CN109406483A - A method of 5-ALA is detected using SERS - Google Patents
A method of 5-ALA is detected using SERS Download PDFInfo
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- CN109406483A CN109406483A CN201811221717.3A CN201811221717A CN109406483A CN 109406483 A CN109406483 A CN 109406483A CN 201811221717 A CN201811221717 A CN 201811221717A CN 109406483 A CN109406483 A CN 109406483A
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- G—PHYSICS
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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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
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N2021/3125—Measuring the absorption by excited molecules
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Abstract
The invention discloses a kind of methods using SERS detection 5-ALA, and the hydrophobic substrate etc. using grafting sulfhydryl compound preparation is material.The 5-ALA solution of various concentration is mixed with nano Au colloid, and is tested using laser Raman spectrometer, the SERS spectra figure of 5-ALA is obtained.Establish the mathematical relationship between Raman spectrum absorption intensity and 5-ALA concentration.It is tested using 5-ALA solution of the laser Raman spectrometer to unknown concentration, the concentration of 5-ALA is extrapolated according to the mathematical relationship.The present invention solves the problems such as current 5-ALA testing cost is expensive, and sample needs multistep to separate, and the advantages of having easily operated, high sensitivity, can save a large amount of economy and time cost, has a wide range of applications.
Description
Technical field
The invention belongs to the technical fields of detection, and in particular to a kind of side using SERS detection 5-ALA
Method.
Background technique
5-ALA can participate in the adjustment process of plant growth and development, additionally it is possible to which selectivity kills dicotyledonous plant
The weeds of species, and it is harmless to monocotyledonous plants such as rice, seeding corn and other crops, the vegetables such as leek.Azoles farm chemicals are one
Class nitrogen-containing heterocycle compound, in addition to there is good bactericidal effect that can have broad-spectrum high efficacy sterilization, toxicity with coordinate plant growth
Low, the advantages that duration is long.
Pesticide provides reliable guarantee for the good harvest of worldwide cereal crops, and being can not in modern agricultural production
Lack.But while bringing big advantages, pesticide especially organic agricultural chemicals is largely used, it is residual to result in serious pesticide
It stays, grave danger is brought to human health.In recent years, food-safety problem causes the concern of the public, because it is to people
Body health has significant damage, while will also result in huge pollution to environment.So guaranteeing that human health is exempted to protect environment
It is endangered, exploring the remaining new tool of pesticide in sensitive, rapid, the convenient and fast test food of one kind becomes the most important thing.
Mainly there is the conventional method of Detecting Pesticide in food: mass spectral analysis, chromatography, is immunized Capillary Electrophoresis
Analytic approach etc., the reliability of these methods is all very high, but all relatively time-consuming, and due to originals such as sample pre-processing process complexity
Cause causes analysis cost comparatively higher, and step is complicated, causes to be difficult to promote and standardize.Raman spectrum can be fine
Reflection molecule structural information, the discovery of Raman enhanced spectrum greatly reduces the concentration of detecting material, to expand
The application range of Raman spectrum, but its detection limit still has received limitation.
Summary of the invention
The problems such as in order to overcome existing 5-ALA detection existing costly, pretreatment is complicated, time-consuming, with
And the problem of Raman detection method detection limit limitation.The object of the present invention is to provide one kind can be with the 5- amino of fast detection of trace
The advantages of method of levulic acid has easily operated, high sensitivity, can save a large amount of economy and time cost.It can be extensive
Detection applied to sample in the fields such as food, drug, environment.
