CN112279909B - Method for reducing oxidized cytochrome c by laser induction - Google Patents
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- 102100030497 Cytochrome c Human genes 0.000 title claims abstract description 67
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- 230000006698 induction Effects 0.000 title 1
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Abstract
本发明公开了一种激光诱导氧化型细胞色素c还原的方法，属于蛋白质还原方法技术领域，在532nm激光照射条件下，K2Ti6O13纳米线可以将氧化型细胞色素c还原成还原型的细胞色素c。方法简单，容易实现；生成物通过拉曼光谱检测，检测灵敏度高，不需要样品前处理，其SERS指纹信息可以鉴定氧化型细胞色素c和还原型细胞色素c的区别。
The invention discloses a method for laser-induced reduction of oxidized cytochrome c, belonging to the technical field of protein reduction methods. Under the condition of 532 nm laser irradiation, K 2 Ti 6 O 13 nanowires can reduce oxidized cytochrome c to reduced form cytochrome c. The method is simple and easy to implement; the resultant is detected by Raman spectroscopy, with high detection sensitivity, no sample pretreatment is required, and the SERS fingerprint information can identify the difference between oxidized cytochrome c and reduced cytochrome c.
Description
技术领域technical field
本发明属于蛋白质还原方法技术领域，具体涉及一种激光诱导氧化型细胞色素c还原的方法。The invention belongs to the technical field of protein reduction methods, in particular to a method for laser-induced reduction of oxidized cytochrome c.
背景技术Background technique
以Ti为基底的材料具有化学稳定性高，表面性能良好，光电性能具有可控性，毒性小，电势低，并且制备方法容易等诸多优点。以Ti为基底的材料在光电耦合，能量的转换与储存，光催化，化学催化，生物催化和表面增强拉曼散射(SERS)等方面得到了广泛的应用。因其生物兼容性和环境友好性在生物学医学领域经常被用来作为一种良好的载体材料和支架材料。M2TinO2n+1(M可以为Na+，K+或者H+)是一种由八个均匀的TiO6单元组成的层状宽带隙半导体，阳离子位于层与层之间，可以广泛地应用于光催化，气体传感和水热处理等领域。钛酸盐纳米线具有特殊的高比表面积，能够提高其在催化和SERS方面的光学活性，在环境净化，碳酸盐分解，氢的制备，生物检测和光电子等领域有很好的应用前景。目前，诸如H2Ti3O7和K2Ti6O13纳米线等钛酸盐层状纳米线结构制备的研究和报道较多，通过控制纳米线晶格距离的厚度，可以很容易的控制带隙，从而可以提高制备钛酸盐纳米线的成功率。Ti-based materials have many advantages, such as high chemical stability, good surface properties, controllable optoelectronic properties, low toxicity, low potential, and easy preparation methods. Ti-based materials have been widely used in photoelectric coupling, energy conversion and storage, photocatalysis, chemical catalysis, biocatalysis, and surface-enhanced Raman scattering (SERS). Because of its biocompatibility and environmental friendliness, it is often used as a good carrier material and scaffold material in the field of biomedicine. M 2 TinO 2n+1 (M can be Na + , K + or H + ) is a layered wide-bandgap semiconductor composed of eight uniform TiO 6 units, with cations located between layers, which can be widely used In the fields of photocatalysis, gas sensing and hydrothermal treatment. Titanate nanowires have a special high specific surface area, which can improve their optical activity in catalysis and SERS, and have good application prospects in the fields of environmental purification, carbonate decomposition, hydrogen production, biodetection, and optoelectronics. At present, there are many studies and reports on the preparation of titanate layered nanowire structures such as H 2 Ti 3 O 7 and K 2 Ti 6 O 13 nanowires. By controlling the thickness of the nanowire lattice distance, it can be easily controlled band gap, which can improve the success rate of preparing titanate nanowires.
