CN112279909B - Method for reducing oxidized cytochrome c by laser induction - Google Patents
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Abstract
The invention discloses a method for reducing oxidized cytochrome c by laser induction, belongs to the technical field of protein reduction methods, and aims to solve the problem that K is generated under the condition of 532nm laser irradiation 2 Ti 6 O 13 The nanowire can reduce oxidized cytochrome c to reduced cytochrome c. The method is simple and easy to realize; the product is detected by Raman spectrum, the detection sensitivity is high, the sample pretreatment is not needed, and the SERS fingerprint information can identify the difference between the oxidized cytochrome c and the reduced cytochrome c.
Description
Technical Field
The invention belongs to the technical field of protein reduction methods, and particularly relates to a method for reducing oxidized cytochrome c by laser induction.
Background
The material using Ti as the substrate has the advantages of high chemical stability, good surface performance, controllable photoelectric performance, low toxicity, low potential, easy preparation method and the like. Materials with Ti as the substrate are widely applied in the aspects of photoelectric coupling, energy conversion and storage, photocatalysis, chemical catalysis, biocatalysis, Surface Enhanced Raman Scattering (SERS) and the like. Due to its biocompatibility and environmental friendliness, it is often used as a good carrier material and scaffold material in the biomedical field. M 2 TinO 2n+1 (M may be Na) + ，K + Or H + ) Is made of eight uniform TiO 6 The unit composed layered wide band gap semiconductor has cations between layers, and can be widely applied to the fields of photocatalysis, gas sensing, hydrothermal treatment and the like. The titanate nanowire has a special high specific surface area, can improve the optical activity of the titanate nanowire in the aspects of catalysis and SERS, and has a good application prospect in the fields of environmental purification, carbonate decomposition, hydrogen preparation, biological detection, photoelectron and the like. At present, such as H 2 Ti 3 O 7 And K 2 Ti 6 O 13 The preparation of titanate layered nanometer wire structure such as nanometer wire is researched and reported more, and the crystal lattice of the nanometer wire is controlledThe distance thickness can easily control the band gap, thereby improving the success rate of preparing titanate nano-wires.
In the research based on SERS technology or Resonance Raman Scattering (RRS), it is found that the metal oxide semiconductor nanoparticles with protein functionalization can generate electron transfer reaction. Protein structural information on metals and semiconductors can be detected using SERS and RRS techniques. To investigate the chemical principles of raman enhancement in semiconductors or proteins, the present team has conducted a series of studies. We can now independently study the raman-enhanced chemical effect of the semiconductor/probe molecule conjugated system, ignoring the electromagnetic effect, because this system is different from the metal-supported system, in which the enhancement of the electromagnetic effect is very small and therefore negligible. This helps us to understand the chemical structure of the protein, as this raman enhancement forms a charge transfer complex between the nanomaterial and the protein. Cyt c is an ideal protein and is widely used in Raman technology research, and recently, reduction of Cyt c and heme enzymes by using certain substances has attracted extensive attention, which will help researchers understand the thermodynamic nature and the kinetic processes thereof. After the heme protein is combined with a certain substance, the compound has higher charge transfer rate and sensing performance, is beneficial to the research on the aspects of biomolecular structure change, physical and chemical processes, metabolic processes, energy conversion and the like, and the most important of the compound is beneficial to researching the physiological function and action mechanism of the compound in vivo and developing a novel biosensor.
Disclosure of Invention
The invention provides a method for reducing oxidized cytochrome c by laser induction. Under the condition of 532nm laser irradiation, K 2 Ti 6 O 13 The nanowire can reduce oxidized cytochrome c to reduced cytochrome c. Based on the charge transfer mechanism, 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 is adsorbed to K by hydrophobic or electrostatic interaction 2 Ti 6 O 13 The surface of the nanowire is conjugated and combined with the nanowire, and the Raman spectrum is enhanced. And, at K 2 Ti 6 O 13 In the presence of the nanowires, oxidized cytochrome c is reduced to reduced cytochrome c by laser light having a wavelength of 532 nm.
In order to achieve the above object, the present invention provides a method for reducing oxidized cytochrome c induced by laser, comprising the steps of:
(1) preparation K 2 Ti 6 O 13 Nanowire and method of manufacturing the same
Weighing 14.00g of KOH, putting into a clean beaker, and pouring distilled water till 25mL, so that the KOH is fully dissolved to form 10mol/L alkaline solution; 2.00g of TiO are weighed 2 (P25), putting the mixture into the beaker to mix with an alkali solution, injecting the uniformly mixed liquid into a 25mL high-pressure reaction kettle attached with polytetrafluoroethylene lining, then placing the high-pressure reaction kettle into an electric heating constant-temperature air-blast drying box, setting the temperature at 140-.
