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WO1997005489A1 - Matieres complexantes metalliques solubles et polymeres destinees a la mesure de sucres et de molecules apparentees dans une solution - Google Patents

Matieres complexantes metalliques solubles et polymeres destinees a la mesure de sucres et de molecules apparentees dans une solution Download PDF

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Publication number
WO1997005489A1
WO1997005489A1 PCT/US1996/010065 US9610065W WO9705489A1 WO 1997005489 A1 WO1997005489 A1 WO 1997005489A1 US 9610065 W US9610065 W US 9610065W WO 9705489 A1 WO9705489 A1 WO 9705489A1
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WIPO (PCT)
Prior art keywords
metal complex
triazacyclononane
polymerizable
styryl
detection system
Prior art date
Application number
PCT/US1996/010065
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English (en)
Inventor
Frances H. Arnold
Zhibin Guan
Chao-Tsen Chen
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California Institute Of Technology
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Application filed by California Institute Of Technology filed Critical California Institute Of Technology
Priority to AU62732/96A priority Critical patent/AU6273296A/en
Publication of WO1997005489A1 publication Critical patent/WO1997005489A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2650/00—Assays involving polymers whose constituent monomers bore biological functional groups before polymerization, i.e. vinyl, acryl derivatives of amino acids, sugars

Definitions

  • the present invention relates generally to devices and methods used to test for and monitor the presence of sugars and chemically related compounds in solutions. More particularly, the present invention is directed to sensors and methods which rely on metal coordination interactions between electronegative functionalities on the targeted compounds and metal ion complexes to provide detection and/or measurement of samples of sugars or related compounds in aqueous solutions.
  • glucose is by far the most important sugar, and diabetes is the most common disease for which glucose determinations are routinely conducted. Diabetes is a disease of the metabolic system that affects more than 14 million people in the United States and over 100 million people worldwide. It is characterized by an elevated blood-glucose concentration which is caused by a lack of the hormone insulin. Sugars are the primary source of metabolic energy, and the inability to self-regulate the levels of sugar metabolized by the body leads to many other medical problems, including but not limited to blindness, heart disease and kidney failure.
  • Treatment of diabetes involves monitoring of the patient's blood-glucose levels, with insulin injections being given when the glucose concentration rises above normal levels.
  • a simple and accurate method for measuring blood-glucose concentrations is an essential cornerstone of any diabetes treatment protocol, since excessively high blood-glucose levels in diabetes patients can result in coma and even death. Frequent testing and insulin administration can significantly reduce long-term complications of diabetes.
  • the vast majority of sensors which are used currently for glucose monitoring are simple devices based on enzymes such as glucose oxidase or glucose dehydrogenase. These enzyme-based sensors are simple to use and have relatively high sensing selectivity. They are widely used for one-time measurement of blood-glucose concentrations in vitro.
  • the one-time or 'spot' measurement of blood-glucose concentration in vitro is also not optimal, as it requires collection of a blood sample, usually obtained by pricking the finger, which must be done at least several times each day.
  • a blood sample usually obtained by pricking the finger, which must be done at least several times each day.
  • much effort has gone into identifying alternative sources for samples, such as subcutaneous tissue fluid or saliva instead of blood, and for identifying less-painful and more efficient ways to obtain these samples, such as by transdermal extraction or using very thin needles.
  • the development of new, highly sensitive and miniaturizable glucose monitoring technology will make some of these alternative methods more feasible for use at home by patients. Continuous real time measurement of glucose concentrations is most desirable because it can be used for close monitoring and treatment.
  • Continuous monitoring of glucose can also form part of a fully automatic insulin delivery system.
  • a number of different sensor configurations have been proposed for use in either in vitro or in vivo monitoring systems. Continuous glucose monitoring systems are described in a number of publications, for example by E. Wilkins and M.G. Wilkins (J. Biomed. Eng. 1983, Vol. 5, October, pp. 309-315) and J. Pickup (TIBTECH, July, 1993, Vol. 1 1 , pp. 285-291).
  • glucose sensors While nearly all glucose sensors utilize an enzyme for glucose recognition, sensors described in the literature vary widely in their signal transduction mechanisms. Electrodes, optical methods and solid-state electronic devices such as field-effect transistors (FETs) are among the configurations that have been reported. The enzyme electrodes used in these enzyme sensor systems have been amperometric (mediated and non-mediated) and potentiometric. Although some of the above-described glucose sensors have shown promise, none have been found to be entirely satisfactory. There is a continuing need to develop robust sensor systems which can be used to accurately and simply measure the amount of a particular sugar which is present in aqueous solutions and other environments. The need is particularly apparent with respect to in vitro and in vivo glucose determinations which are critical in medical diagnosis and treatment of various metabolic disorders, including diabetes. There is also a strong need to develop robust sensor technology for real-time monitoring of sugars in manufacturing process environments.
  • sensors and systems are presented which are well-suited for use in measuring the presence of a variety of sugars and related compounds (target molecule) in aqueous solutions.
  • the invention is based in part on the discovery that certain metal complexes will chelate sugars and other molecules in alkaline media and can either be used alone in solution, attached to a suitable support surface, or embedded in a polymer to provide a selective sensor which releases a proton when the target molecule binds to the metal complex.
  • the release of protons from the sensor provides a simple, accurate and easily detected indirect measurement of target molecule concentra ⁇ tion in the solution.
  • a sensor that includes metal complexes that bind a given target molecule in aqueous solution wherein binding of said metal complex to the target molecule results in the release of a proton.
  • the metal complexes are preferably attached or embedded in a solid support to provide both anchoring of the complexes and positioning of the metal ions to increase selectivity of the target binding interactions. Attachment or incorporation of the metal complexes in a porous solid support can also reduce fouling of the sensor from other components in the biological sample (e.g. proteins) and provide selectivity in terms of molecular size.
  • the sensor may be used to measure the target molecule concentration in a wide variety of aqueous solutions so long as the solutions are initially alkaline, are treated with a suitable base to make them alkaline prior to contact with the sensor, or the local environment of the metal complex is maintained in an alkaline state.
  • the sensor is especially well-suited for use in measuring the concentration of glucose in blood or serum and other bodily fluids.
  • the sensor when appropriately configured, is suitable for measuring the concentration of a wide variety of other sugars and chemically-related compounds that also chelate the metal complexes and release protons.
  • the metal complex contains a polymerizable functionality, which allows it to be copolymerized with other monomers and crosslinking agents to provide incorporation of the metal complex into a polymer support matrix.
  • the copolymerization may further be conducted with the target molecule or another molecule (referred to as the template) bound to the polymerizable metal complex to improve the polymerization process and to provide imprinting of the resulting polymer matrix. If the template binds only to one metal complex, polymerization in the presence of the template and subsequent removal of the bound template molecules can result in a polymer with greater shape and size selectivity for that molecule or its structural analogs.
  • the template binds more than one metal complex
  • the resulting imprinted polymer matrix holds and positions the metal complexes in a spatial orientation which increases the selectivity of the sensor for the template molecule or its structural analogs.
  • the template serves to direct the formation of a selective polymer. It has also been discovered that the template serves to improve the extent of polymerization, providing more rigid polymeric materials that can be obtained in the absence of the target molecule or other templates which bind the metal ions.
