CN101360755A - Redox mediators - Google Patents

Redox mediators Download PDF

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Publication number
CN101360755A
CN101360755A CNA2006800485415A CN200680048541A CN101360755A CN 101360755 A CN101360755 A CN 101360755A CN A2006800485415 A CNA2006800485415 A CN A2006800485415A CN 200680048541 A CN200680048541 A CN 200680048541A CN 101360755 A CN101360755 A CN 101360755A
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iii
pyridine
cooh
title complex
integer
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休·奥利弗·艾伦·希尔
克里斯托弗·保罗·纽曼
洛特·洛克·翁
劳太褚
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F Hoffmann La Roche AG
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Oxford Biosensors Ltd
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Abstract

The present application is directed to complexes according to Formulae (I) or (II) as described herein, and their use as redox mediators in electrochemical biosensors.

Description

Redox mediators
The present invention relates to new ruthenium or osmium title complex and their application in biosensor.Particularly, the present invention relates to be in the application of the ruthenium complexe of ruthenium (III) state as redox mediators, described ruthenium complexe has the total charge that is less than 3+ containing on the ruthenium kind.
Biosensor is that biochemistry is discerned component or sensing member and physical transformation device bonded analysis tool.They and have in food and drink industry widely and use in the monitoring of for example personal health monitoring of different fields, environmental screening and monitoring, bioprocess.
The bio-sensing element can be enzyme, antibody, dna sequence dna and even microorganism.This biochemical component is used for optionally catalyzed reaction or promotion keying action (event).It is operation in the body fluid in complicated sample matrix that the selectivity of biochemical recognition reaction has allowed biosensor.Transmodulator is transformed into measurable signal with biochemical action, thereby the means that are used to detect it are provided.The scope of measurable effect is from the quality change during to biochemical complexing owing to the spectrum change of the production of the product/substrate of enzyme reaction and consumption.
Usually, transmodulator has many forms, and their indicate the physico-chemical parameter that will measure.Thereby transmodulator can be based on optical, measures for example optical absorption, fluorescence or change of refractive.It can be a quality-base, measures the quality change of following the biological association reaction that drives.In addition, it can be the variation of (measure enthalpy (heat)) of hot radical, or follows the interactional amperometry of analyte/biology-identification layer or impedance (measuring the variation of electrical property).
Biosensor provides the convenience and the facility of distributed measurement, that is, make chemical examination reach the potential ability of point-of-care (the point of concern or care).By suitably designing and making, scale operation biosensor device expediently.Yet, exist the restriction of many uses for biosensor.These comprise that transmodulator is to dirt (foulant) and interferential vulnerability.
That enzyme base biosensor is widely used for is clinical, the detection of the analyte in environment, agricultural and the biotechnology applications.The analyte that can measure in the clinical assay of people's body fluid comprises, for example, and glucose, lactic acid, cholesterol, bilirubin and amino acid.The biofluid for example level of these analytes in the blood is important for the diagnosis and the monitoring of disease.
Provide the transmitter that can be generally used for the enzyme based system as (the point of care) of point-of-care or (the over the counter) device that can sell nowhere.They can be used to test finger thorn (finger prick) whole blood sample of fresh unmodified, (annotate: this time was unfixing to 2 minutes this sample being joined 1 in the device, but can noticeable change) within, determine the concentration of total cholesterol, triglyceride level, HDL and LDL.Proved clinically that these 4 parameters with array mode provide the very good indication to the risk of human adult heart disease.As everyone knows, hypercholesterolemia is asymptomatic, thereby recommends each adult to test, to estimate their risk.If find their risk height, then can be by diet individually, or the correct processing that combines with medicine of diet and significantly reduction.
In an example of such enzyme base biosensor, adopt electrochemical assay to detect the analyte of being discussed.Utilized the variation of the oxidation state of amboceptor, the enzyme interacting of the analyte response that described amboceptor is same and to be determined.Oxidation state to amboceptor is selected, and makes it when adding substrate, only be in with the state of enzyme interacting.Analyte is by the amboceptor reaction of enzyme and stoichiometric concentration.This causes the oxidized or reduction (depending on enzyme reaction) of amboceptor, and can measure by being determined at the electric current that produces under the given current potential in this variation on the amboceptor level.
Electrochemical assay is typically carried out in having the battery of two or three electrodes, and it comprises at least one measurement or working electrode and a reference electrode.In the system of 3 electrodes, the 3rd electrode is right-electrode.In the system of 2 electrodes, reference electrode also plays a part right-electrode.Electrode is by for example potentiostat connection of circuit.Measurement or working electrode are carbon or metallic conductor.When electric current passed through working electrode, oxydo-reductase was by electrooxidation or electroreduction.Described enzyme is specific for analyte to be determined or for the product of analyte.The turnover ratio of enzyme typically with testing liquid in enzyme itself or the concentration of its product relevant (preferred but inessential linear dependence).
The electrooxidation of enzyme or electroreduction often by exist in solution or electrode on redox mediators promoted.Redox mediators helps the telecommunication (electricalcommunication) between working electrode and the enzyme usually.Redox mediators can be dissolved in the fluid to be analyzed that contacts with electrolysis.Is under the specific situation of catalysis at enzyme for the analyte or its product of needs, can be for example by making useful device with the film that comprises redox mediators and described enzyme is electrode coated.Solvable or insoluble diffusible oxydation reduction amboceptor in water transports electronics and works by coming and going between for example enzyme and electrode.In any case, when the substrate of enzyme during by electrooxidation, redox mediators is transported to electrode with electronics from substrate reductive enzyme; When substrate during by electroreduction, redox mediators is transported to electronics the enzyme of substrate oxidation from electrode.
The enzyme based electrochemical transmitter of many previous uses has used many different redox mediators, for example monomer ferrocene, the quinoid-compound that comprises quinine (for example, benzoquinones), cyclohexane sulfamic acid nickel and ruthenium amine (ruthenium amines).In most cases, these redox mediators have one or more of following restriction; The solubleness of redox mediators in testing liquid is low, their chemistry, light, heat or pH poor stability, or they not with enzyme or electrode or described both rapid enough ground exchange electronics.In addition, many redox potentials of the redox mediators of these reports are carried out such oxidation, make that at the reductive amboceptor solution component except that analyte is also by electrooxidation under the current potential of electrooxidation on the electrode; In other cases, they carry out such reduction, make solution component for example dissolved oxygen also by rapid electroreduction.As a result, it is not fully specific utilizing the transmitter of this amboceptor.
Ruthenium basigamy compound had been utilized in the past as the redox mediators in the reaction that comprises cholesterol dehydrogenase for example.For example, be equilibrated at [the Ru arbitrarily that the electrode of suitable potential will exist II(NH 3) 6] 2+Plant and be converted into [Ru III(NH 3) 6] 3+: electric current that passes through and the Ru that forms by enzyme reaction II(NH 3) 6The amount of planting is proportional.Yet more or less, the kind of such altitudinal belt electric charge is [Ru for example III(NH 3) 6] 3+Usually form title complex with ion-right form, electronegative group is on enzyme and electrode surface, and the necessary reaction of analytic process is effective also to be taken place efficiently thereby stop.
Therefore, need utilize such redox mediators, the component of itself and analysis of mixtures and electrode form not too strong title complex, or do not form title complex, thus cause from described amboceptor the measurement response reliable more, stable and can reappear.
According to a first aspect of the invention, provide the purposes of the title complex of formula I as redox mediators,
[M(A) x(B) y] m(X z) n
Formula I
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 1 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is monodentate or the bidentate aromatic ligand that contains 1 or 2 nitrogen-atoms;
B is the part of independently electing as except that the heterocycle containing n-donor ligand that is fit to arbitrarily;
X is the counter ion that are fit to arbitrarily;
Wherein, A is optional is replaced by 1 to 8 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl ,-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group (arylcarboxamido), alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio; Wherein, the quantity of ligating atom is 6.
Part A can be by one or more CO 2R 2The unidentate ligand that group replaces, or optional by one or more CO 2R 2Bidentate or tridentate ligand that group replaces.
Radicals R 2Can elect H as.
Part A can be selected from nicotinic acid, Yi Yansuan, 2,2 '-dipyridyl, 2,2-dipyridyl-5,5 '-dicarboxylic acid, 2,2-dipyridyl-4,4 '-dicarboxylic acid or 5-chloro-oxine.
According to a second aspect of the invention, provide the purposes of formula II title complex as redox mediators:
[M(A) x(B) y] m(X z) n
Formula II
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 0 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is bidentate, three teeth, four teeth, five teeth or sexadentate ligand, and it can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) qOr [(RNC 2H 4) (RNC 3H 6)] s, wherein w is 1 to 5 integer, and v is 3 to 6 integer, and p and q are 1 to 3 integers, and thus, the summation of p and q is 4,5 or 6, and s is 2 or 3, and wherein R and R 1Be hydrogen or methyl;
B independently elects the part that is fit to arbitrarily as;
X is the counter ion that are fit to arbitrarily;
Wherein, B is optional is replaced by 1 to 8 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl ,-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, the aryl formamido group, the alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio;
Wherein, the quantity of ligating atom is 6.
Part A can be bidentate, three teeth or tetradentate ligands, and it can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) q, [(RNC 2H 4) (RNC 3H 6)] sWherein w is 1 to 3 integer, and v is 3 or 4 integer, and p and q are 1 to 3 integers, and thus, the summation of p and q is 4, and s is 2 or 3;
Part A can be selected from 1,4,7-trimethylammonium-1,4,7-7-triazacyclononane, 1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA), 1,2-dimethyl ethylene diamine or 1,1,2,2-tetramethylethylened.
Part B among formula I or the formula II can be selected from for example NH of amine ligand 3, or be selected from CO; CN; Halogen, and acetylacetonate (acac), 3-bromo-acetylacetonate (Bracac), oxalic acid, pyridine, or 5-chloro-oxine.
Part A and B among formula I or the formula II can elect bidentate as, and are the geometries of cis or trans title complex.
The oxidation state of the metal in the title complex of formula I or formula II can be elected 2+, 3+ or 4+ as.
The oxidation state of the metal in the title complex of formula I or formula II can be elected 3+ as.
