阅读说明：本技术 用于光学葡萄糖测定物的经改性的葡聚糖 (Modified dextrans for optical glucose assays ) 是由 特里·T·丹格 瑟伦·奥斯莫 杰斯帕·斯文宁·克里斯滕森 约瑟夫·汉纳 罗伯特·麦金利 于 2018-04-27 设计创作，主要内容包括：本发明涉及竞争性葡萄糖结合亲和力测定物,该竞争性葡萄糖结合亲和力测定物包括用测定荧光团标记的葡萄糖受体(通常为甘露聚糖结合凝集素)和用参考荧光团标记的经改性的葡萄糖类似物(通常为葡聚糖)。在某些实施方案中,葡萄糖类似物是葡聚糖并且偶联到参考荧光团和猝灭剂染料(例如六甲氧基结晶紫-1)两者。任选地,参考荧光团相对于测定荧光团蓝移。(The present invention relates to a competitive glucose binding affinity assay comprising a glucose receptor (typically mannan-binding lectin) labeled with an assay fluorophore and a modified glucose analog (typically dextran) labeled with a reference fluorophore. In certain embodiments, the glucose analog is dextran and is coupled to both the reference fluorophore and a quencher dye (e.g., hexamethoxy crystal violet-1). Optionally, the reference fluorophore is blue-shifted relative to the assay fluorophore.)

1. A glucose sensing complex comprising:

a mannan binding ligand, wherein the mannan binding ligand is coupled to an assay fluorophore;

a reference fluorophore;

a dextran, wherein the dextran is used as a glucose analog in an assay and is coupled to:

the reference fluorophore;

an agent that enhances the hydrophilicity of the glucan; and

a quencher, wherein the assay fluorophore and the quencher form a Forster Resonance Energy Transfer (FRET) pair.

2. The glucose sensing complex of claim 1, wherein the agent that enhances the hydrophilicity of the dextran is the quencher.

3. The glucose sensing complex of claim 2, wherein the quencher is hexamethoxy crystal violet-1 (HMCV 1).

4. The glucose sensing complex of claim 1, wherein the reference fluorophore is Alexa Fluor 647(AF647) or Alexa Fluor 700(AF 700).

5. The glucose sensing complex of claim 1, wherein the fluorophore and/or quencher dye is water-soluble.

6. The glucose sensing complex of claim 1, wherein the dextran is modified with an anhydride compound.

7. The glucose sensing complex of claim 1, wherein the dextran comprises less than 1500 glucose units.

8. The glucose sensing complex of claim 1, wherein the glucose sensing assay complex exhibits a sensor Dose Response (DR) loss of less than 2.5% per day.

9. The glucose-sensing complex of claim 1, wherein the fluorophore has a degree of labeling (DOL) of 4.1 and the quencher dye has a DOL of 5.7.

10. A competitive glucose binding affinity assay comprising:

a glucose receptor labeled with an assay fluorophore; and

a glucose analog labeled with both a reference fluorophore and a quencher dye.

11. The assay of claim 10, wherein:

the glucose receptor is selected from the group consisting of: mannan-binding lectin (MBL), concanavalin a, glucose galactose-binding protein, antibodies, and boronic acid;

the glucose analog is dextran;

the assay fluorophore and the reference fluorophore are Alexa Fluor; and is

The quencher dye is hexamethoxy crystal violet-1 (HMCV 1).

12. The assay of claim 11, wherein the glucose receptor is mannan-binding lectin.

13. The assay of claim 11 wherein the reference fluorophore is Alexa Fluor 647(AF647) or Alexa Fluor 700(AF 700).

14. The assay of claim 11 wherein the dextran is conjugated to a compound that enhances its hydrophilicity.

15. A method of sensing glucose in a solution, comprising:

(a) contacting the solution with a glucose sensing complex comprising:

a mannan binding ligand, wherein the mannan binding ligand is coupled to an assay fluorophore;

a reference fluorophore;

a dextran, wherein the dextran is used as a glucose analog in an assay and is coupled to:

the reference fluorophore; and

an agent that enhances the hydrophilicity of the glucan; and

a quencher, wherein the assay fluorophore and the quencher form a Forster Resonance Energy Transfer (FRET) pair;

(b) observing a signal of the glucose-sensing complex indicative of the presence of glucose; and

(c) the observed signal is correlated with the concentration of glucose.

16. The method of claim 15, wherein the solution comprises interstitial fluid or blood.

17. The method of claim 15, wherein the glucose-sensing complex is disposed in vivo.

18. The method of claim 15, wherein the agent that enhances the hydrophilicity of the dextran is the quencher.

19. The method of claim 15, wherein the quencher is hexamethoxy crystal violet-1 (HMCV 1).

20. The method of claim 15, wherein the dextran is modified with an agent selected to enhance its hydrophilicity.

Technical Field

The present invention relates to optical analyte assays, and in particular to fluorescent competitive binding assays for sensing glucose.

Background

Maintaining normal glucose levels in the body is a key way for diabetics to avoid the long-term problems associated with diabetes, such as retinopathy, circulatory problems and other sequelae. For this purpose, diabetic patients regularly monitor their blood glucose levels, for example to optimize insulin administration. In this context, various systems and methods for monitoring blood glucose levels have been developed. One strategy uses fluorescent compounds to detect glucose levels, for example, by competitive binding assays in which glucose and fluorophore-labeled glucose ligands/analogs compete for the binding site of the glucose receptor and the resulting change in fluorescence is converted to glucose concentration.

In certain competitive glucose binding assays, dextran is used as the displaceable glucose ligand. In such assays, dextran can be labeled with a lipophilic (and cationic) dye such as hexamethoxy crystal violet-1 (HMCV 1). However, the presence of a large number (e.g. more than 10) of lipophilic dye molecules coupled to the flexible poly- (1,6) -glucose backbone of dextran may result in such labeled dextran molecules being in a less water soluble conformation, which may result in precipitation of these molecules. Furthermore, such dyesThe induced conformational change may drive the labeled glucan to a more lipophilic state, which may lead to undesirable changes in the glucan's ability to bind to the glucose receptor and affect its Forster resonance energy transfer(s) ((Resonance Energy Transfer, FRET) efficiency. Furthermore, in conventional systems, the dye may be shielded intramolecularly on the dextran, a phenomenon that can cause the calibration of the assay to change over time, thereby introducing instability in the assay.