  • a target molecule detection system in which a signal transduction system is used to detect the protons which are released from the sensor metal complexes as a result of target molecule binding.
  • a signal transduction system is used to detect the protons which are released from the sensor metal complexes as a result of target molecule binding.
  • the metal ion complexes are designed to 1 ) hold the metal ion tightly, 2) allow at least two coordination sites to be or become available for chelation to the target sugar or other molecule. Further design features are that the chelating ligand from which the metal complexes are formed can be chemically modified to 1 ) have a polymerizable functionality for copolymerization, or 2) have functional groups appropriate for covalent attachment to a solid surface, and 3) provide additional favorable interactions (electrostatic, hydrogen bonding, hydrophobic, etc.) with the target molecule.
  • the metal ion for a particular metal ion complex is chosen such that the target molecule chelates the complex formed by the metal ion and chelating ligand rapidly and reversibly in the presence of the sample solution and that a proton is released upon chelation.
  • the sensors in accordance with the present invention may be used in both the in vitro and in vivo environments.
  • the sensors can be used in spot monitoring of target molecule concentrations, for example, as in monitoring glucose concen ⁇ tration in the blood, serum or subcutaneous tissue fluid sample of a diabetic patient.
  • the senor can be formulated into a continuous monitoring device for continuous measurement of target molecule levels ex vivo or in vivo, for example, as a subcutaneous implant or as part of an ex vivo continuous monitoring system for glucose using subcutaneous tissue fluid, serum or blood.
  • the continuous sensors may be used as part of a feedback device for providing automatic dosing of insulin and other drugs which affect blood-sugar levels.
  • the sensors in accordance with the present invention may also be used to monitor (on a spot or continuous basis) levels of sugars in manufacturing processes such as fermentations, purifications, and for product quality control, where it is desirable to know specific carbohydrate concentrations levels.
  • Sensors in accordance with the present invention can be formulated for additional clinical and manufacturing applications, such as diagnosis of diseases or pathogens characterized by specific carbohydrates or other molecules that will chelate the metal complexes and produce proton signals. Additionally, such sensors can be formulated for monitoring important sugar or other chemical concentrations in other applications in clinical medicine, forensic science, drug testing, and manufacturing where it is important to rapidly obtain accurate measures of such concentrations.
  • FIGS. 1a-h are diagrammatic representations showing exemplary ligands in their free state, in the form of metal complexes and as bound to glucose. The state of the complexes in alkaline aqueous is illustrated for Cu(TACN).
  • FIG. 2 is a diagrammatic representation of an exemplary synthesis of a preferred sugar binding polymer sensor wherein the sensor is a macroporous solid which is formed by co-polymerization of a polymerizable copper complex and crosslinking agent.
  • FIG. 3 is a diagrammatic representation of an exemplary synthesis of a preferred sugar binding polymer sensor wherein the selectivity of the sensor is increased by imprinting the polymer with the sugar (or analog) that is to be detected.
  • FIG. 4 is a schematic representation of an exemplary sugar detection system in accordance with the present invention.
  • FIG. 5 is a schematic representation of a field effect transistor (FET) detector utilizing an imprinted matrix in accordance with the present invention.
  • FET field effect transistor
  • FIG. 6 is a schematic representation of an exemplary implantable (subdermal) continuous glucose monitoring system utilizing fluorescence detection and a microporous sensor material incorporating pH-sensitive fluorescent probe molecules.
  • concentration of an unknown sample of the c/s-diol can be determined by adding a known quantity to one of the metal complex solutions, measuring the resulting depression in pH and comparing that value to these calibration curves prepared using known quantities.
  • concentration of an unknown sample of Me- ?-D-Glc can be determined by adding a known quantity to one of the metal complex solutions, measuring the resulting depression in pH and comparing that value to these calibration curves prepared with known quantities.
  • FIG. 9 is a graph depicting the results of a pH static titration with c s-diol (1 ,4-anhydroerythritol). The graph shows the amount of NaOH required to maintain the pH of the Cu(TACN) metal complex solution at four initial pH values.
  • the concentration of an unknown sample of c/s-diol can be determined by adding a known quantity to Cu(TACN), measuring the amount of NaOH required to maintain constant pH and comparison to these calibration curves prepared using known quantities.
  • FIG. 10 is a graph depicting the results of a pH static titration with Me- -D- Glc, showing amount of NaOH required to maintain the pH of the Cu(TACN) metal complex solution at four initial pH values.
  • concentration of an unknown sample of Me- ?-D-Glc can be determined by adding a known quantity to Cu(TACN), measuring the amount of NaOH required to maintain constant pH and comparison to these calibration curves prepared using known quantities.
  • FIG. 1 1 is a graph showing the results of a titration of an exemplary polymer sensor (Polymer I) with glucose at pH 1 1.50, 10.50 and 10.25. The graph shows total released proton concentration vs total glucose concentration.
  • FIG. 12 is a graph showing the results of static titration of exemplary polymer sensors (Polymers II, III and IV) at a pH of 10.25. The graph show total released proton concentration vs total glucose concentration.
  • FIG. 13 is a graph showing a comparison of protons released from exemplary polymer sensors II and Ile. The graph shows total released proton concentration vs total glucose concentration.
  • FIG. 14 is a graph showing a comparison of protons released from exemplary polymer sensors IV and IVe. The graph shows total released proton concentration vs total glucose concentration.
  • FIG. 15 is a graph showing a comparison of glucose and glucosamine binding to polymer IV at pH 10.25.
  • the sensors in accordance with the present invention may be used to detect and measure the presence of a wide variety of target molecules (sugars and related compounds) in solution.
  • the sensors are suitable for detecting sugars such as glucose, mannose, sialic acid, aminosugars such as glucosamine, disac ⁇ charides, trisaccharides, oligosaccharides, sugar-amino acids, sugar-peptides and giycoproteins.
  • Other target molecules can also be measured using this sensor.
  • Exemplary target molecules include glycerol, dopamine, catechols, ascorbic acid, polyols, diols such as 1 ,4-anhydroerythritol, ethyleneglycol and 3-hydroxy-L- tyrosine.
  • the general characteristics of or requirement for a suitable target molecule is that it must be able to chelate the metal complex and release a proton when used at an appropriate pH.
  • Sensors which are capable of measuring the concentration of glucose in biological samples are preferred because of the importance of glucose in the diagnosis and treatment of diabetes and other disorders.
  • the concentration range which is typically of interest in biological samples is 0-25 mM.
  • the sensors may be used to detect the presence of target molecules in a wide variety of different aqueous solutions.
  • the only requirement is that the immediate environment of the metal complexes be at a pH that will provide ade ⁇ quate binding between the metal complexes in the sensor and the target molecule of interest and will release a proton(s).
  • the pH of the environment can be tailored either by adjusting the sample pH or through the choice of polymer support, co- monomers or crosslinking agents for copolymerization, or by the addition of appropriate counterions.
  • Alkaline solutions are those having a pH of above 7.0.
  • the upper limit for solution pH is not particularly critical provided that the solution is not so alkaline that the target molecules or sensor components are adversely affected.
  • the immediate environment around the metal complex have a pH of between 9 and 13.
