CN101261226B - Surface plasma resonance instrument chip based on polyethyleneglycol - Google Patents
Surface plasma resonance instrument chip based on polyethyleneglycol Download PDFInfo
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- CN101261226B CN101261226B CN 200710064257 CN200710064257A CN101261226B CN 101261226 B CN101261226 B CN 101261226B CN 200710064257 CN200710064257 CN 200710064257 CN 200710064257 A CN200710064257 A CN 200710064257A CN 101261226 B CN101261226 B CN 101261226B
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- 238000000034 method Methods 0.000 claims description 17
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- 239000000126 substance Substances 0.000 claims description 5
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- 238000006243 chemical reaction Methods 0.000 description 18
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
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Images
Abstract
The invention relates to a chip of a surface plasmon resonance instrument which is based on polyethylene glycol and a preparation method. The chip comprises a glass film base and a gold plating film on the glass film base; wherein the gold plating film is provided with an initiator layer which is introduced in the way of self-assembly and a functionalized polyethylene glycol layer which is combined with the initiator layer. The invention uses a quartz crystal microbalance to monitor the growth of the film at the surface of the chip at real time and the chip of the invention effectively solvesthe problem of non-specific absorption of protein. As the surface ornament of the chip is not limited to traditional carboxylic dextran, the density and types of the functional groups at the chip surface are no longer strictly restricted. Furthermore, the chip of the invention also has good performance in terms of the specificity maintenance and quality control of biological signaling molecule and so on. The preparation method of the chip of the invention has few steps and the low manufacturing cost and is more suitable for industrial production. The chip has more potential of deep development and more superior generality.
Description
Technical field
The present invention relates to a kind of surface plasma resonance instrument chip, specifically, relate to a kind of surface plasma resonance instrument chip based on polyglycol, and preparation method thereof.
Background technology
Surface plasma resonance instrument (Surface Plasma Resonance spectroscopy is hereinafter to be referred as SPR) is the sensor of detection of biological interaction of molecules.Proposed and verified the imagination that SPR uses on biomedicine as biology sensor in nineteen eighty-three by people such as Liedberg, on the basis of their a series of activities, the Pharmacia of Sweden has released the business-like SPR instrument of the first generation in nineteen ninety to market.At present, SPR has been widely used in the detection in fields such as biomedicine, clinical diagnosis, new drug development (screening, mechanism research), judicial expertise, environmental monitoring, food security, is the standard detecting method of being accepted extensively by scientific circles and industry member.Compare with enzyme linked immunosorbent assay, the accuracy of SPR detection method is higher, and repeatability is higher.Compare with the vitaminic method of using high pressure liquid chromatography (HPLC) or use microorganism to detect in the food, the SPR detection method is faster easier.In a word, compare with additive method, that the SPR detection method has is highly sensitive, simple to operate, repeatable high, in real time and exempt from advantage such as mark.
Usually, surface plasma resonance instrument is made up of ray machine electricity part and consumptive chip two parts, and chip is the core of this system.Occupied the serial SPR of BIAcore (former Pharmacia) in 90% above market, the whole world at present, it provides the chip that can directly use for the user, as shown in Figure 1, this chip is gold-plated successively rete 2, self-assembled monolayer 3 and a carboxylated glucosan rete 4 on a glass chip 1.When the prism of polarized light by coupling shone this chip on glass at an angle, on the one hand, glass produced reflected light; Simultaneously, the light that glass is crossed in transmission enters Gold plated Layer, and interacts with surface plasma wave that gold surface produces, when incident light is in a certain specific angle, the two can produce resonance, thereby can cause catoptrical intensity minimum to occur in this specific incident angle.Because it is relevant with the refraction constant on gold plate surface that the angle of resonance takes place surface plasma-wave, and this refraction constant is relevant with the material of gold surface, the variation of angle during therefore by detection of reflected light intensity minimum, the variation of detection gold film surface mass that just can be indirect.
