Method for characterizing particle size and composition of nanoparticles by asymmetric flow-field flow combination inductively coupled plasma emission spectrum technology

Abstract

The invention provides a method for characterizing the particle size and composition of nanoparticles by an inductively coupled plasma emission spectrum technology in asymmetric flow-field flow combination. The method comprises the following steps: S1, carrying out anaerobic environment pretreatment on an asymmetric flow field flow instrument; and S2, carrying out sample loading treatment on the sulfate radical solution treated by the sulfate reducing bacteria in an asymmetric flow field flow instrument, and obtaining a characterization result after a sample injection stage, a flushing stage and a flushing stage. According to the method, the sulfate reducing bacteria are matched with the asymmetric flow field flow meter to detect the sulfate radical solution, and the sulfate reducing bacteria are utilized to enable the sulfate radicals to form S, so that effective detection of the sulfate radicals is realized.

Description

Technical Field

[0001] The invention relates to a method for using an asymmetric flow field flow meter, in particular to a method for characterizing the particle size and composition of nanoparticles by using an asymmetric flow-field flow combined with an inductively coupled plasma emission spectroscopy technique. Background Art

[0002] At present, there is no method that can simultaneously separate and qualitatively and quantitatively characterize the particle size distribution and composition of nanoparticles. Therefore, this method is compared with existing nanoparticle characterization methods. Existing nanoparticle characterization methods include: Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES).

[0003] The transmission electron microscope has the resolution capability at the atomic level. It can not only observe the microscopic morphology of the sample, but also characterize the internal structure of the observed area. The principle of TEM is that the high-voltage electron source releases high-speed electrons, which pass through the condenser system to form a parallel electron beam, which is incident on and passes through a local area of ​​the sample. The electron beam interacts with the nanoparticle sample to modulate the phase and amplitude of the incident electron beam to form an outgoing electron beam. The outgoing electron beam passes through the objective lens system and converges on the focal plane of the objective lens to form a diffraction spot, which then propagates further downward to the objective lens image plane to form the first magnified image of the sample. The transmission electron microscope can study the size, shape and crystal structure at the single particle level.

[0004] Dynamic light scattering is a technique that measures the fluctuations in the intensity of scattered light from a sample to obtain information about the particle size of the sample. Because the molecules in the sample are constantly performing Brownian motion, this motion causes changes in the scattered light. The fluctuation of scattered light over time is used to obtain the relevant equation, and then the average movement rate (Dt) of the particles in the sample is obtained, and finally the average hydrodynamic diameter is obtained. The particle size detection of the dynamic light scattering method has the advantages of being fast and simple5, and has certain statistical significance, but because the scattered light intensity is proportional to the sixth power of the particle size, the scattering of large particles is relatively strong, so for a wide distribution sample, the average particle size result tends to be large particles. Therefore, DLS is suitable for particle size detection of monodisperse systems with a distribution that is not too wide, and the dynamic light scattering experiment is easily affected by dust or impurities, so sample filtration and centrifugation are very important.

[0005] Nanoparticle tracking analysis is a method of irradiating a solution of suspended particles with a beam of concentrated laser energy through a glass prism, and using chrome-plated glass to minimize background signals, thereby detecting the intensity of scattered light from each particle, observing the Brownian motion of nanoparticles in the solution, and taking images. By tracking and analyzing the Brownian motion of the particles, the particle size of the nanoparticles is calculated according to the Stokes-Einstein equation, and the concentration is obtained by the number of particles. Nanoparticle tracking analysis is more accurate than dynamic light scattering, but like dynamic light scattering, since particles will scatter light, the signals between particles will block each other, and the particle size will still tend to be large particles. In addition, due to the limitation of the limited shooting focal plane, the movement of the particles in three-dimensional space is actually presented as a two-dimensional motion trajectory on the instrument, so there will still be a certain deviation in the final result.

[0006] Inductively coupled plasma emission spectrometry uses the high temperature formed by ICP plasma to atomize or ionize the element to be tested, forming an excited state and generating a characteristic emission spectrum. By detecting the wavelength and intensity of the spectrum line, it can be determined whether the sample to be tested contains the element to be tested and its content. ICP-OES can achieve simultaneous determination of most metal elements and non-metal elements such as S, and has the advantages of low detection limit and high precision.

