CN103234948B - Application of pyrene and derivative as photosensitive perssad deprotection sensibilization reagent in biological chip production - Google Patents

Abstract

The invention discloses the application of pyrene and its derivatives as photosensitive group deprotection and sensitization reagents in the preparation of biochips. With the re-enhancement of fluorescence, three deprotections of the photosensitive group are realized (the first is the original ultraviolet light, the second is the energy transfer, and the third is the generated fluorescence), which accelerates the deprotection rate of the photosensitive group . At the same time, due to the use of the fluorescent properties of fluorescent compounds, it can effectively avoid the damage of cleavage free radicals caused by general photosensitizers (such as ITX series), and improve the quality of photosensitive group deprotection.

Description

Application of Pyrene and Its Derivatives as Reagents for Deprotection and Sensitization of Photosensitive Groups in Biochip Preparation

technical field

The invention belongs to the technical field of biochips, and specifically relates to the application of pyrene and its derivatives as photosensitive group deprotection and sensitization reagents in the preparation of biochips.

Background technique

Due to the application of photosensitive groups, the development of biochip in situ synthesis technology is more rapid. Some specific chemical groups are protected with photosensitive groups. Under the radiation of specific wavelength light, when the photosensitive group absorbs enough energy, it will be disconnected from the protected active group. This process is called photodeprotection reaction. That is to say, exposure can be used as the control condition for deprotection, and some protected groups in the light-irradiated area can be deprotected in a regular and quantitative manner. This technology is widely used in light-guided in situ synthesis. Fodor et al. first synthesized a polypeptide with a length of 5 amino acids by photolithography. It is also widely used in the synthesis of protein chips, gene chips, etc. . The photodeprotection efficiency of the photosensitive group is one of the factors that determine the success of photolithographic synthesis. In the case of DNA chip synthesis, in the process of repeated photodeprotection and coupling, the leaving time of the photosensitive group must be moderate, otherwise it will take dozens of hours to synthesize a DNA probe with a length of tens of bases. Even days. How to improve the photosensitive efficiency of UV-sensitive groups, especially in the case of low light intensity, has become an increasingly important issue.

In order to improve the leaving efficiency of photosensitive groups, a lot of research work has been carried out. When the photosensitive group and the light intensity are determined, choosing a suitable exposure environment becomes the decisive factor for the deprotection efficiency of the photosensitive group. Most photolithography experiments are carried out in solution, in order to maximize the photosensitivity of the photosensitive group. Therefore, adding a photosensitizer to the solvent becomes one of the options. For example, Woll, D et al. proposed to use the triplet photosensitizer 2-isopropylthioxanthone (ITX) as a sensitizing reagent for the deprotection of the photosensitive group in DNA chip synthesis, which effectively increased the photosensitivity in an oxygen-free environment. rate of deprotection. Nevertheless, the current research has not reached a satisfactory level for wider application.

In the process of in situ synthesis of peptide nucleic acid (PNA) chips by photolithography, it is found that if ITX series photosensitizers are added to the exposure solvent, the exposure process will bring many negative effects, and even destroy all amino active groups, which may It is caused by the generation of destructive free radicals after the ITX series photosensitizers absorb light. Therefore, in order to overcome the negative effects brought by cleavage-type photosensitizers, it is necessary to find a new photosensitizer that can be used in the biochip in situ synthesis process.

Pyrene and some derivatives of pyrene are a class of conjugated aromatic compounds with fluorescent properties, which have high quantum efficiency in dilute solutions and can emit fluorescence close to the excitation wavelength. However, such test substances have only been used as fluorescent labeling reagents and photopolymerization reagents for a long time, or used in the field of organic optoelectronics, and there is no report on the application of pyrene and its derivatives as photosensitive group deprotection and sensitization reagents in the preparation of biochips. .

Contents of the invention

The invention aims to overcome the deficiencies and prejudices of the prior art, and provide a new application of pyrene and its derivatives as photosensitive group deprotection and sensitization reagents in the preparation of biochips.

In order to achieve the above object, the technical solution provided by the invention is:

Application of pyrene and its derivatives as photosensitive group deprotection sensitizing reagents in biochip preparation.

Wherein, the concentration of the pyrene and its derivatives is 0.08-0.12M, preferably 0.1M.

The pyrene derivatives are pyrenemethanol, N-pyrenemethylacetamide, 1,4-dipyrenebenzene, 1-ethynylpyrene, 1,3,6,-tris(trimethylsilyl)pyrene, etc.

The biochip is a peptide nucleic acid biochip.

The preparation of the biochip is to prepare the peptide nucleic acid biochip by light-guided in-situ synthesis method.

