JPS63101403A - Fixing structure composed of multiple monolayers of polymer linkage and its formation - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for forming multiple monolayers of polymer bonds, in which the length of each of the resulting multiple monolayer structures can be controlled so that they are substantially equal. .

Polymer-bound multi-monolayer structures are useful for a variety of purposes, such as chemical or biochemical sensors and molecular leads.

The assembly of molecular stacks has traditionally been evaluated for solid-phase synthesis of peptides. R.B.Fist Merrifield Biochemistry, Volume 3, Fu9.1385-1
p. 390 (1964) describes the steps of pradikinin, a natural nonazuptide, by a step of attaching the terminal amino acid to a support, a decarboxylation step, and a coupling step of attaching the terminal amino acid to the amino acid residue (. A new synthetic method is reported. These steps are repeated until Veptibi with nine amino acid residues is produced.

More recently, this protein synthesis method has been extended to the production of multilayer films of 1,5-hexatecenyltrichlorsilane. Lucie Meklael and Jacob Sajip are J. Am. Kemi Sosa, (J, Am, C
hem, Soc, ), vol. 105, pp. 674-676 (
(1983) reported a two-step method consisting of monolayer absorption followed by chemical activation of the exposed surface to provide polar absorption sites for subsequent monolayer fixation.

The present invention comprises: a) a first bonded reactive moiety 1 to a solid phase;
b) carrying out a wc1 coupling reaction by reacting said bound reactive moiety with an excess of a first polyfunctional reagent or combination of reagents, wherein said reagent or combination of reagents comprises a first polyfunctional group or groups capable of reacting with the first reactive moiety and a second functional group or groups capable of reacting with a second multifunctional reagent or combination of reagents; The coupling reaction produces a first intermediate adduct derived from the first reagent and attached to the solid phase, which adduct is formed from a residue and a second reactive moiety that can be coupled to the second multifunctional reagent. C) performing a second coupling reaction by reacting the first intermediate adduct with an excess of the second polyfunctional reagent to remove residues derived from the first intermediate adduct and residues from the second reagent; forming a first molecular unit consisting of
the molecular unit is reactive with the first polyfunctional reagent; d) the first coupling reaction and the second coupling reaction described above; Repeatedly, one effective sequential polymer bond” (
effective 5equential poly
The present invention provides a method for producing an immobilization structure consisting of multiple monolayers of effective sequential polymeric linkages (1).

According to the present method, a large number of immobilized multilayer monolayer structures can be formed, each structure having a substantially equal length extending from the support.

Another aspect of the invention relates to immobilization structures produced by the method.

In the context of the present invention, the effective sequential polymeric bond 1 is preferably a conductive or semiconducting molecular entity by bond delocalization.
t7) a conjugated moiety that can act as an electrically conductive (and therefore resistive) molecular entity; In any of the preferred examples, biomolecules or other macromolecules or the like may be immobilized by physical or covalent bonding to the terminal functional groups of the multi-monolayer structure.

In the present invention, the solid phase is an immobilized intermediate having a second reactive moiety capable of reacting with a first polyfunctional reagent, taking up at least a portion of that reagent, and reacting with a second polyfunctional reagent. Addition! The result is a reactive moiety formed on the surface.

In the context of the present invention, one reactive moiety 1 is a molecular moiety that can react with other moieties according to classical functional group organic chemistry. Among these, amine groups, carboxylic acids, carbonylimidazoles, aldehydes, carboxylic acid halides::carboxylated esters, carboxylated anhydrides, alcohols or phenols, and other moieties may be mentioned. Among these, amine groups and carbonylimidazole moieties are preferred because of their high reactivity.

FIG. 1 is a plot of radioisotope data obtained after the completion of each reaction cycle adding successive monolayers of phenyl urethane.

FIG. 2 is a plot of immobilized acetylcholinesterase activity at 6 tensions versus polyurethane bond length.

FIG. 3 is a plot of the change in bond length of the immobilization monolayer structure versus the activity (%) of the enzyme immobilized by the multi-monolayer structure.

In the method of the invention, an immobilized structure consisting of multiple monolayers of effective sequential polymeric linkages is formed by alternating reaction sequences carried out one layer at a time from the surface of the solid phase with polyfunctional reagents.