(2) At K 2 Ti 6 O 13 Nanowire surface adsorption oxidation type cytochrome c
The oxidized cytochrome c was dissolved in PBS buffer at a concentration of 10-20. mu.M/L. Will K 2 Ti 6 O 13 Dispersing nanowires in an oxidized cytochrome c solution, incubating at 37 deg.C for 2 hours, centrifuging and washing with PBS buffer to remove unadsorbed oxidized cytochrome c molecules, drying and adding K adsorbed with oxidized cytochrome c 2 Ti 6 O 13 The nanowires are flattened on the surface of the glass slide.
(3) Irradiation of K with laser light having a wavelength of 532nm 2 Ti 6 O 13 And reducing the oxidized cytochrome c to reduced cytochrome c on the surface of the nanowire.
The invention has the advantages that:
the method is simple and easy to realize; the product is detected by Raman spectrum, the detection sensitivity is high, the sample pretreatment is not needed, and the SERS fingerprint information can identify the difference between the oxidized cytochrome c and the reduced cytochrome c.
Drawings
FIG. 1: k 2 Ti 6 O 13 Transmission electron microscopy of nanowires.
FIG. 2: h 2 Ti 3 O 7 Transmission electron microscopy of nanowires.
FIG. 3: TiO 2 2 Transmission electron microscopy of nanoparticles.
FIG. 4: a, oxidized cytochrome c Raman spectrum; b, reduced cytochrome c Raman spectrum; c, adsorption of oxidized cytochrome c to K 2 Ti 6 O 13 SERS spectra of nanowires (oxidized cytochrome c reduced).
FIG. 5: a, Raman spectrum of oxidized cytochrome c; b, reduced cytochrome c Raman spectrum; c, adsorption of oxidized cytochrome c to H 2 Ti 3 O 7 SERS spectra of the nanowires; d, spectrum c is amplified ten times (oxidized cytochrome c is not reduced).
FIG. 6: a, Raman spectrum of oxidized cytochrome c; b, reduced cytochrome c Raman spectrum; c, adsorption of oxidized cytochrome c to TiO 2 (P25) SERS spectrum of the nano-nanoparticles; d, spectrum c is amplified ten times (oxidized cytochrome c is not reduced).
Detailed Description
Example 1: a method for reducing oxidized cytochrome c by laser induction. Adsorption of oxidized cytochrome c to K 2 Ti 6 O 13 And H 2 Ti 3 O 7 And performing SERS spectrum differential analysis on the nanowires.
(1)K 2 Ti 6 O 13 Nanowire preparation
Weighing 14.00g of KOH, putting the KOH into a clean beaker, and pouring distilled water till the volume is 25mL, so that the KOH is fully dissolved to form 10mol/L alkaline solution; weighing2.00g TiO 2 (P25), putting the mixture into the beaker to mix with an alkali solution, injecting the uniformly mixed liquid into 25mL of a high-pressure reaction kettle attached with polytetrafluoroethylene lining, then placing the high-pressure reaction kettle into an electric heating constant-temperature blowing drying box, setting the temperature to be 140-.
(2)H 2 Ti 3 O 7 Preparation of nanowires
Mixing the K prepared in the step (1) 2 Ti 6 O 13 Dispersing the nanowires in 50mL of 0.01mol/L hydrochloric acid solution, pouring the solution into a conical flask, placing a magnet, covering a plug, placing the conical flask on a magnetic stirrer, stirring the solution for 24 hours, repeating the process for 3 times, transferring the obtained solution into a centrifugal test tube, placing the centrifugal test tube in a high-speed centrifuge at the speed of 8000-10000 r/min, then pouring off the supernatant, and finally drying the obtained milky precipitate.
(3) Adsorption of oxidized cytochrome c to K 2 Ti 6 O 13 And H 2 Ti 3 O 7 Nanowire surfaces
Oxidized cytochrome c was dissolved in PBS buffer at a concentration of 10-20. mu.M/L. Will K 2 Ti 6 O 13 And H 2 Ti 3 O 7 Dispersing nanowires in an oxidized cytochrome c solution, incubating for 2 hours at 37 ℃, centrifuging and washing with PBS buffer solution to remove unadsorbed oxidized cytochrome c molecules, drying and adsorbing K of oxidized cytochrome c 2 Ti 6 O 13 And H 2 Ti 3 O 7 The nanowires are flattened on the surface of the glass slide.
(4) SERS detection
Adsorbing K containing oxidized cytochrome c 2 Ti 6 O 13 And H 2 Ti 3 O 7 Performing SERS test on the nanowire slide glass to obtainSERS fingerprint spectrum; the SERS spectrometer is a LabRam Aramis type Raman spectrometer manufactured by JobinYvon France and is provided with an excitation wavelength of 532 nm.
(5) Sample analysis
Under the irradiation of 532nm excitation wavelength, K 2 Ti 6 O 13 The nanowire has reducibility, and can reduce oxidation type cytochrome c, and H 2 Ti 3 O 7 The nanowires are not capable of reducing oxidized cytochrome c.