+ 2 ,+1,0 ,-1 ,-2 and-3 part A and B among formula I or the formula II can be selected from down group:.
Counter ion in the title complex of formula I or formula II can be selected from F -, Cl -, Br -, I -, NO 3 -, NH 4 +, NR 4 +, PF 6 -, CF 3SO 3 -, SO 4 2-, ClO 4 -, K +, Na +, Li +Can use the combination of counter ion.
The title complex of formula I or formula II can be selected from [Ru III(NH 3) 5(pyridine-3-COOH)] (PF 6) 2(CF 3SO 3), [Ru III(2,4-diacetylmethane thing (pentandionate)) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(3-bromo-2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-5,5 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-4,4 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl)] Cl, [Ru III(2,4-diacetylmethane thing) 2(pyridine-4-COOH) (pyridine-4-COO)], [Ru III(5-chloro-oxine) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] (PF 6) (CF 3SO 3), [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2, [Os II(2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru II(2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru II(2,2 ' dipyridyl) 2(C 2O 4)], K[Ru III(C2O 4) 2(pyridine-3-COOH) 2], [Ru III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2
The title complex of formula I or formula II can be selected from [Ru III(2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2Or [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2
This redox mediators can be used for electrochemical sensor.Electrochemical sensor can comprise micro-strip electrode.Electrochemical sensor can be an electrochemica biological sensor.Electrochemica biological sensor can be used for detecting the analyte of body fluid, environmental sample, F﹠B, animal doctor's sample, medicine.
According to a third aspect of the invention we, provide as the purposes of ruthenium complexe in biosensor at the formula I or the formula II of preceding qualification.
It is 6 to 10 that the title complex of formula I or formula II can be used in pH.It is 7 to 9 that the title complex of formula I or formula II can be used in pH.
This biosensor can be used for compatible arbitrarily biochemical analyte.This analyte can be found in the biofluid, and can be selected from any in enzyme, enzyme substrates, antigen, antibody, nucleotide sequence, cholesterol, cholesteryl ester, lipoprotein, triglyceride level or the microorganism.
According to a forth aspect of the invention, provide a kind of detection system that is used for the Measurement and analysis thing, having comprised:
(a) sample that comprises analyte is contacted with the solution that comprises redox mediators, described redox mediators is selected from containing Ru or containing the group of the compound of Os according to formula I or formula II;
(b) causing the sample of cultivating described contact under the condition that enzyme works to analyte;
(c) make step (b) but the cultivation sample stand to produce the condition of the variation of measurement signal; With
(d) measure the signal that produces.
But measurement signal can be electrochemistry, colorimetric, heat, impedance measurement, electric capacity or spectral signal.Measurable signal can be the electrochemical signals of measuring at micro-strip electrode.
According to a fifth aspect of the invention, provide title complex according to formula I,
[M(A) x(B) y] m(X z) n
Formula I
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 1 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is monodentate or the bidentate aromatic ligand that contains 1 or 2 nitrogen-atoms;
B independently elects one or more of the part that is fit to arbitrarily except that the heterocycle containing n-donor ligand as;
X is the counter ion that are fit to arbitrarily;
Wherein, A is optional is replaced by 1 to 8 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl ,-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group, alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio;
Wherein, the quantity of ligating atom is 6.
Part A can be by one or more CO 2R 2The unidentate ligand that group replaces, or optional by one or more CO 2R 2Bidentate or tridentate ligand that group replaces, wherein, described one or more CO 2R 2Group, radicals R 2Can elect H as.
Part A can be selected from nicotinic acid, Yi Yansuan, 5-carboxyl-nicotinic acid, 6-pyridyl-nicotinic acid, 2,2 '-dipyridyl-5,5 '-two-carboxylic acid, 2,2 '-dipyridyl-4,4 '-two-carboxylic acid, 2,2 '-dipyridyl, 1,10-phenanthroline-3, the two carboxylic acids of 9-.
According to a sixth aspect of the invention, provide a kind of title complex according to formula II,
[M(A) x(B) y] m(X z) n
Formula II
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 0 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is bidentate, three teeth, four teeth, five teeth or sexadentate ligand, and it can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) qOr [(RNC 2H 4) (RNC 3H 6)] sWherein w is 1 to 5 integer, and v is 3 to 6 integer, and p and q are 1 to 3 integers, and thus, the summation of p and q is 4, and s is 2 or 3, and wherein R and R 1Be hydrogen or methyl;
B independently elects the part that is fit to arbitrarily as;
X is the counter ion that are fit to arbitrarily;
Wherein, A is optional is replaced by 1 to 7 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl ,-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group, alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio;
Wherein, the quantity of ligating atom is 6.
Part A can be bidentate, three teeth, four teeth, five teeth or sexadentate ligand, and it can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) q, [(RNC 2H 4) (RNC 3H 6)] sWherein w is 1 to 3 integer, and v is 3 or 4 integer, and p and q are 1 to 3 integers, and thus, the summation of p and q is 4, and s is 2 or 3.
Part A can be selected from 1,4,7-trimethylammonium-1,4,7-7-triazacyclononane, 1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA), 1,2-dimethyl ethylene diamine or 1,1,2,2-tetramethylethylened.
Part B can be selected from for example NH of amine ligand 3Or NMe 3, be selected from CO, CN, halogen, acetylacetonate (acac), 3-bromo-acetylacetonate (Bracac), oxalic acid, 1,4,7-triethylene crown ether, oxalic acid or 5-chloro-oxine.
When electing part A or B as bidentate, the geometry of this title complex can be a cis or trans.
The oxidation state of the metal in the title complex of formula I or formula II can be elected 2+ or 3+ as.The oxidation state of the metal in the title complex of formula I or formula II can be elected 3+ as.
+ 2 ,+1,0 ,-1 ,-2 and-3 can select part A and B, make that the whole electric charges on the title complex of formula I or formula II are selected from down group:.
Counter ion can be selected from F -, Cl -, Br -, I -, NO 3 -, NH 4 +, NR 4 +, PF 6 -, CF 3SO 3 -, SO 4 2-, ClO 4 -, K +, Na +And Li +Can use the combination of counter ion.
The title complex of formula I or formula II can be selected from [Ru III(NH 3) 5(pyridine-3-COOH)] (PF 6) 2(CF 3SO 3), [Ru III(2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(3-bromo-2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-5,5 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-4,4 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl)] Cl, [Ru III(2,4-diacetylmethane thing) 2(pyridine-4-COOH) (pyridine-4-COO)], [Ru III(5-chloro-oxine) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] (PF 6) (CF 3SO 3), [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2, [Os II(2,2 '-dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru II(2,2 '-dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru II(2,2 '-dipyridyl) 2(C 2O 4)], K[Ru III(C 2O 4) 2(pyridine-3-COOH) 2], [Ru III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2
Should be appreciated that in each in the title complex of formula I or formula II, metal can be elected ruthenium or osmium as required as.In addition, can change the metal in the above-mentioned specific title complex, to form corresponding ruthenium or osmium title complex.It will be appreciated by those skilled in the art that, in title complex, with Os replace operating potential that Ru will make this title complex change pact-400mV extremely-600mV, and in case of necessity, by changing metal center part on every side, operating potential is accurately adjusted in the opposite direction, until amboceptor reach to the operating potential of Ag/AgCl for-300mV to+300mV.
For example:
[Ru III(acac) 2(py-3-COOH) (py-3-COO) E] 1/2Current potential is-175mV.[Ru III(3-Bracac) 2(py-3-COOH) (py-3-COO)] with [Ru III(acac) 2(py-3-COOH) (py-3-COO)] similar, replace acetylacetonate (acac) and E but have bromo-acetylacetonate (bromo acac) 1/2Current potential is-142mV.
[Ru III(acac) 2(py-4-COOH) (py-4-COO)] with [Ru III(acac) 2(py-3-COOH) (py-3-COO)] similar, still have COO (py-4-COOH rather than py-3-COOH), and have E in different positions 1/2Current potential is-165mV.It will be understood by those skilled in the art that operating potential must be at E 1/2More than about 150mV.
Title complex corresponding to Ru title complex described here is: [Os III(NH 3) 5(pyridine-3-COOH)] (PF 6) 2(CF 3SO 3), [Os III(2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Os III(3-bromo-2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Os III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-5,5 '-(COOH) (COO)], [Os III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-4,4 '-(COOH) (COO)], [Os III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl)] Cl, [Os III(2,4-diacetylmethane thing) 2(pyridine-4-COOH) (pyridine-4-COO)], [Os III(5-chloro-oxine) 2(pyridine-3-COOH) (pyridine-3-COO)], [Os III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] (PF 6) (CF 3SO 3), [Os III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2, [Os II(2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Os II(2,2 '-dipyridyl) 2(C 2O 4)], K[Os III(C 2O 4) 2(pyridine-3-COOH) 2], [Os III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2
When used herein, the described term of following definition:
Term " alkyl " comprises the saturated aliphatic hydrocarbon of straight or branched.The example of alkyl comprise methyl, ethyl, just-propyl group, just-propyl group, sec.-propyl, just-butyl, tert-butyl, cyclopentyl etc.Unless otherwise noted, term " alkyl " comprises alkyl and cycloalkyl.
The alkyl that is connected to the rest part (remainder) of structure by Sauerstoffatom described in term " alkoxyl group ".The example of alkoxyl group comprise methoxyl group, oxyethyl group, just-propoxy-, isopropoxy, butoxy, uncle-butoxy, cyclopentyloxy etc.In addition, unless otherwise noted, term " alkoxyl group " comprises alkoxyl group and cycloalkyloxy.
The undersaturated straight or branched aliphatic hydrocrbon with at least one carbon-to-carbon double bond described in term " alkenyl ".Non-limiting examples of alkenyls comprises vinyl, 1-propenyl, 2-propenyl, 1-butylene base, 2-methyl isophthalic acid-propenyl, cyclopentenyl etc.In addition, unless otherwise noted, term " alkenyl " comprises alkenyl and cycloalkenyl group.
