CN100523789C - Complexes used for detecting the reaction for cleaning free radical with anti-oxidants and detection method there of - Google Patents
Complexes used for detecting the reaction for cleaning free radical with anti-oxidants and detection method there of Download PDFInfo
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- CN100523789C CN100523789C CNB2006100654398A CN200610065439A CN100523789C CN 100523789 C CN100523789 C CN 100523789C CN B2006100654398 A CNB2006100654398 A CN B2006100654398A CN 200610065439 A CN200610065439 A CN 200610065439A CN 100523789 C CN100523789 C CN 100523789C
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Abstract
This invention relates to one compound and its test method to test anti-oxygen to clear free radical, wherein, the formula is as cathode ion conjugate polymer and anode ion free radical quencher for mole proportion as 1:0.01-200 to form the said anode ion free radical quencher as 4-trimethy lammonium iodide-2, 2, 6, 6-tetramethylpiperidine-1- peroxy radical. The invention method comprises the following steps: testing the phosphoric acid buffer liquid cathode ion polymer fluorescence intensity; adding the anode ion free radical quencher to test the intensity; adding the anti-oxygen gas to test match intensity change.
Description
Technical field
The present invention relates to a kind of complex that is used to detect the reaction of antioxidant for clearing free radical, and use this complex to detect the method for the reaction of antioxidant for clearing free radical.
Background technology
In recent years, to polymeric material, the research of the free radical in food and the living things system more and more was subjected to people's attention.Free radical can make polymkeric substance and food degraded, makes DNA, protein and lipid etc. be subjected to oxidisability and destroys, thereby make biosome produce a series of diseases such as cancer.Usually, adopt antioxidant as free radical scavenger, the mechanism of utilizing hydrogen transfer or redox reaction to trap free radical is removed free radical, thereby reaches the purpose that stops the free radical reaction with harmfulness.
When using the reaction of antioxidant for clearing free radical, reaction is monitored in the variation of the fluorescence intensity of the fluorophore that the variation of the ESR signal of use detection free radical, or detection usually links to each other with the free radical covalency.
Because free radical has feature ESR signal, after the adding antioxidant is with radicals scavenging, the intensity and the shape of the ESR signal of free radical just change, so the variation of ESR signal that can be by detecting free radical detects free radical reaction, it is very big that but this ESR analyzes the sample size that needs, and insensitive.
Because free radical is covalently bound on fluorophore, with the complete cancellation of the fluorescence of fluorophore; When add antioxidant with radicals scavenging after, the fluorescence of fluorophore will recover gradually, so the variation of fluorescence intensity that can be by detecting the fluorophore that links to each other with the free radical covalency detects the cleaning reaction of free radical.But this method need be sought a suitable synthetic method, fluorophore and nitroxyl free radical are combined, sometimes this synthetic relatively difficulty, synthesis condition is relatively harsher, and the sample after fluorophore that obtains and the nitroxyl free radical combination is difficult to purifying and preservation.
Summary of the invention
The objective of the invention is to overcome prior art when detecting the reaction of antioxidant for clearing free radical, the sample size that the method for the variation of the ESR signal of use detection free radical needs is very big, and insensitive; Use the method for the variation of the fluorescence intensity that detects the fluorophore that links to each other with the free radical covalency to be difficult to synthetic fluorophore and nitroxyl free radical bond, and this bond is difficult to the defective of purifying and preservation, thereby provide a kind of can be sensitive, simple and be used to detect the complex of the reaction of antioxidant for clearing free radical and detection method thereof reliably.