According to the method of the present invention, a solid phase containing an attached first reactive moiety is contacted with a first polyfunctional reagent or combination of reagents. The contacting method may be any means capable of bringing the reactants into contact with the surface phase.

For example, a support containing a reactive moiety can be reacted with a selected first polyfunctional reagent or combination of reagents in a vapor state in a conventional manner, or by simply mixing the reactants together in the liquid phase. .

When the reaction is carried out in the liquid state, common solvents are often used. In some cases, polyfunctional reagents are susceptible to hydrolysis and therefore aprotic solvents will have to be used. It is generally preferred to use an excess of the multifunctional reagent to avoid termination reactions between the two molecules that produce products in the solid phase that interfere with the formation of the desired polymeric monolayer.

Convenient completion of the first coupling reaction will result in an intermediate adduct containing at least a portion of the first reagent, which adduct will carry at least one second reactive moiety terminally. At this point, it is often preferred to employ an additional step to remove unreacted first polyfunctional reagent, usually by conventional washing methods.

In the second coupling step of the method of the invention, the support to which the intermediate adduct is attached is reacted in a similar manner with a second polyfunctional reagent or combination thereof. This step incorporates at least a portion of the second reagent or combination thereof to form a monolayer of desired polymeric bonds, which monolayer is partially eclipsed and capable of reacting with the first polyfunctional reagent. The cycle of holding at least one species at the end and thus forming a second monolayer is repeated. In this alternating manner, each reaction cycle can be repeated to form the desired number of monolayers, and the ends of the entire structure are partially eclipsed by reactive moieties belonging to either of the two reactive moieties produced. It ends in 1s.

Solid phases useful in the practice of the present invention, having reactive moieties attached to their surfaces, are used as supports for multiple monolayer attachments, fixing the reaction products to the solid phase and removing unreacted components at each step. Any solid phase can be any solid phase from which the polyfunctional reagent can be effectively removed before the next reaction step with another polyfunctional reagent. The selection of these supports depends in part on their physical and chemical properties, such as solubility, functional groups, mechanical stability, surface area, swelling properties, hydrophobicity or hydrophilicity; and their electrical properties, such as Controlled by conductivity, resistivity, etc.

Essentially three main types of supports are highly preferred: inorganic materials, natural polymers and synthetic polymers. Among these, the following may be mentioned: Polymers, such as polyvinyl alcohol, -acrylates and monoacrylic acids, such as polyethylene-acrylic acid,
Polyethylene〇〇-methacrylic acid, polyethylene-a
O-ethyl acrylate, polyethylene-CO-methyl acrylate, polypropylene-CO-acrylic acid, polypropylene-CO-methylacrylic acid, polypropylene-
CO-ethyl acrylate, polypropylene-〇〇-methyl acrylate, polyethylene-co-vinyl acetate, polypropylene-〇〇-vinyl acetate, etc.; and those containing acid anhydride groups, such as polyethylene-CO-maleic anhydride, polypropylene −〇〇−maleic anhydride, etc.;
and natural polymers such as polybutylene, proteins and carbohydrates; metalloids with semiconducting properties such as silicon, germanium.

metals such as platinum, gold, nickel, copper, zinc, tin, palladium, silver.

Particularly useful are metal and metalloyton oxides, Tatoe 1jPt-PtO, G1-8in.

Au-Aug, TlO2, Cu-CuO nato Y: Yes. Inorganic materials with conductive or semiconducting properties are preferred in certain forms of the invention. Among these are silicon/silicon oxide semiconductor devices, or other semiconductor devices such as lithium niobate, gallium arsenide, indium phosphide, and the like.

The supports described above, particularly metalloid 9, can be treated to contain suitable reactive moieties, or can be obtained commercially, optionally already containing reactive moieties. Materials containing reactive moieties on the surface, such as aminosilane, hydroxyl or carboxysilane bonds, are preferred in certain aspects of the invention and can be produced by well-established surface chemistry techniques involving silanization reactions and the like.

Examples of these materials are those with surface oxidized silane moieties that lead to γ-aminopropylsilane and other organic moieties; N-(3-()ethoxysilyl)propyl]phteramicuccinic acid; and bis-(2- Covalently bonded to human 90xethyl)aminopropyltriethoxysilane. Examples of readily available materials containing reactive Nf (difunctional groups) are para-aminophenyltriethoxysilane and γ-aminopropylsilanized controlled porous glass beads (Controlle4 Porous Glas).
s Bead) (obtained from Perth Chemical Co., P.
O, Box 117.61105 Lockford, Illinois).

