BACKGROUND OF THE INVENTION (a) Field of the Invention The present invention relates to a providing of a compound library, more particularly to a providing of a compound library preparation method using an optical modulator and a compound library preparation apparatus.

Furthermore, the present invention provides a compound analyzing method using the above preparation method, and a compound analyzing apparatus.

(b) Description of the Related Art Efforts to synthesize biochemically useful compounds by combining biotechnology and semiconductor preparation technology, and to discover the activities of the synthesized compounds are lately being spread worldwide. After synthesizing a desired peptide or oligonucleotide compound within a minute specific area over a small silicone chip using

semiconductor processing technology, useful information can be easily obtained if these synthesized compounds are biochemically collectively searched. Advanced countries have already been applying these technologies to the Human Genome Project, for example, and various types of DNA (DEOXYRIBONUCLEIC ACID) chips are being developed.

The basic construction of systems that are currently being applied is as follows: appropriate functional groups are employed on a silicone substrate plate, protecting groups which can be decomposed by light before desired compounds are synthesized are bonded to the functional groups, and then light is irradiated only on the necessary area thereof so that the functional groups are exposed. Through bonding specific compounds to specific areas after repeating the above procedures, a compound library is synthesized.

However, there are problems in that a large number of photomasks must be used in order to produce a compound library of inserted biochips, since systems which have been developed thus far require photomasks during every process of protecting group removal by light irradiation. The complication exists that photomasks must be made for each type of biochip, and undesired compounds can be produced if masks and chips are not properly aligned.

SUMMARY OF THE INVENTION The present invention provides a method and apparatus for

effectively preparing varieties of compounds in a simple process without using photomasks, but by using an optical modulator including pluralities of optical switches.

The present invention seeks to decrease error occurrence that is prevalent during the preparation of libraries using photomasks, and to make it easier to automate the compound library preparation process.

The present invention easily provides various compound libraries by controlling the quantity of light, and it provides a method and an apparatus for easily knowing the activities of analyzed compounds according to the difference of light quantity.

The present invention provides a method for the preparation of a compound library contained in pluralities of micro-wells, comprising the steps of: a) removing the protecting group by selectively irradiating light with an optical modulator on the micro-well, seeking to bond the second compound in the pluralities of micro-wells comprising the first compound bonded with a functional group in which a protecting group has been decomposed by light (wherein the optical modulator is of a construction in which pluralities of optical switches are aligned, and each optical switch is a device that is independently rotated to reflect light on a corresponding micro-well, or moves or rotates an opaque masking plate to shield light so that light is selectively irradiated on a specific

micro-well in the pluralities of micro-wells) ; b) employing the second compound of which one terminal end is bonded with a functional group onto the pluralities of micro-wells and reacting at the specific micro-well; and c) varying and repeating the steps a) to b) according to the third compound to be additionally bonded of which one terminal end is bonded with a functional group and the specific micro-well of the final compound libraries in which light is irradiated by the optical modulator (wherein the repetition cycle is varied depending on the objective compound libraries).

In the above method, the step b) can be substituted with a step whereby the reaction at a micro-well occurs by employing and bonding the second compound on a substrate plate by adding a protecting group to the non-reacted functional group.

The method of the present invention can be applied to a variety of compounds such as nucleotides, peptides, saccharides, and phospholipids, as examples.

Furthermore, the present invention provides compound library chips prepared by the above method.

The present invention provides an apparatus for the preparation of compound library chips including pluralities of micro-wells (FIG. 1) comprising: a) an optical irradiator;

b) an optical modulator comprising pluralities of optical switches which are each independently operated to reflect light on the corresponding micro-well, and an address circuit on these optical switches; c) a device which injects a series of compounds into the pluralities of micro-wells in a specific order according to the controller dictates; d) a washing device for injecting a compound into the pluralities of micro-wells that are not to be bonded; and e) a controller comprising at least one optical switch address, irradiating light on a micro-well according to the time, order of compounds to be injected, and information on final compounds produced on each micro-well, and having programs controlling the operating order of the optical irradiator, the compound injecting device, and the washing device.

