US20030008408A1

US20030008408A1 - Detection of analytes in aqueous environments

Info

Publication number: US20030008408A1
Application number: US10/193,244
Authority: US
Inventors: Arthur Colvin
Original assignee: Individual
Current assignee: Individual
Priority date: 2000-08-04
Filing date: 2002-07-12
Publication date: 2003-01-09
Also published as: AU7814501A; DK2292626T3; AU2006204661A1; EP1557422B1; MXPA03001043A; EP1557422A2; TWI313354B; WO2002012251A1; CA2415338A1; KR20030037271A; CA2415338C; BR0112871A; US6794195B2; KR100922146B1; HK1054556A1; HK1076471A1; EP1557422A3; EP1307464A1; CN1446222A; DK1557422T3

Abstract

The invention relates to indicator molecules for detecting the presence or concentration of an analyte in a medium, such as a liquid, and to methods for achieving such detection. More particularly, the invention relates to copolymer macromolecules containing relatively hydrophobic indicator component monomers, and hydrophilic monomers, such that the macromolecule is capable of use in an aqueous environment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of pending U.S. patent application Ser. No. 09/632,624, filed Aug. 4, 2000.[0001]

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to indicator molecules for detecting the presence or concentration of an analyte in a medium, such as a liquid, and to methods for achieving such detection. More particularly, the invention relates to copolymer macromolecules containing relatively hydrophobic indicator component monomers, and hydrophilic monomers, such that the macromolecule is capable of use in an aqueous environment.

2. Description of the Related Art

Indicator molecules for detecting the presence or concentration of an analyte in a medium are known. Unfortunately, many of such indicators, being large organic molecules, are insoluble or sparingly soluble in water. For example, U.S. Pat. No. 5,503,770 (James, et al.) describes a fluorescent boronic acid-containing compound that emits fluorescence of a high intensity upon binding to saccharides, including glucose. The fluorescent compound has a molecular structure comprising a fluorophore, at least one phenylboronic acid moiety and at least one amine-providing nitrogen atom where the nitrogen atom is disposed in the vicinity of the phenylboronic acid moiety so as to interact with the boronic acid. Such interaction thereby causes the compound to emit fluorescence upon saccharide binding. U.S. Pat. No. 5,503,770 describes the compound as suitable for detecting saccharides. See also T. James, et al., J. Am. Chem. Soc. 117(35):8982-87 (1995). However, the compound described in example 2 of U.S. Pat. No. 5,503,770 (having formula (6)) is substantially insoluble in water, and as a practical matter requires the presence of an organic solvent such as methanol in order to work in a liquid environment.

Lack of aqueous solubility is a severe problem when dealing with applications in an aqueous environment, for example, in vivo applications. Thus, there remains a great need for adapting insoluble or sparingly soluble indicators for use in aqueous environments.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to an indicator macromolecule for detecting the presence or concentration of an analyte in an aqueous environment, said macromolecule comprising a copolymer of:

a) one or more indicator component monomers which individually are not sufficiently water soluble to permit their use in an aqueous environment for detecting the presence or concentration of said analyte; and

b) one or more hydrophilic monomers; such that the macromolecule is capable of detecting the presence or concentration of said analyte in an aqueous environment.

In another aspect, the present invention is directed to a method for the production of an indicator macromolecule for detecting the presence or concentration of an analyte in an aqueous environment, said method comprising copolymerizing:

b) one or more hydrophilic monomers; such that the resulting macromolecule is capable of detecting the presence or concentration of said analyte in an aqueous environment.

In another aspect, the present invention is directed to a method for detecting the presence or concentration of an analyte in a sample having an aqueous environment, said method comprising:

a) exposing the sample to an indicator macromolecule, said macromolecule comprising a copolymer of:

i) one or more indicator component monomers which individually are not sufficiently water soluble to permit their use in an aqueous environment for detecting the presence or concentration of said analyte; and

ii) one or more hydrophilic monomers;

such that the resulting macromolecule is capable of detecting the presence or concentration of said analyte in an aqueous environment, and wherein the indicator macromolecule has a detectable quality that changes in a concentration-dependent manner when said macromolecule is exposed to said analyte; and

b) measuring any change in said detectable quality to thereby determine the presence or concentration of said analyte in said sample.