  • the optimum values of pH will depend on the buffer capacity of the sample and the sensor's response at that pH, among other factors. Changes in the local pH resulting from binding to the target molecule are easier to measure at pH values where the sample has little buffering capacity. Similarly, the sensor will be more sensitive to target molecule concen- tration at pH values where the metal complex binds the target molecule most tightly. The optimal pH will also depend on the particular metal complex and target molecule.
  • Cu(ll) complexes with various amino sugars are described by (Kozlowski, H. et al., Carbohydrate Res. 197, 109-1 17 (1990)). Weaker complexes are formed with Ni(ll) and Co(lll).
  • Vanadium complexes of various sugars are described by (Sreedhara, A. et al., Carbohydrate Res. 264, 227-235 (1994)).
  • Complex formation between D- xylo 5-hexulosonic acid and borate, molybdate and tungstate is described by (Caldeira, M.M. et al., Inorg. Chim. Acta 221 , 69-77 (1994)).
  • the ability of Al(lll) to form complexes with glucose and glucoronic acid is reported by (Tonkovic, M. and Bilinski, H., Polyhedron 14, 1025-1030 (1995)).
  • Fe(lll) complexes with eighteen different sugar-type molecules are reported by (Nagy, L. et al., Inorg.
  • D-Glucuronic acid forms complexes with uranium (Tajmir-Riahi, H.A., Inorg. Chim. Acta 1 19, 227-232 (1986)), Ba(ll), Mg(ll) and Sr(ll) (Tajmir-Riahi, H.A., J. Inorg. Biochem., 24, 127-136 (1985)).
  • carbohydrates will bind reversibly to metal complexes immobilized on surfaces during ligand-exchange chromatographic separations.
  • carbohydrates form complexes with electrostatically immobilized rare earth (Tb(IID) and uranyl ions at alkaline pH (Stefannson, M., D. Wsterlund, "Ligand exchange chromatography of carbohydrates at alkaline pH: effects of mobile phase additives and temperature,” J. Chromatogr. Sci. 32, 46-49 (1994)).
  • the sensor includes specific types of metal ion complexes that bind to sugars and other molecules in alkaline aqueous solution.
  • Metal ion complexes which may be used in accordance with the present invention should: 1 ) hold the metal ion tightly; and 2) allow at least two coordination sites to be or become available for chelation to the target sugar or other molecule.
  • the chelating ligand from which the metal complexes are formed may be chemically modified to 1 ) have a polymerizable functionality for copoly ⁇ merization, or 2) have functional groups appropriate for covalent attachment to a solid surface, and 3) provide additional favorable interactions (e.g. electrostatic, hydrogen bonding and hydrophobic) with the target molecule.
  • the metal ion for a particular metal ion complex is chosen such that the target molecule chelates the complex formed by the metal ion and chelating ligand rapidly and reversibly in the presence of the sample solution and that a proton is released upon chelation.
  • FIG. 1 a Four exemplary metal complexes are shown in FIG. 1 a. On the left side of FIG. 1 a, the metal complexes are shown in an unbound state in alkaline aque- ous solution. On the right side of FIG. 1 a, the configurations of the metal com ⁇ plexes when they are bound to glucose are shown. Exemplary metal complexes include copper(ll)-triazacyclononane (Cu(TACN)), copper(ll)-1 ,4-dimethyl triaza- cyclononane (Cu(1 ,4-dimethyl-TACN)) (not shown in Fig.
  • Cu(TACN) copper(ll)-1 ,4-dimethyl triaza- cyclononane
  • M is copper or iron
  • X is H or a functional group selected from styrene, methacrylate, acrylate, vinyl ether, vinyl acetate, trialkoxysilane, dialkyl- chlorosilane, epoxy and alkylhydroxyl or alkylamine groups having from 1 to 3 carbon atoms.
  • Y is H or a functional group selected from styrene, methacrylate, acrylate, vinyl ether, vinyl, vinyl acetate, trialkoxysilane, dialkylchlorosilane, epoxy and alkyl, alkylhydroxyl or alkylamine groups having from 1 to 3 carbon atoms.
  • Z is H or a functional group selected from styrene, methacrylate, acrylate, vinyl ether, vinyl acetate, trialkoxysilane, dialkylchlorosilane, epoxy and alkyl, alkylhydroxyl or alkylamine groups having from 1 to 3 carbon atoms. Additional exemplary metal complexes are listed below and classified by their coordination number and the donor atoms. Polymerizable functional groups for these complexes are also selected from styrene, methacrylate, acrylate, vinyl, vinyl ether, vinyl acetate, trialkoxysilane, dialkylchlorosilane and epoxy.
  • Ethylenediamine which can form a five-membered ring with metal ions by chelation, shows relatively strong complexation with metal ions (M) such as Cu + , Cu 2 + , Co 3 + , Ni 2 + , Fe 3 + and Hg 2 + .
  • M metal ions
  • the geometry of the complexes is either square planar, tetrahedral or octahedral. Even when there is no vacant coordination site left in the complex, the deproteinated sugar is able to replace one of ethylene ligands at the equatorial positions under strongly basic conditions.
  • the coordinating amine can be either primary or secondary or even tertiary, as long as steric strain is minimized.
  • Polymerizable functional groups can be attached either through nitrogen or carbon atoms.
  • Other diamines including linear and macrocyclic forms can serve the same purpose, as long as they can chelate the metal ion to form stable metal complexes with the features listed above.
  • Exemplary chemical formulas for this class of complexes are set forth in FIG. 1 b where R is the polymerizable functional group. The binding of the complexes to glucose is also shown wherein " «»»» « « represents glucose.
  • Linear triamines bind metal ions more tightly than the bidentate open chain diamines.
  • the ligands which are able to form either five-membered ring or six- membered rings are suitable. Facial and meridional coordination stereoisomers are commonly observed for the octahedronal complexes for most metal complexes.
  • the negatively-charged oxygens from sugar molecules are able to displace one of the ligands to a bind metal ion at its equatorial positions.
  • polymerizable functional groups can be incorporated at the nitrogen or carbon atoms. In the following example, only derivatization on the nitrogen is shown.
  • Exemplary metal complexes belonging to this class are shown in FIG. 1 c.
  • the chemical formula for the ligand alone is shown along with exemplary formulas for the ligand bound to a metal ion to form the metal complex.
  • An exemplary binding of the metal complex to glucose is also shown.
  • Derivatization on the nitrogen atom only is shown. As mentioned above, derivatization on the carbon atom is also possible.
  • M includes the same metal ions as the Class I complexes and R', R" is H, CH 3 or another other functional group that confers greater stability to the complex.
  • one or more of the R groups is selected from styrene, methacrylate, acrylate, vinyl, vinyl ether, vinyl acetate, trialkoxysilane, dialkylchlorosilane or epoxy.
  • FIG. 1 d The structure of exemplary ligand from this class is shown in FIG. 1 d.
  • the ligand structure is shown as well as the structure of the ligand after binding to a metal ion (M) and subsequent binding to glucose ( w ).
  • M metal ion
  • the four nitrogen atoms can be either primary or secondary or tertiary amines. Ethylene units or propyl units between coordinating nitrogens are suitable.
  • R, R', R", R" is H, CH 3 or another other functional group that confers greater stability to the complex.