[0007] The existing transmission electron microscopy method may lead to a lack of overall statistical results due to the small amount of nanopowder used for electron microscopy observation; since the surface activity of nanoparticles is very high and they are easy to agglomerate, ultrasonic dispersion must be performed before dropping the sample; it is difficult to obtain accurate results for some nanoparticle samples that are not resistant to strong electron beam bombardment. In addition, the nanoparticles must be electron transparent and able to withstand the high vacuum and beam energy used in the characterization process. In particular, due to the presence of high-energy electron beams, sample damage is common.

[0008] In the existing dynamic light scattering method, since the scattered light intensity is proportional to the sixth power of the particle size, the scattering is stronger for large particles, resulting in that for samples with a wide distribution, the average particle size results will be biased towards large particles. The results may not be accurate or representative, and are easily affected by dust or impurities, which will increase the error of the results and increase the complexity and difficulty of the operation.

[0009] In the existing nanoparticle tracking analysis method, due to the light scattering of particles, the signals between particles will be blocked by each other, and the obtained particle size will tend to be large particles. In addition, due to the limitation of the limited shooting focal plane, the movement of particles in three-dimensional space is actually presented as a two-dimensional motion trajectory on the instrument, resulting in a certain deviation in the final result.

[0010] The existing inductively coupled plasma emission spectrometry has complex spectral lines, serious spectral interference, large sample requirements, and requires acidification and other pre-treatments when used alone, which destroys the sample structure.

[0011] In order to overcome the shortcomings of existing technologies such as complex operation, over-reliance on separation and extraction technology and relatively single characterization capabilities, and to achieve efficient separation and characterization of nanoparticles, this method uses an asymmetric flow field flow instrument (AF4, hereinafter referred to as field flow instrument) combined with ICP-OES to separate and characterize nanoparticles. It has the advantages of convenient operation, simultaneous determination of multiple elements, simple pretreatment, low destructiveness and low detection limit. Summary of the invention

[0012] The invention provides a method for characterizing the particle size and composition of nanoparticles by using an asymmetric flow-field flow coupled inductively coupled plasma emission spectroscopy technique, so as to realize the detection and characterization of a sulfate radical solution treated with sulfate-reducing bacteria.

[0013] The present invention provides a method for characterizing the particle size and composition of nanoparticles by using an asymmetric flow-field flow coupled inductively coupled plasma emission spectroscopy technique, comprising:

[0014] S1. Pre-treating the asymmetric flow field flow instrument in an oxygen-free environment;

[0015] S2. The sulfate solution treated with sulfate-reducing bacteria is loaded into an asymmetric flow field flow instrument, and after the injection stage, the flushing stage and the rinsing stage, the characterization result is obtained.

[0016] Furthermore, the sulfate solution treated with sulfate-reducing bacteria to be tested also contains metal ions, and the metal ions are Fe 2+ , Fe 3+ , Cu 2+ 、Al 3+ One or more of .

[0017] Furthermore, the concentration of the metal ions is 100-400 μmol·L -1 .

[0018] Furthermore, the preparation method of the sulfate solution treated with sulfate-reducing bacteria to be tested is:

[0019] S101. Take sulfate solution;

[0020] S102. Prepare a bacterial solution with OD600 = 0.8 using the anaerobic background solution, mix the bacterial solution and the Cu stock solution in a volume ratio of 1:1, and react with horizontal oscillation for 24 hours;

[0021] S103. Take the solution treated in S102 and centrifuge it to obtain a solution to be tested.

[0022] Furthermore, the anaerobic background solution is a 1 mmol / L Na2SO4 anaerobic background solution, and the temperature is 30±1°C.

[0023] Furthermore, the horizontal oscillation reaction is specifically: reacting at 30±1°C and 150rpm for 24h.

[0024] Furthermore, the S1. pre-treating the asymmetric flow field flow instrument in an oxygen-free environment comprises:

[0025] Prepare 25 μM NaCl solution with oxygen-free water, select a low flow rate of 0.05 mL / min, and control the asymmetric flow field flow meter to run for 12-36 hours to ensure that the asymmetric flow field flow meter is an anaerobic environment.

[0026] Furthermore, the S2. subjecting the sulfate solution treated with sulfate-reducing bacteria to a sample loading process in an asymmetric flow field flow instrument, and obtaining characterization results after a sample loading phase, a flushing phase and a rinsing phase, including:

[0027] S201. In the anaerobic workstation, take 2.5 mL of the sulfate solution treated with sulfate-reducing bacteria after 24 hours of reaction into a 1 mL injection bottle for measurement;

[0028] S202. After the injection stage, the flushing stage and the rinsing stage, the characterization result is obtained.

