U.S. patent application number 17/276760 was filed with the patent office on 2021-11-11 for oxidation-reduction polymer including transition metal complex, and electrochemical biosensor using same.
This patent application is currently assigned to I-SENS, INC.. The applicant listed for this patent is I-SENS, INC.. Invention is credited to Sumin GWAK, In Seok JEONG, Young Jea KANG, Jinseon LEE, Hyunseo SHIN, Bona YANG, Hyunhee YANG.
Application Number | 20210347926 17/276760 |
Document ID | / |
Family ID | 1000005786552 |
Filed Date | 2021-11-11 |
United States Patent
Application |
20210347926 |
Kind Code |
A1 |
KANG; Young Jea ; et
al. |
November 11, 2021 |
OXIDATION-REDUCTION POLYMER INCLUDING TRANSITION METAL COMPLEX, AND
ELECTROCHEMICAL BIOSENSOR USING SAME
Abstract
The present disclosure relates to an oxidation-reduction polymer
including a transition metal complex, which has a unique structure,
and so can be prepared in a simpler step compared to a conventional
method, and can increase the immobilization rate of the transition
metal complex and facilitates the introduction of a functional
group or a linker, a method for preparing the same and an
electrochemical biosensor comprising the oxidation-reduction
polymer.
Inventors: |
KANG; Young Jea; (Seoul,
KR) ; SHIN; Hyunseo; (Seoul, KR) ; YANG;
Bona; (Gyeonggi-do, KR) ; JEONG; In Seok;
(Seoul, KR) ; LEE; Jinseon; (Gyeonggi-do, KR)
; GWAK; Sumin; (Incheon, KR) ; YANG; Hyunhee;
(Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
I-SENS, INC. |
Seoul |
|
KR |
|
|
Assignee: |
I-SENS, INC.
Seoul
KR
|
Family ID: |
1000005786552 |
Appl. No.: |
17/276760 |
Filed: |
May 9, 2019 |
PCT Filed: |
May 9, 2019 |
PCT NO: |
PCT/KR2019/006000 |
371 Date: |
March 16, 2021 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/32 20130101; C08F
226/06 20130101; C08F 2810/00 20130101; G01N 27/3272 20130101; C12Q
1/26 20130101 |
International
Class: |
C08F 226/06 20060101
C08F226/06; C12Q 1/26 20060101 C12Q001/26; C12Q 1/32 20060101
C12Q001/32; G01N 27/327 20060101 G01N027/327 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 2018 |
KR |
10-2018-0111633 |
Claims
1. An oxidation-reduction polymer for an electron transport medium
of an electrochemical biosensor, having any one structure of the
following Chemical Formulas 1 to 4: ##STR00020## ##STR00021##
wherein, M is a transition metal selected from the group consisting
of Os, Rh, Ru, Ir, Fe and Co; L.sub.1 and L.sub.2 are combined with
each other to form a bidentate ligand selected from the following
Chemical Formulas 5 to 7; L.sub.3 and L.sub.4 are combined with
each other to form a bidentate ligand selected from the following
Chemical Formulas 5 to 7; L.sub.5 and L.sub.6 are each combined
with each other to form a bidentate ligand selected from the
following Chemical Formulas 5 to 7; ##STR00022## the R.sub.1,
R.sub.2 and R'.sub.1 are each independently selected from the group
consisting of a substituted or unsubstituted alkyl group having 1
to 10 carbon atoms, a substituted or unsubstituted alcohol group
having 1 to 20 carbon atoms, a substituted or unsubstituted
alkylhalogen group having 1 to 20 carbon atoms, a substituted or
unsubstituted thiol group having 1 to 20 carbon atoms, a
substituted or unsubstituted alkyl azide group having 3 to 20
carbon atoms, a substituted or unsubstituted aryl azide group
having 7 to 30 carbon atoms, a substituted or unsubstituted alkenyl
group having 2 to 40 carbon atoms, a substituted or unsubstituted
alkynyl group having 2 to 40 carbon atoms, a cyano group, a halogen
group, deuterium and hydrogen, the R.sub.3 to R.sub.20 are each
independently selected from the group consisting of a substituted
or unsubstituted alkyl group having 1 to 10 carbon atoms, a
substituted or unsubstituted cycloalkyl group having 3 to 40 carbon
atoms, a substituted or unsubstituted aryl group having 6 to 50
carbon atoms, a substituted or unsubstituted heteroaryl group
having 3 to 50 carbon atoms, a substituted or unsubstituted alkoxy
group having 1 to 20 carbon atoms, a substituted or unsubstituted
alcohol group having 1 to 20 carbon atoms, a substituted or
unsubstituted alkylhalogen group having 1 to 20 carbon atoms, a
substituted or unsubstituted thiol group having 1 to 20 carbon
atoms, a substituted or unsubstituted alkyl azide group having 3 to
20 carbon atoms, a substituted or unsubstituted aryl azide group
having 7 to 30 carbon atoms, a substituted or unsubstituted aryloxy
group having 6 to 30 carbon atoms, a substituted or unsubstituted
alkylamino group having 1 to 20 carbon atoms, a substituted or
unsubstituted arylamino group having 6 to 30 carbon atoms, a
substituted or unsubstituted alkylsilyl group having 1 to 20 carbon
atoms, a substituted or unsubstituted arylsilyl group having 6 to
30 carbon atoms, a substituted or unsubstituted arylalkylamino
group having 1 to 50 carbon atoms, a substituted or unsubstituted
alkenyl group having 2 to 40 carbon atoms, a substituted or
unsubstituted alkynyl group having 2 to 40 carbon atoms, a cyano
group, a halogen group, deuterium and hydrogen; the Ad is selected
from the group consisting of a primary and secondary amine group,
an ammonium group, a halogen group, an epoxy group, an azide group,
an acrylate group, an alkenyl group, an alkynyl group, a thiol
group, isocyanate, an alcohol group, and a silane group; the X is a
counter ion; the a is an integer of 1 to 15; the b is an integer of
1 to 15; the c is an integer of 1 to 15; the m is an integer of 10
to 600; the n is an integer of 10 to 600; and the o is an integer
of 0 to 600.
