CN113567398A - Lead ion concentration detection method based on dark field spectrum detection technology - Google Patents
Lead ion concentration detection method based on dark field spectrum detection technology Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 27
- 238000001228 spectrum Methods 0.000 title claims abstract description 21
- RVPVRDXYQKGNMQ-UHFFFAOYSA-N lead(2+) Chemical compound [Pb+2] RVPVRDXYQKGNMQ-UHFFFAOYSA-N 0.000 title claims abstract description 20
- 238000005516 engineering process Methods 0.000 title claims description 5
- 239000002105 nanoparticle Substances 0.000 claims abstract description 36
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000010931 gold Substances 0.000 claims abstract description 24
- 229910052737 gold Inorganic materials 0.000 claims abstract description 24
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 22
- 239000011521 glass Substances 0.000 claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 15
- 150000002500 ions Chemical class 0.000 claims abstract description 13
- 229960003180 glutathione Drugs 0.000 claims abstract description 10
- 238000004611 spectroscopical analysis Methods 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims abstract description 7
- 108010024636 Glutathione Proteins 0.000 claims abstract description 5
- 239000006059 cover glass Substances 0.000 claims abstract description 3
- 238000005406 washing Methods 0.000 claims abstract description 3
- 238000001446 dark-field microscopy Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 5
- 238000003384 imaging method Methods 0.000 claims description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 3
- 230000003100 immobilizing effect Effects 0.000 claims description 2
- 239000010842 industrial wastewater Substances 0.000 claims description 2
- 239000008399 tap water Substances 0.000 claims description 2
- 235000020679 tap water Nutrition 0.000 claims description 2
- 239000002699 waste material Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000012742 biochemical analysis Methods 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract 1
- 238000005070 sampling Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 9
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 238000005530 etching Methods 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000252506 Characiformes Species 0.000 description 1
- 206010012559 Developmental delay Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000003968 anodic stripping voltammetry Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000004641 brain development Effects 0.000 description 1
- 239000008364 bulk solution Substances 0.000 description 1
- 230000001808 coupling effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- GUGNSJAORJLKGP-UHFFFAOYSA-K sodium 8-methoxypyrene-1,3,6-trisulfonate Chemical compound [Na+].[Na+].[Na+].C1=C2C(OC)=CC(S([O-])(=O)=O)=C(C=C3)C2=C2C3=C(S([O-])(=O)=O)C=C(S([O-])(=O)=O)C2=C1 GUGNSJAORJLKGP-UHFFFAOYSA-K 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By Optical Means (AREA)
Abstract
The invention relates to the technical field of biochemical analysis by utilizing dark field spectroscopy, in particular to a novel method for detecting lead ion concentration. The invention discloses a lead ion concentration detection method based on dark field spectroscopy, and particularly relates to a method for rapidly detecting lead ions by taking gold nanoparticles modified by glutathione as a probe under a dark field microscope, which comprises the following steps: (1) modifying the gold nanoparticles by using glutathione; (2) fixing gold nanoparticles on a glass substrate; (3) dropwise adding a lead-containing sample on a glass base, and washing after 5 minutes; (4) dropwise adding gold nanoparticles modified by glutathione on a glass substrate, and then covering a cover glass; (5) and detecting the red shift amount of the scattering light spectrum of the gold nanoparticles under a dark field microscope system, and quantifying the concentration of the lead ions according to the red shift amount of the spectrum. The invention has the advantages of novelty, rapidness, high sensitivity, wide detection concentration range and less sampling, and can be widely used for the actual lead ion environmental sample detection.
Description
Technical Field
The invention relates to the technical field of biochemical analysis by utilizing dark field spectroscopy, in particular to a novel method for detecting lead ion concentration.
Background
Lead ion (Pb)2+) Has great harm to human health and environment. Even at very low concentrations of Pb2+It also causes children brain development disorder, bone marrow related hematopoietic system and nervous system diseases. Conventional Pb2+Although detection methods (e.g., inductively coupled plasma mass spectrometry and anodic stripping voltammetry) have high sensitivity and good specificity, these techniques require tedious and time-consuming proceduresSample pre-processing and professional operators, resulting in inefficient testing. In recent years, Pb2+The colorimetric detection is explosively developed due to the characteristics of simple operation, high efficiency and the like. However, these colorimetric sensors are bulk solution measurements, which limits their lower detection limit. Based on this, Dark Field Microscopy (DFM) was used to increase Pb due to the ability to perform single nanoparticle level detection2+Lower limit of detection of (1). Detection of Pb by gold nanoparticles (AuNPs) as dark-field imaging probes under DFM has been reported2 +The lower detection limit reaches 0.2 pM. However, this method of AuNPs etching limits the detection speed and is sensitive to temperature. In order to improve the detection speed and enlarge the detection range, the invention provides a Single Particle Dark Field Spectroscopy (SPDFS) based on the combination of DFM and Glutathione (GSH) -functionalized AuNPs (GSH-AuNPs), the lower limit value of the detection is 3.5pM, and the linear range of the detection is 10pM to 10 mu M.