The objective of the invention is to realize by the following technical solutions:
The complex that is used to detect the reaction of antioxidant for clearing free radical provided by the invention, it forms complex with the mol ratio of 1:0.01～200 by electrostatic interaction for negative ion conjugated polymer and radical cation quencher,
The structural formula of described negative ion conjugated polymer is suc as formula shown in the I
m＝1～10，n＝20～100
R
1，R
2，R
3，R
4＝-C
xH
2x+1，-O(C
xH
2x+1)，x＝1～1.0
R
5＝-COONa，-PO
3Na，-SO
3 -Na
+
Formula I
Described radical cation quencher is a 4-trimethyl ammonium iodide-2,2,6,6-tetramethyl piperidine-1-oxygen radical (hereinafter to be referred as CAT1), and its structural formula is suc as formula shown in the II
Formula II
The complex that is used to detect the reaction of antioxidant for clearing free radical provided by the invention is a kind of fluorescence probe of radicals scavenging, and the radical cation quencher in the complex is the fluorescence of cancellation negative ion conjugated polymer effectively.With negative ion conjugated polymer PFP-SO
3 -(poly-{ 2,7-[9,9-two (4 '-phenoxy group butyl sulfonic acid sodium salt)] fluorenes-altogether-(1,4-benzene) }) and radical cation quencher CAT1 are example with the complex of mol ratio 1:1 by electrostatic interaction formation, the Stern-Volmer cancellation constant K of this reaction
SvBe 2.3 * 10
7M
-1, the kinetic rate constant is 8.85 * 10
16M
-1s
-1, show that this is a cancellation process based on static cancellation, be to form the right result of electric charge between negative ion conjugated polymer and the radical cation.When adding other antioxidant, by hydrogen transfer or oxidation-reduction process, the paramagnetism nitroxyl free radical of radical cation quencher CAT1 has changed into diamagnetic hydramine, thus negative ion conjugated polymer PFP-SO
3 -Fluorescence owing to not being restored by cancellation.By detecting negative ion conjugated polymer PFP-SO
3 -The fluorescence light signal, the hydrogen transfer reaction process of detection that can be quantitative from the antioxidant to the free radical, and free radical is by the redox process of antioxidant, ground that promptly can be quantitative detects the oxidation resistance of antioxidant.
The method of using the complex that is used to detect the reaction of antioxidant for clearing free radical provided by the invention to detect the reaction of antioxidant for clearing free radical is specially:
1) be in the 5mM phosphate buffer in the 2970uL ionic strength, add 3uL concentration and be the negative ion conjugated polymer of the complex of the 1mM reaction that is used for detecting the antioxidant for clearing free radical of the present invention, measure the fluorescence intensity of (1uM) negative ion conjugated polymer under this concentration conditions;
2) add the radical cation quencher of the complex of the reaction that is used for detecting the antioxidant for clearing free radical of the present invention then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:0.01～200; Detect the fluorescence intensity of formed complex;
3) add antioxidant to be measured, mix the back room temperature and leave standstill, on luminoscope, detect the fluorescence intensity of complex this moment; The variation of complex fluorescent intensity can obtain in this complex the added value of the complex fluorescent intensity under this antioxidant concentration condition before and after relatively antioxidant added then.
Use above-mentioned detection method can the antioxidant such as the ascorbic acid with superpower oxidation resistance be detected, its detectability is 5nM, based on this, the complex that is used to detect the reaction of antioxidant for clearing free radical provided by the invention can be used as the ascorbic acid fluorescent optical sensor of a high sensitivity and high selectivity.
Compare with some micromolecular compounds, the electronic structure of negative ion conjugated polymer provided by the invention is made up of many absorptive units, when fluorescence excitation is to quencher, can produce the fluorescent quenching effect of amplifying, promptly this conjugated polymer optical signalling is extremely sensitive; In addition, owing to can use radicals scavenging reversibly to regulate and control the fluorescent quenching of negative ion conjugated polymer, thereby make to use the detection technique of fluorescence of this conjugated polymer have simplicity and high sensitivity, can survey the process that hydrogen transfer process from the antioxidant to the free radical and free radical are reduced by antioxidant.
With negative ion conjugated polymer PFP-SO
3 -With the complex of radical cation quencher CAT1 (mol ratio 1:1) be example, research the present invention use radical cation quencher CAT1 to negative ion conjugated polymer PFP-SO
3 -Carry out the ability of cancellation, by containing 1.0 * 10
-6The PFP-SO of M (calculating) by single repetitive (RUs)
3 -Phosphate buffer solution (5mM, pH 7.4) in add continuously CAT1, detect negative ion conjugated polymer PFP-SO then
3 -By the fluorescence emission spectrum after the cancellation (excitation wavelength is 376nm), as shown in Figure 1, along with the concentration of CAT1 increases to 2.0 * 10 from 0
-6M, PFP-SO
3 -Reduced to 94 by the fluorescence excitation intensity after the cancellation from 695, can calculate the Stem-Volmer constant K according to formula (1) at last
Sv
I
0/I＝1+K
sv[Q] (1)
In the formula (1): I
0Represent that there is PFP-SO down in no quencher
3 -Fluorescence excitation intensity, I represents to have quencher to have PFP-SO down
3 -Fluorescence excitation intensity, the concentration of [Q] expression quencher CAT1.Under low concentration, the Stern-Volmer constant K
SvBe 2.3 * 10
7M
-1
According to formula (2), by PFP-SO
3 -K
SvCan calculate dynamics cancellation rate constants k with fluorescence lifetime τ
q
PFP-SO
3 -Fluorescence lifetime approximately be 0.26ns, thereby dynamics cancellation rate constant is 8.85 * 10
16M
-1s
-1, this value is the several magnitude of diffusion control cancellation constant, and therefore, in the method, static cancellation is main cancellation mechanism, and this also can be by PFP-SO
3 -/ CAT1 forms electric charge to proving.
