CN103674925B - 4-mercaptophenyl boronic acid modify golden nanometer particle and with its detection cell surface sugar labelling method - Google Patents
4-mercaptophenyl boronic acid modify golden nanometer particle and with its detection cell surface sugar labelling method Download PDFInfo
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Abstract
The present invention provides the golden nanometer particle that a kind of 4 mercaptophenyl boronic acid are modified and the method detecting cell surface sugar labelling with it.The present invention provides the method for detection carbohydrate metabolism labelling and provides the label closer to natural product, and realizes the direct detection to label on the basis of label does not occurs chemical reaction.Under conditions of without poisoned catalyst etc., use nanometer gold surface enhanced Raman technique to realize the cell surface sugar to special Raman signal label labelling and detect.
Description
Technical field
The present invention relates to chemistry glycobiology field, particularly relate to the gold nano that 4-mercaptophenyl boronic acid is modified
Particle and the method with its detection cell surface sugar labelling.
Background technology
Bio-orthogonal reaction has utilization widely in chemical biology field, is a kind of two step chemistry
Labelling method.In glycobiology field, the first step, by methodology of organic synthesis on natural monosaccharide molecule
The orthogonal group of modified biological (such as: nitrine or Terminal Acetylenes, the most commonly used), then by organism certainly
Body metabolism is integrated in biological sugar chain；Second step, after the integration of previous step, in natural sugar chain
With a chemistry report group, with a molecule carrying the complementary group of bio-orthogonal, pass through
Target molecule is detected by the mode of chemical reaction, imaging and separation.Although this two step chemistry marks
Notation in terms of detection, imaging and the separation of biomolecule, especially biomolecule dynamically change aspect,
Achieve important progress；But, the method still suffers from a series of problem needs researchers to solve.
One, the orthogonal group on synthetic modification has certain change to the physicochemical property of molecule monomer itself；Two,
The biocompatibility issues of follow-up chemical reaction detection system；Three, live body is dynamically labeled the most restricted.
For these problems, the resolving ideas of researchers concentrates on the bio-compatible improving chemical reaction
Property aspect；With Ac4As a example by ManNAz biological metabolism labelling, its general plan is:
As a example by copper catalysis nitrine with alkynes [3+2] cycloaddition reaction.This catalytic reaction Cu (I)
Cytotoxicity very strong, on the one hand researchers are placed on center of gravity exploitation part to reduce its toxicity,
And improve (the most commonly used with BTTAA) in reaction rate, on the other hand it is placed on activation alkynes (with ring
Octyne and derivant thereof are the most successful).Although the method for part can add fast response speed largely
Rate, but the toxicity of Cu (I) can only obtain a certain degree of reduction；And the method activating alkynes does not uses
Cu (I) catalyst, only reduces the activation energy of cycloaddition reaction by the way of ring strain and conjugation,
Realize improving the purpose of reaction rate.It is the most multiple that the shortcoming of the method maximum of activation alkynes is exactly the activation of alkynes
Miscellaneous, synthesis cost is high, it is high, slow relative to rate of catalysis reaction to adsorb background and biocompatibility
Problem is not well solved yet.
Raman (Raman) is imaging and the detection method of a kind of non-chemically labelling, can be to specifically
The group with Raman signal carries out detecting and imaging.In the Raman imaging of cell, exist
Region (the 1800-2800cm that one section of Raman signal is reticent-1), i.e. there is no appointing of cell in this region
What signal.So, the Targeting groups in reticent region spreads out as natural sugar at this to use Raman signal
(such as, the Raman signal of alkynyl is at 2100cm for biological modification group-1Left and right) carry out cell
The even Raman of vivisection detects and imaging.But, Raman signal is relative to fluorescence signal
For the most weak, its on cell membrane rich in the detection of phospholipid and imaging all seem some difficulty
(needing to use the stronger laser instrument of power), then a small amount of containing Raman for surface of cell membrane
Detection and the imaging of the non-natural sugar of signal group can not realize especially.
Surface-enhanced Raman (SERS) is that the metal surfaces such as Au, Ag are for the one of Raman signal
Plant and strengthen phenomenon, typically can realize 106-1012Reinforced effects.This enhancing is likely to allow for
The detection of a small amount of sugar of the non-natural containing Raman signal group of surface of cell membrane and imaging become can
Energy.