The multifunctional reagents useful in the method of the invention react in an alternating manner to form at least one molecular unit of the desired effective polymer bond, and this alternating reaction system is repeated until the desired number of molecular units is produced. It is something that can be used. The combinations of the first and second multifunctional reagents can be varied to obtain a wide variety of polymer bonds ranging in electrical properties from nonconductors to semiconductors and conductors. The terminal functional groups of the obtained multi-monolayer structure may be further reacted with biomolecules, other polymers, and the like. A combination of first reagents can also be used with a second combination of multifunctional reagents to obtain branched polymeric linkages and the like.

In general, a first multifunctional reagent or a combination thereof is incorporated into an immobilized molecular unit to form an intermediate addition containing a small number of terminal functional groups that can react with a second multifunctional reagent. @2 Upon reaction with a polyfunctional reagent or a combination thereof, a small amount of this second reagent (
Both are partially incorporated into the immobilization structure, yielding one completed molecular unit containing at least one terminal group capable of reacting with the @l polyfunctional reagent. This cycle is then repeated. Generally, it is preferred that the end group(s) of the completed molecular unit be the same reactive moiety as originally applied on the support.

In the method of the invention, the first multifunctional reagent or combination thereof is selected according to the type of multi-monolayer structure desired to be achieved and may be either heterofunctional or homofunctional.

The first reagent or combination thereof reacts with the reactive moiety attached to the solid phase.

It is stated that examples of polyfunctional reagents suitable for use alone or in combination as the first reagent are those that react with reactive moieties of the support by nucleophilic substitution and are represented by the general formula: Can be done.

(2) In the formula, R is easily substituted by a nucleophile and is preferably an aromatic heterocyclic residue or a halogen atom.

Other examples of reagents suitable as the first multifunctional reagent are those capable of reacting in a condensation/elimination manner with reactive moieties of the support and are represented by general formula II and below.

■ In the formula, R1 is a phenyl group, polyphenyl group, fused cyclic aromatic residue, alkyl group, alkene residue, aromatic heterocyclic residue, or organometallic compound residue; R2 is H, a halogen atom, OR4, OH, SH; R3 is H, a halogen atom, OR4, OH, or SH, where R4 is an aliphatic or aromatic hydrocarbon residue.

R2-N-R1-N-R3 ■ In the formula, R1 is a phenyl group, a polyphenyl group, a fused cyclic aromatic residue, an alkyl group, an alkene residue, an aromatic heterocyclic residue, or an organometallic compound residue. Yes; R-N-+! I(O-
N-, 0-N-, X-N2+-; R3-N- is H○-
N-, 0-N-, and X-N2+-.

The second polyfunctional reagent reacts with the terminal functional group (one type or two or more types) of the intermediate adduct produced in the first reaction.

The second polyfunctional reagent (one or more) may be heterofunctional or homofunctional, and can be expressed by the following formula:
selected from a group of reagents represented by

Xl-R-X2 ■ In the formula, R is a phenyl group, a polyphenyl group, a fused cyclic aromatic residue, an alkyl group, an alkene residue, an aromatic heterocyclic residue, or an organometallic compound residue; and x2
are the same or different, Nl2゜NHOH, or OH
; X3 is Nl2. NHOH, OH.

Maf, :HaH;
There is tH-c-.

The first and second reagents are selected according to the polymer bond desired to be formed. When the first multifunctional reagent is selected from general formula I, the 1M2 multifunctional reagent is further combined with the terminal functional moiety(s) of the intermediate adduct formed by the first coupling reaction by a substitution reaction. It must be chosen to contain a reactive nucleophilic group. The first reagent has the general formula ■ or ■
When selected from among the above, the second polyfunctional reagent must be one that reacts by condensation/elimination. Generally the conjugate type second
A reaction cycle performed using a combination of a reagent and a first reagent that reacts by nucleophilic substitution will produce semi-conjugated polymeric bonds with potentially semiconducting properties. On the other hand, non-conjugated reagents produce non-conjugated bonds.

A reaction cycle performed with a first reagent acting by condensation/elimination and a conjugated second reagent will produce a conjugated polymer bond that is potentially electrically conductive. Non-conjugated type 2
The reaction cycle performed with the reagent will produce an unconjugated reagent.