2. The oxidation-reduction polymer for an electron transport medium
according to claim 1, wherein the compound of Chemical Formula 1
has a structure represented by the following Chemical Formula 8 or
9. ##STR00023##
3. A method for preparing an oxidation-reduction polymer as set
forth in claim 1, the method comprising the steps of: (i)
functionalizing polyvinylpyridine or polyimidazole to produce a
polyvinylpyridine precursor or a polyimidazole precursor; (ii)
functionalizing the transition metal complex; and (iii) reacting
the polyvinylpyridine precursor or polyimidazole precursor produced
in step (i) and the functionalized transition metal complex
produced in step (ii) by a click reaction to prepare the
oxidation-reduction polymer as set forth in claim 1.
4. The preparation method according to claim 3, wherein the click
reaction of step (iii) is azide-alkyne Huisgen cycloaddition or
thiol-ene reaction.
5. A composition for an electrochemical biosensor comprising: an
enzyme capable of subjecting a liquid biological sample to an
oxidation-reduction reaction; and the oxidation-reduction polymer
as set forth in claim 1.
6. The composition according to claim 5, wherein the enzyme
comprises: at least one oxidoreductase selected from the group
consisting of dehydrogenase, oxidase, and esterase; or at least one
oxidoreductase selected from the group consisting of dehydrogenase,
oxidase, and esterase, and at least one cofactor selected from the
group consisting of flavin adenine dinucleotide (FAD), nicotinamide
adenine dinucleotide (NAD), and pyrroloquinoline quinone (PQQ).
7. The composition according to claim 5, wherein the enzyme is at
least one selected from the group consisting of flavin adenine
dinucleotide-glucose dehydrogenase (FAD-GDH) and nicotinamide
adenine dinucleotide-glucose dehydrogenase.
8. The composition according to claim 5, further comprising one or
more additives selected from the group consisting of surfactants,
water-soluble polymers, and thickeners, and a crosslinking
agent.
9. An electrochemical biosensor comprising the oxidation-reduction
polymer according to claim 1.
10. The electrochemical biosensor according to claim 9, further
comprising a sensing layer, a diffusion layer, a protection layer,
two or more electrodes, an insulator and a substrate, comprising
the oxidation-reduction polymer as set forth in claim 1.
11. The electrochemical biosensor according to claim 10, wherein
the electrode is a 2-electrode consisting of an working electrode
and a counter electrode, or a 3-electrode consisting of an working
electrode, a counter electrode and a reference electrode.
12. The electrochemical biosensor according to claim 9, wherein the
biological sample is blood.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This application claims the benefit of Korean Patent
Application No. 10-2018-0111633 filed on Sep. 18, 2018 with the
Korean Intellectual Property Office, the disclosure of which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present disclosure relates to an oxidation-reduction
polymer including a transition metal complex, which can be prepared
in a simpler step compared to a conventional method, and can
increase the immobilization rate of the transition metal complex
and facilitates the introduction of a functional group or a linker,
and a method for preparing the same.
BACKGROUND ART
[0003] Recently, interest in the development of biosensors is
increasing day by day for quantitative and qualitative analysis of
target analytes from the medical field to the environment and food
fields. In particular, an enzymatic biosensor is a chemical sensor
used for selectively detecting and measuring chemical substances
contained in a sample by utilizing a biological sensing function in
which functional substances of living organisms or organisms such
as microorganisms react sensitively with a specific substance, and
it has been mainly developed for medical measurement applications
such as blood glucose sensors, and is also being actively studied
even in applications in the fields of food engineering and
environmental measurement.
[0004] Periodic measurement of blood glucose is very important in
the management of diabetes. Therefore, a wide variety of blood
glucose level measuring devices are being prepared so that blood
glucose levels can be easily measured using a portable measuring
device. The operating principle of such a biosensor is based on an
optical method or an electrochemical method. Such an
electrochemical biosensor can reduce the influence of oxygen,
unlike a biosensor using a conventional optical method, and has the
advantage that it can be used without separate pretreatment even if
the sample becomes turbid. Therefore, various types of
electrochemical biosensors with accuracy and precision are widely
used.
[0005] Currently commercialized electrochemical blood glucose
sensors mainly use enzyme electrodes. More specifically, it has a
structure in which a glucose oxidase is immobilized on an electrode
capable of converting an electrical signal by a chemical or
physical method. These electrochemical blood glucose sensors are
based on the principle of measuring the electric current generated
by transferring electrons generated by the enzyme oxidation of
glucose in analytes such as blood by enzymes to electrodes, thereby
measuring the glucose concentration in the analyte. In the case of
a biosensor using an enzyme electrode, there is a problem that
since the distance from the active center of the enzyme is too far,
it is not easy to transfer electrons generated by oxidation of the
substrate directedly to the electrode. Therefore, in order to
easily carry out such an electron transfer reaction, an
oxidation-reduction medium, that is, an electron transfer medium is
essentially required. Therefore, it is the type of enzyme used and
the characteristics of the electron transfer medium that have the
greatest influence on the characteristics of the electrochemical
biosensor that measures blood sugar.
[0006] In order to block changes in the measured values due to
differences in oxygen partial pressure (pO.sub.2) that varies
depending on the blood (venous blood, capillary blood, etc.), the
development trend of the blood glucose sensor has been changed into
the use of GDH, requiring no oxygen in the in the enzymatic
reaction, instead of GOX, including oxygen involved in the
enzymatic reaction with glucose in the blood. In addition, in the
case of electron transfer mediums, organic compounds such as
quinone derivatives (phenanthroline quinone, quineonediimine, etc.)
and organometallic compounds such as Ru complex (ruthenium
hexamine, etc.) or osmium complex, which are excellent in stability
according to temperature and humidity, replace the ferricyanide,
which is sensitive to stability according to humidity.
[0007] A most commonly used electron transfer medium is potassium
ferricyanide [K.sub.3Fe(CN).sub.6]. Because it is inexpensive and
has a good reactivity, it can be useful for all sensors using
FAD-GOX, PQQ-GDH or FAD-GDH. However, the sensor using the
above-mentioned electron transfer medium causes a measurement error
due to the interfering substances such as uric acid or gentisic
acid present in the blood and is easily deteriorated due to
temperature and humidity. Thus, it must be carefully prepared and
stored. However, it is difficult to accurately detect low
concentration of glucose due to changes in background current after
long storage.
