CN115308144B - Optical fiber miRNA sensor based on graphene oxide coated gold nanoparticles, material, probe and application of optical fiber miRNA sensor - Google Patents
Optical fiber miRNA sensor based on graphene oxide coated gold nanoparticles, material, probe and application of optical fiber miRNA sensor Download PDFInfo
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Abstract
The invention discloses an optical fiber miRNA sensor based on graphene oxide coated gold nanoparticles, a material, a probe and application thereof. Based on electrostatic interaction, the GO-AuNPs composite particles are prepared, and the graphene oxide can amplify the LSPR effect of the composite particles, so that the sensitivity and stability of the sensor are improved. And fixing the composite material on the surface of the optical fiber sensing probe by using a chemical crosslinking method, and combining a light source and a spectrometer to prepare the miRNA optical fiber sensor. When the GO-AuNPs-RNA probe 1 fixed on the surface of the optical fiber probe performs hybridization reaction with target RNA and GO-AuNPs-RNA probe 2, the distance between GO-AuNPs particles is reduced, the local surface plasma effect of the particles is influenced, and the local surface plasma peak position is changed. The invention overcomes the defects of time consumption, high cost and the like of the traditional method and lays a foundation for the application of the optical fiber LSPR sensor in the biomedical diagnosis field.
Description
Technical Field
The invention relates to the field of optical fiber sensing, in particular to an optical fiber miRNA sensor based on gold nanoparticles coated by graphene oxide, a material, a probe and application thereof.
Background
MiRNAs are widely involved in evolutionary processes such as tumor development, invasion and metastasis, drug resistance, etc., and are considered as biomarkers for early tumor discovery, classification and predictive diagnosis. However, the short sequence and high similarity characteristics of mirnas greatly limit the development of their detection means. Therefore, research and development of a rapid, sensitive, label-free, high-stability, low-cost miRNA detection technology has become an important topic in the clinical medicine field.
The optical fiber biosensor is an analysis and measurement device which selectively and continuously converts biological information of a detection object into an optical signal which is easy to be measured by an analysis instrument, can detect single (multiple) objects on line in real time, and is an important detection means for various biomass, molecules and ions. Compared with other sensors, the sensor has the advantages of high detection precision, strong electromagnetic interference resistance, quick response, capability of realizing probe miniaturization, capability of being used for remote sensing and the like. Although the existing optical fiber biosensors are quite abundant in types, the research on the optical fiber miRNAs sensors at home and abroad is still blank, because miRNA is single-stranded RNA and is extremely unstable in natural environment, the method has great requirements on selecting a proper sensitive material; meanwhile, the mode of combining the sensitive material with the optical fiber also directly influences the detection range and the sensitivity of the sensor.
Disclosure of Invention
Aiming at the problems of complex detection means, expensive instrument, long detection time consumption and the like of the existing miRNA, the optical fiber miRNA sensor, material, probe and application thereof based on the graphene oxide coated gold nanoparticles are solved.
The technical scheme adopted by the invention is as follows:
An optical fiber miRNA sensor based on graphene oxide coated gold nanoparticles comprises a broadband light source, a Y-shaped optical fiber, an optical fiber probe and a spectrometer, wherein the side surface of the sensing probe is coated with graphene oxide-gold nano-RNA composite material (GO-AuNPs-RNA probe), and the tail end of the optical fiber probe is coated with a reflecting silver mirror; light enters the optical fiber sensing probe through the Y-shaped optical fiber, and excites graphene oxide-gold nano-sensitive materials on the side face of the probe to generate a local surface plasma resonance phenomenon.
Preferably, when the GO-AuNPs-RNA probe 1 fixed on the surface of the optical fiber probe performs hybridization reaction with the target miRNA and the GO-AuNPs-RNA probe 2, the distance between graphene oxide and gold nano composite particles is reduced, the local surface plasma effect of the particles is influenced, and the local surface plasma peak position is changed, so that the detection of the target RNA is realized.
Preferably, the diameter of the fiber optic probe is 125-1000 μm;
The light source is a halogen tungsten lamp broadband light source, and the working wavelength is as follows: 360-1200 nm, and the working wavelength of the spectrometer is 320-1050 nm.
Preferably, the graphene oxide-gold nano-RNA composite material on the surface of the optical fiber probe is fixed by a chemical crosslinking method.