A general format using various first and second bifunctional reagents is shown in the table below.

A general mode of synthesis of branched multi-monolayer structures in which the resulting polymer or oligomer is a potential semiconductor and can be used as a two-dimensional molecular conductor is shown below.

+2) H2N-@-NH2 It should be recognized that various combinations of multiple monolayer units with different electrical properties may be used to form various electrical components at the molecular level. For example, 1 electric switch 1.
1 resistor 1, etc. are included within the intended scope of the present invention. A general format for forming a Molecule error switch is shown below.

As mentioned above, semiconjugated or conjugated bonds (in this case polybenziimine) are formed on a conductive support with a substitutable metal-containing compound that can act as a molecular switch inserted between the parts of these bonds. administered. When sufficient voltage or other stimulation is applied to this structure, the metal will be displaced from the molecular switch and current will be conducted.

A first polymer linkage is applied to the support according to the method of the present invention to provide a terminal reactive group capable of reacting with the metalloporphyrin. Further polymer monolayers are then formed, layer by layer, from the metalloporphyrin molecular switch, and the end of the second bond is then reacted with the reactive moiety on the support to which it is attached.

Compounds suitable for use as molecular switches are those that can respond to an applied voltage or other stimulus (eg, photoactivation, etc.) in a manner that either prevents or allows the passage of electrons. What is useful in this regard is
These compounds include metalloporphyrins, phthalocyanines, and hemi'quinones.

Charge transfer current, e.g. T TF −T CN Q,
(Tetrathiafulvalene-tetracyanoquinodimethane)
There are also formats that appear to be reliable for capturing.

For example, diamino-TTF can be covalently attached within the main body of a solid-phase synthesized conjugated polymer and then chelated to TTF' units from solution to form TTF-TCNQ charge transfer salts at specific positions in the molecular circuit. You can do it. For compounds that can act as molecular switches, please refer to the following literature: Pryon M. Hoffmann and James N. Epels, 'Bolf 49 Molecular Metals 1, Ace, C.
hem.

Res, 1983, 16.15-21); T. Daburiney Palette et al., Conductivity in Phthalocyanines Modulated by Circularly Polarized Light 1. Nature.

Volumes 301 and 24; and the 2nd International Symposium on Molecule Electronics Devices, Naval Research.
Lab, Washington DC, April 13-15 (198
3 years).

The non-conjugated region, which essentially acts as a molecular resistor, is further coupled to the molecular conductor portion and the molecular switch to modulate the current passing therethrough. The format for a simple molecular circuit is shown below. Here, a polybencyimine molecular conductor is conjugated with a metal porphyrin inch parallel to the n-propylamine resistor.

It should be appreciated that the stepwise monolayer addition of the method of the invention can provide supports with multiple multilayer structures starting from the surface and a narrow distribution of total leather layer lengths. In some configurations, the accuracy achieved is on the order of a few angstroms. This is particularly useful in the field of conductive or semiconducting polymers. Uniform length of the oligomer or polymer may be beneficial for forming Molecule error level circuits, etc., as described above. This is because it allows for careful control over the length of each component forming the circuit. Such uniformity is not readily achievable with the techniques currently available in this field.

It should also be recognized that the number of multiple monolayer structures formed on a support is governed by the number of initial reaction sites on the support surface, as well as other factors, such as steric hindrance between the monolayer bonds themselves. It is. The initial number of reaction sites is such that an effective layer is formed over all or part of the support surface, and the layer thickness is governed by the number of completed reaction cycles, forming a sequential monolayer on each structure. be done. The method of the present invention thus also provides an alternative to conventional film forming methods.

Along these same directions, by adjusting the effectiveness of the in-phase reactive moieties, the spacing between the multiple monolayer structures of the ridges can be controlled. In certain forms of the invention, the spacing between each bond is on the order of about 23 A units.

In this case, the effective rate on the face is about 50% Passivesi A7￥
This spacing can be increased to approximately 50 angstroms by increasing the spacing. In a preferred embodiment, where multiple monolayers of phenylurethane bonds are prepared, the spacing of the NH2 groups on the solid phase is determined by passivating about 50% of the available sites with dichlorodimethylsilane prior to the monolayer synthesis process. It can be changed.