Reference dyes are also used in certain fluorescence assays to track changes in the experimental setup, such as light source fluctuations, changes in the light path (coupling light into the light guide), mechanical perturbations (e.g., bending), temperature changes, and the like. Traditionally, optical or fluorescence-based sensing systems have utilized a reference fluorophore that is red-shifted relative to the assay fluorophore. However, by exciting the fluorophore with a lower wavelength of light having more energy than required, the risk is increased that an electron in the fluorescent molecule will transition from the ground electronic state (S0) to the second excited state (S2) instead of the first excited electronic state (S1). The molecule at S2 is much more likely to break down than the same molecule at S1. Thus, if the fluorophore is excited to S2 instead of just S1, faster photobleaching will occur.

Accordingly, there is a need in the art for optical glucose assays that utilize reagents and materials selected to improve assay stability. The invention disclosed herein meets this need, for example, by using assays designed to include multiply labeled glucose analogs/ligands (e.g., dextran conjugated/labeled with reagents that promote hydrophilicity) and/or blue-shifted reference fluorophores. As discussed below, glucose assays designed to include multiple labeled glucose analogs and/or blue-shifted reference fluorophores exhibit improved material properties such as assay stability.

Disclosure of Invention

The present invention provides optimized materials and methods for glucose assays. As discussed in detail below, the components in certain fluorescent glucose assays may be selected and/or modified, for example, to optimize the hydrophilic-hydrophobic balance of the population of components used to form the assay complex. In doing so, improved assays are produced in which undesirable conformational changes in the assay components are less likely to occur over time. Exemplary modifications of the glucose assay complex include those that modify the molecule by coupling the dextran to an agent selected to prevent the dextran from becoming unduly negatively or positively charged. Such modifications result in assays that are more stable than their unmodified equivalents. In addition, in embodiments where the reference fluorophore is selected for hydrophilic/hydrophobic characteristics and/or is blue-shifted relative to the assay fluorophore or indicator fluorophore (rather than the typical red-shifted reference fluorophore which may become photolabile due to low wavelength excitation), the overall stability of the reference fluorophore and the assay complex may be improved.

Embodiments of the invention include competitive glucose binding affinity assays, and methods of making and using these assays. Typically, the assay comprises a glucose receptor labeled with an assay fluorophore and a glucose analog labeled with both a reference fluorophore and a quencher dye. In an embodiment of the invention, the glucose receptor may be selected from the group consisting of: mannan-binding lectin (MBL), concanavalin a, glucose galactose-binding protein, antibodies, and boronic acid. In typical embodiments, the glucose receptor is a mannan-binding lectin. In an exemplary embodiment, the glucose analog is dextran, and the assay fluorophore and the reference fluorophore are Alexa Fluors^TMAnd the quencher dye is hexamethoxy crystal violet-1 (HMCV 1). In some embodiments, the assay fluorophore and the reference fluorophore are each selected from the group consisting of: alexa Fluor^TM647(AF647) and Alexa Fluor^TM700(AF700), and the assay fluorophore and the reference fluorophore are different. Typically, in such embodiments, one or more components of the complex (e.g., dextran) are coupled to or labeled with an agent selected to have a charge and/or hydrophilic/hydrophobic characterThe hydrophobic/sexual characteristics contribute to the stability of the agent within the complex and thus to the overall stability of the glucose sensing complex. Embodiments of the invention also include methods of making and using these improved glucose assays.

In other embodiments of the invention, the assay comprises a glucose receptor labeled with an assay fluorophore and a glucose analog labeled with a reference fluorophore, wherein the reference fluorophore is blue-shifted relative to the assay fluorophore. In some embodiments, the glucose analog is also labeled with a quencher dye (e.g., hexamethoxy crystal violet-1, HMCV 1). The glucose receptor may be selected from the group consisting of: mannan-binding lectin (MBL), concanavalin a, glucose galactose-binding protein, antibodies, and boronic acid. In one example, the glucose receptor is a mannan-binding lectin. Typically, the glucose analog is dextran, and the assay fluorophore and the reference fluorophore are Alexa Fluors^TM. Typically, the fluorophore and quencher dye form a Forster Resonance Energy Transfer (FRET) pair. The fluorophore and/or quencher dye is also typically water soluble. In a typical embodiment, the assay fluorophore and the reference fluorophore are each selected from the group consisting of: alexa Fluor 594(AF594), Alexa Fluor 647(AF647) and Alexa Fluor 700(AF700), where the reference fluorophore has a shorter wavelength than the assay fluorophore.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating some embodiments of the invention, are given by way of illustration and not limitation. Many changes and modifications may be made within the scope of the present invention without departing from the spirit thereof, and the invention includes all such modifications.

Drawings

Referring now to the drawings in which like reference numbers represent corresponding parts throughout:

FIGS. 1A-C illustrate having fluorescence in accordance with one or more embodiments of the inventionPrecisense of ligand^TMOptical sensor (Precisense)^TMOptical). Fig. 1A shows the change of the quencher from dye to fluorophore and the reference fluorophore is omitted. Fig. 1B is a graph showing changes in absorbance and emission spectrum. Fig. 1C is a graph showing glucose concentration measurements and sensor dose response loss (DR loss).

FIGS. 2A-C illustrate Precisense with a combination ligand according to one or more embodiments of the invention^TMAn optical sensor. FIG. 2A shows a reference fluorophore labeled onto the same ligand as a dye quencher, eliminating the need for a reference carrier. Fig. 2B is a graph showing changes in absorbance and emission spectrum. Fig. 2C is a graph showing glucose concentration measurements and sensor dose response loss (DR loss).

