CN1687777A - Aldehyde group modified substrate of protein chip and preparation method - Google Patents

Abstract

This invention is about a kind of protein slug chip which is dressed with the aldehyde group and the producing method. It use the simple glass chip or silicon dice as the carrier and it leads the active functional group aldehyde group onto the chip's surface, to produce the protein slug chip which can efficiently and firmly fix the protein. The process includes hydroxylation, self packing of APTES and terephthalic aldehyde packing. One of the two aldehyde groups of the terephthalic aldehyde will act with amino group on the face of the chip and will combine together with C=N double bond. The other aldehyde group will expose outside the chip and will act with the amino group of the protein, producing C=N double bond to fix the protein on the surface of the glass chip or silicon dice. The merit is that it uses the glass chip and silicon dice as the carrier of the protein slug, so that the cost can be decreased. What is more, by covalent bond we can fix the protein on the surface of the glass chip or silicon dice, so that the protein will not be pulled off during the action process. Besides, the producing method is easy and the nature is stable, and it can be produced in a large amount.

Description

A kind of aldehyde group modified substrate of protein chip and preparation method thereof

Technical field

The present invention relates to a kind of aldehyde group modified substrate of protein chip and preparation method thereof, this aldehyde group modified substrate of protein chip can be fixed on chip surface by covalent bond with protein.

Technical background

Present protein-chip is meant at the fixing a large amount of albumen probes (can be antigen, antibody, acceptor, part, enzyme, substrate etc.) in solid support (carrier) surface, forms the protein array that high density is arranged.Utilize this chip and the liquid (body fluid or cell and tissue extract) that contains agnoprotein matter to carry out incubation reaction, the reaction back is detected with corresponding detecting system, last appliance computer analysis and comparison respective detection protein expression situation.Protein-chip can be divided into the Ag-Ab chip according to interaction principle, receptor-ligand chip, enzyme-substrate chip and polypeptide chip etc.

The making of protein-chip is more more complicated than the making of DNA chip, it is synthetic that protein is difficult in carrier surface, synthetic sequence is also very short, and the very difficult prediction of the space structure of synthetic peptide sequence, thereby the present protein-chip methods that adopt at the direct point sample of substrate surface more.A wherein important step is how the proteinaceous solid of these point samples to be fixed on chip surface.Because kinds of protein is various, character is different, and carrier surface can only be modified with a kind of chemistry or biological group, and this brings difficulty with combining of carrier surface group for probe.This is the problem that at first will solve in the preparation protein-chip process.

There are multiple diverse ways and label (the compound chain of a Tag weak point) to be used for protein fixing on the solid support surface.Absorption and covalent bonds as non-covalent bond, affinity capture ((1) Hoyer-Hansen G with streptavidin, Hamers M J, Pedersen A N, et al.Loss of ELISAspecificity due to biotinylation of monoclonal antibodies.[J] J.Immunol.Methods 2000,235 (1-2): 91-9; (2) Rowe C A, Tender L M, Feldstein M J, et al.Array biosensor for simultaneous identification of bacterial, viral, and proteinanalytes.Anal.Chem.1999,71,3846-3852).These protein fixedly technology respectively have relative merits, the absorption of non-covalent bond has high-adsorption-capacity, the space structure that keeps protein preferably, lower advantages such as non-specific adsorption, but the quantity of the uncontrollable protein adsorption of non-covalent absorption of physics and the directivity of protein adsorption, thereby the reaction efficiency of chip, the accuracy of reaction and the aspects such as reappearance of reaction all are affected.

