CN110879243A - Preparation method and application of functional biological inorganic nano composite membrane - Google Patents
Preparation method and application of functional biological inorganic nano composite membrane Download PDFInfo
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Abstract
A preparation method and application of a functional biological inorganic nano composite membrane belong to the technical field of material chemistry and biological detection. The invention forms a heterogeneous nano-channel structure by combining a nano-channel formed by gold nanoparticles based on the pre-modification of a biological aptamer with an anodic aluminum oxide substrate; transferring the gold film to the same anode alumina substrate repeatedly for one time or multiple times to obtain the functional biological inorganic nano composite film. Transferring the gold film to the same anode alumina substrate repeatedly for many times to obtain the biological inorganic nano composite film formed by multiple layers of gold films. The application of the Chinese traditional medicine is as follows: based on the ion current rectification characteristic of the heterogeneous nano-channel, the nano-channel is modified through hybridization of the biological aptamer and the target object so as to influence ion transmission, and the rectification ratio is used as a method for quantitatively detecting the target object. The invention endows the artificial nanochannel intelligent biosensor with the functions by a method with simple and convenient control and intuitive result.
Description
Technical Field
The invention relates to a preparation method and application of a functional biological inorganic nano composite membrane, belonging to the technical field of material chemistry and biological detection.
Background
Biomimetic nanochannels have greater dimensional flexibility, chemical stability and mechanical support than biological channels, while artificial nanochannels have been shown to possess ion transport properties similar to biological channels, such as ion selectivity, current rectification and ion gating, which can be effectively tuned by controllable parameters to obtain stable characterization results. Therefore, this new, well-controlled nanofluidic phenomenon has become the basis for many promising applications, such as bioenergy conversion systems, nanofluidic sensing devices, and the like.
The regulation of ion channels for ions to enter and exit cell membranes has important significance on a plurality of physiological functions, so that the potential of the bionic nano channel serving as the basis of biological, physiological and chemical research and the prospect of the flow control phenomenon thereof on the aspect of practical application can be predicted, and the development of various intelligent nano channels is promoted.
The invention provides innovative insight for the design of functional nanofluid devices based on the application of ionic current rectification characteristics of a synthetic nanofluid system.
Disclosure of Invention
Compared with other detection methods which need to undergo a series of complex chemical reactions and have poor controllability, the method of the invention endows the artificial nanochannel intelligent biosensor with functions by a method with simple and convenient control and intuitive result.
The technical scheme of the invention is a preparation method of a functional biological inorganic nano composite membrane, which forms a heterogeneous nano channel structure by compounding a nano channel formed by gold nano particles based on the pre-modification of a biological aptamer and an anodic aluminum oxide substrate; transferring the gold film to the same anode alumina substrate repeatedly for one time or multiple times to obtain the functional biological inorganic nano composite film. Repeatedly transferring the gold film to the same anodic alumina substrate for many times to obtain a biological inorganic nano composite film formed by multiple layers of gold films; in the process of repeatedly transferring the gold film, each layer of gold film is added and is subjected to heat fixation.
The nano-channel is a heterogeneous structure formed by covering a single layer or multiple layers of gold nano-particle films on the surface of an anodic aluminum oxide substrate.
In one implementation method of the invention, the size of the gold nanoparticles is applicable to be in a range of 15-55 nm.
In one embodiment of the present invention, the gold nanoparticles are synthesized by a sodium citrate reduction method.
In one embodiment of the present invention, the gold nanoparticle film is a regularly arranged array structure film formed by an oil-water self-assembly method.
In one implementation method of the invention, the oil-water self-assembly method is a process of mixing gold nanoparticle solution and n-hexane for layering, then dripping an inducer absolute ethyl alcohol to destroy the stability of particles in the gold nanoparticle solution so as to transfer the particles to an oil-water interface for array arrangement, and after the n-hexane volatilizes, a gold film stably exists in the gas-liquid interface.
In one embodiment of the invention, the gold film is a highly ordered small pore monolayer gold nanoparticle array.