The optical fiber probe sensitive material is a GO-AuNPs-RNA probe composite material, and the preparation process comprises the following steps:
S1, preparing gold nano composite particles coated with graphene oxide by an electrostatic method, wherein the gold nano particles are coated in the graphene oxide, and the particle size of the gold nano particles is 10-70nm;
s2, fixing the RNA probe on the surface of the gold nano composite particle coated by graphene oxide by utilizing a sulfhydryl group through a chemical crosslinking method to obtain a GO-AuNPs-RNA probe composite material;
the sensing RNA probes are GO-AuNPs-RNA probe 1 and GO-AuNPs-RNA probe 2, and respectively conform to the base complementary pairing principle only with the target miRNA, and do not conform to the base complementary pairing principle.
Preferably, the gold nanoparticles are prepared by adopting a water phase reduction method, graphene Oxide (GO) is synthesized by adopting a Hummers method, and the GO-AuNPs composite particles specifically comprise the following steps:
(1) Cleaning glassware required in experiments by using aqua regia, washing with deionized water, and drying for later use; the cleaning of the reaction environment can avoid introducing impurities in the growth process of AuNPs;
(2) Adding a trisodium citrate solution into the boiled chloroauric acid solution, and continuing the reaction when the color of the mixed solution is observed to be changed from light yellow to black and finally to be changed into wine red so as to obtain AuNPs;
(3) The preparation method of the GO-AuNPs composite material comprises the following steps: and mixing the synthesized AuNPs solution with the GO solution, and stirring to obtain the GO-AuNPs composite particles, wherein oxygen-containing groups on the surface of GO can promote the embedding of GO into AuNPs, so that the GO-AuNPs composite particles are formed.
Preferably, in the step (2), the AuNPs particle size ranges from 10 to 70nm;
in the step (3), the ratio of AuNPs to GO is (1-5): 1.
Preferably, the preparation steps of the GO-AuNPs-RNA probe composite material are as follows:
step one preparation of GO-AuNPs-RNAp: dispersing the prepared GO-AuNPs solution in PBS buffer solution after washing for a plurality of times, respectively adding TCEP aqueous solution into RNA probe 1 and RNAp probe 2 to activate sulfhydryl at the end of the solution, adding the activated RNA probe into the GO-AuNPs solution, and incubating at room temperature;
Step two, salinization of GO-AuNPs-RNAp: adding NaCl into the GO-AuNPs-RNA probe 1 and GO-AuNPs-RNA probe 2 solutions respectively, incubating after ultrasonic treatment, repeatedly adding NaCl for multiple times, and continuing incubating;
Step three, sample washing: the incubation solution was centrifuged with PBS, and unbound RNA was removed from the solution and dispersed in buffer PBS.
An optical fiber miRNA sensor sensing probe based on graphene oxide coated gold nanoparticles comprises the following preparation steps:
step (one), silanizing the surface of the optical fiber: after the organic cladding on one end surface of the optical fiber is removed, the optical fiber is marked as an optical fiber sensing end which is an S end, and the S end of the optical fiber which is cleaned by acetone, alcohol and deionized water is inserted into an HF acid solution (VHF: V water=1:1) for corrosion so as to enhance an evanescent field of the optical fiber in leakage; after the corroded optical fiber is cleaned, the optical fiber is inserted into piranha solution (V hydrogen peroxide: V sulfuric acid=3:7) for soaking for a plurality of hours, the soaking time range can be 2-10 hours, the cleaned optical fiber is placed in a vacuum drying oven at 110 ℃ for heating for about 1 hour, and the hydroxyl groups on the surface of the optical fiber are activated; then, soaking the S end of the optical fiber in APTES solution (3-aminopropyl triethoxysilane) for several hours to convert hydroxyl on the surface of the optical fiber into amino;
Step (two) immobilization of GO-AuNPs on optical fiber: soaking the S end of the optical fiber in the GO-AuNPs composite particle solution, wherein the carboxyl of GO can be combined with the amino on the optical fiber, so that a GO-AuNPs composite particle monolayer film can be formed on the surface of the optical fiber;
And (3) silver mirror reaction end capping: coating a silver film on the end face of the S end of the optical fiber treated in the second step through silver mirror reaction, so as to ensure that light is reflected into a spectrometer;
Diluting the RNA probe1 solution by using PBS buffer solution to obtain RNA probe1 diluent;
step five, soaking the GO-AuNPs functionalized fiber S end treated in the step three in RNA probe1 diluent for 12 h, so that the GO-AuNPs composite particles on the surface of the fiber and the RNA probe1 are fixed together through covalent bonds; washing the unbound RNA molecules on the surface of the optical fiber by using absolute ethyl alcohol and nuclease-free water;
passivating non-specific sites on the surface of the optical fiber, which are not combined with GO-AuNPs composite particles, by utilizing BSA, and soaking the S end of the optical fiber in a BSA solution;
and (seven) obtaining the optical fiber LSPR MIRNA-126 sensor based on the GO-AuNPs-RNAp1 for detecting the miRNA-126.