基于SERS技术或共振拉曼散射(RRS)的研究中，发现带有蛋白质功能化的金属氧化物半导体纳米颗粒可以发生电子转移反应。在金属和半导体上的蛋白质结构信息可以采用SERS和RRS技术检测出来。为了研究在半导体或蛋白质中拉曼增强的化学原理，本团队已经开展了一系列研究。我们现在可以在忽略电磁效应的前提下，独立的研究半导体/探针分子共轭体系的拉曼增强化学效应，因为此体系不同于金属支撑体系，在此体系中，电磁效应增强非常小，因此可以忽略。因为这种拉曼增强在纳米材料和蛋白质之间形成了电荷转移复合体，所以这有助于帮助我们理解蛋白质的化学结构。Cyt c作为一种理想的蛋白质，它被广泛的应用于拉曼技术的研究中，最近，通过使用某些物质对cyt c和亚铁血红素酶进行还原，已经引起了广泛的关注，这将有助于研究者了解热力学本质及其动力学过程。将亚铁血红素蛋白和某种物质结合后，具有较高的电荷转移率和传感性能，有益于对生物分子结构变化，物理和化学过程，新陈代谢过程和能量转换等方面的研究，其中最重要是有助于探究其在体内的生理功能和作用机理，并开发新型生物传感器。In studies based on SERS technology or resonance Raman scattering (RRS), it was found that metal oxide semiconductor nanoparticles with protein functionalization can undergo electron transfer reactions. Protein structural information on metals and semiconductors can be detected using SERS and RRS techniques. In order to study the chemistry of Raman enhancement in semiconductors or proteins, the team has carried out a series of studies. We can now independently study the Raman-enhanced chemical effect of the semiconductor/probe-molecular conjugated system under the premise of ignoring the electromagnetic effect, because this system is different from the metal support system, in which the electromagnetic effect enhancement is very small, so Can be ignored. Because this Raman enhancement forms a charge-transfer complex between the nanomaterial and the protein, it helps us understand the chemical structure of the protein. As an ideal protein, Cyt c has been widely used in Raman technology research. Recently, the reduction of cyt c and heme by using certain substances has attracted extensive attention, which will It is helpful for researchers to understand the nature of thermodynamics and its kinetic process. After combining heme protein with a certain substance, it has high charge transfer rate and sensing performance, which is beneficial to the research on changes in biomolecular structure, physical and chemical processes, metabolic processes and energy conversion, among which the most It is important to help explore its physiological function and mechanism of action in vivo, and to develop new biosensors.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种激光诱导氧化型细胞色素c还原的方法。在532nm激光照射条件下，K2Ti6O13纳米线可以将氧化型细胞色素c还原成还原型的细胞色素c。在电荷转移机理的基础上，cyt c可以作为一种新型的探针用来研究发生在基质和被吸附的生物分子之间的电荷转移。发现cyt c通过疏水作用或者静电相互作用吸附在K2Ti6O13纳米线的表面，两者共轭结合后，拉曼光谱得到了增强。并且，在K2Ti6O13纳米线存在的情况下，用波长为532nm的激光将氧化型细胞色素c还原成还原型的细胞色素c。The invention provides a method for laser-induced reduction of oxidized cytochrome c. Under the condition of 532nm laser irradiation, K 2 Ti 6 O 13 nanowires can reduce oxidized cytochrome c to reduced cytochrome c. Based on the mechanism of charge transfer, cyt c can be used as a novel probe to study the charge transfer between the substrate and the adsorbed biomolecules. It was found that cyt c was adsorbed on the surface of K 2 Ti 6 O 13 nanowires through hydrophobic interaction or electrostatic interaction, and the Raman spectra were enhanced after the two were conjugated. Then, in the presence of K 2 Ti 6 O 13 nanowires, oxidized cytochrome c was reduced to reduced cytochrome c with a laser light having a wavelength of 532 nm.