The method comprises the following specific steps:
(1) placing 7mL of aptamer-modified gold nanoparticle solution in a beaker, adding 1.5mL of n-hexane, slowly dripping absolute ethyl alcohol along the wall after layering, gradually forming a gold nano array on an oil-water interface, stopping dripping the absolute ethyl alcohol after the array is assembled into a large-area gold film, and stably enabling the gold film to exist in the gas-liquid interface after the n-hexane is completely volatilized;
(2) taking an anodic aluminum oxide substrate to support a gold film on a gas-liquid interface, and completely covering the substrate with the gold film to obtain a biological inorganic nano composite film; and (3) placing the obtained biological inorganic nano composite membrane in a drying and ventilating environment of 65 ℃ for thermal fixation for 3h to obtain the functional biological inorganic nano composite membrane.
(3) Repeating the steps (1) and (2) on the same alumina substrate to obtain the biological inorganic nano composite membrane formed by multiple layers of gold membranes.
In one embodiment of the present invention, the overall transfer time of the gold film transfer process is 20-30s, so as to avoid mechanical damage to the film caused by too fast operation.
The pre-modification method of the gold nanoparticles modifies the gold nanoparticles according to the effective modification ratio, so that the morphology state of the gold nanoparticles is stable, and particularly, the film forming property of the gold nanoparticles is not influenced.
The gold nanoparticles are firstly modified by polyethylene glycol to enhance the stability of the particles and the mechanical strength of the gold nanoparticle film; the polyethylene glycol is covalently bonded with the gold nanoparticles through sulfydryl, and the modification effect is stable.
The preparation process of the aptamer modified gold nanoparticle solution is as follows:
(1) adding 10mL of 4g/L chloroauric acid solution into 400mL of ultrapure water, uniformly stirring, heating at 350 ℃ for boiling, adding 2.8mL of nano gold seed solution and 2mL of 1% sodium citrate solution after boiling, uniformly mixing, continuously heating and stirring to keep a boiling state, stopping heating when the color of the solution is changed into wine red and is not changed any more, and stirring at a low speed until the solution is cooled to room temperature to obtain a uniform 30nm gold nanoparticle solution;
(2) centrifuging 1mL of the synthesized gold nanoparticle solution for 10min at 5000rpm, removing supernatant, taking a precipitate, and suspending the precipitate in 100 mu L of 10mM PB buffer solution;
(3) collecting the concentrated gold nanoparticle solution, adding a polyethylene glycol solution according to the proportion of 1:50 to enable the final concentration of polyethylene glycol to be 500nM, incubating for 30min, centrifuging for 10min at 5000rpm, discarding the supernatant, taking the precipitate, and resuspending under the same conditions in an equal proportion, wherein the excess sulfhydryl is eliminated;
(4) collecting gold nanoparticle solution with fixed polyethylene glycol, adding protein aptamer according to the ratio of 1:10 to enable the final concentration of the aptamer to be 100nM, incubating for 12h, centrifuging for 10min at 5000rpm, discarding supernatant, taking precipitate, resuspending in the same condition at equal ratio, and removing excessive aptamer to obtain the final gold nanoparticle solution modified by the aptamer.
The synthesis method of the nano gold seed solution comprises the following steps: adding 2mg of trisodium citrate into 30mL of ultrapure water, dissolving, adding 500 mu L of 4g/mL chloroauric acid solution, uniformly mixing, adding 600 mu L of 100mM sodium borohydride solution under rapid stirring, and continuously stirring for 20s to obtain orange nanogold seed solution.
In one embodiment of the invention, the centrifugal speed of the gold nanoparticle solution is 5000rpm, and the time is 10 min.
In one embodiment of the present invention, the concentration ratio of the gold nanoparticles is 10 times.
In one embodiment of the invention, the polyethylene glycol has a molecular weight of 5000.
In one embodiment of the present invention, the polyethylene glycol is modified at one end with a thiol group (-SH) and at one end with a carboxyl group (-COOH).
In one embodiment of the present invention, the protein aptamer of step (4) is a mucin aptamer; the mucin aptamer is 32 nucleotides in length (5'-ttt ttt tgc agt tga tcc ttt gga tac cct gg-3').
