Enhanced 3D gold nano-tree and electrochemical preparation method thereof and application of enhanced 3D gold nano-tree to hazardous substance detection and in-situ degradation

Abstract

The invention discloses an enhanced 3D gold nano tree and a preparation method and application thereof. The preparation method comprises the steps that a gold nano film serves as a working electrode, EDTA, K2HPO4, Na2SO3 and HAuCl4. 3H2O are used for preparing an electrolyte solution, the pH value of the electrolyte solution is 5.5-7, electrochemical deposition is carried out at the constant voltage ranging from-0.65 V to-0.85 V, the 3D gold nano tree is obtained, and the deposition time ranges from 300 s to 500 s. And continuously applying a constant voltage of-0.55 V to-0.8 V to carry out secondary electrochemical deposition to obtain the final enhanced 3D gold nano-tree, wherein the deposition time is 200-500 seconds. The preparation method is short in preparation time, and the prepared enhanced 3D gold nano tree can detect the concentration of hazardous substances with high sensitivity, high stability and high detection speed and perform in-situ degradation on the hazardous substances.

Description

Technical Field

[0001] The invention belongs to the field of compound detection, and is an enhanced 3D gold nanotree and an electrochemical preparation method thereof, and is used for hazardous substance detection and in-situ degradation. Background Art

[0002] In the process of fish farming, cases of illegal addition of antibiotics often occur. Eating aquatic products containing antibiotics will cause certain harm to the human body. Therefore, rapid and accurate detection of antibiotics is of great significance to ensuring food safety. At present, the main method for detecting antibiotics is chromatography. Although it can realize the detection of antibiotics, it also has disadvantages such as expensive instruments and cumbersome steps. Spectrophotometric detection has the advantages of simplicity and speed, but low sensitivity. Electrochemical detection is easily interfered by complex matrices. Surface enhanced Raman spectroscopy (SERS) technology, as a fingerprint spectroscopy technology, has the characteristics of fast response, strong specificity and high sensitivity, and is expected to achieve rapid detection of antibiotics.

[0003] SERS substrate is the core component of SERS technology. In the SERS detection of antibiotics, the traditional monodisperse nanoparticle SERS substrate is prone to coffee ring effect during the detection process, resulting in unstable SERS signal. The currently commonly used solid-state SERS substrate has problems such as low detection sensitivity, poor SERS signal stability, and complex substrate preparation steps when detecting antibiotics.

[0004] Electrochemical technology has been widely used in the preparation of various solid-state SERS substrates. For example, the template method is used to electrochemically deposit noble metal materials in anodized aluminum oxide and silicon dioxide nanochannels with adjustable pore size. After removing the template, the size-adjustable metal nanorod structure can be synthesized. Secondly, the nanostructure is prepared by photolithography technology, and then the noble metal material is deposited on its surface by electrochemical methods to reduce the gap between the nanostructures and improve the SERS enhancement effect at the "hot spot".

[0005] However, the above solid-state SERS substrate has certain limitations. The nanostructures synthesized directly on the electrode often have poor SERS effects. The process of synthesis by template method often involves multi-step synthesis, and expensive and complex instruments and professional personnel are required to operate, which is expensive. Therefore, it is of great significance to realize the rapid detection of harmful substances by electrochemically synthesizing SERS substrates with good SERS effects without templates. Summary of the invention

[0006] The present invention provides a method for preparing an enhanced 3D gold nanotree. The preparation method has a short preparation time. The prepared enhanced 3D gold nanotree can detect the concentration of harmful substances with higher sensitivity, stronger stability and faster detection speed and degrade the harmful substances in situ.

[0007] The present invention provides a method for preparing an enhanced 3D gold nanotree, comprising:

[0008] (1) Using the gold nanofilm as a working electrode, an electrolyte solution was prepared using EDTA, K2HPO4, Na2SO3 and HAuCl4·3H2O, wherein the pH value of the electrolyte solution was 5.5-7, and electrochemical deposition was performed at a constant voltage of -0.65V--0.85V, and the deposition time was 300-500s to obtain a 3D gold nanotree;

[0009] (2) Continue to apply a constant voltage of -0.55V to -0.8V in the system obtained in step (1) to perform secondary electrochemical deposition of enhanced 3D gold nanotrees, wherein the deposition time is 200-500s.