为实现上述目的，本发明所述的一种激光诱导氧化型细胞色素c的还原方法，包括以下步骤：In order to achieve the above object, the method for reducing laser-induced oxidized cytochrome c according to the present invention comprises the following steps:
(1)制备K2Ti6O13纳米线(1) Preparation of K 2 Ti 6 O 13 nanowires
称量14.00g KOH，投入干净的烧杯中，再倒入蒸馏水直到25mL，使其充分溶解形成10mol/L的碱性溶液；称取2.00g TiO2(P25)，投入上述烧杯中与碱溶液混合，将混合均匀的混合液体注入25mL附有聚内衬四氟乙烯的高压反应釜中，之后将高压反应釜置于电热恒温鼓风干燥箱中，设置温度为140-180℃，保温48-96h，然后取出反应釜使其自然冷却至室温，得到乳白色沉淀，取出沉淀，将其用去离子水洗涤3次，将所得溶液放入离心试管，高速离心机进行离心，速度为8000-10000转/分钟，然后倒掉上层清夜，最后将所得乳白色沉淀进行干燥。Weigh 14.00g KOH, put it into a clean beaker, pour in distilled water until 25mL, make it fully dissolved to form an alkaline solution of 10mol/L; weigh 2.00g TiO 2 (P25), put it into the above beaker and mix with the alkaline solution , inject the mixed liquid into a 25mL autoclave with poly-lined tetrafluoroethylene, then place the autoclave in an electric heating constant temperature blast drying oven, set the temperature to 140-180°C, and keep the temperature for 48-96h. , then take out the reactor and let it cool to room temperature naturally to obtain a milky white precipitate, take out the precipitate, wash it with deionized water 3 times, put the obtained solution into a centrifugal test tube, and centrifuge with a high-speed centrifuge at a speed of 8000-10000 rpm/ minutes, then the supernatant was poured off, and finally the resulting milky white precipitate was dried.
(2)在K2Ti6O13纳米线表面吸附氧化型细胞色素c(2) Adsorption of oxidized cytochrome c on the surface of K 2 Ti 6 O 13 nanowires
将氧化型细胞色素c溶解在PBS缓冲液中，氧化型细胞色素c的浓度为10-20μM/L。将K2Ti6O13纳米线分散在氧化型细胞色素c溶液中，在37℃下孵育2小时，然后离心并用PBS缓冲液洗涤以去除未吸附的氧化型细胞色素c分子，最后干燥并将吸附有氧化型细胞色素c的K2Ti6O13纳米线压平在载玻片表面。The oxidized cytochrome c was dissolved in PBS buffer, and the concentration of oxidized cytochrome c was 10-20 μM/L. K 2 Ti 6 O 13 nanowires were dispersed in oxidized cytochrome c solution, incubated at 37 °C for 2 h, then centrifuged and washed with PBS buffer to remove unadsorbed oxidized cytochrome c molecules, and finally dried and put The K 2 Ti 6 O 13 nanowires adsorbed with oxidized cytochrome c were flattened on the glass slide surface.
(3)用波长为532nm的激光照射K2Ti6O13纳米线表面，将氧化型细胞色素c还原成还原型的细胞色素c。(3) The surface of K 2 Ti 6 O 13 nanowires is irradiated with a laser with a wavelength of 532 nm to reduce oxidized cytochrome c to reduced cytochrome c.
本发明优点在于：The advantages of the present invention are:
方法简单，容易实现；生成物通过拉曼光谱检测，检测灵敏度高，不需要样品前处理，其SERS指纹信息可以鉴定氧化型细胞色素c和还原型细胞色素c的区别。The method is simple and easy to implement; the resultant is detected by Raman spectroscopy, with high detection sensitivity, no sample pretreatment is required, and the SERS fingerprint information can identify the difference between oxidized cytochrome c and reduced cytochrome c.
附图说明Description of drawings
图1：K2Ti6O13纳米线的透射电镜图。Figure 1 : TEM image of K2Ti6O13 nanowires .
图2：H2Ti3O7纳米线的透射电镜图。Figure 2 : TEM image of H2Ti3O7 nanowires.