In another aspect, the present invention provides a macromolecule which is capable of exhibiting an excimer effect, which comprises a copolymer of:

a) one or more excimer forming monomers, the molecules of which are capable of exhibiting an excimer effect when suitably oriented with respect to each other; and

b) one or more other monomers;

such that the resulting macromolecule exhibits said excimer effect.

In yet another aspect, the present invention provides a method for producing a macromolecule which is capable of exhibiting an excimer effect, which method comprises copolymerizing:

b) one or more other monomers;

such that the resulting macromolecule exhibits said excimer effect.

In yet another aspect, the present invention provides a method for detecting the presence or concentration of an analyte in a sample, said method comprising:

i) one or more indicator component monomers, the molecules of which are capable of exhibiting an excimer effect when suitably oriented with respect to each other, and which are also capable of detecting the presence or concentration of an analyte; and

ii) one or more other monomers;

such that the resulting macromolecule exhibits said excimer effect, and wherein the indicator macromolecule has a detectable quality that changes in a concentration-dependent manner when said macromolecule is exposed to said analyte; and

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. [0034] 1-2 illustrate the emission spectra of several indicator macromolecules of the present invention as described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a way to utilize, in an aqueous environment, indicator component monomers which by themselves are insoluble or sparingly soluble in an aqueous environment. Such indicators are, in effect, copolymerized with one or more monomers which are sufficiently hydrophilic such that the resulting indicator macromolecule is sufficiently hydrophilic overall so as to overcome the hydrophobic contribution of the indicator component monomers. [0035]
Suitable indicator components include indicator molecules which are insoluble or sparingly soluble in water, and whose analyte is at least sparingly soluble in water. Suitable analytes include glucose, fructose and other cis-diols; oxygen; carbon dioxide; various ions such as zinc, potassium, hydrogen (pH measurement), carbonate, etc. [0036]
Many such indicator components are known. For example, the compounds depicted in U.S. Pat. No. 5,503,770 are useful for detecting saccharides such as glucose, but are sparingly soluble to insoluble in water. Other classes of indicators include the lanthanide chelates disclosed in copending U.S. application Ser. No. 09/265,979 filed Mar. 11, 1999 (and published as PCT International Application WO 99/46600 on Sep. 16, 1999), incorporated herein by reference; polyaromatic hydrocarbons and their derivatives; etc. [0037]
The indicator components of the present invention will generally have a detectable quality that changes in a concentration-dependent manner when the macromolecule is exposed to the analyte to be measured. Many such qualities are known and may be used in the present invention. For example, the indicator may include a luminescent (fluorescent or phosphorescent) or chemiluminescent label, an absorbance based label, etc. The indicator may comprise an energy donor moiety and an energy acceptor moiety, each spaced such that there is a detectable change when the macromolecule is bound to the analyte. [0038]
Preferably, the detectable quality is a detectable spectral change, such as changes in fluorescent decay time (determined by time domain or frequency domain measurement), fluorescent intensity, fluorescent anisotropy or polarization; a spectral shift of the emission spectrum; a change in time-resolved anisotropy decay (determined by time domain or frequency domain measurement), etc. [0039]
Suitable hydrophilic monomers should be sufficiently hydrophilic so as to overcome the sum of the hydrophobic indicator component monomers, such that the resultant indicator macromolecule is capable of functioning in an aqueous environment. It will be readily apparent that a wide variety of hydrophilic monomers are suitable for use in the present invention. For example, suitable hydrophilic monomers include methacrylamides, methacrylates, vinyls, polysaccharides, polyamides, polyamino acids, hydrophilic silanes or siloxanes, etc., as well as mixtures of two or more different monomers. [0040]
Suitable hydrophilic monomers for a given application will vary according to a number of factors, including intended temperature of operation, salinity, pH, presence and identity of other solutes, ionic strength, etc. It would be readily apparent that the degree of hydrophilicity of the indicator macromolecule can be increased by adding additional functional constituents such as ions (e.g., sulfonate, quartenary amine, carboxyl, etc.), polar moieties (e.g., hydroxyl, sulfhydryl, amines, carbonyl, amides, etc.), halogens, etc. [0041]
It will be appreciated that the molar ratio of hydrophilic monomer to indicator component monomer may vary widely depending on the specific application desired. Preferred ratios of hydroplilic monomer:indicator component monomer range from about 2:1 to about 500:1, more preferably from about 5:1 to about 50:1. [0042]