Experimental result shows that CAT1 is 10
-7During M, cancellation efficient can reach 80%, and promptly under very low concentration, CAT1 is effective cancellation PFP-SO just
3 -Fluorescence, be a kind of very sensitive quencher.
Use to detect the variation of the ESR signal of free radical with prior art, or the method that the reaction of antioxidant for clearing free radical is monitored in the variation of the fluorescence intensity of the fluorophore that links to each other with the free radical covalency of detection is compared, the complex of the reaction that is used to detect the antioxidant for clearing free radical provided by the invention and the advantage of detection method thereof are: need not synthetic fluorophore and nitroxyl free radical bond, so very simple; This method is highly sensitive and reliable, can survey the process that hydrogen transfer process from the antioxidant to the free radical and free radical are reduced by antioxidant.
Description of drawings
Fig. 1 is for using radical cation quencher CAT1 to negative ion conjugated polymer PFP-SO
3 -Carry out the spectrogram of cancellation; Detection is containing 1.0 * 10
-6The PFP-SO of M (calculating) by single repetitive (RUs)
3 -Phosphate buffer solution (5mM, pH 7.4) in, add the CAT1 of variable concentrations, PFP-SO
3 -By the fluorescence emission spectrum after the cancellation (excitation wavelength is 376nm); Wherein, from top to bottom, 9 curves are represented respectively and are added 0,1.0 * 10
-8M, 2..0 * 10
-8M, 4.0 * 10
-8M, 1.0 * 10
-7M, 2.0 * 10
-7M, 7.0 * 10
-7M, 1.6 * 10
-6M, 3.5 * 10
-6The CAT1 of M concentration;
Fig. 2 is being for to have in the presence of oxygen and the no oxygen, and ascorbic acid (ascorbic acid) add with situation about not adding under, PFP-SO
3 -Excitation spectrum; All detections are carried out in phosphate buffer solution (5mM, pH 7.4), and excitation wavelength is 376nm.[PFP-SO
3 -]=1.0 * 10
-6M (calculating) by single repetitive (RUs), [CAT1]=5.0 * 10
-5M, [ascorbic acid]=2.0 * 10
-4M.
Fig. 3 removes the spectrogram of radical cation for using trolox; At no trolox with PFP-SO in the presence of the trolox arranged
3 -And PFP-SO
3 -The excitation spectrum of/CAT1; Excitation wavelength is 376nm, measures [PFP-SO in phosphate buffer solution (5mM, pH 7.4)
3 -]=1.0 * 10
-6M (calculating) by single repetitive (RUs), [CAT1]=5.0 * 10
-5M, [trolox]=1.0 * 10
-4M.
Embodiment:
Embodiment 1, preparation negative ion conjugated polymer PFP-SO
3 -Complex I with radical cation CAT1
In the 2970uL ionic strength is in the phosphate buffer of 5mM, and adding 3uL concentration is the PFP-SO of 1mm
3 -(can reference literature: Burrows, H.D.; Lobo, V.M.M.; Pina, J.; Ramos, M.L.; De Melo, J.S.; Valente, A.J.M.; Tapia, M.J.; Pradhan, S.; Scherf, U.Macromolecules 2004,37,7425-7427 is synthetic, or directly buys), adding 1.0uL concentration then is the radical cation quencher CAT1 of 30uM, can obtain the complex I of 1:0.01 mol ratio after mixing.
The present invention uses ultraviolet spectrometer (UVS) Jasco V-550 type, and fluorescence spectrophotometer is F-4500 type (Hitachi), is being excitation source with the xenon lamp; Used fluorescence pond is the polystyrene fluorescence pond of 3mL, excitation wavelength detects under the condition of 376nm, and (5mM, pH 7.4) all carried out in all experiments in phosphate buffer solution, institute's water is through Millipore filtering system purifying, and the fluorescence intensity that records this complex I is 497.