Typical configurations of an optical modulator include the following constructions: The first example is an optical modulator having a separate medium structure, comprising: a) a micro-mirror which can rotate to at least two states, where light is reflected on each corresponding micro-well and where light is not reflected on each corresponding micro-well; b) a substrate plate which supports the micro-mirror, aligned on a

plane, placed separately from the micro-mirror, and comprising a circuit for controlling the rotation of an address electrode on each micro-mirror, a landing electrode, and the micro-mirror; and c) a hinge which can rotate the micro-mirror by connecting the micro-mirror with the substrate plate.

Another example is an optical modulator comprising : a) a micro-mirror which is designed to make rotation possible; b) a substrate plate which is placed under the micro-mirror, which comprises an address circuit for each micro-mirror and a circuit controlling the micro-mirror rotation; c) pluralities of electrodes formed on the surface of the substrate plate; and d) at least one supporting post of which one end is formed on a substrate plate between the electrodes, the other end being integrated with the micro-mirror so that the micro-mirror is placed at a prescribed position with respect to the electrodes.

Another example is an optical modulator irradiating light only at the desired micro-wells, comprising : a) a masking plate that is installed to make transfer or rotation possible ; b) a substrate plate comprising an address circuit on the masking plate and a circuit controlling the masking plate operation;

c) a window which is installed so that light passes through it, under the masking plate; and d) pluralities of operating devices generating forces which can move the masking plate.

Furthermore, the present invention provides a compound analyzing method comprising the steps of: a) irradiating light on the pluralities of micro-wells included in the compound libraries prepared by the aforementioned methods; b) injecting the target compound of which activities are to be measured into the compound libraries; and c) ascertaining a degree of reaction with the reacted micro-well.

Furthermore, the present invention provides a compound analyzing apparatus (FIG. 1) comprising: a) the aforementioned compound library preparation device ; b) a device confirming a degree of reaction with the reacted micro- well address when a specific compound is reacted with a compound library; and c) a device sending an address of a micro-well at which the reaction occurs, and a degree of the reaction, to a controller having information on compound libraries.

Exemplary compounds of the present invention include varieties of monomers and polymers.

The term"wall"as used in the present invention not only refers to

the meaning usually used in the field of the present invention but also as a general term of a divisional means in which a compound used in the present invention is divided from other extant compounds.

The example where a micro-mirror is used as an optical switch consists of pluralities of micro-mirrors (a) on a substrate plate (b) (FIG. 2), wherein these micro-mirrors are individually adjusted with electrical signals.

Each micro-mirror can irradiate light only on a particular micro-well of the substrate plate, which comprises corresponding pluralities of micro-wells of the same magnitude, so that functional groups are activated only at the desired position. Furthermore, other libraries can be obtained depending on the quantity of light irradiated since even the same method can control the reflecting time of a micro-mirror. The present invention is based on an idea that various types of biochips can be effectively manufactured if the micro-mirror is used instead of a photomask as in the previous compound library preparation method.

An optical modulator can two-dimensionally align a micro-mirror (a) on a substrate plate (b). Each micro-mirror has rotation angles of at least two states, when light is reflected on a micro-well (FIG. 3) and when light is not reflected on a micro-well (FIG. 4). In FIG. 3 and FIG. 4, a illustrates a micro-mirror, c illustrates the incident light, d illustrates the reflected light, and e illustrates a micro-well.

The embodiment examples of an optical modulator are as follows.

In the first embodiment example, micro-mirror a is connected to pillar c by

torsion spring b, wherein pillar c is fixed to substrate plate d as shown in FIG.

5. Micro-mirror a is tilted by applying voltage to one side of electrode e, and therefore the direction of a reflected light is changed accordingly. At least two types of individually operated micro-mirrors are tilted by electrical signals, and the tilting time is controlled.

In the second embodiment example, micro-mirror b is manufactured by cladding reflective metal over cantilever a, and micro-mirror b can be driven by cladding piezoelectric material or bimetal over cantilever a, as shown in FIG. 6.

