Application of nanochannel membrane in detection of glutathione and construction of drug release system

Abstract

The application discloses application of a nanochannel film in detection of glutathione and construction of a drug release system; and is based on a super-assembly method to prepare a periodic mesoporous organosilica / anodic aluminum oxide (PMO / AAO) hetero film; an electrochemical test device is built, and after the PMO / AAO film is reacted with glutathione, electrochemical test is carried out in an electrolyte, and the PMO / AAO film is used for detection of glutathione; and doxorubicin drugs (DOX) are loaded on the hetero film, and the hetero film is used for glutathione-responsive drug release. The PMO / AAO film prepared through the super-assembly method has the advantages of rich organic functional groups, a simple reaction process, high-flux ion transmission, multifunction and the like compared with other sensing methods, and has good reference value for intelligent nanofluidic nanochannel devices in application of biomolecule sensing and drug release.

Description

Technical Field

[0001] This invention belongs to the field of nano-ion channel technology, specifically relating to the application of a nanochannel membrane in the detection of glutathione and the construction of a drug delivery system. Background Technology

[0002] Glutathione (GSH), an important cellular thiol, plays a crucial role in many physiological and pathological processes. However, glutathione deficiency can lead to cellular oxidative stress and other diseases such as Parkinson's disease and diabetes. Therefore, glutathione detection is essential for disease diagnosis and treatment. Integrating sensing and controlled drug release capabilities into therapeutic nanoplatforms for diagnosis and treatment has attracted increasing attention. Nanofluidic sensing is a promising approach that relies on changes in ion transport signals through nanofluidic membranes, enabling high-throughput and high-sensitivity detection of a wide range of targets. Furthermore, nanochannel membranes possess abundant functional groups and pores, which can be used for efficient drug loading. Therefore, developing a stable and efficient glutathione sensing and glutathione-responsive drug release system based on nanochannel membranes is of great significance. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention aims to provide an application of nanochannel thin films in glutathione detection and the construction of drug delivery systems. This invention utilizes a superassembly method to prepare periodic mesoporous organoporous silica / anodic alumina (PMO / AAO) heterostructure thin films; an electrochemical testing device is constructed, and after the PMO / AAO thin film reacts with glutathione, electrochemical testing is performed in an electrolyte to detect glutathione; doxorubicin (DOX) is loaded onto the heterostructure thin film for glutathione-responsive drug release. This invention, through the superassembly method, prepares periodic mesoporous organoporous silica / anodic alumina heterostructure thin films with abundant organic functional groups, a simple reaction process, high-throughput ion transport, and multifunctionality, providing valuable reference for the application of intelligent nanofluidic nanochannel devices in biomolecular sensing and drug delivery.

[0004] The objective of this invention can be achieved through the following technical solutions.

[0005] An application of a nanochannel membrane in the detection of glutathione and the construction of a drug delivery system is disclosed. The nanochannel membrane, designated PMO / AAO heterostructure, has a tight bilayer structure: a lower porous anodic alumina (AAO) membrane layer and an upper sulfide-bridged layer of periodically porous organosilicone PMO nanoparticles. The method for detecting glutathione is as follows: Step 1: Place the PMO / AAO heterostructure between the anodic and cathodic conductivity cells of the electrochemical testing device, with the PMO layer facing the anode side. The anodic and cathodic conductivity cells are filled with electrolyte. Measure the current before the PMO / AAO heterostructure reacts with the glutathione solution. Step 2: Prepare PBS solutions containing glutathione at different concentrations, with the pH of the PBS between 7.2 and 7.4. Immerse the PMO / AAO heterostructure in the glutathione solution for reaction. Then place the PMO / AAO heterostructure after reaction with glutathione between the anodic and cathodic conductivity cells of the electrochemical testing device. Repeat Step 1 to test the current of the PMO / AAO heterostructure after reaction with the glutathione solution. Step 3: Compare the current changes before and after the PMO / AAO heterofilm reacts with glutathione solutions of different concentrations, and plot a standard curve between glutathione concentration and current change to achieve the detection of glutathione concentration in the test solution.

[0006] In this invention, in step 1, the electrolyte in the anolyte and the cathode conductivity cell is a potassium chloride solution of the same concentration of 0.01-1 mol / L, and both the anode and the cathode use Ag / AgCl electrodes.