[0008] Hexaamine ruthenium chloride [Ru(NH.sub.3).sub.6Cl.sub.3]
has higher redox stability than the ferricyanide. Biosensor using
this electron transfer medium has advantages in easy manufacturing
and storage and has high stability due to small change of
background current even when it is stored for a long time. However,
it cannot match the reactivity of FAD-GDH, when it is used with
FAD-GDH, and thus, it cannot be manufactured as a commercially
useful sensor.
[0009] Further, in the case of using such a biosensor, obtaining
accurate and rapid measurement values with a small amount of
samples is a very important problem in terms of maximizing user
convenience.
[0010] Therefore, the development of a new electron transport
medium capable of achieving the reduction of the disadvantages and
measurement time of the conventional electron transport medium is
still required.
[0011] Meanwhile, a continuous glucose monitoring (CGM) system is
used to continuously monitor blood glucose levels and manage
diseases such as diabetes, and existing enzyme sensors that collect
blood from the fingertips induce a considerable pain due to a
needle during blood collection and thus limits the measurement
frequency and cannot be used for such CGM. In order to solve these
problems, an improved version of a continuous blood glucose
monitoring enzyme sensor that can adhere to the body and thus
minimize invasion has recently been developed. In the case of such
as continuous blood glucose monitoring enzyme sensor, since part of
the sensor enters the human body, the electron transfer medium
containing transition metals and the like is absorbed into the
human body and fixed with a polymer backbone such as
polyvinylpyridine or polyvinylimidazole so that toxicity and side
effects do not occur, whereby it is intended to prevent problems
caused by the loss of the electron transfer medium in the human
body.
[0012] In this way, in the case of the oxidation-reduction polymer
to which the electron transfer medium is connected, conventionally,
coupling reaction using N-hydroxysuccinimide (NHS), an active
ester, was mainly used in order to efficiently fix the transition
metal complex to the main polymer backbone. However, in the case of
such an existing synthesis method, it has to go through very
complicated steps until it is synthesized into the final
oxidation-reduction polymer. In addition, when the coupling
reaction using NHS proceeds, the immobilization efficiency of the
transition metal complex due to hydrolysis is not actually high,
and there was a problem that it was difficult to introduce other
types of linkers or functional groups into the main backbone of the
polymer. Therefore, there is an increasing demand for the
development of oxidation-reduction polymers for a biosensor that
can solve these problems.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
[0013] The present disclosure has been devised to solve the
above-mentioned problems, and thus, it is an object of the present
disclosure to provide an oxidation-reduction polymer including a
transition metal complex, which can be prepared in a simpler step
compared to a conventional method, can increase the immobilization
rate of the transition metal complex and facilitates the
introduction of a functional group or a linker, and a method for
preparing the same.
[0014] It is another object of the present disclosure to provide an
electrochemical biosensor including an oxidation-reduction polymer
including a transition metal complex.
Technical Solution
[0015] In order to achieve the above objects, the present
disclosure provides an oxidation-reduction polymer including a
transition metal complex, which can be easily synthesized by
utilizing an azide-alkyne cycloaddition reaction using copper
catalyst and heat, and a thiol-ene click reaction using light,
exhibits an improved immobilization rate of the transition metal
complex, and facilitates the introduction of a functional group or
a linker, and an electrochemical biosensor, such as a blood glucose
sensor.
[0016] The oxidation-reduction polymer including the transition
metal complex according to the present disclosure includes a
transition metal complex including polymer backbones such as
poly(vinylpyridine) (PVP) or poly(vinylimidazole) (PVI), and
transition metals such as osmium, ruthenium, iridium, rhodium,
iron, or cobalt, and a ligand thereof, and a linker structure
connecting the polymer backbone and the transition metal complex.
Specifically, the oxidation-reduction polymer has the structure of
the following Chemical Formulas 1 to 4:
##STR00001## ##STR00002##
[0017] wherein,
[0018] M is a transition metal selected from the group consisting
of Os, Rh, Ru, Ir, Fe and Co;
[0019] L.sub.1 and L.sub.2 are combined with each other to form a
bidentate ligand selected from the following Chemical Formulas 5 to
7;
[0020] L.sub.3 and L.sub.4 are combined with each other to form a
bidentate ligand selected from the following Chemical Formulas 5 to
7;
[0021] L.sub.5 and L.sub.6 are each combined with each other to
form a bidentate ligand selected from the following Chemical
Formulas 5 to 7;
##STR00003##
[0022] the R.sub.1, R.sub.2 and R'.sub.1 are each independently
selected from the group consisting of a substituted or
unsubstituted alkyl group having 1 to 10 carbon atoms, a
substituted or unsubstituted alcohol group having 1 to 20 carbon
atoms, a substituted or unsubstituted alkylhalogen group having 1
to 20 carbon atoms, a substituted or unsubstituted thiol group
having 1 to 20 carbon atoms, a substituted or unsubstituted alkyl
azide group having 3 to 20 carbon atoms, a substituted or
unsubstituted aryl azide group having 7 to 30 carbon atoms, a
substituted or unsubstituted alkenyl group having 2 to 40 carbon
atoms, a substituted or unsubstituted alkynyl group having 2 to 40
carbon atoms, a cyano group, a halogen group, deuterium and
hydrogen,
[0023] the R.sub.3 to R.sub.20 are each independently selected from
the group consisting of a substituted or unsubstituted alkyl group
having 1 to 10 carbon atoms, a substituted or unsubstituted
cycloalkyl group having 3 to 40 carbon atoms, a substituted or
unsubstituted aryl group having 6 to 50 carbon atoms, a substituted
or unsubstituted heteroaryl group having 3 to 50 carbon atoms, a
substituted or unsubstituted alkoxy group having 1 to 20 carbon
atoms, a substituted or unsubstituted alcohol group having 1 to 20
carbon atoms, a substituted or unsubstituted alkylhalogen group
having 1 to 20 carbon atoms, a substituted or unsubstituted thiol
group having 1 to 20 carbon atoms, a substituted or unsubstituted
alkyl azide group having 3 to 20 carbon atoms, a substituted or
unsubstituted aryl azide group having 7 to 30 carbon atoms, a
substituted or unsubstituted aryloxy group having 6 to 30 carbon
atoms, a substituted or unsubstituted alkylamino group having 1 to
20 carbon atoms, a substituted or unsubstituted arylamino group
having 6 to 30 carbon atoms, a substituted or unsubstituted
alkylsilyl group having 1 to 20 carbon atoms, a substituted or
unsubstituted arylsilyl group having 6 to 30 carbon atoms, a
substituted or unsubstituted arylalkylamino group having 1 to 50
carbon atoms, a substituted or unsubstituted alkenyl group having 2
to 40 carbon atoms, a substituted or unsubstituted alkynyl group
having 2 to 40 carbon atoms, a cyano group, a halogen group,
deuterium and hydrogen;
[0024] the Ad is selected from the group consisting of a primary
and secondary amine group, an ammonium group, a halogen group, an
epoxy group, an azide group, an acrylate group, an alkenyl group,
an alkynyl group, a thiol group, isocyanate, an alcohol group, and
a silane group;
[0025] the X is a counter ion;
[0026] the a is an integer of 1 to 15;
[0027] the b is an integer of 1 to 15;
[0028] the c is an integer of 1 to 15;
[0029] the m is an integer of 10 to 600;
[0030] then is an integer of 10 to 600; and
[0031] the o is an integer of 0 to 600.