Disclosure of Invention
The invention aims to solve the problems mentioned in the background technology and provides an ultra-sensitive method for rapidly detecting lead ions.
The technical scheme adopted by the invention is as follows:
firstly, AuNPs are subjected to surface modification by GSH to form GSH-AuNPs probe, and the probe is added into Pb2+Under the action of the nano particles, GSH-AuNPs form dimers or polymers, and the scattering spectrum of the GSH-AuNPs is red-shifted due to the coupling effect among the nano particles. DFM is used for detecting red shift quantity (delta lambda) of scattering spectrum of GSH-AuNPs, and the size of delta lambda and Pb2+Concentration of Pb is related to the amount of Pb2+The concentration of (c).
The method comprises the following steps: (1) utilizing GSH to modify AuNPs as a specific recognition probe and dark field imaging particles of lead ions; (2) immobilizing GSH-AuNPs on a glass substrate; (3) dropwise adding a lead-containing sample on a glass base, and washing after 5 minutes; (4) dropwise adding GSH-AuNPs on a glass base, and then covering a cover glass; (5) lead ions were detected by dark field microscopy.
Wherein the amount of Pb is determined2+The equation for the concentration of (a) is: Δ λ ═ Δ λmax*cn)/(Kn+cn) And Δ λ representsThe spectrum red shift amount of the gold nano particles, the concentration of lead ions is c, and the maximum red shift amount is delta lambdamax41.15nm, hill coefficient n 0.32, and binding constant K25.94.
Wherein the lead-containing sample comprises tap water, lake water, industrial wastewater and laboratory waste liquid.
Wherein the step of fixing gold nanoparticles on a glass substrate comprises modifying thiol groups on glass.
Wherein, the dark field microscope system detects the lead ion concentration by utilizing the red shift amount of the peak value of the nanoparticle scattering spectrum.
Wherein, the red shift of the gold nanoparticle scattering spectrum is the average value of the red shift of all the gold nanoparticle scattering spectra in the dark field.
Wherein, the average value of the red shift of all gold nanoparticle scattering spectra in the dark field is calculated by Gaussian fitting.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. in the invention, the single-particle dark field spectroscopy is used for detecting the lead ions, the method is novel and simple, the sensitivity is high, the detection range is wide, and the detection is completed within 10 minutes.
2. In the present invention, Pb is quantified using the shift of the dark field spectrum of the nanoparticles, as compared with the previously reported method of using the dark field colorimetric method and the method of using the dark field microscope to detect the variation of the scattered light intensity of the nanoparticles2+The concentration is more accurate and faster, because the dark field colorimetric method limits the detection range, and the detection method of the variation of the scattered light intensity of the nano particles needs to use gold nano particle etching, so the method is long in time consumption and sensitive to temperature.
Drawings
FIG. 1 is a schematic diagram of a dark field microscopy system for detecting lead ions according to the present invention;
FIG. 2 shows the spectral red shift (. DELTA.. lamda.) of gold nanoparticles of the present invention as Pb2+Plot of concentration as a function of time.
The labels in the figure are: 1. white light emitted from a halogen lamp; 2. a monochromator; 3. quasi-monochromatic light; 4. a dark field condenser; 5. a glass slide; 6. gold nanoparticles; 7. scattering light of gold nanoparticles; 8. a microscope objective lens; 9. scattered light collected by a microscope objective; 10. an industrial camera.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The specific detection steps of the invention comprise:
(1) surface modification of gold nanoparticles: first, 500. mu.L of 0.05mg/mL AuNPs were centrifuged at 1687g for 15 minutes. The supernatant was discarded, and the AuNPs were then dispersed in 1mL of 1mM GSH (pH 8). Next, the mixture was incubated at room temperature for 2 hours. Unbound GSH was then removed from the solution by centrifugation at 1687g for 10 minutes. The supernatant was discarded and the GSH-AuNPs were gently dispersed in deionized water. The centrifugation step was repeated three times. GSH-AuNPs prepared by the above method were diluted 1: 6 in deionized water for further experiments.