The combination of covalent bond is at the carrier surface active chemical group of deriving, as aldehyde radical, epoxy radicals, amino, Acibenzolar ((3) MacBeath G, Schreiber S L.Printing proteins as microarrays forhigh-throughput function determination.Science 2000,289,1760-1763. (4) Ziauddin M, Sabatini D M.Microarrays of cell expressing defined cDNAs.Nature 2001,411,107-110.).And nickel-sodium nitrilo triacetate chelate ((5) Templin M F, Stoll D, Schrenk M, et al.Protein microarray technology.Trends Biotechnol2002 20:160-166) etc., is used for fixing protein.The short chain two ends respectively have the silane of a functional group can be used for protein molecule fixing at solid phase surface, its functional group can with the hydroxyl reaction of glass surface, and another one functional group (aldehyde radical or epoxy radicals) can react with the amino of protein, can also be to the further chemical modification of this functional group to reach the specificity of reaction.But epoxy group modified substrate process is comparatively complicated, and fixed efficiency is not high, and is though amido modified substrate is comparatively simple, undesirable in conjunction with fixed effect.

Make protein-chip and at first will select the carrier of suitable substrate as protein, the carrier of protein-chip will satisfy the requirement of several aspects: 1) substrate wants to be fit to the requirement of high density point sample; 2) Gu Ding protein difficult drop-off in hybridization, wash-out and analytic process; 3) homogeneity is good between the different batches substrate and between the same substrate each point; 4) signal to noise ratio (S/N ratio) is preferably arranged during the Analysis of Complex protein mixture; 5) protein probe has the shelf-life preferably; 6) can keep activity of proteins after the probe stationary.

Summary of the invention

The object of the present invention is to provide a kind of aldehyde group modified substrate of protein chip and preparation method thereof, it is a kind ofly to carry out aldehyde group modifiedly at glass sheet or silicon chip surface, is used for fixing protein, the substrate of protein chip that is made into.

The key issue that the present invention solves is how glass substrate or silicon chip to be carried out a series of chemical surface treatment, and the functional group aldehyde radical of activity is incorporated into substrate surface, provides a kind of confession enough effectively to firmly fix the chip or the silicon chip substrate of protein.

It is abundant, with low cost that this substrate of protein chip and preparation method thereof has material source, and method for making and flow process are simple, protein is firmly fixing, and characteristics such as fixed efficiency height are fit to the production of large-scale protein matter chip substrate.

The method for making of a kind of aldehyde group modified substrate of protein chip of the present invention comprises the assembling of hydroxylation, 3-aminopropyl-3 Ethoxysilane and three steps of assembling of terephthalaldehyde, and concrete steps are:

(1) at first is 98% H with concentration ₂SO ₄With 30% H ₂O ₂Surface treatment is carried out in the washing lotion immersion that is 7: 3 ratio preparation by volume, makes the substrate hydroxylation;

(2) the hydroxylated substrate of step (1) is cleaned dry up after, in the ethanol solution of 3-aminopropyl-3 Ethoxysilane, assemble; Forming one deck end is amino self-assembled monolayer;

(3) the assembling meron toasts in baking oven through rinsing with dry up, make 3-aminopropyl-3 Ethoxysilane fully (APTES) assemble with chip;

(4) with the substrate after the step assembling, in terephthalaldehyde solution, assemble again; The surface forms active aldehyde radical;

(5) take out rinsing, dry up, keep in Dark Place.

Described substrate is common glass substrate or silicon chip, and cleaning is a ultrasonic cleaning, uses washed with de-ionized water then.

The ethanol solution concentration of assembling 3-aminopropyl-3 Ethoxysilane of usefulness in above-mentioned steps (2) is 10-15mmol/L, and built-up time is 30-45 minute.

At the hydroxylated substrate described in the above-mentioned steps (1) is to use washed with de-ionized water earlier, and dries up with nitrogen.

At the baking oven baking temperature described in the above-mentioned steps (3) is 100-110 ℃, and the time is 45 minutes.

At the described assembling post rinse of above-mentioned steps (3) is with absolute ethyl alcohol rinsing in shaking table, dries up with nitrogen again; The described assembling post rinse of step (5) is with deionized water rinsing in shaking table, dries up with nitrogen again.

Assemble in the described terephthalaldehyde solution of above-mentioned steps (4), its solution concentration is 10-15mmol/L, and built-up time is 45-60 minute.

The aldehyde group modified substrate of protein chip of made of the present invention is characterized in that substrate surface has highdensity active aldehyde radical, and proteinaceous solid fixes on substrate surface.