FIG. 3 is a graph showing ORS SITS data for an AF594-MBL/AF647-dex assay according to one or more embodiments of the invention.

FIG. 4 is a graph showing ORS SITS data for an AF594-MBL/AF647-HMCV1-dex assay in accordance with one or more embodiments of the invention. This assay was used in rat experiments 76 and 77 as shown in FIGS. 5-7 below.

Fig. 5 is a graph showing results from a rat study 76 according to one or more embodiments of the present invention: graph of data for rat #1, ORS sensor # 2. The sensor is calibrated during a low-high transition, the first jaw.

Fig. 6A-D show images from a rat study 76 according to one or more embodiments of the invention: data for rat #1, ORS sensor #2 corresponding to jaw 1 (fig. 6A), jaw 2 (fig. 6B), and jaw 3 (fig. 6C). Fig. 6D is a picture of explants.

Figures 7A-B show data from a rat study 77 according to one or more embodiments of the invention. Figure 7A shows baselines for the assay fluorophore and the reference fluorophore. Note the stable assay baseline and the reference baseline. Figure 7B shows glucose concentration measurements from rat study 77.

FIG. 8 is a graph showing data from an AF594/AF647 assay in accordance with one or more embodiments of the invention.

FIG. 9 is a graph showing assays from AF594MBL/AF647-Dex and AF594/AF647-HMCV1-Dex according to one or more embodiments of the invention: graph of data for the optimal ratio between AF594 and AF647 fluorescence.

FIG. 10 is a graph illustrating data from filter configurations for AF594-MBL/647-Dex and AF594-MBL/AF647-HMCV1-Dex assays according to one or more embodiments of the invention.

FIG. 11 shows a filter configuration for an AF594/647 assay in accordance with one or more embodiments of the invention.

FIGS. 12A-C provide examples of a degree of labeling (DOL) assay for multi-labeled dextran (MLD) according to one or more embodiments of the invention. Fig. 12A is a graph showing normalized absorbance spectra for HMCV1 and AF 647. Fig. 12B is a graph showing the absorbance spectra of conjugates #478(HMCV 1X 5 and AF 647X 15) and #479(HMCV 1X 15 and AF 647X 5). Fig. 12C is a graph showing the linear combination of the conjugate 479(HMCV 1X 15 and AF 647X 5) spectra and the HMCV1 and AF647 absorbance spectra. DOL of MLD conjugates carrying HMCV1 and AF647 include: DOL #472(X10/X10) ═ 5.7/4.1; DOL #478(X5/X15) ═ 3.6/9.4; and DOL #479(X15/X5) 8.1/2.1.

Figure 13 is a graph illustrating single labeled dextran performance according to one or more embodiments of the present invention. The graph shows the Dose Response (DR) development for five groups of sensors, all constructed using singly labeled dextran in the glucose response assay. DR was calculated as the difference between the normalized intensity at 40mg/dL glucose and 40mg/dL glucose, relative to the normalized intensity at 400mg/dL glucose. The loss of DR relative to the initial DR is between 3% and 6% per day. Dextran labeled with HMCV1 showed only a large Dose Response (DR) loss.

Fig. 14 is a graph showing the performance of multi-labeled dextran (HMCV1-AF 647-dextran) according to one or more embodiments of the present invention.

Fig. 15 is a graph showing the performance of multi-labeled dextran (HMCV1-AF 700-dextran) according to one or more embodiments of the invention.

FIG. 16 is a graph showing the performance of multi-labeled dextran (HMCV1-HMCV 3-dextran) according to one or more embodiments of the present invention.

Fig. 17 is a graph showing the performance of multi-labeled dextran (HMCV 1-dextran-succinylation) according to one or more embodiments of the invention.

Fig. 18 is a graph illustrating normalized absorption and emission spectra of different dyes and fluorophores in a capsule sensor according to one or more embodiments of the present invention.

Fig. 19 is a table illustrating combinatorial ligands with different DOL values, their DR and intensity levels in capsule sensors according to one or more embodiments of the invention.

Detailed Description

Unless otherwise defined, all terms of art, notations and other scientific words or terms used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention belongs. In some instances, terms having commonly understood meanings are defined herein for clarity and/or for ease of reference, and such definitions included herein should not be construed to represent a substantial difference over what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood and often employed by those skilled in the art using conventional methods. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. In the description of the exemplary embodiments, reference is made to the accompanying drawings, which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. It is to be understood that other embodiments may be utilized and structural changes may be made without departing from the scope of the present invention.

Blood glucose is typically monitored by diabetics through the use of commercially available calorimetric test strips or electrochemical biosensors (e.g., enzyme electrodes), both of which require periodic use of a lancet-type instrument to draw an appropriate amount of blood each time a measurement is taken. This places a considerable burden on the diabetic in terms of economy and in terms of pain and discomfort, especially in long-term diabetics, who must use blood lancets to draw blood from the fingertips on a regular basis. Accordingly, many proposals have been made regarding glucose measurement techniques that do not require blood to be drawn from the patient. It has been observed that the concentration of analytes in subcutaneous body fluids correlates with the concentration of analytes in blood, and thus there have been several reports on the use of glucose monitoring devices located at subcutaneous locations. The use of competitive binding assays for glucose that can be interrogated remotely is of particular interest.

A typical method of measuring competitive binding is to use a proximity-based signal-generating moiety/signal-modulating moiety pair (see, e.g., U.S. patent No. 6,232,120), which is typically an energy transfer donor-acceptor pair (comprising an energy donor moiety and an energy acceptor moiety). The energy donor moiety is photoluminescent (typically fluorescent). In such methods, an energy transfer donor-acceptor pair is contacted with a sample to be analyzed, such as a subcutaneous body fluid. The sample is then irradiated and the resulting emission detected. The energy donor or energy acceptor moiety of the donor-acceptor pair binds to the acceptor support, while the other moiety of the donor-acceptor pair (bound to the ligand support) competes with any analyte present for binding sites on the acceptor support. When the donor and acceptor are brought together, energy transfer occurs between the donor and acceptor, which produces a detectable lifetime change (decrease) in the fluorescence of the energy donor moiety. In addition, a proportion of the fluorescent signal emitted by the energy donor moiety is quenched. Lifetime variation is reduced or even eliminated by competitive binding to the analyte. Thus, the amount of analyte in a sample can be determined by measuring the apparent luminescence lifetime, for example by phase modulation fluorometry or time-resolved fluorometry (see Lakowicz, Principles of fluorescence Spectroscopy), Plenum Press,1983, Chapter 3.