The in-situ synthesis method adopts 10-13mW/cm ² , 365nm ultraviolet light, preferably 12mW/cm ² , 365nm ultraviolet light when preparing the peptide nucleic acid biochip.

The present invention will be further described below in conjunction with principle:

The present invention uses pyrene and its derivatives as Zeng-sensitizing reagents to accelerate the deprotection rate of photosensitive groups when preparing peptide-nucleic acid biochips by in-situ synthesis.

The ITX series of photosensitizers are mainly used for the deprotection of the hydroxyl group protected by the photosensitive group, and play a better role in DNA synthesis. However, the ultraviolet light used in the previous report of peptide synthesis is relatively strong, reaching about 35mW, and does not need to be used. Photosensitizer. The inventors of the present invention used relatively weak ultraviolet light (10-13mW/cm ² , ultraviolet light at 365nm) when synthesizing PNA (the photosensitive group is connected to the amino group), therefore, considering the use of a photosensitizing enhancing reagent, the result It is found that there are many side effects of ITX series. After the ITX series of photosensitizers are excited, the energy that is not transmitted will crack ITX and generate free radicals, thereby destroying the reaction system; and pyrene and its derivatives release excess energy in the form of fluorescence, thereby avoiding negative effects. The wavelength difference between the excitation light and the emission light of the substance is very small, that is, both of them are in the wavelength band that the photosensitive group can sense. One time is the fluorescence generated).

The photosensitive group is irradiated by ultraviolet light of a certain wavelength. When the photon energy is sufficient, the photosensitive group will be disconnected from the protected active group, thereby realizing deprotection. But in the case of constant or low light intensity, in order to speed up the rate of photodeprotection, it is necessary to speed up the energy absorption efficiency of the photosensitive group. In a solvent environment, the photosensitizer pyrene is excited to a triplet state under the irradiation of a certain wavelength band, and through the collision with the photosensitive group, the triplet energy is transferred to the photosensitive group to accelerate photodeprotection. Electrons in the triplet state usually eventually lose their energy due to thermal collisions and thus do not emit light. However, due to the fluorescence properties of pyrene, it can not only be excited to the triplet state, but also can be excited to the singlet state and emit fluorescence. This is because after the electrons are excited to the singlet state, after vibrational relaxation and internal transfer, the electrons reach the excited singlet state The lowest layer (V=0) of the state (S1), and then transitions to the ground state (S0) in the form of radiation, emitting fluorescence. The fluorescence wavelength is very close to the excitation wavelength and can be reabsorbed by the photosensitive group, that is to say, under the joint action of the radiation ultraviolet light and the singlet and triplet energy of the photosensitizer, the energy absorption of the photosensitive group is increased to achieve three deprotection Effects (the first is the original UV light, the second is the energy transfer, and the third is the generated fluorescence), which in turn increases the deprotection rate of the photosensitive group.

Not only pyrene has fluorescent properties, but pyrene derivatives also have similar properties, such as N-pyrene methylacetamide, which is used for the deprotection of photosensitive groups, which significantly improves the photodeprotection rate, and because N-pyrene methyl acetamide The fluorescence generated by acetamide is close to the excitation wavelength of the photosensitive group and the fluorescence intensity is slightly higher than that of pyrene (as shown in Figure 3), which is more conducive to the deprotection of the photosensitive group.

The invention discloses the use of pyrene, a compound capable of emitting fluorescence, and derivatives of pyrene as a sensitizing reagent for deprotection of the photosensitive group. The third deprotection of the photosensitive group is realized by utilizing the enhancement of fluorescence again (the first time) is the original ultraviolet light, the second is the energy transfer, and the third is the generated fluorescence), which accelerates the deprotection rate of the photosensitive group. At the same time, due to the use of the fluorescent properties of fluorescent compounds, it can effectively avoid the damage of cleavage free radicals caused by general photosensitizers (such as ITX series), and improve the quality of photosensitive group deprotection (as shown in Figure 2).

In a word, the present invention proposes for the first time that the fluorescence of the fluorescent photosensitizer is used to accelerate the deprotection of the photosensitive group, which produces unexpected effects. Pyrene and its derivatives are used in the deprotection of photosensitive groups. On the one hand, these reagents can absorb ultraviolet light of a certain wavelength and transfer energy to the photosensitive group; Fluorescence with a similar wavelength of light, which can provide secondary exposure to the photosensitive group, accelerate the deprotection rate of the photosensitive group, and overcome the negative effects of free radicals caused by photocleavage caused by general photosensitive reagents.