" reactive group " is the functional group of such molecule, its can with another kind of compound reaction, be connected to this molecule with at least a portion with this other compound.Reactive group comprises carboxyl, activatory ester, sulfonic acid halide (sulfonyl halide), sulphonate, isocyanic ester, lsothiocyanates, epoxide, aziridine, halogenide, aldehyde, ketone, amino, acrylamide, mercaptan, acylazide, hydrazine, azanol, haloalkane, imidazoles, pyridine, phenol, alkyl, sulphonate, halo triazine, imido-ester, maleimide, hydrazides, hydroxyl and light-reaction nitrine aryl.As understood in the art, the activatory ester generally includes succinimido, benzotriazole base, or by the electron-withdrawing group ester of the aryl that replaces of sulfo group, nitro, cyano group or halogen group for example.
Term " acac " is meant as 2, the acetylacetonate negatively charged ion of the conjugate base of 4-diacetylmethane.
" replacement " functional group (for example, the alkyl of replacement, alkenyl or alkoxyl group) comprises at least a substituting group that is selected from following groups: halogen, alkoxyl group, sulfydryl, aryl, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH ,-NH 2, alkylamino, dialkyl amido, trialkyl ammonium, alkanoylamino, two alkanoylamino, aryl formamido group, diazanyl, alkylthio, alkenyl and reactive group.
" biofluid " be wherein can the Measurement and analysis thing any body fluid or body fluid derivative, for example blood, tissue juice, blood plasma, skin liquid (dermal fluid), sweat and tears.
" electrochemical sensor " is that configuration is used for the existence by the analyte of electrochemical oxidation or reduction reaction test sample, or the device of the concentration of Measurement and analysis thing or amount.These reactions typically can be converted into the electrical signal that can be associated with the amount or the concentration of analyte.
" redox mediators " is a kind of electron transfer agent, and it is used for directly or by one or more other electron transfer agents, transports electronics between the enzyme of analyte or analyte-reductive or analyte-oxidation and electrode.
" electrolysis " is directly on electrode, or the electrooxidation or the electroreduction of the compound by one or more electron transfer agents (for example, redox mediators or enzyme).
Term " reference electrode " unless otherwise noted, comprising: a) reference electrode, and b) can also play the reference electrode (that is right/reference electrode) of counter electrode effect.
Term " counter electrode " unless otherwise noted, comprising: a) counter electrode, and b) can also play the counter electrode (that is right-reference electrode) of reference electrode effect.
Term " measurable signal " is meant the signal that can easily be measured, for example electropotential, fluorescence, spectral absorption, luminous, scattering of light, NMR, IR, mass spectrum, thermal distortion, or piezoelectricity changes.
Term " biochemical analyte " comprises any measurable chemistry or the biochemical substances that may be present in the biofluid, and comprises any enzyme, antibody, dna sequence dna or microorganism.
Has the implication that they are accepted usually in the art according to monodentate of the present invention, bidentate and three teeth.That is, unidentate ligand is defined as chemical part or group with a potential ligating atom.Surpass a potential ligating atom, be called as polydentate ligand, wherein, the quantity of potential ligating atom is by expressions such as term bidentate, three teeth.
For example, known biosensor that can be used according to the invention can be made up of the band (strip) with four reagent ponds (reagent well) and common plan reference (pseudo reference); And each pond has the little band working electrode of tubulose of itself.The sensory package of band (component) is that the reagent by the different special preparation of drying provides, described reagent comprise with each pond in sample in interactional at least a enzyme of specific analyte and amboceptor.Potentially, owing to can in each pond, add and dry different reagent, be clear that therefore many-analyte is tested can to use one sample to finish.The quantity in pond is variable, thereby the quantity of unique test is variable, for example, can use the transmitter that adopts 1 to 6 pond.
Can use the typical case to have the conventional microelectrode of work microelectrode and reference electrode.Working electrode is made by palladium, platinum, gold or carbon usually.Counter electrode is typically carbon, Ag/AgCl, Ag/Ag 2SO 4, palladium, gold, platinum, Cu/CuSO 4, Hg/HgO, Hg/HgCl 2, Hg/HgSO 4Or Zn/ZnSO 4
In a preferred microelectrode, working electrode is in the wall of the storage that forms described microelectrode.The example of microelectrode that can be used according to the invention is those disclosed among the WO 03/097860.
Only pass through embodiment, and with reference to the accompanying drawings, the embodiment of the present invention will now be described, in described accompanying drawing:
10mM amboceptor [Ru among the 0.1M KCl in Fig. 1 display buffer liquid II(py- 3COOH) (NH 3) 5] (PF 6) 2The voltammetry of solution, this voltammetry are by at 100mVs -1Scanning speed under, use standard pool electrode (well electrode) record described in the WO200356319;
Fig. 2 is presented at new the amboceptor [(Ru of the 10mM among the 0.1M KCl II(py- 3COOH) (NH 3) 5] (PF 6) 2Repeated oxidation test (with respect to Ag/AgCl be+0.25V), this repeated oxidation is tested and to be write down in (blueness) environment of deoxidation (redness) and oxidation;
The tris buffer (containing 0.1M KCl) that Fig. 3 is presented at 0.1M pH 9 is (a) with at the new amboceptor [Ru of 0.1M KCl solution (being made by water) in (b) II(py- 3COOH) (NH 3) 5] (PF 6) 2Cyclic voltammetry;
Fig. 4 a is presented at the new amboceptor Ru among the 0.1M KCl II(py- 3COOH) (NH 3) 5] (PF 6) 2Repeated oxidation test (with respect to Ag/AgCl be+0.25V), this repeated oxidation is tested and is write down in well-oxygenated environment.Fig. 4 b shows the Ru with respect to the mediator concentration of electric current 2+Calibration data;
Fig. 5 is presented at and does not exist (black-(a)) and has under the situation of (grey-(b)) notatin Ru II(py- 3-COOH) (NH 3) 5] (PF 6) 2Cyclic voltammogram;
Fig. 6 is presented in the total cholesterol mixture described in the WO200356319, [Ru III(NH 3) 5(py-3-COOH) (PF 6) 2(CF 3SO 3) cyclic voltammogram;
Fig. 7 A is presented under the situation of blood plasma as testing liquid, has new amboceptor [Ru III(NH 3) 5(py-3-COOH) (PF 6) 2(CF 3SO 3) the total cholesterol mixture be the oxidation of 118s in the time;
Fig. 7 B is presented under the situation of blood plasma as testing liquid, has amboceptor [Ru III(NH 3) 5(py-3-COOH-pyridine) (PF 6) 2(CF 3SO 3) the total cholesterol mixture at the reduction current of time=118s;
Fig. 7 C is presented under the cholesterol situation of different concns, amboceptor [Ru III(NH 3) 5(3-COOH-pyridine) (PF 6) 2(CF 3SO 3) time point experiment (time point experiment), this experiment shows averaged oxygen galvanic current over time;
Fig. 8 A shows with amboceptor [Ru III(NH 3) 5(3-COOH-pyridine) (PF 6) 2(CF 3SO 3) in the time time point experiment of the oxidation current of 118s;
Fig. 8 B shows with amboceptor [Ru III(NH 3) 5(3-COOH-pyridine) (PF 6) 2(CF 3SO 3) in the time time point experiment of the oxidation current of 202s;
Fig. 8 C shows with amboceptor [Ru III(NH 3) 5(3-COOH-pyridine) (PF 6) 2(CF 3SO 3) in the time time point experiment of the reduction current of 202s;
Fig. 8 D is presented under the cholesterol situation of different concns, Ru amboceptor [Ru (NH 3) 5(py-3-COOH)] (PF 6) 2(CF 3SO 3) time point experiment, it shows averaged oxygen galvanic current over time;
Fig. 9 is presented at by 10mM[Ru III(acac) 2(py-3-COOH) (py-3-COO)], in the solution that 0.1MKCl, 16mM Chaps and 0.1M tris buffer (pH 9.0) are formed, the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 10 shows use [Ru III(acac) 2(py-3-COOH) (py-3-COO)] as the oxidation current of amboceptor graphic representation with respect to total cholesterol (TC) concentration of different people plasma sample in the pond.Electric current is that the oxidizing potential (with respect to the Ag/AgCl reference) at general+0.15V is applied to the later record of working electrode;
Figure 11 is presented at by 10mM[Ru III(3-Bracac) 2(py-3-COOH) (py-3-COO)], in the solution that 0.1MKCl, 16mM Chaps and 0.1M tris buffer (pH 9.0) are formed, the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 12 is presented at and does not exist (black-(a)) and has (grey-(b)) 0.75mM NADH and 0.03mg ml -1Under the situation of PdR, by 0.5mM[Ru III(3-Bracac) 2(py-3-COOH) (py-3-COO)] the uv-visible absorption spectra of the solution of Zu Chenging;
Figure 13 shows use [Ru III(3-Bracac) 2(py-3-COOH) (py-3-COO)] as amboceptor, oxidation current is with respect to the graphic representation of total cholesterol (TC) concentration of freeze-dry blood serum samples different in the pond.Electric current is that the oxidizing potential (with respect to the Ag/AgCl reference) at general+0.15V is applied to the later record of working electrode;
