CN106749034A - Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker - Google Patents

Ratio-type fluorescent labeling reagent and its synthetic method and application are answered to bisulfite and hypochlorite double-bang firecracker Download PDF

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CN106749034A
CN106749034A CN201611069757.1A CN201611069757A CN106749034A CN 106749034 A CN106749034 A CN 106749034A CN 201611069757 A CN201611069757 A CN 201611069757A CN 106749034 A CN106749034 A CN 106749034A
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concentration
hso
clo
acetonitrile
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尤进茂
窦昆
纪仲胤
孙志伟
李国梁
吕政贤
路帅敏
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Dongying Guangli Lingang Industrial Park Co ltd
Dongying Guangli Port Park Operation Co ltd
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Qufu Normal University
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Abstract

The present invention relates to fluorescent labeling reagent, and in particular to a kind of to answer Ratio-type fluorescent labeling reagent and its synthetic method and application to bisulfite and hypochlorite double-bang firecracker.Described fluorescent labeling reagent, with phenanthro- imidazoles as fluorophor, is reaction active groups with the C=C double bonds for activating, and its chemical name is 2 (4 (base of 1 methyl 1H phenanthro- [9,10d] imidazoles 2) benzylidene) malononitrile.Its synthetic method is to obtain 4 benzimidazole benzaldehydes by phenanthrenequione, to two benzaldehydes, ammonium acetate and acetic acid back flow reaction, 4 tolimidazole benzaldehydes are obtained again, finally 2 (4 (the base benzylidene of 1 methyl 1H phenanthro- [9,10d] imidazoles 2) malononitrile are obtained with malononitrile reaction.The mark of fluorescent labeling reagent of the invention is swift in response, selectivity high, test limit is extremely low, simple synthetic method, has further promoted what biological micromolecule acted in life entity to probe into.

Description

Ratio-type fluorescent labeling reagent and its conjunction are answered to bisulfite and hypochlorite double-bang firecracker Into methods and applications
Technical field
The present invention relates to fluorescent labeling reagent, and in particular to a kind of to answer Ratio-type to bisulfite and hypochlorite double-bang firecracker Fluorescent labeling reagent and its synthetic method and application.
Background technology
Nearly ten years, some biological micromolecules adjust organismic internal environment because of wide participation life vivo oxidation reduction reaction Balance and be potentially applied to clinical medicine and receive much concern.Among these, redox equilibrium active oxygen (ROS) and reduction Sulfur species (RSS) play the part of pivotal player, such as cell propagation, differentiation and apoptosis in bioprocess is adjusted, and wherein representational Material surely belongs to HSO3 -And ClO-.Although in recent years, people are in the response path of these biological micromolecules of extensive discussions, physiology work( The various performances such as energy, but, the effect that these materials are played the part of in life system still has many unknown aspects.Therefore, very It is necessary that set up a kind of instant detection method completes to distinguish and detect to these biological micromolecules.
Up to the present, High Performance Liquid Chromatography/Mass Spectrometry, electrochemical analysis, capillary electrophoresis analysis and with organic probes It is used for detecting that biological micromolecule is widely used with the analytic approach such as the fluorescence based on nano material, Raman.In these analysis sides In method, fluorescence probe has super high sensitivity, detects and be applied to the unique advantages such as living organism system immediately.Additionally, phase Compare switching mode fluorescence organic probes, Ratiometric fluorescent probe greatly reduces by the shadow from external environment, instrument and equipment etc. Ring.
Most of all, current also can be used to detect HSO simultaneously without a kind of Ratiometric fluorescent probe3 -And ClO-, therefore, Double-bang firecracker proposed by the invention answers fluorescent molecular probe for further appreciating that HSO3 -And ClO-The mechanism of action in body has Far reaching significance.
The content of the invention
The purpose of the present invention be a kind of mark be swift in response, to HSO3 -And ClO-Selectivity is high, test limit is extremely low, synthesis side Method it is easy Ratio-type fluorescent labeling reagent is answered to bisulfite and hypochlorite double-bang firecracker；Present invention simultaneously provides its synthesis side Method and application.
It is of the present invention that Ratio-type fluorescent labeling reagent is answered to bisulfite and hypochlorite double-bang firecracker, it is with phenanthro- miaow Azoles is fluorophor, is reaction active groups with the C=C double bonds for activating, and its chemical name is：2- (4- (1- methyl isophthalic acid H phenanthro-s [9,10d] imidazoles -2 base)-benzylidene) malononitrile, its chemical structural formula is：
The described synthetic method for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent, including with Lower step：
(1) by phenanthrenequione, to two benzaldehydes it is added in flask, adds ammonium acetate and acetic acid, is heated to reflux being reacted, Reaction terminates, and treats that solution is cooled to room temperature, and suction filtration washs the solid for obtaining with acetic acid, and the filtrate that suction filtration is obtained is added to frozen water In, stirring obtains yellow solid, and yellow solid is merged with the solid after acetic acid washing, obtains intermediate product 4- benzimidazolyls Benzaldehyde；
(2) the intermediate product 4- benzimidazole benzaldehydes anhydrous acetonitrile dissolving for obtaining step (1), adds potassium carbonate It is heated to reflux with potassium hydroxide, after being down to room temperature, adds the anhydrous acetonitrile of iodomethane, temperature rising reflux reaction, cooling, mistake Filter, intermediate product 4- methyl-benzoimidazole benzaldehydes are obtained after filtrate is collected through rotary distillation, purifying；
(3) the intermediate product 4- methyl-benzoimidazole benzaldehydes that will be obtained are dissolved in anhydrous pyridine, are heated up and are stirred, Malononitrile reaction is added, after the completion of reaction, cooling, filtering obtains red colored crystalline thing, i.e. target product 2- (4- (1- methyl isophthalic acids H Phenanthro- [9,10d] imidazoles -2 base-benzylidene) malononitrile (MPIBA).
