A method for preparing functionalized magnetic beads with surface-coupled photoaffinity chains

Abstract

The invention discloses a preparation method of functionalized magnetic beads with surface-coupled photoaffinity chains. The method comprises the following steps: activating carboxyl magnetic beads, incubating the activated carboxyl magnetic beads with photoaffinity linking chains to obtain carboxyl magnetic beads loaded with the photoaffinity linking chains, and blocking the surfaces of the carboxyl magnetic beads loaded with the photoaffinity linking chains with a blocking liquid to obtain the functionalized carboxyl magnetic beads. The photoaffinity linking chains use trifluoromethylphenyldiaziridine as photosensitive groups and have good chemical stability. Carbene intermediates generated after photolysis can be irreversibly combined with most small molecules and are relatively stable. A large number of different types of small molecules can be directly covalently docked to the surface of the functionalized carboxyl magnetic beads without the need for structure-activity relationship (QSAR) research data and corresponding chemical modifications. The small molecules can be used to fix a large number of small molecules with different structures in a complex system. The coupling of the small molecules with the functionalized magnetic beads can be achieved by only ultraviolet irradiation in the whole process. The method is fast and efficient, simple to operate, and has wider applicability.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for preparing functionalized magnetic beads with surface-coupled photoaffinity chains. Background Art

[0002] Magnetic beads refer to superparamagnetic microspheres with tiny particle sizes. They can be directed and concentrated under the action of an external magnetic field. After the external magnetic field is removed, they can restore their original structure and state, making complex liquid-solid separation technology quick and simple. Targeted substances with high purity can be obtained by simple elution. After the surface functionalization of magnetic beads, they can not only increase their dispersibility in the solution, but also have the effect of coupling with various biological macromolecules or small molecules. In recent years, functionalized magnetic beads have developed rapidly and are widely used in biomedical fields such as environmental monitoring, gene classification, medical imaging technology and drug transport, protein extraction and purification. At present, the functionalized magnetic beads on the market mainly include amino magnetic beads, carboxyl magnetic beads, hydroxyl magnetic beads, epoxy magnetic beads, etc. Usually, the biological macromolecules or small molecules coupled with the above-mentioned functionalized magnetic beads need to be specifically chemically modified, and the modification process cannot destroy the original active site of the molecule. This requirement is very demanding and greatly limits the application of functionalized magnetic beads. Therefore, in order to overcome this difficulty, it is necessary to design and synthesize a universal functionalized magnetic bead that is suitable for coupling more components and easy to operate, so as to further improve the use value of the functionalized magnetic beads.

[0003] With the continuous progress of photochemical technology, people have discovered a series of chemical groups with photosensitivity, such as benzyl trifluorobisaziridine, which has good chemical stability and a photolysis wavelength (about 360nm) greater than the ultraviolet absorption wavelength of protein. This compound produces a highly reactive intermediate carbene under ultraviolet light, which can undergo photocrosslinking reactions with a variety of chemical bonds, such as OH, CH, NH, etc. It is fast, simple and time-controlled, and is widely used in many research fields such as chemistry, biology, medicine and materials. At present, there are no reports on this method of functionalizing carboxyl magnetic beads with surface-coupled photoaffinity linkers.

[0004] The present invention provides a novel method for preparing functionalized magnetic beads with surface-coupled photoaffinity linker chains. The obtained novel functionalized magnetic beads can generate highly active intermediates under the irradiation of light of a specific wavelength, and form a specific irreversible covalent bond chemical reaction with most compounds, thereby greatly expanding the application scope of the functionalized magnetic beads to a certain extent. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing functionalized carboxyl magnetic beads with surface-coupled photoaffinity linker chains. The functionalized carboxyl magnetic beads are independent of specific functional groups and can be used for non-selective fixation of compounds, thereby constructing a universal active molecule fixation method that can be used to study the direct interactions of complex systems.