Figure 14 is presented at by 1mM[Ru III(acac) 2(2,2 ' bpy-5,5 '-(COOH) (COO)], in the solution formed of 0.1MKCl, 16mM Chaps and 0.1M tris buffer (pH 9.0), the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 15 shows use [Ru III(acac) 2(2,2 ' bpy-5,5 '-(COOH) (COO)] as amboceptor, oxidation current is with respect to the calibration graph of total cholesterol (TC) concentration of freeze-dry blood serum samples different in the pond.Electric current is that the oxidizing potential (with respect to the Ag/AgCl reference) at general+0.15V is applied to the later record of working electrode;
Figure 16 is presented at by 10mM[Ru III(acac) 2(2,2 '-bpy)] in the solution formed of Cl, 0.1M KCl, 16mMChaps and 0.1M tris buffer (pH 9.0), the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 17 is presented at and does not exist (dark-(a)) and has (light-(b)) 0.017mgml -1Under the situation of PdR, by 1mM[Ru III(acac) 2(2,2 '-bpy)] uv-visible absorption spectra of the solution of Cl and 1mM NADH;
Figure 18 shows use [Ru III(acac) 2(2,2 '-bpy)] Cl is as amboceptor, and oxidation current is with respect to the graphic representation of total cholesterol (TC) concentration of freeze-dry blood serum samples different in the pond.Electric current is that the oxidizing potential (with respect to the Ag/AgCl reference) at general+0.15V is applied to the later record of working electrode;
Figure 19 is presented at by 10mM[Ru III(acac) 2(py-4-COOH) (py-4-COO)], in the solution that 0.1MKCl, 16mM Chaps and 0.1M tris buffer (pH 9.0) are formed, the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 20 shows use [Ru III(acac) 2(py-4-COOH) (py-4-COO)] as amboceptor, oxidation current is with respect to the graphic representation of human plasma samples' different in the pond total cholesterol (TC) concentration.Electric current is that the oxidizing potential (with respect to the Ag/AgCl reference) at general+0.15V is applied to the later record of working electrode;
Figure 21 is presented at by 10mM[Ru III(5-Cl-Quin) 2(py-3-COOH) (py-3-COO)], in the solution that 0.1M KCl, 16mM Chaps and 0.1M tris buffer (pH 9.0) are formed, the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 22 is presented on the carbon microelectrode band of silk screen printing, contains (a) 0, (b) 0.1, (c) 0.5, (d) 1 and (e) 5mgml what have a 10mM NADH -1In the 0.1M tris buffer of PdR (pH 9.0), [Ru III(5-Cl-Quin) 2(py-3-COOH) (py-3-COO)] cyclic voltammogram (1mM) is (at 10mVs -1);
Figure 23 is presented at and does not exist (dark-(a)) and has (light-(b)) 0.033mg ml -1Under the situation of PdR, contain 0.25mM[Ru III(5-Cl-Quin) 2(py-3-COOH) (py-3-COO)] and the uv-visible absorption spectra of the solution of 0.25mMNADH;
Figure 24 is presented at by 5mM[Ru III(Me 6-tet) (acac)] (PF 6) (CF 3SO 3), in the solution formed of 0.1M KCl, 16mM Chaps and 0.1M tris buffer (pH 9.0), the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 25 is presented at and does not exist (dark-(a)) and has (light-(b)) 0.033mgml -1Under the situation of PdR, by 5mM[Ru III(Me 6-tet) (acac)] (PF 6) (CF 3SO 3) and the uv-visible absorption spectra of the solution formed of 0.5mM NADH;
Figure 26 shows use [Ru III(Me 6-tet) (acac)] (PF 6) (CF 3SO 3) as amboceptor, oxidation current is with respect to the graphic representation of human plasma samples' different in the pond total cholesterol (TC) concentration.Electric current be applying+oxidizing potential (with respect to the Ag/AgCl reference) of 0.15V after record;
Figure 27 is presented at by 10mM[Os II(2,2 '-bpy) 2 (acac)] in the solution formed of Cl, 0.1M KCl, 16mMChaps and 0.1M tris buffer (pH 9.0), the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 28 is presented on the carbon microelectrode band of silk screen printing, contains (a) 0, (b) 0.1, (c) 0.5, (d) 1 and (e) 5mgml what have a 10mM NADH -1In the 0.1M tris buffer of PdR (pH 9.0), [Os II(2,2 '-bpy) 2(acac)] cyclic voltammogram of Cl (1mM) is (at 10mVs -1);
Figure 29 is presented at by 1mM[Os II(2,2 '-bpy) 2(acac)] in the solution that Cl, 0.1M KCl, 16mM Chaps and 0.1M tris buffer (pH 9.0) are formed, the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 10mVs in scanning speed -1Situation under write down;
Figure 30 is presented on the carbon microelectrode band of silk screen printing, contains (a) 0, (b) 0.5, (c) 1.25 and (d) 2.5mgml what have 0.1M glucose -1In the 0.1M Tutofusin tris of notatin (pH 7.0), [Ru (2,2 '-bpy) 2(acac)] cyclic voltammogram of Cl (1mM) is (at 10mVs -1);
Figure 31 is presented on the carbon microelectrode band of silk screen printing, and what have 0.1M glucose contains (a) 0, (b) 0.25, (c) 1.25 and (d) 5mg ml -1In the 0.1M tris buffer of notatin (pH 7.0), [Ru II(2,2 '-bpy) 2(acac)] cyclic voltammogram of Cl (1mM) is (at 10mVs -1);
Figure 32 is presented on the carbon microelectrode band of silk screen printing, is having the comprising of 0.1M glucose (a) 0 and (b) 5mg ml -1In the 0.1M Tutofusin tris of notatin (pH 7.0), K[Ru III(C 2O 4) 2(py-3-COOH) 2] (5mM) cyclic voltammogram is (at 10mVs -1);
Figure 33 shows [Ru II(Me 3TACN) (acac) (py)] PF 6At CH 3ESI mass spectrum among the CN (+ve pattern);
Figure 34 shows [Ru III(Me 3-TACN) (acac) (py)] (NO 3) 2ESI mass spectrum in methyl alcohol (+ve pattern);
Figure 35 is presented at and comprises 10mM[Ru III(Me 3-TACN) (acac) (py)] (NO 3) 2, 0.1M KCl and 0.1M tris buffer (pH 9.0) solution in, the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 36 is presented at and does not exist (dark-(a)) and has (light-(b)) 0.033mg ml -1Under the situation of PdR, comprise 2mM[Ru III(Me 3-TACN) (acac) (py)] (NO 3) 2Uv-visible absorption spectra with the solution of 5mM NADH;
Figure 37 shows for containing 1mg ml -1The 10mM[Ru of PdR III(Me 3-TACN) (acac) (py)] (NO 3) 2Solution is with respect to the graphic representation of the oxidation current of NADH concentration;
Figure 38 is presented at and contains 3.3mM[Ru III(acac) 2(2,2 '-bpy-4,4 '-(COOH) (COO)], in the solution of 0.1M KCl and 0.1M tris buffer (pH 9.0), the cyclic voltammogram of the carbon microelectrode band of silk screen printing, this cyclic voltammogram are to be 100mVs in scanning speed -1Situation under write down;
Figure 39 is presented on the carbon microelectrode band of silk screen printing of standard, is having under the situation of 50mMNADH, contains (a) 0 and (b) 10mgml -1In the solution that contains 0.1M KCl and 0.1M tris buffer (pH 9.0) of PdR, [Ru III(acac) 2(2,2 '-bpy-4,4 '-(COOH) (COO)] (3.3mM) cyclic voltammogram is (at 10mVs -1);
Figure 40 shows the reduction (left side) and oxidation (right side) form of the 10mM solution of amboceptor of the present invention in 0.1M KCl; With
Figure 41 shows the example according to ruthenium complexe of the present invention.
All solution all is to use from the preparation of the Milli-Q reagent water of Millipore Synergy 185 water purification systems or SILVER REAGENT solvent.All solids former state under situation about not being further purified is used.Use enzyme mediation (enzyme mediation) or use enzyme cascade (enzyme cascade) to test the mediation (mediation) of amboceptor.Be used for the electrode of enzyme cascade and the common pending application WO200356319 that preparation is described in the inventor.
Synthetic following amboceptor:
[Ru III(NH 3) 5(py-3-COOH)] (PF 6) 2(CF 3SO 3); [Ru III(acac) 2(py-3-COOH) (py-3-COO)]; [Ru III(3-Bracac) 2(py-3-COOH) (py-3-COO)]; [Ru III(acac) 2(2,2 ' bpy-5,5 '-(COOH) (COO)]; [Ru III(acac) 2(2,2 '-bpy-4,4 '-(COOH) (COO)]; [Ru III(acac) 2(2,2 '-bpy)] Cl; [Ru III(acac) 2(py-4-COOH) (py-4-COO)]; [Ru III(5-Cl-Quin) 2(py-3-COOH) (py-3-COO)]; [Ru III(Me 6-tet) (acac)] (PF 6) (CF 3SO 3); [Ru III(Me 6-tet) (acac)] Cl 2[Os II(2,2 '-bpy) 2(acac)] Cl; [Ru (2,2 '-bpy) 2(acac)] Cl; [Ru II(2,2 '-bpy) 2(C 2O 4)]; K[Ru III(C 2O 4) 2(py-3-COOH) 2]; [Ru III(Me 3-TACN) (acac) (py)] (NO 3) 2
Wherein, py=pyridine; 3-Bracac=3-bromo-2,4-diacetylmethane thing; Acac=2,4-diacetylmethane thing; 2,2 '-bpy=2,2 ' dipyridyl; 2,2 ' bpy-5,5 '-(COOH) 2=2,2 '-dipyridyl-5,5 '-dicarboxylic acid; Me 6-tet=1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA); Me 3-TACN=1,4,7-trimethylammonium-1,4,7-7-triazacyclononane, and 5-Cl-Quin=5-chloro-oxine.
For title complex each, particularly, can follow identical synthesis program, and initial compounds be changed over corresponding ruthenium or osmium compound simply, thereby prepare the title complex of corresponding ruthenium or osmium.
[Ru II(NH 3) 5(py-3-CO 2H)] (PF 6) 2[Ru III(NH 3) 5(py-3-CO 2H)] (PF 6) 2(CF 3SO 3) synthetic
Figure A20068004854100261
Material
RuCl 3·3H 2O(Heraeus)
One hydrazine hydrate (80%, RDH)
Nicotinic acid (aldrich (Aldrich))
Tosic acid silver (aldrich (Aldrich))
Silver trifluoromethanesulfonate (aldrich (Aldrich))
Ammonium hexafluorophosphate (aldrich (Aldrich))
Chlorine five amine ruthenium (III) muriates, [Ru (NH 3) 5Cl] Cl 2, be according to literature method (A.D.Allen, inorganic synthetic (Inorg.Synth.) 1970,12,2) from the ruthenium trichloride preparation, and by at 40 ℃ from 0.1M HCl recrystallize and purifying.
