CN114518348A - Method and device for detecting allergic protein based on coffee ring effect - Google Patents
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Abstract
本发明涉及生物分析检测技术领域,尤其涉及一种检测过敏蛋白的方法和装置。该方法基于咖啡环效应对过敏蛋白进行检测,无需复杂仪器和操作即可快速检测出是否含有待测过敏蛋白,准确性好、灵敏度高,可实现非专业人员在现场任何地方进行食物中有无待测过敏蛋白的简单、便捷、快速检测。The invention relates to the technical field of biological analysis and detection, in particular to a method and device for detecting allergic proteins. The method detects allergenic proteins based on the coffee ring effect, and can quickly detect whether the allergenic protein to be tested is contained without complex instruments and operations. Simple, convenient and rapid detection of allergenic proteins to be tested.
Description
技术领域technical field
本发明涉及医学分析检测技术领域,尤其涉及一种检测过敏蛋白的方法和检测装置。The invention relates to the technical field of medical analysis and detection, in particular to a method and a detection device for detecting allergic proteins.
背景技术Background technique
鱼类过敏是由于鱼类中富含大量的异种蛋白,这些异种蛋白直接或间接地激活免疫细胞,引起化学介质的释放,继而产生一系列复杂的生物化学反应。过敏反应程度可以从轻微的局部症状到严重的全身性反应,严重时可能产生过敏性休克,甚至危及生命。小清蛋白(Parvalbumin,PV)是鱼类主要致敏原,90%的鱼类过敏患者会对PV产生过敏反应。目前,该过敏蛋白的检测方法主要有ELISA(酶联免疫吸附测定)法和PCR(聚合酶链式反应)法。这些方法需要高昂的仪器设备以及专业的操作人员,操作要求技术性强,且较费时。因在便捷程度、检测速度等方面的局限性而难以满足市场的应用需求。现阶段已报道多种基于纳米金的比色传感器,但是易受到干扰,灵敏度较低。Fish allergy is due to the abundance of a large number of heterologous proteins in fish, which activate immune cells directly or indirectly, causing the release of chemical mediators, and then producing a series of complex biochemical reactions. Allergic reactions can range from mild local symptoms to severe systemic reactions, which can lead to anaphylactic shock and even life-threatening reactions. Parvalbumin (PV) is the main allergen in fish, and 90% of fish allergy patients will have an allergic reaction to PV. At present, the detection methods of the allergic protein mainly include ELISA (enzyme-linked immunosorbent assay) method and PCR (polymerase chain reaction) method. These methods require expensive equipment and professional operators, and the operation is highly technical and time-consuming. Due to limitations in convenience and detection speed, it is difficult to meet the application needs of the market. A variety of colorimetric sensors based on gold nanoparticles have been reported at this stage, but they are susceptible to interference and have low sensitivity.
“咖啡环效应”是指液滴蒸发时,溶质会在边缘沉积出一个颜色较深的环状沉积渍的现象。该现象普遍存在于胶体颗粒、纳米颗粒及小分子体系之中。大量研究表明,通过液滴的“咖啡环效应”,可成功实现多种待检目标物的鉴定及定量检测,在诊断分析、生物传感技术等领域有着巨大的应用前景。The "coffee ring effect" refers to the phenomenon in which the solute will deposit a darker ring-shaped deposit on the edge as the droplet evaporates. This phenomenon generally exists in colloidal particles, nanoparticles and small molecule systems. A large number of studies have shown that the identification and quantitative detection of various targets to be detected can be successfully achieved through the "coffee ring effect" of droplets, which has great application prospects in the fields of diagnostic analysis and biosensing technology.