[0032] The oxidation-reduction polymer including the transition
metal complex provided in the present disclosure has a unique
linker structure, and so it has an advantage that the number of
synthesis steps is reduced compared to the existing case and so the
production can be carried out in a simple step, the immobilization
rate of the transition metal complex is increased, and the
introduction of a functional group or a linker is easy. In
addition, it is preferable that the oxidation-reduction polymer
including the transition metal complex provided in the present
disclosure has three kinds of bidentate ligands. Therefore, the
electrochemical biosensor including such an oxidation-reduction
polymer, preferably a continuous blood glucose monitoring sensor,
and has advantages of being prepared economically, significantly
reducing toxicity and side effects due to transition metals, and
having a high yield during production.
[0033] An embodiment of the present disclosure relates to an
electrochemical biosensor, which is prepared by applying an enzyme
capable of subjecting a liquid biological sample to an
oxidation-reduction with the oxidation-reduction polymer having
Chemical Formulas 1 to 4 onto a substrate having at least two
electrodes, and then drying the same. Examples of the electrode may
be a working electrode and a counter electrode. For example,
enzymes and transition metal polymers can be applied to or placed
in close proximity to the working electrode.
[0034] In specific embodiments, although a biosensor for measuring
glucose level is presented as an example of the applicable
electrochemical biosensor, but the present disclosure can be
applied to a biosensor for quantification of various substances
such as cholesterol, lactate, creatinine, hydrogen peroxide,
alcohol, amino acid, and glutamate by varying the types of enzymes
contained in the reagent composition of the present disclosure.
[0035] Hereinafter, the present disclosure will be described in
more detail.
[0036] Preferably, in the oxidation-reduction polymer of Chemical
Formulas 1 to 4 according to the present disclosure, the counterion
of X may be selected from the group consisting of an anion, for
example, a halide which may be selected from the group consisting
of F, Cl, Br and I, sulfate, phosphate, hexafluorophosphate,
tetrafluoroborate, or a cation (preferably monovalent cations), for
example lithium, sodium, potassium, tetraalkylammonium and
ammonium. More preferably, X may be chloride.
[0037] Preferably, the a may be an integer of 2 to 10.
[0038] Preferably, the b may be an integer of 2 to 10.
[0039] Preferably, the c may be an integer of 2 to 10.
[0040] Preferably, the m may be an integer of 15 to 550.
[0041] Preferably, the n may be an integer of 15 to 550.
[0042] Preferably, the o may be an integer of 0 to 300.
[0043] Preferably, the oxidation-reduction polymer medium according
to the present disclosure may have a structure represented by the
following Chemical Formula 8 or 9, without being limited
thereto.
##STR00004##
[0044] The transition metal complex in the oxidation-reduction
polymer having a structure selected from Chemical Formulas 1 to 4
according to the present disclosure may include, specifically, an
osmium complex, for example, a trivalent osmium complex and a
divalent osmium complex, and the osmium complex may preferably be
an oxidized compound (trivalent Os compound). The oxidizing agent
used in the oxidation treatment of the present disclosure is not
particularly limited, and specific examples may be one or more
selected from the group consisting of NaOCl, H.sub.2O.sub.2,
O.sub.2, O.sub.3, PbO.sub.2, MnO.sub.2, KMnO.sub.4, ClO.sub.2,
F.sub.2, Cl.sub.2, H.sub.2CrO.sub.4, N.sub.2O, Ag.sub.2O,
OsO.sub.4, H.sub.2S.sub.2O.sub.8, ceric ammonium nitrate (CAN),
pyridinium chlorochromate, and 2,2'-dipyridyldisulfide. In
addition, in the case of a mixture containing a compound in an
oxidized state and a reduced state as a transition metal complex,
the transition metal complex in an oxidized state or the chloride
compound of a transition metal complex in an oxidized state can be
provided by the oxidation treatment.
[0045] The transition metal complex according to the present
disclosure may be in the form of a salt having suitable counter
ions and ions. The salt compound are more preferable because they
have high solubility in water or other aqueous solutions or organic
solvents. When it consists of small counter anions such as F-, Cl-
and Br- in the chloride compound, it tends to be dissolved well in
water or various aqueous solutions, and groups consisting of large
counter anions such as I-, hexafluorophosphate (PF.sub.6.sup.-) and
tetrafluoroborate (BF.sub.4.sup.-) tend to dissolve well in organic
solvents. Thus, the counter anion may be one or more salt compounds
selected from the group consisting of a halide, which may be
selected from the group consisting of F, Cl, Br and I,
hexafluorophosphate and tetrafluoroborate, and the counter cation
may be one or more salt compounds selected from the group
consisting of Li salt, Na salt, K salt, Rb salt, Cs salt, Fr salt,
tetraalkylammonium and ammonium, without being limited thereto.