In one embodiment of the invention, the aptamer is covalently linked to the surface of the gold nanoparticle with a gold-sulfur bond.
The application of the functional biological inorganic nano composite membrane is based on the ion current rectification characteristic of a heterogeneous nano channel, the nano channel is modified through hybridization of a biological aptamer and a target object so as to influence ion transmission, and the rectification ratio is used as a method for quantitatively detecting the target object.
The ion rectifying property refers to a phenomenon in which an ion current is increased or decreased due to a difference in interaction force of a heterostructure with respect to various ions.
In the application of the functional biological inorganic nano composite membrane, after the functional biological inorganic nano composite membrane and an actual protein standard sample are incubated for 20min, two organic glass electrolytic cells with the external through hole diameter of 1mm are combined, 2mL of 10mM KCl solution is added into the organic glass electrolytic cells, and an external Ag/AgCl electrode is applied with voltage, so that an ion communication loop is formed; setting the linear scanning range to be-2V and the scanning speed to be 0.05V/s, placing the composite membrane between the outer through holes of the two electrolytic cells, and measuring the ionic current under bias voltage.
The invention has the beneficial effects that: the heterogeneous structure of the modified aptamer has selectivity on electrolyte ions under different biases, and is particularly represented by ionic current rectification characteristics.
The method determines the optimal modification amount of the aptamer, realizes that the nano channel has special response to various environmental stimuli, and has great significance for realizing accurate regulation and control of the nano channel.
Compared with the existing nano-channel for biological detection, the method has the advantages of easy chemical modification, simple and convenient operation and strong controllability.
The invention realizes the quantity control and accurate detection of mucin, the detection limit is reduced along with the increase of the number of layers of gold films of heterogeneous films, and the detection range is enlarged along with the increase of the number of layers of gold films of heterogeneous films.
The invention provides a novel method for utilizing heterogeneous nanochannel ion transport characteristics, utilizes simple electrochemical signals to respond to a target object, and abandons complex chemical reactions and complicated operation means.
Drawings
FIG. 1 is a transmission electron microscope image of gold nanoparticles with and without modified aptamers. a. C, ultraviolet-visible spectrum shows the influence of the aptamer on the light absorption intensity of the gold nanoparticles, and d, ultraviolet-visible spectrum shows the influence of the aptamer on the absorbance of the gold nanoparticles.
FIG. 2 is a structural representation of a monolayer modified gold film.
FIG. 3 is a scanning electron micrograph of an anodized aluminum substrate.
FIG. 4 is a scanning electron micrograph of a lateral cross-section of a heterogeneous structure with a single gold film covering an anodized aluminum substrate.
FIG. 5 is a graph showing the ionic current profile through a heterogeneous membrane composed of a monolayer of modified and unmodified aptamer gold under an applied voltage.
FIG. 6 is a graph showing the comparison of the rectifying strength of 1-10 layers of gold films modified under an applied voltage. a. And b, comparing the intensity of the current and the intensity of the rectification ratio.
FIG. 7 is a current-voltage diagram of a functional bio-inorganic nanocomposite film composed of a single gold film. a. And b, detecting the linear range of the mucin standard sample by a heterogeneous membrane formed by covering a single-layer modified gold membrane on an anodized aluminum substrate.
FIG. 8 shows a heterogeneous membrane assay mucin standard formed by 8-layer modified gold membranes. a. And b, detecting the linear range of the mucin standard sample by using a heterogeneous membrane formed by covering 8 layers of modified gold membranes on the anodized aluminum substrate.
FIG. 98 shows the comparison of different proteins detected by a heterogeneous membrane formed by a layer of modified gold. a. The rectification degree of the composite membrane incubated with different proteins is strong and weak, and a b, current-voltage diagram.
Detailed Description
The present invention will be further illustrated below with reference to specific examples and comparative examples.