The indicator macromolecules of the present invention may generally be synthesized by simply copolymerizing at least one indicator component monomer with at least one hydrophilic monomer. Optimum polymerization conditions (time, temperature, catalyst, etc.) will vary according to the specific reactants and the application of the final product, and can easily be established by one of ordinary skill. [0043]
It will be appreciated that the indicator macromolecules of the present invention may have any desired extent of water solubility. For example, the indicator macromolecule of Examples 1 and 2 below are very soluble, readily dissolving in aqueous solution. On the other hand, indicator macromolecules containing, for example, the hydrophilic monomer HEMA (hydroxyethyl methacrylate) or other common hydrogel constituents, can be non-soluble yet hydrophilic. [0044]
The soluble indicator macromolecules may be used directly in solution if so desired. On the other hand, if the desired application so requires, the indicator macromolecule may be immobilized (such as by mechanical entrapment or covalent or ionic attachment) onto or within an insoluble surface or matrix such as glass, plastic, polymeric materials, etc. When the indicator macromolecule is entrapped within, for example, another polymer, the entrapping material preferably should be sufficiently permeable to the analyte to allow suitable interaction between the analyte and the indicator components in the macromolecule. [0045]
Many uses exist for the indicator macromolecules of the present invention, including uses as indicators in the fields of energy, medicine and agriculture. For example, the indicator macromolecules can be used as indicator molecules for detecting sub-levels or supra-levels of glucose in blood or urine, thus providing valuable information for diagnosing or monitoring such diseases-as diabetes and adrenal insufficiency. Medical/pharmaceutical production of glucose for human therapeutic application requires monitoring and control. [0046]
Uses for the present invention in agriculture include detecting levels of an analyte such as glucose in soybeans and other agricultural products. Glucose must be carefully monitored in critical harvest decisions for such high value products as wine grapes. As glucose is the most expensive carbon source and feedstock in fermentation processes, glucose monitoring for optimum reactor feed rate control is important in power alcohol production. Reactor mixing and control of glucose concentration also is critical to quality control during production of soft drinks and fermented beverages, which consumes the largest amounts of glucose and fermentable (cis-diol) sugars internationally. [0047]
When the indicator macromolecules incorporate fluorescent indicator substituents, various detection techniques also are known in the art that can make use of the macromolecules of the present invention. For example, the macromolecules of the invention can be used in fluorescent sensing devices (e.g., U.S. Pat. No. 5,517,313) or can be bound to polymeric material such as test paper for visual inspection. This latter technique would permit, for example, glucose measurement in a manner analogous to determining pH with a strip of litmus paper. The macromolecules described herein may also be utilized as simple reagents with standard benchtop analytical instrumentation such as spectrofluorometers or clinical analyzers as made by Shimadzu, Hitachi, Jasco, Beckman and others. These molecules would also provide analyte specific chemical/optical signal transduction for fiber optic-based sensors and analytical fluorometers as made by Ocean Optics (Dunedin, Florida), or Oriel Optics. [0048]
U.S. Pat. No. 5,517,313, the disclosure of which is incorporated herein by reference, describes a fluorescence sensing device in which the macromolecules of the present invention can be used to determine the presence or concentration of an analyte such as glucose or other cis-diol compound in a liquid medium. The sensing device comprises a layered array of a fluorescent indicator molecule-containing matrix (hereafter “fluorescent matrix”), a high-pass filter and a photodetector. In this device, a light source, preferably a light-emitting diode (“LED”), is located at least partially within the indicator material, or in a waveguide upon which the indicator matrix is disposed, such that incident light from the light source causes the indicator molecules to fluoresce. The high-pass filter allows emitted light to reach the photodetector, while filtering out scattered incident light from the light source. [0049]
The fluorescence of the indicator molecules employed in the device described in U.S. Pat. No. 5,517,313 is modulated, e.g., attenuated or enhanced, by the local presence of an analyte such as glucose or other cis-diol compound. [0050]
In the sensor described in U.S. Pat. No. 5,517,313, the material which contains the indicator molecule is permeable to the analyte. Thus, the analyte can diffuse into the material from the surrounding test medium, thereby affecting the fluorescence emitted by the indicator molecules. The light source, indicator molecule-containing material, high-pass filter and photodetector are configured such that at least a portion of the fluorescence emitted by the indicator molecules impacts the photodetector, generating an electrical signal which is indicative of the concentration of the analyte (e.g., glucose) in the surrounding medium. [0051]