图3：TiO2纳米粒子的透射电镜图。Figure 3: TEM image of TiO2 nanoparticles.
图4：a,氧化型细胞色素c拉曼光谱图；b,还原型细胞色素c拉曼光谱图；c,氧化型细胞色素c吸附于K2Ti6O13纳米线的SERS光谱图(氧化型细胞色素c被还原)。Figure 4: a, Raman spectrum of oxidized cytochrome c; b, Raman spectrum of reduced cytochrome c; c, SERS spectrum of oxidized cytochrome c adsorbed on K 2 Ti 6 O 13 nanowires (oxidation type cytochrome c is reduced).
图5：a,氧化型细胞色素c拉曼光谱图；b,还原型细胞色素c拉曼光谱图；c,氧化型细胞色素c吸附于H2Ti3O7纳米线的SERS光谱图；d,光谱c放大十倍(氧化型细胞色素c未被还原)。Figure 5: a, Raman spectrum of oxidized cytochrome c; b, Raman spectrum of reduced cytochrome c; c, SERS spectrum of oxidized cytochrome c adsorbed on H 2 Ti 3 O 7 nanowires; d , the spectrum c is magnified tenfold (oxidized cytochrome c is not reduced).
图6：a,氧化型细胞色素c拉曼光谱图；b,还原型细胞色素c拉曼光谱图；c,氧化型细胞色素c吸附于TiO2(P25)纳米纳米粒子的SERS光谱图；d,光谱c放大十倍(氧化型细胞色素c未被还原)。Figure 6: a, Raman spectrum of oxidized cytochrome c; b, Raman spectrum of reduced cytochrome c; c, SERS spectrum of oxidized cytochrome c adsorbed on TiO 2 (P25) nanoparticles; d , the spectrum c is magnified tenfold (oxidized cytochrome c is not reduced).
具体实施方式Detailed ways
实施例1：一种激光诱导氧化型细胞色素c的还原方法。氧化型细胞色素c吸附在K2Ti6O13和H2Ti3O7纳米线的SERS光谱区别分析。Example 1: A laser-induced reduction method of oxidized cytochrome c. Differential analysis of SERS spectra of oxidized cytochrome c adsorbed on K 2 Ti 6 O 13 and H 2 Ti 3 O 7 nanowires.
(1)K2Ti6O13纳米线制备(1) Preparation of K 2 Ti 6 O 13 nanowires
称量14.00g KOH，投入干净的烧杯中，再倒入蒸馏水直到25mL，使其充分溶解形成10mol/L的碱性溶液；称取2.00g TiO2(P25)，投入上述烧杯中与碱溶液混合，将混合均匀的混合液体注入25mL附有聚内衬四氟乙烯的高压反应釜中，之后将高压反应釜置于电热恒温鼓风干燥箱中，设置温度为140-180℃，保温48-96h，然后取出反应釜使其自然冷却至室温，得到乳白色沉淀，取出沉淀，将其用去离子水洗涤3次，将所得溶液放入离心试管，高速离心机进行离心，速度为8000-10000转/分钟，然后倒掉上层清夜，最后将所得乳白色沉淀进行干燥。Weigh 14.00g KOH, put it into a clean beaker, pour in distilled water until 25mL, make it fully dissolved to form an alkaline solution of 10mol/L; weigh 2.00g TiO 2 (P25), put it into the above beaker and mix with the alkaline solution , inject the mixed liquid into a 25mL autoclave with poly-lined tetrafluoroethylene, then place the autoclave in an electric heating constant temperature blast drying oven, set the temperature to 140-180°C, and keep the temperature for 48-96h. , then take out the reactor and let it cool to room temperature naturally to obtain a milky white precipitate, take out the precipitate, wash it with deionized water 3 times, put the obtained solution into a centrifugal test tube, and centrifuge with a high-speed centrifuge at a speed of 8000-10000 rpm/ minutes, then the supernatant was poured off, and finally the resulting milky white precipitate was dried.