In this way, the bonds can be concentrated to a center of about 5 OA, which may be advantageous in applications such as molecular electronics.

The electrical activity of the conductive or semiconducting polymers prepared according to the above reaction mode is generally p or n)
- Can be enhanced using the ping method. For example, the process commonly used to toppe polyacetylene is
A dopant such as ξ can be used to prepare the conjugated bonds produced by the present method.

After the desired number of monolayers of a specific molecule have been produced,
The ends of the multi-monolayer structure can also be reacted with various molecules. Useful in this regard are macromolecules, such as chelated crown ethers, or biological molecules, such as enzymes, hormones, antigens, antibodies, membrane receptors, and the like. Immobilization of biomolecules by employing an immobilized monolayer structure increases the stability of biomolecules and therefore represents an alternative to direct immobilization on supports. In certain wing configurations, the stability of individual enzymes is directly proportional to the length of the multi-monolayer structure.

Monolayers of about 4 or more are generally preferred, and monolayers of about 10 or more are particularly preferred. One skilled in the art can easily optimize the stability of a biomolecule of interest by testing its stability on monolayers of various lengths.

When immobilizing biomolecules, it is preferable to employ a covalent bonding method. General techniques that can be employed are reported in the art. One of the documents of this kind is 1. Immobilized enzymes, antigens, antibodies and peptides, preparation and characterization 1.
Edited by Hard H. Questal, Marcel Detzker (1975).

The following describes a preferred reaction mode according to the invention for producing multi-monolayer structures consisting of electroactive polyphenylurethane linkages.

The support containing the reactive moiety (preferably NF2) is first reacted with carbodiimidazole in a solvent.

Particularly useful solvents in this regard are aprotic in order to avoid the side reaction of carbonyldiimidazole hydrolysis. Preferred solvents include dimethylformamide and dimethyl sulfoxide9.
, benzene, chloroform, acetone, ether, etc.

The amount of carbodiimidazole used in this first coupling step can vary widely and must be sufficient to avoid the formation of termination reaction products that interfere with the formation of the desired polymer monolayer. . For this reason, an excess of reagent is generally used.

For example, from about 1 meq to about 10 meq or more carbonyldiimidazole can be used, from about 6 to about 10 meq.
Millimeter bonds are more preferable. The coupling reaction can be carried out in conventional manner, for example by simply mixing the reactants together. Reaction times can vary widely;
Generally about 15 to about 60 minutes. The temperature at which the reaction is carried out can also vary widely and is generally carried out at room temperature for convenience.

It is preferred to employ an additional step to remove unreacted carbonyldiimidazole. This can be achieved by conventional filtration and washing techniques, preferably with an aqueous solution followed by washing with an aprotic solvent to destroy unreacted carbonyldiimidazole and remove residual products from the solid phase.

The coupling reaction described above produces a support-bound imidazole derivative, which is reactive towards the second polyfunctional reagent, preferably a diamine, such as 1,4-phenylenediamine. Again, an additional step to remove excess of this second reagent is employed to minimize biomolecule termination reactions that can generate products on the solid phase that interfere with the formation of the desired polymeric monolayer. It is preferable to keep it to .

The imidazole derivative and the second reagent are brought into contact by a conventional method. Contact times vary widely, with at least about 15 minutes being suitable for this reaction.

When the second coupling reaction is completed, one phenyl urethane monolayer is formed. This alternating reaction pattern is then repeated as many times as necessary to obtain the overall structure of the desired length. The terminal group ends with an amine function or a hemicarbonyldiimidazole function.

In a particularly preferred embodiment, the multi-monolayer structure thus produced is used to immobilize specific enzymes and other proteins. Immobilization is carried out by conventional methods. For example, contact of the first multifunctional reagent with the terminal portion of the multimonolayer structure would produce an intermediate adduct, which would then react with amino groups contained in the enzyme. Covalent attachment of the enzyme to the monolayer structure thus takes place.

Examples of diverse applications of the immobilized multi-monolayer structures described above include applications in the area of biological and chemical sensors. Any quantity of antibodies, enzymes, hormones, membrane receptors, etc. can be combined into a multi-monolayer structure, resulting in an optimal molecular error design for a particular detection unit. Certain forms of the invention In some embodiments, the stability of a particular immobilized enzyme can be increased by increasing the distance between the support and the enzyme through the use of multiple monolayer structures.