  • one or more of the R groups is selected from styrene, methacrylate, acrylate, vinyl, vinyl ether, vinyl acetate, trialkoxysilane, dialkylchlorosilane or epoxy.
  • Ligands used in the metal complexes can be improved by making them more rigid, with the coordinating groups correctly preoriented for coordination to the metal ions. Macrocyclic rings provide this advantageous feature.
  • the tridentate macrocyclic ligands display strong complexation with most transition metal ions, chelating in a facial arrangement with two equatorial sites open for sugar binding. As before, structural variations on the ring can be made through the carbon or nitrogen atoms.
  • a preferred ligand for the metal complex is triazacyclononane. This ligand tightly complexes metal ions such as copper(ll), leaving coordination sites appropriately positioned for sugar chelation. The binding of this type of ligand to a metal ion is shown in FIG. 1 a and in more detail in FIG. 1 e.
  • triazacyclononane under alkaline conditions can be improved by alkylating the nitrogens.
  • a preferred metal ion complex is therefore a polymerizable derivative of di-alkylated triazacyclononane.
  • the triazacyclononane can be methylated, for example, at the 1 and 4 nitrogens (synthesis shown in Example 2), while a polymerizable group can be added at the remaining unalkylated nitrogen (using the procedures of Example 3).
  • M is a metal ion as described above and R, R', R" is H, CH 3 or another other functional group that confers greater stability to the complex.
  • R, R', R" is H, CH 3 or another other functional group that confers greater stability to the complex.
  • one or more of the R groups is selected from styrene, methacrylate, acrylate, vinyl, vinyl ether, vinyl acetate, trialkoxy- silane, dialkylchlorosilane or epoxy.
  • This class of ligands is very similar to the ligands in Class III. The only difference is that the functional groups with hydrogen bonding capability, such as hydroxyl, phosphinic, amide and carboxylic acid groups, are introduced to the nitrogen atoms.
  • the functional groups with hydrogen bonding capability such as hydroxyl, phosphinic, amide and carboxylic acid groups.
  • the close proximity of hydrogen donors (acceptors) on the pendant arms can provide additional hydrogen bonding interactions besides metal-hydroxyl interactions.
  • the chemical structure of this type of ligand and the resulting metal complex as bound to glucose is shown is FIG. 1 f.
  • one of the R groups can be hydroxyl, phosphinic, amide or carboxylic acid group.
  • aromatic moieties or alkyl chains are incorporated on the ligands to provide hydrophobic interactions with sugar molecules.
  • Other favorable interactions e.g. electrostatic, etc.
  • one or more of the R groups is selected from styrene, methacrylate, acrylate, vinyl ether, vinyl acetate, trialkoxysilane, dialkylchlorosilane or epoxy.
  • Class VI Nonrogen-based tetradentate macrocyclic ring
  • the macrocyclic ring is bigger than the tridentate macrocyclic rings.
  • Most metals can sit in the plane consisting of four nitrogens atoms, and the conforma ⁇ tion of these macrocyclic rings is important for the binding geometry. If four nitrogens adopt square planar geometry around the metal ion, this metal complex cannot interact with a sugar molecule effectively. If two nitrogen atoms occupy two equatorial sites and the other two atoms coordinate to metal from axial positions and leave two equatorial coordination sites available, the resulting metal complex can bind sugar molecules more efficiently.
  • the chemical structure of this class of ligand is shown in FIG. 1 g.
  • Noncoordinating functional groups can be introduced on the rings to provide additional interactions similar to class V discussed earlier.
  • Tridentate ring ligands consisting of pyridine or pyrazole or imidazole rings are also suitable for forming metal complexes which may be used in detector systems in accordance with the present invention and are illustrated in FIG 1 h.
  • the coordination geometry is similar to the saturated nitrogen donors set forth above.
  • Other functional groups which can provide additional interactions with the target molecules can be introduced, as described previously.
  • FIG. 1h illustrates the tridentate ring ligands
  • « ⁇ ⁇ " is a polymerizable functional group which can be the same as the other classes.
  • B is boron.
  • metal complexes which utilize copper as the metal ion are preferred.
  • other metal ions which may be used in certain metal complexes include lead, vanadium, iron, mercury, nickel, cobalt, aluminum, uranium, borate, calcium, barium, Y 3 + , La 3 + .
  • a proton upon binding of glucose to the metal complex, a proton is released into solution.
  • the release of protons provides a direct indication of the amount of sugar which is available to bind to the metal complexes.
  • the released protons may be measured directly using proton detec ⁇ tion equipment. It is preferred that the released protons be measured by monitoring their effect on solution pH. As is well known, release of protons into solution results in lowering of the solution pH. Accordingly, measurement of pH provides a relatively simple and straightforward method for qualitatively observing the binding reaction between sugar or other target molecule and the metal complex.
  • FET field effect transistor
  • LAPS light-addressable potentiometric sensor
  • the probe then provides an optical signal which can be read to determine the sugar concentration.
  • Static titration involves addition of a suitable base, such as sodium hydroxide, to the solution to maintain a constant pH level.
  • a suitable base such as sodium hydroxide
  • the amount of base which must be added to the solution to maintain a constant pH provides a direct indication of the amount of protons which are released due to sugar binding.
  • the advantage of static titration is that the pH level remains the same so that the apparent binding constant between the target molecule and metal complex will also remain unchanged.
  • the metal complexes of the present invention be anchored to a support surface.
  • the present invention does cover the use of metal complexes which are in solution and not anchored to a support.
  • the free metal complexes may be used as a liquid sensor to detect the presence of sugar in those situations where solid sensors are either undesirable or not practical.
  • attachment of the metal complex to ligand support structures is preferred since the support can provide the sensor with additional selectivity for the target molecule and can be used to connect the metal complexes with the appropriate proton detection system (e.g. field effect transistor (FET), light addressable potentiometric sensor (LAPS), pH-sensitive chromophore with optical wave guide).
  • FET field effect transistor
  • LAPS light addressable potentiometric sensor
  • pH-sensitive chromophore with optical wave guide e.g. pH-sensitive chromophore with optical wave guide.
  • the metal complex be attached to a ligand support structure. Any number of methods may be used to attach the metal complexes, and any number of different support materials and physical forms may be utilized.
  • Exemplary ligand support materials include silicon, glass, quartz, ceramics, organic or inorganic polymers, and zeolites and other inorganic materials.
  • the ligand support structure can be almost any solid form which provides a surface to which the metal complex may be attached. Exemplary forms include beads, porous polymer beads, particles or membranes, plates, threads, fibers and solid-state electronic devices such as FETs or LAPS devices and the like.
  • the metal complexes may be attached to these materials and forms covalently or noncova- lently, using methods well known to those practiced in the art.
  • the metal complexes include a polymerizable moiety which allows the complex to be copolymerized with monomers and crosslinking agents to form porous polymeric materials.
  • Polymerizable metal complexes are preferred since they can be incorporated via co-polymerization directly into the support structure.
  • the polymer can be formulated into appropriate forms or configurations (membrane, beads, etc.) using methods well known to those practiced in the art.
  • appropriate pH-sensitive chromophores or fluorophores can be incorporated during polymerization for optical detection of target molecule binding.
  • the resulting polymer whether it be in the form of a powder, micro beads or a larger structure, can be used directly as a sensor in conjunction with a pH monitoring system.
