis and an aromatic amino acid required for growth and metabolism in animals and humans. The lack of L-tryptophan can lead to cataracts, diabetes, depression and other diseases, and L-tryptophan cannot be synthesized in the body, it needs to be obtained through diet or supplementation. Therefore, the analysis and detection of L-tryptophan is of great significance in pharmaceutical, environmental, clinical and industrial research. Fluorescent probe is a new type of detection reagent, which is favored by people because of its advantages of high sensitivity, low detection cost, simple sample processing, convenient operation, rapid determination and real-time detection. However, so far, the use of sulfur quantum dots as fluorescent probes for the detection of L-tryptophan has not been reported.
发明内容Contents of the invention
基于上述背景，本发明提供了一种用于L-色氨酸检测的荧光探针及其制备。本发明利用硫量子点构建荧光探针，通过荧光分析法构建线性关系曲线，进而实现对L-色氨酸的定量检测。该方法成本低廉、灵敏度高、线性关系好、操作简便易行、选择性好，可用于氨基酸注射液中L-色氨酸的定量检测，具有较好的临床应用前景。Based on the above background, the present invention provides a fluorescent probe for L-tryptophan detection and its preparation. The invention utilizes sulfur quantum dots to construct fluorescent probes, constructs a linear relationship curve through a fluorescence analysis method, and then realizes the quantitative detection of L-tryptophan. The method has the advantages of low cost, high sensitivity, good linear relationship, simple operation and good selectivity, and can be used for the quantitative detection of L-tryptophan in amino acid injections, and has good clinical application prospects.
为实现上述目的，本发明是通过以下技术方案来实现：To achieve the above object, the present invention is achieved through the following technical solutions:
本发明提供了一种用于L-色氨酸检测的荧光探针，所述荧光探针为β-环糊精硫量子点。The invention provides a fluorescent probe for detecting L-tryptophan, and the fluorescent probe is a β-cyclodextrin sulfur quantum dot.
进一步地，所述β-环糊精硫量子点的制备方法为：将β-环糊精溶于NaOH溶液中，再加入升华硫粉和超纯水，经加热、透析处理，得到所述β-环糊精硫量子点。Further, the preparation method of the β-cyclodextrin sulfur quantum dots is: dissolving the β-cyclodextrin in NaOH solution, adding sublimation sulfur powder and ultrapure water, heating and dialysis to obtain the β-cyclodextrin sulfur quantum dots. - Cyclodextrin sulfur quantum dots.
进一步地，所述NaOH溶液的浓度为1.8-2.0mol/L。Further, the concentration of the NaOH solution is 1.8-2.0mol/L.
进一步地，所述β-环糊精与升华硫粉的质量比为3:1-2.5:1。Further, the mass ratio of the β-cyclodextrin to the sublimed sulfur powder is 3:1-2.5:1.
进一步地，所述加热的方式为油浴加热。Further, the heating method is oil bath heating.
进一步地，所述加热的时间为120-144h，温度为60-80℃。Further, the heating time is 120-144h, and the temperature is 60-80°C.
进一步地，所述透析的具体过程为：在截留分子量为3500Da的透析袋中透析12-24h。Further, the specific process of the dialysis is: dialysis in a dialysis bag with a molecular weight cut off of 3500Da for 12-24 hours.
本发明还提供了上述荧光探针在L-色氨酸检测中的应用。The present invention also provides the application of the above-mentioned fluorescent probe in the detection of L-tryptophan.
进一步地，应用于氨基酸注射液中L-色氨酸的检测测定。Further, it is applied to the detection and determination of L-tryptophan in amino acid injection.
进一步地，向待检测液中加入β-环糊精硫量子点，并加入Tris-HCl缓冲液，然后以固定激发波长340nm进行荧光光谱测定。Further, β-cyclodextrin sulfur quantum dots are added to the liquid to be detected, and Tris-HCl buffer solution is added, and then the fluorescence spectrum is measured at a fixed excitation wavelength of 340 nm.
与现有技术相比，本发明的有益效果如下：Compared with the prior art, the beneficial effects of the present invention are as follows:
(1)本发明以廉价的硫粉为硫源，β-环糊精为刻蚀剂，通过一锅法合成β-环糊精硫量子点，制备过程操作简单、绿色无污染。(1) The present invention uses cheap sulfur powder as a sulfur source and β-cyclodextrin as an etchant to synthesize β-cyclodextrin sulfur quantum dots through a one-pot method. The preparation process is simple, green and pollution-free.