A method of 5-ALA being detected using SERS, is included the following steps:
(1) copper sheet: being cut into the small copper sheet of 2cm × 5cm rectangle, remove the oxidation film on surface by the pretreatment of copper sheet, is rinsed
It is dried after clean；
(2) nano silver is plated on copper sheet；
(3) be grafted sulfhydryl compound: the plating nano silver metal copper sheet of preparation is 1.0 × 10 in concentration-3mol/L-1.0×
10-2It is impregnated 3-12 hours in the ethanol solution of the sulfhydryl compound of mol/L, after taking-up, then with ethyl alcohol flushing to get detection base
Bottom material；
(4) preparation of nano Au colloid: by HAuCl4Solution is heated to boiling, and citric acid three sodium solution is added, continues to keep
After boiling 15 minutes, it is centrifuged after natural cooling, takes lower layer's golden yellow precipitate, the nano Au colloid as prepared；
(5) detection of standard curve: weighing 5-ALA, prepares the 5- glycyl third of 5-6 concentration gradient
The quasi- solution example of acidity scale；The 5-ALA standard solution of various concentration gradient and isometric step (4) are obtained
Nano Au colloid uniformly mixes, centrifuge separation, collects sediment fraction, evenly laid out on the base material of step (3) preparation, and benefit
It is tested with FT-Raman and confocal Raman instrument, obtains the SERS spectra figure of 5-ALA；
The nano Au colloid for taking step (4) to prepare individually carries out Raman spectrum test, obtains nano Au colloid background Raman signal；
Use nano Au colloid background Raman signal as internal standard to 5-ALA standard solution surface-enhanced Raman light
Spectrum carries out intensity normalization, establishes 5-ALA Surface enhanced Raman spectroscopy relative line intensity-concentration standard control
Working curve diagram；
(6) unknown sample concentration is detected
The nano Au colloid that the 5-ALA solution to be determined of unknown concentration and isometric step (4) are obtained
Uniformly mixing, is then tested using laser Raman spectrometer, and carry out spectral strength normalized and obtain unknown concentration
5-ALA Surface enhanced Raman spectroscopy spectrogram；The concentration of 5-ALA is calculated by formula.
Further, the specific steps of the preparation of step (4) nano Au colloid are as follows: accurately pipetting 50mL concentration is 1.0
×10-4g/mL-1.0×10-3The HAuCl of g/mL4Solution is placed in heat collecting type constant temperature blender with magnetic force in the beaker of 200mL,
Reaction temperature is set as 100 DEG C；It is heated to boiling under strong stirring, the citric acid three that 1.5mL-6mL mass fraction is 1% is added
Sodium solution keeps boiling 15 minutes, is centrifuged after natural cooling, takes lower layer's golden yellow precipitate, the nano Au colloid as prepared.
Further, the concentration changing value of the 5-ALA standard solution sample of the 5-7 concentration gradient is
10-4-10-10Between mol/L.
Further, sulfhydryl compound described in step (3) is n-butyl mercaptan, in n- dodecyl mereaptan, positive stearylmercaptan
One or two kinds of mixture.
Further, the pretreatment specific steps of step (1) copper sheet are as follows: it is small that copper sheet is cut into 2cm × 5cm rectangle
Copper sheet is successively immersed in each ultrasound 10min in acetone, ethyl alcohol and ultrapure water, copper sheet is finally immersed in the HNO of 0.1mol/L3
Inside remove the oxidation film on surface；The copper sheet ultrapure water that pretreatment finishes is clean, and room temperature is dried.
Further, the specific steps of nano silver are plated on step (2) copper sheet are as follows: the copper after handling step (1) well
SnCl of the piece in 0.02mol/L2It is impregnated 1 minute in the hydrochloric acid mixed solution of 0.02mol/L, drying at room temperature；Secondly, by copper sheet
In the AgNO of 0.01mol/L3With mass fraction to impregnate 3 minutes in the mixed solution of 0.2% polyvinylpyrrolidone, room temperature is dry
It is dry；The above two step 4 periods are repeated, finally by substrate in SnCl2It is impregnated 3 minutes in solution, uses ultrapure water.
The invention has the following beneficial effects:
Base material prepared by the present invention has super-hydrophobic effect, can play the role of concentration, with conventional method phase
Than there is higher sensitivity, the limting concentration for detecting 5-ALA of the invention is 10-10Mol/L is realized super
Trace detection, accuracy with higher and sensitivity are 100 times of the prior art, as long as testing cost liquid phase-mass spectrometry
/ 50th of Instrument measuring method.
Detailed description of the invention
Fig. 1 is the Surface enhanced Raman spectroscopy figure of the various concentration 5-ALA measured in embodiment 1；
Fig. 2 is that Fig. 1 wave number is 930cm-1The Surface enhanced Raman spectroscopy figure at place；
Fig. 3 is that Fig. 1 wave number is 1296cm-1The Surface enhanced Raman spectroscopy figure at place；
Fig. 4 is to measure the Surface enhanced Raman spectroscopy of various concentration 5-ALA in 930cm in embodiment 1-1Wave
Linear relationship curve at number between absorption peak strength and concentration；
Fig. 5 is that wave number is 930.00cm-1Remove 10 in place-10Raman absorption intensity (y) and concentration after mg/mL data point
(x) the mathematical relationship curve between；
Fig. 6 is to measure the Surface enhanced Raman spectroscopy of various concentration 5-ALA in 1296cm in embodiment 1-1Wave
The linear relationship curve of absorption peak strength and concentration at number.