[0006] The technical solution of the present invention is as follows:

[0007] A method for preparing functionalized magnetic beads with surface-coupled photoaffinity chains comprises the following steps:

[0008] (1) Preparation of oleic acid-coated magnetic nanoparticles

[0009] Under the protection of inert gas, a mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O was added to ammonia water, reacted at 50°C for 0.5h, then separated by magnetic attraction, washed, and Fe3O4 magnetic nanoparticles were obtained; the obtained Fe3O4 magnetic nanoparticles were dispersed in water, oleic acid was added under the protection of inert gas, reacted at 50°C for 1h, then separated by magnetic attraction, washed, and dried with N2 to obtain oleic acid-coated magnetic nanoparticles;

[0010] The preferred molar ratio of FeCl3·6H2O to FeCl2·4H2O is 2:1;

[0011] Preferably, in the mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O, the concentration of FeCl3·6H2O is 11.2mM, and the concentration of FeCl2·4H2O is 5.6mM; preferably, the volume amount of ammonia water accounts for 8% to 10% of the mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O; preferably, the volume amount of oleic acid accounts for 1% to 2% of the mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O;

[0012] The solvent and detergent used in the above operation are all deoxygenated water; the stirring speed during the reaction is 500-600 rpm;

[0013] (2) Preparation of carboxylated magnetic nanoparticles

[0014] The oleic acid-coated magnetic nanoparticles obtained in step (1) are dispersed in n-hexane, ethyl acetate and acetonitrile are added and mixed, and an aqueous sodium periodate solution is added under stirring, and the mixture is reacted at room temperature for 2 h, followed by magnetic separation, washing, and drying with N2 to obtain carboxylated magnetic nanoparticles;

[0015] Preferably, the content of oleic acid-coated magnetic nanoparticles dispersed in n-hexane is 10 mg / mL;

[0016] Preferably, the volume ratio of ethyl acetate:acetonitrile:n-hexane is 1:1:1;

[0017] Preferably, the mass fraction of sodium periodate in the sodium periodate aqueous solution is 2% to 3%, and the volume ratio of the sodium periodate aqueous solution to n-hexane is 1.5:1;

[0018] The stirring speed during the preferred reaction is 600 rpm;

[0019] (3) Activation of carboxylated magnetic nanoparticles

[0020] Adding an activation solution to the carboxylated magnetic nanoparticles obtained in step (2), stirring and activating at room temperature for 2 h, and then vortex washing with N,N-dimethylformamide to obtain activated carboxylated magnetic beads;

[0021] The activation solution is a solution of N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in N,N-dimethylformamide, and the molar ratio of NHS to EDC is 1:1;

[0022] The N,N-dimethylformamide used in the above operation steps needs to be added with molecular sieves and allowed to stand for more than 24 hours, mainly for the purpose of removing water;

[0023] (4) Preparation of functionalized carboxyl magnetic beads loaded with photoaffinity linker

[0024] Add the activated carboxyl magnetic beads obtained in step (3) to the N,N-dimethylformamide solution containing N,N-diisopropylethylamine and the photoaffinity linker, stir and react for 8 to 12 hours at room temperature in the dark, then separate by magnetic attraction, wash by vortexing with N,N-dimethylformamide, add the washed magnetic beads to the blocking solution, stir for 1 hour at room temperature in the dark, then separate by magnetic attraction, wash, and vacuum dry to obtain the functionalized carboxyl magnetic beads loaded with the photoaffinity linker (stored in a -20°C refrigerator);

[0025] Preferably, in the N,N-dimethylformamide solution containing N,N-diisopropylethylamine and the photoaffinity linker, the concentration of N,N-diisopropylethylamine is 50 mM, and the concentration of the photoaffinity linker is 10 mM; the photoaffinity linker is N-[2-[2-(2-Aminoethoxy)ethoxy]ethyl]-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzamide, and the structural formula is as follows:

[0026]

[0027] The blocking solution was 1 M ethanolamine in N,N-dimethylformamide;

[0028] The N,N-dimethylformamide used in the above operation steps needs to be added with molecular sieves and allowed to stand for more than 24 hours, mainly for the purpose of removing water.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention adopts activated carboxyl magnetic beads and photoaffinity linker chains for coupling to obtain functionalized carboxyl magnetic beads. The photoaffinity linker chains and the carboxyl magnetic beads are combined together by covalent bonds. A large number of different kinds of small molecules do not need structure-activity relationship (QSAR) research data and corresponding chemical modifications, and can be directly covalently docked. The method can be used to fix many chemical molecules with different structures in complex systems. The method is fast, efficient and easy to operate. The whole process only requires ultraviolet irradiation to achieve coupling of small molecules and matrix.