(2)本发明制备得到的β-环糊精硫量子点作为荧光探针具有良好的荧光性能和光稳定性，其在激发波长为312nm时，在420nm波长处具有最强的荧光强度，且荧光发射峰的峰形良好。(2) The β-cyclodextrin sulfur quantum dots prepared by the present invention have good fluorescence performance and photostability as a fluorescent probe. When the excitation wavelength is 312nm, it has the strongest fluorescence intensity at a wavelength of 420nm, and the fluorescence The peak shape of the emission peak was good.
(3)本发明提供了一种高灵敏、高选择性、快速检测L-色氨酸的新方法。将β-环糊精硫量子点应用于L-色氨酸检测中，可显著增强其位于420nm处的荧光发射，这是由于L-色氨酸与β-环糊精硫量子点形成了稳定的包合物，在L-色氨酸与β-环糊精硫量子点共存时，硫量子点的荧光会显著增强。基于此，β-环糊精硫量子点可实现对L-色氨酸的定量检测，线性范围为50-500nmol/L，检出限为9.3nmol/L。(3) The present invention provides a new method for detecting L-tryptophan with high sensitivity, high selectivity and rapidity. The application of β-cyclodextrin sulfur quantum dots in the detection of L-tryptophan can significantly enhance its fluorescence emission at 420nm, which is due to the stable formation of L-tryptophan and β-cyclodextrin sulfur quantum dots. When L-tryptophan and β-cyclodextrin sulfur quantum dots coexist, the fluorescence of sulfur quantum dots will be significantly enhanced. Based on this, β-cyclodextrin sulfur quantum dots can realize the quantitative detection of L-tryptophan, with a linear range of 50-500nmol/L and a detection limit of 9.3nmol/L.
附图说明Description of drawings
图1为β-环糊精硫量子点的红外光谱图。Figure 1 is the infrared spectrum of β-cyclodextrin sulfur quantum dots.
图2为β-环糊精硫量子点的荧光激发和发射谱图。Figure 2 is the fluorescence excitation and emission spectra of β-cyclodextrin sulfur quantum dots.
图3为pH对β-环糊精硫量子点稳定性的影响。Figure 3 shows the effect of pH on the stability of β-cyclodextrin sulfur quantum dots.
图4为离子强度对β-环糊精硫量子点稳定性的影响。Figure 4 shows the effect of ionic strength on the stability of β-cyclodextrin sulfur quantum dots.
图5为β-环糊精硫量子点检测L-色氨酸的高选择性。Figure 5 shows the high selectivity of β-cyclodextrin sulfur quantum dots to detect L-tryptophan.
图6为L-色氨酸的浓度与体系荧光强度变化的线性关系图。Fig. 6 is a graph showing the linear relationship between the concentration of L-tryptophan and the change of the fluorescence intensity of the system.
具体实施方式Detailed ways
为了便于理解本发明，下面将对本发明进行更全面的描述。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。In order to facilitate the understanding of the present invention, the following will describe the present invention more fully. However, the present invention can be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided for the purpose of making the disclosure of the present invention more thorough and comprehensive.
实施例1Example 1
β-环糊精硫量子点的制备Preparation of β-cyclodextrin sulfur quantum dots
将β-环糊精(3.7g)溶于盛有20mL NaOH(2.0mol/L)溶液的圆底烧瓶中，室温搅拌至完全溶解，然后向溶液中加入升华硫粉(1.4g)和超纯水(30mL)；再将溶液置于70℃的油浴中搅拌144h，经截留分子量为3500Da的透析袋透析24h后，即得β-环糊精硫量子点。Dissolve β-cyclodextrin (3.7g) in a round bottom flask filled with 20mL NaOH (2.0mol/L) solution, stir at room temperature until completely dissolved, then add sublimed sulfur powder (1.4g) and ultrapure water (30mL); then the solution was stirred in an oil bath at 70°C for 144h, and dialyzed through a dialysis bag with a molecular weight cut-off of 3500Da for 24h to obtain β-cyclodextrin sulfur quantum dots.
如图1所示，由上到下依次分别为升华硫粉、β-环糊精硫量子点和β-环糊精的红外光谱图，在3410cm-1、1633cm-1处出现的吸收峰分别对应-OH的伸缩振动和弯曲振动。2926cm-1为C-H的伸缩振动特征吸收峰。在1155cm-1、1030cm-1处出现的吸收峰分别对应β-环糊精的C-O伸缩振动和O-H弯曲振动。通过对比其红外谱图，可以发现β-环糊精硫量子点和β-环糊精在1155cm-1处均有较强的吸收峰，而升华硫粉在该处没有吸收峰。由此可知，β-环糊精成功附着在硫量子点的表面。As shown in Figure 1, from top to bottom are the infrared spectra of sublimated sulfur powder, β-cyclodextrin sulfur quantum dots and β-cyclodextrin respectively. The absorption peaks appearing at 3410cm -1 and 1633cm -1 respectively Corresponding to the stretching vibration and bending vibration of -OH. 2926cm -1 is the characteristic absorption peak of CH stretching vibration. The absorption peaks at 1155cm -1 and 1030cm -1 correspond to the CO stretching vibration and OH bending vibration of β-cyclodextrin, respectively. By comparing their infrared spectra, it can be found that both β-cyclodextrin sulfur quantum dots and β-cyclodextrin have strong absorption peaks at 1155 cm -1 , while sublimed sulfur powder has no absorption peaks there. It can be seen that β-cyclodextrin was successfully attached to the surface of sulfur quantum dots.