  • Exemplary functional groups which are attached to the metal complex to form polymerizable metal complexes are set forth in detail above.
  • Exemplary monomers and cross-linkers which may be co-polymerized with the polymerizable metal complex include styrene, methyl methacrylate, 2- hydroxyethyl methacrylate, 2-hydroxyethyl acrylate, methyl acrylate, acrylamide, vinyl ether, vinyl acetate, divinylbenzene, ethylene glycol dimethacrylate, ethylene glycol diacrylate, pentaerythritol dimethacrylate, pentaerythritol diacrylate, N,N'- methylenebisacrylamide, N,N'-ethylenebisacrylamide, N,N'-(1 ,2-dihydroxyethy- lene)bis-acrylamide, trimethylolpropane trimethacrylate, etc.
  • co ⁇ monomer and cross-linker will be dictated by the chemical (hydrophilicity, local pH, chemical stability, degree of crosslinking, ability to graft to other surfaces, interactions with other molecules, etc.) and physical (porosity, morphology, mechanical stability, etc.) properties desired for the polymeric sensor material.
  • An exemplary synthesis is shown in FIG. 2 for forming a sugar sensing polymer by co-polymerization of a polymerizable copper(ll) complex with a cross ⁇ linking agent to form a macroporous polymer solid.
  • the cross-linker and polymerizable metal complex are initially polymerized (step 1 ) to form the macroporous polymer sensor wherein the metal complexes are exposed for interaction with glucose molecules in solution.
  • step 2 the glucose binds to the metal complex which is in turn bound by polymerization to the polymer support structure which is represented by the shaded region in FIG. 2.
  • the procedures and conditions which are use to copolymerize the polymerizable metal complex and cross-linking agent are conventional.
  • the relative amounts of each monomer can be varied to achieve desired concentra- tions of metal complexes in the polymer support structure.
  • the amount of chelating monomer will be on the order of 5-10% weight percent of the cross ⁇ linking monomer.
  • the solvent, temperature and means of polymerization e.g. free radical initiation, 7-radiation
  • the solvent will also be chosen based on its ability to solubilize all the various components of the reaction mixture.
  • pH-sensitive indicators such as fluorescent probes or other optical probes can be incorporated into these polymers for detection of glucose binding by optical methods.
  • Polymerizations are generally conducted in bulk solution of a polymerizable metal complex, a template molecule (if used), and a cross-linker by the free radical method. Similar methodology can be applied to surface grafting and particle coating with the polymer, as described in "Surface Grafting of Functional Polymers to Macroporous PolyCTrimethylolpropane Trimethacrylate," P. K. Dhal, S. Vidyasankar and F. H. Arnold, Chemistry of Materials 7, 154-162 (1995) and
  • aqueous/organic mixed solvent which serves as a porogen to create a porous structure.
  • AIBN azo-bis(isobutyronitrile)
  • aqueous/organic mixed solvent which serves as a porogen to create a porous structure.
  • the solution is heated at 60 - 70° C for 24 hrs.
  • the resulting cross- linked polymer is cut into pieces or ground into a powder and washed thoroughly with the same solvent used for the polymerization.
  • Polymerizations can be carried out by a sol-gel process when an alkoxy- silane type of polymerizable metal complex is used.
  • the alkoxysilane metal chelating monomer is mixed with tetramethoxysilane or tetraethoxysilane in aqueous solution.
  • the sol-gel condensation can be conducted in acidic or basic conditions using procedures well known to those practiced in the art.
  • the selectivity of the resulting metal complexing sensor polymer is enhanced by utilizing a polymerizable metal complex which is initially bound to the target molecule (or an analog of the target molecule) to form an imprinted polymer matrix.
  • the target molecule (or its analog) serves as a template to assist in the formation of selective binding cavities in the polymer.
  • Imprinting of polymers using metal complexes is a well-known technique which is described in detail in United States Patent No. 5,310,648. The imprinting process is shown schematically in FIGS. 3a and b. Referring to
  • the polymerizable copper metal complex is allowed to bind a sugar (methyl- ⁇ -D-glucopyranoside) to form an imprinting polymerizable monomer/ template complex which is composed of one metal complex attached to the glucose analog.
  • This imprinting monomer/template complex is then co-pol ⁇ mer- ized with a suitable crosslinking agent to form a porous polymer structure which is schematically shown at 10 in FIG. 3a.
  • the polymer 10 includes the sugar analog and metal complex which is fixed in a cavity formed by the surrounding polymer support structure.
  • the polymer support structure is shown as the shaded region in FIG. 3a.
  • the template molecule i.e. the glucose analog
  • the template molecule is removed. Removal of the template molecule leaves a macroporous polymer with complementary molecular cavities which include metal complexes that have metal ions which are positioned for specific binding with the template sugar.
  • the polymerizable copper metal complex is allowed to bind a sugar (glucose) to form an imprinting polymerizable monomer/template complex which is composed this time of two metal complexes attached to the glucose.
  • This imprinting complex is then co-polymerized with a suitable crosslinking agent to form the polymer structure which is schematically shown at 1 1 in FIG. 3b.
  • the polymer 1 1 includes the sugar and the polymerizable metal complexes which are fixed in a three-dimensional spatial distribution within the surrounding polymer support structure.
  • the polymer support structure is shown as the shaded region in FIG. 3b.
  • the glucose template is removed in the final step of sensor formation. Removal of the template molecule leaves a macroporous polymer (13) with complementary molecular cavities which include metal complexes which are positioned for specific binding with the template sugar or its analogs.
  • the imprinted polymers (12 and 13) can be prepared in a wide variety of forms ranging from powders to beads to macro structures such as plates, rods, membranes or coatings on other materials.
  • pH-sensitive indicators such as fluorescent probes or other optical probes can be incorporated into these polymers for detection of glucose binding by optical methods.
  • the same polymerizable metal complexes and cross-linking monomers described previously may be used.
  • the same polymerization conditions described above can be used, except a sugar template molecule such as methyl- ⁇ -D-glucopyranoside, ⁇ -D-glucose, a disaccharide or oligosaccharide, is first equilibrated with the polymerizable metal complex in aqueous solution at pH > 9 to form the polymerizable template complex.
  • a sugar template molecule such as methyl- ⁇ -D-glucopyranoside, ⁇ -D-glucose, a disaccharide or oligosaccharide
  • other non-sugar template molecules such as a diol or dopamine, can be used.
  • the morphology and selectivity of the polymer for binding the target molecule may be improved by altering the solvent, polymerization temperature, choice of crosslinking agent, as described by Sellergren, B., Shea, K.J., "Influence of polymer morphology on the ability of imprinted network polymers to resolve enantiomers," . Chromatogr. A 1993, 635:31 -49).
  • photoinitiation at low temperature should promote high selectivity and strong binding by materials that rely on temperature sensitive interactions for recognition.
  • FIG. 4 An exemplary detection system for measuring the presence of sugar in an alkaline aqueous solution is shown in FIG. 4.
  • the solution which is being measured for sugar content is shown at 20.
  • the solution is introduced into the testing vessel 22 via sample introduction line 24.
  • the sample may be blood, urine or other biological fluid.
  • the sample solution introduced through inlet 24 can be any aqueous solution such as a fermentation broth or process stream.