(2)H2Ti3O7纳米线的制备(2) Preparation of H 2 Ti 3 O 7 nanowires
将步骤(1)制备的K2Ti6O13纳米线分散在50mL，0.01mol/L的盐酸溶液中，倒入锥形瓶，放入磁石，盖上塞子，置于磁力搅拌器之上，搅拌24h，此过程重复3次，将所得溶液转入离心试管，在置于高速离心机中，速度为8000-10000转/分钟，然后倒掉上层清夜，最后将所得乳白色沉淀进行干燥。Disperse the K 2 Ti 6 O 13 nanowires prepared in step (1) in 50 mL of 0.01 mol/L hydrochloric acid solution, pour into a conical flask, put in a magnet, cover with a stopper, and place it on a magnetic stirrer, Stir for 24h, repeat this process three times, transfer the obtained solution into a centrifuge test tube, place it in a high-speed centrifuge at a speed of 8000-10000 rpm, then pour off the supernatant, and finally dry the obtained milky white precipitate.
(3)氧化型细胞色素c吸附在K2Ti6O13和H2Ti3O7纳米线表面(3) Adsorption of oxidized cytochrome c on the surface of K 2 Ti 6 O 13 and H 2 Ti 3 O 7 nanowires
氧化型细胞色素c溶解在PBS缓冲液中，浓度为10-20μM/L。将K2Ti6O13和H2Ti3O7纳米线分散在氧化型细胞色素c溶液中孵育2小时，37℃，然后离心并用PBS缓冲液洗涤以去除未吸附的氧化型细胞色素c分子，最后干燥并将吸附有氧化型细胞色素c的K2Ti6O13和H2Ti3O7的纳米线压平在载玻片表面。Oxidized cytochrome c was dissolved in PBS buffer at a concentration of 10-20 μM/L. K 2 Ti 6 O 13 and H 2 Ti 3 O 7 nanowires were dispersed in oxidized cytochrome c solution and incubated for 2 h at 37°C, then centrifuged and washed with PBS buffer to remove unadsorbed oxidized cytochrome c molecules , and finally dried and flattened the nanowires of K 2 Ti 6 O 13 and H 2 Ti 3 O 7 adsorbed with oxidized cytochrome c on the glass slide surface.
(4)SERS检测(4) SERS detection
将放有氧化型细胞色素c吸附的K2Ti6O13和H2Ti3O7的纳米线载玻片进行SERS测试，得到SERS指纹图谱；SERS光谱测试仪器为法国JobinYvon公司生产的LabRam Aramis型拉曼光谱仪，配备532nm激发波长。The nanowire glass slides with K 2 Ti 6 O 13 and H 2 Ti 3 O 7 adsorbed by oxidative cytochrome c were subjected to SERS test, and the SERS fingerprint was obtained; the SERS spectrum test instrument was LabRam Aramis produced by JobinYvon, France Raman spectrometer with 532nm excitation wavelength.
(5)样品分析(5) Sample analysis
在532nm激发波长照射下，K2Ti6O13纳米线具有还原性，可以将氧化型细胞色素c还原，而H2Ti3O7纳米线不能将氧化型细胞色素c的还原。Under the excitation wavelength of 532nm, K 2 Ti 6 O 13 nanowires have reducing properties and can reduce oxidized cytochrome c, while H 2 Ti 3 O 7 nanowires cannot reduce oxidized cytochrome c.
实施例2：一种激光诱导氧化型细胞色素c的还原方法。氧化型细胞色素c吸附在K2Ti6O13纳米线和TiO2(P25)纳米粒子的SERS光谱区别分析。Example 2: A laser-induced reduction method of oxidized cytochrome c. Differential analysis of SERS spectra of oxidized cytochrome c adsorbed on K 2 Ti 6 O 13 nanowires and TiO 2 (P25) nanoparticles.