图2是β-环糊精硫量子点的荧光激发和发射谱图。由图2可知，β-环糊精硫量子点的激发波长为312nm，发射波长为420nm。Figure 2 is the fluorescence excitation and emission spectra of β-cyclodextrin sulfur quantum dots. It can be seen from FIG. 2 that the excitation wavelength of the β-cyclodextrin sulfur quantum dot is 312nm, and the emission wavelength is 420nm.
实施例2Example 2
β-环糊精硫量子点的稳定性测试Stability Test of β-Cyclodextrin Sulfur Quantum Dots
为了考察β-环糊精硫量子点的荧光稳定性，固定β-环糊精硫量子点浓度，加入不同pH的0.1mol/L PBS缓冲液，固定激发波长340nm进行420nm处荧光强度的测定。图3是pH条件对β-环糊精硫量子点稳定性的影响。由图3可知，在较宽的pH(3.0-12)值范围内，β-环糊精硫量子点稳定性好，具有良好的荧光性能。In order to investigate the fluorescence stability of β-cyclodextrin sulfur quantum dots, the concentration of β-cyclodextrin sulfur quantum dots was fixed, 0.1mol/L PBS buffer solution with different pH was added, and the excitation wavelength was fixed at 340nm to measure the fluorescence intensity at 420nm. Figure 3 is the effect of pH conditions on the stability of β-cyclodextrin sulfur quantum dots. It can be seen from Figure 3 that in a wide range of pH (3.0-12), the β-cyclodextrin sulfur quantum dots have good stability and good fluorescence properties.
固定β-环糊精硫量子点浓度，加入等体积的不同浓度的NaCl溶液，最后用pH 7.0的0.1mol/L Tris-HCl缓冲液定容，考察离子强度对β-环糊精硫量子点稳定性的影响，结果见图4。由图4可知，氯化钠浓度增加至1.0mol/L，β-环糊精硫量子点荧光性能几乎也不受影响，可见，β-环糊精硫量子点稳定性好，具有良好的荧光性能。Fix the concentration of β-cyclodextrin sulfur quantum dots, add an equal volume of NaCl solution of different concentrations, and finally use 0.1mol/L Tris-HCl buffer solution with pH 7.0 to make up the volume, and investigate the effect of ionic strength on the concentration of β-cyclodextrin sulfur quantum dots. The effect of stability, the results are shown in Figure 4. It can be seen from Figure 4 that the fluorescence performance of β-cyclodextrin sulfur quantum dots is hardly affected when the concentration of sodium chloride is increased to 1.0mol/L. It can be seen that β-cyclodextrin sulfur quantum dots have good stability and good fluorescence performance.
实施例3Example 3
β-环糊精硫量子点的选择性测试Selectivity Test of β-Cyclodextrin Sulfur Quantum Dots
为了保证手性β-环糊精硫量子点在分析复杂样品中L-色氨酸的准确性，在相同的测试条件下，确定了一些潜在的干扰物种包括L-Val、L-His、L-Glu、L-Leu、L-Cys、L-Ala、L-Arg、L-Asn、Thr、L-Phe、D/L-Ser、Gly、D-Trp氨基酸对环糊精硫量子点荧光强度的影响。在Tris-HCl缓冲液中分别加入上述干扰物种后再加入β-环糊精硫量子点溶液，固定激发波长340nm进行420nm处荧光强度测定。图5为等浓度的上述氨基酸及L-色氨酸分别在30℃的水浴条件下孵育24h后的荧光强度变化图。由图5可知，只有加入L-色氨酸后，β-环糊精硫量子点的荧光强度显著增强。而加入其他氨基酸后，环糊精硫量子点的荧光强度基本未发生明显变化，说明其他氨基酸对β-环糊精硫量子点的荧光强度无影响。由此可见，环糊精硫量子点对L-色氨酸具有较高的选择性。In order to ensure the accuracy of chiral β-cyclodextrin sulfur quantum dots in the analysis of L-tryptophan in complex samples, under the same test conditions, some potential interfering species including L-Val, L-His, L- -Glu, L-Leu, L-Cys, L-Ala, L-Arg, L-Asn, Thr, L-Phe, D/L-Ser, Gly, D-Trp amino acid fluorescence intensity of cyclodextrin sulfur quantum dots Impact. The above-mentioned interfering species were added to the Tris-HCl buffer solution, and then the β-cyclodextrin sulfur quantum dot solution was added, and the excitation wavelength was fixed at 340nm to measure the fluorescence intensity at 420nm. Fig. 5 is a graph showing changes in fluorescence intensity after incubation of equal concentrations of the above amino acids and L-tryptophan in a water bath at 30°C for 24 hours. It can be seen from Figure 5 that only after adding L-tryptophan, the fluorescence intensity of β-cyclodextrin sulfur quantum dots is significantly enhanced. After adding other amino acids, the fluorescence intensity of cyclodextrin sulfur quantum dots basically did not change significantly, indicating that other amino acids had no effect on the fluorescence intensity of β-cyclodextrin sulfur quantum dots. It can be seen that cyclodextrin sulfur quantum dots have higher selectivity to L-tryptophan.