When surface plasma resonance instrument is used for the sensor of detection of biological interaction of molecules, in order to improve the sensitivity of SPR, normally at the sulfhydryl compound formation self-assembled monolayer of gold surface by terminal band carboxyl, modify the last layer host material then---carboxylated glucosan rete, thereby increase its specific surface area, in order to fixing more signaling molecule.The principle of work synoptic diagram of SPR in the BIAcore system as shown in Figure 2, the chip of its core is the gold-plated glass sheet of one side, carboxylated glucosan rete by modifying on gold surface, the fixing a pair of part (as one of part and acceptor) that the molecule of specific action can take place.To contain the golden film surface that above-mentioned another part medium transport that the molecule of specific action can take place arrives modified by the miniflow induction system.When MEDIA FLOW during through chip, the molecular specificity combination of specific action to taking place in this, make the local concentration on golden film surface change, the material that is gold surface changes, thereby cause its refraction constant to change, the resonance angle that final SPR measures surface plasma-wave change (changing to resonance angle II) from resonance angle I.At document 1:
S; Johnsson, B.J.Chem.Soc., Chem.Commun., 1990, verified among the 1526-1528, the variation of this resonance angle is directly to be directly proportional with the molecular mass of receiving gold surface, in the BIAcore system, the angle of the corresponding 0.0001 ° of minimum intensity of a reacton (resonance units is hereinafter to be referred as RU) changes.To the most protein sample, the variation of a RU is equivalent at chip surface 1pg/mm take place
2Variation.
But this class uses the technology of carboxylated glucan-modified chip that following shortcoming is arranged: the step during (1) preparation chip is various, makes the controllability of chip and repeatability be affected; (2) fixedly used traditional " being transplanted to " (" grafting to ") strategy during polymer matrix at chip surface, made the density and the thickness of the carboxylated glucosan layer modified all be restricted, thereby further have influence on the molecular detection of being fixed; (3) employed raw material---the source of glucosan hydrogel is limited, makes that the manufacturing cost of chip is higher, and its performance that prevents the protein non-specific adsorption can not reach best; (4) owing to used the glucosan hydrogel, the density and the kind of functional group are restricted, and the difficulty of deriving is big; (5) lack connection space (promptly lacking Spacer) between bio signal molecule and matrix, thereby it is bigger to make experimental result influenced by the fixed form of bio signal molecule.
Summary of the invention
The objective of the invention is to overcome the many disadvantages of surface plasma resonance instrument chip of the prior art, a kind of method of having used the growth of the modified membrane that QCM (Quartz Crystal Microbalance) (hereinafter to be referred as QCM) comes real-time chip monitoring surface is provided, and the performance based on the surface plasma resonance instrument chip of polyglycol of this method preparation all is better than the performance of this class existing chip in every respect.Chip of the present invention has solved the problem of the non-specific adsorption of protein effectively.Because the modification of chip surface of the present invention is not limited to traditional carboxylated glucosan, make the density and the kind of functional group of chip surface no longer be subjected to strict restriction; In addition, at aspects such as the specificity maintenance of bio signal molecule, quality controls, chip of the present invention also has superior performance.Chip production method step of the present invention is few, and production cost is low, is more suitable for suitability for industrialized production.But the potentiality of the deep development of this chip are bigger, and versatility is more superior.
The objective of the invention is to realize by the following technical solutions:
The invention provides a kind of surface plasma resonance instrument chip based on polyglycol, as shown in Figure 3, it comprises a glass chip 1 and the gold-plated rete 2 on it, the initiating agent layer of on described gold-plated rete, introducing 5 with the method for self assembly, and and the initiating agent layer in conjunction with the polyglycol layer 6 of the functionalization of (chemical bonding-surface initiated polymerization).
Described initiating agent layer is initiating agent bromo acid-ω-sulfydryl undecyl ester (ω-mercaptoundecylbromoisobutyrate) and thinning agent ω-sulfydryl undecane (self-assembled monolayer of ω-mercapto-undecanthiol) mix, wherein, initiating agent bromo acid-ω-sulfydryl undecyl ester proportion is 5-100%, be preferably 5%, 30%, 45%, 60% and 100%;
The thickness of the polyglycol layer of described functionalization is 3～150 nanometers;
The polyglycol of described functionalization is the oligomeric ethylene glycol methyl methacrylate, and its chemical formula is
(CH
2＝CH)CH
3COO(CH
2CH
2O)
nR
Wherein, n=6,12, terminal R is hydroxyl (OH), carboxyl (COOH), aldehyde radical (CHO), amino (NH
2), sulfydryl (SH) or nitrilotriacetic acid (nitrilotriacetic acid closes group hereinafter to be referred as the NTA-huge legendary turtle).