[Ru II(NH 3) 5(py-3-CO 2H)](PF 6) 2
[Ru (NH in 40mL water 3) 5Cl] Cl 2(1.0g, and adding 2 normal tosic acid silver in suspension 3.4mmol) (1.9g, 6.8mmol).After this mixture of stirring at room 1h, solution is filtered, to remove AgCl.In the pale yellow solution of the gained of using argon-degassed, add 4 times of excessive nicotinic acid (py-3-CO 2H, 1.67g is 13.6mmol) with 12-15 sheet mossy zinc mercury alloys (about 10g).Under Ar, after this mixture of stirring at room 2hr, solution is filtered.In the reddish orange solution that obtains, add ammonium hexafluorophosphate (NH 4PF 6, 3g), and this mixture remained on 4 ℃ spend the night.After acetone-ether filtration and this crude product of recrystallize, obtain [the Ru of 0.6g II(NH 3) 5(py-3-CO 2H)] (PF 6) 2(yield: 29.5%)
[Ru III(NH 3) 5(py-3-CO 2H)](PF 6) 2(CF 3SO 3)
[Ru in 30mL acetone II(NH 3) 5(py-3-CO 2H)] (PF 6) 2(160mg adds 1.3 normal silver trifluoromethanesulfonate (AgCF in solution 0.27mmol) 3SO 3, 89mg), and, this mixture is stirred 1h in the dark in room temperature.By the centrifugal silver that removes, to obtain pale yellow solution.Under condition of stirring, the solution that obtains is dropwise joined in the 250ml ether, and this mixture was kept 3 hours at 4 ℃, to obtain light-yellow precipitate.Product is cleaned with ether, and from acetone and ether recrystallize.(yield: 90mg, 44%).
Cis-[Ru II(acac) 2(py-3-COOH) 2] synthetic
Under argon gas, with the Ru in the ethanol (acac) 3(200mg, 0.5mmol) and nicotinic acid (494mg, mixture 4mmol) and several mossy zinc mercury alloys backflow 5h.The red-brown mixture that obtains is cooled off, and remove Zn/Hg by filtration.The red-brown deposition is collected by filtering, and clean with 0.1M HCl, water and ether.(crude product yield: 250mg, 91%, contain the Zn/Hg fragment).
Cis-[Ru III(acac) 2Synthesizing (py-3-COOH) (py-3-COO)]
With cis-[Ru II(acac) 2(py-3-COOH) 2] (200mg) be dissolved in 0.1M NH 3In, filter then.This reddish-brown filtrate is stirred spend the night (about 18 hours) in air, to obtain mulberry solution.Filter and this solution of evaporate to dryness.Collect the mulberry resistates, and clean, then dry air with acetone and ether.(yield: 150mg)
Cis-[Ru III(3-Bracac) 2Synthesizing (py-3-COO) (py-3-COOH)]
Figure A20068004854100281
At 2ml H 2Dissolve cis-[Ru among the O III(acac) 2(py-3-COO) (py-3-COOH)] (93mg, 0.17mmol).Bromine water (the 0.17mmol Br that adds the 0.041M of 4.3ml then 2).The hyacinthine suspension that obtains stirred in air spend the night.The blueness precipitation is collected and the water cleaning.Yield: 25%; With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.15V.
[Ru III(acac) 2(5,5 '-(COO) (COOH)-2,2 '-bpy)] synthetic
Figure A20068004854100291
Under argon gas, with the Ru in the ethanol of 30ml (acac) 3(200mg is 0.5mmol) with 5,5 '-(COOH) 2-2, (122mg, red mixture 0.5mmol) reflux with several spongy Zn/Hg amalgam and spend the night 2 '-bpy.Cool off the brown mixture that obtains, and remove zinc amalgam with tweezers.Collect this brown precipitate, and clean with ethanol and ether.Then, this brown solid is dissolved in the 0.1M NH of 50ml 3In, and filter.The filtrate of green stirred in air spend the night, to obtain red solution.This solution is filtered, then evaporate to dryness.Collect purple-red precipitate, and clean, then dry air with acetone and ether.Yield: 22%; With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.18V.
[Ru III(acac) 2(2，2’-bpy)](PF 6)
Figure A20068004854100301
With [Ru II(acac) 2(2,2 '-bpy)] (75mg 0.165mmol) is dissolved in the methylene dichloride (15ml).Under the situation of vigorous stirring, in room temperature, with respect to [Ru II(acac) 2(2,2 '-bpy)] small portion of solution is added in ferrocene hexafluorophosphate (ferroceniumhexafluorophosphate) (54.6mg, 0.165mmol) solution in the methylene dichloride (10ml).Behind the several minutes, the color of solution becomes blush from deep green.After further stirring 10 minutes, solution is filtered, and add ether (60ml), by centrifugal collecting precipitation, and clean with ether.(yield: 50%).
[Ru III(acac) 2(2,2 '-bpy)] Cl synthetic
Under condition of stirring, in room temperature, with [the Bu in the acetone (15ml) 4N] (463mg, 1.667mmol) solution dropwise joins [the Ru in the 20ml acetone to Cl II(acac) 2(2,2 '-bpy)] (PF 6) (200mg is 0.333mmol) in the solution.The purple precipitation is collected by filtering, clean with acetone and ether, then dry air.To precipitate again and be dissolved in the acetonitrile, and come purifying by crystallization, described crystallization is to be undertaken by the vapor diffusion of using ether.(yield=50%)
[Ru II(acac) 2(py-4-COOH) 2] synthetic
Figure A20068004854100311
Under argon gas, with the Ru in the 40ml ethanol (acac) 3(200mg, 0.5mmol) and py-4-COOH (124mg, the spongy Zn/Hg amalgam of red mixture 1mmol) and several refluxed 4 hours.The intense violet color mixture that obtains is cooled off, and remove zinc amalgam with tweezers.The precipitation of burgundy is collected by filtering, and clean with 0.1M HCl, water and ether subsequently.(yield=250mg of crude product, 91%, contain the Zn amalgam)
[Ru III(acac) 2Synthesizing (py-4-COOH) (py-4-COO)]
With Ru II(acac) 2(py-4-COOH) 2(250mg) be dissolved in 0.1M NH 3In, filter then.Reddish-brown filtrate stirred in air spend the night, to obtain purple solution.This solution is filtered, then evaporate to dryness.Collection purple precipitation is cleaned with acetone and ether, then dry air.Yield=50%; With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.12V.
[Ru III(5-Cl-Quin) 2Synthesizing (py-3-COOH) (py-3-COO)]
Figure A20068004854100321
Chemical formula: C 30H 18Cl 2K 2N 4O 6Ru 2-
Accurate mass: 779.89
Under argon gas, with the nicotinic acid in the ethanol (25ml) (92.3mg, 0.75mmol) and Ru (5-Cl-Quin) 3(200mg, solution 0.37mmol) and several zinc amalgams refluxed 24 hours.Light brown solid by isolated by filtration obtains cleans with ethanol, and dry air.This brown solid is suspended in the water (15ml), and adding KOH (0.2g, 3.57mmol).After stirring 0.5h, the dark brown solution that obtains is filtered, and the rotation evaporate to dryness.With ethanol erase residual thing, and by be dissolved into then it slowly is evaporated in 1: 1 the methanol/ethanol initial volume that is about it about 50% and recrystallize.With solid 60 ℃ of vacuum-dryings.Yield: 57% (150mg, 0.21mmol).MS：m/z?634(M+1)。With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.097V.
[Ru II(Tet-Me 6) (acac)] (PF6) synthetic
[Ru(Me6-2，2，2-tet)(acac)] 2+
Figure A20068004854100322
Chemical formula: C 18H 37F 9N 4O 5PRuS
Molecular weight: 724.60
Ultimate analysis: C, 29.84; H1 5.15; F, 23.60; N, 7.73; O, 11.04; P, 4.27; Ru, 13.95; S, 4.43
With the cis in the 10ml ethanol-[Ru III(Tet-Me 6) Cl 2] (PF 6) (100mg, 0.18mmol) and Li (acac) (40mg 0.36mmol) refluxes and to spend the night.With the brown solution cooling that obtains, filter then.Filtrate is concentrated into about 1ml, adds ether, and brown precipitate is filtered, clean with ether, then dry air.Yield=85%.For RuC 17H 37N 4O 2PF 6Calculated: C, 35.48%; H, 6.48%; N, 9.73%: actual measurement: C, 35.39%; H, 6.37%; N, 9.60%
[Ru III(Tet-Me 6) (acac)] (PF 6) (CF 3SO 3) synthetic
With AgCF 3SO 3(67mg 0.26mmol) joins [Ru in the 10ml acetone II(Tet-Me 6) (acac)] (PF 6) (125mg is in solution 0.22mmol).Brown solution becomes blueness immediately, and this mixture was stirred 30 minutes in the dark.By the centrifugal silver metal that removes in the solution, then this blue solution is slowly joined in about 80ml ether.Blueness precipitation is collected by filtering, clean, then dried overnight in a vacuum with ether.Yield=70%; With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.18V.
[Ru III(Tet-Me 6) (acac)] Cl 2Synthetic
[Ru (Me in MeOH 6-tet) (acac)] (PF 6) (CF 3SO 3) solution in add [n-Bu 4N] Cl, produce [Ru (Me 6-tet) (acac)] Cl 2Precipitation, with described sedimentation and filtration, and dried overnight in a vacuum.
[Os II(acac) (bipy) 2] Cl synthetic
Figure A20068004854100341
Acetylacetonate (1ml) is joined water (20ml) and [Os of alcohol in (10ml) II(bipy) 2Cl 2] (0.25g, 0.44mmol) in, and at excess amount of Ca CO 3Under the existence (0.5g), with the mixture 6h that refluxes.Volatile matter is evaporated, and with chloroform (30ml) extracted residues.Dark red brown chloroform extraction liquid is filtered, and use anhydrous Na 2SO 4Drying is evaporated to little volume, and when adding ether, [Os II(bpy) 2(acac) Cl] as dark orange-brown plates crystallization.Then these are filtered and dry air.Yield: 63% (0.176g, 0.28mmol).MS：m/z?603(M)。With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.31V.