The detection method comprises the following steps:
the validity detection method comprises the following steps: the effect of the heterogeneous film composed of a single-layer gold film, which is modified or unmodified, on the rectification of the ion current under an applied voltage was observed to be compared, and the effect was good if the effect was modified to be equal to or greater than that of the film without modification, the effect was general if the effect was slightly smaller than that of the film without modification, and the effect was poor if the effect was much smaller than that of the film without modification.
The stability detection method comprises the following steps: soaking a heterogeneous membrane consisting of a gold membrane modified with an aptamer in an electrolyte solution in the same detection environment for 2 days, observing the surface change and the electrochemical signal change of the heterogeneous membrane, wherein if the surface change and the electrochemical signal change do not change, the stability of the heterogeneous membrane is good, if the surface change and the electrochemical signal change do not change, the stability of the heterogeneous membrane is general, and if the surface change and the electrochemical signal change are obvious, the stability of the heterogeneous membrane is poor.
The characterization method comprises the following steps: and performing structural characterization on the material for forming the heterogeneous film and the heterogeneous film by means of a transmission electron microscope, a scanning electron microscope, various spectra and the like. In addition, the biosensor intelligence of the nano-channel of the heterogeneous membrane system is represented by using an electrochemical signal.
Example 1: preparation of aptamer modified gold nanoparticles
(1) Adding 10mL of 4g/L chloroauric acid solution into 400mL of ultrapure water, uniformly stirring, heating at 350 ℃ for boiling, adding 2.8mL of nano gold seed solution and 2mL of 1% sodium citrate solution after boiling, uniformly mixing, continuously heating and stirring to keep a boiling state, stopping heating when the color of the solution is changed into wine red and is not changed any more, and stirring at a low speed until the solution is cooled to room temperature to obtain a uniform 30nm gold nanoparticle solution.
The synthesis method of the nanogold seed solution comprises the following steps: adding 2mg of trisodium citrate into 30mL of ultrapure water, dissolving, adding 500 mu L of 4g/mL chloroauric acid solution, uniformly mixing, adding 600 mu L of 100mM sodium borohydride solution under rapid stirring, and continuously stirring for 20s to obtain orange nanogold seed solution.
(2) 1mL of the synthesized gold nanoparticle solution was centrifuged at 5000rpm for 10min, the supernatant was discarded to collect the pellet, and the pellet was resuspended in 100. mu.L of 10mM PB buffer.
(3) Collecting the concentrated gold nanoparticle solution, adding a polyethylene glycol solution according to the proportion of 1:50 to enable the final concentration of polyethylene glycol to be 500nM, incubating for 30min, centrifuging for 10min at 5000rpm, removing the supernatant, taking the precipitate, and resuspending under the same conditions in an equal proportion, wherein the aim of removing the redundant sulfydryl is to remove the excessive sulfydryl.
(4) Collecting gold nanoparticle solution with immobilized polyethylene glycol, adding protein aptamer according to the ratio of 1:10 to make the final concentration of the aptamer 100nM, incubating for 12h, centrifuging at 5000rpm for 10min, discarding supernatant, taking precipitate, and resuspending under the same conditions at equal ratio to remove excess aptamer.
The characterization results were as follows: the transmission electron microscope result shows that the size and the shape of the gold nanoparticles for modifying the aptamer are unchanged, and the uniformity and the consistency are still kept (see figure 1 a); the results of the particle size analysis showed that there was no major change in the size of the aptamer-modified gold nanoparticles (see fig. 1 b); the UV-Vis spectrum shows that the aptamers do not interfere significantly with the light absorption of the nanoparticles (see FIG. 1 c).
Example 2: preparation of functional biological inorganic nano composite film
(1) Placing 7mL of aptamer-modified gold nanoparticle solution in a beaker, adding 1.5mL of n-hexane, slowly dripping absolute ethyl alcohol along the wall after layering, gradually forming a gold nano array on an oil-water interface, stopping dripping the absolute ethyl alcohol after the array is assembled into a large-area gold film, and stably enabling the gold film to exist in the gas-liquid interface after the n-hexane is completely volatilized.
(2) And taking the anodic aluminum oxide substrate to support the gold film on the gas-liquid interface, and completely covering the substrate with the gold film to obtain the biological inorganic nano composite film.