[0046] In a specific embodiment, the oxidation-reduction polymer
according to the present disclosure may be synthesized by reacting
a polymer backbone and a transition metal complex by a click
reaction. Specifically, the compound according to Chemical Formula
1 of the oxidation-reduction polymer according to the present
disclosure may be prepared by the following azide-alkyne Huisgen
cycloaddition reaction, which is represented by Reaction Scheme 1,
without being limited thereto.
##STR00005## ##STR00006##
[0047] Further, the compound of Chemical Formula 2 may be prepared
by the following thiol-ene reaction, which may be represented by
Reaction Scheme 2, without being limited thereto.
##STR00007##
[0048] Preferably, as a starting material for preparing an
oxidation-reduction polymer according to Reaction Scheme 1 or 2,
polyvinylpyridine or polyvinylimidazole can be functionalized to
give a polyvinylpyridine or polyvinylimidazole precursor. The
functionalized polyvinylpyridine or polyvinylimidazole precursor
may have a structure represented by the following Chemical Formula
10 or 11.
##STR00008##
[0049] In the structure of the polymer precursor, R.sub.a and
R.sub.b are selected from the group consisting of a substituted or
unsubstituted alkyl group having 1 to 10 carbon atoms, a
substituted or unsubstituted alcohol group having 1 to 10 carbon
atoms, a substituted or unsubstituted alkylhalogen group having 1
to 20 carbon atoms, a substituted or unsubstituted thiol group
having 1 to 20 carbon atoms, a substituted or unsubstituted alkyl
azide group having 3 to 20 carbon atoms, a substituted or
unsubstituted aryl azide group having 7 to 30 carbon atoms, a
substituted or unsubstituted alkenyl group having 2 to 40 carbon
atoms, and a substituted or unsubstituted alkynyl group having 2 to
40 carbon atoms.
[0050] The functionalized polyvinylpyridine or polyvinylimidazole
precursor can be synthesized, for example, as shown in the
following Reaction Scheme 3.
##STR00009##
[0051] Preferably, as a starting material for preparing an
oxidation-reduction polymer according to Reaction Scheme 1 or 2,
the transition metal complex may be functionalized. The
functionalized transition metal complex can be synthesized, for
example, as shown in the Reaction Schemes 4 and 5.
##STR00010##
##STR00011##
[0052] Therefore, in a further embodiment, the present disclosure
relates to a method for preparing an oxidation-reduction polymer of
Chemical Formula 1 or 2 comprising the steps of:
[0053] (i) functionalizing polyvinylpyridine or polyimidazole to
produce a polyvinylpyridine precursor or a polyimidazole
precursor;
[0054] (ii) functionalizing the transition metal complex; and
[0055] (iii) reacting the polyvinylpyridine precursor or
polyimidazole precursor produced in step (i) and the functionalized
transition metal complex produced in step (ii) by a click reaction
to prepare the oxidation-reduction polymer of Chemical Formula 1 or
2.
[0056] Specific embodiments of each step are as described
above.
[0057] The transition metal complex of the oxidation-reduction
polymer according to the present disclosure not only enables
accurate, reproducible, rapid and continuous analysis of the target
material, but also can be produced easily and economically in high
yields, and has the advantage that the problems of toxicity and
side effects that can be caused by the outflow of transition metals
are significantly low.
[0058] The oxidation-reduction polymer according to the present
disclosure may be applied or laminated on the working electrode or
may be located around the working electrode (for example, a
structure surrounding the electrode in a solution) to transfer
electrons between the working electrode and the substance to be
analyzed via an enzyme. The oxidation-reduction polymer can form
non-filterable coatings on the working electrode within the
electrochemical biosensor.
[0059] A further embodiment of the present disclosure relates to a
composition for an electrochemical biosensor comprising an enzyme
capable of subjecting a liquid biological sample to an
oxidation-reduction reaction, and the oxidation-reduction
polymer.
[0060] The oxidoreductase is a generic term for an enzyme that
catalyzes the redox reaction in a living organism. In the case of a
target substance to be measured in the present disclosure, such as
a biosensor, the oxidoreductase refers to an enzyme that is reduced
by reacting with a target substance to be measured. The enzyme
reduced in this way reacts with the electron transport medium and
generate signal such as current change, and the metabolite is
quantified by measuring the signal such as the current change
occurring at this time. The oxidoreductase usable in the present
disclosure may be at least one selected from the group consisting
of various dehydrogenase, oxidase, esterase, and the like.
Depending on the redox reaction or detection target material, an
enzyme using the substrate as the target material may be selected
and used among enzymes belonging to the enzyme group.
[0061] More specifically, the oxidoreductase may be one or more
selected from the group consisting of glucose dehydrogenase,
glutamate dehydrogenase, glucose oxidase, cholesterol oxidase,
cholesterol esterase, lactate oxidase, ascorbic acid oxidase,
alcohol oxidase, alcohol dehydrogenase, bilirubin oxidase, and the
like.
[0062] Meanwhile, the oxidoreductase can also include a cofactor
that plays a role of storing hydrogen deprived by the
oxidoreductase from the target substance (e.g., metabolite) to be
measured. For example, the cofactor may be one or more selected
from the group consisting of flavin adenine dinucleotide (FAD),
nicotinamide adenine dinucleotide (NAD), pyrroloquinoline quinone
(PQQ) and the like.
[0063] For examples, when measuring the blood glucose
concentration, glucose dehydrogenase (GDH) may be used as an
oxidoreductase, and may include flavin adenine dinucleotide-glucose
dehydrogenase (FAD-GDH) containing FAD as the cofactor and/or
nicotinamide adenine dinucleotide-glucose dehydrogenase containing
FAD-GDH as the cofactor.