According to preparation method and the detection method of embodiment 1, prepare a series of complexs of the present invention.Its negative ion conjugated polymer, and and the mol ratio of radical cation quencher, the fluorescence intensity of complex is listed in table 1.
The composition of table 1, complex of the present invention and fluorescence intensity
Complex among embodiment 13, the use embodiment 1 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 1
3 -, measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 695;
2) add radical cation quencher CAT1 among the embodiment 1 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:0.01; Detect the fluorescence intensity 497 (second curve from top to bottom as shown in Figure 1) of formed complex;
3) add 10uM ascorbic acid (antioxidant) 1.5uL to be measured, mix the back room temperature and left standstill 10 minutes, on luminoscope, detect the fluorescence intensity 530 of complex this moment; The variation of the fluorescence intensity of complex can obtain before and after relatively ascorbic acid added, and in this complex, concentration is that the ascorbic acid of 5nM makes the fluorescence intensity of complex increase by 33.
Complex among embodiment 14, the use embodiment 1 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 1
3 -, measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 695;
2) add radical cation quencher CAT1 among the embodiment 1 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:50; Detect the fluorescence intensity 239 of formed complex;
3) adding concentration to be measured is 0.1M ascorbic acid (antioxidant) 6uL, mixes the back room temperature and leaves standstill 10 minutes, and the fluorescence intensity that detects complex this moment on luminoscope was 1570 (as shown in Figure 2); The variation of the fluorescence intensity of complex can obtain before and after relatively ascorbic acid added, and in this complex, concentration is that the ascorbic acid of 0.2mM makes the fluorescence intensity of complex increase by 1331.
Complex among embodiment 15, the use embodiment 2 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 2
3 -, measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 695;
2) add radical cation quencher CAT1 among the embodiment 2 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:50; The fluorescence intensity that detects formed complex is 239;
3) adding concentration to be measured is that 0.02M trolox (antioxidant) volume is 15uL, mixing the back room temperature left standstill 35 minutes, the fluorescence intensity that detects complex this moment on luminoscope is respectively 812, the variation of the fluorescence intensity of complex before and after relatively trolox adds, can obtain, in this complex, concentration is that the trolox of 0.1mM makes the fluorescence intensity of complex increase by 573.(as shown in Figure 3)
Complex among embodiment 16, the use embodiment 3 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 3
3 -, measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 695;
2) add radical cation quencher CAT1 among the embodiment 3 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:200; The fluorescence intensity that detects formed complex is 65;
3) adding concentration to be measured is that 1mM halfcystine (antioxidant) volume is 15uL, mixes the back room temperature and leaves standstill 35 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 90.The variation of the fluorescence intensity of complex can obtain before and after relatively halfcystine added, and in this complex, concentration is that the halfcystine of 5uM makes the fluorescence intensity of complex increase by 25.
Complex among embodiment 17, the use embodiment 4 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 4
3 --II (poly-2,7-[4-methyl-5-ethoxy-9,9-two (4 '-phenoxy group nonyl sulfonate sodium)] fluorenes-altogether-[1,4-(2-octyl group-5-penta alkoxy) benzene] }), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 675;
2) add radical cation quencher CAT1 among the embodiment 4 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:100; The fluorescence intensity that detects formed complex is 97;
3) adding concentration to be measured is that 0.01M halfcystine (antioxidant) volume is 21uL, mixes the back room temperature and leaves standstill 35 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 150.The variation of the fluorescence intensity of complex can obtain before and after relatively halfcystine added, and in this complex, concentration is that the halfcystine of 70uM makes the fluorescence intensity of complex increase by 53.
Complex among embodiment 18, the use embodiment 5 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 5
3 --III (poly-[2,7-(4-methoxyl-9,9-two (4 '-phenoxy group hexyl sulfonate sodiums)] fluorenes-altogether-[1,4-(2-ethyl-5-alkoxy in the ninth of the ten Heavenly Stems) benzene] }), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 730;
2) add radical cation quencher CAT1 among the embodiment 5 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:150; The fluorescence intensity that detects formed complex is 75;
3) adding concentration to be measured is that 1mM glutathione (antioxidant) volume is 15uL, mixes the back room temperature and leaves standstill 35 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 165.The variation of the fluorescence intensity of complex can obtain before and after relatively paddy Guang peptide added, and in this complex, concentration is that the glutathione of 5uM makes the fluorescence intensity of complex increase by 90.