[0029] Furthermore, the injection stage includes: taking the sulfate solution treated with sulfate-reducing bacteria in the injection bottle, setting the program of the asymmetric flow field flow instrument to a detector flow rate of 0.5 mL / min; during the focusing process: injection speed: 0.20 mL / min, injection time 5 min, injection volume: 200 μL, lateral flow rate: 1.00 mL / min, focusing pump: 1.3 mL / min.

[0030] Furthermore, the flushing stage includes: setting the elution process conditions as follows: (1) lateral flow rate 1.00 mL / min, mode constant, time 15 min; (2) lateral flow rate 1.00 mL / min, mode Power, Exponent 0.20, time 30 min; (3) lateral flow rate 0.05 mL / min, mode Power, Exponent 0.80, time 10 min; (4) lateral flow rate 0.00 mL / min, mode constant, time 30 min.

[0031] Compared with the prior art, the present invention uses sulfate-reducing bacteria in combination with an asymmetric flow field flow meter to detect sulfate solution, and uses sulfate-reducing bacteria to convert sulfate into S, so as to achieve effective detection of sulfate. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The results of AF4-UV of nanosphere standards of different sizes in the embodiments of the present invention are shown below;

[0033] Figure 2 The element content in MNPs was determined by ICP-OES in combination with AF4 of the present invention. The ordinate represents the emission spectrum signal intensity value (cps); among them, (a) Cu; (b) Fe; (c) S; (d) Mn. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments.

[0035] Asymmetric flow field instrument (Poatnova, AF2000MT, GERMAN), hereinafter referred to as field flow instrument. Field flow instrument separation involves an asymmetric flow field. Under the action of the asymmetric flow field force and the opposing diffusion field, the sample forms different equilibrium layers in the channel. In the injection stage, that is, before the separation begins, after the sample enters the channel from the injection port, it will be subjected to the same-direction cross-flow flow field force from the top of the channel. The pressure generated by the cross-flow will cause the colloidal particles of the sample to be retained on a dialysis membrane that can only pass small molecular solutes. At this time, the sample will diffuse in the vertical direction of the channel, and the diffusion coefficient depends on factors such as the size and density of different colloidal particles. Colloidal particles with large particle size or density have a small diffusion coefficient and gather in the lower part of the channel near the membrane, while those with smaller particle size or density will be close to the middle position of the channel space. At the same time, the sample will also be subjected to the focusing flow field force opposite to the elution direction. Under the combined action of the channel flow and the focusing flow, the sample will be gathered near the injection port, and only small molecular solutes can be eluted through the dialysis membrane. After the injection is completed, the flow rate of the focused flow returns to zero, and the colloidal particles are eluted with the channel flow and then flow to the detector. According to the boundary layer theory, for the fluid on the same cross-section in the channel, the fluid flow rate near the boundary is slower, and the fluid flow rate near the center of the channel is faster. The fluid flow rate is a parabolic streamline along the cross section. Therefore, smaller particles are flushed out first with the faster fluid and enter the detector first, while larger particles are flushed out one after another as the cross-flow gradually decreases.

[0036] Field flow meter can be coupled with many detectors, such as UV-visible spectrophotometer (UV-vis), multi-angle static laser particle size analyzer (MALS), fluorescence detector, differential detector, ICP-OES, etc. Different characteristics of colloidal particles can be analyzed by different detectors, and then the colloidal particles can be detected, separated and characterized simultaneously online.

[0037] By combining field flow analysis and ICP-OES, nanoparticles of different sizes are separated and then characterized for their elemental composition and particle size. The ability of ICP-OES to simultaneously determine multiple elements, such as most metal elements and non-metal elements such as S, can not only explore the relationship between the particle size and elemental composition of nanoparticles, but also determine the binding status of nanoparticles. This has important reference value for the study of the environmental effects and biotoxicity of nanoparticles at the nanoscale.

[0038] To further illustrate the solution of the present invention, specific embodiments are now disclosed as follows.

[0039] The experiment used sulfate-reducing bacteria (SRB) and Cu 2+ SRB is a common characteristic microbial population in sulfur-rich anaerobic sediment habitats in mining areas. It can generate sulfide through sulfate reduction reaction and combine with heavy metal ions to mediate the formation of metal nanoparticles (MNPs). Using asymmetric flow field flow separation coupled characterization technology, the binding of nanoparticles can be judged and proved by the characterization results of Cu, Fe, Mn and S elements.