In accordance with other possible embodiments for using the indicator macromolecules of the present invention, sensing devices also are described in U.S. Pat. Nos. 5,910,661, 5,917,605 and 5,894,351, all incorporated herein by reference. [0052]
The macromolecules of the present invention can also be used in an implantable device, for example to continuously monitor an analyte in vivo (such as blood glucose levels). Suitable devices are described in, for example, co-pending U.S. patent application Ser. No. 09/383,148 filed Aug. 26, 1999, as well as U.S. Pat. Nos. 5,833,603, 6,002,954 and 6,011,984, all incorporated herein by reference. [0053]
The macromolecules of the present invention have unique advantages when used in absorbance-based assays. For example, absorbance of a sample is directly proportional to both the concentration of the absorber and the sample path length. Thus, it is apparent that for a given level of absorbance, the sample path length may be greatly reduced if the absorber concentration is greatly increased. That desirable increase in concentration may be accomplished by decreasing the ratio of the hydrophilic monomer:indicator component monomer. In effect, the present invention allows the localized concentration of much more absorber component into a limited space, thereby increasing the absorbance per unit thickness. Thus the present invention additionally allows use of much smaller equipment when performing absorbance-based assays. [0054]
As a further aspect of the present invention, it has been discovered that certain macromolecules exhibit an excimer effect. By way of background, when two planar molecules with aromatic structure (such as is common with fluorophores) are concentrated to a point where their pi electron orbital lobes may overlap, a resonance condition can then occur for some species where the resonance from overlap results in a hybrid (couplet) structure which is energy favorable and stable. These two planar molecules become oriented in a coplanar configuration like two slices of bread on a sandwich with their electron clouds overlapping between them. For fluorescent planar species, a characteristic downfield emission occurs relative to the uncoupled species at wavelength of substantially lower energy than the parent species. Molecules able to form such favorable resonant configurations are known as excimers. As used herein, an excimer effect refers to the resulting characteristic longer wavelength emission from excimers. [0055]
Some examples of typical excimer-forming polyaromatic hydrocarbons include anthracene and pyrene. There are many others. An example is the anthracene derivative (boronate included), the indicator component used in Examples 1 and 2 of the present application. Although anthracene is known to form excimers in solution, one must be able to concentrate the molecule to sufficiently high levels to observe any excimer character. In the case of the anthracene derivative of Examples 1 and 2, the molecule is insoluble in water and insufficiently soluble in a solvent such as methanol to observe excimer characteristics. In the present examples, the relative concentration of the anthracene derivative monomer was increased in proportion to the hydrophilic monomer in the copolymer from 500:1, 400:1, 200:1, 100:1, 50:1, 25:1, 15:1 and then 5:1. All have the characteristic blue emission at 417 nm of the anthracene derivative except at 5:1 ratio, a green emission suddenly appears. This green emission is that of an excimer hybrid and the emission has been shifted downfield by approximately 100+nanometers (˜515-570 nm, green). The concentration of the overall solution does not need to be high since the distance between planar species is being controlled by placement along the polymer backbone rather than soluble concentration in 3-D space. [0056]
Surprisingly, it has been found that the excimer emission region is not responsive to changes in analyte concentration, but is responsive to all other aspects of the system analyzed, such as excitation intensity, temperature, and pH. As a result, the present indicator macromolecules may serve as both an indicator and an internal reference. For example, an ideal referencing scheme is one where the emission intensity at an indicator wavelength is divided optically using select bandpass filters, by the emission intensity at the excimer wavelength. The resultant value corrects for interfering factors which affect fluorescent emission properties, such as fluorescent quenching by, e.g., oxygen, drift and error in pH, power factors and drift affecting LED intensity, ambient temperature excursions, etc. [0057]
It will be readily appreciated that the macromolecules of the present invention which exhibit an excimer effect will be useful in both aqueous and non-aqueous environments. Consequently, those macromolecules, as well as the component monomers (excimer-forming and other monomer), may range from hydrophilic to hydrophobic, depending upon the desired application. [0058]
Also, when the excimer macromolecules of the present invention are used to detect the presence or concentration of an analyte, the macromolecule may be used directly in solution, or may be immobilized as described above. [0059]
The macromolecules of the present invention can be prepared by persons skilled in the art without an undue amount of experimentation using readily known reaction mechanisms and reagents, including reaction mechanisms which are consistent with the general procedures described below. [0060]