促进与抑制“咖啡环效应”对于实际的技术应用具有重要意义:DNA沉积物进行光谱分析时,沉积物越均匀分析越准确,抑制咖啡环效应可以得到更精确的分析图谱;利用咖啡环筛分颗粒的功能,可以实现有机大分子的分离和提纯;根据血液蒸干后图案的差异可以快速检测疾病,适用于医疗条件较差地区;利用咖啡环效应可以制备微型电路或是透明电路。同时,通过液滴蒸发对颗粒进行组装不需要额外提供能量,且简单易操作,可用来分离不同类型的血细胞诊断疾病,在分选生物大颗粒、浓缩样品、均匀喷涂和增强分析信号等领域均有用武之地。Promoting and inhibiting the "coffee ring effect" is of great significance for practical technical applications: when DNA sediments are subjected to spectral analysis, the more uniform the sediment is, the more accurate the analysis is, and the more accurate analysis patterns can be obtained by inhibiting the coffee ring effect; the use of coffee ring sieving The function of particles can realize the separation and purification of organic macromolecules; diseases can be quickly detected according to the difference in the pattern of blood after drying, which is suitable for areas with poor medical conditions; microcircuits or transparent circuits can be prepared by using the coffee ring effect. At the same time, the assembly of particles by droplet evaporation does not require additional energy, and is simple and easy to operate. It can be used to separate different types of blood cells to diagnose diseases. Useful place.
发明内容SUMMARY OF THE INVENTION
有鉴于此,本发明提供了一种检测过敏蛋白的方法和装置。该方法基于咖啡环效应对过敏蛋白进行检测,准确性好、灵敏度高,可供非专业人员无需复杂仪器和操作即可快速检测出是否含有待测过敏蛋白。In view of this, the present invention provides a method and device for detecting allergic proteins. The method is based on the coffee ring effect for the detection of allergenic proteins, with good accuracy and high sensitivity, and can be used by non-professionals to quickly detect whether the allergenic protein to be detected is contained without complex instruments and operations.
为了实现上述发明目的,本发明提供以下技术方案:In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供一种基于咖啡环效应检测过敏蛋白的方法,包括:The present invention provides a method for detecting allergic proteins based on the coffee ring effect, comprising:
将待测样本与检测试剂混合均匀,获得混合液;Mix the sample to be tested and the detection reagent evenly to obtain a mixed solution;
将所述混合液滴至玻璃载玻片,静置,待液滴蒸发形成“咖啡环”沉淀;Put the mixed droplets onto a glass slide, let stand, and wait for the droplets to evaporate to form a "coffee ring" precipitate;
对所述沉淀进行紫外照射,根据荧光信号判断待测样本中是否含有过敏蛋白;Perform ultraviolet irradiation on the precipitate, and determine whether the sample to be tested contains an allergic protein according to the fluorescent signal;
所述检测试剂为修饰有核酸适配体的纳米金颗粒;The detection reagent is gold nanoparticles modified with nucleic acid aptamers;
所述核酸适配体为特异性结合待测过敏蛋白的核酸适配体。The nucleic acid aptamer is a nucleic acid aptamer that specifically binds to the allergic protein to be tested.
“咖啡环效应”是指液滴蒸发时,由于液滴的蒸发通量的不均匀分布,边缘处的蒸发通量较大,导致液滴内部浓度不均匀,因而液滴内部会产生由内向外的补偿流动,这种流动被称为毛细流动。在蒸发过程中由于毛细流动的作用,液滴内部大部分的颗粒会被带到液滴边缘三相接触线处,并且堆积到一起形成沉积,将接触线钉扎,最终颗粒会在边缘沉积出一个颜色较深的环状沉积的现象。"Coffee ring effect" means that when the droplet evaporates, due to the uneven distribution of the evaporation flux of the droplet, the evaporation flux at the edge is larger, resulting in uneven concentration inside the droplet, so the inside of the droplet will produce a flow from the inside to the outside. compensating flow, which is called capillary flow. During the evaporation process, due to the action of capillary flow, most of the particles inside the droplet will be brought to the three-phase contact line at the edge of the droplet, and they will accumulate together to form a deposit, pin the contact line, and finally the particles will be deposited on the edge. A phenomenon of darker annular deposits.