The invention provides a kind of preparation method of above-mentioned surface plasma resonance instrument chip based on polyglycol, this method uses QCM (Quartz Crystal Microbalance) to come the growth of each tunic of real-time chip monitoring surface, comprises following step:
1) adopts the self-assembled monolayer technology, immerse immediately after the glass sheet cleaning with commercially available surface gold-plating and contain in the ethanolic solution of initiating agent and thinning agent, after 24 hours, the distribution density that connects the surface on the golden membranous layer of this glass sheet is the initiating agent unimolecular layer of 5-100% density;
Concrete steps are according to document 3:Ma, H.; Wells, M.; Bebees, T.; Chilkoti, A.; Adv.Funct.Mater.2006, middle reported method: gold surface is carried out the ozone ultraviolet ray with the UV/Ozone cleaning machine clean, remove surface impurity; Immerse immediately after the cleaning and contain in the ethanolic solution of initiating agent and thinning agent, the sulfydryl of initiating agent and gold reaction, can obtain having covered the chip of self-assembled monolayer of initiating agent of the density of 5-100% after 24 hours, this chip is inserted in the sensor cavities of QCM (Quartz Crystal Microbalance), feed nitrogen protection 15 minutes;
In the described ethanolic solution that contains initiating agent and thinning agent, described initiating agent is bromo acid-ω-sulfydryl undecyl ester, described thinning agent is ω-sulfydryl undecane, and the mol ratio of initiating agent and thinning agent is 1: 9-0, and the two total concentration in ethanolic solution is 1 mM;
Because it is different to contain the reactivity of the initiating agent of sulfydryl and thinning agent,, need to measure according to disclosed method in the document 3 by surface chemistry analyser (XPS) so its blending ratio in ethanolic solution is different from its density from the teeth outwards; In the technical program, the mol ratio of initiating agent and thinning agent is 1: 9 o'clock, and corresponding initiating agent is 5% in superficial density; The mol ratio of initiating agent and thinning agent is 1: 1 o'clock, and corresponding initiating agent is 30% in superficial density; The mol ratio of initiating agent and thinning agent is 1: 0.4 o'clock, and corresponding initiating agent is 45% in superficial density; The mol ratio of initiating agent and thinning agent is 1: 0.25 o'clock, and corresponding initiating agent is 60% in superficial density; The mol ratio of initiating agent and thinning agent is 1: 0 o'clock, and corresponding initiating agent is 100% in superficial density;
2) feed and only to contain solution and monomer, do not contain the incomplete reaction liquid 0 to 10 minute of catalyzer, QCM reaches stable initial baseline; Then with 70mL h
-1Speed feeds the complete reaction liquid of the catalyzer that contains catalytic amount, after complete reaction liquid entered fully in 2 minutes, the complete reaction flow velocity is dropped to 20mL h
-1, 20 ℃ of reactions; Complete reaction liquid is switched to incomplete reaction liquid, cessation reaction; Take out chip, the thickness of the polyglycol layer of measurement functionization; Repeating step 2), makes the linear corresponding curve of frequency change (Δ F)-thickness (Thickness), and calculate the k value of its slope;
3) according to step 2) the k value and the following formula that calculate its slope determine the pairing frequency change Δ of required film thickness F
N ' is overtone number (Overtone number) in the formula, n '=3, thickness obtains for the ellipsometer test data model fits, Poly (OEGMA) adopts the Cauchy model, (An Bn) is (1.46,0.01), the thickness of sample value is the average of three position measurements, and the report result is a mean+SD;
Feed and only to contain solution and monomer, do not contain the incomplete reaction liquid 0 to 10 minute of catalyzer, QCM reaches stable initial baseline; Then with 70mL h
-1Speed feeds the complete reaction liquid of the catalyzer that contains catalytic amount, after complete reaction liquid entered fully in 1-2 minute, the complete reaction flow velocity is dropped to 3mL h
-1, react to the pairing frequency change Δ of required film thickness F at 20 ℃; Complete reaction liquid is switched to incomplete reaction liquid, cessation reaction; Take out chip, obtain the surface plasma resonance instrument chip based on polyglycol of the present invention.