[0031] Fe3O4 magnetic nanoparticles were prepared by coprecipitation method. Oleic acid-modified Fe3O4 magnetic nanoparticles were prepared by oxidizing the C=C bonds on the surface of oleic acid to carboxyl groups using sodium periodate to obtain carboxyl magnetic beads. The raw materials used were cheap and easily available. The preparation process was simple and highly operable, and carboxyl magnetic beads with superparamagnetism could be quickly obtained.

[0032] The dual activators 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide were used to activate the carboxyl magnetic beads in equal volumes at a molar concentration ratio of 1:1. N-hydroxysuccinimide is a chemical modifier that converts the carboxyl groups on the surface of the magnetic beads into amine-reactive NHS esters, which can improve the coupling efficiency mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and improve the activation effect of the carboxyl magnetic beads.

[0033] Benzyltrifluorobisaziridine is used as the photoaffinity group of the photoaffinity linker chain, which has good chemical stability and can withstand acid, alkali and other environments. The carbene intermediates produced after photolysis can irreversibly bind to most small molecules and are relatively stable, which is convenient for subsequent research. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention provides a preparation route for functionalized carboxyl magnetic beads with surface-coupled photoaffinity linker chains.

[0035] Figure 2 Scanning electron microscopy (a), XRD (b), infrared characterization (c) and conductivity titration (d) images of carboxyl-functionalized magnetic nanoparticles.

[0036] Figure 3 Figure 1 is a TLC characterization diagram of the supernatant and the washing solution after the photoaffinity linker chain is fixed. The spot at the Rf value of 0.815 is the characteristic spot of the photoaffinity linker chain, where 1 is the photoaffinity linker chain solution before fixation, 2 is the supernatant after the photoaffinity linker chain is fixed, and 3 is the washing solution after the photoaffinity linker chain is fixed.

[0037] Figure 4The liquid phase detection diagram (a) and mass spectrometry characterization diagram (b) of the supernatant before and after the photoaffinity linker was fixed, where the peak at m / z 383.1 is the [M+Na] + The fragment ion peak at m / z 361.1 is the [M+H] of the photoaffinity linker. + fragment ion peaks.

[0038] Figure 5 This is a liquid phase detection diagram of ginsenoside Re in Example 2 before and after reaction with functionalized magnetic beads. DETAILED DESCRIPTION

[0039] The present invention is further described below by means of specific embodiments, but the protection scope of the present invention is not limited thereto.

[0040] Embodiment 1

[0041] A method for preparing functionalized magnetic beads with surface-coupled photoaffinity chains comprises the following steps:

[0042] (1) Preparation of Fe3O4 magnetic nanoparticles coated with oleic acid:

[0043] 150mL of aqueous solution containing 11.2mM FeCl3·6H2O and 5.6mM FeCl2·4H2O was heated to 50°C, and then added to 12.5mL of ammonia water (25%-28%) under N2 flow, and stirred in a 50°C water bath for 0.5h at a speed of 500rpm. After the reaction, magnetic separation was performed to fully wash. The washed Fe3O4 magnetic nanoparticles were ultrasonically dispersed in 150mL of deoxygenated water, and heated to 50°C in a water bath under N2 flow and stirring, and then 2mL of oleic acid was added to stir and react for 1h. After the reaction, magnetic separation was performed to fully wash with anhydrous ethanol, and then dried with nitrogen for use.