Wherein：
Phenanthrenequione, it is 1 to the mol ratio of two benzaldehydes, ammonium acetate, acetic acid in step (1):3:7:10.
The time of back flow reaction is 50 minutes in step (1).
In step (2), the mol ratio of potassium carbonate, potassium hydroxide, iodomethane and intermediate product 4- benzimidazole benzaldehydes It is 1:1:1.2:1.
In step (2), add potassium carbonate and potassium hydroxide to be heated to reflux 30 minutes, after being down to room temperature, add iodomethane Anhydrous acetonitrile, temperature rising reflux reacts 30 minutes.
In step (3), malononitrile is 1.5 with the mol ratio of intermediate product 4- methyl-benzoimidazole benzaldehydes:1.
In step (3), it is warming up to 70 DEG C and stirs 10 minutes, adds malononitrile to react 1 hour.
The described application for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent is thin for detecting The exogenous HSO of born of the same parents3 -And ClO-Concentration, detection endogenous cellular HSO3 -And ClO-Concentration and fluorescence imaging.
Specifically include following steps：
First, the exogenous HSO of detection cell3 -And ClO-Concentration：
(1) pH=7.40 is prepared, concentration is the PBS buffer salt solutions of 10mM；Compound concentration is the HSO of 1mM3 -Acetonitrile mark Quasi- solution, concentration are the ClO of 1mM-Acetonitrile standard liquid and concentration for 0.05mM 2- (4- (1- methyl isophthalic acid H phenanthro-s [9, 10d] base of imidazoles -2)-benzylidene) and malononitrile acetonitrile solution；
(2) 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L, 25 μ L are taken respectively Concentration is the HSO of 1mM3 -And ClO-Totally 22 parts of acetonitrile standard liquid, be added separately in fluorescence cuvette, be separately added into 100 μ L concentration is the PBS buffer salt solutions of 10mM, and the 2- that 100 μ L concentration are 0.05mM is then separately added into again, and ((1- methyl isophthalic acids H is luxuriant and rich with fragrance for 4- And [9,10d] imidazoles -2 base)-benzylidene) malononitrile acetonitrile solution, be finally separately added into acetonitrile constant volume to 1mL, mixing is equal It is even；
Fluorescence intensity is tested by sepectrophotofluorometer, fluorescence intensity ratio is obtained, ClO is detected-Fluorescence intensity swash Hair wavelength is 440nm, detection HSO3 -The excitation wavelength of fluorescence intensity is respectively 330nm, 410nm；
(3) respectively with HSO3 -And ClO-Concentration be abscissa, with fluorescence intensity ratio as ordinate, obtain on cell Exogenous HSO3 -And ClO-The linear equation of concentration, fluorescence intensity ratio；
(4) by testing sample and acetonitrile by volume 1:After 4 mixing, 900mL is taken, and is added thereto to 100 μ L concentration and be The acetonitrile solution of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.05mM, according to outer Source property HSO3 -And ClO-Concentration, the linear equation of fluorescence intensity ratio obtain HSO3 -And ClO-Concentration；
2nd, for detecting living cells endogenous HSO3 -And ClO-Concentration：
(1) compound concentration is the HSO of 1mM3 -Acetonitrile standard liquid, concentration for 1mM ClO-Acetonitrile standard liquid, dense Spend the acetonitrile solution, concentration of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile for 0.5mM LPS solution, the SO that concentration is the PMA solution of 1 μ g/ml, concentration is 1mM for 1 μ g/ml2Donor solution；
(2) living cells Hela cells are inserted in culture medium and is cultivated, be divided into and do not cultivate 10 groups, inoculum concentration in every group of culture medium It is 2 × 107~9 × 107Individual/mL, cultivates 24h, is first separately added into 2- (4- (the 1- methyl isophthalic acid H phenanthro-s that 10 μ L concentration are 0.5mM [9,10d] imidazoles -2 base)-benzylidene) malononitrile acetonitrile solution, then be separately added into 0 μ L, 7.5 μ L, 15 μ L, 22.5 μ L, 30 μ L concentration is the HSO of 1mM3 -Acetonitrile standard liquid and ClO-Acetonitrile standard liquid, cultivate 15min altogether in 37 DEG C, insert altogether Lower observation imaging is focused on, the fluorescence intensity of different colours optical channel is collected, so as to carry out fluorescence intensity ratio, obtained on cell Endogenous HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation；
(3) living cells Hela cells are inserted in culture medium and is cultivated, be divided into and do not cultivate 4 groups, inoculum concentration in every group of culture medium It is 2 × 107~9 × 107Individual/mL, cultivates 24h；
Used to the PMA solution that 100 μ L concentration are the LPS solution of 1 μ g/ml, 100 μ L concentration are 1 μ g/ml is added in first group To stimulate cell to produce endogenic ClO-, add 2- (4- (1- methyl isophthalic acid H phenanthro-s [9,10d] that 10 μ L concentration are 0.5mM The base of imidazoles -2)-benzylidene) acetonitrile solution of malononitrile cultivated, is imaged, and second group is not added with LPS and PMA, only plus 10 μ L Concentration is made for the acetonitrile solution of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.5mM It is control；
To the SO for adding 100 μ L concentration to be 1mM in the 3rd group2Donor is used for stimulating cell to produce endogenic HSO3 -, then Add the second of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile that 10 μ L concentration are 0.5mM Nitrile solution is cultivated, imaging, and the 4th group is not added with SO2Donor, only adds 10 μ L concentration to be 2- (4- (the 1- methyl isophthalic acids H phenanthrene of 0.5mM And [9,10d] imidazoles -2 base)-benzylidene) malononitrile acetonitrile solution as control；
The burnt lower observation imaging of copolymerization is inserted, the fluorescence intensity of different colours optical channel is collected, so as to carry out fluorescence intensity ratio Value, according to endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation, so as to obtain endogenous cellular HSO3 -And ClO-Concentration.