[0007] In this invention, in step 3, the concentration of glutathione in the test solution is between 0.01 and 0.1 mmol / L.

[0008] In this invention, the PMO / AAO heterostructure film is prepared by first preparing sulfide-bridged periodic mesoporous organosilicon PMO nanoparticles, and then using a super-assembly strategy to disperse the sulfide-bridged periodic mesoporous organosilicon PMO nanoparticles in an organic solution containing a binder and spin-coating them onto the surface of porous anodic alumina (AAO) to form a PMO layer.

[0009] In this invention, the drug release system constructed from nanochannel membranes is a glutathione-responsive drug release system. By fixing a PMO / AAO heterostructure membrane loaded with drug molecules between two conductivity cells, glutathione-responsive drug controlled release is achieved in a PBS solution containing glutathione.

[0010] In this invention, the preparation method of the drug-loaded PMO / AAO heterostructure film is as follows: First, thioether-bridged periodic mesoporous organosilica PMO nanoparticles are prepared. Then, 8-12 mg of thioether-bridged periodic mesoporous organosilica PMO nanoparticles and 4-6 mg of drug molecules are mixed in 5-10 mL of PBS solution. After stirring in the dark for 10-30 h, the mixture is centrifuged, washed, and dried to obtain drug-loaded thioether-bridged periodic mesoporous organosilica PMO nanoparticles. Then, using a super-assembly strategy, the drug-loaded thioether-bridged periodic mesoporous organosilica PMO nanoparticles are dispersed in an organic solution containing a binder and then spin-coated onto the surface of porous anodic alumina (AAO) to construct a PMO layer.

[0011] In this invention, the drug molecule is doxorubicin (DOX).

[0012] In this invention, the concentration of glutathione in the PBS solution is 5-20 mmol / L.

[0013] In this invention, the method for preparing sulfide-bridged periodic mesoporous organosilica PMO nanoparticles is as follows: (1) First, 0.1-0.2 g of hexadecyltrimethylammonium bromide (CTAB) was added to an ethanol aqueous solution containing ammonia and stirred at 30℃-40℃ to prepare a template agent solution; then, using bis[3-(triethoxysilyl)propyl]tetrasulfide (TESPTS) and tetraethyl orthosilicate (TEOS) as silicon precursors, 0.05-0.20 mL of bis[3-(triethoxysilyl)propyl]tetrasulfide and 0.1-0.4 mL of tetraethyl orthosilicate (TEOS) were mixed and added to the template agent solution while stirring. The mixture was stirred at 30℃-40℃ for 20-30 h; then, the mixture was centrifuged, washed, and nanospheres were obtained; the template agent was hexadecyltrimethylammonium bromide (CTAB). (2) The nanospheres were subjected to hydrothermal reaction to obtain shell-core structured nanoparticles; the hydrothermal reaction temperature was 150℃-180℃ and the hydrothermal reaction time was 10-16h. (3) The nanoparticles were repeatedly washed with acidic ethanol solution at a temperature of 60-90℃ to remove the template agent and create pores, resulting in periodic mesoporous organosilicon PMO nanoparticles with a shell-core structure and sulfide bridging.

[0014] In this invention, the organic solution containing the binder is an acetone solution of polyvinylidene fluoride (PVDF)-hexafluoropropylene (HFP) with a concentration of 3-4 wt%; the mass ratio of periodic mesoporous organosilicon (PMO) nanoparticles to PVDF-HFP is 1:2 to 1:5; and the spin coating speed is 400-600 rpm.

[0015] The present invention further provides a nanochannel membrane for detecting glutathione and constructing a drug delivery system, and a method for preparing the same.

