CN113125403B

CN113125403B - Raman-fluorescence dual-mode detection method for calcium ions based on dual-mode nano probe

Info

Publication number: CN113125403B
Application number: CN202110441854.3A
Authority: CN
Inventors: 马小媛; 李晨彪; 王周平
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2022-04-05
Anticipated expiration: 2041-04-23
Also published as: CN113125403A

Abstract

The invention discloses a Raman-fluorescence dual-mode nanoprobe which is used for detecting calcium ions in vitro and living cells or imaging. The detection method is based on the 'on-off' conversion of surface enhanced Raman scattering and fluorescence resonance energy transfer, and Ca is assembled on the nano-Venus²⁺And constructing the dual-mode nanoprobe by using the specific enzyme chain and the partially complementary substrate chain for modifying the fluorescent group. The method combines the advantages of SERS ultrasensitiveness and visual and visible fluorescence, has good specificity and sensitivity, and has good universality in various cell lines.

Description

Raman-fluorescence dual-mode detection method for calcium ions based on dual-mode nano probe

Technical Field

The invention relates to the field of nano materials and life science, in particular to a method for carrying out Raman-fluorescence dual-mode detection on calcium ions based on a dual-mode nano probe.

Background

Ca²⁺Not only are they pivotal among many second messengers, but they are also involved in or coordinate the metabolism of other second messengers and the regulation of cellular physiological functions. A large number of studies have found intracellular Ca²⁺Dyshomeostasis is a conserved biochemical event in apoptosis suggesting Ca²⁺Participate in the signal transduction process in apoptosis. When the cells are stimulated, the cells store Ca²⁺Release to cytosol to make Ca in cytosol²⁺Increased concentrations stimulate the relevant pathways and induce apoptosis. About Ca²⁺The recognition of the relationship to apoptosis also presents a number of problems that need to be addressed, thus tracking intracellular Ca²⁺Dynamic change of concentration and knowledge of Ca²⁺The role of the signal in the cellular pathway is very important. At present, intracellular Ca²⁺The concentration measuring method comprises electrode method, isotope tracer method, nuclear magnetic resonance method, ion indicator method, and fluorescence indicator methodAnd the like. The electrode method does not require an indicator, but the reaction rate is slow and cellular Ca cannot be measured²⁺And the penetration of the electrode through the cell membrane would damage the cell while not being suitable for too small cells. The isotope tracing method has high sensitivity but certain radioactive effect. The nuclear magnetic resonance method requires a fluorine-containing indicator, is non-destructive and nondestructive, and has a high measurement cost. The fluorescent indicator is widely applied to intracellular Ca due to the characteristics of convenience, high use safety and the like²⁺Detection and imaging.

Liulina et al (establishment of a method for detecting the concentration of calcium ions in primary neurons in rats by photoelectric combination, laboratory animal science, 2020, 37(06):7-10) utilizes a fluorescent probe fluo-4AM to mark calcium ions, electrically stimulates the neurons, and detects the rapid change of the intracellular fluorescence intensity by a turntable confocal technology, thereby displaying the concentration change of the calcium ions in the cells. However, acetoxymethyl ester (AM) probe is easily hydrolyzed and needs to be kept dry when used. In this case, novel Ca is designed and synthesized²⁺Probes are of great significance.

Disclosure of Invention

In order to solve the technical problems, the invention provides a dual-mode nanoprobe, which integrates fluorescence and SERS signals onto the same nanoparticle, so that the particle has fluorescence and SERS imaging capabilities at the same time, and can be quickly positioned and imaged through the fluorescence signals at first, and then multi-target tracking and quantitative research are carried out through the SERS technology, thereby effectively solving the problems of the fluorescence and SERS imaging technologies.

The invention discloses a dual-mode nanoprobe, which comprises nanogold and Ca²⁺Specific deoxyribozymes, surface ligand one and surface ligand two, Ca²⁺The specific deoxyribozyme, the surface ligand I and the surface ligand II are wrapped on the surface of the nano-gold star; ca²⁺The specific deoxyribozyme comprises a substrate chain and a polymerase chain, wherein the substrate chain is partially complementary with the polymerase chain, the enzyme cutting site is positioned on the substrate chain, and the polymerase chain can be modified by Ca²⁺Activating and acting on a restriction enzyme site and cutting a substrate chain; modifying fluorescent group at 5 'end or 3' end of substrate chain, and modifying sulfydryl at 5 'end or 3' end of enzyme chain; surface ligand oneComprises methoxy polyethylene glycol thiol (SH-mPEG), carboxyl polyethylene glycol thiol (SH-PEG-COOH) and amino polyethylene glycol thiol (SH-PEG-NH)₂) One or more of polyethylene glycol thiol (SH-PEG) and Bovine Serum Albumin (BSA), and the surface ligand II comprises one or more of 6-mercapto-1-hexanol (MCH), thioglycolic acid (TGA) and mercaptodibutanoic acid (MSA); the polymerase chain, the surface ligand I and the surface ligand II are connected with the nano-Au star through Au-S covalent bonds.