(1)K2Ti6O13纳米线制备(1) Preparation of K 2 Ti 6 O 13 nanowires
称量14.00g KOH，投入干净的烧杯中，再倒入蒸馏水直到25mL，使其充分溶解形成10mol/L的碱性溶液；称取2.00g TiO2(P25)，投入上述烧杯中与碱溶液混合，将混合均匀的混合液体注入25mL附有聚内衬四氟乙烯的高压反应釜中，之后将高压反应釜置于电热恒温鼓风干燥箱中，设置温度为140-180℃，保温48-96h，然后取出反应釜使其自然冷却至室温，得到乳白色沉淀，取出沉淀，将其用去离子水洗涤3次，将所得溶液放入离心试管，高速离心机进行离心，速度为8000-10000转/分钟，然后倒掉上层清夜，最后将所得乳白色沉淀进行干燥。Weigh 14.00g KOH, put it into a clean beaker, pour in distilled water until 25mL, make it fully dissolved to form an alkaline solution of 10mol/L; weigh 2.00g TiO 2 (P25), put it into the above beaker and mix with the alkaline solution , inject the mixed liquid into a 25mL autoclave with poly-lined tetrafluoroethylene, then place the autoclave in an electric heating constant temperature blast drying oven, set the temperature to 140-180°C, and keep the temperature for 48-96h. , then take out the reactor and let it cool to room temperature naturally to obtain a milky white precipitate, take out the precipitate, wash it with deionized water 3 times, put the obtained solution into a centrifugal test tube, and centrifuge with a high-speed centrifuge at a speed of 8000-10000 rpm/ minutes, then the supernatant was poured off, and finally the resulting milky white precipitate was dried.
(2)氧化型细胞色素c吸附在K2Ti6O13纳米线和TiO2(P25)纳米粒子表面(2) Adsorption of oxidized cytochrome c on the surface of K 2 Ti 6 O 13 nanowires and TiO 2 (P25) nanoparticles
氧化型细胞色素c溶解在PBS缓冲液中，浓度为10-20μM/L。将K2Ti6O13纳米线和TiO2(P25)纳米粒子分散在氧化型细胞色素c溶液中孵育2小时，37℃，然后离心并用PBS缓冲液洗涤以去除未吸附的氧化型细胞色素c分子，最后干燥并将吸附有氧化型细胞色素c的K2Ti6O13纳米线和TiO2(P25)纳米粒子压平在载玻片表面。Oxidized cytochrome c was dissolved in PBS buffer at a concentration of 10-20 μM/L. K 2 Ti 6 O 13 nanowires and TiO 2 (P25) nanoparticles were dispersed in oxidized cytochrome c solution and incubated for 2 hours at 37°C, then centrifuged and washed with PBS buffer to remove unadsorbed oxidized cytochrome c Molecules, finally dried and flattened on the glass slide surface with K 2 Ti 6 O 13 nanowires and TiO 2 (P25) nanoparticles adsorbed with oxidized cytochrome c.
(3)SERS检测(3) SERS detection
将放有氧化型细胞色素c吸附的K2Ti6O13纳米线和TiO2(P25)纳米粒子载玻片进行SERS测试，得到SERS指纹图谱；SERS光谱测试仪器为法国JobinYvon公司生产的LabRamAramis型拉曼光谱仪，配备532nm激发波长。The K 2 Ti 6 O 13 nanowires and TiO 2 (P25) nanoparticles on which oxidized cytochrome c were adsorbed were subjected to SERS test to obtain the SERS fingerprint; Raman spectrometer equipped with 532 nm excitation wavelength.
(4)样品分析(4) Sample analysis
在532nm激发波长照射下，K2Ti6O13纳米线具有还原性，可以将氧化型细胞色素c还原，而TiO2纳米粒子不能将氧化型细胞色素c的还原。Under the excitation wavelength of 532nm, K 2 Ti 6 O 13 nanowires have reducing properties and can reduce oxidized cytochrome c, while TiO 2 nanoparticles cannot reduce oxidized cytochrome c.
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