Described high density active aldehyde radical, because terephthalaldehyde is a rigid molecule, can not twist arbitrarily, in its molecule two aldehyde radicals can only have one with substrate surface on the amino reaction and close with the two bonds of C=N, another is exposed to substrate surface, react with the amino group of protein, generate the two keys of C=N, proteinaceous solid is fixed on glass or the silicon chip surface.

The quality evaluating method of described aldehyde group modified chip substrate:

The quality of chip substrate is mainly estimated by fluorescence background, three parameters of fixed efficiency He Lisi coefficient of detecting substrate.Fluorescence background is a numerical value, refers to that chip substrate scans the mean fluorecence signal intensity of gained with the confocal scanning instrument under certain scanning intensity.In order not influence in use the interpretation of fluorescence signal, the fluorescence background signal should be lower as far as possible.Fixed rate is a chip substrate after point sample is fixing, the number percent of fluorescence signal intensity before cleansing solution washing back fluorescence signal intensity and the washing, and it has reflected the fixed efficiency of point sample district protein.Coefficient of dispersion is fixed rate standard deviation S and the ratio of fixed rate mean F, represents with percentage, and homogeneity of its size reflection substrate, coefficient of dispersion is more little, illustrates that the homogeneity of substrate is good more.

The present invention is intended to set forth the detection method of chip substrate fluorescence background and fixed rate, and the fixing means of the IgG of FITC mark on aldehyde group modified chip substrate provides the respective detection result by the aldehyde group modified protein-chip of embodiment 1,2,3 preparations simultaneously.(seeing table 1 for details)

(1) detection of fluorescence background

Protein-chip being inserted scanner Genepix 4000B, adopt the 532nm wavelength to detect, is 750 at PMT, and laser intensity is under 100% the condition whole point samples zone of substrate to be scanned, and is with from scanner then and reads the fluorescence background value the software.

(2) point sample and fixing

Point sample (shown in figure two) in 5 point sample sub-districts setting on the substrate of protein chip with point sample instrument Cartesian-prosy 5510-A, evenly put the matrix of system 5 * 5 in each sub-district, the albumen that point sample is used is the IgG of FITC mark, and concentration is 0.09 μ g/ μ l.

Substrate behind the point sample, at 37 ℃, relative humidity is that lucifuge is fixed 12 hours in 75% the wet tank, fixing back IgG and chip substrate combine synoptic diagram such as figure three.

(3) fixed rate

Protein-chip with point sample after fixing is with scanner Genepix 4000B scanning, scanning wavelength is 532nm, PMT is 750, laser intensity is 100%, preserve the picture (as shown in Figure 4) in each point sample district, carrying the fluorescence signal intensity of each sampling point of software analysis with scanner, is the fluorescence signal intensity PO of this sampling point before washing.

Protein-chip after the scanning is placed 0.5%Tween-20 PBS damping fluid rinsing 5 minutes, place PBS damping fluid washing 5 minutes again, scan with said method then, the gained fluorescence signal intensity is the fluorescence intensity P1 of washing back revise point.

The fixed rate Fi of each point of sample calculates by formula 1

The fixed rate of whole substrate is the average fixed rate F of all sampling points

$F=\frac{\underset{i=1}{\overset{125}{\mathrm{\Σ}}}\mathrm{Fi}}{125}----\left(2\right)$

(4) coefficient of dispersion Cv (calculating) by formula 3

$\mathrm{Cv}=\frac{S}{F}\×100\%----\left(3\right)$

Wherein F is the average fixed rate, and S is a standard deviation

$S=\sqrt{\frac{\underset{i=1}{\overset{125}{\mathrm{\Σ}}}{(\mathrm{Fi}-F)}^2}{124}}----\left(4\right)$

Advantage of the present invention and beneficial effect are:

1, the base material that adopts of the present invention is simple glass sheet or silicon chip, has advantages such as the source is abundant, cheap, and method and the technological process adopted are simple, and the equipment of use is simple, suitable large-scale production.