In addition to the lifetime of the excited state, the intensity of the emitted fluorescence is also related to the glucose concentration. Harmonizing longevityThe measurements of the emission fluorescence are in contrast influenced by the intensity of the light source and the coupling between the assay and the optical system. Therefore, intensity measurement requires the incorporation of an internal reference fluorophore into the assay. The reference fluorophore must be different from the assay fluorophore in such a way that the emitted fluorescence from the assay and the emitted fluorescence from the reference can be separated from each other, for example by having different absorption or emission spectra. The reference fluorophore may be, for example, Alexa Fluor labeled onto Human Serum Albumin (HSA) or another macromolecule that does not substantially bind to the glucose acceptor^TM 594(AF594)。Alexa Fluor^TM700(AF700) may be excited simultaneously with AF594 because their absorption spectra spectrally overlap. The emission spectrum of AF594 is slightly blue-shifted with respect to AF700, which makes it possible to detect their respective fluorescence emissions in separate wavelength regions. Any change in light source intensity will equally scale the fluorescence from AF594 and AF700 when they are excited by the same light source at the same time. Thus, any effects from the variation in the intensity of the light source can be counteracted. Excitation and detection of the emitted fluorescence of the assay and reference follow the same optical path from the optical system to the assay. Thus, the signal detected from the reference is used as a measure of the optical coupling between the optical interrogation system and the assay. Any effects from changes in optical coupling (e.g., collimation) can be cancelled out.

A special fluorescent property known as Forster Resonance Energy Transfer (FRET) occurs when the energy of the excited electron of one fluorophore (i.e., the donor) is transferred to a nearby acceptor dye, i.e., a quencher (non-emitting chromophore) or another fluorophore with an excitation spectrum that overlaps with the emission spectrum of the donor. Energy transfer occurs in the absence of photons and is the result of a long-range dipole-dipole interaction between the donor and acceptor. An important feature of FRET is that it occurs at distances comparable to the size of biological macromolecules. The distance at which FRET efficiency is 50% (known as the Foster distance) is typically atWithin the range of (1). Ranging from 20 toThe Foster distance of (A) facilitates competitive binding studies. WO91/09312 describes subcutaneous methods and devices employing a glucose-based affinity assay (incorporating an energy transfer donor-acceptor pair) that is remotely interrogated by optical means. Examples WO1997/19188, WO2000/02048, WO2003/006992 and WO2002/30275 each describe glucose sensing by energy transfer which generates an optical signal that can be read remotely.

The invention disclosed herein relates generally to optical or fluorescence-based assays and analyte sensing compositions. In exemplary embodiments of the invention, the assays, compositions, systems and methods of the invention are described with reference to glucose as the analyte whose level/concentration is to be determined. However, this is merely illustrative and not restrictive, as the principles, devices, systems, and methods of the present invention may be used to sense and/or determine the levels of various other physiological parameters, agents, characteristics, and/or compositions.

As described herein, embodiments of the present invention provide sensors that are designed to include a composition disposed in a specific area of the sensor in order to provide the sensor with enhanced functionality and/or material properties. The present disclosure also provides methods of making and using such sensors. Embodiments of the invention described herein, such as those discussed in the immediately following paragraphs, may be adapted to and implemented with a variety of components in a sensor having a sensing complex that produces an optical signal that can be correlated with the concentration of an analyte, such as glucose. Many such sensors and compositions are disclosed, for example, in U.S. patent nos. 6,6761,527, 7,228,159, 7,884,338, 7,567,347, 8,305,580 and 8,691,517, and U.S. patent application publication nos. 2008/0188723, 2009/0221891, 2009/018708, 2009/0131773, 2013/0060105 and 2014/0200336, the contents of each of which are incorporated herein by reference.

The invention disclosed herein has many embodiments. One exemplary embodiment is a glucose-sensing complex comprising a reference fluorophore, a mannan-binding ligand (e.g., Alexa Fluor 647(AF647) or Alexa Fluor 700(AF700)) coupled to an assay fluorophore; dextran used as a glucose analog in the assay and coupled to a reference fluorophore, an agent that enhances dextran hydrophilicity, and a quencher (e.g., hexamethoxy crystal violet-1 (HMCV 1)). In this embodiment, the assay fluorophore and quencher form a Forster Resonance Energy Transfer (FRET) pair. Typically, the fluorophore and/or quencher dye is water soluble. Optionally, the fluorophore has a degree of labeling (DOL) of at least 4.1 and the quencher dye has a DOL of at least 5.7. In some embodiments of the invention, the agent that enhances the hydrophilicity of dextran is a quencher. Optionally, the dextran is modified with an anhydride compound (e.g., to couple the dextran to a moiety that modulates the hydrophilicity of the dextran). Typically, glucans comprise less than 1500 glucose units. In some embodiments of the invention, the glucose sensing assay complex exhibits a sensor Dose Response (DR) loss of less than 2.5% per day.

Embodiments of the invention may include a competitive glucose binding affinity assay comprising a glucose receptor labeled with an assay fluorophore, and a glucose analog labeled with both a reference fluorophore (e.g., Alexa Fluor 647(AF647) or Alexa Fluor 700(AF700)) and a quencher dye. In certain embodiments of the invention, the glucose receptor is selected from the group consisting of: mannan-binding lectin (MBL), concanavalin a, glucose galactose-binding protein, antibody, and boronic acid, the glucose analog is dextran, the assay fluorophore and the reference fluorophore are alexafluors, and the quencher dye is a hexamethoxy crystal violet compound. In typical embodiments, the dextran is coupled to or modified by a compound that enhances its hydrophilicity.