[0016] In this invention, glutathione can induce the redox reaction of disulfide (SS) bonds in the thioether-bridged organosilicon framework of the PMO / AAO film, generating thiol groups (-SH), making the film more hydrophilic, thereby enabling the regulation of ion transport within the nanochannels and resulting in changes in current. Simultaneously, after the DOX-PMO / AAO film loaded with the drug DOX reacts with glutathione, the disulfide bonds in the framework break, disrupting the hydrophobic interaction between the periodic mesoporous organosilicon nanoparticles (PMOs) and DOX, achieving glutathione-responsive drug release.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention fabricates a PMO / AAO heterostructure thin film using a superassembly method. The abundant mesoporous channels of the shell-core structure PMO and the three-dimensional interstitial channels formed by the PMO array result in high ion flux, improving detection sensitivity. Simultaneously, the sulfide bridging of the SS bonds in the organosilicon framework within the film enables a glutathione response, suitable for sensing and intelligent drug release applications. Compared to other nanofluidic sensors, this heterostructure nanochannel thin film offers advantages such as no post-modification required, a simple reaction process, high-throughput ion transport, and multifunctionality, providing valuable reference for the application of intelligent nanofluidic nanochannel devices in biomolecular sensing and drug release. Attached Figure Description

[0018] Figure 1 This is a diagram illustrating the application mechanism of PMO / AAO.

[0019] Figure 2 The current diagram shows the effect of glutathione concentration on the example.

[0020] Figure 3 The image shows the contact angle test results of the heterogeneous films before and after the reaction in the example.

[0021] Figure 4 The UV-Vis spectra of PMOs, DOX, and DOX-PMOs are shown.

[0022] Figure 5 Zeta potential diagrams for PMOs, DOX, and DOX-PMOs.

[0023] Figure 6 The drug release is shown in the graphs for PBS solutions with different reaction times (a) and different GSH concentrations (b). Detailed Implementation

[0024] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0025] Figure 1 This is a diagram illustrating the application mechanism of PMO / AAO.

[0026] A method for preparing a sensing platform with GSH detection and drug release functions includes the following steps: Step 1: Prepare periodic mesoporous organosilicon / anodic aluminum oxide (PMO / AAO) heterostructure films with high porosity and adjustable thickness based on the superassembly method; Step 1 specifically includes the following steps: Step 1-1: Thioether-bridged shell-core structured mesoporous organosilicon nanoparticles (PMOs) were prepared using a surfactant assembly process. Steps 1-2: Hybrid membranes were prepared using PMOs as fillers via a superassembly method. First, PMOs were dispersed in an acetone solution containing 3-4 wt% PVDF-HFP by ultrasonication (0.5-1 h) to obtain a uniform suspension, wherein the mass ratio of PMOs to PVDF-HFP was 1:2 to 1:5. Steps 1-3: Finally, spin-coat the mixed solution onto the AAO membrane (400-600 rpm), and vacuum dry overnight at 80℃-90℃ to obtain the hybrid membrane.

[0027] Step 2: Place the PMO / AAO heterostructure between the anode and cathode cells of the electrochemical testing device, with the PMO layer facing the positive electrode. The conductivity cell is filled with KCl electrolyte solution. Measure the current before the PMO / AAO heterostructure reacts with the glutathione solution. The anolyte in the anode tank is a 0.1 mol / L potassium chloride solution, and the catholyte in the cathode tank is a 0.1 mol / L potassium chloride solution. A potassium chloride solution of mol / L, wherein the electrodes placed in the anode and cathode cells are both Ag / AgCl electrodes; Step 3: Prepare a PBS (pH 7.2-7.4) solution containing glutathione. Immerse the PMO / AAO membrane in the glutathione solution and react for 0-60 min. Repeat step 2 and test the current after the PMO / AAO heterostructure membrane reacts with the glutathione solution to complete the detection of glutathione. Step 4: Investigate the effect of different concentrations of glutathione on electric current; The method for studying the effect of different concentrations of glutathione on current includes: preparing multiple glutathione solutions of different concentrations and obtaining the corresponding current curves using the same method as steps 2-3.

[0028] Step 5: Subsequently, DOX-PMO / AAO membranes loaded with doxorubicin (DOX) were prepared to construct a glutathione-responsive drug release system. 8-12 mg of PMOs and 4-6 mg of DOX were mixed in PBS (pH 7.2-7.4), stirred in the dark for 24 h, centrifuged, washed, and dried to obtain DOX-PMOs. Step 6: A hybrid membrane was prepared using DOX-PMOs as fillers via a super-assembly method. First, DOX-PMOs were dispersed into an acetone solution containing PVDF-HFP by ultrasound to obtain a uniform suspension. The mixed solution was then spin-coated onto an AAO membrane to obtain the hybrid membrane. The ultrasonic time was 0.5-1h, the PVDF-HFP concentration was 3-4wt%, the mass ratio of DOX-PMO to PVDF-HFP was 1:2-1:5, and the spin coating speed was 400-600rpm.