'A multilevel operating mirror device'disclosed in the U. S. Patent No.

5,083,857, can be referred to as the third embodiment example. In FIG. 7, a is a micro-mirror, b is an address electrode, c is a landing electrode, d is a hinge supporting post, and e is an electrode supporting post. f1 and f2 illustrate the states of tilt when a voltage is applied to an electrode.

Korean Patent Application No. 96-20611 can be referred to as the fourth embodiment example. A prescribed patterned electrode (b) is formed on a top surface of a substrate plate (a) comprising an address scanning circuit (which is not drawn) in FIG. 8. The electrode (b) has a strip shape, and there is at least one pair of them. A post (f) is formed on the substrate plate (a) between these electrodes (b). This post is rotationally connected to and arranged perpendicularly with the substrate plate (a) so as not to be influenced by the electrode. A micro-mirror is rotationally connected to the top of this post. A prescribed perforative slot (g) is formed on the top

surface of a micro-mirror, and the post-integrated perforative slot is formed.

In the fifth embodiment example, light can be irradiated through a window installed under a masking plate by transferring only the desired masking plate itself as shown in FIG. 9, or rotating only the desired masking plate itself as shown in FIG. 10. An address circuit for the masking plate and an operating device which moves a masking plate rotation controlling circuit and the masking plate, are contained in the substrate plate. (a) of FIG. 9 and FIG. 10 is a state in which light of two windows is masked, and (b) is a state in which light of the right side windows is irradiated.

A library preparing method where light is irradiated using an optical modulator instead of a photo mask as disclosed, in U. S. Patent No.

5,753,788 is referred to in the present invention as a part of the present invention.

A protecting group of the present invention means a material that is removed by an activator such as light after being in the state of bonding with a functional group. Examples of protecting groups include a-methyl-2- nitrophenyloxycarbonyl chloride, nitroveratryloxycarbonyl chloride (NVOC- CI), nitropiperonyl, pyrenylmethoxy-carbonyl, nitroveratryl, nitrobenzyl, dimethyldimethoxybenzyl, 5-bromo-7-nitroindolinyl, ortho-hydroxy-a-methyl cinnamoyl, and 2-oxymethyleneanthraquinone.

Examples of an activator of the present invention preferably include light (such as ultraviolet rays), ion beams, electron beams, x-rays, electric

fields, and electron fields.

The controller program is an important component in the apparatus for preparing and analyzing a compound library of the present invention, because the final library to be manufactured or analyzed is dependant on it.

This program should at least include: the types of compounds injected; whether light is shielded or irradiated at an address which is given to each optical switch through a code which identifies each optical switch; the time of light irradiation; a code which enables operating orders for the optical modulator, compound injector, washing device, and analyzing device; and the final compound composition for each address. The program should only be replace when other libraries are manufactured or analyzed. An additional apparatus includes a circuit that deciphers a controller code.

The prepared compound libraries are used to measure the activity of a specific compound. The measuring example comprises the steps of i) irradiating light on a compound library chip, ii) reacting a fluorescent marked target compound with a compound library, and iii) measuring fluorescence generated from the reacted micro-well together with its intensity.

One example of a detecting device for carrying out this is disclosed in the U. S. Patent No. 5,753,788.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention, and many of the attendant advantages thereof, will be readily apparent as the same becomes

better understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, wherein: FIG. 1 illustrates a compound library preparation apparatus and a compound analyzer comprising the same; FIG. 2 is an optical modulator in which pluralities of micro-mirrors are configured by an optical switch; FIG. 3 illustrates a state in which light is reflected on a micro-well through a micro-mirror; FIG. 4 illustrates a state in which a micro-mirror does not reflect light on a micro-well; FIG. 5 to FIG. 10 are embodiment examples of an optical modulator; FIG. 11 (a) and FIG. 11 (b) are front and side scanning electron microscope photographs of a micro-mirror array according to one example of the present invention; FIG. 12 illustrates a direction in which a mirror array pixel bloc is driven in order to test a micro-mirror ; FIG. 13 is a microscope photograph in which a practically driven mirror appearance is confirmed using the brightness difference of light <BR> <BR> reflected from each mirror according to a driving signal illustrated in FIG. 12 ; FIG. 14 illustrates a procedure in which polynucleotide library is prepared according to the present invention; and FIG. 15 illustrates a procedure in which a polypeptide library is prepared according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following detailed description, only the preferred embodiments of the invention have been shown and described, simply by way of illustration of the best mode contemplated by the inventor (s) of carrying out the invention. As will be realized, the invention is capable of modification in various obvious respects, all without departing from the invention.

Accordingly, the description is to be regarded as illustrative in nature, and not restrictive. <BR> <BR> <BR> <BR> <BR> <P> <EXAMPLE1><BR> <BR> <BR> <BR> <BR> <BR> Fabricating and driving a micro-mirror array An micro-mirror array having a magnitude of 50 x 50/w11 was fabricated using an aluminum micro machining process. A distance between a micro-mirror and a substrate plate was 4.5 ßn, and the mirror was rotationally fixed to the substrate plate using a supporting pillar and a hinge.

Front and side scanning electron microscope photographs of these fabricated micro-mirror arrays are illustrated in FIG. 11 (a) and FIG. 11 (b) respectively.

While driving these manufactured micro-mirror arrays using a driving circuit, the following method was used to determine whether a mirror was normally driven or not. First, a mirror was fixed at the XY-stage of a microscope, and the brightness difference of light reflected from the mirror was measured with the light obliquely projected to the left of a mirror array

using a fiber optic light guide. At this time, the mirror was either in a flat state, a leftward tilted state or a rightward tilted state according to the driving circuit signals, and the reflected light brightness was determined with respect to each state.

FIG. 12 illustrates a direction in which a mirror array pixel bloc is driven, and FIG. 13 is a microscope photograph in which the practically driven mirror appearance is confirmed using the brightness difference of light reflected from each mirror according to a driving signal illustrated in FIG.

12. As illustrated in FIG. 13, it can be known that the block image progresses from bottom left to top right as time passes, and it can be confirmed that with the bias voltage applied the line mirror is driven properly, and the mirror is tilted toward the right and left respectively according to the address voltage.

<EXAMPLE 2> Polvnucleotide library preparation a. Employment of functional groups and protecting groups After washing the floor of a micro-well made of glass with sulfuric <BR> <BR> <BR> <BR> <BR> acid, 0.1 M Y-aminopropyl-triethoxysilane (-APS) was put into a chloroform solution, and reacted at 50°C for 12 hours. After the reaction, the floor of the micro-well was washed with the chloroform solution and dried.

To the terminal end amine group a protecting group that can be selectively decomposed by light was bonded. When a 360 nm level of light is

irradiated on it this protecting group is decomposed, exposing the amine group, where specific nucleotides or amino acids can be reacted at those exposed areas using a-methyl-2-nitrophenyloxycarbonyl chloride of the following Chemical Formula 1, or nitroveratryloxycarbonyl chloride (NVOC- Cl) of the following Chemical Formula 2, as examples, after they are dissolve in acetonitrile and reacted for 30 minutes. The amine group is dyed with a bromophenol blue reagent before the reaction, so reaction termination can be confirmed when the color is removed.

[Chemical Formula 1] [Chemical Formula 2]

b. Removal of protecting groups

A microchip employing an amine protecting group was mounted on a system and washed with dried acetonitrile. Ultraviolet rays of 360 nm were irradiated on a micro-well to be reacted through a micro-mirror for 15 minutes in order to selectively remove the protecting group. After washing with dried acetonitrile, an oligonucleotide succinic acid monoester of which a 5'position was protected by a nitroveratryloxycarbonyl group was coupled with a BOP/HOBt/acetonitrile solution for 3 hours. The 3'position of nucleotide used in the reaction can be easily combined with an amino group on a chip due to its carboxylic acid shape, and the 5'position has a protecting group which is decomposed by light (Chemical Formula 3). After the first nucleotide was coupled and washed with acetonitrile for 5 minutes, it was treated with an acetic anhydride/2, 6-lutidine/ tetrahydrofuran (1: 1: 8) solution for 5 minutes, capping a unreacted amino group.