[0044] (2) Preparation of carboxylated magnetic nanoparticles:

[0045] 100 mg of the magnetic particles in step (1) were dispersed in 10 mL of n-hexane, and 20 mL of a mixed solution of ethyl acetate / acetonitrile with a volume ratio of 1:1 was added, and 15 mL of a sodium periodate aqueous solution (0.41 g of sodium periodate) was added under stirring, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the mixture was magnetically separated and washed with ethanol and ultrapure water in turn, and dried with N2 to obtain carboxyl functionalized magnetic nanoparticles.

[0046] Characterization Figure 2 . Figure 2In the figure, a is a 200,000-fold electron microscope image of carboxyl magnetic nanoparticles; b is an XRD pattern of carboxyl magnetic nanoparticles, which has obvious absorption peaks at diffraction angles of 35.6°, 43.5°, 53.9° and 67.6°, which is highly consistent with the Fe3O4 standard powder diffraction data card (98-000-0294). By comparing the standard card, it is known that the corresponding crystal planes are 211, 400 and 511, respectively, indicating that the prepared magnetic material is ferroferric oxide crystals; c is an infrared spectrum of carboxyl magnetic nanoparticles, and the lower curve 3424cm -1 The broad peak is the stretching vibration absorption peak of -OH, 1630cm -1 The absorption peak of C=O is at about 3007cm in the upper curve, indicating the formation of carboxyl group. -1 There are absorption peaks of different intensities at , which is the characteristic absorption peak of C=C double bond, indicating that the surface of magnetic Fe3O4 particles is successfully coated with oleic acid molecules, and the absorption peak at this point in the curve below almost disappears, indicating that the double bond is almost completely oxidized. The results show that the method synthesizes oleic acid-coated magnetic Fe3O4 particles and oleic acid is oxidized to carboxyl groups, that is, the surface of the magnetic particles contains carboxyl groups. d is the conductivity titration diagram of carboxyl magnetic nanoparticles, and the results show that the carboxyl content is 2.061*10 -7 mol / mg.

[0047] (3) Preparation of functionalized carboxyl magnetic beads loaded with photoaffinity linker:

[0048] Activation of carboxyl magnetic beads: Prepare a N,N-dimethylformamide solution containing N-hydroxysuccinimide (1.15 g, 200 mM) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (1.53 g, 200 mM) passed through a molecular sieve and allowed to stand for more than 24 hours. The purpose of this step is mainly to remove water, that is, the required activation solution. Add the activation solution to the carboxyl magnetic beads prepared in step (2) and stir at room temperature for 2 hours at a speed of 500 rpm. After the reaction is completed, add N,N-dimethylformamide passed through a molecular sieve and allowed to stand for multiple vortex washings to obtain activated carboxyl magnetic beads.

[0049] Fixation of the photoaffinity chain on the activated carboxyl magnetic beads: To ensure the stability of the trifluoromethylphenylbis(aziridine) photoactive group in the photoaffinity linker chain, subsequent operations need to be performed under light-proof conditions, and the N,N-dimethylformamide used must be passed through a molecular sieve and left to stand for more than 24 hours. Add N,N-diisopropylethylamine (41 μL, 50 mM) and the photoaffinity linker chain (20 mg, 10 mM) to the activated and washed carboxyl magnetic beads, add N,N-dimethylformamide to 10 mL, stir the reaction overnight at room temperature in the dark, stir at 300-400 rpm, separate magnetically, remove the reaction solution, add N,N-dimethylformamide, and vortex wash several times to obtain the carboxyl magnetic beads loaded with the photoaffinity linker chain.

[0050] Add 1.5 mL of ethanolamine to a 25 mL volumetric flask, and dilute with N,N-dimethylformamide to obtain a 1 M ethanolamine blocking solution. Transfer the above photoaffinity linker-loaded carboxyl magnetic beads to a round-bottom flask filled with blocking solution, stir and react for 1 h in the dark at a stirring speed of 300-400 rpm. After stirring, perform magnetic separation, remove the blocking solution, wash with ultrapure water and anhydrous ethanol in turn, and vacuum dry at 41°C to obtain functionalized carboxyl magnetic beads loaded with photoaffinity linkers, which are stored in a -20°C refrigerator.