[0040] The reaction was carried out at pH = 6, 1 mmol / L Na2SO4 anaerobic background solution at 30 ± 1 °C. First, prepare a bacterial solution with OD600 = 0.8 using the background solution, add 20 mL of the bacterial solution to a 50 mL centrifuge tube, and then add 20 mL of Cu stock solution (specifically 200 μmol / L Cu(NO3)2 solution) to make the copper concentration in the system 200 μmol / L and the total volume of the solution 40 mL. The reaction was kept at 30 ± 1 °C and 150 rpm for 24 hours. After the reaction, 5 mL was centrifuged in a 3 kDa (about 1 nm) ultrafiltration centrifuge tube (Microsep Advance, Pall) to ensure complete separation of nanoparticles from dissolved Cu2+ and other metal ions.

[0041] Before separation, prepare a 25μM NaCl background solution with oxygen-free water and run it overnight at a low flow rate of 0.05mL / min to ensure an anaerobic environment during separation. In the anaerobic workstation, take 2.5mL of the sample after 24h of reaction and put it into the 1mL injection bottle of AF4 for measurement. 2+ The products after the interaction were separated using an asymmetric flow field instrument (AF2000MT, Postnova, Germany). The operating method was set up on the field flow instrument software. The operating method was divided into three steps, namely the injection stage, the flushing stage and the rinsing stage.

[0042] Specific method parameter settings: detector flow rate 0.5mL / min. During the focusing process: injection speed: 0.20mL / min, injection time 5min, injection volume: 200μL, lateral flow rate: 1.00mL / min, focusing pump: 1.3mL / min. Elution process: (1) lateral flow rate 1.00mL / min, mode constant, time 15min. (2) lateral flow rate 1.00mL / min, mode Power, Exponent 0.20, time 30min. (3) lateral flow rate 0.05mL / min, mode Power, Exponent 0.80, time 10min. (4) lateral flow rate 0.00mL / min, mode constant, time 30min. The total running time for one sample is 90min.

[0043] UV-vis is used to obtain ultraviolet signals and ICP-OES is used to determine the Cu, Fe, Mn and S elements to obtain spectral signals, and MALS is used to obtain particle signals. Then, by running the latex standard sample for calibration, the relationship between the appearance time of the product signal value and the particle size is obtained, so that the particle size of the separated sample and the distribution of Cu particles of different particle sizes can be obtained. AF4-UV-ICP-OES coupling technology can separate SRB and Cu in different states 2+ The products after interaction were analyzed, and the particle size of the products was determined by the peak time of standard samples with different particle sizes. The distribution of Cu, Fe, Mn and S elements in products of different sizes was analyzed by ICP-OES.

[0044] In order to conduct particle size analysis on the separation results, we first need to explore the separation and characterization of nanosphere standards of different sizes under the parameter setting method of this experiment, and obtain the corresponding peak time of nanosphere standards. This experiment selected nanosphere standards provided by POSTNOVA of Germany, with sizes of 20nm, 200nm, 600nm and 900nm. The standards are all thick and have trace surfactants to prevent coagulation and enhance stability, and are equipped with NIST calibration traceability certificates. The AF4-UV results of nanosphere standards of different sizes are shown below. Figure 1 As shown, it can be seen that the peak particle size at 1 to 10 minutes is 20nm, the peak particle size at 20 minutes is between 200nm, the peak at 30 minutes represents a particle size of 600nm, and the peak at about 60 minutes is 900nm. This result will be used for particle size analysis.

[0045] The field flow meter can effectively separate nanoparticles of different sizes from other dissolved substances. ICP-OES detection shows that the signal curves of Cu and S are basically consistent, and the signal values ​​show a proportional correlation. The signal curves of the two metal ions Fe and Mn are generally consistent with S.

[0046] It was observed that the first significant signal peak appeared between 25 and 30 min ( Figure 2 -a, b, d), indicating that Cu is formed in the system x S nanoparticles, and a small amount of Mn x S nanoparticles, the particle size of this part is 200~600nm. The system has a second signal peak at 30~40min ( Figure 2 -a, b, d), where the signals of Cu and S are more significant, while that of Mn is not obvious. The particle size of the nanoparticles is 600-900 nm. In addition, around 30 min and 50 min, Fe ions show significant signal peaks ( Figure 2 -b), which proves that Fe-containing nanoparticles are formed in the system, but cannot prove the combination of Fe and S. Especially between 30 and 40 min, the Fe signal is not significant, indicating that the proportion of Fe in the nanoparticles with a particle size of 600 to 900 nm formed in the system is not high.

