KR100497673B1 - Method and Apparatus for Compound Library Preparation Using Optical Modulator - Google Patents

Abstract

The present invention relates to providing a chemical library, and more particularly, to a chemical library manufacturing method using an optical modulator and a chemical library manufacturing apparatus used therefor. Furthermore, the present invention provides a chemical analysis method using the above method and a chemical analysis device used therein.

Description

Method and apparatus for manufacturing chemical library using optical modulator {Method and Apparatus for Compound Library Preparation Using Optical Modulator}

The present invention relates to providing a chemical library, and more particularly, to a chemical library manufacturing method using an optical modulator and a chemical library manufacturing apparatus used therefor. Furthermore, the present invention provides a chemical analysis method using the above method and a chemical analysis device used therein.

 Recently, efforts are being made to combine biotechnology and semiconductor manufacturing technology to synthesize biochemically useful chemicals and to reveal their activities. Using semiconductor processing techniques on small silicon chips, it is easy to obtain useful information by synthesizing the desired peptide or oligonucleotide chemicals in specific microscopic areas and biochemically searching them. Accordingly, advanced countries are already applying these technologies to the Human Genome Project, and various types of DNA chips are coming out.

The basic configuration of current systems is to introduce the appropriate functional group on the silicon substrate, combine the protecting group with a functional group that can be decomposed by light before synthesizing the desired chemical, and then irradiate the light only where necessary. . In this way, the functional groups revealed only in specific parts combine various chemical reactions with desired chemicals. Thus, by repeating the process of removing the protecting group by light irradiation and the chemical coupling reaction, a chemical library in which a specific chemical is bound to a specific portion is synthesized.

However, the systems developed so far have a disadvantage in that a large number of photomasks have to be used to produce a biochip containing a chemical library since a photomask is required for each process of removing a protecting group by light irradiation. The production of various types of biochips requires as many photomasks as possible, and if the masks and chips are not properly arranged, there is a complicated problem of generating unwanted chemicals.

The present invention provides a method and apparatus for effectively providing various chemical libraries in a simple process using an optical modulator including a plurality of optical switches without using a photo mask.

The present invention seeks to reduce errors that occur in library fabrication by the use of photo masks.

The present invention seeks to facilitate the automation of the chemical library manufacturing process.

The present invention is to provide a library of various chemical substances by easily adjusting the amount of light, and to provide a method and apparatus for easily knowing the activity of analytical chemicals according to the difference in the amount of light.

A method for preparing a chemical library included in a plurality of microwells, the method comprising: a) binding a second chemical among a plurality of the microwells comprising a first chemical bound to a protecting group capable of being decomposed by light; Selectively irradiating the microwell with light by an optical modulator to remove the protector, wherein the optical modulator has a structure in which a plurality of optical switches are arranged, and each optical switch rotates independently to correspond to the corresponding micro-well. A device that allows light to be selectively irradiated to a particular microwell of a plurality of said microwells by reflecting light into the wells or by moving or rotating an opaque shielding plate, b) one end coupled to a protector The second chemical into the plurality of microwells Reacting in a particular micro-well, and c) the final chemical desired for the particular micro-well that is irradiated with the light modulator with a third chemical to be further bound, one end of which is bound to a protecting group Repeating steps a) to b) with changes according to the material library, wherein the number of repetitions is changed according to the desired chemical library.

In place of step b) of the above method, a second chemical may be introduced onto the substrate to react in the micro-well, and may be replaced by adding a protecting group to a functional group that does not occur.

  In the present invention, the chemical is mainly applicable to nucleotides or peptides, or other various chemicals such as saccharides and phospholipids.

The present invention also provides a chemical library chip prepared by the above method.