The polymerase chain, the SH-mPEG and the MCH are firmly wrapped on the surfaces of the nano-Au stars, so that mutual contact and aggregation among the nano-Au stars are prevented, MCH micromolecules occupy unoccupied sites of the polymerase chain and the SH-mPEG in a supplementing manner, and the stability of the dual-mode nano-probe is improved.

Further, the molar ratio of the nanogold, the substrate chain, the enzyme chain, the surface ligand I and the surface ligand II is 0.5-1.5: 1600-.

Further, the sequence of the substrate strand is specifically:

5 '-GTCACGAGTCCATATrAGGAAGATGGCGAAA-3'; wherein rA is an enzyme cutting site.

Further, the sequence of the enzyme chain is specifically:

5’-AAAAAAAAAGCCATCTTTTCTCACAGCGTACTCGCTAAGGTTGTTAGTGACTCGTGAC-3’。

furthermore, the nano-gold star is in a star shape, and the particle size is 60-70 nm. The method can generate a high electric field effect at the tail end of the gold needle tip structure and has a good surface enhanced Raman effect.

Further, the fluorophore includes one or more of Cy5, Cy5.5, Cy3, and ROX.

The preparation method of the dual-mode nanoprobe comprises the following steps:

(1) adding a thiol reducing agent into the PCR solution for reaction for 1-2h, adding nanogold, then carrying out oscillation reaction for 2-4h at 35-40 ℃, adding sodium chloride until the final concentration is 200-400mM, continuing the reaction for 11-13h, adding a surface ligand I after the reaction is finished, carrying out centrifugal washing, and adding a surface ligand II to obtain PCR functionalized nanogold;

(2) and (2) adding a substrate chain solution into the polymerase chain functionalized nano-Venus obtained in the step (1), incubating for 3-4h at 35-40 ℃, and centrifuging to remove redundant substrate chains to obtain the dual-mode nano-probe.

Further, in step (1), the concentration of the enzyme chain solution is 170-.

Further, in step (1), the final concentration of sodium chloride was 200-400 mM. The aging is completed by adding sodium chloride with proper concentration, so that the nano-gold star is not aggregated, the electrostatic repulsion between nucleic acid and the nano-gold star can be overcome, the connection between DNA and AuNSs is completed, and the degradation of nuclease to the DNA is prevented by the high-concentration salt environment around the nucleic acid, thereby protecting the stability of the dual-mode nano-probe.

Further, in step (1), the thiol reducing agent comprises TCEP and/or DTT. The purpose of adding thiol reducing agents is to cleave the dimercapto bond formed between the enzyme chains.

Further, in the step (2), the concentration of the substrate strand solution is 30 to 50. mu.M.

The invention relates to a calcium ion dual-mode detection method for non-diagnosis and non-treatment purposes, which comprises the following steps:

uniformly mixing the dual-mode nanoprobes on a calcium-free minimal medium, co-culturing the dual-mode nanoprobes with a cell to be detected for 6-7h, sucking out the calcium-free minimal medium and cleaning, adding the toxin-containing calcium-free minimal medium for co-culturing for 30-90min, sucking out the calcium-free minimal medium and cleaning, dyeing and fixing the cell, and carrying out SERS spectrum detection, SERS imaging or fluorescence imaging to obtain Ca in the cell²⁺The detection result of (3);

or mixing the dual-mode nanoprobe with Ca²⁺The solution is mixed and reacted, and after centrifugation, SERS spectrum detection, SERS imaging or fluorescence imaging is carried out to obtain in vitro Ca²⁺The detection result of (1).

Further, the toxin includes T-2 toxin. Under normal conditions, the intracellular calcium ion concentration is stable and mainly located in an intracellular calcium reservoir, cells begin to die under the induction of toxin, and the intracytoplasmic calcium ion concentration is increased, so that the calcium ion concentration in the intracellular die process is monitored.

Further comprisesIn a toxin-free minimal calcium medium, the toxin concentration preferably corresponds to the IC at the cultivation time₅₀The value is obtained. The concentration can ensure that the toxin generates certain toxicity on cells to be detected and certain cell survival rate is maintained.

Further, the concentration of T-2 toxin is 5-15. mu.M, preferably 11. mu.M.

Further, when the dual-mode nanoprobe is mixed with the calcium-free minimal medium, the concentration of the dual-mode nanoprobe is 0.1-0.5nM, preferably 0.25 nM. This concentration allows for higher cell viability and better detection.

Further, the calcium-free minimal medium is preferably RPMI-1640 calcium-free minimal medium.

Furthermore, the cells to be detected comprise one or more of human cervical cancer cell Hela, human liver cancer cell HepG2, human breast cancer cell MCF-7 and human colorectal adenocarcinoma cell Caco-2.