Another embodiment of a method of sensing glucose in a solution (e.g., interstitial fluid or blood) includes contacting the solution with a glucose-sensing complex comprising a reference fluorophore, a mannan binding ligand coupled to the assay fluorophore, a glucan selected to function as a glucose analog in the assay and further coupled to the reference fluorophore. The complex also includes an agent that enhances the hydrophilicity of the dextran, and a quenching agent. Optionally, the agent that enhances the hydrophilicity of the glucan is a quencher. In this embodiment, the assay fluorophore and quencher form a Forster Resonance Energy Transfer (FRET) pair. The method includes observing a signal of the glucose-sensing complex indicative of the presence of glucose and then correlating the observed signal with a glucose concentration.

Yet another embodiment of the invention is a glucose sensing complex comprising a glucose binding agent (e.g., mannan-binding lectin) coupled to an assay fluorophore and a glucose analog coupled to a reference fluorophore. In this embodiment, the reference fluorophore selected for the complex is blue-shifted relative to the assay fluorophore. Typically, the reference fluorophore has a lower wavelength than the assay fluorophore. Optionally, the assay fluorophore exhibits a wavelength at least 50 nanometers greater than the wavelength of the reference fluorophore. Optionally, the glucose analog is further coupled to a quencher (e.g., a hexamethoxy crystal violet compound). In certain embodiments, the glucose receptor is selected from the group consisting of: mannan-binding lectin (MBL), concanavalin a, glucose galactose-binding protein, antibodies, and boronic acid; and/or the glucose analog is dextran (e.g., dextran having a molecular weight between 90kDa and 110 kDa). In certain embodiments, the assay fluorophore and the reference fluorophore are each selected from the group consisting of: alexa Fluor 594(AF594), Alexa Fluor 647(AF647), and Alexa Fluor 700(AF 700). Typically, the reference fluorophore and the quencher are water soluble, forming a Forster Resonance Energy Transfer (FRET) pair.

Another embodiment of the invention is a method of making a glucose-sensing complex comprising forming a glucose-sensing complex by coupling a glucose-binding agent (e.g., mannan-binding lectin (MBL)) to an assay fluorophore and coupling a glucose analog (e.g., dextran) to a reference fluorophore. In this embodiment, the reference fluorophore and the assay fluorophore are selected such that the reference fluorophore emits light at a wavelength that is blue-shifted relative to the light emitted by the assay fluorophore. Typically, the assay fluorophore exhibits a wavelength at least 50 nanometers greater than the wavelength of the reference fluorophore. Typically, the assay fluorophore exhibits a wavelength of no more than 100 nanometers compared to the wavelength of the reference fluorophore. In some cases, the glucose analog is further treated with succinic anhydride. Typically, the dextran is about 100 kDa. In certain embodiments, the composition exhibits a sensor Dose Response (DR) loss of less than 2.5% per day. In one example, the fluorophore has a degree of labeling (DOL) of at least 4.1 and the quencher dye has a DOL of at least 5.7.

Another embodiment of the invention is a method of sensing glucose in a solution (e.g., interstitial fluid or blood) comprising contacting the solution with a glucose-sensing complex having a glucose-binding agent coupled to an assay fluorophore and a glucose analog coupled to a reference fluorophore, wherein the reference fluorophore is selected to be blue-shifted relative to the assay fluorophore. The method includes observing a signal of the glucose-sensing complex indicative of the presence of glucose and correlating the observed signal with a glucose concentration. Optionally, the method comprises exciting the reference fluorophore and the assay fluorophore with two different light sources. Typically, the assay fluorophore exhibits a wavelength of no more than 100 nanometers compared to the wavelength of the reference fluorophore, and the assay fluorophore exhibits a wavelength at least 50 nanometers greater than the wavelength of the reference fluorophore.

There are many variations of the present invention. As described herein, the analyte receptor is typically a lectin, which includes any carbohydrate-binding protein. In typical embodiments, the glucose receptor is a fluorophore-labeled mannan-binding lectin (MBL, also known as mannose/mannan binding protein, Sheriff et al, Structural Biology,1: 789-. Typically, the lectin provides a stable signal in the assay for at least 10 days, more typically for at least 14 days. It is particularly preferred that the sensor provides a stable signal when implanted in the human body. Surprisingly, MBL has been found to be stable in glucose assays for at least 17 days.

Other analyte receptors or analyte binding moieties may also be used in the assays and sensor systems described herein. For example, The analyte receptor may be ｃA human lectin derived from The human body, including human lung surfactant protein A (SP-A, Allen et al, Infection and Immunity,67: 4563-. Alternatively, the lectin may be a recombinantly produced lectin or a humanized animal lectin, such as a humanized bovine lectin. The lectin may alternatively be an animal lectin, bird lectin, fish lectin, vertebrate lectin, invertebrate lectin (e.g. insect lectin) or plant lectin. Suitable animal lectins include coaggrectin, collectin-43 (e.g., bovine CL-43), lung surfactant protein (lung collectin), PC-lectin (US2003/0216300, US 2004/0265898), CTL-1(US 2010/179528), keratinocyte membrane lectin (Parfuemerie und Kosmetik 74,164-80), CD94(Eur J Immunol 25,2433-7), p35 (synonym: human L-Figelin, group of lectins) (Immunol Lett 67,109-12), ERGIC-53 (synonym: MR60) (Mol Biol Cell,7,483-93), HIP/PAP (Eur J Biochem 267,1665-71), CLECSF8(Eur JImmunel 34,210-20), DCL (group of lectins) (application No. 00231996/US) and GLUT family proteins, in particular GLUT1, GLUT4 and GLUT11(PNAS 97,1125-30). Additional suitable animal Lectins are shown in appendices A, B and C of Handbook of animal proteins: Properties and biological Applications, David C.Kilparick, Wiley 2000. Suitable phytohemagglutinin or Phytohemagglutinin (PHA) include concanavalin a and those derived from pea (pisum sativum), sweet pea (lathyrus odoratus), lentil (lentil), narcissus pseudonarcissus (narcissus pseudonarcissus), broad bean (vicia faba) and wild pea (vicia sativa). The analyte receptor may also be a periplasmic glucose/galactose binding receptor, an antibody raised against a glucose-like molecule, or a boronic acid.