  • an important aspect of the present invention is that either the test solution 20 be alkaline or that the sensor material 32 be able to provide a local alkaline environment.
  • an inlet 26 is provided for allowing input of either acid or basic solutions into the sample solution 24 in order to pre-adjust it to desired pH levels. PH levels of 9 to 13 are preferred.
  • the pH in the test solution 20 is monitored by way of pH measuring probe 28 which is connected to pH meter 30.
  • the probe field effect transistor, electrode
  • metering equipment utilized to measure and monitor pH are well known.
  • a sugar sensor in accordance with the present invention is shown at 32.
  • the sugar sensor material can be in the form of a plate, block, beads or other macro structure.
  • a preferred sensor type is a macroporous polymer which has been formed by the polymer imprinting process shown in FIGS. 3a or b.
  • the pH of the test solution 20 is continuously monitored while it is present in the test cell 22.
  • One way to measure the glucose concentration is to monitor the drop in pH which occurs as the test solution 20 is allowed to interact with sensor 32.
  • the drop in pH provides an accurate measurement of sugar concentration in the solution when compared to a calibration curve, provided the levels are below saturation in the pH response.
  • a titrator shown schematically at 34 is provided which adds known amounts of basic solution to the test vessel 22 in order to maintain the reaction solution 20 a constant pH.
  • Feedback loop 36 is provided between the pH meter 30 and titrator 34 to provide automated operation of the system by providing constant pH level input into the titrator to allow control of the amount of basic solution being added.
  • a similar device can be used for continuous monitoring of sugar levels in a continuous sample stream 24.
  • an effluent stream 38 removes sample continuously at a rate equal to streams 24 plus 26, such that a constant sample level is maintained in vessel 22.
  • the pH, or amount of basic solution required to maintain constant pH can be monitored continuously in order to obtain a measure of the glucose concentration in the inflowing stream 24.
  • FIG. 5 Another exemplary sensor device utilizing a field effect transistor (FET) is shown generally at 50 in FIG. 5.
  • Chemical sensors based on pH detection by FETs are well known (Saito, A. et al., "An ISFET glucose sensor with a silicone rubber membrane for undiluted serum monitoring," Sensors and Actuators, B 20, 125-129 (1994); Reinhoudt, D. and E. J. R. Sudholter, "The transduction of Host- Guest interactions into electronic signals by molecular systems," Advanced Materials, 2,23-32 (1990)).
  • the imprinted polymer membrane 52 is attached to the gate oxide surface 54 of the semiconductor chip 56.
  • the semiconductor chip 56 is preferably p-type silicon.
  • the FET 50 also includes an encapsulant 58 and n- type source/drain 60 as is well-known in the art. An appropriate reference device can be made without the imprinted polymer membrane.
  • the protons released upon glucose binding to the sensor material could be measured using a light addressable potentiometric sensor (LAPS), as described by McConnell, H. M. et al., "The cytosensor microphysio ⁇ meter: biological applications of silicon technology,” Science, 257, 1906-1912 (1992).
  • LAPS light addressable potentiometric sensor
  • the sensor material would be applied such that it would be in diffusive contact with the pH-sensitive surface of the LAPS chip.
  • FIG. 4 and FIG. 5 The simplified schematic systems set forth in FIG. 4 and FIG. 5 will be understood by those of ordinary skill in the art as being merely representative of the type of detection system for which the metal complex sensors are well suited.
  • a wide range of devices may be prepared, and the type of device will depend on the conditions of use (e.g. spot monitoring, continuous monitoring, implantable sensor, process monitoring, whether used in a hospital or at home, disposable, etc.)
  • Another type of device appropriate for spot monitoring of glucose concentrations in clinical or process samples would be a simple colorimetric stick device or paper, in which the protons released by glucose binding would result in a color change of a pH-sensitive dye incorporated into the sensor material. When contacted with the biological sample, an estimate of the glucose concentration can be obtained from the resulting color of the indicator.
  • Additional exemplary systems include micro detection devices which can be implanted into a patient to provide continual in vivo glucose monitoring.
  • An example of such a system is shown in FIG. 6 in which a biocompatible sugar sensor 70 is implanted under the skin 72.
  • the sugar sensor material 74 is made such that the local environment of the metal complexes embedded in the material is alkaline. Variations in the local glucose concentration in the tissue will result in the release or uptake of protons in the immediate vicinity of the sugar sensor device.
  • the material can be made so that it provides an optical signal (e.g. by incorporating pH-sensitive fluorophores 76 inside the sensor material) that varies with pH.
  • the sensing material i.e. metal complex
  • This optical signal can be detected through the skin by interrogation with red light (which readily penetrates skin) and using an appropriate detector to measure either the steady state fluorescence or fluorescence lifetimes, as has been described previously (Bambot, S.B. et al. "Potential applications of lifetime-based phase-modulation fluorimetry in bioprocess and clinical monitoring," TIBTECH 13, 106-1 15 (1995)).
  • the light source is shown in FIG. 6 as laser 80 and the detector is shown at 82.
  • a device can be used ex vivo, connected to a continuous method for drawing a glucose sample from the patient, for example by microdialysis of subcutaneous tissue fluid (Meyerhoff, C. et al. 'Use of the microdialysis technique in the monitoring of subcutaneous tissue glucose concentration, Int'l J. of Art. Organs 16, 268-275 (1993)), by using a needle to continuously draw subcutaneous tissue fluid, or by transdermal extraction
  • These continuous monitors may be connected to insulin delivery devices by appropriate feed back systems to provide automatic insulin delivery to maintain desired blood glucose levels.
  • This example shows the use of several metal complexes in accordance with the present invention in measuring the concentration of sugars and other related molecules in aqueous solution.
  • PDN propylenediamine
  • IDA iminodiacetate
  • DIEN diethylenetriamine
  • TACN triazacyclononane
  • Chelation of the target molecule to the metal complex to form a ternary complex results in the release of a proton, which can be used to measure the concentration of the bound molecule either by the change in solution pH or by titration and comparison to a calibration curve.
  • the corresponding trans-d ⁇ o ⁇ does not chelate and therefore does not result in a change in solution pH, demonstrating the selectivity of these complexes.
  • This example also demonstrates how a metal complex is evaluated for its suitability to recognize a particular target molecule in the current invention.
  • FIG. 7 shows the pH curves for titration of the various metal complexes CuL by the c/s-diol.
  • Glc of concentration 0.10 M and pH of 9.0, 10.0, 1 1.0, or 12.0 were prepared.
  • 5.00 mL of the Cu(TACN) solution was introduced into the titration vessel of a Brinkman pH titrator. The vessel was sealed, purged thoroughly with N 2 , and equilibrated to 25 °C by a constant temperature water bath. The pH of the Cu(TACN) solution was adjusted to a desired value (9.0, 10.0, 1 1.0, or 12.0) with the addition of 0.10 N sodium hydroxide solution. Then a target molecule solution of the same pH as the Cu(TACN) solution in the titration vessel was titrated into the Cu(TACN) solution.
  • FIG. 9 shows the titration data for the Cu(TACN)/c/s-diol system at various values of pH.