2, used the molecular self-assembled monolayer technology, organic molecule has been fixedly secured at glass substrate or silicon chip surface, difficult drop-off in hybridization, cleaning and testing process with crossing covalent bond.

3, this method can guarantee the high density and the homogeneity of surperficial aldehyde radical functional group, thereby guarantees that substrate has the characteristics of high fixed rate, low coefficient of dispersion.

4, adopt the rigid molecule terephthalaldehyde as aldehyde radical reagent, avoided the possibility of all reacting with lip-deep amino, guaranteed that the chip substrate surface has highdensity active aldehyde radical functional group with two aldehyde radicals in a part.

5, the chip substrate background fluorescence of this method preparation is lower, and chip surface has certain hydrophobicity, and sampling point does not obviously enlarge in protein example point sample process, and homogeneity is good.

Description of drawings

The preparation process substrate surface structural change synoptic diagram of the aldehyde group modified substrate of protein chip of Fig. 1

(1)---hydroxylation among the figure

(2)---the APTES self assembly

(3)---the assembling of terephthalaldehyde

The combination synoptic diagram of Fig. 2 protein and aldehyde group modified chip substrate

Fig. 3 point sample square formation fluorescence signal testing result

Wherein left side figure is the testing result before the cleansing solution washing, and right figure is the testing result after the cleansing solution washing.

Specific embodiment

The present invention is further illustrated below in conjunction with embodiment:

The preparation of one aldehyde group modified substrate of protein chip

Embodiment 1

Aldehyde group modified substrate of protein chip adopts is common flint glass sheet or cuts the suitable silicon chip of back size, and it is of a size of: the thick 1mm of the wide 52mm of long 75mm, the thick 0.4mm of silicon chip.Its preparation process mainly comprises the assembling of hydroxylation, APTES and three steps of assembling of terephthalaldehyde, this process substrate surface the structural change synoptic diagram as shown in Figure 1.The preparation process of aldehyde group modified substrate of protein chip is:

(1) with absolute ethyl alcohol with glass or silicon chip ultrasonic cleaning 5 minutes twice, and then with deionized water ultrasonic cleaning 5 minutes twice, the flush away substrate surface sticked thing.

(2) substrate after will cleaning is at the Piranha solution (98%H of new preparation ₂SO ₄: 30%H ₂O ₂=7: 3 volume ratios) soak 30Min in, make the complete hydroxylation of surface of glass slide, take out a large amount of washed with de-ionized water of substrate, and dry up with nitrogen stream.

(3) substrate of surface hydroxylation is assembled 30Min in the ethanol solution of 10mmol/LAPTES (3-aminopropyl-3-Ethoxysilane), take out substrate absolute ethyl alcohol rinsing twice in shaking table, and dry up with nitrogen stream.

(4) above-mentioned substrate is placed on baking oven.Baking 45Min makes the complete and substrate assembling of APTES (3-aminopropyl-3-Ethoxysilane) under 100 ℃ of conditions.

(5) substrate after will assembling is assembled 30Min in the acetone soln of the terephthalaldehyde of 10mmol/L, takes out the back with deionized water rinsing twice in shaking table, and nitrogen dries up.

(6) room temperature keeps in Dark Place and modifies good chip.

By the aldehyde group modified protein-chip that present embodiment provides, the fixing back of protein result numbers 1-3 and make substrate as shown in Figure 3, and its fixing main performance index is listed in the table 1.

Embodiment 2

The modification process process is with embodiment 1, and only the concentration of employed APTES is 12mmol/L, and the concentration of terephthalaldehyde is 15mmol/L, protein and aldehyde group modified glass sheet combination as shown in Figure 2, fixed efficiency is listed in (numbering 4-6) in the table 1.