[0064] In an embodiment, the available oxidoreductase may be at
least one selected from the group consisting of FAD-GDH (e.g., EC
1.1.99.10 etc.), NAD-GDH (e.g., EC 1.1.1.47 etc.), PQQ-GDH (e.g.,
EC1.1.5.2 etc.), glutamate dehydrogenase (e.g., EC 1.4.1.2 etc.),
glucose oxidase (e.g., EC 1.1.3.4 etc.), cholesterol oxidase (e.g.,
EC 1.1.3.6 etc.), cholesterol esterase (e.g., EC 3.1.1.13 etc.),
lactate oxidase (e.g., EC 1.1.3.2 etc.), ascorbic acid oxidase
(e.g., EC 1.10.3.3 etc.), alcohol oxidase (e.g., EC 1.1.3.13 etc.),
alcohol dehydrogenase (e.g., EC 1.1.1.1 etc.), bilirubin oxidase
(e.g., EC 1.3.3.5 etc.), and the like.
[0065] The composition according to the present disclosure may
contain 20 to 700 parts by weight, for example, 60 to 700 parts by
weight or 30 to 340 parts by weight of an oxidation-reduction
polymer, based on 100 parts by weight of the oxidoreductase. The
content of the oxidation-reduction polymer may be appropriately
adjusted in accordance with the activity of the redox enzyme.
[0066] In addition, the composition according to the present
disclosure may further include a crosslinking agent.
[0067] Meanwhile, the composition according to the present
disclosure may further include one or more additives selected from
the group consisting of surfactants, water-soluble polymers,
quaternary ammonium salts, fatty acids, thickeners, etc., for the
role of a dispersant during reagent dissolution, an adhesive during
reagent production, a stabilizer for long-term storage, and the
like.
[0068] The surfactant may play a role in allowing the composition
to spread evenly over the electrodes and be dispensed with a
uniform thickness when dispensing the reagents. As the surfactant,
at least one selected from the group consisting of Triton X-100,
sodium dodecyl sulfate, perfluorooctane sulfonate, sodium stearate,
etc. may be used. In order to properly perform the role of
spreading the reagent uniformly on the electrodes and dispensing
the reagent with uniform thickness when dispensing the reagent, the
reagent composition according to the present disclosure may contain
the surfactant in an amount of 3 to 25 parts by weight, for example
10 to 25 parts by weight, based on 100 parts by weight of the
oxidoreductase. For example, when using an oxidoreductase with an
activity of 700 U/mg, the reagent composition may contain 10 to 25
parts by weight of a surfactant based on 100 parts by weight of the
oxidoreductase. When the activity of the oxidoreductase is higher
than that, the content of the surfactant can be adjusted to lower
level.
[0069] The water-soluble polymer may serve to stabilize and
disperse enzymes as a polymer support for the reagent composition.
The water-soluble polymers used herein may include at least one
selected from the group consisting of polyvinyl pyrrolidone (PVP),
polyvinyl alcohol (PVA), polyfluoro sulfonate, hydroxyethyl
cellulose (HEC), and hydroxypropyl cellulose (HPC), carboxy methyl
cellulose (CMC), cellulose acetate, polyamide, and the like. The
reagent composition according to the present disclosure may contain
the water-soluble polymer in an amount of 10 to 70 parts by weight,
for example 30 to 70 parts by weight based on 100 parts by weight
of the oxidoreductase, in order to sufficiently and appropriately
exhibit the role of assisting the stabilization and dispersing of
oxidoreductase. For example, when using an oxidoreductase having an
activity of 700 U/mg, the composition may contain 30 to 70 parts by
weight of a water-soluble polymer based on 100 parts by weight of
the oxidoreductase. If the activity of the oxidoreductase is higher
than that, the content of the water-soluble polymer can be adjusted
to lower level.
[0070] The water-soluble polymer may have a weight average
molecular weight of about 2,500 to 3,000,000, for example, about
5,000 to 1,000,000, in order to effectively assist the
stabilization and dispersion of a support and an enzyme.
[0071] The thickener serves to firmly adhere the reagent to the
electrode. As the thickener, at least one selected from the group
consisting of natrosol, diethylaminoethyl-dextran hydrochloride,
and the like may be used. The electrochemical sensor according to
the present disclosure may contain the thickener in an amount of 10
to 90 parts by weight, for example, 30 to 90 parts by weight, based
on 100 parts by weight of the oxidoreductase, in order to ensure
that the oxidation-reduction polymer according to the present
disclosure is firmly attached to the electrode. For example, when
using an oxidoreductase having an activity of 700 U/mg, it may
contain 30 to 90 parts by weight of a thickener based on 100 parts
by weight of the oxidoreductase, and when the activity of the
oxidoreductase is higher than that, the content of the thickener
can be adjusted to lower level.
[0072] In a further embodiment, the present disclosure provides an
electrochemical biosensor including the oxidation-reduction
polymer.
[0073] Specifically, the type of the electrochemical biosensor is
not limited, but a continuous blood glucose monitoring sensor can
be preferably used.
[0074] In the configuration of such a continuous blood glucose
monitoring sensor, the present disclosure may include, for example,
an electrode, an insulator, a substrate, a sensing layer, a
diffusion layer, a protection layer, and the like which include the
oxidation-reduction polymer and the oxidoreductase. In the case of
an electrode, it may include two types of electrodes such as a
working electrode and a counter electrode, and it may also include
three types of electrodes such as a working electrode, a counter
electrode, and a reference electrode. In one embodiment, the
biosensor according to the present disclosure may be an
electrochemical biosensor prepared by coating a reagent composition
containing an electron transfer medium and an enzyme capable of
subjecting a liquid biological sample to an oxidization-reduction,
onto a substrate having at least two, preferably two or three
electrodes, and then drying it. For example, there is provided a
planar electrochemical biosensor, characterized in that in the
electrochemical biosensor, an working electrode and a counter
electrode are provided on opposite surfaces of a substrate, and a
sensing layer containing the oxidation-reduction polymer according
to the present disclosure is stacked on the working electrode, and
an insulator, a diffusion layer and a protective film are
sequentially stacked on both sides of a substrate having an working
electrode and a counter electrode.
[0075] In a specific embodiment, the substrate may be made of one
or more materials selected from the group consisting of
polyethylene terephthalate (PET), polycarbonate (PC), and polyimide
(PI).
[0076] Further, as the working electrode, a carbon, gold, platinum,
silver or silver/silver chloride electrode may be used.