Further, Ca was carried out²⁺Also includes Pb during detection²⁺The blocking agent can be selected from L-cysteine, acetate, thioglycolic acid (TGA), dimercaptobutanol (BAL), MCH, etc., Pb²⁺The concentration of the blocking agent is 1-10 mM. Most deoxyribozymes can be substituted by Pb²⁺Activating and adding Pb²⁺The blocking agent can block Pb²⁺To exclude interference in detection without affecting Ca²⁺Detection of (3).

The detection method of the present invention is based on the "on-off" conversion of Surface Enhanced Raman Scattering (SERS) and Fluorescence Resonance Energy Transfer (FRET). By assembling Ca on AuNSs²⁺The specific enzyme chain EtNa-Enz and the partially complementary substrate chain EtNa-Sub for modifying the fluorescent group construct the dual-mode nanoprobe, the ultraviolet absorption peak of AuNSs is obviously overlapped with the emission spectrum part of the fluorescent group, so the AuNSs can quench the fluorescence thereof. The bimodal nanoprobes are capable of entering cells when Ca is present in the cells²⁺When it is Ca²⁺The activity of the specific deoxyribozyme is activated, the substrate chain is cut into two sections by the enzyme chain, the substrate chain marked by the fluorescent group is cracked and released from the surface of AuNSs, the enhancement of the fluorescent signal and the reduction of the Raman signal are realized, and the Ca is further realized²⁺Detection of dual signals。

Further, the nano-gold star is prepared by the following steps:

(1) boiling the chloroauric acid solution, adding the sodium citrate solution, stirring vigorously, and cooling to obtain a gold seed solution;

(2) and (2) uniformly mixing the silver nitrate solution and the ascorbic acid solution, quickly adding the mixture into the gold seed solution obtained in the step (1), and centrifuging to obtain the nano-Au star.

By the scheme, the invention at least has the following advantages:

(1) the invention combines the dual advantages of Raman and fluorescence, after the probe enters the cell, the initial fluorescence signal is quenched, the fluorescence background signal is very weak, the fluorescence signal is recovered along with the apoptosis, and the process of calcium ion concentration increase can be reflected and monitored more intuitively, thereby realizing the Ca concentration increase in the apoptosis process²⁺The concentration change is monitored in real time, the preparation is simple, the property is stable, and the biocompatibility is high.

(2) The SERS-fluorescence dual-mode probe based on the specificity recognition function and the enzyme digestion activity of DNA enzyme not only can detect Ca²⁺Has excellent selectivity and sensitivity, and can realize Ca in the process of apoptosis²⁺The real-time monitoring of the concentration change is beneficial to knowing Ca²⁺The role of the signal in the cellular pathway.

(3) The dual-mode nanoprobe has low toxicity and no damage to cells, is easy to load into cells, and has low synthesis cost and detection cost.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is a TEM image and UV-visible absorption spectrum of a gold seed prepared in example 1;

FIG. 2 is a TEM micrograph and UV-VIS spectrum of AuNSs prepared in example 1;

FIG. 3 is a statistical plot of the NanoMeasurer software versus the particle size of AuNSs prepared in example 1;

FIG. 4 shows the optimization results of the enzyme chain (EtNa-Enz) concentration;

FIG. 5 shows the optimization results of the substrate strand (EtNa-Sub) concentration;

FIG. 6 shows the stability test results of the dual-mode nanoprobe;

FIG. 7 shows the application of a dual-mode nanoprobe for in vitro Ca²⁺Raman detection results;

FIG. 8 is at 1366cm^-1A linear plot of SERS intensity;

FIG. 9 shows the application of dual-mode nanoprobes for in vitro Ca²⁺Detecting the result of fluorescence;

FIG. 10 is a graph of the linear relationship of fluorescence intensity at 670 nm;

FIG. 11 shows the selective results of Raman signals of the dual-mode nanoprobe;

FIG. 12 shows the selective results of the fluorescence signal of the dual-mode nanoprobe;

FIG. 13 shows the results of different concentrations of T-2 toxin (20. mu.M, 16. mu.M, 12. mu.M, 10. mu.M, 8. mu.M, 6. mu.M, 4. mu.M, 2. mu.M, 0. mu.M) infecting HeLa cells;

FIG. 14 shows the results of the CCK-8 method for detecting cell viability in different culture media;

FIG. 15 is an optical microscope to observe the effect of different media on cell morphology and adherence;

FIG. 16 shows cytotoxicity of dual-mode nanoprobes with Hela cells in different concentrations of AuNSs (0.5nM, 0.25nM, 0.125nM) for different periods of co-culture;

FIG. 17 is a TEM image showing the uptake of the bimodal nanoprobe by Hela cells;

FIG. 18 is a diagram showing ICP-MS for evaluating the uptake of the bimodal nanoprobe by Hela cells;

FIG. 19 is a diagram of a dual mode nanoprobe for confocal laser imaging in living cells;

FIG. 20 shows Fluo-4/AM for confocal laser imaging in living cells;

FIG. 21 is a comparison of fluorescence intensity of Fluo-4/AM and a bimodal nanoprobe;

FIG. 22 is a diagram of a dual mode nanoprobe for SERS imaging in living cells;