  • the apparent binding strength greatly depends on the pH of the solution. At pH 9, the binding is very weak and saturation conditions are approached only at higher target molecule concentrations. However, at pH 1 1 or 12, saturation occurs at lower concentrations of target molecule, corresponding to a molar ratio of target molecule to Cu(TACN) of approximately 2. Thus there is a trade-off between the range of easily measurable concentrations and sensitivity of the sensor. At lower pH, the concentration range is greatest, but the sensitivity in terms of protons released is lowest.
  • FIG. 10 shows the pH static titration data for Cu(TACN)/Me- ?-D-Glc system at various values of pH. Again, the apparent binding strength increases at higher pH.
  • the concentration of an unknown sample of c/s-diol or Me- ?-D-Glc can be determined by adding a known quantity to one of the metal complex solutions, measuring the resulting depression in pH and comparing that value to these calibration curves prepared with known quantities.
  • the product can be stirred in refluxing acetone for 30 minutes.
  • 1 ,4,7-Triazacyclononane-N-monotosylated-dihydrobromide (42.0 g) was dissolved in minimum amount of H 2 0 (ca. 50 mL) and to this solution was added NaOH carefully to pH 7.
  • 75 mL of an aqueous solution of formaldehyde (37 % CH 2 0) and 75 mL of concentrated formic acid (HCOOH) were added to the solution and the resultant mixture was brought to reflux for 15 hours.
  • the reaction was cooled to room temperature and 30 mL of concentration. HCl added and the excess solvent was removed on a rotovapor to yield a solid.
  • This salt was dissolved in 200 mL of water and to this solution was added 20 g of NaOH and 500 mL of toluene. The mixture was then refluxed with dean stark apparatus to remove all the water. The resultant solution was then filtered and the filtrate was concentrated on a rotovap to yield a yellow oil which can be stored under Ar at 0°C indefinitely.
  • the complex is made polymerizable and loaded with copper(ll) by the procedures outlined in Example 3.
  • This example shows the synthesis of polymerizable metal complexes containing triazacyclononane in accordance with the present invention.
  • Triazacyclononane (2 g, 15.5 mmol), powdered NaOH (0.62 g, 15.5 mmol) and dry acetonitrile (35 mL) were placed in a 100 mL 2-neck round-bottomed flask charged with a reflux condenser. The reaction mixture was brought to reflux under argon for an hour. To this suspension was added dropwise a solution of chloromethyl styrene (1.84 g, 12.4 mmol) in 10 mL of acetonitrile over a period of 8 hours, during which the consumption of the starting material was closely monitored by thin layer chromatography on a neutral alumina oxide gel plate with 20% MeOH/CHCI 3 as eluent.
  • EXAMPLE 4 Preparation Of A Polymeric Sensor For Methyl- ⁇ -D-glucopyranoside
  • This example shows the preparation of a sensor in accordance with the present invention which includes a polymer support structure imprinted with a glucopyranoside.
  • the example also demonstrates the ability of the sensor to rebind with the template molecule and produce protons upon rebinding.
  • This example uses Cu(ll)-[1 ,4-bis(4'-vinylbenzyl)-1 ,4,7-triazacyclononane] (Dsty-TACN- Cu + ) as the polymerizable metal complex.
  • the solution was polymerized at 60°C for 7 hrs. After polymerization was complete, the polymer was ground into a fine powder using a mortar and washed three times with 50/50 water/methanol. The resin was equilibrated with 100 mM EDTA solution at 60°C for several hrs to strip off most of the Cu(ll) ion and the template sugar molecule. The polymer resin was then washed with water to remove extra EDTA, reloaded Cu(ll) with 20 mM CuS0 4 solution, washed again with water to remove free Cu(ll), and finally frozen and lyophilized.
  • the amount of sugar added to the solution is directly related to the amount of NaOH which must be added in order to maintain a constant pH. Accordingly, the sensor provided an indirect measure of the amount of sugar added to the solution.
  • Polvmer preparation 30 mg (0.058 mmol) of [Dsty-TACN-Cu 2 + ]S0 4 2" was dissolved in a mixed solvent composed of 1.1 1 mL water and 2.22 mL methanol. The pH of the solu ⁇ tion was adjusted to 1 1.50 with addition of 1 N sodium hydroxide. After equi ⁇ librating at room temperature for half an hour, 1 .0 g (6.49 mmol) of N,N'- methylene bisacrylamide and 10 mg of 2,2'-azobisisobutyronitrile (AIBN) were added into the solution. After purging the solution with argon, the flask was sealed and heated at 60°C.
  • AIBN 2,2'-azobisisobutyronitrile
  • the solution was polymerized at 60°C for 7 hours. After polymerization was complete, the polymer was ground into a fine powder using a mortar and washed three times with 50/50 water/methanol. The resin was equilibrated with 100 mM EDTA solution at 60°C for several hours to strip off most Cu(ll) ion. The polymer resin was then washed with water to remove extra EDTA, reloaded Cu(ll) with 20 mM CuS0 4 solution, washed again with water to remove free Cu(ll), and finally frozen and lyophilized.
  • This example demonstrates the ability of the imprinted metal-complexing polymer to bind glucose and produce protons. This example also shows that the system can be configured such that the proton release is linearly proportional to
  • Msty-TACN-Cu 2+ As The Polymerizable Metal Complex
  • This example demonstrates the ability of the metal-complexing polymer to bind glucose and produce protons. This example also shows that the system can be configured such that the proton release is proportional to glucose concentra ⁇ tion.
  • This example further demonstrates the use of Cu(ll)-[1-(4'-vinylbenzyU- 1 ,4,7-triazacyclononane] (Msty-TACN-Cu 2 + ) as the polymerizable metal complex.
  • Imprinted polvmer preparation Imprinted polvmer preparation:
  • Rebinding of glucose to the imprinted polvmer 0.5 g of polymer resin was suspended in 5.0 mL pH 1 1.50 NaOH solution in the pH titration vessel, which was kept at 25 °C by a constant temperature water bath. The pH of the suspension was adjusted to 1 1.50 by addition of 6N sodium hydroxide solution. A solution of D-( + )-glucose with concentration of 0.208 M and pH of 1 1.50 was prepared. The glucose solution was titrated into the polymer suspension sequentially. After each addition, 0.10 N sodium hydroxide solution was added to maintain pH of the system at 1 1 .50. The system was kept stirring for a few minutes until equilibrium was reached. The volumes of the glucose solution injected and of the 0.10 N sodium hydroxide solution added were recorded for data analysis. The data for the static titration are given in Table 4. TABLE 4 Titration of sensor with glucose at pH 11.52.
  • the proton release from the sensor is proportional to the glucose concentration.
  • Polymers I to IV are rigid polymers prepared using four different ratios of functional monomer Msty-TACN-Cu 2 + (Cu(ll)-[1-(4'-vinylbenzyl)-1 ,4,7-triazacyclo- nonane]) to crosslinker MBA (N,N'-methylenebisacrylamide), as listed in Table 5.