Specific embodiment
In order to better understand the present invention, below by embodiment to the present invention into further explanation, embodiment is served only for
It explains the present invention, any restriction can't be constituted to the present invention.The following examples are intended to illustrate the invention, but is not limited to this
The range of invention.Unless otherwise specified, the conventional hand that technological means used in embodiment is well known to those skilled in the art
Section, raw materials used is commercial goods.
Embodiment 1
(1) pretreatment of copper sheet
Copper sheet is cut into the small copper sheet of 2cm × 5cm rectangle, is immersed in each ultrasound 10min in acetone, ethyl alcohol and ultrapure water,
Copper sheet is finally immersed in the HNO of 0.1mol/L3Inside remove the oxidation film on surface.The copper sheet finished is pre-processed to be rushed with ultrapure water
Wash clean, room temperature are dried.
(2) nano silver is plated on copper sheet
By copper sheet 0.02mol/L SnCl2It is impregnated 1 minute in the hydrochloric acid mixed solution of 0.02mol/L, room temperature is dry
It is dry；Secondly, by copper sheet 0.01mol/L AgNO3With mass fraction be 0.2% polyvinylpyrrolidone mixed solution in soak
Bubble 3 minutes, drying at room temperature；The above two step 4 periods are repeated, finally by substrate in SnCl2It is impregnated 3 minutes in solution, with super
Pure water rinsing.
(3) it is grafted sulfhydryl compound
The plating nano silver metal copper sheet of preparation is 3.0 × 10-3It is impregnated 5 hours in the ethanol solution of the n-butyl mercaptan of mol/L,
After taking-up, then with ethyl alcohol flushing to get detection base material.
(4) preparation of nanogold
Accurately pipette 50mL 2.5 × 10-4The HAuCl of g/mL4Solution is placed on heat collecting type constant temperature magnetic in the beaker of 200mL
In power blender, reaction temperature is set as 100 DEG C.It is heated to boiling under strong stirring, the lemon that 4mL mass fraction is 1% is added
Lemon three sodium solutions of acid, keep boiling 15 minutes, are centrifuged after natural cooling, take lower layer's golden yellow precipitate, what is as prepared receives
Rice gold size.
(5) detection of standard curve
It is 10 that 5-ALA concentration is prepared by solvent of ultrapure water-4-10-10(specific concentration is 10 to mol/L- 4Mol/L, 10-5Mol/L, 10-6Mol/L, 10-7Mol/, 10-8Mol/L, 10-9Mol/L, 10-10Mol/L solution) is as to be measured
Object, take respectively 7 of the 20 above-mentioned preparations of μ L different gradient concentrations 5-ALA standard solution and isometric step
(4) nano Au colloid for obtaining plating nano silver metal copper sheet uniformly mixes, and is centrifugated, and collects sediment fraction, is placed on step (3) system
On standby base material, and (the model thunder of used Raman instrument is tested using FT-Raman and confocal Raman instrument
The Buddhist nun invia that continues is copolymerized burnt microscopic Raman, time of integration 10s, and acquisition is primary；The laser of the 785nm of used exciting light),
The SERS spectra figure (as shown in Figure 1) of 5-ALA is obtained, using origin data processing software to detected
Various concentration 5-ALA SERS spectra carries out general peak intensity normalized, with 5-ALA 930cm-1Wave
Strongest SERS Pu Feng does 5-ALA aqueous solution as X-axis as Y-axis, 5-ALA concentration at number
The standard working curve that the general peak relative intensity of SERS spectra changes with concentration, as shown in Figure 2.Each concentration samples acquire 5 light
Spectrum, then average spectral line is done, draw standard working curve.Regression equation are as follows: Y=1052.65X+10374.47, coefficient R2=
0.9900。
(6) it detects
By 10-6The 5-ALA aqueous solution of mol/L takes 20 μ L above-mentioned to be measured using unknown concentration as solution to be measured
Solution is uniformly mixed with the nano Au colloid that isometric step (4) obtains, and is then tested using laser Raman spectrometer, according to
The mathematics between 5-ALA Raman spectrum absorption intensity and 5-ALA concentration obtained according to step (5) closes
The concentration that system extrapolates 5-ALA is 9.98 × 10-7mol/L。
Embodiment 2
(1) pretreatment of copper sheet
Copper sheet is cut into the small copper sheet of 2cm × 5cm rectangle, is immersed in each ultrasound 10min in acetone, ethyl alcohol and ultrapure water,
Copper sheet is finally immersed in the HNO of 0.1mol/L3Inside remove the oxidation film on surface.The copper sheet finished is pre-processed to be rushed with ultrapure water
Wash clean, room temperature are dried.