Beneficial effects of the present invention are as follows：
(1) it with phenanthro- imidazoles is parent ring that Ratio-type fluorescent labeling reagent of the invention is, with the C=C double bonds for being activated It is reaction site, makes probe to HSO3 -And ClO-There is superior selectivity, and have obvious fluorescence signal and read.
(2) Ratio-type fluorescent labeling reagent response of the invention is sensitive, to ClO-Response time within the several seconds, to HSO3 - Response within 40s.
(3) Ratio-type fluorescent labeling reagent test limit of the invention is low, is compared to commercialized fluorescent labeling reagent, this Invention proposed to HSO3 -And ClO-Test limit be respectively 3.5nm/L and 7.5nm/L, well below intracellular both things The content that matter is present.
(4) Ratio-type fluorescent labeling reagent of the invention, the switching mode that compares fluorescent labeling reagent, with the ratio of fluorescence intensity On the basis of value, rather than direct fluorescence intensity, the influence from external environment and instrument etc. can be greatly reduced.
(5) present invention be so far the first for simultaneously distinguish and detection HSO3 -And ClO-Organic fluorescence probe.
(6) due to the change of ultra-violet colors after reaction, open hole detection is available for, it is convenient and swift.
(7) present invention is applied to the detection of living cells, the spy for further having promoted biological micromolecule to be acted in life entity Study carefully.
Brief description of the drawings
Fig. 1 is the synthetic route chart of MPIBA；
Fig. 2 is the mass spectrogram in embodiment 1；
Wherein：The mass spectrogram of A, MPIBA；B, MPIBA and ClO-The mass spectrogram of reaction；C, MPIBA and HSO3 -The matter of reaction Spectrogram；
Fig. 3 is the nuclear-magnetism H spectrums of MPIBA in embodiment 1；
Fig. 4 is the nuclear-magnetism C spectrums of MPIBA in embodiment 1；
Fig. 5 is MPIBA to HSO3 -Fluorescence intensity and Linear equations；
Fig. 6 is MPIBA to ClO-Fluorescence intensity and Linear equations；
Fig. 7 is the fluorogram of sample detection；
Wherein：HSO in A, detection rainwater3 -The fluorogram of concentration；ClO in B, detection running water-The fluorogram of concentration；
Fig. 8 is MPIBA to HSO3 -Chromatic graph is researched and analysed and compared to the selectivity of molecule；
Wherein：A、HSO3 -To metal ion；B、HSO3 -To other reduction class sulfur species of anion；
Fig. 9 is MPIBA to ClO-Chromatic graph is researched and analysed and compared to the selectivity of molecule；
Wherein：A、ClO-To metal ion；B、ClO-To other active oxygens of anion and active nitrogen；
Figure 10 is to probe into pH value to MPIBA and the influence figure of substance reaction effect；
Wherein：A、HSO3 -；B、ClO-；
Figure 11 is the response time figure for probing into MPIBA to material；
Wherein：A、HSO3 -；B、ClO-；
Figure 12 is intracellular Fluorescence Linear equations；
Wherein：A、HSO3 -；B、ClO-；
Figure 13 is detection endogenous relevant cell figure；
Wherein：A、HSO3 -；B、ClO-；
Figure 14 is cell survival rate block diagram.
Specific embodiment
The present invention is described further with reference to embodiments.
Embodiment 1
As shown in figure 1, of the invention to HSO3 -And ClO-Double-bang firecracker answers the Ratio-type fluorescent labeling reagent synthesis step to have 3 steps, With phenanthrenequione and to two benzaldehydes as raw material, specific synthetic operation is as follows：
(1) synthesis of intermediate 4- benzimidazoles benzaldehyde：
600mg is added in the round-bottomed flask of 50ml to two benzaldehydes, the ammonium acetate of 315mg phenanthrenequione and 2.15g, Ran Houjia Enter the glacial acetic acid of 25ml, be heated with stirring to backflow 50 minutes, be cooled to room temperature, suction filtration obtains solid, is washed with glacial acetic acid and obtained Solid, the filtrate that suction filtration is obtained pour into frozen water stir, stirring obtain yellow solid, filter and merge above washing after Solid, obtains intermediate 4- benzimidazole benzaldehyde 395.5mg, and yield is 96%.