本发明以咖啡环效应为理论基础对过敏蛋白进行检测,以小清蛋白为例进行说明。当待测样本中在存在小清蛋白时,核酸适配体与小清蛋白特异性结合后形成的复合物与金颗粒相比阻力较大、迁移率较低,所以金颗粒在毛细流动作用下堆积在液滴边缘,而核酸适配体-蛋白质复合物则在中间部分形成沉淀;由于纳米金是一种较强的能量受体,具有荧光共振能量转移(FRET)效应,是一种良好的荧光猝灭剂,在携带荧光基团的核酸适配体与纳米金颗粒组合后,荧光基团因靠近金纳米颗粒导致荧光淬灭。而当纳米金-核酸适配体探针与小清蛋白识别后,核酸适配体与小清蛋白特异性结合形成复合物,使核酸适配体-纳米金组装体解组装,从而使核酸适配体上的荧光基团远离纳米金颗粒,荧光基团恢复荧光。The invention detects allergenic proteins based on the coffee ring effect, and takes parvalbumin as an example for illustration. When parvalbumin is present in the sample to be tested, the complex formed by the specific binding of nucleic acid aptamer and parvalbumin has greater resistance and lower mobility than gold particles, so the gold particles can be affected by capillary flow. It accumulates on the edge of the droplet, and the nucleic acid aptamer-protein complex forms a precipitate in the middle part; because gold nanoparticles are a strong energy acceptor, they have the effect of fluorescence resonance energy transfer (FRET), which is a good Fluorescence quencher, after the nucleic acid aptamer carrying a fluorophore is combined with gold nanoparticles, the fluorophore is close to the gold nanoparticles, resulting in fluorescence quenching. When the nano-gold-aptamer probe recognizes parvalbumin, the aptamer specifically binds to parvalbumin to form a complex, which disassembles the aptamer-nano-gold assembly, thereby making the nucleic acid suitable for The fluorophore on the ligand moves away from the gold nanoparticles, and the fluorophore restores fluorescence.
本发明中,待测样本为食物样本,对食物来源没有任何限定,本领域常见的均可一些实施方案中,对食物形态没有任何限定,将食物样本制成溶液后进行检测。一些实施方案中,所述待测样本包括鱼肉类生食、鱼类熟食、含鱼类零食及调味品等食物样本。In the present invention, the sample to be tested is a food sample, and there is no restriction on the source of the food, which is common in the art. In some embodiments, there is no restriction on the form of the food, and the food sample is prepared into a solution for detection. In some embodiments, the samples to be tested include food samples such as raw fish food, cooked fish food, fish-containing snacks and condiments.
一些实施方案中,所述过敏蛋白包括小清蛋白、牛血清蛋白、卵清蛋白等常见过敏蛋白或中的至少一种。一些具体实施例中,所述过敏蛋白为小清蛋白。In some embodiments, the allergenic protein includes at least one of common allergenic proteins such as parvalbumin, bovine serum albumin, ovalbumin, or the like. In some specific embodiments, the allergenic protein is parvalbumin.
一些实施方案中,所述待测过敏蛋白的核酸适配体连接有荧光基团。其中,荧光基团的种类没有特殊限定,本领域熟知的种类能通过紫外灯照射后有荧光反应均可。In some embodiments, the nucleic acid aptamer of the allergenic protein to be tested is linked with a fluorescent group. The types of fluorescent groups are not particularly limited, and those well-known in the art can be irradiated with an ultraviolet lamp and have a fluorescent reaction.
采用本发明提供的方法进行过敏蛋白检测之前,取样之后,将样本制成溶液,获得所述待测样本。一些实施方案中,待测样本和检测试剂均匀混合。Before using the method provided by the present invention to detect allergic proteins, and after sampling, the sample is made into a solution to obtain the sample to be tested. In some embodiments, the sample to be tested and the detection reagent are uniformly mixed.
一些实施方案中,所述静置的时间为20~40min。经过静置,液滴蒸发,然后形成“咖啡环”沉淀。In some embodiments, the standing time is 20-40 min. Upon standing, the droplets evaporate and then form a "coffee ring" precipitate.