(2) nano silver is plated on copper sheet
By copper sheet 0.02mol/L SnCl2It is impregnated 1 minute in the hydrochloric acid mixed solution of 0.02mol/L, room temperature is dry
It is dry；Secondly, by copper sheet 0.01mol/L AgNO3With mass fraction be 0.2% polyvinylpyrrolidone mixed solution in soak
Bubble 3 minutes, drying at room temperature；The above two step 4 periods are repeated, finally by substrate in SnCl2It is impregnated 3 minutes in solution, with super
Pure water rinsing.
(3) it is grafted sulfhydryl compound
The plating nano silver metal copper sheet of preparation is 4.5 × 10-3It is impregnated 5 hours in the ethanol solution of the n-butyl mercaptan of mol/L,
After taking-up, then with ethyl alcohol flushing to get detection base material.
(4) preparation of nanogold
Accurately pipette 50mL3.5 × 10-4The HAuCl of g/mL4Solution is placed on heat collecting type constant temperature magnetic in the beaker of 200mL
In power blender, reaction temperature is set as 100 DEG C.It is heated to boiling under strong stirring, it is 1% that 3.5mL mass fraction, which is added,
Citric acid three sodium solution keeps boiling 15 minutes, is centrifuged after natural cooling, takes lower layer's golden yellow precipitate, as prepare
Nano Au colloid.
(5) detection of standard curve
It is 10 that 5-ALA concentration is prepared by solvent of ultrapure water-5-10-9The solution of mol/L as determinand,
Take respectively 5 of the 20 above-mentioned preparations of μ L different gradient concentrations 5-ALA standard solution and isometric step (4)
The nano Au colloid for obtaining plating nano silver metal copper sheet uniformly mixes, and is centrifugated, and collects sediment fraction, is placed on step 3 preparation
It on base material, and is tested using FT-Raman and confocal Raman instrument, obtains the SERS spectra figure of 5-ALA
(as shown in Figure 1), using origin data processing software to detected various concentration 5-ALA SERS spectra
General peak intensity normalized is carried out, with 5-ALA 930cm-1Strongest SERS Pu Feng is as Y-axis, 5- ammonia at wave number
Base levulic acid concentration does what the general peak relative intensity of 5-ALA aqueous solution SERS spectra changed with concentration as X-axis
Standard working curve, as shown in Figure 2.Each concentration determination 5 times, is averaged, and draws standard working curve.Regression equation are as follows: Y
=9752.13X+20586.65, coefficient R2=0.9954.
(6) it detects
By 10-6The 5-ALA standard aqueous solution of mol/L takes 20 μ L above-mentioned using unknown concentration as solution to be measured
Solution to be measured is uniformly mixed with the nano Au colloid that isometric step (4) obtains, and is then surveyed using laser Raman spectrometer
Examination, between the 5-ALA Raman spectrum absorption intensity and 5-ALA concentration of foundation step (5) acquisition
The concentration that mathematical relationship extrapolates 5-ALA is 9.98 × multiplication sign 10-7mol/L。
Embodiment 3
(1) pretreatment of copper sheet
Copper sheet is cut into the small copper sheet of 2cm × 5cm rectangle, is immersed in each ultrasound 10min in acetone, ethyl alcohol and ultrapure water,
Copper sheet is finally immersed in the HNO of 0.1mol/L3Inside remove the oxidation film on surface.The copper sheet finished is pre-processed to be rushed with ultrapure water
Wash clean, room temperature are dried.
(2) nano silver is plated on copper sheet
By copper sheet 0.02mol/L SnCl2It is impregnated 1 minute in the hydrochloric acid mixed solution of 0.02mol/L, room temperature is dry
It is dry；Secondly, by copper sheet 0.01mol/L AgNO3With mass fraction be 0.2% polyvinylpyrrolidone mixed solution in soak
Bubble 3 minutes, drying at room temperature；The above two step 4 periods are repeated, finally by substrate in SnCl2It is impregnated 3 minutes in solution, with super
Pure water rinsing.
(3) it is grafted sulfhydryl compound
The plating nano silver metal copper sheet of preparation is 2.5 × 10-3It is small that 4.5 are impregnated in the ethanol solution of the n-butyl mercaptan of mol/L
When, after taking-up, then with ethyl alcohol flushing to get detection base material.