[0010] The present invention electrochemically operates the gold nanofilm to grow enhanced 3D gold nanotrees with smaller spacing on the surface of the gold nanofilm. The enhanced 3D gold nanotree provided by the present invention is star-shaped in top view and is evenly and independently dispersed on the gold nanofilm. The shape of the gold nanostructure is tree-like. The surface of the enhanced 3D gold nanotree is composed of multiple branches and tips of different sizes. The morphology and structural characteristics of the enhanced 3D gold nanotree are determined by the interaction between EDTA, K2HPO4, Na2SO3 and HAuCl4·3H2O. Na2SO3 promotes Au 3+ Reduction to Au + , the introduced EDTA can react with Au + The ions are chelated to form a stable Au-EDTA complex. K2HPO4 as a secondary complexing agent can bind and stabilize Au + Ions help to form enhanced 3D gold nanotrees with good spacing, height and structure. By changing the pH and voltage of the electrolyte, the morphology of the enhanced 3D gold nanostructure can be controlled. Under the condition of pH 5.5, due to the high deposition rate, more growth sites are easily generated to form multi-core gold nanostructures. When the pH value increases to 6.5, the nanostructure is transformed into a highly ordered enhanced 3D gold nanotree. As the pH further increases, the deposition rate is greatly reduced.

[0011] Preferably, after obtaining the 3D gold nanotree, a secondary electrochemical deposition is performed, and a constant voltage of -0.55V--0.8V is applied to obtain the final enhanced 3D gold nanotree, wherein the deposition time is 200s-500s. Controlling the deposition time can control the size of the enhanced 3D gold nanotree. As the deposition time increases, the height of the enhanced 3D gold nanotree increases significantly, while the width remains essentially unchanged, and during the entire deposition process, the thickness of the Au film at the bottom layer does not change.

[0012] After the secondary electrochemical deposition provided by the present invention, the spacing between the enhanced 3D gold nanotrees becomes significantly smaller, and the size of the gold nanotrees tends to increase. The small gap is conducive to the coupling between the enhanced 3D gold nanotrees, so that multiple SERS "hotspot" areas are distributed on the enhanced 3D gold nanotrees, indicating that it has high SERS enhancement performance.

[0013] Since the enhanced 3D gold nanotrees are prepared with smaller spacing, they can be used as SERS substrates, resulting in denser localized surface plasmon resonance "hot spots" and higher enhancement effects. The electric field strength at the edge of the enhanced 3D gold nanotrees is significantly stronger than that of ordinary 3D gold nanotrees, thus significantly improving the SERS enhancement factor. SERS signals were collected at 200 different locations at 1361 cm -1 The surface enhanced Raman scattering signal recorded at 4.72% remained stable, with a relative standard deviation of 4.72%, indicating that the enhanced 3D gold nanotree substrate is stable and sensitive in quantitative analysis experiments. The special 3D gold nanotree shape can absorb a large amount of visible light, and the absorbed visible light can generate more hot carriers during the relaxation process, which can then be directly used for photoelectric degradation.

[0014] Preferably, the concentration of the EDTA solution is 0.01-0.02 mol / L, the concentration of the K2HPO4 solution is 0.1-0.3 mol / L, the concentration of the Na2SO3 solution is 1-1.5 mol / L, and the concentration of the HAuCl4·3H2O solution is 0.02-0.04 mol / L. 3+ Reduction to Au + , the introduced EDTA can react with Au + The ions are chelated to form a stable Au-EDTA complex. K2HPO4 as a secondary complexing agent can bind and stabilize Au + ions help to form 3D gold nanotrees with good spacing, height and structure.

[0015] Preferably, the pH of the electrolyte is adjusted to 5.5-7 with hydrochloric acid.

[0016] Preferably, before adding the gold nanofilm to the electrolyte, the gold nanofilm is cleaned with a plasma cleaner.

[0017] Further preferably, the cleaning time of the plasma cleaner is 1-10 min.

[0018] Preferably, steps (1) and (2) also include a reference electrode and a counter electrode, which together with the working electrode of the gold nanofilm form a three-electrode system, wherein the reference electrode is Ag / AgCl and the counter electrode is a platinum wire.

[0019] On the other hand, the present invention also provides an enhanced 3D gold nanotree, which is prepared by the enhanced 3D gold nanotree preparation method.