一些实施方案中,所述判断的方法为:In some embodiments, the method of judging is:
若检测到荧光信号,则判定待测样本中含有过敏蛋白;若未检测到荧光信号,则判定待测样本中不含过敏蛋白。If a fluorescent signal is detected, it is determined that the sample to be tested contains an allergic protein; if no fluorescent signal is detected, it is determined that the sample to be tested does not contain an allergic protein.
本发明还提供一种基于咖啡环效应检测过敏蛋白的装置,包括试剂管、玻片、紫外灯和检测试剂;所述检测试剂为修饰有待测过敏蛋白的核酸适配体的纳米金颗粒。The invention also provides a device for detecting allergic proteins based on the coffee ring effect, comprising a reagent tube, a glass slide, an ultraviolet lamp and a detection reagent; the detection reagent is gold nanoparticles modified with nucleic acid aptamers of the allergic protein to be detected.
本发明提供的基于咖啡环效应的过敏蛋白的检测方法和装置在存在过敏蛋白(如小清蛋白)时,金颗粒与携带荧光基团的核酸适配体-蛋白复合物在咖啡环效应作用下有效分离,让光照后荧光反应更明显,便于结果的鉴定。另外,操作流程简单无需复杂工具,可供非专业人员在现场进行检测,且灵敏度高,结果判定不受主观因素影响,可以定性确定过敏蛋白——小清蛋白的有无。The detection method and device for allergenic proteins based on the coffee ring effect provided by the present invention When the allergenic proteins (such as parvalbumin) are present, the gold particles and the nucleic acid aptamer-protein complex carrying a fluorescent group are under the effect of the coffee ring effect. Effective separation makes the fluorescence reaction more obvious after illumination, which is convenient for the identification of results. In addition, the operation process is simple and does not require complex tools, which can be detected by non-professionals on site, and the sensitivity is high. The result judgment is not affected by subjective factors, and the presence or absence of the allergenic protein - parvalbumin can be qualitatively determined.
附图说明Description of drawings
图1示本发明检测过敏蛋白的操作流程示意图;Fig. 1 shows the operation flow schematic diagram that the present invention detects allergic protein;
图2示液滴蒸发时毛细流动示意图;Figure 2 shows a schematic diagram of capillary flow when droplets evaporate;
图3示存在小清蛋白时液滴内溶质颗粒流向示意图;Figure 3 shows a schematic diagram of the flow of solute particles in a droplet in the presence of parvalbumin;
图4示纳米金-核酸适配体探针与靶蛋白结合机理示意图;Figure 4 shows a schematic diagram of the binding mechanism between the nano-gold-aptamer probe and the target protein;
图5示荧光“OFF-ON”探针示意图;Figure 5 shows a schematic diagram of a fluorescent "OFF-ON" probe;
图6示环状沉淀进行紫外线光照后的反应示意图;Fig. 6 shows the reaction schematic diagram after ring-shaped precipitation carries out ultraviolet irradiation;
图7示根据荧光反应鉴别结果的示意图。FIG. 7 is a schematic diagram showing the identification results according to the fluorescence reaction.
具体实施方式Detailed ways
本发明提供了一种检测过敏蛋白的方法和装置。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。The present invention provides a method and device for detecting allergic proteins. Those skilled in the art can learn from the content of this document and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.
如无特殊说明,本发明采用的试材皆为普通市售品,皆可于市场购得。Unless otherwise specified, the test materials used in the present invention are all common commercial products and can be purchased in the market.