EXAMPLE 1

a) Preparation of 9-[(Methacryloylaminopropylamino) Methyl]anthracene [0061]
(A) One-Phase [0062]
To a suspension of N-(3-aminopropyl) methacrylamide hydrochloride (Polysciences, #21200) (11.82 g, 0.066 mole, 3.0 eq) and a trace of inhibitor DBMP (2,6-di-t-butyl -4-methylphenol) (10 mg) in chloroform (250 mL) stirring in an ice-water bath, diisopropylethylamine (25 mL, 18.55 g. 0.144 mole, 6.5 eq) was added dropwise in 20 minutes. The mixture was allowed to warm up to room temperature and cooled again in ice-water bath. A clear solution of 9-chloromethylanthracene (5.0 g, 0.022 mole) in chloroform (100 mL) was added dropwise over 1 hour. It was run at 25° C. for 1 hour, 50° C. for 12 hours and then 70° C. for 2 hours. [0063]
The mixture was washed with water (60 mL×4), and the aqueous layer was extracted with methylene chloride. The organic layers were combined, dried over Na[0064] ₂SO₄, separated, and the solvent was removed under reduced pressure at 40° C. The crude material was then chromatographed on silica gel with 2-5% methanol in methylene chloride to give 2.44 g (33.4%) of product as a solid. TLC (silica gel): R_f0.39 (MeOH/CH₂Cl₂={fraction (1/9)}), a single spot.
(B) Two-Phase [0065]
To a clear solution of N-(3-aminopropyl) methacrylamide hydrochloride (788 mg, 4.41 mmole, 10 eq) and a trace of inhibitor MEHQ (methylether hydroquinone) (2 mg) in a mixture of water (30 mL) and tetrahydrofuran (30 mL) stirring in an ice-water bath. A Na[0066] ₂CO₃/NaHCO₃buffer (66 mL, 0.2 M, pH 10) was added in 1 hour and a solution of 9-chloromethylanthracene (100 mg, 0.441 mmole) in chloroform (100 mL) was added in 3 hours. It was run at 25° C. for 7 hours and then 55° C. for 12 hours.
The organic layer was separated, washed with water (50 mL×4), and the aqueous layers were extracted with methylene chloride. The organic layers were combined, dried over Na[0067] ₂SO₄, separated, and the solvent was removed with reduced pressure at 45° C. The crude material (270 mg) was then chromatographed on silica gel with 10-20% methanol in methylene chloride to give 28.7 mg (19.6% of product as a solid TLC (silica gel): R_f0.77 (MeOH/CH₂Cl₂={fraction (3/7)}), a single spot.
b) Preparation of 9-[[N-methacryloylaminopropyl-N-(o- boronobenzyl)amino]methyllanthracene [0068]
To a solution of the product obtained in step a) above (2.440 g, 0.00734 mole) and a trace of inhibitor DBMP (10 mg) in chloroform (200 mL) stirring in an ice-water bath, DIEA (diisopropylethylamine) (2.846 g, 3.84 mL, 0.022 mole, 3.0 eq) was added by portions in 10 minutes, and then a solution of 2,2-dimethylpropane-1,3-diyl[o-(bromomethyl)phenyl]boronate (2.492 g, 0.00881 mole, 1.2 eq) in chloroform (15 mL) was added in 30 minutes. The reaction was run at room temperature for 20 hours. [0069]
The mixture was washed with water, separated and the aqueous layers were extracted with methylchloride. The organic layers were combined, dried over Na[0070] ₂SO₄, separated and the solvent was removed with reduced pressure at 25° C. The semi-solid (4.75 g) was then chromatographed on silica gel with 2-5% methanol in methylene chloride to give 2.50 g (76.3%) of product as a lightly yellow crystalline solid, mp 72-73° C., TLC (silica gel): R_f0.36 (MeOH/CH₂Cl₂={fraction (1/9)}). It is soluble in CH₂Cl₂, CHCl₃, THF, CH₃OH, and C₂H₅OH. Limited solubility in H₂O and ether.
c) Preparation of Water-Soluble-Copolymeric Solutions of MAPTAC and 9-[[N-methacryloylaminopropyl-N-(o- boronobenzvl)amino]methyl]anthracene [0071]
(50:1) solution: To a solution of the monomer (42.3 mg, 0.0908 mmole) in ethanol (100%, 1.5 mL), MAPTAC [3-(methacryloylamino)propyl]trimethylammonium chloride (2.0 mL, 1.0 g, 4.54 mmole, 50 eq) and an AIBN (azobisisobutyl nitrile) ethanolic solution (0.183 M, 0.2 mL) as radical initiator were added, a clear solution was obtained. It was saturated with nitrogen and then heated to 70° C. in 1 hour, and kept at 70° C. for 80 minutes, and a viscous liquid was obtained. [0072]
The liquid obtained was treated with water (26 mL) and filtered through a microfilter (0.45 um) to give a clear solution. After dialysis through a cellulose acetate membrane (MWCO 3500) with water 5 L×4), it was concentrated with polyethylene glycol (MW 20 K) to a clear solution (34.54 g). Concentration: 24.0 mg solid in 1.0 g solution, total solid 829 mg, yield 79.5%. [0073]
Similar procedures were applied to prepare copolymeric solutions of 500:1, 400:1, 200:1, 100:1, 50:1, 25:1, 15:1, and 5:1 molar ratios of hydrophilic monomer:indicator. [0074]
Glucose Modulation of 50:1 and 25:1 Co-polymers [0075]
The modulation of the fluorescence of the 50:1 and 25:1 indicator macromolecules by glucose solutions having various concentrations is shown below in Tables 1 and 2. Table 1 shows the results using two different concentrations (15 mg/ml and 25 mg/ml) of the 25:1 indicator macromolecule of this example with four different glucose concentrations. Table 2 shows the results using two different concentrations (10 mg/ml and 20 mg/ml) of the 50:1 indicator macromolecule of this example with four different glucose concentrations. In both Tables, I/Io is the ratio of the emitted intensities at 420 nm after and before exposure to glucose (365 nm excitation). [0076]