FIG. 23 is a diagram of a dual-mode nanoprobe for SERS spectroscopy in living cells;

FIG. 24 is Ca in different cell lines for dual-mode nanoprobes²⁺Fluorescence imaging;

FIG. 25 shows Ca in different cell lines for dual-mode nanoprobes²⁺SERS imaging;

FIG. 26 is a SERS spectrum of dual mode nanoprobes in different cell lines stimulated for 0min by T-2 toxin;

FIG. 27 is a SERS spectrum of dual mode nanoprobes in different cell lines stimulated for 30min by T-2 toxin.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

(1) Boiling 100mL of 1mM chloroauric acid solution, adding 15mL of 1% sodium citrate solution, stirring vigorously, reacting for 10min, turning the solution into wine red, stopping the reaction, and cooling to room temperature to obtain gold seed solution. The gold seeds were stored at 4 ℃ until use. And (3) characterizing the gold seeds by using a TEM (transmission electron microscope) image and an ultraviolet-visible absorption spectrogram, wherein the result is shown in FIG. 1, A is the TEM image, and B is the ultraviolet-visible absorption spectrogram.

(2) mu.L of a 3mM silver nitrate solution and 100. mu.L of a 0.1M ascorbic acid solution were mixed well and quickly added to 20mL of a 0.25mM chloroauric acid solution already containing 200. mu.L of gold species. The reaction solution was centrifuged at 4000rpm for 15min to obtain Nanoguang (AuNSs), which was resuspended in 2mL of 0.05% Tween 20 (Tween-20). And (3) characterizing the nano-Au by using a TEM (transmission electron microscope) image and an ultraviolet-visible absorption spectrogram, wherein the result is shown in FIG. 2, A is the TEM image, and B is the ultraviolet-visible absorption spectrogram. And the particle size of the nano-star is measured and analyzed by using the NanoMeasurer1.2 software (measuring no less than 50 nano-particle samples on an electron microscope image), and the result is shown in FIG. 3.

(3) mu.L of the enzyme chain (EtNa-Enz) solution was added with 1. mu.L of 20mM TCEP (Ready-made) and reacted at room temperature for 1 hour; adding 100 mu L of AuNSs solution prepared above, shaking for reaction at 37 ℃ for 3h, then adding 2M sodium chloride solution for multiple times until the final concentration is 300mM, mixing uniformly, and reacting for 12h under continuous shaking. To stabilize the nanoprobes, 1mM SH-mPEG (5k) was added to the solution and left for 30 min. The solution was then washed by centrifugation (4000rpm, 15min) and resuspended in Tris-HCl buffer (10mM, pH 8.0) containing 0.01% Tween-20. The AuNSs was then surface-passivated with 0.1mM 6-mercapto-1-hexanol (MCH) at 37 ℃ for 10min, washed three times by centrifugation and resuspended in Tris-HCl buffer to obtain the enzyme chain functionalized Nanogonging (EtNa-Enz-AuNSs).

To obtain the maximum loading, the concentrations of the EtNa-Enz solutions were modified to 60. mu.M, 80. mu.M, 100. mu.M, 120. mu.M, 160. mu.M, 180. mu.M, 190. mu.M and 200. mu.M, respectively, for optimization, to determine the decrease in absorbance (. DELTA.A) at 260nm of the supernatant after centrifugation₂₆₀) The nanoparticles were demonstrated to be modified by DNA. FIG. 4 is a graph showing the decrease in absorbance (. DELTA.A) at 260nm for EtNa-Enz solution₂₆₀) And (6) obtaining the result.

As can be seen from FIG. 1, the gold seeds are spherical particles with a particle size of about 13nm, and the nanoparticles are uniformly distributed and have good morphology and dispersion degree; the obtained nano ion sol shows wine red, and the maximum absorption wavelength is 519 nm.

As can be seen from FIG. 2A, the synthesized AuNSs has uniform particle size, a large number of irregular spikes are distributed on the surface, and the appearance is star-shaped; as can be seen from fig. 2B, the ultraviolet absorption peak of AuNSs is around 800nm, and there is a significant overlap with the emission spectrum of Cy5, which indicates that AuNSs can quench the fluorescence of Cy5.

Fig. 3 shows that the AuNSs mean particle size is 66.84 nm.

FIG. 4 shows that as the concentration of EtNa-Enz increases, Δ A₂₆₀Is remarkably increased. When the concentration reached 180. mu.M,. DELTA.A₂₆₀Hardly changed. Therefore, the optimal concentration of EtNa-Enz was determined to be 180. mu.M.

EXAMPLE 2 hybridization experiment of substrate chain (EtNa-Sub) with EtNa-Enz-AuNSs

A concentration of EtNa-Sub was added to a solution of 1nM EtNa-Enz-AuNSs. The mixture was incubated at 37 ℃ for 3h, and then the mixture was subjected to high speed centrifugation (5000rpm, 15min) to remove excess substrate strands, and resuspended in 10mM Tris-HCl buffer (80mM LiCl, 0.01% Tween-20, pH 8.0) to obtain a dual-mode nanoprobe. To obtain the maximum loading, the EtNa-Sub solution concentrations were modified to 5 μ M, 10 μ M, 15 μ M, 20 μ M, 25 μ M, 35 μ M and 50 μ M for optimization, demonstrating the hybridization of EtNa-Sub to EtNa-Enz-AuNSs with SERS enhancement of Cy5 at AuNSs. The results are shown in FIG. 5.