Description of drawings

Fig. 1 is a scanning result diagram of a photosensitive group exposure efficiency comparison experiment in which pyrene acts as a photosensitizer to accelerate the synthesis of a peptide nucleic acid chip;

Fig. 2 is a scanning result diagram of a comparison experiment of photosensitive group exposure efficiency in pyrene and ITX acting as a photosensitizer to accelerate the synthesis of a peptide nucleic acid chip;

Figure 3 is the fluorescence spectrum of the dioxane solution of pyrene, N-pyrenemethylacetamide and ITX at a concentration of 10 ^-5 M;

Fig. 4 The scanning result diagram of the comparison experiment of pyrene and pyrene derivative N-pyrene methylacetamide exposure link.

Detailed ways:

Example 1

Pyrene Acting as Photosensitizer to Accelerate the Experiment of Exposure in the Synthesis of Peptide Nucleic Acid Chip

Using light-guided in situ synthesis of peptide nucleic acid chips, the process of deprotecting the photosensitive group is the key link in the synthesis. To synthesize peptide nucleic acid chip probes generally 13-17 bases in length, you need to carry out 13- There are 17 exposures, and new bases are coupled after each exposure, that is to say, the efficiency of exposure directly affects the synthesis efficiency of the entire peptide nucleic acid chain. In this experiment, the light intensity is 365nm ultraviolet light of 12mW/cm ² , the monomer photosensitive group used in the synthesis is 2-(2-nitrobenzene) propoxycarbonyl (NPPOC), in order to prove the sensitization effect of pyrene, The slides modified with NPPOC-protected amino groups were subjected to exposure experiments under the same time gradient in different solvents (1,4-dioxane and pyrene) (that is, the deprotection process of the photosensitive group), after exposure Treat with 1 mM rhodamine isothiocyanate in N-methylpyrrolidone solution for 30 minutes, then ultrasonically wash with N-methylpyrrolidone, methanol, dichloromethane, and aqueous solution containing 0.3% Tween for 5 minutes, and then Rinse with double distilled water, blow dry with nitrogen, and detect and analyze the fluorescence signal by Genepix Pro 4000B scanner. The exposure results in the organic solvent 1,4-dioxane (Dixoane) were compared with the results in the dixoane solution added with 0.1M pyrene, and the scanning results are shown in Figure 1.

Under the irradiation of 365nm ultraviolet light and exposure in dioxane, the saturation phenomenon appeared in about 10 minutes, but the overall fluorescence intensity was low. It can be seen that in the case of only using dioxane solvent, the photosensitive group does not necessarily achieve 100% deprotection. Or the departure of the photosensitive group and the destruction of the amino group produced in the exposure reaction occur prematurely in balance. Saturation occurred at 17 minutes in the dioxane solution added with photosensitizer pyrene, but the overall signal intensity was stronger than exposure in dioxane, that is to say, in the case of low light intensity, adding an appropriate amount of pyrene can make the illuminated area Achieve maximum deprotection. This is a key step in the synthesis of biochips. On the basis of complete exposure, coupling and re-exposure can ensure the accuracy of biochip synthesis. This example shows that the photosensitizer pyrene does play a role in improving the photodeprotection efficiency in the exposure solvent .

Example 2

Exposure comparison experiment of photosensitizer 2-isopropylthioxanthone (ITX) and pyrene in peptide nucleic acid (PNA) chip synthesis

The photosensitizer ITX has a certain effect in accelerating the hydroxyl group protected by the photosensitive group in the synthesis of DNA chips. Therefore, the photosensitizer ITX and the photosensitizer pyrene were exposed in the synthesis of peptide nucleic acid (PNA). In this experiment, the light intensity is 365nm ultraviolet light of 12mW/cm ² , the monomer photosensitive group used in the synthesis is 2-(2-nitrophenyl)propoxycarbonyl (NPPOC), and the amino group modified with NPPOC protected The slides were subjected to exposure experiments with the same time gradient in different solvents (photosensitizers ITX and pyrene) (that is, the deprotection process of the photosensitive group), and 1 mM N-methyl rhodamine isothiocyanate was used after exposure. Treat with pyrrolidone solution for 30 minutes, then ultrasonically wash with N-methylpyrrolidone, methanol, dichloromethane, and aqueous solution containing 0.3% Tween for 5 minutes, then rinse with double distilled water, blow dry with nitrogen, and the fluorescence signal is obtained by Genepix Pro 4000B scanner detection and analysis. The experimental results of the exposure of the DMF solution of 0.1M photosensitizer ITX and the DMF solution of 0.1M photosensitizer pyrene were compared and analyzed, and the scanning results are shown in Figure 2.