[0051] For the photoaffinity linker, the supernatant before and after fixation and the wash solution after fixation were collected for thin layer characterization analysis. The results are as follows Figure 3 As shown: Figure 3 1 is the photoaffinity linker solution before fixation, 2 is the supernatant after photoaffinity linker fixation, and 3 is the washing solution after photoaffinity linker fixation. Developing agent: DCM:MeOH=8:2, UV lamp 254nm color development.

[0052] The Rf values ​​of the characteristic spots of the photoaffinity linker solution before fixation and the supernatant after fixation are basically the same. Compared with before fixation, the fluorescence of the photoaffinity linker becomes lighter after the photoaffinity linker is fixed to the surface of the magnetic beads, and the fluorescent spots of the washing solution disappear after fixation, indicating that the photoaffinity linker synthesized by the present invention has good reactivity, and it is preliminarily judged that the photoaffinity linker is fixed to the surface of the magnetic beads.

[0053] In order to confirm the specific situation of the photoaffinity linker fixed on the magnetic beads, the supernatant before and after the photoaffinity linker was collected for LC-MS characterization. The liquid phase conditions were: octadecylsilane bonded silica gel as filler (Aglient Eclipse C18, 4.6mm×250mm, 5.0μm); column temperature 25℃; flow rate 1.0mL·min-1; detection wavelength 230nm; injection volume 10μL; acetonitrile-pure water as mobile phase for elution, and the gradient elution conditions are shown in the following table:

[0054]

[0055] The results are as follows Figure 4 As shown, there is a strong [M+Na]+ fragment ion peak at m / z 383.0 in the supernatant mass spectra before and after fixation of the photoaffinity linker chain, and a strong [M+H]+ fragment ion peak at m / z 361.0, indicating that the structure of the photoaffinity linker chain has not changed and can be stably fixed on the surface of the activated carboxyl magnetic beads; there is a difference in the peak area of ​​the supernatant before and after fixation of the photoaffinity linker chain, which further confirms that the photoaffinity linker chain is cross-linked with the activated carboxyl magnetic beads.

[0056] Embodiment 2

[0057] This embodiment provides a method for preparing ginsenoside Re magnetic microspheres, which is specifically carried out according to the following steps:

[0058] (1) Preparation of Ginsenoside Re Sample Solution

[0059] Weigh 0.5 mg of ginsenoside Re and add methanol to make up to 2 mL to prepare a 0.25 mg / mL ginsenoside Re solution.

[0060] (2) Bonding of functionalized magnetic beads with surface-coupled photoaffinity chains to ginsenoside Re

[0061] Accurately weigh 20 mg of surface-coupled photoaffinity functionalized magnetic beads into a weighing bottle, and add 1 mL of ginsenoside Re solution in (1). Shake gently to spread the magnetic beads as evenly as possible on the bottom. Place in a 45°C vacuum dryer to remove excess solvent, and transfer the dried special magnetic beads to a UV spectrometer and irradiate at 365 nm for 3 hours. After taking out, shake and wash with methanol several times, place the washed sample in a 45°C vacuum drying oven to dry to obtain ginsenoside Re loaded magnetic microspheres, and store in a -20°C refrigerator.

[0062] (3) Liquid chromatography analysis

[0063] Sample: Take the ginsenoside Re solution in (1) as the sample before cross-linking, combine the washing liquid in (2) and evaporate the solvent in a solvent evaporator, add methanol to re-dissolve, and prepare the sample.