Embodiment 3

The modification process process is with embodiment 1, and only the concentration of employed APTES is 15mmol/L, and built-up time is 45Min.Baking temperature is 110 ℃, and the time is 45 minutes.The concentration of terephthalaldehyde is 12mmol/L, and built-up time is 60Min.With protein and aldehyde group modified glass sheet combination as shown in Figure 2, fixed efficiency is listed in (numbering 7-9) in the table 1

Table 1 is fluorescence background, fixed rate and the coefficient of dispersion testing result of pressing the aldehyde group modified protein-chip of embodiment 1,2,3 preparations.Has low fluorescence background, the characteristic of high fixed rate and low coefficient of dispersion according to the prepared aldehyde group modified protein-chip of the present invention as can be seen from this table result.

Table 1

The substrate numbering	????1	????2	????3	????4	????5	????6	????7	????8	????9
										Fluorescence background	??3781	??3920	??3842	??3657	??4167	??4526	??4089	??3915	??4233
Fixed rate (%)	??41.2	??38.13	??47.53	??45.26	??39.48	??42.14	??45.81	??37.45	??38.55
										Coefficient of dispersion (%)	??6.24	??7.38	??7.23	??8.09	??8.43	??9.63	??5.04	??7.89	??9.13

Claims (9)

1. the method for making of an aldehyde group modified substrate of protein chip is characterized in that comprising the assembling of hydroxylation, 3-aminopropyl-3 Ethoxysilane and three steps of assembling of terephthalaldehyde, and concrete steps are:

(1) at first is 98% H with concentration ₂SO ₄With 30% H ₂O ₂Surface treatment is carried out in the washing lotion immersion that is 7: 3 ratio preparation by volume, makes the substrate hydroxylation;

(2) the hydroxylated substrate of step (1) is cleaned dry up after, in the ethanol solution of 3-aminopropyl-3 Ethoxysilane, assemble; Forming one deck end is amino self-assembled monolayer;

(3) the assembling meron toasts in baking oven through rinsing with dry up, make 3-aminopropyl-3 Ethoxysilane fully and chip assemble;

(4) with the substrate after the step assembling, in terephthalaldehyde solution, assemble again; The surface forms active aldehyde radical;

(5) take out rinsing, dry up, keep in Dark Place.

2. by the method for making of the described aldehyde group modified substrate of protein chip of claim 1, it is characterized in that described substrate is common glass substrate or silicon chip, cleaning is a ultrasonic cleaning, uses washed with de-ionized water then.

3. by the method for making of the described aldehyde group modified substrate of protein chip of claim 1, it is characterized in that the ethanol solution concentration of 3-aminopropyl-3 Ethoxysilane of step (2) assembling usefulness is 10-15mmol/L, built-up time is 30-45 minute.

4. by the method for making of the described aldehyde group modified substrate of protein chip of claim 1, it is characterized in that the hydroxylated substrate described in the step (1) is to use washed with de-ionized water earlier, and dry up with nitrogen.

5. by described aldehyde group modified substrate of protein chip of claim 1 and preparation method thereof, it is characterized in that the baking oven baking temperature described in the step (3) is 100-110 ℃, the time is 45 minutes.

6. by the method for making of the described aldehyde group modified substrate of protein chip of claim 1, it is characterized in that the described assembling post rinse of step (3) is with absolute ethyl alcohol rinsing in shaking table, dries up with nitrogen again; The described assembling post rinse of step (5) is with deionized water rinsing in shaking table, dries up with nitrogen again.

7. by described aldehyde group modified substrate of protein chip of claim 1 and preparation method thereof, it is characterized in that assembling in the described terephthalaldehyde solution of step (4), its solution concentration is 10-15mmol/L, and built-up time is 45-60 minute.

8. by the aldehyde group modified substrate of protein chip of any claim made in the method for making of the described aldehyde group modified substrate of protein chip of claim 1-7, it is characterized in that substrate surface has highdensity active aldehyde radical, proteinaceous solid fixes on substrate surface.

9. by the described aldehyde group modified substrate of protein chip of claim 8, it is characterized in that the high density active aldehyde radical can only have one with substrate surface on amino reaction and close with the two bonds of C=N, another is exposed to substrate surface, react with the amino group of protein, generate the two keys of C=N, proteinaceous solid is fixed on glass or the silicon chip surface.

Images