The present invention provides an apparatus for manufacturing a chemical library chip comprising a plurality of microwells, the apparatus comprising: a) a light irradiator, b) a plurality of optical switches that operate independently of each other and reflect light to the corresponding microwells; An optical modulator comprising an address circuit for a switch, c) a device for injecting a series of chemicals into said plurality of microwells in turn upon command of a controller, d) injecting unintegrated chemicals from said plurality of microwells A device for cleaning, and e) information about an address of an optical switch that irradiates light into the microwells at least over time, the sequence of chemicals injected, and the final chemical produced in each microwell. And a controller having a program for controlling the operation sequence of the chemical injection device and the cleaning device. And a chemical library production apparatus (1) to.

A typical configuration of the optical modulator has the following structure. In a first example, a) the micromirror is rotatable in at least two states, with and without light reflecting for each corresponding microwell, b) supporting and arranging the micromirrors in a plane, A substrate that is separate from the micromirrors, each comprising an address electrode for the micromirror, a landing electrode, and a circuit for controlling the rotation of the micromirror, and c) connecting the micromirror to the substrate. While there is a light modulator having a separate intermediate structure including a hinge that can rotate the micro mirror.

As another example, a) a rotatable micromirror, b) a substrate positioned below the micromirror, the circuit including an address circuit for each micromirror and a circuit for controlling the rotation of the micromirror, c) the upper surface of the substrate A plurality of electrodes formed on the substrate; And d) at least one post, one end of which is formed on a substrate between the electrodes and the other end of which is integrally formed with the micromirror to support the micromirror at a predetermined position on the electrode. There is an optical modulator.

As another example, a) a shield plate installed to be movable or rotatable, b) a substrate comprising an address circuit for the shield plate and a circuit for controlling the operation of the shield plate, c) allowing light to pass under the shield plate. There is an optical modulator, wherein the light is irradiated only to a desired microwell, including an installed window, and d) a plurality of actuators for generating a force for moving the shield plate.

Further, the present invention provides a method for producing a light source, the method comprising: a) irradiating light to a plurality of microwells comprising a chemical library prepared by the methods described above, and b) injecting a chemical to be measured for activity into the chemical library. And c) provides a chemical analysis method comprising the step of confirming the degree of reaction with the microwells in which the reaction occurred.

In another aspect, the present invention provides a device for producing a chemical library described above, b) a device for confirming the address of the microwells and the degree of the reaction when a specific chemical is reacted with the chemical library, and c) A chemical analysis device is provided that includes a device that sends the address of the microwell and the degree of reaction to a controller having information about the chemical library (FIG. 1).

In the present invention, the chemical substance is a concept including various monomers, polymers, compounds, and the like.

Wells used in the present invention is a general term for the partition means for allowing the chemicals used in the present invention to be distinguished from other chemicals in addition to the meanings commonly used in the art.

An example of using a micro mirror as an optical switch is composed of a plurality of micro mirrors (a) on a substrate (b) (FIG. 2), and these micro mirrors are individually adjusted to electrical signals. Each micromirror can irradiate light only to a corresponding microwell of a substrate including a plurality of corresponding microwells of the same size, thereby activating a functional group only at a desired position. In addition, even the same compound can control the reflection time of the micromirror, so that different libraries can be obtained depending on the amount of light irradiated. The present invention is based on the idea that various kinds of biochips can be efficiently produced by using the micromirror instead of the photo mask in the conventional compound library manufacturing method.

As shown in FIG. 2, the optical modulator may arrange the micromirrors a two-dimensionally on the substrate b. Each micromirror has a rotation angle of at least two states, a state of reflecting light into the microwell (FIG. 3) and a state of non-reflecting (FIG. 4). 3 and 4, a shows a micromirror, c shows incident light, d shows reflected light, and e shows a micro well.