(4) preparation of nanogold
Accurately pipette 50mL 5 × 10-4The HAuCl of g/mL4Solution is placed on heat collecting type constant temperature magnetic force in the beaker of 200mL
In blender, reaction temperature is set as 100 DEG C.It is heated to boiling under strong stirring, the lemon that 2.5mL mass fraction is 1% is added
Lemon three sodium solutions of acid, keep boiling 15 minutes, are centrifuged after natural cooling, take lower layer's golden yellow precipitate, what is as prepared receives
Rice gold size.
(5) detection of standard curve
It is 10 that 5-ALA concentration is prepared by solvent of ultrapure water-5-10-9The solution of mol/L as determinand,
Take respectively 5 of the 20 above-mentioned preparations of μ L different gradient concentrations 5-ALA standard solution and isometric step (4)
The nano Au colloid for obtaining plating nano silver metal copper sheet uniformly mixes, and is centrifugated, and takes sediment fraction, drips the base prepared in step 3
It on bottom material, and is tested using FT-Raman and confocal Raman instrument, obtains the SERS spectra figure of 5-ALA (such as
Shown in Fig. 1), it is 5-ALA characteristic peak 930cm-1Linearity curve between peak height and its concentration, as shown in Figure 2.Each
Sample acquires 5 spectrum, then does average spectral line, draws standard working curve.Regression equation are as follows: Y=1342.14X+9372.44,
Coefficient R2=0.9925.
(6) it detects
By 10-6The 5-ALA standard aqueous solution of mol/L as solution to be measured, take the above-mentioned solution to be measured of 20 μ L with
The nano Au colloid that isometric step (4) obtains uniformly mixes, and is then tested using laser Raman spectrometer, according to step
(5) it is 9.97 that linear relationship, which extrapolates the concentration of 5-ALA, between the 5-ALA peak height obtained and its concentration
×10-7mol/L。
Embodiment 4
(1) pretreatment of copper sheet
Copper sheet is cut into the small copper sheet of 2cm × 5cm rectangle, is immersed in each ultrasound 10min in acetone, ethyl alcohol and ultrapure water,
Copper sheet is finally immersed in the HNO of 0.1mol/L3Inside remove the oxidation film on surface.The copper sheet finished is pre-processed to be rushed with ultrapure water
Wash clean, room temperature are dried.
(2) nano silver is plated on copper sheet
By copper sheet 0.02mol/L SnCl2It is impregnated 1 minute in the hydrochloric acid mixed solution of 0.02mol/L, room temperature is dry
It is dry；Secondly, by copper sheet 0.01mol/L AgNO3With mass fraction be 0.2% polyvinylpyrrolidone mixed solution in soak
Bubble 3 minutes, drying at room temperature；The above two step 4 periods are repeated, finally by substrate in SnCl2It is impregnated 3 minutes in solution, with super
Pure water rinsing.
(3) it is grafted sulfhydryl compound
The plating nano silver metal copper sheet of preparation is 3.5 × 10-3It is small that 6 are impregnated in the ethanol solution of the n- dodecyl mereaptan of mol/L
When, after taking-up, then with ethyl alcohol flushing to get detection base material.
(4) preparation of nanogold
Accurately pipette 50mL 3.5 × 10-4The HAuCl of g/mL4Solution is placed on heat collecting type constant temperature magnetic in the beaker of 200mL
In power blender, reaction temperature is set as 100 DEG C.It is heated to boiling under strong stirring, the lemon that 3mL mass fraction is 1% is added
Lemon three sodium solutions of acid, keep boiling 15 minutes, are centrifuged after natural cooling, take lower layer's golden yellow precipitate, what is as prepared receives
Rice gold size.
(5) detection of standard curve
It is 10 that 5-ALA concentration is prepared by solvent of ultrapure water-5-10-9The solution of mol/L as determinand,
Take respectively 5 of the 20 above-mentioned preparations of μ L different gradient concentrations 5-ALA standard solution and isometric step (4)
The nano Au colloid for obtaining plating nano silver metal copper sheet uniformly mixes, and is centrifugated, and takes sediment fraction, drips the base prepared in step 3
It on bottom material, and is tested using FT-Raman and confocal Raman instrument, obtains the SERS spectra figure of 5-ALA (such as
Shown in Fig. 1), it is 5-ALA characteristic peak 930cm-1Linearity curve between peak height and its concentration, as shown in Figure 2.Each
Sample acquires 5 spectrum, then does average spectral line, draws linearity curve.Regression equation are as follows: Y=8902.33X+98582.15, phase
Close coefficients R2=0.9914.