Described monomer is the oligomeric ethylene glycol methyl methacrylate, and its chemical formula is
(CH
2＝CH)CH
3COO(CH
2CH
2O)
nCH
2R，
Wherein, n=6,12, terminal R is hydroxyl (OH), carboxyl (COOH), aldehyde radical (CHO), amino (NH
2), sulfydryl (SH) or the NTA-huge legendary turtle close group.
It is that polymer-based material replaces the decorative layer in the existing SPR chip that the present invention has adopted the polyglycol that prevents the protein non-specific adsorption that generally acknowledge, the best, has used QCM (Quartz Crystal Microbalance) to monitor the growth of film in real time simultaneously.These measures make chip provided by the invention have following advantage:
(1) ability that prevents non-specific adsorption of polyglycol matrix is better than glucosan matrix.Under the solution system of complexity, as cow's serum, clasmatosis liquid, body fluid etc., the non-specific adsorption of polyglycol matrix is zero (being the detection limit of instrument).For example, by cow's serum the time, glucosan matrix has the RU more than 200 to change, and polyglycol matrix does not then have.
(2) polyglycol matrix is than the easier functionalization of glucosan matrix.Realized as hydroxyl (OH), carboxyl (COOH), aldehyde radical (CHO), amino (NH
2), (SH), functional group such as NTA is fixing for sulfydryl.
(3) under the same thickness, polyglycol matrix is than the functional group density height of glucosan matrix, the sensitivity that has improved SPR.
(4) density of functional group can be regulated and control by the ratio that changes monomer easily.
(5), can control the thickness (3～150 nanometer) of polyglycol matrix membrane in the larger context accurately by the QCM monitoring.
Description of drawings
Fig. 1 is the chip structure synoptic diagram of BIAcore series SPR; Wherein, 1 glass chip, 2 gold-plated retes, 3 self-assembled monolayers, 4 carboxylated glucosan retes;
Fig. 2 is the principle of work synoptic diagram of BIAcore series SPR;
Fig. 3 is the structural representation of the surface plasma resonance instrument chip based on polyglycol of the present invention; Wherein, 1 glass chip, 2 gold-plated retes, 5 initiating agent layers, 6 polyglycol layers.
Fig. 4 is frequency change (Δ F)-linear corresponding diagram of thickness (Thickness).
Embodiment
Initiating agent bromo acid-ω-sulfydryl undecyl ester ω-mercaptoundecylbromoisobutyrate that the present invention uses, oligomeric ethylene glycol the methyl methacrylate ((CH of functionalization
2=CH) CH
3COO (CH
2CH
2O)
nCH
2R, n=6,12, terminal R is hydroxyl (OH), carboxyl (COOH), aldehyde radical (CHO), amino (NH
2), sulfydryl (SH), NTA) is all available from the big Bioisystech Co., Ltd in Hangzhou east.
(specification is 10 * 10 * 0.5mm to the glass sheet of used surface gold-plating among the present invention
3, the gold of surface plating 50 nanometers) and available from the big Bioisystech Co., Ltd in east, Hangzhou.
The UV/Ozone cleaning machine that uses among the present invention is BioForce Nanosciences, IA, USA.QCM (Quartz Crystal Microbalance) is a Q-Sense E4 sensor, Q-Sense, Gothenburg, Sweden.
(J.A.Woollam Co. Inc) measures thickness, and taking measurement of an angle is 65 °, and 70 ° and 75 °, measuring wavelength is that 400nm is to 800nm by M-2000V type beam split ellipsometric measurement instrument among the present invention.
The surface plasma resonance instrument chip of the polyglycol matrix of 100% carboxyl-functional of embodiment 1, preparation 100% initiating agent density
According to document 3:Ma, H.; Wells, M.; Bebees, T.; Chilkoti, A.; Adv.Funct.Mater.2006, middle reported method adopts the self-assembled monolayer technology, and the gold surface of the glass sheet of commercially available surface gold-plating is carried out ozone ultraviolet ray cleaning with the UV/Ozone cleaning machine, removes surface impurity; Immerse immediately after the cleaning and contain in the ethanolic solution that concentration is 1 mM bromo acid-ω-sulfydryl undecyl ester, the sulfydryl of initiating agent and gold reaction can obtain having covered the chip of 100% initiating agent self-assembled monolayer after 24 hours.