Preferably, the concentration of APTES in step (one) is 7% -11% and the soaking time is 2-10 hours.
The optical fiber miRNA sensor based on the graphene oxide coated gold nano particles senses or the optical fiber probe sensitive material or the probe is used for quantitatively detecting RNA, and the detection steps are as follows:
(1) Mixing the GO-AuNPs-RNAp2 compound with the same volume with miRNA-126 solutions with different concentrations, heating in a water bath, naturally cooling to room temperature, and incubating for a period of time to enable the pairing base between the compound and the target RNA to fully react;
(2) Placing the GO-AuNPs-RNAp1 functionalized optical fiber sensing probe in the mixed solution for a period of time, and recording the change of an optical fiber LSPR spectrogram; the test was repeated three times for each concentration of sample to complete the test.
The invention provides an optical fiber miRNA sensor based on graphene oxide coated gold nanoparticles (GO-Au, GO-AuNPs). The evanescent field released from the fiber core excites the GO-AuNPs nano material fixed on the fiber core, and a plasma resonance phenomenon occurs, so that the characteristic absorption spectrum (LSPR spectrum) of the nano material is generated in spectrum, and the nano material is sensitive to surrounding media, and when the surrounding media change, the intensity or displacement of the LSPR spectrum changes. Provides a method and means with high sensitivity, low detection limit, high stability and microminiaturization for miRNAs detection, and has important scientific significance and great practical value for promoting the development and application of optical fiber sensing technology.
The GO-AuNPs composite particles are prepared based on electrostatic action, and the graphene oxide can amplify the LSPR effect of the composite particles and increase the sensitivity and stability of the sensor. GO-AuNPs-RNA probe 1 and GO-AuNPs-RNA probe 2 prepared based on a chemical crosslinking method respectively accord with the base complementary pairing principle only with the target miRNA, and do not accord with the base complementary pairing principle. And fixing the composite material on the surface of the optical fiber sensing probe by using a chemical crosslinking method, and combining a light source and a spectrometer to prepare the miRNA optical fiber sensor. When graphene oxide-gold nanocomposite particle-RNA 1 probes fixed on the surface of the optical fiber probe perform hybridization reaction with target RNA and GO-AuNPs-RNA probe 2, the distance between GO-AuNPs particles is reduced, the local surface plasma effect of the particles is affected, and the local surface plasma peak position is changed. Based on the LSPR effect of the composite material, the rapid quantitative detection of miRNA can be realized. The invention constructs the optical fiber miRNA sensor of the GO-AuNPs-RNA probe composite material and the gold nano particles coated by the graphene oxide, overcomes the defects of time consumption, high cost and the like of the traditional method, realizes the rapid, sensitive and stable detection of the target miRNA based on the LSPR effect of the nano material and the sandwich hybridization reaction, further widens the application range of the sensor, and lays a foundation for the application of the optical fiber LSPR sensor in the biomedical diagnosis field.
The invention has the beneficial effects that:
(1) By utilizing the characteristics and advantages of the GO-AuNPs composite particles, the optical fiber LSPR sensor based on the GO-AuNPs composite particles is prepared and used for detecting miRNA-126, so that the application range of the sensor is further widened, and a foundation is laid for the application of the optical fiber LSPR sensor in the biomedical diagnosis field.
(2) The optical fiber sensor for detecting miRNA-126 provided by the invention has the advantages of economy, rapidness, sensitivity, stable performance, simple structure, easiness in preparation and the like, and shows good selectivity. Provides a new approach and strategy for developing a stable and economical detection probe for early diagnosis markers of cancers.