(3) The obtained heterogeneous membrane was subjected to heat fixation for 3 hours in a dry and ventilated environment at 65 ℃ to enhance the stability of the heterogeneous membrane.
(4) Repeating the steps (1) to (3) on the same anodized aluminum substrate to obtain a biological inorganic nano composite membrane structure consisting of a plurality of layers of gold membranes.
And (4) carrying out stability detection and characterization on the functional biological inorganic nano composite membrane obtained in the step (4).
Validity detection shows that the ionic current rectification effect of the composite membrane of the modified aptamer is stronger than that of an unmodified membrane, which indicates that the validity is good (see figure 5).
Stability detection shows that the appearance and the electrochemical properties of the composite membrane have no obvious change after the composite membrane is soaked in an electrolyte solution for 2 days, which indicates that the composite membrane has good stability.
The characterization results were as follows: the results of transmission electron microscope and scanning electron microscope of the bio-inorganic nano composite film show that the gold film is composed of highly ordered and regularly and uniformly arranged single-layer nano arrays (see fig. 2-4).
The electrochemical signal detection result shows that the rectification degree of the composite membrane is enhanced along with the increase of the number of the gold membrane layers (see figure 6).
Example 3: application of functional biological inorganic nano composite membrane in detection of actual protein standard sample
After the functional biological inorganic nano composite membrane and an actual protein standard sample are incubated for 20min, two organic glass electrolytic cells with the external through hole diameter of 1mm are combined, 2mL of 10mM KCl solution is added into the cells, and an external Ag/AgCl electrode is applied with voltage, so that an ion communication loop is formed. Setting the linear scanning range to be-2V and the scanning speed to be 0.05V/s, placing the composite membrane between the outer through holes of the two electrolytic cells, and measuring the ionic current under bias voltage.
The characterization results were as follows: the current-voltage diagram of the functional biological inorganic nano composite membrane formed by the single-layer gold membrane shows that the linear range of the response of the functional biological inorganic nano composite membrane to the target object is 100 fg/mL to 10 pg/mL (see figure 7); the current-voltage diagram of the functional biological inorganic nano composite membrane consisting of 8 layers of gold membranes shows that the linear range of the response of the functional biological inorganic nano composite membrane to a target object is 1 fg/mL to 10 pg/mL (see figure 8). With the increase of the number of the modified aptamer gold film layers, the detection limit of the functional biological inorganic nano composite film is reduced, and the detection range is expanded. The selective binding between the aptamer and the target protein confers specificity to the composite membrane as evidenced by the strong and weak rectification of the composite membranes incubated with the different proteins (see FIG. 9).
Comparative example 1
Step (4) of example 1 was replaced as follows: collecting gold nanoparticle solution with immobilized polyethylene glycol, adding protein aptamer according to the ratio of 1:20 to make the final concentration of the aptamer 200 nM, incubating for 12h, centrifuging at 5000rpm for 10min, discarding supernatant, taking precipitate, and resuspending at the same condition and equal ratio.
The characterization results were as follows: the obtained gold nanoparticle solution for modifying the aptamer has poor film forming property, is difficult to form a large-area uniform nano gold array, is easy to disperse and break and has poor stability.
Claims (9)
1. A preparation method of a functional biological inorganic nano composite membrane is characterized by comprising the following steps: the functional biological inorganic nano composite membrane is obtained by compounding a nano-pore channel formed by gold nano-particles based on the pre-modification of a biological aptamer with an anodic aluminum oxide substrate to form a heterogeneous nano-channel structure.
2. The method for preparing the functional bio-inorganic nanocomposite membrane according to claim 1, wherein: transferring the gold film to the same anode alumina substrate repeatedly for many times to obtain the biological inorganic nano composite film formed by multiple layers of gold films.