应理解,本发明提到的一个或多个方法步骤并不排斥在所述组合步骤前后还存在其他方法步骤或在这些明确提到的步骤之间还可以插入其他方法步骤;还应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。而且,除非另有说明,各方法步骤的编号仅为鉴别各方法步骤的便利工具,而非为限制各方法步骤的排列次序或限定本发明可实施的范围,其相对关系的改变或调整,在无实质变更技术内容的情况下,当亦视为本发明可实施的范畴。为了更清楚地说明本发明实施例的技术方案,下面将结合本发明的附图说明,对本发明实施例中的技术方案进行清楚、完整地描述,应当理解,所描述的实施例仅仅是本发明的一部分实施例,而不是全部的实施例,因此不应被看作是对保护范围的限定。基于本发明中的实施例,本领域普通技术工作人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。It should be understood that the one or more method steps mentioned in the present invention do not exclude that there are other method steps before and after the combination step or other method steps may be inserted between these explicitly mentioned steps; it should also be understood that these The examples are only used to illustrate the present invention and not to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the arrangement order of each method step or limiting the scope of the present invention. In the case where the technical content is not substantially changed, it should also be regarded as the scope in which the present invention can be implemented. In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following will describe the technical solutions in the embodiments of the present invention clearly and completely with reference to the accompanying drawings. It should be understood that the described embodiments are only the present invention. Some of the embodiments, but not all of the embodiments, should not be construed as limiting the scope of protection. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.
下面结合实施例,进一步阐述本发明:Below in conjunction with embodiment, the present invention is further elaborated:
实施例1Example 1
本发明检测装置包括试剂管、玻片、紫外灯和检测试剂;所述检测试剂为携带荧光基团并修饰有小清蛋白的核酸适配体的纳米金颗粒。The detection device of the present invention includes a reagent tube, a glass slide, an ultraviolet lamp and a detection reagent; the detection reagent is a nano-gold particle carrying a fluorescent group and modified with a parvalbumin nucleic acid aptamer.
(1)检测原理:由于液滴的蒸发通量的不均匀分布,边缘处的蒸发通量较大,导致液滴内部浓度不均匀,因而液滴内部会产生由内向外的补偿流动,这种流动被称为毛细流动。在蒸发过程中由于毛细流动的作用,液滴内部大部分的颗粒会被带到液滴边缘三相接触线处,并且堆积到一起形成沉积,将接触线钉扎,如图2。最终颗粒会在边缘沉积出一个颜色较深的环状沉积的现象。在存在过敏蛋白情况下,核酸适配体与过敏蛋白特异性结合后形成的复合物与金颗粒相比阻力较大、迁移率较低,所以金颗粒在毛细流动作用下堆积在液滴边缘,而核酸适配体-蛋白质复合物则在中间部分形成沉淀,如图3;由于纳米金是一种较强的能量受体,能够通过荧光共振能量转移(FRET)效应淬灭荧光分子,在携带荧光基团的核酸适配体与纳米金颗粒组合后,荧光基团因靠近金纳米颗粒导致荧光淬灭。而当纳米金-核酸适配体探针与过敏蛋白识别后,核酸适配体与过敏蛋白特异性结合形成复合物,使核酸适配体-纳米金组装体解组装,如图4。从而使核酸适配体上的荧光基团远离纳米金颗粒,荧光基团恢复荧光。如图5。(1) Detection principle: Due to the uneven distribution of the evaporation flux of the droplet, the evaporation flux at the edge is large, resulting in uneven concentration inside the droplet, so the inside of the droplet will generate compensation flow from the inside to the outside. The flow is called capillary flow. During the evaporation process, due to the action of capillary flow, most of the particles inside the droplet will be brought to the three-phase contact line at the edge of the droplet, and accumulate together to form a deposit, pinning the contact line, as shown in Figure 2. Eventually the particles will deposit a darker ring around the edges. In the presence of allergen, the complex formed by the specific binding of nucleic acid aptamer and allergen has higher resistance and lower mobility than gold particles, so gold particles accumulate on the edge of the droplet under the action of capillary flow. The nucleic acid aptamer-protein complex forms a precipitate in the middle part, as shown in Figure 3; since gold nanoparticles are a strong energy acceptor, they can quench fluorescent molecules through the fluorescence resonance energy transfer (FRET) effect, and carry After the nucleic acid aptamer of the fluorophore is combined with the gold nanoparticles, the fluorescence quenches due to the proximity of the fluorophore to the gold nanoparticles. When the nano-gold-aptamer probe recognizes the allergy protein, the nucleic acid aptamer specifically binds to the allergy protein to form a complex, and the nucleic acid aptamer-nano-gold assembly is disassembled, as shown in Figure 4. Thus, the fluorophore on the nucleic acid aptamer is kept away from the gold nanoparticles, and the fluorophore restores fluorescence. Figure 5.