[0029] Step 7: The DOX-PMO / AAO membrane was immobilized between two conductivity cells, and the in vitro release of glutathione in response to DOX was tested in PBS solution (pH 7.2-7.4) containing 0-10 mM (5 mL). The PBS solution was collected at regular time intervals, and UV-Vis spectroscopy was performed at 490 nm. Step 8: Investigate the effects of different concentrations of glutathione and different reaction times on drug release rate.

[0030] The drug release rate is calculated using the formula: Drug release rate (%) = Drug release amount / Maximum drug load * 100. The method for studying the effects of different concentrations of glutathione and different reaction times on the drug release rate includes: preparing glutathione solutions of different concentrations and obtaining the corresponding spectra at different reaction times using the same method as in step 7.

[0031] The following is a specific example. Example

[0032] A method for preparing a glutathione detection and responsive drug release system, comprising the following steps: (i) Preparation of periodic mesoporous organosilicon / anodic oxide with high porosity and adjustable thickness based on superassembly method Aluminum (PMO / AAO) heterostructure thin films; specific methods include: (1) ① First, add 0.16g of hexadecyltrimethylammonium bromide (CTAB) to an ethanol-water solution containing ammonia, prepared by mixing 1mL of ammonia, 30mL of ethanol, and 75mL of deionized water. Stir the ethanol-water solution containing ammonia at 35°C to prepare the template agent solution; then add 0.1mL of bis[3-(triethoxysilyl)propyl]tetrasulfide and 0.25mL of tetraethyl orthosilicate (TEOS). Mix and add to the template agent solution while stirring. Continue stirring at 35℃ for 24h, then centrifuge and wash to obtain nanospheres; ② Perform hydrothermal reaction treatment on the nanospheres, and centrifuge to obtain shell-core structured nanoparticles; the hydrothermal reaction temperature is 150℃ and the hydrothermal reaction time is 12h; ③ Wash the nanoparticles repeatedly at 60℃ with an acidic ethanol solution prepared by mixing 37% concentrated hydrochloric acid and ethanol at a volume ratio of 1:500 to remove the template agent and create pores to obtain sulfide-bridged periodic mesoporous organosilicon nanoparticles (PMOs) with a shell-core structure; (2) A hybrid membrane was prepared using PMOs as fillers by superassembly. First, PMOs were dispersed in an acetone solution containing (3.8 wt%) PVDF-HFP by ultrasound, wherein the mass ratio of PMO to PVDF-HPF was 2:5. The mixture was sonicated for 0.5 h to obtain a uniform suspension. The suspension was then spin-coated onto an AAO membrane (500 rpm) and vacuum dried overnight at 85 °C to obtain the hybrid membrane.

[0033] (ii) Prepare 0.1 M potassium chloride solution as anolyte and catholyte respectively. Use the same electrochemical testing device as CN202010640001.8 (including picoammeter and a pair of Ag / AgCl electrodes, and the other device details are the same) to test the electrochemical performance (record the current value at voltage +2V). The PMO / AAO heterofilm is installed at the connection channel between anolyte cell 1 and catholyte cell 2, with the PMO layer facing the anode side. Measure the current of PMO / AAO before reaction with glutathione. (III) Prepare a PBS (pH 7.4) solution containing glutathione. Immerse PMO / AAO in the glutathione solution and react for 60 min. Repeat step (II) and measure the current of PMO / AAO after reacting with glutathione. It can be seen that the current increases, thus completing the detection of glutathione.

[0034] (iv) Study the effect of different concentrations of glutathione on current: Prepare multiple glutathione solutions of different concentrations and obtain the corresponding current curves using the same method as in steps (ii) to (iii).

[0035] (V) Subsequently, DOX-PMO / AAO membranes loaded with DOX drugs were prepared to construct a glutathione-responsive drug release system. 10 mg of PMOs prepared in step (I) and 5 mg of DOX drug molecules were mixed in PBS (pH 7.4) solution, stirred in the dark for 24 h, and then centrifuged, washed and dried to obtain DOX-PMOs.