[Ru II(bipy) 2(acac)] Cl's is synthetic
Figure A20068004854100342
Chemical formula: C 25H 23ClN 4O 2Ru
Accurate mass: 548.06
(1ml 9.70mmol) joins [Ru (bipy) that is suspended in water (20ml) and the alcohol (20ml) with acetylacetonate 2Cl 2] (300mg, 0.62mmol) in, and at excess amount of Ca CO 3Existence under, mixture was refluxed 6 hours.Filtering mixt, and with filtrate rotary evaporation drying.Resistates is extracted with chloroform (30ml), and filter.With filtrate at MgSO 4Last dry, be evaporated to about 5ml then.When adding ether, [Ru II(bipy) 2(acac)] Cl is as the slow crystallization of burgundy crystalline solid.In a vacuum, at 60 ℃ of drying solids.Yield: 70% (236mg, 0.43mmol).MS：m/z513(M)。With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.71V.
Ru II(bipy) 2Ox's is synthetic
Figure A20068004854100351
With Ru (bipy) 2Cl 2(0.2g 0.41mmol) is suspended in water (20ml) and the ethanol (10ml), and with mixture boiled 2 minutes.(52.1mg 0.41mmol), and heats 2h with mixture under refluxing, produce light red solution to add two oxalic acid hydrate potassium.When cooling, obtain the green crystal solid, with described green crystal solid filtering, water and ether clean, and dry air.Yield: 94%, 0.19g.MS：m/z?503.1(M)。With respect to the Fc/Fc among the 0.1M TBHP in acetonitrile +, E 0=32.5mV.
K[Ru III(ox) 2(py-3-COOH) 2] synthetic
Will be at 50ml H 2K among the O 3[Ru (Ox) 3] (500mg, 1.0mmol) and py-3-COOH (255mg, solution 2.1mmol) reflux in air and spend the night.With the brown solution cooling that produces, filter then.Filtrate is concentrated into about 1ml, adds acetone, with the light brown sedimentation and filtration, and with acetone and ether cleaning.Make crude product from H 2O/ acetone recrystallize 3 times.Yield: 70%.ESI-MS：m/z＝602(M+K) +。With respect to the NHE in phosphate buffered saline buffer (pH 8.05), Ru III/IIE 1/2=0.16V.
[Ru II(DMSO) 4Cl 2] preparation
Backflow three hydrate ruthenium trichlorides (1.0g) are 5 minutes in methyl-sulphoxide (5mL).In a vacuum volume is reduced to half, adds acetone (20mL), produce yellow mercury oxide.Filter out isolating yellow title complex, clean with acetone and ether, and vacuum-drying.
[Ru III(L) Cl 3] preparation
Under condition of stirring, the Ru in dehydrated alcohol (25mL) II(DMSO) 4Cl 2(1.0g adds L (0.80g, 4.7mmol) (L=1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) in mixture 2.1mmol).This suspension is heated to 60 ℃ reaches 1h, up to obtaining clarifying dark red brown solution, then with its backflow 2h.By rotary evaporation, under reduced pressure remove solvent.Handle orange-red resistates with dense HCl, and in the presence of air, it was heated 30 minutes under refluxing.Collect orange microcrystalline solids by filtering, and water, ethanol and ether cleaning, and dry air.
[Ru III(L) (acac) (OH)] PF 6H 2The preparation of O
In envrionment temperature, under condition of stirring, with solid Ru III(L) Cl 3(2.0g; 5.0mmol) (sodium 2,4-pentanedionate) (acac) (3.0g to join methyl ethyl diketone sodium in the water (60mL) on a small quantity; In～24mmol) the solution.Mixture is stirred 3.5h, until obtaining clarifying red solution.Add H 2NaPF among the O (5mL) 6(2.0g) solution, and be cooled to 0 ℃, cause the precipitation of orange crystallite, it is collected by filtering, clean with ether, and dry air.
[Ru II(L) (acac) (py)] PF 6Preparation
In the presence of 10 Zn amalgam, will dehydrated alcohol/pyridine (5mL) (4: 1, contain [Ru in v/v) III(L) (acac) (OH)] PF 6(105mg, solution 0.20mmol) is reflux 4h under argon gas atmosphere.Be cooled to after the envrionment temperature, collect red crystallite precipitation, clean with ether by filtering, and dry air.With this product from the acetone recrystallize.Yield: (94mg, 79%) ESI/MS (holotype): m/z=451, [M] +With respect at CH 3Fc among the 0.1M TBAH among the CN +/0, Ru III/IIE 1/2=-0.18V.
[Ru III(L) (acac) (py)] (NO 3) 2Preparation
Figure A20068004854100371
With the AgCF in the acetone (1mL) 3SO 3(42mg, solution 0.16mmol) slowly join and comprise [Ru II(Me 3-TACN) (acac) (py)] PF 6(90mg is in the orange acetone soln (3mL) 0.15mmol).Stir after 5 minutes, add solid [n-Bu 4N] NO 3(304mg 1mmol), and with the purple sedimentation and filtration, cleans with ether with acetone then.With product from the methanol recrystallize.Yield: (64mg, 87%) ESI/MS (holotype): m/z=451.0, [M]+; 225.4, [M] 2+With respect to the NHE in pH 8.05 phosphate buffer solns, Ru III/IIE 0=0.2V.
[Ru III(acac) 2(4，4’-(COO)(COOH)-2，2’-bpy)]
Under argon gas, with the Ru in the 30ml ethanol (acac) 3(200mg is 0.5mmol) with 4,4 '-(COOH) 2-2, (122mg, red mixture 0.5mmol) reflux with several spongy Zn/Hg amalgam and spend the night 2 '-bpy.Cool off the brown mixture that produces, and remove zinc amalgam with tweezers.Collect brown precipitate, and clean with ethanol and ether.Then brown solid is dissolved in the 0.1M NH of 50ml 3In, filter and pricked to stir in the air and spend the night, to produce purplish red solution.Solution is filtered and evaporate to dryness.The collection purple precipitates, and cleans with acetone and ether, then dry air.Yield: 45%.With respect to the NHE at pH=8 (phosphate buffered saline buffer), E 0=0.21V.
Electrochemical test
Use cyclic voltammetry (PdR) test mediation
In order to test with reduction form synthetic amboceptor, that is, and [Ru III(5-Cl-Quin) 2(py-3-COOH) (py-3-COO)] and [Os II(2,2 '-bpy) 2(acac)] Cl has carried out one group of experiment, wherein, and in the presence of NADH, with Ru 2+[Ru II(5-Cl-Quin) 2(py-3-COOH) (py-3-COO)] or Os 2+[Os II(2,2 '-bpy) 2(acac)] Cl electrochemical oxidation.The adding of putidaredoxin reductase enzyme (PdR) (mediation enzyme) causes electrocatalysis, and this is discerned by the increase greatly of oxidation current and the shortage of the reduction peak in the reverse scan.
Use the formulations prepared from solutions that is used to test mediation (PdR) of cyclic voltammetry
Prepare a series of putidaredoxin reductase enzymes (Biocatalysts Ltd. (biological catalyst company), Wales) solution by the following method: the Trizma that PdR is dissolved into the pH 9 that makes 0.1M
Figure A20068004854100381
In preset (Pre-set) crystal (Sigma-aldrich company limited (Sigma-Aldrich Company Ltd)), obtaining the liquid storage of 10mg/mL, and dilute described liquid storage subsequently, to obtain having the solution of different PdR concentration.Then with these solution and the NADH (Sigma-aldrich company limited (Sigma-Aldrich Company Ltd)) that contains amboceptor or 1: 1 (v: v) mix of TNADH (Oriental Yeast company, Japan) solution.Whole final solutions of test have 1mM amboceptor, 5mMTNADH (or 10mM NADH), and the PdR of different concns (5mg ml -1To 0.1mg ml -1).
Use the formulations prepared from solutions that is used to test amboceptor (GOx) of cyclic voltammetry
Prepare a series of notatins (GOx) (Sigma-aldrich company limited (Sigma-Aldrich Company Ltd)) solution by the following method: the Trizma that GOx is dissolved into the pH 7 that makes 0.1M In the preset crystal (Sigma-aldrich company limited (Sigma-Aldrich CompanyLtd)), obtaining the liquid storage of 10mg/mL, and dilute described liquid storage subsequently, to obtain having the solution of different GOx concentration.Then with these solution and the NADH (Sigma-aldrich company limited (Sigma-Aldrich Company Ltd)) that contains amboceptor or 1: 1 (v: v) mix of TNADH (Oriental Yeast company, Japan) solution.Whole final solutions of test have 1mM amboceptor, 5mMTNADH (or 10mM NADH), and the GOx of different concns (2.5mg ml -1To 0.5mgml -1).
Experiment 1
[Ru (II) is (NH (py-3-COOH) in order to measure new amboceptor 3) 5] (PF 6) 2Electrochemical response, the amboceptor solution of 10mM (using non--de-oxygenised water preparation) is placed on the standard electrode, and uses the cyclic voltammetry test.
The results are shown among Fig. 1, described Fig. 1 is under round-robin situation between-0.35V and the 0.5V, with 100mVs -1The cyclic voltammogram of the 10mM amboceptor solution (oxidation) on standard electrode, tested of scanning speed.Voltammogram has clearly illustrated the oxidation of amboceptor and the specific peak of reductive subsequently, and described specific peak has relatively little peak separation.
Experiment 2
In order to test new amboceptor [Ru II(py-3-COOH) (NH 3) 5] (PF 6) 2The reduction form for the stability of the direct oxidation of dissolved oxygen, come testing sensor by repeated oxidation (using identical solution) in anaerobic and aerobic these two kinds of conditions.
The repeated oxidation that Fig. 2 obtains when being presented at testing sensor in glove box (square) and the open air (circle) (with respect to Ag/AgCl ,+0.25V) result.
The result shows, the oxidation current of new amboceptor with repeated test reduce-under the situation of repeated oxidation 11 times, reduce about 12%, but with Ru (NH 3) 6Cl 2In the time of relatively, the effect of dissolved oxygen significantly reduces.