[0020] Preferably, the diameter of the enhanced 3D gold nanotree is 50-300 nm, and the tree gap is 20-80 nm. The enhanced 3D gold nanotree has multiple SERS "hotspot" areas, indicating that it has the potential for high SERS enhancement performance.

[0021] On the other hand, the present invention also provides the use of the enhanced 3D gold nanotree in detecting the content of levofloxacin, including:

[0022] The enhanced 3D gold nanotrees were immersed in a solution with a concentration of 5×10 -7 mol / L-1×10 -4 In a levofloxacin solution of 1.447 W / mol / L, the SERS intensity measured by the spectrum was collected by Raman spectroscopy microscope, and the SERS intensity was substituted into the levofloxacin content-SERS intensity standard curve to obtain the levofloxacin content.

[0023] Preferably, the levofloxacin content-SERS intensity standard curve is:

[0024] I=1118.2×lg C+7389.5,R 2 =0.9836, where I is the SERS intensity and C is the levofloxacin concentration.

[0025] On the other hand, the present invention also provides the use of the enhanced 3D gold nanotree in detecting malachite green, including:

[0026] The enhanced 3D gold nanotree is immersed in a 1.37-13.70 ng / L malachite green solution, the SERS intensity measured by the spectrum is collected by a Raman spectroscopic microscope, and the SERS intensity is substituted into the malachite green content-SERS intensity standard curve to obtain the malachite green content.

[0027] Preferably, the malachite green content-SERS intensity standard curve is:

[0028] I = 1928.1C-1492.1, R2 = 0.9927, where I is the SERS intensity and C is the malachite green concentration.

[0029] On the other hand, the present invention also provides the use of the enhanced 3D gold nanotree in in-situ degradation of harmful substances, including:

[0030] The enhanced 3D gold nanotree is inserted into a sample to be tested containing harmful substances, a voltage of 1.0-1.6V is applied to the enhanced 3D gold nanotree, and the harmful substances are degraded in situ under light of λ>400nm.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention synthesizes enhanced 3D gold nanotrees by an electrochemical method. The synthesis time is short, and the spacing between the synthesized enhanced 3D gold nanotrees is narrow. The surface of the enhanced 3D gold nanotree SERS substrate contains a large number of SERS "hotspot" areas, so it is suitable for use as a SERS substrate to detect hazards. The prepared SERS sensor has the characteristics of low detection limit and wide linear detection range. Therefore, the enhanced 3D gold nanotrees prepared by the electrochemical method provided by the present invention can ensure the rapid and simple determination of hazard residues in complex food matrices.

[0033] (2) The template-free enhanced 3D gold nanotree provided by the present invention has the advantages of short material synthesis time, high sensitivity, high stability, etc., and can be used for SERS quantitative analysis of harmful substances in samples and in-situ degradation of harmful substances. This provides a basis for establishing a SERS detection platform that does not require templates, is simple and fast, and can degrade harmful substances in situ to achieve the reuse of SERS substrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a scanning electron microscope image of the enhanced 3D gold nanotree prepared in Example 1, a scanning electron microscope image of the enhanced 3D gold nanotree magnified 20,000 times, wherein the local magnified image is a scanning electron microscope image of the enhanced 3D gold nanotree magnified 50,000 times;

[0035] Figure 2 The SERS spectrum and linear relationship of the enhanced 3D gold nanotree prepared in Example 1 for detecting levofloxacin solution, Figure 2 a is the collection concentration of 5×10 -7 mol / L-1×10 -4 SERS spectrum of 1 mol / L levofloxacin solution. Figure 2 b is the use of levofloxacin at 1405 cm -1 The SERS intensity at is linearly related to the concentration;

[0036] Figure 3 The enhanced 3D gold nanotree prepared in Example 1 detects the content of levofloxacin in fish meat at 1405 cm -1 The SERS intensity at is linearly related to the concentration;

[0037] Figure 4 This is the SERS signal collection diagram of different regions of the enhanced 3D gold nanotree prepared in Application Example 1. Figure 4 a is the SERS spectrum collected at 200 sites, Figure 4 b is the characteristic peak at 1361cm -1 The RSD value of the intensity at the