(6) it detects
By 10-6The 5-ALA standard aqueous solution of mol/L takes 20 μ L above-mentioned using unknown concentration as solution to be measured
Solution to be measured is uniformly mixed with the nano Au colloid that isometric step (4) obtains, and is then surveyed using laser Raman spectrometer
Examination, linear relationship extrapolates 5-ALA between the 5-ALA peak height obtained according to step (5) and its concentration
Concentration be 9.99 × 10-7mol/L。
Embodiment 5
(1) pretreatment of copper sheet
Copper sheet is cut into the small copper sheet of 2cm × 5cm rectangle, is immersed in each ultrasound 10min in acetone, ethyl alcohol and ultrapure water,
Copper sheet is finally immersed in the HNO of 0.1mol/L3Inside remove the oxidation film on surface.The copper sheet finished is pre-processed to be rushed with ultrapure water
Wash clean, room temperature are dried.
(2) nano silver is plated on copper sheet
By copper sheet 0.02mol/L SnCl2It is impregnated 1 minute in the hydrochloric acid mixed solution of 0.02mol/L, room temperature is dry
It is dry；Secondly, by copper sheet 0.01mol/L AgNO3With mass fraction be 0.2% polyvinylpyrrolidone mixed solution in soak
Bubble 3 minutes, drying at room temperature；The above two step 4 periods are repeated, finally by substrate in SnCl2It is impregnated 3 minutes in solution, with super
Pure water rinsing.
(3) it is grafted sulfhydryl compound
The plating nano silver metal copper sheet of preparation is 3.5 × 10-35.5 are impregnated in the ethanol solution of the n- dodecyl mereaptan of mol/L
Hour, after taking-up, then with ethyl alcohol flushing to get detection base material.
(4) preparation of nanogold
Accurately pipette 50mL 5 × 10-4The HAuCl of g/mL4Solution is placed on heat collecting type constant temperature magnetic force in the beaker of 200mL
In blender, reaction temperature is set as 100 DEG C.It is heated to boiling under strong stirring, the lemon that 1.0mL mass fraction is 1% is added
Lemon three sodium solutions of acid, keep boiling 15 minutes, are centrifuged after natural cooling, take lower layer's golden yellow precipitate, what is as prepared receives
Rice gold size.
(5) detection of standard curve
It is 10 that 5-ALA concentration is prepared by solvent of ultrapure water-5-10-9The solution of mol/L as determinand,
Take respectively 6 of the 20 above-mentioned preparations of μ L different gradient concentrations 5-ALA standard solution and isometric step (4)
The nano Au colloid for obtaining plating nano silver metal copper sheet uniformly mixes, and is centrifugated, and takes sediment fraction, drips the base prepared in step 3
It on bottom material, and is tested using FT-Raman and confocal Raman instrument, obtains the SERS spectra figure of 5-ALA (such as
Shown in Fig. 1), it is 5-ALA characteristic peak 930cm-1Linearity curve between peak height and its concentration, as shown in Figure 2.Each
Sample acquires 5 spectrum, then does average spectral line, draws linearity curve.Regression equation are as follows: Y=1142.71X+50372.58, phase
Close coefficients R2=0.9961.
(6) it detects
By 10-6The 5-ALA standard aqueous solution of mol/L takes 20 μ L above-mentioned using unknown concentration as solution to be measured
Solution to be measured is uniformly mixed with the nano Au colloid that isometric step (4) obtains, and is then surveyed using laser Raman spectrometer
Examination, linear relationship relationship extrapolates 5- glycyl between the 5-ALA peak height obtained according to step (5) and its concentration
The concentration of propionic acid is 9.99 × 10-7mol/L。
Comparative examples
2.1 chromatographic condition
Chromatographic column: ZORBAX SB-C18 column (150mm × 4.6mm, 5 μm)；Mobile phase: acetonitrile (A) -20mmol/L, phosphoric acid
Potassium dihydrogen (B) (pH) 2.0, perfluorooctane sulfonate containing 2.0mmol/L)；Gradient elution program: 0-5min 18%A, 5-10min
18%A, 10-15min 55%A, 15-20min 55%A-18%A；Flow velocity: 0.5mL/min；Detection wavelength: 205nm；Column temperature:
25℃；Sample volume: 20 μ L.