Drawings
FIG. 1 is a schematic diagram of a miRNA-126 fiber sensor based on GO-AuNPs-RNAp complex;
FIG. 2 is a device diagram of the fiber LSPR MIRNA-126 sensor;
FIG. 3 (a) reflectance spectra of fiber optic sensors at different concentrations of miRNA-126; (b) relationship of ΔA to miRNA-126 concentration.
The selectivity of the fiber LSPR MIRNA-126 sensor of FIG. 4;
Response time of the fiber LSPR MIRNA-126 sensor of FIG. 5;
Fig. 6 GO, stability of AuNPs and GO-AuNPs;
FIG. 7 AuNPs effect of particle size on sensor probe performance;
FIG. 8 AuNPs effect on sensor probe performance of GO ratio;
FIG. 9 effect of crosslinker concentration on sensor probe performance;
FIG. 10 effect of modification time on sensor probe performance;
FIG. 11 effect of time to crosslink sensitive material on sensor probe performance.
Detailed Description
The invention will be further illustrated with reference to specific examples. The present invention will be described in further detail with reference to examples, but is not limited to these examples.
Example 1
The method for detecting the optical fiber miRNA-126 of the graphene oxide coated gold nanoparticle comprises the following steps:
Step 1: gold nanoparticles were prepared by aqueous phase reduction, and Graphene Oxide (GO) was synthesized by Hummers method. And cleaning glassware required in the experiment by using aqua regia, washing with deionized water, and drying for later use. The reaction environment is cleaned to avoid introducing impurities during the growth of AuNPs. To a boiling chloroauric acid solution (0.01% (w/w), 50 mL) was added a solution of 0.75% mL trisodium citrate (1% (w/w), and when the color of the mixture was observed to change from pale yellow to black and finally to reddish wine, the reaction was continued for 30 min to obtain AuNPs having a particle size of about 37 nm.
Step 2: and preparing the graphene oxide coated gold nanocomposite by utilizing the electrostatic bonding effect. The synthesized AuNPs solution and GO solution are mixed according to the proportion of 2:1, and are stirred for 2h and then are reserved for standby. The oxygen-containing groups on the surface of the GO promote the embedding of the GO into the AuNPs, so that the GO-AuNPs composite particles are formed.
Step 3: as shown in fig. 1, the surface of the optical fiber is silanized. After removing the organic cladding layer on the surface of one end of the optical fiber with the fiber core diameter of 600 micrometers, which is denoted as an optical fiber sensing end (S end), the S end of the optical fiber cleaned by acetone, alcohol and deionized water is inserted into an HF acid solution (VHF: V water=1:1) for corrosion for 4 hours, so as to enhance the evanescent field of the optical fiber leakage. After cleaning the corroded optical fiber, immersing the optical fiber in piranha solution (V hydrogen peroxide: V sulfuric acid=3:7) for a plurality of hours, and then heating the cleaned optical fiber in a vacuum drying oven at 110 ℃ for about 1 hour to activate hydroxyl groups on the surface of the optical fiber. Thereafter, the S-terminus of the fiber was immersed in a 9% APTES solution for 8 hours to convert the hydroxyl groups on the surface of the fiber into amino groups. The S end of the optical fiber is soaked in the GO-AuNPs composite particle solution for 6 hours, and the carboxyl of GO can be combined with the amino on the optical fiber, so that a GO-AuNPs composite particle monolayer film can be formed on the surface of the optical fiber. A silver film was coated on the end face of the S-end of the optical fiber by a silver mirror reaction to ensure that light was reflected into the spectrometer.
Step 4: the RNAp1 solution was diluted to the appropriate concentration with PBS buffer (10 mM,1M NaCl,pH =6.8). Wherein, naCl is introduced to improve the ionic strength, shorten the Debye length and enhance the adsorption capacity of the nucleotide on the surface of GO-AuNPs particles. And soaking the S end of the GO-AuNPs functionalized optical fiber in the diluent for 12 h, so that the GO-AuNPs composite particles on the surface of the optical fiber and the RNAp1 are fixed together through covalent bonds. Thereafter, unbound RNA molecules on the surface of the fiber were washed away with absolute ethanol and nuclease-free water. The non-specific sites of the GO-AuNPs composite particles which are not combined with the surface of the optical fiber are passivated by utilizing BSA (bovine serum albumin), the S end of the optical fiber is soaked in 2mg/mL BSA (10 mM, pH=6.8) solution for 1 h so as to block the non-specific electrostatic adsorption of the GO-AuNPs-RNAp2 composite and the optical fiber in the detection process, but the hybridization reaction activity between the RNA probe molecules and target RNA is not affected.