Furthermore, the bond length of the electrically active bond bonded to the electrode and semiconductor surface is formed with an accuracy of several angstroms,
By varying this, a Molecki error conducting wire can be manufactured. Along these same lines, the combination of these single-layer structures with compounds that act as switches could be useful for creating Molecker-scale electronic circuits.

The following examples illustrate specific embodiments of the invention:
This should not be considered a limitation.

Example 1 Preparation of a multi-monolayer structure consisting of polyphenylurethane bonds Controlled porous glass (CPG) beads 19 were washed with 4-5 volumes of dried dimethylformamide (DMF) on a glass funnel and then placed in a flask. Transfer to 0.0% in DMF'.
25 M carbonyldiimidazole (CDI) 5,0-
was added. The mixture was shaken at room temperature for 15 minutes, then filtered and washed with water and 4-5 volumes of DMF. C
Transfer the DI-treated CPG to a flask and add 0.25 M-c-p
-Add phenylenediamine 5,0-100-50
0 DPM/micromol diamine), shaken for 80 minutes and
did. The mixture was filtered and washed with DMF until no 14C was detected in the F solution.

This process was repeated for up to 18 cycles. Samples from each cycle were counted per 140 and analyzed for NH2 end groups. The radioisotope data were added sequentially to the structure, one monolayer per completion of each reaction sequence, with a correlation coefficient of 0.94.
It was shown that A similar correlation coefficient of 0.9932 was obtained for the formation of the first eight monolayers (see Figure @1).

The surface area per 7g of CPG beads is approximately 70 rn”/9
It is known that According to the routine calculation method, the packing density of phenylurethane bonds is given by the name 1 linkage u1.
528A2, and the interval between adjacent bonds was calculated to be 23A.

CPG-Perth 23909 Controlled Porous Glass, Aminopropyl DMF-Haas 20673 Dimethylformamide) #p-7 22-diamine-Aldrich P-2゜3
96-2, recrystallized from toluene, melting point 143-14C-
p-phenylenediamine-Pathfinder 15.03
Hc/micromolmol l-noses 20220-
N,Nl-Carbonyldiimidazole Example 2 Acetylcholinesterase Immobilization and Stability Test Using the series of glass beads described above with phenylurethane bonds of various bond lengths, eex-type amoacetylcholinesterase was immobilized with these bonds. The terminal amino group was immobilized with carbonyldiimidazole (CDI). Immobilized enzyme - stored in a glass pea refrigerator (approximately 10°C)
. Assays were performed periodically to determine the extent to which initial enzyme activity was retained over a 6-month period. The data is summarized below.

Bond length vs. AChE enzyme activity (%) Bond length (angstroms)... 2135 4
The effect of the 960A immobilization bond length on enzyme stability is quite significant. Minimal bonding after 6 months (21A)
is 30 for 65% activity for the longest bond (60A)
% of enzyme activity.

This is more than twice as much hatred (see Figure 2).

It should be recognized that there is a mathematical correlation between immobilization bond length and long-term stability of the immobilized enzyme.

In fact, there is a linear relationship between enzyme activity (%) versus immobilization bond length or monolayer length. In other data manipulations, % enzyme activity was plotted against % change in total bond length per continuous monolayer of phenyl urethane. This plot is very linear, with a satisfactory correlation coefficient of 0.9992 (Figure 3).

[Brief explanation of the drawing]

FIG. 1 is a plot of radioisotope data obtained after the completion of each reaction cycle adding successive monolayers of phenyl urethane. FIG. 2 is a plot of immobilized acetylcholinesterase activity after 6 months versus polyurethane bond length. FIG. 3 is a plot of the change in bond length of the identification monolayer structure versus the activity (%) of the enzyme immobilized by the multi-monolayer structure. (4 other people) Forgetfulness, wallet / 1 / Agoka Ri's 3 colors A± (14C So 4 Yu Z now) Rukame's activation (2 →

Claims (18)