[0036] (vi) A hybrid membrane was prepared using DOX-PMOs as fillers via a super-assembly method. First, DOX-PMOs were dispersed in an acetone solution containing 3.8 wt% PVDF-HFP by ultrasonication (0.5 h), with a mass ratio of DOX-PMO to PVDF-HFP of 2:5, to obtain a uniform suspension. The mixed solution was then spin-coated onto an AAO membrane (500 rpm) to obtain the hybrid membrane.

[0037] (vii) The DOX-PMO / AAO membrane was immobilized between two conductivity cells, and the in vitro release of glutathione in response to DOX was tested in PBS solutions (pH 7.4) containing 0, 5, and 10 mM (5 mL). A circular window with a diameter of 4 mm was located between the two conductivity cells, representing the actual membrane area to be tested; the drug release rate in this area was measured. PBS solutions were collected at regular time intervals, and UV-Vis spectroscopy was performed at 490 nm.

[0038] (viii) Study the effects of different concentrations of glutathione and different reaction times on drug release rate: Prepare glutathione solutions of different concentrations and obtain the corresponding spectra at different reaction times using the same method as in step 7.

[0039] from Figure 2 It can be seen that the ion current passing through the PMO / AAO membrane increases after contact with glutathione. Different concentrations of glutathione were prepared, and the current gradually increased with increasing glutathione concentration. The current change at 2V showed a good linear relationship with the glutathione concentration in the range of 0.01-0.1 mM.

[0040] like Figure 3 The image shows the contact angle measurements of the heterojunction nanochannels before and after the reaction. It can be seen that the contact angle decreased from 41.9±0.3° to 28.8±0.5° after reacting with 10 mM glutathione. This indicates that the change in wettability is the main factor affecting the change in ion current. The increase in current in PMO / AAO may be due to the redox reaction of the disulfide (SS) bonds in the thioether-bridged organosilicon framework induced by glutathione breaking, generating thiol groups (-SH), which lead to channel hydrophilicity, regulate ion transport behavior, and result in a high ion current.

[0041] The unique shell-core structure and abundant porous channels make PMOs excellent nanocarriers. Figure 4 As can be seen, after loading DOX, the UV-Vis spectrum of DOX-PMOs shows the characteristic absorption peaks of DOX. From... Figure 5 It can be seen that the zeta potential of PMOs loaded with positively charged drugs changes from negative to positive, confirming the successful loading of DOX into PMO nanoparticles.

[0042] from Figure 6 It can be seen that, over time, the DOX absorption intensity gradually increases at 10 mM glutathione, indicating a gradual release of DOX. In contrast, the drug release rates are lower at 0 mM and 5 mM glutathione over the same time period. These results demonstrate that the DOX-PMO / AAO membrane system can achieve a sustained-release effect. After the DOX-PMO / AAO membrane reacts with glutathione, the disulfide bonds in the framework break, disrupting the hydrophobic interaction between PMOs and DOX, thus achieving glutathione-responsive drug release.

[0043] In summary, the PMO / AAO prepared in the embodiments of the present invention can achieve glutathione detection and intelligent responsive drug release.

Claims

1. The application of a nanochannel membrane in detecting glutathione and constructing a drug delivery system, characterized in that, The nanochannel film is designated as a PMO / AAO heterostructure film, which has a tight bilayer structure. The lower layer is a porous anodic alumina (AAO) film layer, and the upper layer is a sulfide-bridged layer of periodic mesoporous organosilicon PMO nanoparticles. The method for detecting glutathione is as follows: Step 1: Place the PMO / AAO heterostructure between the anodic and cathodic conductivity cells of the electrochemical testing device, with the PMO layer facing the anode side. The anodic and cathodic conductivity cells are filled with electrolyte. Measure the current before the PMO / AAO heterostructure reacts with the glutathione solution. Step 2: Prepare PBS solutions containing glutathione at different concentrations, with the pH of the PBS between 7.2 and 7.

4. Immerse the PMO / AAO heterostructure in the glutathione solution for reaction. Then place the PMO / AAO heterostructure after reaction with glutathione between the anodic and cathodic conductivity cells of the electrochemical testing device. Repeat Step 1 to test the current of the PMO / AAO heterostructure after reaction with the glutathione solution. Step 3: Compare the current changes before and after the PMO / AAO heterofilm reacts with glutathione solutions of different concentrations, and plot a standard curve between glutathione concentration and current change to achieve the detection of glutathione concentration in the test solution.