  • Glucose rebinding studies a) Glucose rebinding to polymer I at pH 11.50, 10.50 and 10.25 by pH static titration:
  • Polymer I (0.5 g) was suspended in 5 mL of pH 1 1.50, 10.50 or 10.25 NaOH solution in the pH titration vessel maintained at 25 °C by a constant temperature water bath. The pH of the suspension was adjusted as necessary by addition of 6N sodium hydroxide solution to maintain the starting pH. A solution of D-( + )-glucose with concentration of 0.208 M and pH of 1 1.50, 10.50 or 10.25 was titrated into the polymer suspension sequentially. After each addition, 0.10 N sodium hydroxide solution was added to maintain pH of the system constant. The system was kept stirring for a few minutes until equilibrium was reached. The volumes of the glucose solution injected and of the 0.10 N sodium hydroxide solution added were recorded for data analysis. The data in terms of the total concentration of released protons versus glucose concentration are plotted in FIG. 1 1 for examples performed at the three values of pH.
  • Polymer II (0.100 g) was suspended in 0.980 mL of pH 10.25 NaOH solution in a pH titration vessel, which was kept at 25 °C by a constant temperature water bath. The pH of the suspension was adjusted to 10.25 by addition of 6N and 0.1 N sodium hydroxide solution and pH 10.25 NaOH solution (0.020 mL total). A solution of D-( + )-glucose with concentration of 0.500 M and pH of 10.25 was titrated into the polymer suspension sequentially. After each addition, 0.10 N sodium hydroxide solution was added to maintain pH of the system at 10.25. The system was kept stirring for a few minutes until equilibrium was reached. The volumes of the glucose solution injected and of the 0.10 N sodium hydroxide solution added were recorded for data analysis. The titration data are plotted in FIG. 12.
  • Polymer III (0.100 g) was suspended in 0.980 mL of pH 10.25 NaOH solution in a pH titration vessel, which was kept at 25 °C by a constant temperature water bath. The pH of the suspension was adjusted to 10.25 by addition of 6N and 0.1 N sodium hydroxide solution and pH 10.24 NaOH solution (0.020 mL total). A solution of D-( + )-glucose with concentration of 0.500 M and pH of 10.25 was titrated into the polymer suspension sequentially. After each addition, 0.10 N sodium hydroxide solution was added to maintain pH of the system at 10.25. The system was kept stirring for a few minutes until equilibrium was reached.
  • Polymer IV (0.100 g) was suspended in 0.980 mL of pH 10.25 NaOH solution in a pH titration vessel, which was kept at 25 °C by a constant temperature water bath. The pH of the suspension was adjusted to 10.25 by addition of 6N and 0.1 N NaOH and pH 10.25 NaOH solution (0.020 mL total).
  • a solution of D-( + )-glucose with concentration of 0.500 M and pH of 10.25 was titrated into the polymer suspension sequentially. After each addition, 0.10 N sodium hydroxide solution was added to maintain pH of the system at 10.25. The system was kept stirring for a few minutes until equilibrium was reached. The volumes of the glucose solution injected and of the 0.10 N sodium hydroxide solution added were recorded for data analysis. The titration data are plotted in FIG. 12.
  • FIG. 12 compares the titration results for polymers II to IV at pH 10.25 in terms of protons released versus glucose concentration.
  • the largest total signal per unit weight of polymer is provided by polymer IV, made with the largest amount of polymerizable metal complex. This polymer also gives the most linear response, with respect to protons released versus glucose concentration.
  • Polymers Ile and IVe are rigid polymers prepared using the same polymerization conditions and ratios of functional monomer Msty-TACN-Cu 2 +
  • Polymers of molar ratio Msty-TACN-Cu 2 + monomer to crosslinker MBA were prepared as described in Example 8 for polymers II and IV. The only differences was that ethylene glycol was used as the template instead of methyl- 5 7-D-glucopyranoside. Polymer Ile was prepared using 0.101 1 g of Msty-TACN-
  • Polymer IVe was prepared using 0.101 1 g Msty-TACN-Cu 2 + , 21.0 mL (0.375 mmol) of ethylene glycol, 0.3854 g MBA and 9 mg AIBN.
  • Glucose rebinding was measured as described in Example 8. The titration data are plotted in FIGS. 13 and 14. Also plotted, for comparison, are the titration data for polymers II and IV, made using methyl- 7-D-glucopyranoside as
  • This example shows the binding of several substrates to glucopyranoside- imprinted polymer IV of Example 8 and the protons released upon binding.
  • the substrates investigated are D-glucosamine, urea, lactic acid and the amino acid alanine. These are compounds commonly found in serum and other biological samples that might be monitored for glucose by the current invention.
  • example illustrates a method by which potential competing signals from compo ⁇ nents in the sample other than glucose can be evaluated under different operating conditions and with different polymers. Furthermore, this example demonstrates the potential application of the current invention to the detection and measure ⁇ ment of different compounds.
  • Polymer IV (0.100 g) from Example 8 was suspended in 0.980 mL of pH 10.25 NaOH solution in a pH titration vessel, which was kept at 25 °C by a constant temperature water bath.
  • the pH of the suspension was adjusted to 10.25 by addition of 6N and 0.1 N sodium hydroxide solution, and pH 10.25 NaOH solution (0.020mL total).
  • a solution of D-glucosamine with concentration of 0.500 M and pH of 10.25 was titrated into the polymer suspension sequen ⁇ tially. After each addition, 0.10 N sodium hydroxide solution was added to maintain pH of the system at 10.25. The system was kept stirring for a few minutes until equilibrium was reached.
  • the volumes of the glucose solution injected and of the 0.10 N sodium hydroxide solution added were recorded for data analysis.
  • the titration data are plotted in FIG. 15, along with the data for glucose binding.
  • D-Glucosamine binds well to polymer IV (2) at pH 10.25. However, it releases fewer protons than glucose does at the same concentration (FIG. 15).
  • glucosamine can release protons upon binding to the polymer, this method is useful for monitoring its concentration in solution, provided large concentrations of other, competing compounds such as glucose are not present.
  • Other conditions of pH or the use of other metal complexes selective for glucosamine can be investigated in order to enhance the proton release.

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Abstract

L'invention porte sur un détecteur utilisé pour déceler la présence de sucres et de molécules apparentées dans des solutions aqueuses. Ce détecteur est constitué d'un complexe métallique se fixant à un sucre ciblé ou à une autre molécule ciblée dans une solution aqueuse, ce qui débouche sur la libération d'un proton. La mesure de la libération du proton par le détecteur fournit une indication indirecte de la concentration de molécules cible. Les complexes métalliques peuvent être reliés pour soutenir des structures afin d'assurer à la fois l'ancrage et le positionnement d'ions métalliques en vue d'accroître la sélectivité d'interactions sucre/complexe métallique. Cette invention concerne également des systèmes de détection dans lesquels on fait appel au pH comme indicateur d'une libération de proton, ainsi que des procédés de surveillance des concentrations de sucres et de molécules apparentées, procédés mettant en oeuvre les détecteurs à complexe métallique.
PCT/US1996/010065 1995-07-31 1996-06-11 Matieres complexantes metalliques solubles et polymeres destinees a la mesure de sucres et de molecules apparentees dans une solution WO1997005489A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997033177A1 (fr) * 1996-03-04 1997-09-12 California Institute Of Technology Detecteurs de sucres et d'autres analytes se liant a des metaux
WO2008104992A1 (fr) * 2007-02-26 2008-09-04 Council Of Scientific & Industrial Research Nouveau capteur potentitiométrique de cholestérol destiné à l'estimation quantitative de cholestérol total dans le sérum sanguin humain

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