(2)操作过程:从食物中取样配置待测蛋白溶液,与提前制备好的特异性试剂(携带荧光基团的纳米金-核酸探针)均匀混合,纳米金-核酸适配体探针与待检测蛋白相互识别和作用,将混合液滴到玻璃片上,静置蒸发,待后续检测。如图1。在液滴蒸发后,对其环状沉淀进行紫外线光照,如图6。通过沉淀是否呈现荧光反应来判断是否含有过敏蛋白。结果分析:液滴蒸发后咖啡环效应可将金颗粒与适配体-蛋白质复合物物分离,金颗粒大多在环上,而适配体-蛋白质复合物会聚集沉淀大多均匀分布在中间部分,其中的荧光基团因在光照后出现荧光反应即为含有过敏蛋白(阳性);若不含过敏蛋白,纳米金-核酸适配体位于环上,因适配体未远离金颗粒,适配体上携带的荧光基团因纳米金颗粒的荧光淬灭能力,在光照后不会出现荧光反应(阴性)如图7。(2) Operation process: sample the protein solution to be tested from the food, and mix it with the specific reagent (gold nanoparticles-nucleic acid probes carrying fluorescent groups) prepared in advance. The gold nanoparticles-nucleic acid aptamer probes are mixed with The proteins to be detected recognize and interact with each other, and the mixture is dropped onto the glass slide and left to evaporate for subsequent detection. Figure 1. After the droplets evaporated, their ring-shaped precipitates were subjected to UV irradiation, as shown in Figure 6. The presence of allergenic proteins was judged by whether the precipitate exhibited a fluorescent reaction. Analysis of results: The coffee ring effect can separate gold particles from aptamer-protein complexes after droplet evaporation. Most of the gold particles are on the ring, while the aptamer-protein complexes will aggregate and precipitate and most of them are evenly distributed in the middle part. The fluorescent group in it contains allergenic protein (positive) because of the fluorescent reaction after light exposure; if there is no allergic protein, the nano-gold-nucleic acid aptamer is located on the ring, because the aptamer is not far from the gold particles, the aptamer Due to the fluorescence quenching ability of gold nanoparticles, the fluorophore carried on it will not show a fluorescence reaction (negative) after illumination, as shown in Figure 7.
(3)反应条件分析:核酸适配体与蛋白质特异性结合、核酸适配体-纳米金组装体解组装、液滴蒸发生成环状沉淀均为自然现象,不需要额外提供外力、能量。且整个过程不需要高端设备和严苛的环境,可直接在现场取物快速检测。(3) Analysis of reaction conditions: the specific binding of nucleic acid aptamers to proteins, the disassembly of nucleic acid aptamer-gold nanoparticles, and the formation of ring-shaped precipitates by droplet evaporation are all natural phenomena, and do not require additional external force or energy. And the whole process does not require high-end equipment and harsh environments, and can be directly taken on-site for rapid testing.
实施例2鱼类食物中小清蛋白的检测Embodiment 2 Detection of parvalbumin in fish food
从鱼类食物中取样配置出小清蛋白溶液。具体步骤如下:The parvalbumin solution was prepared by sampling from fish food. Specific steps are as follows:
(1)将小清蛋白溶液与特异性试剂加入试剂管中均匀混合1.5h,其中,小清蛋白溶液浓度为100μg/mL;小清蛋白核酸适配体浓度为0.1mM;纳米金溶液浓度为20nM。(1) Add the parvalbumin solution and the specific reagents into the reagent tube and mix them evenly for 1.5 hours, wherein the concentration of the parvalbumin solution is 100 μg/mL; the concentration of the parvalbumin aptamer is 0.1 mM; the concentration of the nanogold solution is 20nM.