An example of an optical modulator is as follows. As a first example, as shown in Fig. 5, the micromirror a is supported on the pillar c by the torsional or bending spring b, and the pillar c is fixed to the substrate d. The micro mirror a is inclined by applying one voltage of the electrode e, and thus the direction of reflected light is changed. At least two individually operated micromirrors are tilted by an electrical signal and the tilted time is adjusted. As a second example, as shown in FIG. 6, the cantilever a is coated with metal for reflection to manufacture the micro mirror b, and the cantilever a is coated with piezoelectric material or bimetal to drive the micro mirror b. As a third example, reference may be made to the 'multilevel movable mirror device' disclosed in US Pat. No. 5,083,857. In Figure 7, a is a micromirror, b is an address electrode, c is a landing electrode, d is a hinge support post, and e is an electrode support post. f1 and f2 show a state inclined by the voltage applied to the electrode. As a fourth example, reference may be made to Korean Patent Application No. 96-20611. In FIG. 8, an electrode b having a predetermined pattern is formed on the upper surface of the substrate a including the address scanning circuit (not shown). The electrode b has a stripe shape and is provided with at least one pair. A post f is formed on the substrate a between the electrodes b. This post is pivotally coupled to the substrate, and is disposed perpendicular to the substrate a when not affected by the electrode. On the upper part of this post, a micro mirror is rotatably coupled. The upper surface of the micro mirror is formed with a predetermined through groove g, so that the through groove is integrally formed with the post. As a fifth example, as shown in FIG. 9, only a desired shield plate may be moved, or as shown in FIG. 10, only a desired shield plate may be rotated to irradiate light through a window installed under the shield plate. The substrate includes an address circuit to the shield plate, a circuit for controlling the rotation of the shield plate, and an actuator for moving the shield plate. 9 and 10 (a) is a state in which both the light of the window is shielded, (b) is a state in which the light of the right window is irradiated.

For methods of fabricating a library other than irradiating light with an optical modulator instead of using a photo mask, the disclosure of US Pat. No. 5,753,788 is incorporated herein by reference and forms part of the invention.

In the present invention, the protecting group refers to a substance which is combined with a functional group and removed by an activator such as light. Examples of protecting groups include α-methyl-2-nitrophenyl-oxycarbonyl chloride, nitroveratriloxycarbonyl chloride (NVOC-CI), nitropiperonyl, pyrenylmethoxy-carbonyl, nitroveratril, nitrobenzyl , Dimethyl dimethoxybenzyl, 5-bromo-7-nitroindolinyl, ortho-hydroxy-alpha-methyl cinnamoyl, and 2-oxymethylene anthraquinone.

In the present invention, the activator preferably includes light (including ultraviolet rays), ion beams, electron beams, x-rays, electric fields, electromagnetic fields and the like.

 In the apparatus for preparing and analyzing the compound library of the present invention, it is important to design a program that can be mounted on the controller according to the final library to be manufactured or analyzed. This program includes: whether the light is opened or closed by the address (code distinguishing each optical switch) given to each optical switch, and the type of compound injected over time; Code coding an operation sequence between the light modulator, the compound injector, the cleaning device, and the analysis device; And the composition of the final compound for each address. If you want to prepare or analyze another library, you only need to replace the program. And devices other than the controller include circuitry for decoding the code of the controller.

The prepared library of compounds is used to determine the activity for a particular compound. As an example of the measurement, i) irradiating light to the compound library chip, ii) reacting a chemical substance labeled with a fluorescent substance with the compound library, and i) measuring the fluorescence generated in the microwell in which the reaction occurred, together with its intensity. It includes a step. An example of a detection device for doing this is disclosed in US Pat. No. 5,753,788.

<Example 1>

Build and Drive Micrometer Arrays

A micromirror array having a size of 50 × 50 μm was manufactured using an aluminum micromachining process. The distance between the micromirror and the substrate was 4.5 μm, and the mirror was rotatably attached to the substrate using a support pillar and a hinge. Front and side SEM photographs of the micromirror array thus manufactured are shown in FIGS. 11A and 11B, respectively.

While driving the micromirror array fabricated as described above using a driving circuit, it was confirmed whether the mirror was normally driven in the following manner. First, the mirror was fixed to the XY-stage of the microscope, and the difference in brightness of the light reflected from the mirror was measured while obliquely entering light from the left side of the mirror array using a fiber optic light guide. At this time, the mirror is in a flat state, inclined left or right according to the signal of the driving circuit, and the brightness of reflected light varies according to each state.