Step 5: as shown in fig. 2, an optical path diagram is constructed. Equal volumes of GO-AuNPs-RNAp2 complex are mixed with miRNA-126 solutions with different concentrations (the final concentration of target RNA is 10 < -1 > nM, 100 nM, 101 nM, 102 nM, 103 nM and 104 nM respectively), and the mixture is placed in a water bath kettle at 60 ℃ for heating, then naturally cooled to room temperature and incubated for a period of time, so that the pairing base between the complex and the target RNA is fully reacted. Then, the GO-AuNPs-RNAp1 functionalized optical fiber sensing probe is placed in the mixed solution for a period of time, and the change of an optical fiber LSPR spectrogram is recorded. The samples at each concentration were tested in triplicate and then averaged to reduce experimental error.
As shown in fig. 3, when the optical fiber probe fixed with GO-AuNPs-RNAp is immersed in the miRNA-126 solution, GO-AuNPs-RNAp2-miRNA-126 adsorbs to GO-AuNPs-RNAp1 on the surface of the optical fiber through RNA base complementary pairing reaction, so that the distribution of composite particles on the optical fiber is changed, the LSPR peak value of the material is changed, and the absorption peak intensity is increased. We measured the sensitivity of the sensor by how much the intensity of the absorbance peak varies with miRNA-126 in solution.
As shown in FIG. 4, we prepared several other miRNAs solutions (miRNA-21, miRNA-106, miRNA-148a and random sequences) at equal concentrations to evaluate the specificity of the fiber LSPR MIRNA-126 sensor for miRNA-126 detection. Control experiments were performed with GO-AuNPs-RNAp2 complex solution without any RNA added as a blank. It can be seen that equal concentrations of miRNA-21, miRNA-106, miRNA-148a and random sequence samples do not bridge the two RNA probes together due to base unpairement with RNAp1 and RNAp2, and therefore the corresponding LSPR spectral patterns do not change significantly. In contrast, only miRNA-126 base paired with both RNA probes can cause the free GO-AuNPs-RNAp2 complex to adsorb near the GO-AuNPs-RNAp1 on the fiber surface by hybridization reaction and cause significant changes in LSPR. By comparing the LSPR spectral pattern changes of each group of sample solutions, the optical fiber biosensor has good selectivity.
As shown in FIG. 5, the response time of the fiber LSPR MIRNA-126 sensor is defined as the time from the time the fiber-optic probe immersed in the miRNA-126 solution responds until the sensor receives a stable signal. GO-AuNPs-RNAp2-miRNA-126 is adsorbed to GO-AuNPs-RNAp1 on the surface of the optical fiber through RNA base complementary pairing reaction, so that the distribution of the complex on the optical fiber is changed, and the LSPR peak value of the complex is changed. After 40 min the sensor signal tended to stabilize and the response time of the sensor was 40 min.
Example 2
The introduction of GO in the optical fiber miRNA-126 detection method of graphene oxide coated gold nanoparticles can improve the stability of the composite particles GO-AuNPs.
Adding trisodium citrate solution (1% (w/w) with the volume of 0.75mL into boiled chloroauric acid solution (0.01% (w/w) and 50 mL), preparing gold nanoparticles with the particle size of 37: 37 nm, mixing the synthesized AuNPs solution and GO solution according to the ratio of 2:1 for 2 h to obtain composite particles GO-AuNPs, and researching the stability of the composite particles. The potential difference between the continuous phase in solution and the particles dispersed therein defines the Zeta potential, the absolute value of which is directly related to the stability of the solution system. In general, the positive and negative values of the Zeta potential of the sample represent the positive and negative of the charge, the absolute value of the Zeta potential represents the quantity of the charge, and the larger the absolute value of the potential is, the more the charges of the particles in the sample system are, the repulsive force among the particles is enhanced, and the whole system becomes more stable. To evaluate the dispersion stability of the prepared GO-AuNPs composite particle solution, zeta potential detection was performed by a laser particle sizer Zetasizer Nano ZS, and the test results are shown in fig. 6. From the graph, the Zeta potential of the GO-AuNPs composite particles is higher than the potential value of the independent AuNPs, which shows that the dispersion stability of the synthesized GO-AuNPs composite particles is higher than that of the independent AuNPs, and the dispersion stability of the metal particles is improved by introducing GO, so that the possibility is provided for improving the sensing performance of the sensor.