(2) Manufacturing a sensor chip: the whole manufacturing process of the sensor is divided into two steps. First, thiol groups are modified on a glass slide. The method comprises the following specific steps: the slides were immersed in "piranha solution" (H)2SO4∶H2O2(30%) 7: 3 by volume) for 15 minutes, and then rinsed with deionized water and absolute ethanol, respectively. The slides were then immediately immersed in a 6% MPTS absolute ethanol solution for 15 minutes. The slides were then ultrasonically cleaned 3 times for 3 minutes each with an absolute ethanol solution. Finally, the slides were placed in an oven at 120 ℃ for 2 hours. In the second step, GSH-AuNPs were immobilized on slides. First, 20. mu.L of GSH-AuNPs solution was dropped on a slide glass and reacted for 30 minutes. Then rinsed with deionized water and dried at room temperature. Finally, the slides were stored in a sealed box at 4 ℃ until use.
(3) Detection of Pb using dark field microscope2+: 20 mu L of the solution containing Pb2+The sample solution was dropped onto the sensor chip and reacted for 5 minutesA clock. Then rinsed with deionized water and the residual solution was pipetted off. Next, 5. mu.L of GSH-AuNPs solution was added dropwise to the sensor chip and immediately covered with a cover slip. The sensor chip can now be tested under DFM. The detection is carried out under a dark field microscopic imaging system, and the working principle is as follows as shown in reference to fig. 1: white light 1 emitted by the halogen lamp is spectrally filtered by the monochromator 2 and illuminates the sample 6, and scattered light 7 of the sample is collected by the objective lens 8 and recorded by the industrial camera 10. The samples herein are referred to as GSH-AuNPs. Images recorded under different illumination wavelengths are combined into an image series, and the scattering spectrum of a single nanoparticle is fitted by using an image collection. The specific method of fitting the spectra is as follows: and extracting single particle spectra of all the nanoparticles by calculating the intensity of each image, wherein the peak value of the single nanoparticle scattering spectrum is calculated by a Lorentz function. From this, the average red shift Δ λ of all nanoparticles in the field of view was then calculated. Finally, the concentration value of the lead ions is obtained according to the function relation between the delta lambda and the lead ion concentration, and the reference is made to fig. 2. The exposure time and frame rate of the camera 10 in the experiment were 0.267s and 3.75fps, respectively. The scanning range, step length and interval time of the light source controlled by the monochromator 2 are 500nm to 700nm, 1nm and 0.3s respectively. In the experiment, the LSPR resonance peak position of AuNP is calculated by analyzing and processing a dark field image by utilizing Matlab R2007a software.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (7)
1. A method for detecting lead ion concentration based on dark-field spectroscopy detection technology, the method comprising the following steps: (1) gold nanoparticles modified by glutathione are used as a specific recognition probe and a dark field imaging particle of lead ions; (2) fixing gold nanoparticles on a glass substrate; (3) dropwise adding a lead-containing sample on a glass base, and washing after 5 minutes; (4) dropwise adding gold nanoparticles modified by glutathione onto a glass substrate, and then covering a cover glass; (5) lead ions were detected by dark field microscopy.
2. The method for detecting lead ions based on the dark-field spectroscopy detection technology as claimed in claim 1, wherein: the lead ion concentration in the sample was determined based on the following equation: Δ λ ═ Δ λmax*cn)/(Kn+cn) Wherein Delta lambda represents the spectrum red shift of the gold nanoparticles, the concentration of lead ions is c, and the maximum red shift is Delta lambdamax41.15nm, hill coefficient n 0.32, and binding constant K25.94.
3. The method of claim 1 wherein the lead-containing sample comprises tap water, lake water, industrial waste water, laboratory waste.
4. The method of claim 1, wherein: the step of immobilizing the gold nanoparticles on the glass substrate includes modifying thiol groups on the glass.
5. The method of claim 1, wherein: the dark field microscope system is used for detecting the lead ion concentration by utilizing the red shift amount of the peak value of the scattering spectrum of the nano particles.
6. The method of claim 5, wherein the red shift of the gold nanoparticle scatter spectrum is an average of the red shifts of all gold nanoparticle scatter spectra in the dark field view.
7. The method of claim 6, wherein the average of the red shift of the scattering spectra of all gold nanoparticles in the dark field view is calculated using a Gaussian fit.
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