[0038] Figure 5 The enhanced 3D gold nanotree prepared in Application Example 2 detects the content of malachite green in fish meat at 1612 cm -1 The SERS intensity at is linearly related to the concentration;

[0039] Figure 6 The enhanced 3D gold nanotree prepared in Example 3 was used as an electrode. The C of malachite green in the real sample during the degradation process t / C0 change curve. DETAILED DESCRIPTION

[0040] Example 1

[0041] This embodiment provides a method for preparing an enhanced 3D gold nanotree, comprising:

[0042] (1) Cleaning the electrode: Place the gold nanofilm in a plasma cleaner for 2 minutes;

[0043] (2) Preparation of enhanced 3D gold nanotrees: The electrochemical deposition of enhanced 3D gold nanotrees was performed using a three-electrode system consisting of a gold nanofilm as a working electrode, Ag / AgCl as a reference electrode, and a platinum wire as a counter electrode. The electrolyte solution included 0.017 mol / L EDTA, 0.17 mol / L K2HPO4, 1.27 mol / L Na2SO3, and 0.029 mol / L HAuCl4·3H2O, and the pH of the electrolyte was adjusted to 6.5 with hydrochloric acid. At a constant potential of -0.8 V, the electrochemical deposition time was 500 s, and 3D gold nanotrees were grown on the gold nanofilm. A voltage of -0.65 V was continued to be applied, and the deposition time was 500 s to obtain an enhanced 3D gold nanotree. Subsequently, it was rinsed with deionized water and dried with nitrogen.

[0044] like Figure 1 As shown, the diameter of the synthesized enhanced 3D gold nanotree is 141.22±23.55nm, and the tree gap is 14.72±4.06nm. In the enlarged top view, the gold nanostructure is star-shaped, evenly and independently dispersed on the Au film, and the shape of the gold nanostructure is tree-like. The surface of the enhanced 3D gold nanotree is composed of multiple branches and tips of different sizes, indicating that the enhanced 3D gold nanotree has the potential for high SERS enhancement performance.

[0045] Application Example 1

[0046] Drawing of working curve: The enhanced 3D gold nanotree was immersed in different concentrations of levofloxacin, with specific concentrations of 0.025, 0.075, 0.126, 0.176, and 0.251 μg / L. The test conditions were: laser wavelength of 638 nm, power of 0.66 mW, acquisition time of 120 s, magnification of 50 times, and an average of 3 measurements each time. The surface enhanced Raman scattering signal was monitored at different spot positions.

[0047] like Figure 2 a and Figure 2 As shown in b, the SERS spectrum of levofloxacin is at 1405 cm -1 、1569cm -1 and 1626cm -1 A strong SERS characteristic peak appears at 1405cm -1 The characteristic peaks were attributed to the stretching vibration of the quinolone ring and were selected as the characteristic peaks for the detection of levofloxacin.

[0048] With the increase of levofloxacin content, the SERS intensity increases linearly; when different concentrations of levofloxacin are placed at 1405 cm -1 The intensity corresponding to the Raman shift at the position (SERS Intensity) is used as the ordinate, and the logarithm of the levofloxacin concentration (lg(C Levofloxacin / mol / L)) as the horizontal axis, draw a standard curve as shown in Figure 3 As shown, I = 10683C + 704.33, R2 = 0.994, where I is the SERS intensity and C is the levofloxacin concentration. The calculated detection limit is 1×10 -7 mol / L. The method has a wide linear detection range, and the detection sensitivity conforms to the national standard (GB 31656.3-2021) method (GB 31656.3-2021 National Food Safety Standard Determination of Norfloxacin, Ciprofloxacin, Enrofloxacin, Ofloxacin, Oxolinic Acid, and Flumequine Residues in Aquatic Products by High Performance Liquid Chromatography).

[0049] like Figure 4 a and Figure 4 As shown in b, at 1361cm -1 The SERS signals recorded at 40°C remained stable, and the relative standard deviation (RSD) obtained was 4.72%, which showed that the prepared enhanced 3D gold nanotree substrate was stable, reliable, and sensitive in quantitative analysis experiments.