The sensor cavities and the peristaltic pump of liquid storage tank, QCM (Quartz Crystal Microbalance) are used pipeline connection successively, said chip is inserted in the sensor cavities of QCM (Quartz Crystal Microbalance), feed nitrogen protection 15 minutes.
Be ready to only contain solution and monomer simultaneously, do not contain the incomplete reaction liquid (hereinafter to be referred as IRM) of catalyzer and contain the complete reaction liquid (hereinafter to be referred as CRM) of the catalyzer of catalytic amount.IRM forms according to following proportional arrangement: the mixed liquor of water and methyl alcohol (15mL, the volume ratio of water and methyl alcohol is 1: 2), and oligomeric ethylene glycol the methacrylate ((CH of monomer-carboxylic acidization
2=CH) CH
3COO (CH
2CH
2O)
12CH
2COOH) (8mmol 0.35M), and is kept in the inert gas; CRM is for adding catalyzer on the basis of IRM, CuBr (36mg, 0.25mmol) and dipyridine (78mg 0.5mmol) disposes and forms, and is kermesinus liquid, is kept in the inert gas.All earlier logical nitrogen deoxygenation was 15 minutes before IRM and CRM used.
With the IRM liquid storage tank of packing into, start peristaltic pump, make IRM enter in the sensor cavities of QCM (Quartz Crystal Microbalance), QCM reaches stable initial baseline after 0 to 10 minute; Then with 70mL h
-1Speed feeds CRM, and CRM entered fully in 2 minutes, and the CRM flow velocity is dropped to 3mL h
-1, 20 ℃ of reactions; CRM is switched to IRM, cessation reaction; Take out chip, use methyl alcohol, washed with de-ionized water flows down at nitrogen and to dry up the thickness (Thickness) of the polyglycol layer of use ellipsometer test Measurement and analysis functionalization.Repeat above-mentioned steps, in 1,2,4,8,12,16 hours different reaction time cessation reaction, make the linear corresponding curve of frequency change (Δ F)-thickness (Thickness), as shown in Figure 4, and the k value that calculates its slope is 0.0643.
When needs obtain 25nm oligomeric ethylene glycol methacrylate polymeric membrane, can be according to k value and following formula, calculating the pairing frequency change Δ of required film thickness F is 389Hz.
N ' is overtone number (Overtone number) in the formula, n '=3, k=0.0643, thickness obtains for the ellipsometer test data model fits, and Poly (OEGMA) adopts Cauchy model, (An, Bn) be (1.46,0.01), the thickness of sample value is the average of three position measurements, the report result is a mean+SD;
Again feed and only contain solution and monomer, do not contain the incomplete reaction liquid 0 to 10 minute of catalyzer, QCM reaches stable initial baseline; Then with 70mL h
-1Speed feeds the complete reaction liquid of the catalyzer that contains catalytic amount, after complete reaction liquid entered fully in 2 minutes, the complete reaction flow velocity is dropped to 20mL h
-1, react at 20 ℃ and to the qcm sensor chamber, to observe Δ F when being 389Hz, complete reaction liquid is switched to incomplete reaction liquid, cessation reaction; Take out chip, obtain the surface plasma resonance instrument chip based on polyglycol of the present invention, the thickness of the polyglycol layer of its functionalization is 25nm, and the density of initiating agent is 100%, and the carboxylated density of film is 100%.
Embodiment 2, the chip that uses embodiment 1 to make carry out SPR and detect
In the BIAcore3000 instrument, use the chip that obtains among this method embodiment 1.According to standard operating procedure (SOP), (PBS buffer pH=7.4), obtains baseline at first to flow through phosphate buffer.Because the film density that this method obtains is higher, shows as RU and reaches 30000～40000.The RU of general glucosan chip only is 15000.Then by N-hydroxysuccinimide (NHS, N-maloyl imines, 0.1mM) and 1-[3-(dimethylamino)-propyl]-3-ethylcarbodiimide hydrochloride (EDC, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 0.1mM) mixed solution 30 minutes, make activated carboxylic, at last by bovine serum albumin (BSA).The density that obtains COOH owing to this method is higher, shows as to connect more BSA, under the identical condition (protein concentration, temperature, time etc.), because the RU that connection BSA obtains reaches 2000, one ten times increase is arranged with 200 of glucosan.