TABLE 1

I/Io for 15 mg/ml I/Io for 25 mg/ml

Glucose indicator indicator

concentration macromolecule macromolecule

(mM) (25:1) (25:1)

0 1.00 1.00

50 1.44 1.50

100 1.75 1.90

200 2.13 2.33
[0077]

TABLE 2

I/Io for 10 mg/ml I/Io for 20 mg/ml

Glucose indicator indicator

concentration macromolecule macromolecule

(mM) (50:1) (50:1)

0 1.00 1.00

50 1.40 1.48

100 1.70 1.79

200 2.04 2.22

EXAMPLE 2

This example demonstrates a surprising and useful eximer effect present in connection with the 5:1 indicator macromolecule prepared in Example 1. [0078]
FIG. 1 depicts the emission spectra of the 5:1 indicator macromolecule when exposed to three concentrations of glucose (0 mM, 30 mM and 60 mM) after excitement by light at 365 nm. Also shown in the shaded region of FIG. 1 is the emission of the non-excimer 25:1 indicator macromolecule from example 1. The excimer emission region shows an “isosbestic region” rather than an isosbestic point. It can be seen from FIG. 1 that the excimer emission region (the region where the 0 mM, 30 mM and 60 mM glucose lines overlap) is not responsive to changes in glucose concentration (just like an isosbestic point). The excimer emission region begins approximately 100 nm downfield from the peak responsive wavelength of the anthracene derivative modulation. Except for glucose, the excimer is responsive to all other aspects of the system, such as excitation intensity, temperature, and pH. Therefore, an ideal referencing scheme is one where the amplitude or signal value at 415 nm is divided electronically by the amplitude or signal value at 515 nm or another wavelength or range of wavelengths within the excimer emission region, and the resultant value will be corrected for drift and error in pH, power factors and drift affecting LED intensity, ambient temperature excursions, etc. That is demonstrated below. [0079]
Demonstration of Excitation Intensity, Temperature and pH Correction [0080]

The glucose modulation of the 5:1 indicator macromolecule was measured with three different glucose solutions (0 mM, 100 mM and 200 mM). The emission spectra were determined for each of the glucose solutions at three different spectrophotometer slit configurations for source and emitted light (1.5 being narrower and 3 being wider). The data are shown in Table 3. In the Table, the ratio of the emission intensity at 420 nm to the emission intensity at 550 nm is relatively independent of slit configuration.