The preparation of 2.2 solution
2.2.1 contrast solution is mixed
Take phthalimide, phthalic acid, phthalimide methyl ester levulinate reference substance each 10mg, 5-
Amino-laevulic acid, levulic acid reference substance each 200mg, it is accurately weighed.It sets in same 25mL measuring bottle, adds acetonitrile-water (volume
Than 18:82) dissolution, constant volume, prepare mixing control stock solution.Precision measures mixing control stock solution 1mL, sets in 10mL measuring bottle,
Add acetonitrile-water (volume ratio 18:82) constant volume, it is sub- containing phthalimide, phthalic acid, phthalyl to be configured to 1mL
The mixing control of amine methyl ester levulinate reference substance each 40 μ g, 5-ALA and each 0.8mg of levulic acid reference substance are molten
Liquid.
2.2.2 test solution is mixed
Take laboratory self-control 5-ALA, levulic acid, phthalimide, phthalic acid, adjacent benzene two
Carboximide methyl ester levulinate test sample is appropriate, accurately weighed.With acetonitrile-water (volume ratio 18:82) dissolution, constant volume, prepare
Solution at 1mL containing 5-ALA and levulic acid, phthalimide, phthalic acid and phthalyl are sub-
The mixing test solution of each 40 μ g of amine methyl ester levulinate.
2.35- amino-laevulic acid standard reserving solution
Accurately weighed 5-ALA reference substance is configured to acetonitrile-water (volume ratio 18:82) dissolution, constant volume
5-ALA standard reserving solution.
Test:
The Raman spectrum of sample is by Reinshaw inVia micro confocal laser Raman spectrometer (Reinshaw company, Britain
Renishaw) acquisition obtains.(the wave number precision of measurement can equipped with Leca microscope for micro confocal laser Raman spectrometer
To reach 0.1cm-1), have the function of real time correction；Detector, grating and optical filter can realize full-automatic accurate positioning；Using
WiRETM2.0 softwares are as matched spectra collection software.
From the Raman absorption intensity found out in Fig. 2 and Fig. 3 under corresponding wave number, data are listed with Origin and carry out linear fit
Obtain Fig. 4 and Fig. 5.From the point of view of the linear relationship in Fig. 4,930.00cm-1The linear relationship at place is y=-1447.87x+
11691.85, R values are 0.88, do not reach 0.9 or more, linear poor.If by 10-10It is sent out after the removal of mg/mL data point
Existing, the obtained mathematical relationship between Raman spectrum absorption intensity (y) and concentration (x) is y=-1052.65x+10374.47 (ginseng
See Fig. 5), R value reaches 0.99, this explanation may be since the concentration of 5-ALA is 10-10When mg/mL, surface increases
Strong Raman spectrum identifies it beyond its detection limit, can not detect, 10 on the other hand can be found out from Fig. 2 and Fig. 3-10Mg/mL does not have
Characteristic peak is occurred, this also demonstrates 10-10Mg/mL exceeds the detection limit of 5-ALA.
Fig. 6 show that wave number is 1295.95cm-1Mathematical relationship be y=-1490.83x+11560.45, R value be 0.94.Always
From the point of view of on body, the correlation of information collection is good, from the two on the preferable characteristic peak of concentration dependence, Fig. 4, Fig. 5, Fig. 6
It can be concluded that a conclusion:, can be by 5-ALA about the method for quick detection 5-ALA
Surface enhanced Raman spectroscopy carries out characteristic peak and determines, obtains 5- glycyl under the good wave number of 5-ALA repeatability
Relationship between propionic acid Raman spectrum absorption intensity and 5-ALA concentration, as the wave number determined in the application is
930.00cm-1、1295.95cm-1, establish the number between Raman spectrum absorption intensity (y) and 5-ALA concentration (x)
Relationship.It is tested using 5-ALA solution of the laser Raman spectrometer to unknown concentration, obtains above-mentioned repetition
The corresponding 5-ALA Raman spectrum absorption intensity of the good wave number of property.According to the Raman spectrum absorption intensity established
(y) the mathematical relationship formula between 5-ALA concentration (x), such as (930.00cm-1) y=-1052.65x+
10374.47 (1295.95cm-1) y=-1490.83x+11560.45, unknown 5- can be calculated by substituting into relationship
The concentration of amino-laevulic acid, unknown concentration test should choose three data, substitute into average value after formula, so that it may obtain 5- to be measured
The concentration of amino-laevulic acid.
The application is simultaneously by the method for embodiment 1 and use embodiment 1, embodiment 2, embodiment 3, embodiment 4 and reality
Raman peak values are applied in example 5 in 930.00cm-1The linear relationship curve at place, carrying out test sample concentration is 5 × 10-5mol/L、5×
10-6Ol/L and 5 × 10-7Mol/L is detected and is calculated the rate of recovery.