As described herein, an analyte analog can include multiple carbohydrate moieties or carbohydrate mimetic moieties that bind to a binding site of an analyte receptor. The analyte analog should have a molecular weight high enough to prevent escape from the sensor, but low enough so that precipitation does not occur when the analyte analog binds to the analyte receptor. The analyte analog may have a weight in the range of 25 to 250kDa, more typically between 90 to 120 kDa. In typical embodiments where glucose is the analyte, dextran is used as the replaceable glucose analog/ligand. Dextran is a flexible macromolecule consisting of up to 1500 glucose units. In some cases, dextran consists of approximately 600 glucose units (-100 kDa), or 500-700 glucose units.

Other analyte analogs and ligands can also be used in the exemplary assays and sensor systems described herein. The analyte analog may be a synthetic polymer comprising different carbohydrate moieties or carbohydrate mimetic moieties with different affinities for MBL and similar lectins. Alternatively, the analyte analogue may be a carbohydrate-protein conjugate or a carbohydrate-dendrimer conjugate. Examples of suitable carbohydrates for such conjugates are mono-and oligosaccharides. Suitable monosaccharides are optionally derivatized tetrasaccharides, pentoses, hexoses, heptoses or higher homologous aldoses or ketoses, for example optionally derivatized D-glucose, D-mannose, N-acetyl-D-glucosamine, L-fucose, D-fructose, D-tagatose or D-sorbitol. Suitable oligomers may be linear or branched homo-or mixed oligomers, for example containing from 2 to 50 carbohydrate units.

As described herein, a fluorophore or fluorochrome is a chemical compound capable of absorbing light energy of a particular wavelength and re-emitting light energy at a longer wavelength. Fluorophores can also be used to quench the fluorescence of other fluorescent dyes or to transmit their fluorescence at even longer wavelengths (FRET). Usually, Alexa Fluor^TM(AF)594, 647 and/or 700 are used as reference and assay fluorophores for labeling glucose analogs and glucose receptors, respectively. It will be appreciated by those skilled in the art that other fluorophores suitable for optical glucose assays may also be used, such as coumarins, rhodamines, xanthenes, cyanines, and others that cover other excitation and emission wavelengthsAlexa Fluor (e.g., AF350, AF405, AF488, AF532, AF546, AF555, AF568, AF594, AF680, AF 750).

Energy acceptors that do not emit fluorescence are referred to as quenching moieties. The HMCV dyes described in WO05/059037 are suitable energy acceptor moieties for use in the present invention. These dyes are stabilized carboxycarbenium ions. In a typical embodiment, hexamethoxy crystal violet-1 (HMCV1) is used as the quencher/acceptor dye. Alternatively, QSY 21 may function as an energy acceptor moiety, while AF594 functions as an energy donor moiety.

The bound assay that produces the optical signal should generally be reversible so that continuous monitoring of the level of analyte fluctuations can be achieved. This reversibility is a particular advantage of using binding assay formats that do not consume components of the assay. Typically, the sensor is adapted to detect or measure glucose in a body fluid (e.g. subcutaneous body fluid). It is desirable that the sensor is suitable for use in vivo. Typically, the assay is capable of measuring blood glucose concentrations in at least a portion of the range of 0 to 35mM glucose, typically in the range of 2 to 10mM glucose. Suitable detection techniques include FRET, fluorescence energy transfer, fluorescence polarization, fluorescence quenching, phosphorescence, luminescence enhancement, luminescence quenching, diffraction, or plasmon resonance. Typically, the sensors of the present invention incorporate an assay that uses FRET techniques to generate an optical readout.

As discussed above, there is a need in the art for optical or fluorescence-based assays with enhanced stability and that require lower optical sensor calibration frequencies. In one aspect of the invention, an analyte sensing composition having significantly improved stability and solubility is provided. The analyte sensing composition includes an analyte analog labeled with both a fluorophore and a quencher dye. In typical embodiments, the analyte sensing composition is a glucose sensing composition that includes a fluorophore (e.g., Alexa Fluor)^TM 647、Alexa Fluor^TM700) And a quencher dye (e.g., hexamethoxy crystal violet-1, HMCV1) labeled multiple labeled glucose analogs (e.g., dextran).

In another aspect of the invention, methods based on the analysis are providedA competitive analyte binding affinity assay of the substance sensing composition. The competitive analyte binding affinity assay includes an analyte receptor labeled with an assay fluorophore and an analyte analog labeled with both a reference fluorophore and a quencher dye. In some cases, the reference fluorophore is blue-shifted relative to the assay fluorophore or indicator fluorophore, which improves the stability of the assay, and more particularly the reference fluorophore. In typical embodiments, the competitive analyte binding affinity assay is a competitive glucose binding affinity assay that includes the use of an assay fluorophore (Alexa Fluor)^TM647, AF647) and a glucose receptor/lectin (e.g., mannan-binding lectin, MBL) labeled with a reference fluorophore (e.g., Alexa Fluor)^TM594, AF594) and a quencher dye (e.g., HMCV1) labeled multi-labeled glucose analogs (e.g., dextran).

In embodiments of the invention, the binding between MBL and glucose-like molecules (e.g. dextran) is reversible. In the absence of glucose, MBL and dextran will primarily bind together. When glucose is added to the assay, the glucose will compete with a portion of the dextran population, causing the assay to enter a new equilibrium state. This equilibrium state always corresponds to the glucose concentration. To determine this equilibrium state, MBL was labeled with a fluorophore (e.g., AF647, AF700) and dextran was multiply labeled with a quencher dye (e.g., HMCV1) and a reference fluorophore (e.g., AF 594). The donor assay fluorophore and the acceptor quencher dye together form a Forster Resonance Energy Transfer (FRET) pair, i.e., the emission spectrum of the assay fluorophore and the absorption spectrum of the quencher dye overlap. It should be noted that fluorophores and dyes are generally water soluble, as they will function in an aqueous environment.