ITX is used as a photosensitizer, the overall signal intensity is not high from the beginning of exposure, and as the exposure time increases, the fluorescence intensity continues to be low, and the scanning intensity is lower than the background, which is due to the exposure of ITX to the exposure solvent. After the light exposure, the energy is not completely transferred to the photosensitive group, and the free radicals are produced by self-cleavage, which damages the surface arm molecules to a certain extent; pyrene is used as a sensitizing reagent, and within the exposure time of 20 minutes, the exposure signal intensity varies with time. The gradient rises and reaches a saturated state in about 15 minutes. This is due to the fact that after pyrene absorbs ultraviolet light, it performs energy transfer and sensitization, and transmits ultraviolet energy directly to the photosensitive group. On the other hand, pyrene absorbs ultraviolet light. After excitation, it emits fluorescence that is very close to the wavelength of the excited ultraviolet light, and is similar to the sensitive wavelength of the photosensitive group, and performs secondary exposure to the photosensitive group. As can be seen from the fluorescence spectra of pyrene and pyrene derivatives and ITX shown in Figure 3, ITX has almost no fluorescence, which proves that pyrene has more advantages in the comparison of promoting photodeprotection with the photosensitizer ITX, and its fluorescence characteristics can be effective. Avoid the destructive effect of cracking free radicals caused by energy accumulation.

Example 3

Comparison experiment of pyrene derivative N-pyrene methylacetamide and pyrene exposure

(a) Synthesis of N-pyrene methylacetamide: the first step in the synthesis is the formylation of pyrene. At room temperature, pyrene is added to the mixture of phosphorus trioxide and N-methylformanilide, and the solution is heated to 100°C, under nitrogen protection, to obtain 1-pyrene formaldehyde; the second step is to react 1-pyrene formaldehyde with hydroxylammonium hydrochloride to generate 1-pyrene formaldehyde oxime intermediate; the third step is to reduce 1-pyrene with zinc powder under formic acid conditions Methyloxime obtains 1-pyrenemethylamine: the fourth step is to carry out polycondensation reaction of amino group and carboxyl group, the reactant is dissolved in glacial acetic acid, reacts with acetic anhydride, and refluxes to obtain the final product N-(1-pyrenemethyl)acetamide .

(b) Pyrene and pyrene derivatives are a class of substances with fluorescent properties. When corresponding groups are connected to the structure of pyrene, they even have stronger fluorescent properties than pyrene. In this embodiment, the effects are compared with the exposure experiment of NPPOC-protected amino groups. In this experiment, the light intensity is 365nm ultraviolet light of 12mW/cm ² , the monomer photosensitive group used in the synthesis is 2-(2-nitrophenyl)propoxycarbonyl (NPPOC), and the amino group modified with NPPOC protected The slides were subjected to exposure experiments with the same time gradient in different solvents (pyrene and pyrenemethylacetamide) (ie, the deprotection process of the photosensitive group), and 1 mM rhodamine isothiocyanate was used after exposure. Methylpyrrolidone solution was treated for 30 minutes, and then ultrasonically washed with N-methylpyrrolidone, methanol, dichloromethane, and aqueous solution containing 0.3% Tween for 5 minutes, then rinsed with double distilled water, and dried with nitrogen. The fluorescence signal was obtained by Genepix Pro 4000B scanner detection analysis. The photodeprotection experiments were carried out in 0.1M pyrene in N,N-dimethylformamide (DMF) solution and 0.1M N-pyrenemethylacetamide in DMF solution respectively, and the scanning results are shown in Figure 4. It can be seen that the exposure in the solvent containing pyrene acetamide is better than the result in the solvent containing pyrene, which is manifested by the increase of the overall fluorescence intensity and the advance of the saturation time. This example can prove that pyrene derivatives have the same or better exposure sensitization effect.

Claims (6)

1. pyrene and derivant thereof are as the application of photosensitive group deprotection enhanced sensitivity reagent in biochip preparation; The concentration of described pyrene and derivant thereof is 0.08-0.12M; Described biochip is peptide nucleic acid biochip.

2. apply as claimed in claim 1, it is characterized in that, the concentration of described pyrene and derivant thereof is 0.1M.

3. apply as claimed in claim 1, it is characterized in that, described pyrene derivatives is pyrene methyl alcohol, N-pyrene methylacetamide, Isosorbide-5-Nitrae-two pyrene benzene, 1-ethinyl pyrene or 1,3,6 ,-three (trimethyl silicane) pyrene.

4. apply as claimed in claim 1, it is characterized in that, the preparation of described biochip be adopt photoconduction to in-situ synthesis prepare peptide nucleic acid biochip.

5. apply as claimed in claim 4, it is characterized in that, when described in-situ synthesis prepares peptide nucleic acid biochip, adopt 10-13mW/cm ², 365nm ultraviolet light.

6. apply as claimed in claim 5, it is characterized in that, when described in-situ synthesis prepares peptide nucleic acid biochip, adopt 12mW/cm ², 365nm ultraviolet light.