[0050] Sample pretreatment: After adding levofloxacin standard to the fish meat, the fish meat was pretreated. The fish meat was crushed into a homogenate with a blender. 5g of fish meat homogenate was weighed. The extraction method was referred to the national standard (GB 31656.3-2021). The supernatant after extraction was passed through a C18 solid phase extraction column. The eluate was collected and dried with 50°C nitrogen. 5mL of acetonitrile was added for dissolution and then passed through a 0.22μm microporous filter membrane for SERS and spectrophotometry measurement. After adding levofloxacin standard sample to the aquaculture water, it was directly passed through a 0.22μm filter membrane, evaporated to near dryness, and fixed to 5mL with acetonitrile.

[0051] Determination of levofloxacin concentration in samples: Different known concentrations of levofloxacin were added to the samples, with specific concentrations of 0.10, 0.19, 0.18, and 0.038 μg / g. The spectra were collected using a Raman spectroscopic microscope, and the concentration of levofloxacin in the sample to be tested was obtained based on the established standard curve. As shown in Table 1, it is shown that there is a very high consistency between the spiked concentration and the measured content, and the recovery rates in aquaculture water and fish meat are in the range of 91.78% to 97.80% and 85.22% to 93.59%, respectively. The good recovery rate and relative standard deviation values ​​in the table also indicate that there is no potential matrix interference, so it has the reliability of analyzing complex food matrices.

[0052] Table 1. Analysis of levofloxacin in fish meat and aquaculture water after pretreatment using this method

[0053]

[0054] Note: The values ​​are the mean ± standard deviation of three repeated experiments

[0055] Application Example 2

[0056] Working curve drawing: The enhanced 3D gold nanotree prepared in Example 1 was immersed in different concentrations of malachite green, specifically 1.37, 4.11, 6.85, 9.59, and 13.70 ng / L. The test conditions were: laser wavelength of 638 nm, power of 0.66 mW, acquisition time of 120 s, magnification of 50 times, and an average of 3 measurements each time. Different concentrations of malachite green were immersed in 1612 cm -1 The intensity corresponding to the Raman shift (SERS Intensity) is used as the ordinate, and the malachite green concentration (C Malachite green / mol / L) as the horizontal axis, draw the standard curve as Figure 5 As shown, I = 1928.1C-1492.1, R2 = 0.9927, where I is the SERS intensity and C is the malachite green concentration. The calculated detection limit is 5×10 -10 mol / L;

[0057] Sample pretreatment: same as (5) in Application Example 1;

[0058] Determination of malachite green concentration in samples: Different known concentrations of malachite green were added to the samples, with specific concentrations of 1.37, 1.64, and 6.85 ng / g. The spectra were collected using a Raman spectroscopic microscope, and the concentration of malachite green in the sample to be tested was obtained based on the established standard curve. As shown in Table 2, it shows that there is a very high consistency between the spiked concentration and the measured content, and the recovery rates in aquaculture water and fish meat are in the range of 92.73% to 98.57% and 78.01% to 95.50%, respectively.

[0059] Table 2. Analysis of malachite green in pre-treated fish and aquaculture water using this method

[0060]

[0061] Note: The values ​​are the mean ± standard deviation of three repeated experiments

[0062] Application Example 3

[0063] Same as (1)-(2) in Example 1;

[0064] The prepared enhanced 3D gold nanotree electrode was inserted into the actual aquaculture water containing malachite green, and the real-time concentration of malachite green was measured by SERS technology. The SERS spectrum acquisition conditions were: laser wavelength of 638nm, power of 33mW, acquisition time of 0.1s, multiple of 5 times, and average of three measurements each time. -1 The peak intensity measured at is used as a signal, and the real-time concentration of malachite green in the aquaculture water is calculated based on the established working curve.

[0065] After detection, the mode was switched to photoelectric mode, 1.2V voltage was applied to the electrode, and malachite green was degraded in situ under light of λ>400nm. Every 20 minutes, the mode was switched to SERS mode for real-time measurement of pollutant concentration. The results are shown in Figure 2. Figure 6 As shown, Figure 6 For the enhanced 3D gold nanotrees as electrodes, the C of malachite green during degradation in real samples t / C0 changes, with the increase of degradation time, the concentration of malachite green in the aquaculture water body continues to decrease. The concentration of malachite green in the in situ degradation process was monitored by the electrode. The results showed that when the degradation time was 0, 20, 40, and 60 minutes, the concentration of malachite green decreased to 97.04±3.54, 44.51±0.61, 15.66±1.21, and 4.50±0.24μmol / L, respectively.