Embodiment 3, use method of the present invention prepare the polyglycol matrix of 60% amino functional of 45% initiating agent density
According to document 3:Ma, H.; Wells, M.; Bebees, T.; Chilkoti, A.; Adv.Funct.Mater.2006, middle reported method adopts the self-assembled monolayer technology, and the gold surface of the glass sheet of commercially available surface gold-plating is carried out ozone ultraviolet ray cleaning with the UV/Ozone cleaning machine, removes surface impurity; Immerse immediately after the cleaning in the ethanolic solution of the binary mixing that contains bromo acid-ω that total concentration is 1 mM-sulfydryl undecyl ester and ω-sulfydryl undecane (thinning agent), wherein initiating agent bromo acid-ω-sulfydryl undecyl ester and the undecanoic mol ratio of thinning agent ω-sulfydryl are 1: 0.4, sulfydryl and gold reaction, (ratio of two sulfydryl chemical combination is different with the ratio of final lip-deep two sulfhydryl compounds in the solution can to obtain having covered the chip of 45% initiating agent self-assembled monolayer after 24 hours, can draw by surface chemistry analyser (XPS), as disclosed method in the document 3).
The sensor cavities and the peristaltic pump of liquid storage tank, QCM (Quartz Crystal Microbalance) are used pipeline connection successively, said chip is inserted in the sensor cavities of QCM (Quartz Crystal Microbalance), feed nitrogen protection 15 minutes.
Be ready to only contain solution and monomer simultaneously, do not contain the incomplete reaction liquid (hereinafter to be referred as IRM) of catalyzer and contain the complete reaction liquid (hereinafter to be referred as CRM) of the catalyzer of catalytic amount.IRM forms according to following proportional arrangement: the mixed liquor of water and methyl alcohol (15mL, the volume ratio of water and methyl alcohol is 1: 2), and oligomeric ethylene glycol the methacrylate ((CH of monomer-propylhomoserinization
2=CH) CH
3COO (CH
2CH
2O)
12CH
2NH
2) (4mmol is 0.17M) with methylated oligomeric ethylene glycol methacrylate ((CH
2=CH) CH
3COO (CH
2CH
2O)
12CH
3) (4mmol, 3: 2 mixed liquors 0.17M), and be kept in the inert gas; CRM is for adding catalyzer on the basis of IRM, CuBr (36mg, 0.25mmol) and dipyridine (78mg, 0.5mmol),, configuration forms, and is kermesinus liquid, is kept in the inert gas.All earlier logical nitrogen deoxygenation was 15 minutes before IRM and CRM used.
With the IRM liquid storage tank of packing into, start peristaltic pump, make IRM enter in the sensor cavities of QCM (Quartz Crystal Microbalance), QCM reaches stable initial baseline after 0 to 10 minute; Then with 70mL h
-1Speed feeds CRM, and CRM entered fully in 2 minutes, and the CRM flow velocity is dropped to 20mL h
-1, 20 ℃ of reactions; CRM is switched to IRM, cessation reaction; Take out chip, use methyl alcohol, washed with de-ionized water flows down at nitrogen and to dry up the thickness (Thickness) of the polyglycol layer of use ellipsometer test Measurement and analysis functionalization.Repeat above-mentioned steps,, make the linear corresponding curve of frequency change (Δ F)-thickness (Thickness), and the k value that calculates its slope is 0.0615 in 1,2,4,8,12,16 hours different reaction time cessation reaction.
When needs obtain 35nm oligomeric ethylene glycol methacrylate polymeric membrane, can be according to k value and following formula, calculating the pairing frequency change Δ of required film thickness F is 569Hz.
N ' is overtone number (Overtone number) in the formula, n '=3, k=0.0615, thickness obtains for the ellipsometer test data model fits, and Poly (OEGMA) adopts Cauchy model, (An, Bn) be (1.46,0.01), the thickness of sample value is the average of three position measurements, the report result is a mean+SD;
Again feed and only contain solution and monomer, do not contain the incomplete reaction liquid 0 to 10 minute of catalyzer, QCM reaches stable initial baseline; Then with 70mL h
-1Speed feeds the complete reaction liquid of the catalyzer that contains catalytic amount, after complete reaction liquid entered fully in 2 minutes, the complete reaction flow velocity is dropped to 20mL h
-1, react at 20 ℃ and to the qcm sensor chamber, to observe Δ F when being 569Hz, complete reaction liquid is switched to incomplete reaction liquid, cessation reaction; Take out chip, obtain the surface plasma resonance instrument chip based on polyglycol of the present invention, the thickness of the polyglycol layer of its functionalization is 35nm, and the density of initiating agent is 45%, and the aminated density of film is 60%.