(2) synthesis of intermediate 4- methyl-benzoimidazoles benzaldehyde：
30ml anhydrous acetonitriles are added in 100ml round-bottomed flasks, 336mg intermediate 4- benzimidazolyl benzene first is added Aldehyde, 56mg KOH, 150mg K2CO3, be heated with stirring to backflow 30 minutes, be cooled to after room temperature added with constant pressure funnel it is molten There are 0.1ml CH3The acetonitrile solution of I, completion of dropping is warming up to reflux state and reacts 30 minutes, and cold filtration collects filtrate simultaneously Revolving, crude product is further purified, with (v n-hexanes：V ethyl acetate=10:1) be eluant, eluent, obtain greenish yellow solid 4- methyl- Benzimidazole benzaldehyde 280mg, yield 80.5%.
(3) target product 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile (MPIBA) Synthesis：
Take 176mg intermediate product 4- methyl-benzoimidazole benzaldehydes to be put into 25ml round-bottomed flasks, be dissolved in 10ml anhydrous In pyridine, it is warming up to 70 DEG C and stirs 10 minutes, add malononitrile 0.05ml to react again 1 hour, treats that slightly cooling is put into refrigerator, treats After solid is separated out, red needle-like solid is directly filtered to obtain, as target product 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles- 2 bases-benzylidene) malononitrile, 158mg, yield 78.3%.
Intermediate 4- benzimidazole benzaldehydes are characterized as below：
1H NMR(DMSO-d6,500MHz),δ(ppm):10.141 (s, 1H), 8.86 (d, J=8.5Hz, 2H), 8.62 (d, J=4.5Hz, 2H), 8.15 (q, J=8.5Hz, 4H), 7.79 (q, J=7.5Hz, 2H), 7.68 (d, J=7.0Hz, 2H),),2.953(s,N-H,1H).
13C NMR(DMSO-d6,500MHz),δ(ppm):(193.42,151.33,130.75,130.20,127.91, 127.71,126.97,126.22,125.98,124.94,124.10,122.35,121.97.MS m/z calcd for 322.37[M+H]+found 323.5.
Intermediate 4- methyl-benzoimidazole benzaldehydes are characterized as below：
1H NMR(DMSO-d6,500MHz),δ(ppm):4.52 (s, 3H) 7.67 (m, 2H), 7.77 (m, 2H), 8.14 (d, J=8.4Hz, 2H), 8.53 (d, J=8.4Hz, 2H), 8.57 (d, J=7.6Hz, 1H), 8.63 (d, J=7.6Hz, 1H), 8.86 (d, J=8.4Hz, 1H), 8.90 (d, J=8.4Hz, 1H), 10.10 (s, 1H)
13C NMR(DMSO-d6,500MHz),δ(ppm):(193.37,151.22,130.75,130.19,127.87, 127.68,126.16,125.92,124.98,124.14,123.53,122.35,122.01)MS m/z calcd for 336.1[M+H]+found 336.5.
(4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base-benzylidene) malononitrile is characterized as below target product 2-：
1H NMR(DMSO-d6,500MHz),δ(ppm):9.0 (d, J=8.5Hz, H), 8.89 (d, J=8.5Hz, 2H), 8.66 (d, J=7.5Hz, 2H), 8.62 (d, J=6.5Hz, 1H), 8.59 (d, J=5.5Hz, 2H), 7.80 (d, J=8.0Hz, 2H), 7.75 (d, J=3.0Hz, 2H), 7.68 (d, J=3.0Hz, H), 7.40 (d, J=3.0Hz, H), 4.37 (s, 3H, N- CH3)
13C NMR(DMSO-d6,500MHz),δ(ppm):(161.01,150.98,137.47,131.24,,130.74, 127.90,127.72,126.24,125.00,122.36,122.07,113.74,82.53,36.90.MS m/z calcd for 384.1[M+H]+found 384.8.
MPIBA is carried out into Mass Spectrometer Method, Fig. 2 is specifically shown in, wherein, A is the mass spectrogram of MPIBA, and B is MPIBA and ClO-Instead The mass spectrogram answered, C is MPIBA and HSO3 -The mass spectrogram of reaction.
MPIBA is carried out into magnetic resonance detection, Fig. 3-4 are specifically shown in, wherein, Fig. 3 is the nuclear-magnetism H spectrums of MPIBA, and Fig. 4 is The nuclear-magnetism C spectrums of MPIBA.