Summary of the invention
It is an object of the invention to overcome drawbacks described above of the prior art, it is provided that 4-mercaptophenyl boronic acid is modified
Golden nanometer particle and with its detection cell surface sugar labelling method, there is not chemical reaction at label
On the basis of realize the direct detection to label.
Specifically, the present invention provides the golden nanometer particle that a kind of 4-mercaptophenyl boronic acid is modified, under it passes through
Prepared by the method for stating:
1) by HAuCl4(gold chloride) aqueous solution is heated to backflow, under agitation adds sodium citrate
Solution；
2) room temperature cooling, it is thus achieved that golden nanometer particle crystal seed；
3) the golden nanometer particle crystal seed that will obtain adds in ultra-pure water, under agitation add sodium citrate,
HAuCl4And hydroxylamine hydrochloride solution, it is stirred at room temperature, it is thus achieved that golden nanometer particle；
4) in the golden nanometer particle obtained, add the ethanol solution of 4-mercaptophenyl boronic acid, be stirred at room temperature
8-24 hour, obtain the golden nanometer particle that described 4-mercaptophenyl boronic acid is modified.
Preferably, step 1) described HAuCl4The mass concentration of aqueous solution is 0.01%, sodium citrate
The mass concentration of solution is 1%.
Preferably, step 3) in the mass concentration of sodium citrate be 1%, HAuCl4Mass concentration
Be 1%, the concentration of hydroxylamine hydrochloride solution be 10mM.
The golden nanometer particle that described 4-mercaptophenyl boronic acid is modified may be used for detecting cell surface sugar mark
Note.
The present invention also provides for a kind of method detecting cell surface sugar labelling, and it comprises the steps of
1) the culture medium culturing cell containing non-natural monosaccharide is used；
2) Jenner's grain of rice that the 4-mercaptophenyl boronic acid cell of cultivation and claim 1 prepared is modified
The solution of son is hatched altogether, and the golden nanometer particle making 4-mercaptophenyl boronic acid modify is attached to the saliva of cell surface
In liquid acid；
3) the Raman tag signal of Raman spectrometer detection surface of cell membrane is used, according to the letter gathered
Number judge whether cell surface exists described non-natural monosaccharide.
Preferably, described non-natural monosaccharide is the non-natural monosaccharide containing nitrine, alkynyl or deuterated methyl.
It is highly preferred that described non-natural monosaccharide is Ac4ManNAz,Ac4ManNAl or d3-Ac4ManNAc。
Preferably, described cell is HeLa and/or Chinese hamster ovary celI.Described Ac4The concentration of ManNAl
Scope is 0.01 μM to 100 μMs.
The Ac of more preferably 50 μMs4When ManNAl cultivates Chinese hamster ovary celI, incubation time is minimum is
34 hours.
The present invention provides golden nanometer particle that 4-mercaptophenyl boronic acid modifies and with its detection cell surface sugar
The method of labelling, detects carbohydrate metabolism labelling, and provides the label closer to natural product, at mark
Sign and on the basis of there is not chemical reaction, realize the direct detection to label.Without poisoned catalyst etc.
Under conditions of, use nanometer gold surface enhanced Raman technique to realize special Raman signal label labelling
The method of cell surface sugar detection.This method is under conditions of without poisoned catalyst etc., and utilization is received
Rice gold surface strengthens Raman technology and realizes the detection of the cell surface sugar to special Raman signal label labelling
Method.Such an approach achieves highly sensitive detection.And the method is applicable to various cell table
The detection of face non-natural sugar.
For above and other objects of the present invention, feature and advantage can be become apparent, cited below particularly
Preferred embodiment, and coordinate accompanying drawing, it is described in detail below.
Accompanying drawing explanation
Fig. 1 is to use nanometer gold surface enhanced Raman technique to realize special Raman signal label labelling
The schematic diagram of cell surface sugar detection.
Fig. 2 is the dark-field imaging of Raman spectrogrph, and the bright spot (shown in arrow) in figure represents Jenner
Rice corpuscles.
Fig. 3 and Fig. 4 is nitrine and the alkynyl detecting surface of cell membrane in CHO and HeLa cell respectively
Raman signal.
Fig. 5 and Fig. 6 is the Raman signal detecting deuterated methyl in CHO and HeLa cell respectively.