Multilabeled analyte analogs

Embodiments of the invention include glucose assay complexes that include dextran as the glucose analog. Dextran is a flexible macromolecule composed of up to 1500 glucose units. In some typical cases, dextran consists of approximately 600 glucose units (-100 kDa). Dextran can be used as a replaceable analyte analog/ligand in a mannose-binding lectin (MBL) based glucose response competitive optical assay. To function in a Forster Resonance Energy Transfer (FRET) assay, dextran is typically (heavily) labeled with a lipophilic (and cationic) dye such as hexamethoxy crystal violet-1 (HMCV 1). The presence of a large number (greater than 10) of lipophilic dyes on the flexible poly- (1,6) -glucose backbone of dextran can cause the HMCV 1-labeled dextran to fold into a less soluble conformation and thus precipitate. Dye-induced conformational changes turn the HMCV 1-labeled dextran into a more lipophilic state, which causes various problems, including adverse changes in the dextran's ability to bind to the glucose receptor and affecting its Forster Resonance Energy Transfer (FRET) efficiency. Since the dye is molecularly shielded on the dextran, an internal filtering effect also occurs, which results in a change in the calibration of the assay, i.e. the assay behaves unstably during this process.

In one aspect of the invention, dextran is co-substituted with typical quenchers (e.g., HMCV1) and dyes or other components with more hydrophilic character, which can achieve better hydrophilic-hydrophobic balance with less change in configuration of the glucose ligand/analog over time. Co-substitution may also include positively and negatively charged substituents that prevent the dextran from becoming fully negative or positive. Exemplary experiments have shown that these factors can enable significantly more stable assays for optical sensors.

In one embodiment, as in fig. 1A, the quencher is changed from a dye to a fluorophore and the reference fluorophore is omitted. This improves the solubility of the assay by improving the solubility of the ligand. It also reduces the complexity of the assay, as AF 647-labeled dextran serves as both receptor and reference. In addition, this improves the photostability of the assay by exciting the reference fluorophore. In this case, blue-shifting the reference fluorophore towards the excitation source will prevent excitation of the fluorophore to the second excited state, a phenomenon that may lead to dye degradation. By shifting the dye towards the light source excitation wavelength, less photobleaching can be achieved (uv light bleaches visible dyes more than visible light). Blue-shifted reference dyes that can be used in embodiments of the invention (depending on, for example, the excitation filter, and the wavelength width of the light source) include Alexa Fluor (AF)546, AF555, AF568, AF594, and Cy3, Cy3B, and Cy 3.5. These are blue-shifted reference dyes compatible with the AF647 donor and the HMCV1 acceptor FRET system.

Various other agents may be coupled to the dextran to improve its conformation and/or hydrophilic-hydrophobic balance, typically for enhancing hydrophilicity. Exemplary reagents include cyclic anhydrides (e.g., phthalic anhydride), tartaric anhydride derivatives (e.g., O-diacetyl-L-tartaric anhydride), and the reagents shown in table 1 below:

in another embodiment, as shown in fig. 2A, the reference fluorophore is labeled/conjugated to the same ligand as the dye quencher, thereby eliminating the need for a reference carrier. This improves the solubility of the assay by improving the solubility of the ligand. This has shown even better stability than fluorescent ligands. It also reduces the complexity of the assay, as AF 647-labeled dextran serves as both receptor and reference. In addition, this improves the photostability of the assay by exciting the reference (where it absorbs more). It also enables further dose response optimization.

It has been found that when using ligands labeled with fluorophores only (e.g., AF647-100dex (d)), increasing (AF647-100dex (d)) concentrations in the assay result in higher Reference (REF) signals (risk of saturating REF) and more extravasation into the Assay (ASY) channel (bleed-over), which reduces sensor Dose Response (DR). Due to the above problems, there are limits on the (dex) concentration and degree of labelling (DOL) of pure AF647 ligand. High DOL HMCV1-dex presents solubility problems and AF647-dex appears to be more soluble in Tris or water. Addition of HCMV1 to AF647 ligand reduced the REF signal and overflow without reducing quenching capacity. The addition of AF647 to the HMCV1 ligand increases solubility. Thus, particular embodiments of the present invention add AF647 to HMCV 1-dextran, thereby forming AF647-HMCV1-Dex ligands with improved solubility, which improves assay stability. AF647 may be replaced by AF 700. The combination ligand, and only the HMCV1-Dex ligand, can be slightly succinylated to further improve assay stability.

In another aspect of the invention, a method of making a multi-labeled/combination glucose analog/ligand is provided. In typical embodiments, the combination ligand is a dextran carrying both a quencher dye and a fluorophore (e.g., both HMCV1 and AF 647). Both dyes were simultaneously stained onto dextran. In one example, 10X HMCV1-SE and 10X AF647-SE are added to 100Dex (d). This is usually followed by purification, dialysis or passage through a small Gel Permeation Chromatography (GPC) column.

The degree of labelling (DOL) of the various dyes on the Multiply Labelled Dextran (MLD) was determined by uv-vis spectroscopy. The DOL of the two dyes was varied to obtain the best fit. Resulting spectra from two dyes on dextranAre various dyesAndand the corresponding dye concentration (Dyex). The dye concentration was determined by solving the following equation (using HMCV1 and AF647 as examples) for all λ in the three recorded spectra.