[0064] The peak area difference between the two groups of samples was determined by high performance liquid chromatography:

[0065] The HPLC conditions are as follows: the chromatographic column is a ZORBAX SB-C18 column (4.6×250 mm, 5 μm); the column temperature is 30° C.; the injection volume is 5 μL; and the flow rate is 0.5 mL·min -1 ; The detection wavelength is 203nm; the mobile phase consists of acetonitrile and pure water; the gradient elution program is:

[0066]

[0067]

[0068] The high performance liquid chromatography determination method is: take the above sample through a 0.22 μm microporous filter membrane, take the subsequent filtrate for liquid phase analysis, record the chromatogram peak area, and compare the difference between the two groups of samples. From the difference in the peak area of ​​the two groups of samples, it can be seen that the peak area before cross-linking is 1184.833, and the peak area after cross-linking is 586.771, indicating that ginsenoside Re is successfully bonded to the magnetic nanoparticles loaded with photoaffinity groups. According to the formula: fixation rate = (peak area before cross-linking - peak area after cross-linking) / peak area before cross-linking × 100%, the ginsenoside fixation rate is calculated to be approximately 50%.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing functionalized magnetic beads with surface-coupled photoaffinity chains, characterized in that: The steps include: (1) Preparation of oleic acid-coated magnetic nanoparticles Under the protection of inert gas, a mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O was added to ammonia water, reacted at 50°C for 0.5h, then separated by magnetic attraction, washed, and Fe3O4 magnetic nanoparticles were obtained; the obtained Fe3O4 magnetic nanoparticles were dispersed in water, oleic acid was added under the protection of inert gas, reacted at 50°C for 1h, then separated by magnetic attraction, washed, and dried with N2 to obtain oleic acid-coated magnetic nanoparticles; In step (1), the molar ratio of FeCl3·6H2O to FeCl2·4H2O is 2:1; in the mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O, the concentration of FeCl3·6H2O is 11.2 mM, and the concentration of FeCl2·4H2O is 5.6 mM; the volume of ammonia water accounts for 8% to 10% of the mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O; the volume of oleic acid accounts for 1% to 2% of the mixed aqueous solution of FeCl3·6H2O and FeCl2·4H2O; (2) Preparation of carboxylated magnetic nanoparticles The oleic acid-coated magnetic nanoparticles obtained in step (1) are dispersed in n-hexane, ethyl acetate and acetonitrile are added and mixed, and an aqueous sodium periodate solution is added under stirring, and the mixture is reacted at room temperature for 2 h, followed by magnetic separation, washing, and drying with N2 to obtain carboxylated magnetic nanoparticles; In step (2), the content of oleic acid-coated magnetic nanoparticles dispersed in n-hexane is 10 mg / mL; Ethyl acetate: The volume ratio of acetonitrile to n-hexane is 1:1:1; the mass fraction of sodium periodate in the sodium periodate aqueous solution is 2% to 3%, and the volume ratio of the sodium periodate aqueous solution to n-hexane is 1.5:1; (3) Activation of carboxylated magnetic nanoparticles Adding an activation solution to the carboxylated magnetic nanoparticles obtained in step (2), stirring and activating at room temperature for 2 h, and then vortex washing with N,N-dimethylformamide to obtain activated carboxylated magnetic beads; In step (3), the activation solution is an N,N-dimethylformamide solution of N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the molar ratio of N-hydroxysuccinimide to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the activation solution is 1:1; (4) Preparation of functionalized carboxyl magnetic beads loaded with photoaffinity linker Add the activated carboxyl magnetic beads obtained in step (3) to an N,N-dimethylformamide solution containing N,N-diisopropylethylamine and a photoaffinity linker, stir and react for 8 to 12 hours at room temperature in the dark, then separate by magnetic attraction, vortex wash with N,N-dimethylformamide, add the washed magnetic beads to a blocking solution, stir for 1 hour at room temperature in the dark, then separate by magnetic attraction, wash, and vacuum dry to obtain functionalized carboxyl magnetic beads loaded with a photoaffinity linker; In step (4), the structural formula of the photoaffinity ligation chain is as follows: In the N,N-dimethylformamide solution containing N,N-diisopropylethylamine and the photoaffinity linker, the concentration of N,N-diisopropylethylamine is 50 mM, and the concentration of the photoaffinity linker is 10 mM; the blocking solution is a 1 M ethanolamine solution in N,N-dimethylformamide.

Images