Embodiment 2
The exogenous HSO of detection cell3 -And ClO-Concentration：
(1) pH=7.40 is prepared, concentration is the PBS buffer salt solutions of 10mM；Compound concentration is the HSO of 1mM3 -Acetonitrile mark Quasi- solution, concentration are the ClO of 1mM-Acetonitrile standard liquid and concentration for 0.05mM 2- (4- (1- methyl isophthalic acid H phenanthro-s [9, 10d] base of imidazoles -2)-benzylidene) and malononitrile acetonitrile solution；
(2) 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L, 25 μ L are taken respectively Concentration is the HSO of 1mM3 -And ClO-Totally 22 parts of acetonitrile standard liquid, be added separately in fluorescence cuvette, be separately added into 100 μ L concentration is the PBS buffer salt solutions of 10mM, and the 2- that 100 μ L concentration are 0.05mM is then separately added into again, and ((1- methyl isophthalic acids H is luxuriant and rich with fragrance for 4- And [9,10d] imidazoles -2 base)-benzylidene) malononitrile acetonitrile solution, be finally separately added into acetonitrile constant volume to 1mL, mixing is equal It is even；
Fluorescence intensity is tested by sepectrophotofluorometer, fluorescence intensity ratio is obtained, ClO is detected-Fluorescence intensity swash Hair wavelength is 440nm, detection HSO3 -The excitation wavelength of fluorescence intensity is respectively 330nm, 410nm；
(3) respectively with HSO3 -And ClO-Concentration be abscissa, with fluorescence intensity ratio as ordinate, obtain on cell Exogenous HSO3 -And ClO-The linear equation of concentration, fluorescence intensity ratio；Fig. 5 is MPIBA to HSO3 -Fluorescence intensity and linear Graph of equation, wherein, A, B are the HSO of 0-22.5 μM of addition3 -Fluorogram, C is HSO3 -Fluorescence Linear equations.Fig. 6 is MPIBA To ClO-Fluorescence intensity and Linear equations；Wherein, A is the ClO of 0-22.5 μM of addition-Fluorogram, B is ClO-Photoluminescence line Property graph of equation.
(4) by testing sample and acetonitrile by volume 1:After 4 mixing, 900mL is taken, and is added thereto to 100 μ L concentration and be The acetonitrile solution of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.05mM, according to outer Source property HSO3 -And ClO-Concentration, the linear equation of fluorescence intensity ratio obtain HSO3 -And ClO-Concentration.
Embodiment 3
Using the HSO in MPIBA detection rainwater3 -With the ClO in running water-Content.
By rainwater sample to be measured and anhydrous acetonitrile by volume 1:4 mixing, take gained liquid 900mL, and be added thereto to 100 μ L concentration is the MPIBA of 0.05mM, and resulting solution preserves 1min, rain is calculated according to the fluorescence intensity of embodiment 2 at room temperature Contain HSO in water sample3 -Content, bring gained fluorescence intensity level (being obtained from Fig. 7 A) into photoluminescence line that Fig. 5 C are set up Property, institute's value is the HSO in 1.9mM, i.e. rainwater3 -Content is 10.5mM.
By originally water sample to be measured and anhydrous acetonitrile by volume 1:4 mixing, take gained liquid 900mL, and be added thereto to 100 μ L concentration are the MPIBA of 0.05mM, and resulting solution preserves 1min at room temperature, are judged originally according to the fluorescence intensity of embodiment 2 Whether contain ClO in water sample-, bring gained fluorescence intensity level (being obtained from Fig. 7 B) into fluorescence that Fig. 6 B are set up linear, Institute's value is the ClO in 2.3mM, i.e. running water-Content is 12.5mM.
Fig. 7 is the fluorogram of sample detection.Wherein, A is HSO in detection rainwater3 -The fluorogram of concentration, B is to detect originally ClO in water-The fluorogram of concentration.
Embodiment 4
To probe into MPIBA to HSO3 -Selectivity, some representational anion Cl-、Br-、I-、CH3COO-、ClO4 -、 SO4 2、H2PO4 -、S2O3 2-、SCN-, reduction sulfur species HS-, GSH, Cys, Hcy and metal ion Na+、K+、Mg2+、Fe3+、Cd2+、Co2 +、Ni2+、Hg2+、Al3+、Mn2+、Ag+、Cu2+、Zn2+Addition gives to be probed into.As seen from Figure 8, several times HSO3 -The ion of concentration It is added in MPIBA, the influence for causing can be ignored.Under 365nm uviol lamps, work as HSO3 -Add it can be found that red glimmering Light disappears, and blue-fluorescence occurs.In Fig. 8, A is that metal ion selectively influences on MPIBA, and bottle is under 365nm uviol lamps The phenomenon of interference solion is added, B is some RSS, and Common Anions select probe Journal of Sex Research, and bottle is corresponding addition Phenomenon of the interfering ion solution under 365nm uviol lamps.
Equally, in order to probe into MPIBA to ClO-Selectivity, except same metal ion, active oxygen (ROS), active nitrogen , including H (RNS)2O2、OH、TBHP、TBO-、KO2、ONOO-、NO2 -、NO3 -, NO is added in solution.From fig. 9, it can be seen that i.e. Make to be several times as much as ClO-The interfering ion of concentration is added, and the influence for causing is also negligible.Equally under the uviol lamp of 365nm, Work as ClO-Add it can be found that red fluorescence is changed into green fluorescence.In Fig. 9, A is that metal ion selectively influences on probe, bottle It is the phenomenon that interference solion is added under 365nm uviol lamps, B is that some ROS, RNS select MPIBA Journal of Sex Research, small Bottle is the corresponding phenomenon for adding interfering ion solution under 365nm uviol lamps.