Detailed description of the invention
Refer to Fig. 1, it is to use nanometer gold surface enhanced Raman technique to realize special Raman signal
The schematic diagram of the cell surface sugar detection of label labelling, non-natural monosaccharide is (with formula I
Ac4As a example by ManNAz) be metabolized cell surface after become non-natural sialic acid (Formula II
NeuNAz), 4-mercaptophenyl boronic acid (MPBA) is modified golden nanometer particle (AuMPBA) and saliva
Upper two hydroxyl reactions in liquid acid 8,9-position generate product as depicted.Pass through golden nanometer particle
(AuNPs) surface-enhanced Raman just can realize the detection to non-natural Raman label.
The synthesis of embodiment 1 AuMPBA
Use the golden nanometer particle of the two-step method synthesis 120nm of reduction of sodium citrate.The first step, 100
The HAuCl of ml0.01%4Aqueous solution is heated to backflow, is rapidly added 1ml1%'s under fast stirring
Sodium citrate solution.After backflow 15min, room temperature cools down the AuNPs crystal seed i.e. obtaining 40nm.
Second step, the crystal seed of the AuNPs of 4ml40nm adds in 56ml ultra-pure water, under agitation adds
The sodium citrate of 0.9ml1%, the HAuCl of 0.9ml1%4Oxammonium hydrochloride. with 1.4ml 10mM
Solution, is stirred at room temperature 1h and i.e. obtains the AuNPs of 120nm.Last use MPBA in ethanol solution
Replace citrate, be the formation of the AuMPBA of 120nm.Concrete grammar is 120nm's
AuNPs3000rpm is centrifuged 10min, abandons supernatant, adds the ethanol solution of the MPBA of 2mM,
Stirred overnight at room temperature (such as 8-24 hour).Before AuMPBA uses, it is centrifuged 10min with 3000rpm,
Then second alcohol and water is respectively washed 3 times, is diluted to required afterwards with solvent used in experiment or buffer solution
Concentration.
Embodiment 2 Raman tag (nitrine and alkynyl) detection in cell line (HeLa and CHO)
AuMPBA modifies cell surface: by containing the Ac of 100 μMs4ManNAz or Ac4ManNAl
DMEM culture medium in incubator 37 degrees Celsius cultivate HeLa or Chinese hamster ovary celI after three days, use phosphorus
Acid buffering solution (PBS, pH=7.4) is washed three times and is washed the non-natural sugar being introduced into cell off, then uses
37 degrees Celsius of incubated cells of the PBS solution of AuMPBA make golden nanometer particle be attached to cell half an hour
Film surface, phosphate buffer solution (PBS, pH=7.4) wash three times after mounting, finally use Raman light
The signal of the Raman label of spectrometer detection surface of cell membrane, uses the 633nm laser instrument of 50% intensity
Gather signal 10s.Its result refer to Fig. 2, Fig. 3 and Fig. 4.Wherein, Fig. 2 is Raman spectrogrph
Dark-field imaging, bright spot (in Fig. 2 shown in arrow) in figure represents golden nanometer particle.Fig. 3 and Fig. 4
It is to be the Raman signal of nitrine and the alkynyl detecting surface of cell membrane in CHO and HeLa cell respectively.
From figs. 3 and 4 it can be seen that the signal (2100cm of nitrine and alkynyl-1Left and right) at HeLa
Can detect well with the surface of Chinese hamster ovary celI and blank does not has.The alkynyl of Chinese hamster ovary celI
With the signal of nitrine 2127 and 2126cm-1；The alkynyl of HeLa cell and the signal of nitrine are 2131
And 2126cm-1。
Non-natural monosaccharide containing nitrine (Az) and alkynyl (Al) has extensively been proved to use existing
The two step chemical labeling methods having technology are expressed on cell membrane.And their the positive benefit of Raman signal
In cell silence region, all at 2100cm-1Left and right.HeLa and Chinese hamster ovary celI are used for proving to fold
Nitrogen and the expression of alkynyl.
Relative to two step chemical labeling methods of prior art, in the application, use AuMPBA auxiliary
SERS detection method need not use Cu (I) poisoned catalyst, biocompatibility substantially carries
High.And said method is the existence rather than such as of the Raman label directly detecting cell surface
Prior art indirectly detects the existence of label like that by the signaling molecule of detection with tag reactant.
So the detection method of the application does not has the absorption background of signaling molecule, the credibility of detection is higher.