FIG. 12 illustrates a direction in which the pixel blocks of the mirror array are to be driven, and FIG. 13 illustrates the actual driving of the mirror according to the driving signal shown in FIG. 12 by using the brightness difference of the light reflected from each mirror. It is the confirmed microscope image photograph. As shown in FIG. 13, it can be seen that as time passes, the image of the block proceeds from the lower left to the upper right. The mirror of the line to which the bias voltage is applied is well driven, and the mirror is right and left according to the address voltage, respectively. You can see that the slope.

<Example 2>

Preparation of Polynucleotide Libraries

a. Introduction of functional groups and protectors

After washing the glass micro-well bottom with sulfuric acid, 0.1 M γ-aminopropyl-triethoxysilane (γ-APS) was added to chloroform solution and reacted at 50 ° C. for 12 hours. After the reaction was washed with chloroform solution and dried. The protecting group which is selectively decomposed by light was introduced to the terminal amine group thus introduced. Such a protecting group is decomposed when irradiated with light of about 360 nm and returns to the amine group, which can be used to react a specific nucleotide or amino acid at a desired site. To introduce a protecting group, α-methyl-2-nitrophenyloxycarb onyl chloride (Formula 1) or nitroveratryloxycarbonyl chloride (NVOC-Cl, Formula 2) was dissolved in acetonitrile and reacted for 30 minutes. Confirmation of the reaction termination can be confirmed by dyeing the amine group before the reaction with bromophenol blue reagent, the color disappears.

  Formula 1 Formula 2

b. Removal of protector

The microchip incorporating the terminal amine group protecting group was mounted in the system and washed with flowing acetonitrile. In order to selectively remove a protecting group, 360 nm UV light was irradiated for 15 minutes through a micromirror to the microwell to be reacted. Washed with dried acetonitrile and oligonucleotide succinic acid monoester protected at 5 'position with NVOC group was coupled for 3 hours using BOP / HOBt / acetonitrile solution. The 3 'position of the nucleotide used in the reaction is in the form of a carboxylic acid so that it can easily bind to the amino group shown on the chip, and the 5' position has a protecting group that is decomposed by light (Formula 3). The first nucleotides were coupled, washed with acetonitrile for 5 minutes, and then treated with acetic anhydride / 2,6-lutidine / THF (1: 1: 8) solution for 5 minutes to cap unreacted amino groups.

  From the second nucleotide, repeat the following process. UV light at 360 nm was irradiated for 15 minutes through a micro mirror to remove the protecting group at the desired position. After washing with dried acetonitrile, the prepared 0.1M nucleotide phosphoramidite / acetonitrile solution and 0.4M 1H-tetrazole / acetonitrile solution were injected into the microwell and reacted for 15 minutes. The 3 'position of the nucleotide used in the reaction has the form of N, N-diisopropylaminoethoxycyanophosphite, which can easily bind to the 5' position hydroxyl group of the nucleotide exposed on the chip, and the 5 'position of the nucleotide has a protecting group that is decomposed by light. . (Formula 4)

                (Formula 3) (Formula 4)

The nucleotides were then coupled and washed with acetonitrile for 5 minutes and then treated with acetic anhydride / 2,6-lutidine / THF (1: 1: 8) for 5 minutes to cap unreacted hydroxy groups. After the reaction was completed, the mixture was washed with acetonitrile for 5 minutes and then oxidized with 0.1M iodine THF solution / pyridine / water (90: 5: 5) solution. This process can be repeated to synthesize oligonucleotide libraries of the desired sequence at the desired location on the chip. 14 shows a process for preparing polynucleotides C-G-T-A and A-G-T-C through the above procedure. Where I is the protecting group.