[0066] Example 2

[0067] Compared with Example 1, the difference is that the concentration of the EDTA solution is 0.015 mol / L, the concentration of the K4HPO4 solution is 0.2 mol / L, the concentration of the Na2SO3 solution is 1.5 mol / L, the concentration of the HAuCl4·3H2O solution is 0.04 mol / L, the pH value of the electrolytic solution is 6, and electrochemical deposition is performed at a constant voltage of -0.85 V to obtain 3D gold nanotrees, and the deposition time is 400 s. Secondary electrochemical deposition is performed at a constant voltage of -0.65 V to obtain enhanced 3D gold nanotrees, and the deposition time is 400 s.

Claims

1. A method for preparing an enhanced 3D gold nanotree, characterized in that: include: (1) Using the gold nanofilm as a working electrode, an electrolyte solution was prepared using EDTA, K2HPO4, Na2SO3 and HAuCl4·3H2O, wherein the pH value of the electrolyte solution was 5.5-7, and electrochemical deposition was performed at a constant voltage of -0.65V--0.85V, and the deposition time was 300-500s to obtain a 3D gold nanotree; (2) Continue to apply a constant voltage of -0.55V to -0.8V in the system obtained in step (1) to perform secondary electrochemical deposition of enhanced 3D gold nanotrees, wherein the deposition time is 200-500s.

2. The method for preparing the enhanced 3D gold nanotree according to claim 1, characterized in that: The concentration of the EDTA solution is 0.01-0.02 mol / L, the concentration of the K2HPO4 solution is 0.1-0.3 mol / L, the concentration of the Na2SO3 solution is 1-1.5 mol / L, and the concentration of the HAuCl4·3H2O solution is 0.02-0.04 mol / L.

3. The method for preparing the enhanced 3D gold nanotree according to claim 1, characterized in that: The deposition time of step (1) is 300-500s, and the constant voltage is -0.65V--0.85V. The deposition time of step (2) is 200-500s, and the constant voltage is -0.55V--0.8V.

4. The method for preparing the enhanced 3D gold nanotree according to claim 1, characterized in that: Before adding the gold nanofilm into the electrolyte, the gold nanofilm is cleaned by a plasma cleaner.

5. The method for preparing the enhanced 3D gold nanotree according to claim 1, characterized in that: In steps (1) and (2), a reference electrode and a counter electrode are also included to form a three-electrode system with the working electrode of the gold nanofilm. The reference electrode is Ag / AgCl, and the counter electrode is a platinum wire.

6. An enhanced 3D gold nanotree, characterized in that: The enhanced 3D gold nanotree is prepared by the method for preparing the enhanced 3D gold nanotree as described in any one of claims 1 to 5.

7. The enhanced 3D gold nanotree according to claim 6, characterized in that: The diameter of the enhanced 3D gold nanotree is 50-300nm, and the tree gap is 20-80nm.

8. An application of the enhanced 3D gold nanotree as claimed in claim 6 or 7 in detecting the content of levofloxacin, characterized in that: include: The enhanced 3D gold nanotrees were immersed in a solution with a concentration of 5×10 -7 mol / L-1×10 -4 In a levofloxacin solution of 1.447 W / mol / L, the SERS intensity measured by the spectrum was collected by Raman spectroscopy microscope, and the SERS intensity was substituted into the levofloxacin content-SERS intensity standard curve to obtain the levofloxacin content.

9. An application of the enhanced 3D gold nanotree as claimed in claim 6 or 7 in detecting malachite green, characterized in that: include: The enhanced 3D gold nanotree is immersed in a 1.37-13.70 ng / L malachite green solution, the SERS intensity measured by the spectrum is collected by a Raman spectroscopic microscope, and the SERS intensity is substituted into the malachite green content-SERS intensity standard curve to obtain the malachite green content.

10. Use of the enhanced 3D gold nanotree as claimed in claim 6 or 7 in in-situ degradation of hazardous substances, characterized in that: include: The enhanced 3D gold nanotree is inserted into a sample to be tested containing harmful substances, a voltage of 1.0-1.6V is applied to the enhanced 3D gold nanotree, and the harmful substances are degraded in situ under light of λ>400nm.

Images