In order to probe into influence of the pH value to ratio fluorescent, in the certain situation of the amount of the material for adding probe and detectable substance Under, it is reaction in the interval of 4.0-10.0 in pH to allow reaction, probes into optimal reaction environment.Fluorescence as shown by Figure 10 A Rate value maintains a higher level in pH is for the environment of 6.0-10.0, and this explanation is surveyed in neutral and weakly alkaline environment Amount is more excellent environment, this and HSO3 -Pka be 7.20 to match.Equally as shown in Figure 10 B, pH be 4.0-6.0 between, instead Answer expression activitiy low, when pH is in 6.0-10.0 environment, reactive ratio is significantly raised.
Figure 11 is the response time figure for probing into MPIBA to material, and wherein A is that MPIBA adds HSO3 -Response time figure, B For MPIBA adds ClO-Response time figure.
Embodiment 5
The HSO of endogenous cellular is detected using MPIBA3 -And ClO-Concentration：
(1) cell culture：This experimental selection Hela cells, the cell recovered is cultivated, and culture medium includes 10% Ox embryo serum, 1% dual anti-, 89%DMEM, in 37 DEG C, 5%CO2Environment in cultivate 24h, the cell for being grown fine is treated With, it is divided into and does not cultivate 14 groups, inoculum concentration is 2 × 10 in every group of culture medium7~9 × 107Individual/mL；
(2) endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation foundation：
1. compound concentration is the HSO of 1mM3 -Acetonitrile standard liquid, concentration for 1mM ClO-Acetonitrile standard liquid, dense It is that the LPS solution of 1 μ g/ml, concentration are that the PMA solution of 1 μ g/ml, concentration are to spend the acetonitrile solution of the MPIBA for 0.5mM, concentration The SO of 1mM2Donor solution；
2. 10 groups of culture mediums are taken, 2- (4- (1- methyl isophthalic acid H phenanthro-s [9,10d] that 10 μ L concentration are 0.5mM are first separately added into The base of imidazoles -2)-benzylidene) malononitrile acetonitrile solution, then be separately added into 0 μ L, 7.5 μ L, 15 μ L, 22.5 μ L, 30 μ L concentration It is the HSO of 1mM3 -Acetonitrile standard liquid and ClO-Acetonitrile standard liquid, cultivate 15min altogether in 37 DEG C, insert copolymerization Jiao under Observation imaging, collects the fluorescence intensity of different colours optical channel, so as to carry out fluorescence intensity ratio, obtains on endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation.
Figure 12 A are intracellular HSO3 -The linear side of concentration and ratio fluorescent (blue-fluorescence strength ratio red fluorescence intensity) Journey.Figure 12 B are intracellular ClO-The linear equation of concentration and fluorescence intensity ratio (green fluorescence intensity is than red fluorescence intensity).
(3) HSO that endogenous cellular is produced3 -And ClO-Content detection：
Take 4 groups of culture mediums, to added in first group 100 μ L concentration be the LPS solution of 1 μ g/ml, 100 μ L concentration be 1 μ g/ The PMA solution of ml is used for stimulating cell to produce endogenic ClO-, 10 μ L concentration are added for the acetonitrile of the MPIBA of 0.5mM is molten Liquid is cultivated, imaging, and second group is not added with LPS and PMA, only adds the acetonitrile solution of the MPIBA that 10 μ L concentration are 0.5mM as right According to；Figure 12 B are endogenous detection ClO-Relevant cell figure.
To the SO for adding 100 μ L concentration to be 1mM in the 3rd group2Donor is used for stimulating cell to produce endogenic HSO3 -, then Add the acetonitrile solution of the MPIBA that 10 μ L concentration are 0.5mM to be cultivated, be imaged, the 4th group is not added with SO2Donor, only adds 10 μ L Concentration is the acetonitrile solution of the MPIBA of 0.5mM as control；Figure 12 A are endogenous detection HSO3 -Relevant cell figure.
The burnt lower observation imaging of copolymerization is inserted, the fluorescence intensity of different colours optical channel is collected, so as to carry out fluorescence intensity ratio Value, according to endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation, so as to obtain endogenous cellular HSO3 -And ClO-Concentration.
Figure 13 A are endogenous HSO3 -Cytological map, in figure from left to right be red channel, blue channel, light field, overlap, Ratio, Figure 13 B are endogenous ClO-Cytological map, in figure from left to right be green channel, red channel, light field, overlap, than Rate.
Embodiment 6
Cell survival rate is tested：
Influence of the main checking MPIBA toxicity of experiment of cell survival rate to cell survival.Added not in cell culture fluid With the MPIBA probes (0M, 5M, 10M, 20M, 30M and 50M) of concentration, in 37 DEG C, 5%CO2Incubator in cultivate 24h, Then 4- methyl thiazolyl tetrazoliums MTT (5mg mL of 25 μ L-1) 4 hours of culture in cell culture fluid are added to.Result passes through MTT cuvettes methods assesses cell survival rate.It is 100% with that group of cell survival for being not added with MPIBA, various concentrations MPIBA adds The experimental group related data for entering, draws opposed cylinder Figure 14.