实施例4Example 4
β-环糊精硫量子点在不同浓度的L-色氨酸存在下的荧光变化Fluorescence changes of β-cyclodextrin sulfur quantum dots in the presence of different concentrations of L-tryptophan
将实施例1制得的β-环糊精硫量子点与L-色氨酸溶液混合，并加入Tris-HCl缓冲液(pH＝7.0)，L-色氨酸溶液的最终浓度范围为0-1.0μM，在340nm激发波长下，测试β-环糊精硫量子点-L-色氨酸体系位于420nm的荧光强度。根据L-色氨酸浓度与β-环糊精硫量子点-L-色氨酸体系相应的荧光变化之间的关系建立了L-色氨酸分析检测的标准曲线，随着L-色氨酸浓度的增加，荧光强度逐渐增强，如图6所示，荧光强度与L-色氨酸浓度在50-500nmol/L范围内呈良好的线性关系，对应的线性回归方程为y＝4.183×10-2x-0.3053，相关系数R2＝0.996，检出限(LOD＝3σ/k)为9.3nmol/L(n＝11)。Mix the β-cyclodextrin sulfur quantum dots prepared in Example 1 with the L-tryptophan solution, and add Tris-HCl buffer solution (pH=7.0), and the final concentration range of the L-tryptophan solution is 0- 1.0 μM, under the excitation wavelength of 340nm, test the fluorescence intensity of the β-cyclodextrin sulfur quantum dots-L-tryptophan system at 420nm. According to the relationship between the concentration of L-tryptophan and the corresponding fluorescence change of the β-cyclodextrin sulfur quantum dots-L-tryptophan system, a standard curve for the analysis and detection of L-tryptophan was established. As the acid concentration increases, the fluorescence intensity gradually increases. As shown in Figure 6, there is a good linear relationship between the fluorescence intensity and the L-tryptophan concentration in the range of 50-500nmol/L, and the corresponding linear regression equation is y=4.183×10 -2 x-0.3053, correlation coefficient R 2 =0.996, limit of detection (LOD=3σ/k) was 9.3 nmol/L (n=11).
实施例5Example 5
氨基酸注射液中L-色氨酸的检测Detection of L-Tryptophan in Amino Acid Injection
将β-环糊精硫量子点加入2.0mL氨基酸注射液中，之后再加入2.0mL Tris-HCl缓冲液(pH＝7.0)，固定激发波长340nm，检测溶液位于420nm处荧光强度测定，由实施例4的线性回归方程计算测得氨基酸注射液中L-色氨酸的含量为108.10nmol/L。在此基础上，对氨基酸注射液进行L-色氨酸的加标回收实验，结果见表，可以看出，L-色氨酸的加标回收率位于99.30％-103.36％之间，RSD小于4.0％(n＝6)。结果表明，本发明提供的β-环糊精硫量子点成功应用于氨基酸注射液中L-色氨酸的定量分析。Add β-cyclodextrin sulfur quantum dots to 2.0mL amino acid injection, then add 2.0mL Tris-HCl buffer solution (pH=7.0), fix the excitation wavelength at 340nm, and measure the fluorescence intensity at 420nm of the detection solution. The linear regression equation of 4 calculated and measured the content of L-tryptophan in the amino acid injection to be 108.10nmol/L. On this basis, the addition and recovery experiment of L-tryptophan was carried out on the amino acid injection. 4.0% (n=6). The results show that the β-cyclodextrin sulfur quantum dots provided by the present invention are successfully applied to the quantitative analysis of L-tryptophan in amino acid injection.
表1氨基酸注射液中L-色氨酸的测定Determination of L-tryptophan in table 1 amino acid injection
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。The technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.
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