Complex among embodiment 19, the use embodiment 6 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-SO in the complex that 3uL concentration is 1mM embodiment 6
3 --IV (poly-[2,7-(4-propoxyl group-5-methoxyl-9,9-two (4 '-phenoxy group heptyl sulfonate sodiums)] fluorenes-(1,4-benzene) altogether }), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 715;
2) add the radical cation quencher CAT1 that implements in 6 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:70; The fluorescence intensity that detects formed complex is 145;
3) adding concentration to be measured is that 1mM glutathione (antioxidant) volume is 3uL, mixes the back room temperature and leaves standstill 35 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 247.The variation of the fluorescence intensity of complex can obtain before and after relatively glutathione added, and in this complex, concentration is that the glutathione of 1uM makes the fluorescence intensity of complex increase by 102.
Complex among embodiment 20, the use embodiment 7 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-PO in the complex that 3uL concentration is 1mM embodiment 7
3 --I (poly-[2,7-(5-oxygen in heptan base-9,9-two (4 '-phenoxy group ethyl phosphonic acid sodium salts)] fluorenes-[1,4-(2-propoxyl group-5-methyl) benzene] altogether }), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 670;
2) add the radical cation quencher CAT1 that implements in 7 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1: 20; The fluorescence intensity that detects formed complex is 350;
3) adding concentration to be measured is that 10mM uric acid (antioxidant) volume is 21uL, mixes the back room temperature and leaves standstill 35 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 460.The variation of the fluorescence intensity of complex can obtain before and after relatively uric acid added, and in this complex, concentration is that the uric acid of 70uM makes the fluorescence intensity of complex increase by 110.
Complex among embodiment 21, the use embodiment 8 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-PO in the complex that 3uL concentration is 1mM embodiment 8
3 --II (poly-[2,7-(4-hexyl-5-ethyl-9,9-two (4 '-the phenoxymethyl sodium ascorbyl phosphate)] fluorenes-[1,4-(2-methyl) benzene] altogether), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 680;
2) add the radical cation quencher CAT1 that implements in 8 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:150; The fluorescence intensity that detects formed complex is 78;
3) adding concentration to be measured is that 10mM glucose (antioxidant) volume is 15uL, mixes the back room temperature and leaves standstill 35 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 95.The variation of the fluorescence intensity of complex can obtain before and after relatively glucose added, and in this complex, concentration is that the glucose of 50uM makes the fluorescence intensity of complex increase by 17.
Complex among embodiment 22, the use embodiment 9 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-PO in the complex that 3uL concentration is 1mM embodiment 9
3 --III (poly-[2,7-(9,9-two (4 '-phenoxy group heptyl sodium ascorbyl phosphate)] fluorenes-[1,4-(2, the 5-dimethoxy) benzene] altogether), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 720;
2) add the radical cation quencher CAT1 that implements in 9 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:50; The fluorescence intensity that detects formed complex is 245;
3) adding concentration to be measured is that 1mM ascorbic acid (antioxidant) volume is 15uL, mixes the back room temperature and leaves standstill 10 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 441.The variation of the fluorescence intensity of complex can obtain before and after relatively ascorbic acid added, and in this complex, concentration is that the ascorbic acid of 5uM makes the fluorescence intensity of complex increase by 196.
Complex among embodiment 23, the use embodiment 10 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-CO in the complex that 3uL concentration is 1mM embodiment 10
3 --I (poly-[2,7-(4-propyl group-5-ethoxy-9,9-two (4 '-phenoxy group undecane-carboxylic acid sodium salt)] fluorenes-[1,4-(2-propoxyl group-5-methoxyl) benzene] altogether), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 650;
2) add radical cation quencher CAT1 among the embodiment 10 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:1; The fluorescence intensity that detects formed complex is 420;
3) adding concentration to be measured is that 0.01M trolox (antioxidant) volume is 3uL, mixes the back room temperature and leaves standstill 10 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 720.The variation of the fluorescence intensity of complex can obtain before and after relatively trolox added, and in this complex, concentration is that the trolox of 10uM makes the fluorescence intensity of complex increase by 300.