2. The application according to claim 1, characterized in that, In step 1, the electrolyte in the anolyte and cathode conductivity cells is a potassium chloride solution of the same concentration, ranging from 0.01 to 1 mol / L, and both the anode and cathode use Ag / AgCl electrodes. In step 3, the glutathione concentration in the test solution is between 0.01 and 0.1 mmol / L.

3. The application according to claim 1, characterized in that, The PMO / AAO heterostructure film was prepared by first preparing sulfide-bridged periodic mesoporous organosilicon PMO nanoparticles, and then using a super-assembly strategy, dispersing the sulfide-bridged periodic mesoporous organosilicon PMO nanoparticles in an organic solution containing a binder and then spin-coating them onto the surface of porous anodic alumina (AAO) to form a PMO layer.

4. The application according to claim 1, characterized in that, The drug release system constructed from nanochannel membranes is a glutathione-responsive drug release system. By fixing a PMO / AAO heterostructure membrane loaded with drug molecules between two conductivity cells, glutathione-responsive drug controlled release is achieved in a PBS solution containing glutathione.

5. The application according to claim 4, characterized in that, The preparation method of drug-loaded PMO / AAO heterostructure film is as follows: First, thioether-bridged periodic mesoporous organosilica PMO nanoparticles are prepared. Then, 8-12 mg of thioether-bridged periodic mesoporous organosilica PMO nanoparticles and 4-6 mg of drug molecules are mixed in 5-10 mL of PBS solution. After stirring in the dark for 10-30 h, the mixture is centrifuged, washed, and dried to obtain drug-loaded thioether-bridged periodic mesoporous organosilica PMO nanoparticles. Then, using a super-assembly strategy, the drug-loaded thioether-bridged periodic mesoporous organosilica PMO nanoparticles are dispersed in an organic solution containing a binder and then spin-coated onto the surface of porous anodic alumina (AAO) to construct a PMO layer.

6. The application according to claim 5, characterized in that, The drug molecule is doxorubicin (DOX).

7. The application according to claim 4, characterized in that, The concentration of glutathione in PBS solution is 5-20 mmol / L.

8. The application according to claim 1, 3, or 5, characterized in that, The preparation method of sulfide-bridged periodic mesoporous organosilica PMO nanoparticles is as follows: (1) First, add 0.1-0.2g of hexadecyltrimethylammonium bromide (CTAB) to an ethanol aqueous solution containing ammonia and stir at 30℃-40℃ to prepare a template agent solution; then, using bis[3-(triethoxysilyl)propyl]tetrasulfide (TESPTS) and tetraethyl orthosilicate (TEOS) as silicon precursors, mix 0.05-0.20mL of bis[3-(triethoxysilyl)propyl]tetrasulfide and 0.1-0.4mL of tetraethyl orthosilicate (TEOS) and add it to the template agent solution while stirring. Continue stirring at 30℃-40℃ for 20-30h; then centrifuge, wash, and obtain nanospheres; (2) The nanospheres were subjected to hydrothermal reaction to obtain shell-core structured nanoparticles; the hydrothermal reaction temperature was 150℃-180℃ and the hydrothermal reaction time was 10-16h. (3) The nanoparticles were repeatedly washed with acidic ethanol solution at a temperature of 60-90℃ to remove the template agent and create pores, resulting in periodic mesoporous organosilicon PMO nanoparticles with a shell-core structure and sulfide bridging.

9. The application according to claim 1 or 5, characterized in that, The organic solution containing the binder is an acetone solution of polyvinylidene fluoride-hexafluoropropylene (PVDF-HFP) with a concentration of 3-4 wt%; the mass ratio of periodic mesoporous organosilicon (PMO) nanoparticles to PVDF-HFP is 1:2-1:5; the spin coating speed is 400-600 rpm.

10. A nanochannel thin film for use in the application described in claim 1 or 5, characterized in that, The nanochannel film is referred to as a PMO / AAO heterostructure film, which has a tight bilayer structure. The lower layer is a porous anodic aluminum oxide (AAO) film layer, and the upper layer is a sulfide-bridged periodic mesoporous organosilicon (PMO) nanoparticle layer.

Images