As shown in FIG. 5, Cy 51366 cm increased with the increase of EtNa-Sub concentration^-1The intensity of the characteristic peak at (a) is significantly increased. When the concentration reached 35. mu.M, the characteristic peak intensity remained approximately unchanged, and thus the optimum concentration of EtNa-Sub was determined to be 35. mu.M.

EXAMPLE 3 stability test of Dual-mode nanoprobes

(1) Characterization of salt stability of Dual-mode nanoprobes by UV-Vis Spectroscopy

The prepared dual-mode nanoprobe was centrifuged, resuspended in PBS buffer solutions containing different NaCl concentrations (200mM, 400mM, 600mM, 1000mM), vortexed, mixed, and left for 10min, and then the change of the spectrum of the dual-mode nanoprobe was detected by an ultraviolet visible absorption spectrometer, with the result shown in fig. 6A.

(2) Fluorescence experiment evaluation of ribozyme and glutathione stability of dual-mode nanoprobe

Various enzymes and sulfhydryl compounds exist in cells, and can cut nucleic acid or compete for thiolated nucleic acid, so that the stability of the probe is an important precondition for ensuring the accuracy of the result of the experiment in the cells. Four groups of the nanoprobes were placed in a 96-well fluorescent plate at 37 ℃ and after the sample solution was stable at equilibrium for 10min, one group was added with 1. mu.L of DNase I (2U/L in the buffer used in the experiment), one group was added with 1. mu.L of 2nM Glutathione (GSH), one group was added with 1. mu.L of 2nM RNaseA, and one group was added with 1. mu.L of 2nM Ca²⁺The fluorescence intensity of the sample was continuously monitored, and the results are shown in FIG. 6B.

(3) Evaluation of Medium stability of Dual-mode nanoprobes by Raman experiment

The prepared dual-mode nanoprobe is suspended in RPMI-1640 calcium-free minimal medium after centrifugation and incubated for 0h, 12h, 24h and 48h, and the change of Cy5 SERS signals is detected, and the result is shown in FIG. 6C.

As shown in FIG. 6A, in NaCl solutions with different concentrations, the absorption peak of the dual-mode nanoprobe is located at the wavelength of 800nm, and the spectral intensity is not obviously changed. The nucleic acid, the PEG and the MCH are firmly wrapped on the surfaces of the nano-gold stars, so that mutual contact and aggregation among the nano-gold stars are prevented, and the stability of the dual-mode nano-probe is improved.

FIG. 6B shows that the fluorescence intensity of the dual-mode nanoprobe in the presence of DNaseI, GSH, and RnaseA remains almost unchanged, probably because AuNSs has a protective effect on DNA strands. In the synthesis process of AuNSs, sodium chloride is added to complete the connection of DNA and AuNSs, and the degradation of nuclease to DNA is prevented by a high-concentration salt environment around nucleic acid, so that the stability of the dual-mode nanoprobe is protected.

As shown in fig. 6C, various substances in the medium have little influence on the stability of the dual mode nanoprobe.

Example 4 use of Dual-mode nanoprobes for in vitro Ca²⁺Detection of

Adding Ca at a certain concentration²⁺The stock solution was added to the bimodal nanoprobe solution prepared above. The reaction was carried out at 37 ℃ for 30min, and after centrifugation, the SERS spectrum and the fluorescence spectrum were measured, and the results are shown in FIGS. 7 and 9, respectively. The centrifuged supernatant was excited with excitation light having a wavelength of 638nm, and data of fluorescence intensity between wavelengths of 650nm and 750nm were collected, and the results are shown in FIG. 10. After the precipitate is resuspended, the precipitate is used for SERS spectrum detection, wherein the excitation wavelength is 633nm, and the scanning range is 1000cm^-1To 1800cm^-1The results are shown in FIG. 8. For the interference substances (Na) possibly existing in the actual environment and cell samples²⁺、K⁺、Mg²⁺、Zn²⁺、Cd²⁺、Ni²⁺、Co²⁺、Cu²⁺、Fe²⁺、Fe³⁺、Pb²⁺、Ba²⁺、Mn ²⁺1 μ M each) were specifically detected by Raman and fluorescence, respectively, and the results are shown in FIGS. 11 and 12。

FIGS. 7-10 show that the following Ca²⁺Increase in concentration, 1366cm^-1When the SERS intensity is weakened, the fluorescence intensity is continuously enhanced; in the concentration range of 2-40 nM, 1366cm^-1The band intensity and fluorescence intensity of (C) are both equal to Ca²⁺There is a linear relationship between concentrations.