Complex among embodiment 24, the use embodiment 11 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-CO in the complex that 3uL concentration is 1mM embodiment 11
3 --II (poly-[2,7-(5-Kui oxygen base-9,9-two (4 '-phenoxy group penta carboxylic acid sodium salt)] fluorenes-[1,4-(2-methoxyl-5-oxygen in heptan base) benzene] altogether), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 630;
2) add radical cation quencher CAT1 among the embodiment 11 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:70; The fluorescence intensity that detects formed complex is 150;
3) adding concentration to be measured is that 0.1M ascorbic acid (antioxidant) volume is 6uL, mixes the back room temperature and leaves standstill 10 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 1350.The variation of the fluorescence intensity of complex can obtain before and after relatively ascorbic acid added, and in this complex, concentration is that the ascorbic acid of 0.2mM makes the fluorescence intensity of complex increase by 1200.
Complex among embodiment 25, the use embodiment 12 detects the reaction of antioxidant for clearing free radical
1) is in the 5mM phosphate buffer in the 2970uL ionic strength, adds the negative ion conjugated polymer PFP-CO in the complex that 3uL concentration is 1mM embodiment 12
3 --III (poly-[2,7-(9,9-two (4 '-phenoxy group hexyl carboxylic acid sodium salt)] fluorenes-(1,4-benzene) altogether), measure (1uM) PFP-SO under this concentration conditions
3 -Fluorescence intensity be 630;
2) add radical cation quencher CAT1 among the embodiment 12 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:30; The fluorescence intensity that detects formed complex is 370;
3) adding concentration to be measured is that 0.01M ascorbic acid (antioxidant) volume is 18uL, mixes the back room temperature and leaves standstill 10 minutes, and the fluorescence intensity that detects complex this moment on luminoscope is 1120.The variation of the fluorescence intensity of complex can obtain before and after relatively ascorbic acid added, and in this complex, concentration is that the ascorbic acid of 60uM makes the fluorescence intensity of complex increase by 850.
Claims (2)
1, a kind of complex that is used to detect the reaction of antioxidant for clearing free radical, it forms complex with the mol ratio of 1:0.01～200 by electrostatic interaction for negative ion conjugated polymer and radical cation quencher,
The structural formula of described negative ion conjugated polymer is suc as formula shown in the I
Formula I
Described radical cation quencher is a 4-trimethyl ammonium iodide-2,2,6,6-tetramethyl piperidine-1-oxygen radical.
2, a kind of method of using the described complex that is used to detect the reaction of antioxidant for clearing free radical of claim 1 to detect the reaction of antioxidant for clearing free radical comprises following step:
1) be in the 5mM phosphate buffer in the 2970uL ionic strength, add 3uL concentration and be the negative ion conjugated polymer of the complex of the described reaction that is used for detecting the antioxidant for clearing free radical of 1mM claim 1, measure the fluorescence intensity of negative ion conjugated polymer under this concentration conditions;
2) add the radical cation quencher of the complex of the described reaction that is used for detecting the antioxidant for clearing free radical of claim 1 then, the negative ion conjugated polymer of adding and the mol ratio of radical cation quencher are 1:0.01～200; Detect the fluorescence intensity of formed complex;
3) add antioxidant to be measured, mix the back room temperature and leave standstill, on luminoscope, detect the fluorescence intensity of complex this moment; The variation of complex fluorescent intensity can obtain in this complex the added value of the complex fluorescent intensity under this antioxidant concentration condition before and after relatively antioxidant added then.
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| CN106501228B (en) * | 2016-10-31 | 2020-06-26 | 华中科技大学 | Chromatographic imaging method |
| CN108088827A (en) * | 2017-12-15 | 2018-05-29 | 中国石油大学(华东) | It is a kind of using carbon quantum dot as the phenol antioxidant content assaying method of fluorescence indicator |
| CN114018887A (en) * | 2021-11-05 | 2022-02-08 | 国网安徽省电力有限公司电力科学研究院 | Method for rapidly detecting antioxidant content in transformer insulating oil |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5354511A (en) * | 1992-11-27 | 1994-10-11 | Alliedsignal Inc. | Unsymmetrically substituted fluorenes for non-linear optical applications |
| JP2005162660A (en) * | 2003-12-02 | 2005-06-23 | Tosoh Corp | Method for producing arylamine derivative having fluorene skeleton and its synthetic intermediate |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5354511A (en) * | 1992-11-27 | 1994-10-11 | Alliedsignal Inc. | Unsymmetrically substituted fluorenes for non-linear optical applications |
| JP2005162660A (en) * | 2003-12-02 | 2005-06-23 | Tosoh Corp | Method for producing arylamine derivative having fluorene skeleton and its synthetic intermediate |
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