[Claims] (1) by a) applying a first bound reactive moiety to a solid phase; b) reacting said bound reactive moiety with an excess of a first polyfunctional reagent or combination of polyfunctional reagents; A first coupling reaction is carried out, wherein the reagent or combination of reagents comprises at least one functional group capable of reacting with the reactive moiety and at least one functional group capable of reacting with the second multifunctional reagent or combination of reagents. a second polyfunctional reagent comprising a second polyfunctional reagent, thereby comprising a residue from a first reagent or combination of reagents, and further capable of being coupled to a second multifunctional reagent.
forming a solid phase-bound intermediate adduct comprising at least one reactive moiety; c) forming a second cup by reacting said intermediate adduct with an excess of a second multifunctional reagent or combination of reagents; A ring reaction is carried out to form a first molecular unit of the active polymer bond;
at least one terminally reactive moiety capable of coupling with a polyfunctional reagent or combination of reagents; d) repeating the above first coupling reaction and second coupling reaction in an effective sequential manner to form a polymer; A method of forming an immobilization structure consisting of multiple monolayers of effective sequential polymeric bonds, comprising the steps of producing an immobilization structure consisting of multiple monolayers of bonds. (2) at least two having substantially the same number of polymer bonds;
2. The method of claim 1, wherein a number of immobilized multi-monolayer structures are produced. (3) The first polyfunctional reagent has general formulas I, II and III ▲There are mathematical formulas, chemical formulas, tables, etc.▼ I , ▲There are mathematical formulas, chemical formulas, tables, etc.▼II, R_2-N-R_1-N-R
_3III (wherein R is easily substituted by a nucleophile and is a heterocyclic aromatic residue or a halogen atom; R_1 is a phenyl group, a polyphenyl group, a fused ring aromatic residue, an alkyl group, an alkene residue) group, aromatic heterocyclic residue, organometallic compound residue; R_2 is H, halogen atom, OR_4, OH
, SH; R_2-N- is HO-N-, O=N-,
X^-N_2^+-; R_3 is H, halogen atom, OR_4, OH or SH; R_3-N- is H
O-N-, O=N-, X^-N_2^+-; where R_4 is an aliphatic or aromatic hydrocarbon residue)
The method according to claim 1, wherein the reagent is selected from the group consisting of reagents. (4) the first polyfunctional reagent is carbonyldiimidazole;
1,4-diardobenzene, 1,1'-diardoferrocene, 1,4-dinitrosobenzene and 1,4-di(
4. The method of claim 3, wherein the polyfunctional reagent is selected from the group of polyfunctional reagents consisting of carboxylic acid chloride)benzene. (5) The method according to claim 4, wherein the first polyfunctional reagent is carbonyldiimidazole. (6) the second polyfunctional reagent is phenylenediamine;
A method according to claim 4. (7) The method according to claim 6, wherein the first polyfunctional reagent is carbonyldiimidazole. (8) The method according to claim 6, wherein the first polyfunctional reagent is 1,4-diardobenzene. (9) The method according to claim 6, wherein the first polyfunctional reagent is 1,1'-diadoferrocene. (10) The method according to claim 4, wherein the second polyfunctional reagent is 1,4-hydroquinone. (11) The support contains an amino functional group, and the first and second
the coupling step is carried out with a mixture of excess 1,4-diardobenzene and phenylenetriamine;
A method according to claim 1. (12) a) applying a bound amino functionality to the solid phase; b) performing a first coupling reaction by reacting the bound amino functionality with excess carbonyldiimidazole to derivate from carbonyldiimidazole; c) forming an intermediate adduct consisting of a carbonyl-containing residue, and a reactive moiety capable of coupling to a second multifunctional reagent comprising at least one amino functional group; performing a second coupling reaction to form a first phenylurethane molecular unit by reacting with a polyfunctional reagent; d) repeating the first and second coupling reactions described above to form at least four layers; e) reacting the terminal amino functionality with carbonyldiimidazole to produce a terminal intermediate adduct capable of coupling with an enzyme; f) An enzyme immobilization method comprising the step of reacting the obtained intermediate adduct with an enzyme to produce an enzyme immobilized on a multilayered structure. (13) The method according to claim 12, wherein the enzyme is acetylcholinesterase. (14) A multi-monolayer structure produced by the method according to claim 1. (15) A branched multilayer monolayer structure produced by the method according to claim 1. (16) A branched multilayer monolayer structure produced by the method according to claim 11. (17) An immobilized enzyme produced by the method according to claim 12. (18) further e) reacting the immobilized structure with a compound capable of acting as a molecular switch; f) reacting the bound molecular switch with a first and second multifunctional reagent alternately to and g) reacting this second structure with a second solid phase for attachment thereto. , the method according to claim 1.