Example 2: a method for reducing oxidized cytochrome c by laser induction. Adsorption of oxidized cytochrome c to K 2 Ti 6 O 13 Nanowire and TiO 2 (P25) SERS spectral discriminatory analysis of nanoparticles.
(1)K 2 Ti 6 O 13 Nanowire preparation
Weighing 14.00g of KOH, putting into a clean beaker, and pouring distilled water till 25mL, so that the KOH is fully dissolved to form 10mol/L alkaline solution; 2.00g of TiO are weighed 2 (P25), putting the mixture into the beaker to mix with an alkali solution, injecting the uniformly mixed liquid into 25mL of a high-pressure reaction kettle attached with polytetrafluoroethylene lining, then placing the high-pressure reaction kettle into an electric heating constant-temperature blowing drying box, setting the temperature to be 140-.
(2) Adsorption of oxidized cytochrome c to K 2 Ti 6 O 13 Nanowire and TiO 2 (P25) surface of nanoparticle
Oxidized cytochrome c was dissolved in PBS buffer at a concentration of 10-20. mu.M/L. Will K 2 Ti 6 O 13 Nanowire and TiO 2 (P25) Dispersion of nanoparticles in oxidized cytochrome c solution incubated for 2 hours at 37 ℃ and then centrifuged and washed with PBS buffer to remove unadsorbed oxidized cytochrome c molecules, and finally driedDrying and adsorbing the oxidized cytochrome c-adsorbed K 2 Ti 6 O 13 Nanowire and TiO 2 (P25) the nanoparticles were flattened onto the slide surface.
(3) SERS detection
Adsorbing K containing oxidized cytochrome c 2 Ti 6 O 13 Nanowire and TiO 2 (P25) carrying out SERS test on the nanoparticle glass slide to obtain an SERS fingerprint; the SERS spectrum tester is a LabRam Aramis type Raman spectrometer manufactured by JobinYvon France and is provided with an excitation wavelength of 532 nm.
(4) Sample analysis
Under the irradiation of 532nm excitation wavelength, K 2 Ti 6 O 13 The nano-wire has reducibility and can reduce oxidation type cytochrome c, and the TiO has the advantages of 2 The nanoparticles are unable to reduce oxidized cytochrome c.
Claims (2)
1. A method for reducing oxidized cytochrome c by laser induction, which is characterized in that the oxidized cytochrome c is adsorbed on K 2 Ti 6 O 13 Reducing oxidized cytochrome c to reduced cytochrome c on the surface of the nanowire under the laser irradiation condition with the wavelength of 532 nm;
at K 2 Ti 6 O 13 The method for adsorbing the oxidized cytochrome c on the surface of the nanowire comprises the following specific steps:
dissolving oxidized cytochrome c in PBS buffer solution, wherein the concentration of the oxidized cytochrome c is 10-20 mu M/L;
will K 2 Ti 6 O 13 Dispersing the nanowires in an oxidized cytochrome c solution, incubating at 37 deg.C for 2 hr, centrifuging, washing with PBS buffer to remove unadsorbed oxidized cytochrome c molecules, drying, and separating K adsorbed with oxidized cytochrome c 2 Ti 6 O 13 The nanowires are flattened on the surface of the glass slide.
2. The method of laser-induced reduction of oxidized cytochrome c according to claim 1,K 2 Ti 6 O 13 the preparation method of the nanowire comprises the following specific steps:
weighing 13.5-14.5 g of KOH, putting into a clean beaker, and pouring distilled water till 25mL, so that the KOH is fully dissolved to form 9.5-10.5 mol/L alkaline solution; 2.00g of TiO are weighed 2 Adding the mixture into the beaker to be mixed with an alkali solution, injecting the uniformly mixed liquid into a 25mL high-pressure reaction kettle attached with polytetrafluoroethylene lining, then keeping the temperature of the high-pressure reaction kettle at 140-180 ℃, keeping the temperature for 48-96h, taking out the reaction kettle, naturally cooling the reaction kettle to room temperature to obtain milky white precipitate, taking out the precipitate, washing, centrifuging and drying to obtain K 2 Ti 6 O 13 A nanowire.
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| CN109540865A (en) * | 2018-11-15 | 2019-03-29 | 江南大学 | A kind of detection method based on Raman-fluorescent dual module formula probe living cells inner cell pigment c |
| CN111175280A (en) * | 2020-02-24 | 2020-05-19 | 江苏师范大学 | Cytochrome detection method based on surface enhanced Raman spectroscopy |
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| CN109540865A (en) * | 2018-11-15 | 2019-03-29 | 江南大学 | A kind of detection method based on Raman-fluorescent dual module formula probe living cells inner cell pigment c |
| CN111175280A (en) * | 2020-02-24 | 2020-05-19 | 江苏师范大学 | Cytochrome detection method based on surface enhanced Raman spectroscopy |
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