Example 3
Conditions such as the proportion of each component in the preparation process of the composite particles affect the sensitivity of the sensor. The parameters were adjusted and the rest was the same as in example 1.
Different volumes (0.3, 0.5, 0.75, 1, 1.75 and mL) of trisodium citrate solution (1% (w/w) are added into boiling chloroauric acid solution (0.01% (w/w) and 50 mL) to prepare gold nanoparticles (18 nm, 23 nm, 37 nm, 56 nm and 76 nm) with different particle sizes, the synthesized AuNPs solution and GO solution are mixed according to the proportion of 2:1,
The fiber etching time was 4 hours, the material crosslinking time was 6 hours, and the procedure of example 1 was followed, except that the fiber was immersed in a 9% APTES solution for 8 hours. As shown in fig. 7, when the particle diameter is too large, steric hindrance to be overcome when the GO-AuNPs composite particles are bonded to the optical fiber becomes large, and the composite particles fixed to the surface of the optical fiber are relatively small, which may result in a decrease in refractive index sensitivity of the optical fiber. These phenomena are determined by the method of synthesis of the GO-AuNPs composite particles and the way in which the GO-AuNPs composite particles are immobilized to the surface of the optical fiber. In addition, in the process of synthesizing AuNPs, the amount of sodium citrate added is different, the amount of citrate adsorbed on the surface of the AuNPs is different, the amount of charge carried on the surface of the AuNPs is different, and the particle stability is different. Therefore, the optimal gold nanoparticle size was 37 nm.
The ratio of AuNPs/GO is adjusted to 0.5, 1,2,3 and 4, and the GO-AuNPs composite particles prepared by the rest methods are fixed on an optical fiber probe and placed in solutions with different refractive indexes prepared by glycerol solution, so that the sensitivity to the change of the refractive index is shown in figure 8. As can be seen from fig. 8, as the AuNPs/GO ratio increases, the refractive index sensitivity of the sensing probe increases, because increasing the concentration of AuNPs increases the generation of LSPR phenomenon, and the response of the sensing probe is more remarkable, but when the content of AuNPs is too large and the content of GO is too low, the effect of reinforcing the AuNPs LSPR cannot be achieved, and the sensitivity of the sensing probe is reduced. Therefore, the composite particles of the GO-AuNPs are synthesized when the ratio of the AuNPs to the GO is 3:1, and are fixed on the surface of the optical fiber so as to ensure that the optical fiber sensing probe with the best performance is obtained.
The fiber probes with immobilized GO-AuNPs composite particles after treatment with APTES concentrations changed to 7%, 8%, 9%, 10%, 11% and modification times changed to 2, 4, 6, 8, 10 h (the rest methods are the same as above) were placed in different refractive index solutions prepared from glycerol solutions, and the change of the refractive index sensitivity was obtained as shown in FIGS. 9 and 10. As can be seen from fig. 9 to 10, as the concentration of the crosslinking agent and the modification time increase, the refractive index sensitivity of the optical fiber probe increases because the concentration of the crosslinking agent and the modification time increase, the more sensitive material fixed to the optical fiber probe increases, the more LSPR phenomenon occurs, and the more the response of the sensing probe becomes apparent. However, the concentration of the cross-linking agent is too large, the modification time is too long, the steric hindrance on the optical fiber is increased, the fixed amount of the sensitive material is reduced, and the refractive index sensitivity of the sensing probe is reduced. Therefore, the optimum concentration of the crosslinking agent is 9% and the optimum modification time is 8 h.
The GO-AuNPs composite particle optical fiber probes (the rest methods are the same) fixed at different crosslinking times (2, 4, 6, 8 and 10 h) are placed in solutions with different refractive indexes prepared from glycerol solutions, and the relation between the refractive index sensitivity and the crosslinking time is shown in figure 11. As the crosslinking time increases, the increase of GO-AuNPs composite particles fixed on the optical fiber probe increases the generation of LSPR phenomenon, and the more obvious the response of the sensing probe is; when the crosslinking time is too long, the GO-AuNPs composite particles are agglomerated on the optical fiber probe, so that the LSPR phenomenon is affected, and the sensitivity of the optical fiber sensing probe is reduced. Thus, the optimal time for crosslinking was 6 h.