In the embodiment of the present invention efficient liquid phase-mass spectral analysis be using the mass spectrometer systems of Agilent 1100 (Agilent, USA), and degasser, quaternary pump, automatic sampler are equipped with, high performance liquid chromatography separation is by Hypersil GOLD C18 Post (2.1mm × 50mm, 1.8 μm of i.d., Agilent, USA) is completed.Fluoroscopic examination is using Hitachi Hitachi F-4600 XRF is carried out, to ClO-Detection excitation wavelength is 420nm, to HSO3 -Detection excite respectively 330 and 440nm, Excite and be 10.0nm, voltage 400V, the nm/min of sweep speed 2400 with transmite slit width.Fluorescence imaging observation is to pass through Olympus Fluo View FV1000 (Japan) copolymerization Jiao is carried out, and is observed with 40 times of object lens.Isolating and purifying for compound be Realized using thin-layer chromatography silicagel column, wherein, filler is 300-400 mesh.

Claims (10)

  1. It is 1. a kind of that Ratio-type fluorescent labeling reagent is answered to bisulfite and hypochlorite double-bang firecracker, it is characterised in that：With phenanthro- miaow Azoles is fluorophor, is reaction active groups with the C=C double bonds for activating, and its chemical name is：2- (4- (1- methyl isophthalic acid H phenanthro-s [9,10d] imidazoles -2 base)-benzylidene) malononitrile, its chemical structural formula is：
  2. 2. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent described in a kind of claim 1 Method, it is characterised in that comprise the following steps：
    (1) by phenanthrenequione, to two benzaldehydes it is added in flask, adds ammonium acetate and acetic acid, be heated to reflux being reacted, reacts Terminate, treat that solution is cooled to room temperature, suction filtration washs the solid for obtaining with acetic acid, and the filtrate that suction filtration is obtained is added in frozen water, stirs Mix and obtain yellow solid, yellow solid is merged with the solid after acetic acid washing, obtain intermediate product 4- benzimidazolyl benzene first Aldehyde；
    (2) the intermediate product 4- benzimidazole benzaldehydes anhydrous acetonitrile dissolving for obtaining step (1), adds potassium carbonate and hydrogen Potassium oxide is heated to reflux, and after being down to room temperature, adds the anhydrous acetonitrile of iodomethane, and temperature rising reflux reaction, cooling is filtered, will Filtrate obtains intermediate product 4- methyl-benzoimidazole benzaldehydes after collecting through rotary distillation, purifying；
    (3) the intermediate product 4- methyl-benzoimidazole benzaldehydes that will be obtained are dissolved in anhydrous pyridine, are heated up and are stirred, and are added Malononitrile reacts, and after the completion of reaction, cooling, filtering obtains red colored crystalline thing, i.e. target product 2- (4- (1- methyl isophthalic acid H phenanthro-s [9,10d] imidazoles -2 base-benzylidene) malononitrile.
  3. 3. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent according to claim 2 Method, it is characterised in that：Phenanthrenequione, it is 1 to the mol ratio of two benzaldehydes, ammonium acetate, acetic acid in step (1):3:7:10.
  4. 4. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent according to claim 2 Method, it is characterised in that：In step (1), the time of back flow reaction is 50 minutes.
  5. 5. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent according to claim 2 Method, it is characterised in that：In step (2), potassium carbonate, potassium hydroxide, iodomethane and intermediate product 4- benzimidazole benzaldehydes Mol ratio be 1:1:1.2:1.
  6. 6. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent according to claim 2 Method, it is characterised in that：In step (2), add potassium carbonate and potassium hydroxide to be heated to reflux 30 minutes, after being down to room temperature, add The anhydrous acetonitrile of iodomethane, temperature rising reflux reacts 30 minutes.
  7. 7. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent according to claim 2 Method, it is characterised in that：In step (3), malononitrile is with the mol ratio of intermediate product 4- methyl-benzoimidazole benzaldehydes 1.5:1。
  8. 8. the synthesis for answering bisulfite and hypochlorite double-bang firecracker Ratio-type fluorescent labeling reagent according to claim 2 Method, it is characterised in that：In step (3), it is warming up to 70 DEG C and stirs 10 minutes, adds malononitrile to react 1 hour.
  9. 9. answer Ratio-type fluorescent labeling reagent to answer bisulfite and hypochlorite double-bang firecracker described in a kind of claim 1 With, it is characterised in that：The exogenous HSO of detection cell3 -And ClO-Concentration, detection endogenous cellular HSO3 -And ClO-Concentration with And fluorescence imaging.