ε_HMCV1(λ_max)＝42000M^-1cm^-1

ε_AF647(λ_max)＝270000M^-1cm^-1

As a performance evaluation on the sensor, the sensor Dose Response (DR) was calculated using the following formula:

wherein I₄₀₀And I₄₀Are normalized intensities at 400mg/dL and 40mg/dL glucose. The loss of sensor dose response (DR loss) was calculated using the following formula:

or from linear regression of DR over time (Excel function slope):

the relative DR loss is used as a key parameter to assess sensor quality. Historically, the term is loss of DR, so the negative development of DR becomes a positive loss, and hence the (-1) multiplication in the "slope" formula. Baseline drift was evaluated, but the drift was much smaller. In an illustrative experiment, as shown in fig. 13-17, singly labeled dextran showed relative DR losses between 3% and 6% per day, while multiply labeled dextran unexpectedly drifted only between 0.5% and 2.5%.

Blue shift reference dyes

Embodiments of the invention include a set of different fluorescent dyes (e.g., a reference fluorophore and a measurement fluorophore) selected for use together due to the wavelength distribution. Reference dyes are needed in (intensity) fluorescence measurements in order to track changes in the experimental setup, such as light source fluctuations, changes in the light path (coupling light into the light guide), mechanical disturbances (e.g. bending), temperature changes, etc. Traditionally, optical or fluorescence-based sensing systems have chosen a reference fluorophore that is red-shifted relative to the assay fluorophore. Using lower wavelength light with more energy than required to excite the fluorophore, there is an increased risk that an electron in the fluorescent molecule will transition from the ground electron state (S0) to the second excited state (S2) rather than to the first excited electron state (S1). The molecules at S2 are much more likely to break down than the same molecules at S1, so if excited to S2, the photobleaching obtained is faster than that obtained when only S1 is occupied. Since the reference dye must be very stable, the use of a reference fluorophore that is red-shifted relative to the assay fluorophore may be suboptimal.

Instead of a reference fluorophore that may appear photolabile, often red-shifted, due to low wavelength excitation, certain embodiments of the invention use a reference fluorophore that is blue-shifted relative to the assay fluorophore or indicator fluorophore. In other words, the reference fluorophore has a shorter wavelength/increased frequency than the assay fluorophore. In visible light, the reference fluorophore is closer to the blue end of the spectrum, while the assay fluorophore is closer to the red end. This improves the stability of the reference fluorophore, a key feature to provide accurate assay measurements. In one or more embodiments, the competitive glucose binding affinity assay comprises the use of an assay fluorophore (Alexa Fluor)^TM647) Labeled glucose receptor/lectin (e.g., mannan-binding lectin) and with a reference fluorophore (e.g., Alexa Fluor)^TM594) And a quencher dye (e.g., hexamethoxy crystal violet-1) labeled multi-labeled glucose analog (e.g., dextran), wherein the reference fluorophore is blue-shifted relative to the assay fluorophore.

With such systems, the spectrum and available light source determine the choice of fluorophore and the design of the optical device, as is known in the art. There are a variety of semiconductor Light Sources (LEDs) that may be used with embodiments of the present invention, such as those found in MIGHTEXSYSTEMS LED Wavelength combinations (MIGHTEX SYSTEMS LED wavelegnth Portfolio). Further, in embodiments of the present invention, the continuous light source (white light source with laser characteristics) may be filtered to select a specific wavelength range for excitation to provide an arbitrary wavelength (range).

Generally, for a red-shifted reference, the concentration of the reference can in principle be increased to reduce the assay fluorophore "red" tail spill effect, and must be separated by about 50nm to avoid the reference fluorophore "blue" tail spill over to the assay fluorophore and thus reduce the dose response. For a blue-shifted reference, the fluorophore must typically be blue-shifted by about 50nm to avoid spilling the reference assay "blue" tail over to the reference fluorophore and thus desensitizing the reference to the assay fluorophore fluorescence level. In this case, the reference fluorophore concentration can be reduced to avoid a reduction in dose response due to overflow of the reference "red" tail into the assay fluorophore emission. For most fluorophore pairs, it is often difficult to separate the fluorophores beyond 100nm and still be able to excite the most red-shifted fluorophore while blue-shifting. One way to avoid this is to use two different light sources for excitation and increase the complexity of the optical system, since the output of the light sources needs to be monitored to compensate for variations in output.

Name of

As described herein and in the drawings, the different dextran conjugates have the following generic nomenclature:

Dye1-Dye2-XXXDex(Y)succ(a/b/Xc)

as described herein and in the drawings, the different MBL conjugates have the following generic nomenclature:

Dye1-zMBL(a)

dye1 and Dye2 are abbreviations for Dye names;

XXX is the molecular weight of dextran in kDa;

y is an ion exchange chromatography (IEX) fraction of aminodextran;

z is "r" for recombinant MBL, "p" for plasma MBL, and "UHP" for ultra-high purified MBL;

succ indicates whether dextran was treated with a molar excess of succinic anhydride of "c". Staining dextran with only one or more dyes if succ is not specified; and

(a/b/Xc) indicates the degree of labelling of the Dye (DOL) and the excess succinic anhydride used. "a" is DOL of Dye1, "b" is DOL or Dye2, and "Xc" is used in molar excess of succinic anhydride. DOL is defined as the number of dyes per dextran, i.e. the dimensionless number.

Illustrative examples are as follows:

HMCV1-AF647-100Dex(d)(5.2/3.9)

the conjugate is a 100kDa dextran IEX peak-d labeled with HMCV1 and AF647 at DOL of 5.2 and 3.9, respectively.

HMCV1-100Dex(c)succ(12.1/X10)

The conjugate was labeled with HMCV1 and 12.1 DOL succinic anhydride with a 10-fold molar excess of the 100kDa dextran IEX peak-c used for modified succinic anhydride.

AF647-rMBL(0.51)

The conjugate was recombinant MBL labelled with AF647 with a DOL of 0.51.

The assay is described as MBL and concentration (PP; DD; RR) from the same named reference conjugate (when required) and each conjugate in square brackets, where PP is the concentration of MBL (rMBL) in μ M, DD is the ligand dextran concentration (Dex) in μ M, and RR is the reference dextran concentration in μ M.

Other aspects and embodiments of the invention are disclosed in the following examples.