Example 4
A method for detecting optical fiber miRNA-21 of graphene oxide coated gold nanoparticles selects RNA probes 1 and 2 combined with MiRNA-21 specific response, and the rest methods are the same as in example 1, so as to construct a sensor for the MiRNA-21 specific response.
Example 5
RNA probe 1 and RNA probe 2 were replaced with each other, RNA probe 2 was immobilized on an optical fiber, and RNA probe 1 and a target RNA probe were mixed, with the same procedure as in example 1.
Example 6
The fiber core diameter was adjusted to 400 μm, and the rest was the same as in example 1.
The above embodiments are merely preferred embodiments of the present application, and should not be construed as limiting the present application, and the embodiments and features of the embodiments of the present application may be arbitrarily combined with each other without collision. The protection scope of the present application is defined by the claims, and the protection scope includes equivalent alternatives to the technical features of the claims. I.e., equivalent replacement modifications within the scope of this application are also within the scope of the application.
Claims (7)
1. An optical fiber miRNA sensor based on graphene oxide coated gold nano composite particles comprises a broadband light source, a Y-type optical fiber, an optical fiber probe and a spectrometer, and is characterized in that the side surface of the optical fiber probe is coated with graphene oxide-gold nano-RNA composite material GO-AuNPs-RNA probe, and the tail end of the optical fiber probe is coated with a reflecting silver mirror; light enters the optical fiber probe through the Y-shaped optical fiber to excite the graphene oxide-gold nano-sensitive material on the side surface of the probe to generate a local surface plasma resonance phenomenon;
the preparation process of the graphene oxide-gold nano-RNA composite material GO-AuNPs-RNA probe comprises the following steps:
S1, preparing gold nano composite particles coated with graphene oxide by an electrostatic method, wherein the gold nano composite particles are coated inside the graphene oxide;
s2, fixing the RNA probe on the surface of the gold nano composite particle coated by graphene oxide by utilizing a sulfhydryl group through a chemical crosslinking method to obtain a GO-AuNPs-RNA probe composite material;
The sensor RNA probes are GO-AuNPs-RNA probe 1 and GO-AuNPs-RNA probe 2, and respectively conform to the base complementary pairing principle only with the target miRNA, and do not conform to the base complementary pairing principle;
the preparation steps of the optical fiber probe are as follows:
Step (one), silanizing the surface of the optical fiber: after removing an organic cladding on one end surface of the optical fiber, marking the optical fiber as an optical fiber sensing end which is an S end, and inserting the optical fiber S end cleaned by acetone, alcohol and deionized water into an HF acid solution for corrosion so as to strengthen an evanescent field of the optical fiber leakage; cleaning the corroded optical fiber, then, immersing the optical fiber in piranha solution for 2-10 hours, heating the cleaned optical fiber in a vacuum drying oven at 110 ℃ for 1 hour, and activating hydroxyl groups on the surface of the optical fiber; then, soaking the S end of the optical fiber in APTES solution for a plurality of hours to convert the hydroxyl on the surface of the optical fiber into amino; the concentration of APTES is 7% -11%, and the soaking time is 2-10 hours;
Step (two) immobilization of GO-AuNPs on optical fiber: soaking the S end of the optical fiber in the GO-AuNPs composite particle solution, wherein the carboxyl of GO can be combined with the amino on the optical fiber, so that a GO-AuNPs composite particle monolayer film can be formed on the surface of the optical fiber;
And (3) silver mirror reaction end capping: coating a silver film on the end face of the S end of the optical fiber treated in the second step through silver mirror reaction, so as to ensure that light is reflected into a spectrometer;
Diluting the RNA probe1 solution by using PBS buffer solution to obtain RNA probe1 diluent;
step five, soaking the GO-AuNPs functionalized fiber S end treated in the step three in RNA probe1 diluent for 12 h, so that the GO-AuNPs composite particles on the surface of the fiber and the RNA probe1 are fixed together through covalent bonds; washing the unbound RNA molecules on the surface of the optical fiber by using absolute ethyl alcohol and nuclease-free water;
passivating non-specific sites on the surface of the optical fiber, which are not combined with GO-AuNPs composite particles, by utilizing BSA, and soaking the S end of the optical fiber in a BSA solution;
Step seven, obtaining an optical fiber LSPR MIRNA-126 optical fiber probe based on GO-AuNPs-RNAp1 for detecting miRNA-126;
when the GO-AuNPs-RNA probe 1 fixed on the surface of the optical fiber probe performs hybridization reaction with the target miRNA and the GO-AuNPs-RNA probe 2, the distance between graphene oxide-gold nano composite particles is reduced, the local surface plasma effect of the particles is influenced, and the local surface plasma peak position is changed, so that the detection of the target RNA is realized.