(2)将反应后的混合液提取5μL滴在玻璃片上水平静置蒸发36min后获得沉淀。沉淀为边缘环状沉淀与中部沉淀。(2) 5 μL of the reacted mixture was extracted and dropped on a glass slide and left to stand horizontally for 36 minutes to obtain a precipitate. The precipitation is an edge ring precipitation and a central precipitation.
(3)用紫外灯照射沉淀,观察到中部沉淀呈现出荧光反应。(3) The precipitate was irradiated with an ultraviolet lamp, and it was observed that the precipitate in the middle showed a fluorescent reaction.
(4)本实施例的检测结果为阳性,说明配置出的溶液中存在小清蛋白。(4) The detection result in this example is positive, indicating that parvalbumin is present in the prepared solution.
实施例3特异性检测Example 3 Specific detection
配置牛血清蛋白溶液。具体步骤如下:Prepare bovine serum albumin solution. Specific steps are as follows:
(1)将牛血清蛋白溶液与特异性试剂加入试剂管中均匀混合1.5h,其中,牛血清蛋白溶液浓度为100μg/mL;其余特异性试剂同实施例2。(1) The bovine serum albumin solution and the specific reagent were added to the reagent tube and mixed evenly for 1.5 hours, wherein the concentration of the bovine serum albumin solution was 100 μg/mL; the other specific reagents were the same as those in Example 2.
(2)将反应后的混合液提取5μL滴在玻璃片上水平静置蒸发38min后获得沉淀。沉淀为边缘环状沉淀。(2) 5 μL of the reacted mixed solution was extracted and dropped on a glass slide and left to stand horizontally for 38 minutes to obtain a precipitate. The precipitate was an edge-ring precipitate.
(3)用紫外灯照射沉淀,无荧光反应。(3) The precipitation was irradiated with an ultraviolet lamp, and there was no fluorescent reaction.
(4)本实施例的检测结果为阴性,说明配置出的溶液中不存在小清蛋白。小清蛋白核酸适配体不会与牛血清蛋白特异性结合,说明本发明试剂盒具有良好的特异性。(4) The test result in this example is negative, indicating that parvalbumin does not exist in the prepared solution. The parvalbumin nucleic acid aptamer does not specifically bind to bovine serum albumin, indicating that the kit of the present invention has good specificity.
实施例4灵敏度检测Example 4 Sensitivity detection
配置不同浓度小清蛋白溶液。具体步骤如下:Prepare different concentrations of parvalbumin solutions. Specific steps are as follows:
(1)将不同浓度的小清蛋白溶液与特异性试剂分别加入不同试剂管中均匀混合1.5h,其中,小清蛋白溶液浓度分别为1、2.5、5、10、20、50、100μg/mL;其余特异性试剂同实施例2。(1) Add the parvalbumin solutions and specific reagents of different concentrations into different reagent tubes and mix them evenly for 1.5 hours, wherein the concentrations of the parvalbumin solutions are 1, 2.5, 5, 10, 20, 50, and 100 μg/mL, respectively. ; Other specific reagents are the same as in Example 2.
(2)将反应后的混合液分别提取5μL滴在玻璃片上水平静置蒸发后获得沉淀。在小清蛋白溶液浓度为10μg/mL以上时沉淀均为边缘环状沉淀与中部沉淀。(2) 5 μL of the reacted mixed solution was extracted and dropped on a glass slide and left to stand horizontally to evaporate to obtain a precipitate. When the concentration of parvalbumin solution was above 10 μg/mL, the precipitates were both edge-ring and central.