[0077] Further, in the case of an electrochemical biosensor having
a 2-electrode, since the counter electrode performs up to the role
of a reference electrode, gold, platinum, silver or silver/silver
chloride electrodes can be used as the counter electrode. In the
case of a 3-electrode electrochemical biosensor including up to the
reference electrode, a gold, platinum, silver, or silver/silver
chloride electrode may be used as the reference electrode, and a
carbon electrode may be used as the counter electrode.
[0078] Nafion, cellulose acetate, silicone rubber can be used as
the diffusion layer, and silicone rubber, polyurethane,
polyurethane-based copolymer, and the like can be used as the
protective film, without being limited thereto.
[0079] As a non-limiting example, in the case of the 2-electrode,
silver chloride or silver may be used because the counter electrode
performs up to the role of the reference electrode, and in the case
of the 3-electrode, silver chloride or silver may be used as the
reference electrode, and a carbon electrode may be used as the
counter electrode.
Advantageous Effects
[0080] The oxidation-reduction polymer according to the present
disclosure has a small number of steps during production, and so is
economical, has an increased immobilization rate of a transition
metal complex, and facilitates the introduction of a functional
group or a linker, whereby biosensors to which this is applied have
the advantages of being simple, quick to detect and economical.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] FIG. 1 is a diagram showing the structure of a biosensor
according to an embodiment of the present disclosure.
[0082] FIG. 2 is a graph showing the results of measuring a cyclic
voltammetry curve using the compound of Chemical Formula 8 which is
the oxidation-reducing polymer for the electron transport medium
according to the present disclosure, and a single Os complex.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0083] Hereinafter, the present disclosure will be described in
more detail.
[0084] The present disclosure will be described in more detail by
way of examples. However, the present disclosure is for
illustrative purposes only and the scope of the present disclosure
is not limited thereto.
Preparation Example 1: Synthesis of an Oxidation-Reduction Polymer
Compound Represented by [Chemical Formula 8]
1-1. Synthesis of 2,2'-biimidazole
##STR00012##
[0086] 79 mL (0.69 mol) of 40% glyoxal aqueous solution was added
to a 500 mL three-neck round-bottom flask, cooled to 0.degree. C.,
and then 370 mL (2.76 mol) of ammonium acetate was slowly added
dropwise through a dropping funnel for 3 hours, while paying
attention to temperature rise (less than 30.degree. C.). After
completion of the dropwise addition, the mixture was stirred
overnight at 45.about.50.degree. C., and then cooled to room
temperature. The resulting solid was filtered, then dissolved in
ethyl glycol, and purified by a hot-filter. Finally,
2,2'-biimidazole was obtained. (10.1 g, yield: 33%)
1-2. Synthesis of N-methyl-2,2'-biimidazole
##STR00013##
[0088] 2 g (15 mmol) of 2,2'-biimidazole was added to a 250 mL
three-neck round-bottom flask, dissolved in 60 mL of anhydrous
dimethylformamide, and then cooled to 0.degree. C. 0.6 g (15 mmol)
of sodium hydride was added little by little, while paying
attention to temperature rise. The mixture was stirred at 0.degree.
C. for 1 hour, and then 1 mL (15 mmol) of iodomethane was slowly
added dropwise through a syringe pump. After completion of the
dropwise addition, the mixture was stirred at room temperature for
12 hours. 100 mL of ethyl acetate was added to the final reaction
solution, and the resulting sodium iodide was removed by
filtration. The filtrate was concentrated under reduced pressure to
remove the solvent, and then the remaining solid was purified by
column chromatography using ethyl acetate and hexane as developing
solvents. Finally, N-methyl-2,2'-biimidazole was obtained. (0.8 g,
yield: 37%)
1-3. Synthesis of N,N'-dimethyl-2,2'-biimidazole
##STR00014##
[0090] 5 g (37 mmol) of 2,2'-biimidazole was added to a 500 mL
three-neck round-bottom flask, dissolved in 60 mL of anhydrous
dimethylformamide, and then cooled to 0.degree. C. 3 g (40 mmol) of
sodium hydride was added little by little, while paying attention
to temperature rise. The mixture was stirred at 0.degree. C. for 1
hour, and then 2.5 mL (40 mmol) of iodomethane was slowly added
dropwise through a syringe pump. After completion of the dropwise
addition, the mixture was stirred at room temperature for 24 hours.
Water was added to the final reaction solution, extracted with
ethyl acetate (200 mL.times.3), and then the organic layer was
collected and dried over magnesium sulfate. The organic layer was
concentrated under reduced pressure to remove the solvent, and then
purified by column chromatography using ethyl acetate and hexane as
developing solvents. Finally, N,N'-dimethyl-2,2'-biimidazole was
obtained. (5.1 g, yield: 84%)
1-4. Synthesis of N-butynyl-N'-methyl-2,2'-biimidazole
##STR00015##
[0092] 1.5 g (10 mmol) of N-methyl-2,2'-biimidazole was added to a
100 mL three-neck round-bottom flask, dissolved in 30 mL of
anhydrous dimethylformamide under nitrogen, and then sodium hydride
0.5 g (13 mmol) was added thereto. The mixture was stirred at room
temperature for 1 hour, and then 1.7 g (13 mmol) of
4-bromo-1-butyne and 1.5 g (10 mmol) of sodium iodide were added
thereto. The reaction solution was heated to 80.degree. C. under
nitrogen and stirred for 24 hours. The final reaction solution was
cooled to room temperature, extracted with water (100 mL) and ethyl
acetate (200 mL.times.3), and then the organic layer was collected
and dried over magnesium sulfate. The organic layer was
concentrated under reduced pressure to remove the solvent, and
purified by column chromatography using ethyl acetate and hexane as
developing solvents. Finally, N-butynyl-N'-methyl-2,2'-biimidazole
was obtained. (1.5 g, yield: 74%)
1-5. Synthesis of [Osmium (III)
(N,N'-dimethyl-2,2'-biimidazole).sub.2
(N-butynyl-N'-methyl-2,2'-biimidazole)](hexafluorophosphine).sub.3
##STR00016##
[0094] A 100 mL three-neck round-bottom flask was equipped with a
reflux condenser, a gas inlet and a thermometer, and 2 g (13 mmol)
of N,N'-dimethyl-2,2'-biimidazole, 3 g (6.5 mmol) of ammonium
hexachloro osmate (IV) and 50 mL of ethylene glycol were stirred
under nitrogen at 140.degree. C. for 24 hours. 1.3 g (6.5 mmol) of
N-butynyl-N'-methyl-2,2'-biimidazole was dissolved in 10 mL of
ethylene glycol, and then added to the reaction mixture using a
syringe. The mixture was again stirred at 140.degree. C. under
nitrogen for 24 hours. After completion of the reaction, the
reaction mixture was cooled to room temperature, and the resulting
red residue was removed by filtration. The filtrate was diluted
with 300 mL of water, and then AG1X4 chloride resin was added and
stirred for 24 hours to sufficiently oxidize in air. The solution
is added dropwise to an aqueous ammonium hexafluorophosphine
solution to obtain a precipitate of the ion-exchanged metal
complex. The resulting solid was filtered, washed several times
with water, and then dried in a vacuum oven to obtain the final
compound osmium (III) complex. (5 g, yield: 67%)
1-6. Synthesis of
poly(4-(2-azidoethyl)pyridinium)-co-(4-(2-aminoethyl)pyridinium)-co-4-vin-
ylpyridine)
##STR00017##
[0096] A 250 mL three-neck round-bottom flask was equipped with a
reflux condenser, a gas inlet and a thermometer, and 20 g of
poly(4-vinylpyridine): number average molecular weight:
.about.160,000 g/mol) was dissolved in 150 mL of dimethylformamide.