Detection limit by above data it is recognised that the rate of recovery of this method is preferable, and in comparative examples is higher, molten
Liquid concentration is 1 × 10-7When mol/L, it can not detect.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (6)
1. a kind of method using SERS detection 5-ALA, which comprises the steps of:
(1) pretreatment of copper sheet；
(2) nano silver is plated on copper sheet；
(3) be grafted sulfhydryl compound: the plating nano silver metal copper sheet of preparation is 1.0 × 10 in concentration-3mol/L-1.0×10- 2It is impregnated 3-12 hours in the ethanol solution of the sulfhydryl compound of mol/L, after taking-up, then with ethyl alcohol flushing to get detection substrate
Material；
(4) preparation of nano Au colloid: by HAuCl4Solution is heated to boiling, and citric acid three sodium solution is added, and continues to keep boiling
It after 15min, is centrifuged after natural cooling, takes lower layer's golden yellow precipitate, the nano Au colloid as prepared；
(5) detection of standard curve: weighing 5-ALA, prepares the 5-ALA mark of 5-6 concentration gradient
Quasi- solution example；The nanometer that the 5-ALA standard solution of various concentration gradient and isometric step (4) are obtained
Gold size uniformly mixes, centrifuge separation, collects sediment fraction, evenly laid out on the base material of step (3) preparation, and utilizes and swash
Light Confocal laser-scanning microscopy instrument is tested, and the SERS spectra figure of 5-ALA is obtained；
The nano Au colloid for taking step (4) to prepare individually carries out Raman spectrum test, obtains nano Au colloid background Raman signal；
Use nano Au colloid background Raman signal as internal standard to 5-ALA standard solution Surface enhanced Raman spectroscopy into
5-ALA Surface enhanced Raman spectroscopy relative line intensity-concentration standard control work is established in the normalization of row intensity
Curve graph；
(6) unknown sample concentration is detected
The 5-ALA solution to be determined of unknown concentration and the nano Au colloid of isometric step (4) acquisition is uniform
Mixing, is then tested using laser Raman spectrometer, and carry out spectral strength normalized and obtain the 5- of unknown concentration
Amino-laevulic acid Surface enhanced Raman spectroscopy spectrogram；The concentration of 5-ALA is calculated by formula.
2. a kind of method using SERS detection 5-ALA according to claim 1, which is characterized in that described
The specific steps of the preparation of step (4) nano Au colloid are as follows: accurately pipetting 50mL concentration is 1.0 × 10-4g/mL-1.0×10-3g/
The HAuCl of mL4Solution is placed in heat collecting type constant temperature blender with magnetic force in the beaker of 200mL, sets reaction temperature as 100 DEG C；
It is heated to boiling under strong stirring, the citric acid three sodium solution that 1.5mL-6 mL mass fraction is 1% is added, keep boiling
15min is centrifuged after natural cooling, takes lower layer's golden yellow precipitate, the nano Au colloid as prepared.
3. a kind of method using SERS detection 5-ALA according to claim 1, it is characterised in that: described
The concentration changing value of the 5-ALA standard solution sample of 5-7 concentration gradient is 10-4-10-10Between mol/L.
4. a kind of method using SERS detection 5-ALA according to claim 1, it is characterised in that: step
(3) sulfhydryl compound described in is one of n-butyl mercaptan, n- dodecyl mereaptan, positive stearylmercaptan or two kinds of mixtures.
5. a kind of method using SERS detection 5-ALA according to claim 1, which is characterized in that described
The pretreatment specific steps of step (1) copper sheet are as follows: copper sheet is cut into the small copper sheet of 2cm × 5cm rectangle, be successively immersed in acetone,
Copper sheet, is finally immersed in the HNO of 0.1 mol/L by each ultrasonic 10 min in ethyl alcohol and ultrapure water3Inside remove the oxidation on surface
Film；The copper sheet ultrapure water that pretreatment finishes is clean, and room temperature is dried.
6. a kind of method using SERS detection 5-ALA according to claim 1, which is characterized in that described
The specific steps of nano silver are plated on step (2) copper sheet are as follows: the SnCl of copper sheet after handling step (1) well in 0.02 mol/L2
1min, drying at room temperature are impregnated in the hydrochloric acid mixed solution of 0.02 mol/L；Secondly, by copper sheet 0.01 mol/L AgNO3
With mass fraction be 0.2% polyvinylpyrrolidone mixed solution in impregnate 3min, drying at room temperature；Repeat above step four
Period, finally by copper sheet in SnCl23min is impregnated in solution, uses ultrapure water.
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