FIGS. 11-12 show Pb removal²⁺In addition, 100nM Ca is contained²⁺The SERS intensity of the sensor is obviously lower than that of other control groups, and the fluorescence intensity is obviously higher than that of other control groups, so that the SERS-fluorescence dual-mode nano-probe provided by the invention is proved to have excellent selectivity. Pb²⁺Most DNAzymes are enzymatically active, and Pb such as L-cysteine, acetate, thioglycolic acid (TGA), dimercaptobutanol (BAL), MCH, etc. can be used²⁺Pair of blocking agent Pb²⁺Masking to exclude Pb²⁺Does not affect Ca at the same time²⁺Detection of (3).

Example 5 cytotoxicity assays for T-2 toxin

The optimal concentration of the T-2 toxin in co-culture with the cells was determined by the CCK-8 method. T-2 toxin is dissolved in DMSO and diluted into different concentrations by using a cell culture solution, and the DMSO concentration is controlled to be below 0.5% during contamination (the DMSO has no obvious influence on the survival rate of Hela cells when the concentration is within 0.5%). Hela cells in the logarithmic phase of growth were digested to prepare a cell suspension, and 100. mu.L of the cell suspension was seeded at a density of 5000 cells/mL in a 96-well plate. The plates were pre-incubated in an incubator for 24h (37 ℃, 5% CO)₂) Thereafter, 10. mu.L of each of the test substances was added to the plate so that the final concentrations of T-2 toxin in the medium were 20. mu.M, 16. mu.M, 12. mu.M, 10. mu.M, 8. mu.M, 6. mu.M, 4. mu.M, 2. mu.M, and 0. mu.M, respectively. After incubating the plates in the incubator for 1h, 10. mu.L of CCK-8 solution was added to each well. After incubation in the incubator for 4h, the absorbance at 450nm was measured. The relative cell viability was calculated using the following formula:

the relative cell survival (RGR) — (absorbance of experimental group-CCK 8 background)/(experimental value of control group-CCK 8 background) × 100%, the results are shown in fig. 13. Fitting IC of T-2 toxin to Hela cells by Origin software₅₀The results are shown in the inset of FIG. 13.

As can be seen in FIG. 13, as the concentration of T-2 toxin increased, the viability of Hela cells decreased in turn. The IC of T-2 toxin on Hela cells can be seen by insetting₅₀The value is about 11.07. mu.M, and the concentration ensures that the T-2 toxin has certain toxicity to Hela cells and maintains certain cell survival rate.

Example 6CCK-8 method for detecting cell viability in different media

Taking Hela cells in logarithmic growth phase, 2 × 10 per well⁴The individual cells were seeded in 96-well plates at 37 ℃ with a volume fraction of 5% CO₂Culturing for 24h under the condition, discarding the culture medium, washing with PBS, adding 3 different basic culture media (RPMI-1640 calcium-free basic culture medium, DMEM basic culture medium and DMEM complete culture medium), respectively culturing for 10h, 16h, 20h and 24h, discarding the culture medium in the wells, adding 10 muL CCK-8 and 50 muL serum-free DMEM basic culture medium into each well, incubating for 2.5h in a cell culture box, and detecting the absorbance at 450nm, wherein the result is shown in figure 14.

As can be seen from FIG. 14, HeLa cells could maintain better cell viability in RPMI-1640 calcium-free minimal medium within 24 h.

Example 7 optical microscopy of the Effect of different media on cell morphology and adherence

After the passage of Hela cells, the cells were completely cultured in DMEM at 37 ℃ with a volume fraction of 5% CO₂Culturing for 24h under the condition, discarding the culture medium, washing with PBS, adding PRMI-1640 calcium-free minimal medium, and observing cell morphology and growth status in PRMI-1640 calcium-free minimal medium under 3 different visual fields at 4 time points when culturing for 4h, 8h, 12h and 24h respectively, as shown in FIG. 15.

As can be seen from FIG. 15, Hela cells could maintain good cell morphology and adherence in RPMI-1640 calcium-free minimal medium within 24 h.

Example 8CCK-8 method for evaluating cytotoxicity of Dual-mode nanoprobes

Hela cells in log phase of growth were digested to prepare a cell suspension, and 100. mu.L of the cell suspension was seeded at 8000 cells/mL in a 96-well plate at 37 ℃ with 5% CO₂Incubating for 24h under air condition to make the cells fully adhere to the wall, and then abandoningThe old culture medium is removed, washed with PBS three times, and then added with probes with final concentrations of 0.5nM, 0.25nM and 0.125nM respectively (the probes are suspended in RPMI-1640 calcium-free basic culture medium) for incubation, and the cell incubation time under the same concentration is set to be 4h, 8h, 12h and 24h respectively. The probe solution was discarded and washed three times with PBS, and then 100. mu.L of DMEM minimal medium and 20. mu.L of CCK-8 reagent were added to each well, and after incubation in an incubator for 4 hours, absorbance at 450nm was measured. The cell activities at different probe concentrations and different action times were calculated, taking the cell activities of the untreated cells as 100%, as shown in FIG. 16.