[0047] In summary, the present invention has the following beneficial effects:

[0048] 1. For the first time, an asymmetric flow field instrument was used in conjunction with inductively coupled plasma emission spectroscopy to characterize nanoparticles, and good characterization results were obtained, which can be used as a means of separating and characterizing nanoparticles;

[0049] 2. Explore the field flow instrument method suitable for the sample, including axial flow velocity, cross flow velocity, injection time, flushing time, etc., to improve the elution and detection effect of the sample;

[0050] 3. Practice has proved that the method parameters of asymmetric flow field flow instrument are effective method parameters for separating colloidal particles and nanoparticles. The specific content of the field flow instrument method parameter settings should be protected.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that after reading the specification of this application, the technicians can still modify or replace the specific implementation mode of the present invention with equivalents, but these modifications or changes do not deviate from the scope of protection of the pending claims of the present application.

Claims

1. A method for characterizing the particle size and composition of nanoparticles using asymmetric flow-field flow coupled inductively coupled plasma emission spectroscopy, characterized in that: include: S1. Pre-treating the asymmetric flow field flow instrument in an oxygen-free environment; S2. The sulfate solution treated with sulfate-reducing bacteria is loaded into an asymmetric flow field flow instrument, and after the injection stage, the flushing stage and the rinsing stage, the characterization result is obtained.

2. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 1, characterized in that: The sulfate solution treated with sulfate-reducing bacteria to be tested also contains metal ions, and the metal ions are Fe 2+ , Fe 3+ , Cu 2+ 、Al 3+ One or more of .

3. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 2, characterized in that: The concentration of the metal ion is 100-400 μmol·L -1 .

4. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 3, characterized in that: The preparation method of the sulfate solution treated with sulfate-reducing bacteria to be tested is: S101. Take sulfate solution as anaerobic background solution; S102. Prepare a bacterial solution with OD600 = 0.8 using the anaerobic background solution, mix the bacterial solution and the Cu stock solution in a volume ratio of 1:1, and react with horizontal oscillation for 24 hours; S103. Take the solution treated in S102 and centrifuge it to obtain a solution to be tested.

5. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 4, characterized in that: The anaerobic background solution is a 1 mmol / L Na2SO4 anaerobic background solution, and the temperature is 30±1°C.

6. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 4, characterized in that: The horizontal oscillation reaction is specifically: reacting at 30±1° C. and 150 rpm for 24 hours.

7. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 1, characterized in that: S1. performing oxygen-free environment pretreatment on the asymmetric flow field flow instrument, comprising: Prepare 25 μM NaCl solution with oxygen-free water, select a low flow rate of 0.05 mL / min, and control the asymmetric flow field flow meter to run for 12-36 hours to ensure that the asymmetric flow field flow meter is an anaerobic environment.

8. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 1, characterized in that: S2. The sulfate solution treated with sulfate-reducing bacteria is subjected to sample loading in an asymmetric flow field flow instrument, and after a sample loading phase, a flushing phase and a rinsing phase, a characterization result is obtained, including: S201. In the anaerobic workstation, take 2.5 mL of the sulfate solution treated with sulfate-reducing bacteria after 24 hours of reaction into a 1 mL injection bottle for measurement; S202. After the injection stage, the flushing stage and the rinsing stage, the characterization result is obtained.

9. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 8, characterized in that: The injection stage includes: taking a sulfate solution treated with sulfate-reducing bacteria in an injection bottle, setting the program of the asymmetric flow field flow instrument to a detector flow rate of 0.5 mL / min; during the focusing process: injection speed: 0.20 mL / min, injection time 5 min, injection volume: 200 μL, lateral flow rate: 1.00 mL / min, focusing pump: 1.3 mL / min.

10. The method for characterizing the particle size and composition of nanoparticles by asymmetric flow-field flow coupled inductively coupled plasma optical emission spectroscopy according to claim 9, characterized in that: The flushing stage includes: setting the elution process conditions as follows: (1) lateral flow rate 1.00 mL / min, mode constant, time 15 min; (2) lateral flow rate 1.00 mL / min, mode Power, Exponent 0.20, time 30 min; (3) lateral flow rate 0.05 mL / min, mode Power, Exponent 0.80, time 10 min; (4) lateral flow rate 0.00 mL / min, mode constant, time 30 min.

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