Experiment 3
In the tris buffer of 0.1M pH 9, under the condition described in the experiment 1, research amboceptor [Ru II(py-3-COOH) (NH 3) 5] (PF 6) 2) voltammetry, whether keep identical to measure it.Cyclic voltammetry is carried out by the following method: use 100mV second -1Scanning speed (sweep rate), from 0V, with respect to Ag/AgCl for+0.5V and-the scanning limit (sweep limit) of 0.35V scans on positive dirction at first.Fig. 3 shows the voltammogram of amboceptor in the tris buffer of water (containing 0.1M KCl) and 0.1M pH 9.Under the situation of similar spike potential and absolute current, voltammogram is very similar.With Ru 2+Ru is compared in the oxidation of planting 3+The littler reduction peak of planting shows that the kind in the solution mainly is Ru 2+With Ru (NH 3) 6Cl 2The similar experiment of being carried out has produced Ru 2+/ Ru 3+The peak current of redox couple is 50: 50 ratios.
Experiment 4
On standard electrode, study amboceptor [Ru II(py-3-COOH) (NH 3) 5] (PF 6) 2To by the stability of oxygen oxidation.At 1% Taurocholic acid sodium salt (NaTC), 50mM MgSO 4, 0.1M KCl tris buffer, among the pH 9, the solution of the new amboceptor of preparation 10,5 and 1mM, then, being+current potential of 0.25V to use multiplicity scheme (repeat timeprotocol) on standard electrode, to test described solution with respect to Ag/AgCl.The results are shown among Fig. 4.Data presentation, in whole mediator concentration of test, new amboceptor is for being stable by the oxidation of oxygen.Compare with the gradient of the 199nA/mM of standard ruthenium hexamine compound (ruthenium hexamine) amboceptor, the working curve of new amboceptor has produced the gradient (writing down identical electrode slice) of 156nA/mM.From the %CV (coefficient of variation) (2.84%) of curve with for the viewed %CV of standard ruthenium hexamine compound (coefficient of variation) (2.81%) much at one.Than the viewed intercept of ruthenium hexamine compound (72nA), the intercept of this figure is 95nA.
Experiment 5
In order to test new amboceptor [Ru II(py-3-COOH) (NH 3) 5] (PF 6) 2Whether can mediate for transfer transport from notatin (GOx), aliquot GOx is joined in (partly by oxygen oxidation) solution of new amboceptor.Other whole conditions are as testing described in 3.Fig. 5 has shown the voltammogram of the generation of writing down under the situation that does not have and exist GOx.Data presentation, new amboceptor can mediate the transfer transport between GOx and the electrode.
Experiment 6
Go up by cyclic voltammetry research amboceptor [Ru at exposed transmitter (bare sensor) III(NH 3) 5(py-3-COOH)] (PF 6) 2(CF 3SO 3).In the tensio-active agent (NaTC or CHAPS) of 0.1M Tutofusin tris (pH9.0), 0.1M KCl and 1%w/v, the amboceptor solution of preparation 50mM.It is the solution of deep yellow that this amboceptor dissolves the generation color easily.Cyclic voltammetry is carried out by the following method: use the scanning speed of 100mV second-1, from 0mV, with respect to Ag/AgCl for+0.7V and-the scanning limit of 0.7V scans on positive dirction at first.Twice scanning is carried out in each pond, and preserved scanning (Fig. 6) for the second time.For each surfactant types, cyclic voltammogram is identical.Voltammogram has shown two reduction peak and an oxidation peak, and the other shoulder on oxidation wave, and this hints that this material may contain some impurity.The oxidizing potential at this peak is than the corrigendum of the hexamine compound of Ru, and electric current experiment will select to be used for oximeter for the current potential of+250mV with respect to Ag/AgCl the time.The current potential of reduction experiment remains unchanged at-300mV.
This amboceptor that use is in 0.1M Tutofusin tris (pH 9.0), 5%CHAPS, the big CHAP of 5% deoxidation (deoxy bigCHAP) and the 66mg/mL cholesterol dehydrogenase (ChDH) prepares the total cholesterol transmitter.Add amboceptor, thereby produce the ultimate density of 48.3mM.Use the determination of plasma transmitter response of thawing of Petex expanded film and 10 μ l.
In first experiment, carry out the measurement of five multiplicity.The gradient of electric current-concentration-response is reasonably high, although intercept is also high.This can show that amboceptor comprises some impurity (for example, some Ru IIKind).Curve (referring to Fig. 7 A-C) with respect to the mean current of time shows, in case reach lowest high-current value, responds just highly stable.The response stability most probable ground of this improvement is owing to the stability of the increase of new Ru amboceptor.Data presentation, [Ru III(NH 3) 5(py-3-COOH)] (PF 6) 2(CF 3SO 3) in the total cholesterol transmitter, play amboceptor, and show the easy and PdR generation electron exchange of new amboceptor.
Experiment 7
In order further to study new amboceptor [Ru III(NH 3) 5(py-3-COOH)] (PF 6) 2(CF 3SO 3) stability of the obvious increase compared with the hexamine title complex, use multiplicity measurements to carry out second and test and reach longer period.
Below shown result in the transmitter response of common Measuring Time (118 seconds) and final Measuring Time (202 seconds).Response much at one, this shows, response and therefore amboceptor be very stable, this is with also consistent at the curve with respect to the mean current of time shown in Fig. 8 A to 8D.
Experiment 8
Fig. 9 has shown the outward appearance of the amboceptor in low oxygen atmosphere (reduction form) and air (oxidised form).Low-oxygen solution is by boil water in container at first, subsequently with its sealing, purges with N2 then and prepares in 20 minutes.Then container is sealed with para cellophane film (parafilm), and transfer to the glove box (＜9ppm O of inert atmosphere 2) in.In case enter inside, just make the KCl of 0.1M with de-oxygenised water, subsequently it is joined in the new amboceptor, to be made into the solution of 10mM.Solution presents redness (following left figure), and the solution (and leaving standstill 4 hours) that uses oxygenated water to make presents yellow (right figure).
The different redox states of amboceptor have distinct colors, and this can be used for spectral measurement.
Title complex of the present invention has low plus or minus electric charge (under pH7-10 ,+2 to-1), and forms not too strong title complex with analysis of mixtures and electrode, or does not form title complex, thereby has caused reliable more, stable and reproducible electrochemical process.In addition, should also be noted that assurance does not have ruthenium (II) to plant by the rapid oxidation of molecular oxygen.
New amboceptor has guaranteed that enzyme reaction is not complicated by the problem institute that the association with protein, enzyme and other electronegative material and electrode interrelates.In other words, the ruthenium kind can be intended to the work of giving effectively: play efficient and effective transfer transport amboceptor.These compounds are keeping for [Ru (NH 3) 6] Cl 3In the time of the quality of the mediation of being reported, allowed the electric charge on the title complex is selected, to be suitable for employed enzyme reaction best.
Under the situation that does not deviate from scope of the present invention described here, can carry out other change and improvement.

Claims (44)

1. the title complex of formula I is as the purposes of redox mediators,
[M(A) x(B) y] m(X z) n
Formula I
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 1 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is monodentate or the bidentate aromatic ligand that contains 1 or 2 nitrogen-atoms;
B is the part of independently electing as except that the heterocycle containing n-donor ligand that is fit to arbitrarily;
X is the counter ion that are fit to arbitrarily;
Wherein, A is optional is replaced by 1 to 8 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, aryl, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group, diazanyl, alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio;
Wherein, the quantity of ligating atom is 6.
2. according to purposes described in the claim 1, wherein A is by one or more CO 2R 2The unidentate ligand that group replaces, or optional by one or more CO 2R 2Bidentate or tridentate ligand that group replaces.
3. according to the purposes described in the claim 2, wherein with R 2Elect H as.
4. according to the purposes described in each of claim 1 to 3, wherein A is selected from nicotinic acid, Yi Yansuan, 2,2 '-dipyridyl, 2,2-dipyridyl-5,5 '-dicarboxylic acid, 2,2-dipyridyl-4,4 '-dicarboxylic acid, 5-chloro-oxine.
5. the title complex of formula II is as the purposes of redox mediators,
[M(A) x(B) y] m(X z) n
Formula II
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n are the integers that independently is selected from 1-6; Y is the integer that is selected from 0-5; M is-5 to+4 integer, and z is-2 to+1 integer;
A is bidentate, three teeth, four teeth, five teeth or sexadentate ligand, and described part can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) qOr [(RNC 2H 4) (RNC 3H 6)] s, wherein w is the integer of 1-5, and v is the integer of 3-6, and p and q are the integers of 1-3, and thus, the summation of p and q is 4,5 or 6, and s is 2 or 3, and wherein R and R 1Be hydrogen or methyl;
B independently elects the part that is fit to arbitrarily as;
X is the counter ion that are fit to arbitrarily;
Wherein, B is optional is replaced by the 1-8 that independently is selected from a following groups group: replace or unsubstituted alkyl alkenyl, or aryl-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, aryl, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group, diazanyl, alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio;
Wherein, the quantity of ligating atom is 6.
6. according to the purposes described in the claim, A is bidentate, three teeth, tetradentate ligands, and it can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) q, [(RNC 2H 4) (RNC 3H 6)] sWherein w is the integer of 1-3, and v is 3 or 4 integer, and p and q are the integers of 1-3, and thus, the summation of p and q is 4, and s is 2 or 3;
7. according to the purposes described in the claim 5, wherein A is selected from 1,4,7-trimethylammonium-1,4,7-7-triazacyclononane or 1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA), 1,2-dimethyl ethylene diamine or 1,1,2,2-tetramethylethylened.
8. according to the purposes described in each of claim 1 to 7, wherein B is selected from for example NH of amine ligand 3Or NMe 3, CO, CN, halogen and acetylacetonate (acac), 3-bromo-acetylacetonate (Bracac), oxalic acid, pyridine or 5-chloro-oxine.
9. according to the purposes described in each of aforementioned claim, wherein when electing A or B as bidentate, the geometry of described title complex is a cis or trans.
10. according to the purposes described in each of claim 1 to 9, the state of oxidation of wherein said metal is elected 2+, 3+ or 4+ as.
11. according to the purposes described in the claim 9, the state of oxidation of wherein said metal is elected 3+ as.
12. according to the purposes described in each of claim 1 to 11, wherein described part A and B are selected, make that the total charge on the described title complex is selected from down group :+3 ,+2 ,+1,0 ,-1 ,-2 and-3.