(3)用紫外灯照射沉淀,在小清蛋白溶液浓度为10μg/mL及以上时均能观察到中部沉淀呈现出荧光反应。浓度为5μg/mL以下时荧光反应不明显。(3) The precipitate was irradiated with ultraviolet light, and the central precipitate showed fluorescent reaction when the concentration of the parvalbumin solution was 10 μg/mL and above. The fluorescence response was not obvious when the concentration was below 5μg/mL.
(4)本实施例的检测结果在小清蛋白溶液浓度为10μg/mL及以上时为阳性,说明本发明检测方法在样本浓度为10μg/mL及以上均能有效检测出结果。(4) The detection result of this embodiment is positive when the concentration of the parvalbumin solution is 10 μg/mL and above, indicating that the detection method of the present invention can effectively detect the result when the sample concentration is 10 μg/mL and above.
实施例5准确性检测Embodiment 5 Accuracy detection
分别配置待测溶液:小清蛋白溶液与牛血清蛋白溶液。具体步骤如下:Prepare the solutions to be tested separately: parvalbumin solution and bovine serum albumin solution. Specific steps are as follows:
(1)将小清蛋白溶液、牛血清蛋白溶液与特异性试剂同时加入试剂管中均匀混合1.5h,其中,小清蛋白溶液浓度为100μg/mL;牛血清蛋白溶液浓度为100μg/mL;其余特异性试剂同实施例2。(1) Add the parvalbumin solution, the bovine serum albumin solution and the specific reagent to the reagent tube at the same time and mix them evenly for 1.5 hours, wherein the concentration of the parvalbumin solution is 100 μg/mL; the concentration of the bovine serum albumin solution is 100 μg/mL; the rest The specific reagents are the same as those in Example 2.
(2)将反应后的混合液提取5μL滴在玻璃片上水平静置蒸发35min后获得沉淀。沉淀为边缘环状沉淀与中部沉淀。(2) 5 μL of the reacted mixture was extracted and dropped on a glass slide and left to stand horizontally for 35 minutes to obtain a precipitate. The precipitation is an edge ring precipitation and a central precipitation.
(3)用紫外灯照射沉淀,观察到中部沉淀呈现出荧光反应。(3) The precipitate was irradiated with an ultraviolet lamp, and it was observed that the precipitate in the middle showed a fluorescent reaction.
(4)本实施例的检测结果为阳性,说明待测溶液中存在小清蛋白。在同时存在目标蛋白与其他蛋白时,本发明仍能有效检测出目标蛋白的存在。(4) The detection result of this embodiment is positive, indicating that parvalbumin exists in the solution to be tested. When the target protein and other proteins exist at the same time, the present invention can still effectively detect the existence of the target protein.
综上,本发明基于咖啡环效应对样品中小清蛋白的存在进行检测,对小清蛋白溶液的检测结果为阳性,对非小清蛋白的其他过敏蛋白呈现阴性结果,具有特异性。对浓度为10μg/mL以上的小清蛋白均能检测出阳性结果,具有较好的灵敏度。当小清蛋白与其他蛋白混合后检测仍能呈现出阳性结果,具有较好的准确性。且操作简单,检测时间较短,无需复杂工具与专业人员即可进行检测。In conclusion, the present invention detects the presence of parvalbumin in the sample based on the coffee ring effect, and the detection result is positive for the parvalbumin solution, and negative for other allergic proteins other than parvalbumin, which is specific. Positive results can be detected for parvalbumin with a concentration of more than 10μg/mL, with good sensitivity. When the parvalbumin is mixed with other proteins, the detection can still show a positive result with good accuracy. And the operation is simple, the detection time is short, and the detection can be carried out without complicated tools and professionals.
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不应理解为必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。此外,本领域的技术人员可以将本说明书中描述的不同实施例或示例进行接合和组合。In the description of this specification, description with reference to the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples", etc., mean specific features described in connection with the embodiment or example , structure, material or feature is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms should not be construed as necessarily referring to the same embodiment or example. Furthermore, the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, those skilled in the art may combine and combine the different embodiments or examples described in this specification.
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.
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