  10. 10. it is according to claim 9 to answer Ratio-type fluorescent labeling reagent to answer bisulfite and hypochlorite double-bang firecracker With, it is characterised in that comprise the following steps：
    First, the exogenous HSO of detection cell3 -And ClO-Concentration：
    (1) pH=7.40 is prepared, concentration is the PBS buffer salt solutions of 10mM；Compound concentration is the HSO of 1mM3 -Acetonitrile standard it is molten Liquid, concentration are the ClO of 1mM-Acetonitrile standard liquid and concentration for 0.05mM 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] miaows The base of azoles -2)-benzylidene) malononitrile acetonitrile solution；
    (2) 0 μ L, 2.5 μ L, 5 μ L, 7.5 μ L, 10 μ L, 12.5 μ L, 15 μ L, 17.5 μ L, 20 μ L, 22.5 μ L, 25 μ L concentration are taken respectively It is the HSO of 1mM3 -And ClO-Totally 22 parts of acetonitrile standard liquid, be added separately in fluorescence cuvette, be separately added into 100 μ L dense The PBS buffer salt solutions for 10mM are spent, 2- (4- (the 1- methyl isophthalic acid H phenanthro-s that 100 μ L concentration are 0.05mM are then separately added into again [9,10d] imidazoles -2 base)-benzylidene) malononitrile acetonitrile solution, be finally separately added into acetonitrile constant volume to 1mL, mixing is equal It is even；
    Fluorescence intensity is tested by sepectrophotofluorometer, fluorescence intensity ratio is obtained, ClO is detected-The excitation wavelength of fluorescence intensity It is 440nm, detection HSO3 -The excitation wavelength of fluorescence intensity is respectively 330nm, 410nm；
    (3) respectively with HSO3 -And ClO-Concentration be abscissa, with fluorescence intensity ratio as ordinate, obtain on cell external source Property HSO3 -And ClO-The linear equation of concentration, fluorescence intensity ratio；
    (4) by testing sample and acetonitrile by volume 1:After 4 mixing, 900mL is taken, and is added thereto to 100 μ L concentration and be The acetonitrile solution of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.05mM, according to outer Source property HSO3 -And ClO-Concentration, the linear equation of fluorescence intensity ratio obtain HSO3 -And ClO-Concentration；
    2nd, for detecting living cells endogenous HSO3 -And ClO-Concentration：
    (1) compound concentration is the HSO of 1mM3 -Acetonitrile standard liquid, concentration for 1mM ClO-Acetonitrile standard liquid, concentration be The acetonitrile solution of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.5mM, concentration are 1 μ The SO that the LPS solution of g/ml, concentration are the PMA solution of 1 μ g/ml, concentration is 1mM2Donor solution；
    (2) living cells Hela cells are inserted in culture medium and is cultivated, be divided into and do not cultivate 10 groups, inoculum concentration is 2 in every group of culture medium ×107~9 × 107Individual/mL, cultivate 24h, be first separately added into 10 μ L concentration be 0.5mM 2- (4- (1- methyl isophthalic acid H phenanthro-s [9, 10d] base of imidazoles -2)-benzylidene) and malononitrile acetonitrile solution, then be separately added into 0 μ L, 7.5 μ L, 15 μ L, 22.5 μ L, 30 μ L Concentration is the HSO of 1mM3 -Acetonitrile standard liquid and ClO-Acetonitrile standard liquid, cultivate 15min altogether in 37 DEG C, insert copolymerization The lower observation imaging of Jiao, collects the fluorescence intensity of different colours optical channel, so as to carry out fluorescence intensity ratio, obtains on intracellular Source property HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation；
    (3) by living cells Hela cells insert in culture medium cultivate, be divided into and do not cultivate 4 groups, in every group of culture medium inoculum concentration be 2 × 107~9 × 107Individual/mL, cultivates 24h；
    It is that the LPS solution of 1 μ g/ml, 100 μ L concentration are the PMA solution of 1 μ g/ml for piercing to 100 μ L concentration are added in first group Swash cell and produce endogenic ClO-, add 10 μ L concentration be 0.5mM 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles- 2 bases)-benzylidene) acetonitrile solution of malononitrile cultivated, is imaged, and second group is not added with LPS and PMA, only plus 10 μ L concentration are The acetonitrile solution of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.5mM is used as control；
    To the SO for adding 100 μ L concentration to be 1mM in the 3rd group2Donor is used for stimulating cell to produce endogenic HSO3 -, add 10 μ L concentration are molten for the acetonitrile of 2- (4- (1- methyl isophthalic acid H phenanthro- [9,10d] imidazoles -2 base)-benzylidene) malononitrile of 0.5mM Liquid is cultivated, imaging, and the 4th group is not added with SO2Donor, only adds 10 μ L concentration to be 2- (4- (the 1- methyl isophthalic acid H phenanthro-s of 0.5mM [9,10d] imidazoles -2 base)-benzylidene) malononitrile acetonitrile solution as control；
    The burnt lower observation imaging of copolymerization is inserted, the fluorescence intensity of different colours optical channel, so as to carry out fluorescence intensity ratio, root is collected According to endogenous cellular HSO3 -And ClO-Concentration and fluorescence intensity ratio linear equation, so as to obtain endogenous cellular HSO3 -With ClO-Concentration.
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CN108169189A (en) * 2017-12-06 2018-06-15 济南大学 A kind of response sulfur dioxide/sulfurous acid（Hydrogen）The ratio fluorescent probe of salt
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CN108484478A (en) * 2018-05-10 2018-09-04 曲阜师范大学 A kind of novel carbazoles fluorescence mercaptan labelled reagent and its synthetic method and application
CN111518066A (en) * 2020-05-25 2020-08-11 河南省农业科学院农业质量标准与检测技术研究所 Bifunctional fluorescent probe for identifying hypochlorite and bisulfite and preparation method and application thereof
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