As can be seen from FIG. 16, the activity of Hela cells was maintained at 85% or more even when the 0.5nM nanoprobe stimulated the cells for 24 h.

Example 9ICP-MS analysis of the amount of Dual mode nanoprobes into cells

To further verify that the cells had indeed swallowed the dual-mode nanoprobes, the gold content of the cells after incubation with the probes was quantitatively analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The method comprises the following steps: and (3) inoculating the Hela cells into a 6-well plate, carrying out adherent growth for 24h, adding a 0.25nM concentration dual-mode nanoprobe, incubating with the cells for different times, discarding the culture medium, carrying out buffer washing for several times by PBS (phosphate buffer solution), adding pancreatin to enable the Hela cells to fall off from the bottom of the dish, counting, and carrying out centrifugal collection. Adding aqua regia to digest cells, performing ultrasonic treatment, incubating overnight, diluting by 100 times, performing ICP-MS analysis, and calculating the number of dual-mode nanoprobes entering the cells, as shown in FIG. 17.

As can be seen from FIG. 17, the absorption of the bimodal nanoprobe by Hela cells was saturated at about 6h and the absorption concentration was about 21 pM.

Example 10TEM imaging Observation of the HeLa cells to the Dual mode nanoprobes

Hela cells were seeded into 6-well plates and incubated with 0.25nM of the dual-mode nanoprobes for 6 h. The cells were then digested and centrifuged. The cells were then fixed by 2.5% glutaraldehyde fixing solution at room temperature, then dehydrated by acetone (50%, 70%, 90%, 100%) with concentration gradient, soaked with pure acetone and embedding solution, embedded, fixed by oven at 37 ℃ and 60 ℃, and finally sectioned, stained and photographed as in fig. 18.

FIG. 18 shows that the bimodal nanoprobes are able to enter Hela cells and are distributed in the cytoplasm mainly in single or multiple form.

Example 11 use of Dual-mode nanoprobes for fluorescence imaging of Living cells

After the cells had grown to a logarithmic growth phase, the cells were grown at 4X 10⁴ Inoculating 500 mu L of the strain/mL of the strain to a special laser confocal dish, culturing cells until the cells are adherent, washing the cells for a plurality of times by PBS, sucking out an old culture medium, adding an RPMI-1640 basic culture medium with uniformly mixed probes and co-culturing the cells for 6 hours, sucking out the culture medium containing the probes, washing the culture medium by PBS, adding a basic culture medium mixed with 11 mu M T-2 toxin, and co-culturing for 0min, 15min, 30min, 45min and 60min respectively; after the reaction was completed, the medium was aspirated and stained with DAPI for 5min, the DAPI was discarded, the cells were fixed with 4% paraformaldehyde, and immediately thereafter fluorescence imaging was performed, the fluorescence intensity of which was determined at an excitation wavelength of 633nm and an emission wavelength of 670nm, as shown in FIG. 19.

As can be seen from FIG. 19, the luminescence intensity of the laser confocal light at 670nm increased with the increase of the incubation time with T-2 toxin.

Example 12Fluo-4/AM for live cell fluorescence imaging

Preparation of pancreatin-digested Hela cells in 4X 10 culture solution⁴one/mL cell suspension was seeded into 500. mu.L laser confocal dishes. After the cells were attached, they were exposed to the toxin 11. mu. M T-2 for various times and then incubated with the calcium indicator Fluo-4/AM for 30 min. Confocal microscope for observing free Ca²⁺The change in level, as in fig. 20. The fluorescence intensity was determined at an excitation wavelength of 488nm and an emission wavelength of 516nm, as shown in FIG. 21.

As shown in FIG. 20, free Ca in cytoplasm was found in HeLa cells²⁺Levels increased in a toxin incubation time-dependent manner. Fluo-4/AM can penetrate cell membranes to enter cells, and then is cleaved by intracellular esterase to form Fluo-4, so that the Fluo-4/AM is retained in the cells. Fluo-4 free ligand is almost non-fluorescent, its fluorescence does not follow Ca²⁺Increased concentration and increaseIs strong. However, when it is associated with intracellular Ca²⁺The binding can produce stronger fluorescence, which can be increased by 60 to 80 times. FIG. 21 shows that the dual-mode nanoprobe shows the same trend in intracellular fluorescence detection compared to Fluo-4/AM, and it can be seen that the dual-mode nanoprobe can be used for intracellular calcium ion concentration monitoring.