TABLE 3


		I420/I550	I420/I550
Slit	I420/I550	100 mM	200 mM
Configuration
0 mM glucose	glucose	glucose

1.5/1.5	3.92	6.18	7.36
1.5/3	3.93	6.12	7.25
3/3	4.00	6.27	7.28

The temperature stability of the 5:1 excimer indicator macromolecule was determined. The ratio of the emissions at 420 nm and 550 nm for a 1 mg/ml solution of the 5:1 excimer indicator macromolecule exposed to 200 mM glucose (pH 7.5) was 7.57 at room temperature and 7.53 at approximately 60° C. [0082]
The pH stability of the 5:1 excimer indicator macromolecule was also determined. The ratio of the emissions at 420 nm and 550 nm for a 1 mg/ml solution of the 5:1 excimer indicator macromolecule at three different pH levels (6.5, 7.0 and 7.5) were determined (excitation light at 370 nm, slits 1.5,3), and are shown in Table 4. The full emission spectra are shown in FIG. 2. The variation over the range tested was statistically insignificant. [0083]

TABLE 4

I420/I550 pH 6.5 I420/I550 pH 7.0 I420/I550 pH 7.5

4.28 ± 0.18 4.60 ± 0.37 4.29 ± 0.19
It is believed that the stability of the excimer pi cloud exceeds that of non-excimer anthracene derivative, and, that the boronate recognition feature, which is able to perturb the pi cloud properties of the non-excimer, and thus make a good indicator, is not able to perturb the more stable excimer cloud and thus the excimer makes a very good reference indicator. The reference molecule is structurally unaltered from the read channel indicator. The polymer matrix may be the same, and in this example is in fact the same macromolecule. The recognition element is open and intact, but the inductive energy influence between recognition element and fluorophore center has been muted. [0084]
The foregoing is quite significant, because it can eliminate the need for separate physical and/or chemical environments between indicator and reference molecules. [0085]

EXAMPLE 3

The synthesis of a suitable lanthanide chelate indicator component monomer is depicted below. Compounds (1) and (2) are commercially available from Macrocyclics, Richardson, Tex. (compound (2) is known as p-NH[0086] ₂-Bz-DOTA) The end product (9) may be co-polymerized with one or more other monomers to form an indicator macromolecule.

Claims

What is claimed is:

1. A macromolecule which is capable of exhibiting an excimer effect, which comprises a copolymer of:

b) one or more other monomers;

such that the resulting macromolecule exhibits said excimer effect.

2. The macromolecule of claim 1, wherein the macromolecule is capable of detecting the presence or concentration of an analyte.

3. The macromolecule of claim 2, wherein

a) the excimer forming monomer individually is not sufficiently water soluble to permit its use in an aqueous environment for detecting the presence or concentration of said analyte; and

b) the other monomer is a hydrophilic monomer; such that the macromolecule is capable of detecting the presence or concentration of said analyte in an aqueous environment.

4. The macromolecule of claim 2, wherein the excimer effect does not substantially change in response to changes in the presence or concentration of the analyte.

5. The macromolecule of claim 4, wherein

i) the molar ratio of other monomer:excimer forming momomer is from about 2:1 to about 15:1,

ii) the excimer forming monomer comprises an N-(o-boronobenzyl)amino]methyl]anthracene derivative, and

iii) the other monomer comprises [3-(methacryloylamino)-propyl]trimethylammonium chloride.

6. The macromolecule of claim 1, wherein the excimer forming monomer is selected from the group consisting of a lanthanide chelate and a polyaromatic hydrocarbon.

7. A method for producing a macromolecule which is capable of exhibiting an excimer effect, which method comprises copolymerizing:

b) one or more other monomers;

such that the resulting macromolecule exhibits said excimer effect.

8. The method of claim 7, wherein the macromolecule is capable of detecting the presence or concentration of an analyte.

9. The method of claim 8, wherein

10. The method of claim 8, wherein the excimer effect does not substantially change in response to changes in the presence or concentration of the analyte.

11. The method of claim 10, wherein

12. The method of claim 7, wherein the excimer forming monomer is selected from the group consisting of a lanthanide chelate and a polyaromatic hydrocarbon.