Claims (6)
1. surface plasma resonance instrument chip based on polyglycol, it comprises a glass chip and the gold-plated rete on it, it is characterized in that: the initiating agent layer of on described gold-plated rete, introducing with the method for self assembly, and and the polyglycol layer of the functionalization of initiating agent layer combination;
Described initiating agent layer is the self-assembled monolayer that bromo acid-ω-sulfydryl undecyl ester and ω-sulfydryl undecane mix, and wherein, bromo acid-ω-sulfydryl undecyl ester proportion is 5-100%;
The thickness of the polyglycol layer of described functionalization is 3～150 nanometers;
The polyglycol of described functionalization is the oligomeric ethylene glycol methyl methacrylate, and its chemical formula is
(CH
2＝CH)CH
3COO(CH
2CH
2O)
nR
Wherein, n=6,12, terminal R is-OH ,-COOH ,-CHO ,-NH
2,-SH or nitrilotriacetic acid.
2. the surface plasma resonance instrument chip based on polyglycol as claimed in claim 1 is characterized in that: described bromo acid-ω-sulfydryl undecyl ester proportion is 5%.
3. the surface plasma resonance instrument chip based on polyglycol as claimed in claim 1 is characterized in that: described bromo acid-ω-sulfydryl undecyl ester proportion is 30%.
4. the surface plasma resonance instrument chip based on polyglycol as claimed in claim 1 is characterized in that: described bromo acid-ω-sulfydryl undecyl ester proportion is 45%.
5. the surface plasma resonance instrument chip based on polyglycol as claimed in claim 1 is characterized in that: described bromo acid-ω-sulfydryl undecyl ester proportion is 60%.
6. the surface plasma resonance instrument chip based on polyglycol as claimed in claim 1 is characterized in that: described bromo acid-ω-sulfydryl undecyl ester proportion is 100%.
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| CN102539777B (en) * | 2010-12-10 | 2014-11-05 | 国家纳米科学中心 | Supramolecular self-assembly biological chip, and preparation method and application thereof |
| CN102866132B (en) * | 2012-09-27 | 2014-10-29 | 广州高通生物技术有限公司 | Chip, production method, application of chip and drug screening method |
| CN102854293B (en) * | 2012-09-27 | 2015-07-08 | 广州高通生物技术有限公司 | Chip, preparation method, application and method for screening drugs |
| CN103898194B (en) * | 2012-12-24 | 2016-04-13 | 深圳国际旅行卫生保健中心 | Gene chip of photo-crosslinking functionalization and preparation method thereof and detection kit |
| CN103409809A (en) * | 2013-07-17 | 2013-11-27 | 国家纳米科学中心 | Small molecule drug screening chip, construction method and application thereof |
| CN103698303B (en) * | 2013-12-23 | 2016-08-10 | 北京科技大学 | A kind of surface plasma body resonant vibration (SPR) sensing chip and its preparation method and application |
| CN104741156B (en) * | 2013-12-31 | 2016-08-24 | 深圳国际旅行卫生保健中心 | Platelet screening chip and preparation method thereof and application |
| CN106117453A (en) * | 2016-06-08 | 2016-11-16 | 广州高通生物技术有限公司 | A kind of macromolecule mother solution and the manufacture method of 3D biochip |
| CN107389619B (en) * | 2017-06-26 | 2020-02-14 | 北京理工大学 | Surface plasma resonance sensor chip for detecting estradiol in water phase and preparation method thereof |
| CN109908982B (en) * | 2019-03-11 | 2021-04-13 | 黔南民族师范学院 | Preparation method and application of three-dimensional chip for screening traditional Chinese medicine components |
| CN113484521A (en) * | 2021-05-19 | 2021-10-08 | 南京医科大学第二附属医院 | ACE2 protein biochip and preparation method and application thereof |
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