<Example 3>

Synthesis of Polypeptides

The procedure for synthesizing peptide libraries on a chip is similar. Compound library chips incorporating protecting groups at the terminal amine groups were mounted in the system and washed with flowing acetonitrile. The specific wells to be reacted were selectively irradiated with UV light at 360 nm through a micromirror for 15 minutes to selectively remove the protecting group and wash the dried NMP solution. The prepared 0.1 M amino acid / NMP solution and 0.5 M BOP / HOBt / NMP solution were injected into the microwells and allowed to react for 60 minutes. After the coupling reaction, the mixture was washed with NMP solution for 10 minutes. Each amino acid used in the coupling reaction has a protecting group in which the amine group portion is decomposed by light. This process was repeated repeatedly to synthesize oligopeptides of the desired sequence at the desired position. 15 shows a process for synthesizing G-E-C-A, H-F-D-B polypeptides through the above procedure. Where I is the protecting group.

In the present invention, using an optical modulator instead of the photo mask used in the prior art has the following advantages.

First, it eliminates the need to replace dozens of photo masks for each process of manufacturing the protecting group, and the optical switches can be operated individually to effectively produce a variety of compound libraries in a simple process.

Second, by irradiating the light with only the optical switch in the fixed state of the optical modulator, it is possible to solve the problem of misalignment of the mask and chip arrangement that can occur during the photo mask replacement process, thereby reducing errors in the library manufacturing.

Third, in addition to the above-described advantages, since the light irradiation position and the amount of light can be easily adjusted by the electrical signal of the controller, the automation of the compound library manufacturing process can be more easily achieved. In addition, since the composition of the compound library and the analysis method of the compound depend on the program to be configured, even if a different configuration is required, only the program may be reconfigured without setting the photo mask.

Fourth, the present invention can easily modify the composition of the same compound by the control of the amount of light, providing a more diverse library of compounds, it is easy to know the aspect of the activity of the analyte compound according to the difference in the amount of light.

1 shows a chemical library manufacturing apparatus and a chemical analyzer including the same.

2 is an optical modulator in which a plurality of micromirrors are arranged as an optical switch.

3 shows a state in which light is reflected to a microwell through a micromirror, which is a form of an optical switch.

4 shows a state in which the micromirror does not reflect light into the microwell.

5 to 10 show an example of implementing an optical modulator.

11 (a) and (b) are front and side SEM images of the micromirror array according to an embodiment of the present invention.

12 illustrates a direction for driving a pixel block of a mirror array to test a micromirror.

FIG. 13 is a microscopic image of the actual mirror driven according to the driving signal shown in FIG. 12 using the brightness difference of the light reflected from each mirror.

14 shows the process for preparing a polynucleotide library in accordance with the present invention.

15 depicts a process for preparing a polypeptide library in accordance with the present invention.

Claims (15)