13. according to the purposes described in each of claim 1 to 13, wherein said counter ion are selected from F -, Cl -, Br -, I -, NO 3 -, NH 4 +, NR 4 +, PF 6 -, CF 3SO 3 -, SO 4 2-, ClO 4 -, OH -, K +, Na +, Li +
14., wherein use the combination of counter ion according to the purposes described in the claim 13.
15. according to the purposes described in each of claim 1 to 14, wherein said title complex is [Ru III(NH 3) 5(pyridine-3-COOH)] (PF 6) 2(CF 3SO 3), [Ru III(2,4-diacetylmethane thing (pentandionate)) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(3-bromo-2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-5,5 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-4,4 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl)] Cl, [Ru III(2,4-diacetylmethane thing) 2(pyridine-4-COOH) (pyridine-4-COO)], [Ru III(5-chloro-oxine) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] (PF 6) (CF 3SO 3), [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2, [Os II(2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru (2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru II(2,2 ' dipyridyl) 2(C 2O 4)], K[Ru III(C 2O 4) 2(pyridine-3-COOH) 2], [Ru III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2In any.
16. according to the purposes described in the claim 15, wherein said title complex is [Ru III(2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2Or [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2
17. the purposes according to described in each of aforementioned claim wherein is used for electrochemical sensor with described redox mediators.
18. according to the purposes described in the claim, wherein said electrochemical sensor comprises micro-strip electrode.
19. according to the purposes described in the claim 18, wherein said electrochemical sensor is an electrochemica biological sensor.
20. according to the purposes described in the claim 19, wherein said electrochemica biological sensor is used for detecting the analyte of body fluid, environmental sample, F﹠B, animal doctor's sample, medicine.
21. according to formula I defined in each of claim 1 to 16 or ruthenium or the purposes of osmium title complex in biosensor of formula II.
22. according to the purposes described in the claim 21, it is 6 to 10 that wherein said title complex is used in pH.
23. according to the purposes described in the claim 22, it is 7 to 9 that wherein said title complex is used in pH.
24. according to the purposes described in each of claim 21 to 23, wherein said biosensor uses for compatible arbitrarily analyte.
25. according to the purposes described in the claim 24, wherein said analyte is found in the biofluid, and can be selected from any in enzyme, enzyme substrates, antigen, antibody, nucleotide sequence, cholesterol, cholesteryl ester, lipoprotein, triglyceride level or the microorganism.
26. a detection system that is used for the Measurement and analysis thing comprises:
(a) sample that comprises described analyte is contacted with the solution that comprises enzyme and redox mediators, described redox mediators is selected from containing Ru or containing the group of the compound of Os according to formula I or formula II;
(b) causing the sample of cultivating described contact under the condition that described enzyme works to analyte;
(c) make described step (b) but the cultivation sample stand to produce the condition that measurement signal changes; With
(e) measure the signal that produces.
27. according to the system described in the claim 26, but wherein said measurement signal is electrochemistry, colorimetric, heat, impedance measurement (impedometric), electric capacity or spectral signal.
28. according to the system described in the claim 27, but the electrochemical signals that wherein said measurement signal is to use micro-strip electrode to measure.
29. according to the system described in the claim 28, wherein said electrochemical signals is to use the micro-strip electrode in the electric current detecting method to detect.
30. the title complex according to formula I,
[M(A) x(B) y] m(X z) n
Formula I
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 1 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is monodentate or the bidentate aromatic ligand that contains 1 or 2 nitrogen-atoms;
B independently elects one or more of the part that is fit to arbitrarily except that the heterocycle containing n-donor ligand as;
X is the counter ion that are fit to arbitrarily;
Wherein, A is optional is replaced by 1 to 8 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, aryl, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group, diazanyl, alkyl diazanyl, hydroxylamino, alkoxy amino, alkylthio;
Wherein, the quantity of ligating atom is 6.
31. according to the title complex described in the claim 30, wherein A is by one or more CO 2R 2The unidentate ligand that group replaces, or optional by one or more CO 2R 2Bidentate or tridentate ligand that group replaces, wherein, with respect to the one or more heteroatomss of described coordinate, described one or more CO 2R 2Position between group is in separately.
32. according to the title complex described in the claim 31, wherein R 2Elect H as.
33. according to the title complex described in each of claim 30 to 32, wherein A is selected from nicotinic acid, Yi Yansuan, 5-carboxyl-nicotinic acid, 6-pyridyl-nicotinic acid, 2,2 '-dipyridyl-5,5 '-two-carboxylic acid, 2,2 '-dipyridyl-4,4 '-two-carboxylic acid, 2,2 '-dipyridyl, 1,10-phenanthroline-3,9-pair-carboxylic acid
34. the title complex according to formula II,
[M(A) x(B) y] m(X z) n
Formula II
Wherein, M is ruthenium or osmium, and has 0,1,2,3 or 4 oxidation state;
X and n independently are selected from 1 to 6 integer; Y is selected from 0 to 5 integer; M is-5 to+4 integer, and z is-2 to+1 integer;
A is bidentate, three teeth, four teeth, five teeth or sexadentate ligand, and it can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) qOr [(RNC 2H 4) (RNC 3H 6)] s, wherein w is 1 to 5 integer, and v is 3 to 6 integer, and p and q are 1 to 3 integers, and thus, the summation of p and q is 4, and s is 2 or 3, and wherein R and R 1Be hydrogen or methyl;
B independently elects the part that is fit to arbitrarily as;
X is the counter ion that are fit to arbitrarily;
Wherein, A is optional is replaced by 1 to 7 group that independently is selected from following groups: replace or unsubstituted alkyl alkenyl, or aryl-F ,-Cl ,-Br ,-I ,-NO 2,-CN ,-CO 2H ,-SO 3H ,-NHNH 2,-SH, aryl, alkoxy carbonyl, alkyl amino-carbonyl, dialkyl amino carbonyl ,-OH, alkoxyl group ,-NH 2, alkylamino, dialkyl amido, alkanoylamino, aryl formamido group, alkyl diazanyl, hydroxylamino, alkoxy amino or alkylthio;
Wherein, the quantity of ligating atom is 6.
35. according to the purposes described in the claim 34, wherein A is bidentate, three teeth, tetradentate ligands, described part can be a straight chain, has formula R 1RN (C 2H 4NR) wR 1, or cyclic, have formula (RNC 2H 4) v, (RNC 2H 4) p(RNC 3H 6) q, [(RNC 2H 4) (RNC 3H 6)] sWherein w is 1 to 3 integer, and v is 3 or 4 integer, and p and q are 1 to 3 integers, and thus, the summation of p and q is 4, and s is 2 or 3;
36. according to the title complex described in the claim 35, wherein A is selected from 1,4,7-trimethylammonium-1,4, and the 7-7-triazacyclononane, or 1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA), 1,2-dimethyl ethylene diamine, or 1,1,2, the 2-tetramethylethylened.
37. according to the title complex described in each of claim 30 to 36, wherein B is selected from for example NH of amine ligand 3CO; CN; Halogen, and acetylacetonate (acac), 3-bromo-acetylacetonate (Bracac), oxalic acid, oxalic acid, or 5-chloro-oxine.
38. according to the title complex described in each of claim 30 to 37, wherein when electing A or B as bidentate, the geometry of described title complex is a cis or trans.
39. according to the title complex described in each of claim 30 to 38, the oxidation state of wherein said metal is elected 2+ or 3+ as.
40. according to the title complex described in each of claim 30 to 39, the oxidation state of wherein said metal is elected 3+ as.
+ 2 ,+1,0 ,-1 ,-2 and-3 41. according to the title complex described in the claim 40, wherein described part A and B are selected, make that the total charge on the described title complex is selected from down group:.
42. according to the title complex described in each of claim 30 to 41, wherein said counter ion are selected from F -, Cl -, Br -, I -, NO 3 -, NH 4 +, NR 4 +, PF 6 -, CF 3SO 3 -, SO 4 2-, ClO 4 -, OH -, K +, Na +, Li +
43., wherein use the combination of counter ion according to the title complex described in the claim 42.
44. according to the title complex described in each of claim 30 to 43, wherein said title complex is [Ru III(NH 3) 5(pyridine-3-COOH)] (PF 6) 2(CF 3SO 3), [Ru III(2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(3-bromo-2,4-diacetylmethane thing) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-5,5 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl-4,4 '-(COOH) (COO)], [Ru III(2,4-diacetylmethane thing) 2(2,2 '-dipyridyl)] Cl, [Ru III(2,4-diacetylmethane thing) 2(pyridine-4-COOH) (pyridine-4-COO)], [Ru III(5-chloro-oxine) 2(pyridine-3-COOH) (pyridine-3-COO)], [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] (PF 6) (CF 3SO 3), [Ru III(1,1,4,7,10,10-hexamethyl Triethylenetetramine (TETA)) (2,4-diacetylmethane thing)] Cl 2, [Os II(2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru (2,2 ' dipyridyl) 2(2,4-diacetylmethane thing)] Cl, [Ru II(2,2 ' dipyridyl) 2(C 2O 4)], K[Ru III(C 2O 4) 2(pyridine-3-COOH) 2], [Ru III(1,4,7-trimethylammonium-1,4,7-7-triazacyclononane) (2,4-diacetylmethane thing) (pyridine)] (NO 3) 2
CNA2006800485415A 2005-12-21 2006-12-21 Redox mediators Pending CN101360755A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102516309A (en) * 2011-10-25 2012-06-27 暨南大学 Ruthenium complex capable of inhibiting tumor angiogenesis and preparation method and application thereof
CN101561407B (en) * 2009-05-13 2013-03-20 哈尔滨工业大学 Method for manufacturing atomic oxygen sensor chip of osmium membrane resistance wire

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101561407B (en) * 2009-05-13 2013-03-20 哈尔滨工业大学 Method for manufacturing atomic oxygen sensor chip of osmium membrane resistance wire
CN102516309A (en) * 2011-10-25 2012-06-27 暨南大学 Ruthenium complex capable of inhibiting tumor angiogenesis and preparation method and application thereof
CN102516309B (en) * 2011-10-25 2014-07-02 暨南大学 Ruthenium complex capable of inhibiting tumor angiogenesis and preparation method and application thereof

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