3. The method for preparing the functional bio-inorganic nano composite membrane according to claim 1, which comprises the following steps:
(1) placing 7mL of the aptamer-modified gold nanoparticle solution in a beaker, adding 1.5mL of n-hexane, slowly dripping absolute ethyl alcohol along the wall after layering, gradually forming a gold nano array on an oil-water interface, stopping dripping the absolute ethyl alcohol after the array is assembled into a large-area gold film, and stably enabling the gold film to exist in the gas-liquid interface after the n-hexane is completely volatilized;
(2) taking an anodic aluminum oxide substrate to support a gold film on a gas-liquid interface, and completely covering the substrate with the gold film to obtain a biological inorganic nano composite film; and (3) placing the obtained biological inorganic nano composite membrane in a drying and ventilating environment of 65 ℃ for thermal fixation for 3h to obtain the functional biological inorganic nano composite membrane.
4. The method for preparing the functional bio-inorganic nanocomposite membrane according to claim 3, wherein: repeating the steps (1) and (2) on the same alumina substrate to obtain the biological inorganic nano composite membrane formed by multiple layers of gold membranes.
5. The method for preparing the functional biological inorganic nano composite membrane according to claim 3, wherein the aptamer-modified gold nanoparticle solution is prepared by the following steps:
(1) adding 10mL of 4g/L chloroauric acid solution into 400mL of ultrapure water, uniformly stirring, heating at 350 ℃ for boiling, adding 2.8mL of nano gold seed solution and 2mL of 1% sodium citrate solution after boiling, uniformly mixing, continuously heating and stirring to keep a boiling state, stopping heating when the color of the solution is changed into wine red and is not changed any more, and stirring at a low speed until the solution is cooled to room temperature to obtain a uniform 30nm gold nanoparticle solution;
(2) centrifuging 1mL of the synthesized gold nanoparticle solution for 10min at 5000rpm, removing supernatant, taking a precipitate, and suspending the precipitate in 100 mu L of 10mM PB buffer solution;
(3) collecting the concentrated gold nanoparticle solution, adding a polyethylene glycol solution according to the proportion of 1:50 to enable the final concentration of polyethylene glycol to be 500nM, incubating for 30min, centrifuging for 10min at 5000rpm, discarding the supernatant, taking the precipitate, and resuspending under the same conditions in an equal proportion, wherein the excess sulfhydryl is eliminated;
(4) collecting gold nanoparticle solution with fixed polyethylene glycol, adding protein aptamer according to the ratio of 1:10 to enable the final concentration of the aptamer to be 100nM, incubating for 12h, centrifuging for 10min at 5000rpm, discarding supernatant, taking precipitate, resuspending in the same condition at equal ratio, and removing excessive aptamer to obtain the final gold nanoparticle solution modified by the aptamer.
6. The method for preparing the functional biological inorganic nano composite membrane according to claim 5, wherein the method for synthesizing the nano gold seed solution comprises the following steps: adding 2mg of trisodium citrate into 30mL of ultrapure water, dissolving, adding 500 mu L of 4g/mL chloroauric acid solution, uniformly mixing, adding 600 mu L of 100mM sodium borohydride solution under rapid stirring, and continuously stirring for 20s to obtain orange nanogold seed solution.
7. The method for preparing the functional bio-inorganic nanocomposite membrane according to claim 5, wherein: the protein aptamer in the step (4) is a mucin aptamer.
8. The use of the functional bio-inorganic nanocomposite membrane prepared by the method of claim 1 or 2, wherein: based on the ion current rectification characteristic of the heterogeneous nano-channel, the nano-channel is modified through hybridization of the biological aptamer and the target object so as to influence ion transmission, and the rectification ratio is used as a method for quantitatively detecting the target object.
9. The use of the functional bio-inorganic nanocomposite membrane prepared by the method of claim 1 or 2, wherein: after the functional biological inorganic nano composite membrane and an actual protein standard sample are incubated for 20min, two organic glass electrolytic cells with the external through hole diameter of 1mm are combined, 2mL of 10mM KCl solution is added into the organic glass electrolytic cells, and an external Ag/AgCl electrode is applied with voltage, so that an ion communication loop is formed; setting the linear scanning range to be-2V and the scanning speed to be 0.05V/s, placing the composite membrane between the outer through holes of the two electrolytic cells, and measuring the ionic current under bias voltage.
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