A method of manufacturing a chemical library included in a plurality of micro wells, a) selectively irradiating light with an optical modulator to the microwell to bind a second chemical from among the plurality of microwells comprising a first chemical bound to a protecting group capable of being decomposed by light; Removing the protector, wherein the optical modulator is a structure in which a plurality of optical switches are arranged, wherein each optical switch rotates independently to reflect light in a corresponding microwell or to move or rotate an opaque shielding plate. To shield light by selectively irradiating light to a particular microwell of the plurality of microwells; b) introducing into said plurality of micro wells said second chemical having one end bound to a protecting group and reacting in said particular micro-well; And c) changing the third chemical to which one end is bound with a protecting group and the specific micro-well irradiated with light by the light modulator according to the desired final chemical library To b) repeating the step, wherein the number of repetitions is changed according to the desired chemical library. Chemical library production method comprising a. The method of claim 1, Wherein said chemical comprises a nucleotide. The method of claim 1, Wherein said chemical comprises an amino acid. A chemical library chip prepared by the method of claim 1, wherein the chemical is a nucleotide or amino acid. A method of manufacturing a chemical library included in a plurality of micro wells, a) selectively irradiating light with an optical modulator to the microwell to bind a second chemical from among the plurality of microwells comprising a first chemical bound to a protecting group capable of being decomposed by light; Removing the protector, wherein the optical modulator is a structure in which a plurality of optical switches are arranged, wherein each optical switch rotates independently to reflect light to a corresponding microwell, or to move an opaque shielding plate or Rotating to shield the light to selectively irradiate light to a particular microwell of the plurality of microwells; b) introducing said second chemical into a plurality of said micro wells and reacting in said particular micro-well; c) adding the or second protecting group to the functional group to which no reaction occurs; And d) repeating steps a) to c) with alteration of the third chemical to be further bound and the specific micro-well irradiated by the light modulator with the desired final chemical library. Where the number of iterations depends on the desired chemical library Chemical library production method comprising a. The method of claim 5, Wherein said chemical comprises a nucleotide. The method of claim 5, Wherein said chemical comprises an amino acid. A chemical library chip prepared by the method of claim 5, wherein the chemical is a nucleotide or amino acid. An apparatus for manufacturing a chemical library chip comprising a plurality of micro wells and irradiated with light by a light irradiator, a) an optical modulator comprising a plurality of optical switches operating independently of each other to irradiate light to corresponding microwells of the plurality of microwells and an address circuit for these optical switches; b) a device for injecting a series of chemicals into said plurality of microwells in sequence upon command of a controller; c) a device for cleaning unbound chemicals injected in said plurality of micro wells; And d) information about the address of an optical switch for irradiating light to said microwells over at least time, the sequence of chemicals injected, and information about the final chemical produced in each microwell, wherein said irradiator, said chemical A controller having a substance injection device, and a program for controlling the operation sequence of the cleaning device; Chemical library chip manufacturing apparatus comprising a. The method of claim 9, The light modulator a) a micromirror rotatable in at least two states, with and without reflecting light, for each corresponding microwell; b) circuitry for supporting and arranging the micromirrors in a plane and positioned separately from the micromirrors, for controlling the address electrode, the landing electrode, and the rotation of the micromirrors for the micromirrors, respectively; A substrate comprising a; And c) having a separate intermediate structure comprising a hinge for pivoting the micromirror while connecting the micromirror to the substrate Chemical Library Chip Manufacturing Equipment. The method of claim 9, The light modulator a) rotatable micromirror; b) a substrate positioned below said micromirror, said substrate comprising circuitry for controlling rotation of said micromirror and said address circuit for each micromirror; c) a plurality of electrodes formed on the upper surface of the substrate; And d) at least one post, one end of which is formed on the substrate between the electrodes and the other end of which is integrally formed with the micromirror to support the micromirror at a predetermined position on the electrode Chemical library chip manufacturing apparatus comprising a. The method of claim 9, The light modulator a) a shield plate installed to be movable or rotatable; b) a substrate comprising said address circuit for said shield plate and circuitry for controlling operation of said shield plate; c) a window installed under the shield plate to allow light to pass therethrough; And d) a plurality of actuators generating a force to move the shield plate; Chemical library chip manufacturing apparatus comprising a. a) irradiating light to a plurality of micro wells comprising a chemical library prepared by the method of claim 1; b) injecting a chemical of interest to measure activity into the chemical library; And c) confirming a reaction degree with the microwell in which the reaction occurred among the plurality of microwells Chemical analysis method comprising a. a) irradiating light to a plurality of micro wells comprising a chemical library prepared by the method of claim 5; b) injecting a chemical of interest to measure activity into the chemical library; And c) confirming a reaction degree with the microwell in which the reaction occurred among the plurality of microwells Chemical analysis method comprising a. a) a chemical library chip manufacturing apparatus according to claim 9, 10, 11, or 12; b) an apparatus for identifying the address of the microwell where the reaction occurred and the extent of the reaction when reacting a specific chemical with a chemical library; c) a device for sending the address of the microwell in which the reaction occurred and the extent of the reaction to a controller having information about the chemical library Chemical analysis device comprising a.