Embodiment 3 Raman tag (deuterated methyl) detection in cell line (HeLa and CHO)
Although the detection of the Raman label in embodiment 2 is able to demonstrate that their own is by dynamic earth's surface
Reach on cell membrane, but owing to their physicochemical property is the most altered, therefore they may pass through and sky
The metabolic pathway that so sugar is different enters and can not represent the Expression of natural sugar on cell membrane.Deuterated
Compound has the physicochemical property identical with native compound, and the Raman signal of deuterated methyl is also
At 2100cm-1Left and right, so the d of formula III3-Ac4ManNAc, is used for detecting the dynamic of natural sugar
Express.
Concrete detection method is similar to Example 2, and institute's difference is, cultivates in cell culture medium
Non-natural monosaccharide be d3-Ac4ManNAc.Concrete testing result refer to Fig. 5 and Fig. 6.From Fig. 5
With in Fig. 6 it will be seen that the Raman signal of deuterated methyl can detect equally well.CHO
It is respectively 2128 and 2125cm with the signal of HeLa cell-1.Thus may certify that natural manna
Sugar is expressed on surface of cell membrane (being converted into sialic acid).
The limit Raman label concentration of embodiment 4 SERS detection and minimum incubation time
The detection method of Raman label is with embodiment 2, wherein by Ac4The concentration of ManNAl is arranged
Ac for a series of variable concentrations4ManNAl(0μM,0.01μM,0.05μM,0.1μM,0.5μM,
1 μM, 5 μMs, 10 μMs, 25 μMs, 50 μMs, 100 μMs), cultivate Chinese hamster ovary celI and hatch after three days
Then AuMPBA detects Raman signal, and result is: the Ac of minimum 0.1 μM4ManNAl is the most permissible
The SERS signal of alkynyl detected.Similarly, the Ac of 50 μMs4ManNAl cultivates Chinese hamster ovary celI
Different time (0h, 22h, 34h, 39h, 48h, 72h) then detects Raman signal, minimum 34h's
Incubation time can detect SERS signal.
Although the present invention discloses as above with preferred embodiment, so it is not limited to the present invention, appoints
What person of ordinary skill in the field, without departing from the spirit and scope of the present invention, when doing some
The change permitted and improvement, therefore protection scope of the present invention is when being as the criterion depending on as defined in claim.
Claims (3)
1. a method for surface-enhanced Raman specific detection cell surface sugar labelling, its feature exists
In, comprise the steps of
1) using the culture medium culturing cell containing non-natural monosaccharide, described non-natural monosaccharide is containing deuterated
The non-natural monosaccharide of methyl；
2) solution of the golden nanometer particle modified with 4-mercaptophenyl boronic acid by the cell of cultivation is hatched altogether,
The golden nanometer particle making 4-mercaptophenyl boronic acid modify is attached on the sialic acid of cell surface；
3) the Raman tag signal of Raman spectrometer detection surface of cell membrane is used, according to the letter gathered
Number judge whether cell surface exists described non-natural monosaccharide.
Method the most according to claim 1, it is characterised in that described non-natural monosaccharide is
d3-Ac4ManNAc。
Method the most according to claim 1 and 2, it is characterised in that described cell is HeLa
And/or Chinese hamster ovary celI.
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| CN106929838A (en) * | 2015-12-31 | 2017-07-07 | 北京大学 | The method that preparation is suitable to the enhancing substrate of cell surface bio-orthogonal Raman image |
| CN105823770B (en) * | 2016-05-25 | 2018-07-27 | 武汉大学 | A kind of Raman labels probe of no optical interference and its preparation method and application |
| CN107064265A (en) * | 2017-05-23 | 2017-08-18 | 中国科学院上海高等研究院 | A kind of electrochemica biological sensor for being used for HbA1c detections of MPBA modifications and its preparation and application |
| CN108254366B (en) * | 2017-09-19 | 2020-08-25 | 中国科学院海洋研究所 | Method for detecting microorganisms based on phenylboronic acid functionalized silver nanoparticles |
| CN110292652B (en) * | 2018-03-23 | 2022-08-12 | 国家纳米科学中心 | Mercaptobenzeneboronic acid activated gold nanoparticles as well as preparation method and application thereof |
| CN110393725B (en) * | 2018-04-20 | 2022-09-02 | 国家纳米科学中心 | Phenylboronic acid with gram selectivity and gold nanoparticle modified by derivative of phenylboronic acid, and preparation method and application of phenylboronic acid and gold nanoparticle |
| CN109001176B (en) * | 2018-06-14 | 2021-08-24 | 福建师范大学 | Preparation method of SERS substrate of Au @ Ag nanoparticles and method for detecting glucose by using substrate |
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