2. The graphene oxide coated gold nanocomposite particle-based optical fiber miRNA sensor according to claim 1, characterized in that the optical fiber probe diameter is 125-1000 μm;
The light source is a halogen tungsten lamp broadband light source, and the working wavelength is as follows: 360-1200 nm, and the working wavelength of the spectrometer is 320-1050 nm.
3. The optical fiber miRNA sensor based on graphene oxide coated gold nanocomposite particles according to claim 1, wherein the graphene oxide-gold nano-RNA composite material on the surface of the optical fiber probe is fixed by a chemical crosslinking method.
4. The optical fiber miRNA sensor based on the graphene oxide coated gold nano composite particles according to claim 1, wherein the gold nano composite particles are prepared by adopting an aqueous phase reduction method, the graphene oxide GO is synthesized by adopting a Hummers method, and the GO-AuNPs composite particles specifically comprise the following steps:
(1) Cleaning glassware required in experiments by using aqua regia, washing with deionized water, and drying for later use; the cleaning of the reaction environment can avoid introducing impurities in the growth process of AuNPs;
(2) Adding a trisodium citrate solution into the boiled chloroauric acid solution, and continuing the reaction when the color of the mixed solution is observed to be changed from light yellow to black and finally to be changed into wine red so as to obtain AuNPs;
(3) The preparation method of the GO-AuNPs composite material comprises the following steps: and mixing the synthesized AuNPs solution with the GO solution, and stirring to obtain the GO-AuNPs composite particles, wherein oxygen-containing groups on the surface of GO can promote the embedding of GO into AuNPs, so that the GO-AuNPs composite particles are formed.
5. The optical fiber miRNA sensor based on graphene oxide coated gold nanocomposite particles according to claim 4, wherein in the step (2), auNPs particle size ranges from 10 to 70nm;
in the step (3), the ratio of AuNPs to GO is (1-5): 1.
6. The optical fiber miRNA sensor based on graphene oxide coated gold nanocomposite particles according to claim 4, wherein the preparation steps of the GO-AuNPs-RNA probe composite material are as follows:
step one preparation of GO-AuNPs-RNAp: dispersing the prepared GO-AuNPs solution in PBS buffer solution after washing for a plurality of times, respectively adding TCEP aqueous solution into RNA probe 1 and RNAp probe 2 to activate sulfhydryl at the end of the solution, adding the activated RNA probe into the GO-AuNPs solution, and incubating at room temperature;
Step two, salinization of GO-AuNPs-RNAp: adding NaCl into the GO-AuNPs-RNA probe 1 and GO-AuNPs-RNA probe 2 solutions respectively, incubating after ultrasonic treatment, repeatedly adding NaCl for multiple times, and continuing incubating;
Step three, sample washing: the incubation solution was centrifuged with PBS, and unbound RNA was removed from the solution and dispersed in buffer PBS.
7. A method for quantitatively detecting RNA, the method comprising the optical fiber miRNA sensor based on graphene oxide coated gold nanocomposite particles according to any one of claims 1 to 6, characterized in that the detection steps are as follows:
(a) Mixing the GO-AuNPs-RNAp2 compound with the same volume with miRNA-126 solutions with different concentrations, heating in a water bath, naturally cooling to room temperature, and incubating for a period of time to enable the pairing base between the compound and the target RNA to fully react;
(b) Placing the GO-AuNPs-RNAp1 functionalized optical fiber sensing probe in the mixed solution for a period of time, and recording the change of an optical fiber LSPR spectrogram; the test was repeated three times for each concentration of sample to complete the test.
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