4.5 g (30 mmol) of 1-azido-2-bromoethane and 6.0 g (30 mmol) of
2-bromoethylamine were added to this solution. The solution was
stirred at 90.degree. C. for 24 hours using a mechanical stirrer.
After completion of the reaction, the reaction mixture was cooled
to room temperature and poured into ethyl acetate solution to form
a precipitate. The solvent was drained off, and the resulting solid
was dissolved again in 300 mL of methanol, concentrated under
reduced pressure (150 mL), and a precipitate was again formed in
diethyl ether. The resulting solid was dried in a vacuum oven to
obtain a polyvinylpyridine precursor. (27 g, yield: 90%)
1-7. Synthesis of Oxidation-Reduction Polymer 1 [Chemical Formula
8]
##STR00018##
[0098] 0.5 g of
poly(4-(2-azidoethyl)pyridinium)-co-(4-(2-aminoethyl)
pyridinium)-co-4-vinylpyridine) was added to a 50 mL culture tube
and dissolved in 10 mL of distilled water. Then, 0.8 g of
[Osmium(III)(N,N'-dimethyl-2,2'-biimidazole).sub.2(N-butynyl-N'-methyl-2,-
2'-biimidazole)](hexafluorophosphine).sub.3 dissolved in 5 mL of
dimethylformamide was added thereto. 25 mg of a copper (I) catalyst
(CuTc: Copper(I)thiophene carboxylate) was added to the reaction
mixture and stirred at room temperature for 12 hours. After
completion of the reaction, the reaction mixture was poured into
ethyl acetate solution to form a precipitate. The solvent was
drained off and the resulting solid was dissolved again in 50 mL of
acetonitrile, and AG1X4 chloride resin and water (150 mL) were
added and stirred for 24 hours. The polymer solution is
concentrated under reduced pressure (50 mL), and then dialyzed to
remove substances of low molecular weight (10,000 g/mol or less).
The dialyzed polymer solution was lyophilized to obtain a final
oxidation-reduction polymer 1. (0.7 g)
Experimental Example: Confirmation of the Electrochemical
Properties of the Oxidation-Reduction Polymer [Formula 8] and Os
Complex for an Electron Transfer Medium According to the Present
Disclosure Using Cyclic Voltammetry
[0099] In order to confirm the performance of the
oxidation-reduction polymer including the Os complex according to
the present disclosure as an electron transfer medium,
electrochemical properties were measured using the cyclic
voltammetry method according to the following experimental
method.
Experimental Method
[0100] {circle around (1)} Two kinds of osmium complexes of the
following Chemical Formulas 12 and 13 (Os(mbim).sub.3,
Os(mbim).sub.3-A), and 20 mg of each of the osmium-polymers of
Chemical Formula 8 according to the present disclosure were
dissolved in deionized water and 5 mL of 0.1M sodium chloride
solution. [0101] {circle around (2)} Degassed with argon for 10
minutes to remove oxygen in the solution. [0102] {circle around
(3)} The working electrode, the reference electrode, and the
counter electrode were connected to the oxygen-degassed solution,
and changes in the electrical signal due to changes in the voltage
were measured under argon. [0103] {circle around (4)} The results
of the experiment are shown in FIG. 2 and Table 1,
respectively.
##STR00019##
[0103] Experimental Materials/Conditions
[0104] Working electrode: glass carbon electrode (dia: 3.0 mm)
[0105] Reference electrode: Ag/AgCl electrode
[0106] Counter electrode: platinum rod
[0107] Test parameters [0108] Equipment: EmStat (PalmSens Co.)
[0109] Technique: cyclic voltammetry [0110] Potential range:
-1.0.about.1.0 V [0111] Scan rate: 10 mV/s
TABLE-US-00001 [0111] TABLE 1 Compound E.sub.pc (V) E.sub.pa (V)
Os(mbim).sub.3-A -0.150 -0.263 Os(mbim).sub.3 -0.165 -0.284
Os-polymer -0.160 -0.290
[0112] As shown in FIG. 2 and Table 1, as a result of measuring the
cyclic voltammetry curve of an oxidation-reduction polymer
[Chemical Formula 8] together with osmium tris(III)
(N,N'-dimethyl-2,2'-biimidazole) and osmium(III)
(N,N'-dimethyl-2,2'-biimidazole).sub.2(N-butynyl-N'-methyl-2,2'-biimidazo-
le), all three compounds showed an oxidation-reduction potential at
approximately the same position. Therefore, it was indirectly
confirmed that the performance of the oxidation-reduction polymer
according to the present disclosure as an electron transport medium
is the same as that of a single osmium complex.
* * * * *