Example 13 Raman detection and imaging of live cells

After culturing the cells until the logarithmic phase of growth, the cells were placed at 5X 10⁴Inoculating 350 mu L of the strain/mL onto a sterile quartz plate, culturing cells until the cells are attached to the wall, removing the old culture medium, washing the cells for several times by PBS, adding an RPMI-1640 basic culture medium with uniformly mixed probes and co-culturing the cells for 6h, sucking out the culture medium containing the probes, washing the culture medium by PBS, adding a basic culture medium mixed with 11 mu M T-2 toxin, co-culturing for 0min, 15min, 30min, 45min and 60min respectively, fixing the cells by 4% paraformaldehyde for 15min, and immediately carrying out SERS spectrum detection and SERS imaging. For SERS imaging, laser excitation was 633nm, power was 4mW, slit was set to 50 μm, exposure time was 0.2 s. Mixing Cy 51366 cm^-1The characteristic peak at (a) is taken as the site of SERS imaging, and the result is shown in fig. 22. The SERS spectrum is shown in fig. 23.

According to FIGS. 22-23, following intracellular Ca²⁺The increase in concentration, the red and green fields of view of Hela cells gradually decreased, which is consistent with the results of SERS spectroscopy.

EXAMPLE 14 Universal testing of Dual-mode nanoprobes for different cell lines HepG2, MCF-7, Caco-2

Human hepatoma cells HepG2, human breast cancer cells MCF-7 and human colorectal adenocarcinoma cells Caco-2 were selected to determine the versatility of the application of the dual-mode nanoprobes of the present invention in human-related cells. After the probe is respectively incubated with HepG2, MCF-7 and Caco-2 for 6h, the fluorescence signal and the SERS signal of Cy5 are respectively shown in FIGS. 24 and 25. After incubating T-2 toxin with HepG2, MCF-7 and Caco-2 for 30min, SERS imaging and laser confocal imaging were performed, and the results before and after incubation are shown in FIGS. 26 and 27, respectively.

As shown in fig. 24-27, the SERS signal of Cy5 is strong, the fluorescence signal is weak, the intracellular fluorescence intensity is significantly increased at 30min, and the red and green visual fields are gradually decreased, which is also consistent with the results of SERS spectroscopy.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

Claims

1. A preparation method of a dual-mode nanoprobe is characterized by comprising the following steps:

(2) adding a substrate chain solution into the polymerase chain functionalized nano-Venus obtained in the step (1), incubating for 3-4h at 35-40 ℃, and centrifuging to remove redundant substrate chains to obtain the dual-mode nano-probe;

wherein the substrate strand and the enzyme chain are partially complementary, the enzyme cutting site is positioned on the substrate strand, and the enzyme chain can be Ca-modified² ⁺Activating and acting on the enzyme cutting site and cutting the substrate chain;

modifying a fluorescent group at the 5 'end or the 3' end of the substrate chain, and modifying a sulfydryl at the 5 'end or the 3' end of the enzyme chain;

the surface ligand I comprises one or more of methoxy polyethylene glycol thiol, carboxyl polyethylene glycol thiol, amino polyethylene glycol thiol, polyethylene glycol thiol and bovine serum albumin, and the surface ligand II comprises one or more of 6-mercapto-1-hexanol, mercaptoacetic acid and mercaptodibutyric acid;

the enzyme chain, the surface ligand I and the surface ligand II are connected with the nanogold through Au-S covalent bonds.

2. The method of claim 1, wherein: in step (1), the concentration of the enzyme chain solution is 170-.

3. The method of claim 1, wherein: in the step (2), the concentration of the substrate strand solution is 30 to 50. mu.M.

4. The method of claim 1, wherein: the molar ratio of the nanogold, the substrate chain, the enzyme chain, the surface ligand I and the surface ligand II is 0.5-1.5:1600-2200:1750-2600: 1000-10000.

5. A bimodal nanoprobe prepared by the preparation method of any one of claims 1 to 4.

6. A method for the dual-mode detection of calcium ions for non-diagnostic, non-therapeutic purposes, comprising the steps of: uniformly mixing the dual-mode nanoprobe of claim 5 on a calcium-free minimal medium, co-culturing the dual-mode nanoprobe with a cell to be detected for 6-7h, sucking out the calcium-free minimal medium and cleaning, adding the toxin-containing calcium-free minimal medium for co-culturing for 30-90min, sucking out the calcium-free minimal medium and cleaning, dyeing and fixing the cell, and carrying out SERS spectral detection, SERS imaging or fluorescence imaging to obtain intracellular Ca²⁺The detection result of (3);

or the dual-mode nanoprobe of claim 5 is mixed with Ca²⁺The solution is mixed and reacted, and after centrifugation, SERS spectrum detection, SERS imaging or fluorescence imaging is carried out to obtain in vitro Ca²⁺The detection result of (1).

7. The method of claim 6, wherein: the toxin includes a T-2 toxin.

8. The method of claim 6, wherein: in a calcium-free minimal medium containing toxins at a concentration corresponding to the culture timeIC of₅₀The value is obtained.

9. The method of claim 6, wherein: when the dual-mode nanoprobe is uniformly mixed with the calcium-free basic culture medium, the concentration of the dual-mode nanoprobe is 0.1-0.5 nM.

10. The method of claim 6, wherein the test cells comprise one or more of human cervical cancer cell Hela, human liver cancer cell HepG2, human breast cancer cell MCF-7, and human colorectal adenocarcinoma cell Caco-2.