The following procedures are repeated for the second nucleotide.

Ultraviolet rays of 360 nm were irradiated through a micro-mirror for 15 minutes, removing a protecting group at a desired position. After washing with dried acetonitrile, it was reacted for 15 minutes by injecting the already prepared 0.1 M nucleotide phosphoramidite/acetonitrile solution and 0.4 M 1 H-tetrazole/acetonitrile solution into a micro-well. The 3'position of nucleotide used in the reaction can be easily combined with the 5'position hydroxy group of the nucleotide exposed on a chip due to its N, N- diisopropylaminoethoxycyanophosphite shape, and the 5'position of nucleotide has a protecting group which is decomposed by light (Chemical Formula 4).

[Chemical Formula 3] Nvoc--O Base 0 0 0 OH 0

[Chemical Formula 4] Nvoc-0 Base O 0 , p, o,, ^CN N

After coupling the nucleotide and washing with acetonitrile for 5 minutes, it was treated with an acetic anhydride/2,6-lutidine/ tetrahydrofuran (1: 1: 8) solution for 5 minutes, capping the unreacted hydroxy group. It was washed with acetonitrile for 5 minutes when the reaction was terminated, and then the oxidation was progressed with a 0.1 M iodine tetrahydrofuran solution/pyridine/water (90: 5: 5) solution. By continuously repeating this

procedure, an oligonucleotide library with desired positions and sequence can be synthesized on a chip. FIG. 14 represents a process in which polynucleotide C-G-T-A and A-G-T-C are prepared through the above procedures, wherein I is a protecting group. <BR> <BR> <P> <EXAMPLE 3><BR> <BR> Synthesizing of Polypeptide A process for synthesizing a peptide library on a chip is similar to the foregoing.

A compound library chip employing a protecting group at a terminal end amine group was mounted on a system and washed with dried acetonitrile. By irradiating ultraviolet rays of 360 nm on a specific well to be reacted through a micro-mirror for 15 minutes, a protecting group was selectively removed and washed with a dried NMP solution. It was reacted for 60 minutes by injecting the already prepared 0.1 M amino acid/NMP solution and 0.5 M BOP/HOBt/NMP solution into a micro-well. After the coupling reaction, it was washed with the NMP solution for 10 minutes.

Each amino acid used in the coupling reaction has a protecting group of which the amine group portion is decomposed by light. By continuously repeating this procedure, an oligopeptide library with desired positions and sequence was synthesized. FIG. 15 represents a process in which polypeptide G-E-C-A and H-F-D-B are synthesized through the above procedures, wherein I is a protecting group.

The present invention has the following merits because of the use of

an optical modulator instead of conventionally used photomasks.

First, various compound libraries can be effectively prepared with a simple process since the process in which tens of sheets of photomask should be replaced during every preparation procedure of a protecting group is omitted, and an optical switch can be individually operated.

Second, problems of alignment incompatibility of masks and chips which can occur during the replacement procedure of photomasks are solved by irradiating light only with an optical switch and a fixed optical modulator, so that errors occurring during the library preparation can be reduced.

Third, an automation of the compound library preparation process can be more easily accomplished since the optical irradiation position and quantity of light can be simply controlled by an electrical signal of a controller. Furthermore, it is appropriate to compose only the program itself even though other configurations are required since the compound library configuration and compound analyzing method depend on programs used.

Fourth, more various compound libraries are provided and the activity aspects of analyzed compounds according to the difference of light quantity can be easily known in the present invention, since even the same compound composition can be easily modified by controlling the quantity of light.

While the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art will appreciate that various modifications and substitutions can be made thereto without departing from the spirit and scope of the present invention as set forth in the appended claims.

WHAT IS CLAIMED IS: