阅读说明：本技术 在光学葡萄糖测定中使用蓝移基准染料 (Use of blue-shifted reference dyes in optical glucose assays ) 是由 瑟伦·奥斯莫 杰斯帕·斯文宁·克里斯滕森 于 2018-04-27 设计创作，主要内容包括：本发明涉及一种竞争性葡萄糖结合亲和测定,该竞争性葡萄糖结合亲和测定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)以及用基准荧光团标记的改性葡萄糖类似物(通常为葡聚糖)。在某些实施方案中,葡萄糖类似物是葡聚糖,并且其偶联到基准荧光团和淬灭剂染料(例如,六甲氧基结晶紫-1)两者。任选地,基准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 it 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 dextrate coupled to an assay fluorophore; and

a glucose analog coupled to a reference fluorophore;

wherein the reference fluorophore is blue-shifted relative to the assay fluorophore.

2. The glucose sensing complex of claim 1, wherein the glucose analog is further coupled to a 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 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.

5. The glucose sensing complex of claim 4, wherein the glucose receptor is a mannan-binding lectin.

6. The glucose-sensing complex according to claim 4, wherein the assay fluorophore and the reference fluorophore are independently selected from the group consisting of Alexa Fluor 594(AF594), Alexa Fluor 647(AF647), and Alexa Fluor 700(AF700), wherein the wavelength of the reference fluorophore is shorter than the wavelength of the assay fluorophore.

7. The glucose sensing complex of claim 2, wherein the reference fluorophore and the quencher dye form a Fluorescence Resonance Energy Transfer (FRET) pair.

8. The glucose sensing complex of claim 2, wherein the fluorophore and/or the quencher dye is water-soluble.

9. The glucose sensing complex of claim 1, wherein the assay fluorophore exhibits a wavelength at least 50 nanometers greater than the wavelength of the reference fluorophore.

10. The glucose sensing complex of claim 4, wherein the dextran is approximately 100 kDa.

11. A method of making a glucose-sensing complex, comprising forming a glucose-sensing complex by:

coupling a dextrate to an assay fluorophore; and

coupling a glucose analog to a reference fluorophore;

wherein the reference fluorophore and the assay fluorophore are selected such that light at a wavelength emitted by the reference fluorophore is blue-shifted relative to light emitted by the assay fluorophore.

12. The method of claim 11, wherein the assay fluorophore exhibits a wavelength at least 50 nanometers greater than the wavelength of the reference fluorophore.

13. The method of claim 11, wherein the assay fluorophore exhibits a wavelength that is up to 100 nanometers greater than the wavelength of the reference fluorophore.

14. The method of claim 11, wherein the glucose receptor comprises mannan-binding lectin (MBL) and the glucose analog is dextran.

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

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

a dextrate coupled to an assay fluorophore; and

a glucose analog coupled to a reference fluorophore;

wherein the reference fluorophore is blue-shifted relative to the assay fluorophore;

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

(c) the observed signal was 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 sensing comprises exciting the reference fluorophore and the assay fluorophore using two different light sources.

19. The method of claim 15, wherein the assay fluorophore exhibits a wavelength that is up to 100 nanometers greater than the wavelength of the reference fluorophore.

20. The method of claim 15, wherein the assay fluorophore exhibits a wavelength at least 50 nanometers greater than the wavelength of the reference fluorophore.

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 an important way for diabetics to avoid the long-term problems associated with diabetes, such as retinopathy, circulatory problems and other sequelae. For this reason, diabetics regularly monitor their blood glucose levels to, for example, optimize insulin administration. In this context, various systems and methods have been developed for monitoring blood glucose levels. One strategy uses fluorescent compounds to detect glucose levels, for example, using a competitive binding assay 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 an alternative glucose ligand. In such assays, dextran can be labeled with lipophilic (and cationic) dyes such as hexamethoxy crystal violet-1 (HMCV 1). However, the presence of a large number (e.g., greater than 10) of lipophilic dye molecules coupled to the flexible poly- (1,6) -glucose backbone of dextran can cause such labeled dextran molecules to adopt a less water-soluble conformation, which can cause these molecules to precipitate. Furthermore, such dye-induced conformational changes may force the labeled dextran to a more lipophilic state, which may cause adverse changes in the dextran's binding capacity to the glucose receptor as well as affect its Fluorescence Resonance Energy Transfer (FRET) efficiency. In addition, in conventional systems, the dye may undergo intramolecular shielding on the dextran, a phenomenon that can cause the calibration of the assay to change over time, thereby rendering the assay unstable.

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

Accordingly, there is a need in the art for optical glucose assays that utilize reagents and materials selected to enhance 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 coupled/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 materials and methods for optimization in glucose assays. As discussed in detail below, certain components of the fluorescent glucose assay may be selected and/or modified to, for example, optimize the hydrophilic-hydrophobic balance of a series of components used to form the assay complex. This results in an improved assay, i.e., an assay in which less undesirable conformational changes of the assay components occur over time. Exemplary modifications to the glucose assay complex include those that modify dextran by: the molecule is coupled to an agent selected to prevent the dextran from carrying too much negative or positive charge. Such modifications result in assays that are more stable than their unmodified equivalents. In addition, in embodiments where the reference fluorophore is selected to achieve its hydrophilic/hydrophobic characteristics and/or is blue-shifted relative to the assay or indicator fluorophore (rather than a typical red-shifted reference fluorophore that may become photolabile due to low wavelength excitation), the stability of the reference fluorophore and the assay complex as a whole may be increased.

Embodiments of the invention include competitive glucose binding affinity assays and methods for making and using these assays. Typically, the assay includes 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 AlexaAnd the quencher dye is hexamethoxy crystal violet-1 (HMCV 1). In some embodiments, the assay fluorophore and the reference fluorophore are independently selected from Alexa647(AF647) and Alexa700(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 character, thereby contributing to the stability of the agent within the complex and thus to the stability of the glucose sensing complex as a whole. Embodiments of the invention also include methods for 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 isThe 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 can be selected from the group consisting of mannan-binding lectin (MBL), concanavalin a, glucose-galactose binding protein, antibodies, and boronic acid. In one instance, the glucose receptor is a mannan-binding lectin. Typically, the glucose analog is dextran, and the assay fluorophore and the reference fluorophore are AlexaTypically, the fluorophore and quencher dye form a Fluorescence 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 independently selected from the group consisting of Alexa Fluor 594(AF594), Alexa Fluor 647(AF647) and Alexa Fluor 700(AF700), wherein the wavelength of the reference fluorophore is shorter than that of 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-1C illustrate a fluorescent ligand according to one or more embodiments of the present inventionAnd (5) Optical. Figure 1A shows the quencher changing from a dye to a fluorophore and omitting the reference fluorophore. Fig. 1B is a graph showing changes in absorption spectrum and emission spectrum. FIG. 1C is a graph showing glucose concentration measurements and sensor dose response loss (DR loss)Graph is shown.

FIGS. 2A-2C illustrate a ligand conjugate according to one or more embodiments of the present inventionAnd (5) Optical. Figure 2A shows a reference fluorophore labeled to the same ligand as the dye quencher, thereby eliminating the need for a reference carrier. Fig. 2B is a graph showing changes in absorption spectrum and emission spectrum. Fig. 2C is a graph showing glucose concentration measurements and sensor dose response loss (DR loss).

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

FIG. 4 is a graph illustrating ORS SITS data for the AF594-MBL/AF647-HMCV1-dex assay, according to one or more embodiments of the invention. This was also used in rat experiments 76 and 77, as shown in figures 5 to 7 below.

Fig. 5 is a graph showing a study from rats 76: graph of data from rat #1, ORS sensor # 2. The sensor is calibrated during the first jaw of the low-to-high transition.

Fig. 6A-6D illustrate a study 76 from rats according to one or more embodiments of the present invention: rat #1, ORS sensor #2 derived data for jaw 1 (fig. 6A), jaw 2 (fig. 6B), and jaw 3 (fig. 6C). Fig. 6D is a picture of an explant.

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

Figure 8 is a graph illustrating data derived from an AF594/AF647 assay, according to one or more embodiments of the present invention.

FIG. 9 is a graph showing measurements from AF594MBL/AF647-Dex and AF594/AF647-HMCV1-Dex, in accordance with one or more embodiments of the present invention: graph of data from an optimal ratio between AF594 and AF647 fluorescence.

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

Fig. 11 shows a filter configuration for AF594/647 assay according to one or more embodiments of the present invention.

Fig. 12A-12C provide examples of degree of labeling (DOL) determinations for multi-labeled dextran (MLD) according to one or more embodiments of the present invention. Fig. 12A is a graph showing normalized absorption spectra of HMCV1 and AF 647. Fig. 12B is a graph showing the absorption 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 absorption 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.

Fig. 13 is a graph showing 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 built using singly labeled dextran in the glucose response assay. DR was calculated as the difference between the normalized intensity at 400mg/dL glucose and 40mg/dL glucose, relative to the normalized intensity at 40mg/dL glucose. Loss of DR relative to the initial DR is between 3% and 6% per day. Dextran labeled with HMCV1 alone showed large Dose Response (DR) losses.

Fig. 14 is a graph showing the performance of multi-labeled dextran-HMCV 1-AF 647-dextran, according to one or more embodiments of the invention.

Fig. 15 is a graph showing the performance of multi-labeled dextran-HMCV 1-AF 700-dextran, in accordance with one or more embodiments of the invention.

Fig. 16 is a graph showing the performance of multi-labeled dextran-HMCV 1-HMCV 3-dextran, according to one or more embodiments of the invention.

Fig. 17 is a graph showing the performance of multi-labeled dextran-succinylated HMCV 1-dextran, 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 invention.

Fig. 19 is a table showing combined ligands with different DOL values in a capsule sensor, their DR and intensity levels, 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 contained herein should not be construed as indicating a substantial difference from 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 using commercially available calorimetric test strips or electrochemical biosensors (e.g., enzyme electrodes), both of which require the 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, particularly for long-term diabetics, who have to use a lancet to draw blood from their fingertips on a regular basis, both from an economic perspective and from a pain and discomfort perspective. Therefore, many proposals have been made for glucose measurement techniques that do not require blood to be drawn from the patient. It has been observed that the concentration of an analyte in subcutaneous fluid is correlated with the concentration of that analyte in blood, and thus several reports have proposed the use of glucose monitoring devices placed 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 based on proximity of a signal generating/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 (usually fluorescent). In such methods, an energy transfer donor-acceptor pair is contacted with a sample to be analyzed, such as a subcutaneous fluid collection. The sample is then illuminated and the resulting emission detected. Either the energy donor moiety or the energy acceptor moiety of the donor-acceptor pair binds to the acceptor carrier, while the other moiety of the donor-acceptor pair (bound to the ligand carrier) competes with any analyte present for binding sites on the acceptor carrier. When the donor and acceptor are brought together, energy transfer occurs between the donor and acceptor, resulting in a detectable change (decrease) in lifetime of the fluorescence of the energy donor moiety. In addition, a proportion of the fluorescent signal emitted by the energy donor moiety is quenched. Competitive binding of the analyte reduces or even eliminates lifetime variation. Thus, the amount of analyte in the 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, PleumPress, 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. In contrast to lifetime measurements, the measured intensity of the emitted fluorescence is 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 distinguished from the assay fluorophore in a manner that results from the assay andthe emitted fluorescent light from the reference may be separated from each other, for example by having different absorption spectra or emission spectra. The reference fluorophore may be, for example, Alexa594(AF594) which labels onto Human Serum Albumin (HSA) or another macromolecule which does not substantially bind to the glucose receptor. Alexa700(AF700) may be excited simultaneously with AF594 because their absorption spectra are spectrally overlapping. The emission spectrum from AF594 is slightly blue-shifted with respect to AF700, which makes it possible to detect its corresponding fluorescence emission in a separate wavelength region. Since they are excited simultaneously by the same light source, any change in light source intensity will scale the fluorescence from AF594 and AF700 equally. Thus, any effects resulting from variations in the intensity of the light source may be counteracted. Excitation and detection of the emitted fluorescence of the assay and the reference follow the same optical path from the optical system to the assay. The detected signal from the reference is therefore used as a measure of the optical coupling between the optical interrogation system and the assay. Any effects resulting from variations in optical coupling (such as alignment) can be cancelled out.

A special property of fluorescence known as Fluorescence 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, either 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 a photon and is the result of a long-range dipole-dipole interaction between the donor and acceptor. An important characteristic of FRET is that it occurs over distances comparable to the size of biological macromolecules. The distance at which FRET is 50% effective (referred to asDistance) is usually inWithin the range of (1). 20 toWithin the range ofThe distance facilitates competitive binding studies. WO91/09312 describes subcutaneous methods and apparatus employing a glucose-based affinity assay (binding 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, the optical signal generated by which can be read remotely.

The invention disclosed herein relates generally to optical or fluorescence-based assay and analyte sensing compositions. In exemplary embodiments of the invention, the assays, compositions, systems and methods of the invention are described in connection with glucose as the analyte whose level/concentration is to be determined. However, this is by way of example only and not by way of limitation, 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 designed to include a composition disposed in a particular region of the sensor to provide the sensor with enhanced functional and/or material properties. The present disclosure also provides methods for making and using such sensors. Embodiments of the invention described herein (such as those discussed in the immediately following paragraph) may be adapted and implemented using a wide variety of elements in a sensor having a sensing complex that generates a light signal that may be correlated with the concentration of an analyte (such as glucose). A number of these sensors and elements are disclosed, for example, in U.S. patents 6,6761,527, 7,228,159, 7,884,338, 7,567,347, 8,305,580 and 8,691,517 and U.S. patent application publications 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 a number of embodiments. One exemplary embodiment is a glucose-sensing complex comprising a mannan-binding ligand coupled to an assay fluorophore, a reference fluorophore (e.g., Alexa Fluor 647(AF647) or Alexa Fluor 700(AF 700)); dextran that acts as a glucose analog in this assay and is coupled to a reference fluorophore, a reagent that enhances the hydrophilicity of dextran, and a quencher (e.g., hexamethoxy crystal violet-1 (HMCV 1)). In this embodiment, the assay fluorophore and quencher form a Fluorescence 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), canavalin a, glucose galactose-binding protein, an antibody, and a boronic acid, the glucose analog is dextran, the assay fluorophore and the reference fluorophore are Alexa Fluors, 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 mannan binding ligand coupled to an assay fluorophore, a reference fluorophore; dextran selected to act as a glucose analog in the assay and further coupled to a reference fluorophore. The complex further comprises 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 Fluorescence 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 concentration of glucose.

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 wavelength of the reference fluorophore is lower than the wavelength of 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, glucosyl 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 independently selected from 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 Fluorescence Resonance Energy Transfer (FRET) pair.

Another embodiment of the invention is a method of making a glucose-sensing complex, the method 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 light emitted by the reference fluorophore at a certain wavelength is blue-shifted with respect 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 up to 100 nanometers greater than the wavelength of the reference fluorophore. In some cases, the glucose analog is further treated with succinic anhydride. Typically, the dextran is approximately 100 kDa. In certain embodiments, the composition exhibits a sensor Dose Response (DR) loss of less than 2.5% per day. In one instance, 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 concentration of glucose. Optionally, the method comprises exciting the reference fluorophore and the assay fluorophore using two different light sources. Typically, the assay fluorophore exhibits a wavelength that is at most 100 nanometers greater than the wavelength of the reference fluorophore, and the assay fluorophore exhibits a wavelength that is at least 50 nanometers greater than the wavelength of the reference fluorophore.

There are many permutations of the present invention. As described herein, the analyte receptor is typically a lectin, which comprises 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 (Structure Biology), Vol.1, pp.789-794, 1994; Dumestre-Perard et al, Molecular Immunology, Vol.39, pp.465-473, 2002). Typically, the lectin provides a stable signal in the assay for at least 10 days, more typically at least 14 days. It is particularly preferred to provide a stable signal when the sensor is 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 alternatively be used in the assay and sensor systems described herein. For example, The analyte receptor may be ｃA human lectin derived from human, including human lung surfactant protein A (SP-A, Allen et al, Infection and Immunity, Vol.67, p.4563-4569, 1999), human lung surfactant protein D (SP-D, Persson et al, The Journal of Biological Chemistry, Vol.265, p.5755-5760, 1990) or CL-43 (human serum albumin). 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 (US 2003/0216300, US 2004/0265898), CTL-1(US 2010/179528), keratinocyte membrane lectin (Parfurerie und Kosmetik [ perfumes and cosmetics ], Vol.74, p.164. sub.180 ], CD94(Eur J Immunol [ European J.Immunol ], Vol.25, p.2433. sub.2437), P35 (also: human L-fibrin, a group of collectins) (Immunol Lett (proceedings of immunology), Vol.67, p.109. sub.112), ERGIC-53 (also: MR60) (Mol Biol [ cytomology Biol ], Vol.7, p.483 ], European Biochem [ Biochem ] 493 ], vol 267, 1665-1671), CLECSF8(Eur JImmunol (journal of European immunology), Vol 34, p 210-220), DCL (lectin panel) (application no 00231996/US) and GLUT family proteins, in particular GLUT1, GLUT4 and GLUT11(PNAS (Proc. Natl. Acad. Sci. USA), Vol 97, p 1125-1130). Other suitable Animal Lectins are listed in the Handbook of Animal Lectins: Properties and Biomedical Applications "(" Handbook of Animal Lectins: Properties and Biomedical Applications "), David C.Kilparick, appendix A, B and C of Wiley 2000. Suitable phytohemagglutinin or Phytohemagglutinin (PHA) include canavalin a (con a) and those derived from pisum sativum (pea), thyrus odoratus (sweet pea), lentils culinaris (lentil), narcissus pseudonarcissus (yellow narcissus), vicia faba (broad bean) and vicia sativa (arrow pea). The analyte receptor may also be a periplasmic glucose/galactose binding receptor, an antibody produced upon stimulation with a glucose-like molecule, or a boronic acid.

As described herein, an analyte analog can comprise a plurality of carbohydrates or carbohydrate mimetic moieties that bind to a binding site for an analyte receptor. The molecular weight of the analyte analog should be 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 and more typically between 90 to 120 kDa. In typical embodiments where glucose is the analyte, dextran is used as the alternative glucose analog/ligand. Dextran is a flexible macromolecule consisting of up to 1500 glucose units. In some cases, the glucan consists of about 600 glucose units (about 100kDa), or 500-700 glucose units.

Other analyte analogs and ligands may alternatively be used in the exemplary assay and sensor systems described herein. The analyte analog can be a synthetic polymer with different carbohydrates or carbohydrate-mimetic moieties with different affinities for MBL and similar lectins. Alternatively, the analyte analog can be a carbohydrate-protein conjugate or a carbohydrate-dendrimer conjugate. Examples of carbohydrates suitable for such conjugates are mono-and oligosaccharides. Suitable monosaccharides are optionally derivatized tetroses, 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 compound capable of absorbing light energy at a particular wavelength and re-emitting the 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). Typically, Alexa(AF)594, 647 and/or 700 are used as reference and assay fluorophores to label glucose analogs and glucose receptors, respectively. Those skilled in the art understand that other fluorophores suitable for optical glucose assays may alternatively be used, such as coumarins, rhodamines, xanthenes, cyanines, and Alexa Fluor dyes that cover other excitation and emission wavelengths (e.g., AF350, AF405, AF488, AF532, AF546, AF555, AF568, AF594, AF680, AF 750).

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

The binding assay that generates the optical signal should generally be reversible so that continuous monitoring of fluctuating levels of analyte 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 bodily fluid (e.g., subcutaneous fluid). It is desirable that the sensor is suitable for use in vivo. Typically, the assay is capable of measuring blood glucose for 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 assays that use FRET techniques to generate optical readouts.

As discussed above, there is a need in the art for optical or fluorescence-based assays that have enhanced stability and require lower calibration frequencies for optical sensors. In one aspect of the invention, an analyte sensing composition having significantly improved stability and solubility is provided. The analyte sensing composition comprises an analyte analog labeled with both a fluorophore and a quencher dye. In typical embodiments, the analyte sensing composition is a glucose sensing composition comprising a fluorophore (e.g., Alexa)647、Alexa700) 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, competitive analyte binding affinity assays based on analyte sensing compositions are provided. Competitive analyte binding affinity assays include analyte receptors labeled with an assay fluorophore and analyte analogs 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 specifically 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)647, AF647) and a glucose receptor/lectin (e.g., mannan-binding lectin, MBL) labeled with a reference fluorophore (e.g., Alexa594, AF594) and a quencher dye (e.g., HMCV 1).

In embodiments of the invention, the binding between MBL and glucose-like molecules (e.g., dextran) is reversible. In the absence of glucose, mainly MBL and dextran are bound together. When glucose is added to the assay, the glucose will compete off a portion of the dextran population, causing the assay to enter a new equilibrium state. The 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 Fluorescence 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 because they must function in an aqueous environment.

Multilabeled analyte analogs

Embodiments of the invention include a glucose assay complex comprising dextran as a glucose analog. Dextran is a flexible macromolecule composed of up to 1500 glucose units. In certain typical cases, dextran consists of about 600 glucose units (about 100 kDa). Dextran can be used as an alternative analyte analogue/ligand in a glucose response competitive optical assay based on mannan-binding lectin (MBL). To function in Fluorescence Resonance Energy Transfer (FRET) assays, dextran is often (heavily) labeled with lipophilic (and cationic) dyes 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 allows the HMCV 1-labeled dextran to fold into a less water-soluble conformation, resulting in precipitation. Dye-induced conformational changes convert HMCV 1-labeled dextran to a more lipophilic state, which can cause various problems, including adverse changes in the binding capacity of dextran to the glucose receptor and affecting its Fluorescence Resonance Energy Transfer (FRET) efficiency. As the dye undergoes intramolecular shielding on the dextran, an intrinsic leaching effect also occurs, which causes a change in the calibration of the assay, i.e. the assay behaves unstably during the process.

In one aspect of the invention, complex substitution of dextran with typical quenchers (e.g., HMCV1) along with dyes or other components with more hydrophilic character, achieves better hydrophilic-hydrophobic balance with less conformational change of glucose ligand/analog over time. The complex substitutions may also include positively and negatively charged substituents that prevent the dextran from becoming completely negative or positive. Exemplary experiments have demonstrated that these factors allow for a significantly more stable determination of the optical sensor.

In one embodiment, as shown in figure 1A, the quencher changes from a dye to a fluorophore and the reference fluorophore is omitted. This improves the stability of the assay by improving the solubility of the ligand. This also reduces the complexity of the assay, as AF 647-labeled dextran acts as both receptor and benchmark. In addition, this improves the photostability of the assay by exciting the reference fluorophore. In this context, a blue shift of the reference fluorophore towards the excitation source will prevent the fluorophore excitation from reaching the second excited state, a phenomenon that may cause dye degradation. By shifting the dye towards the light source excitation wavelength, we can achieve less photobleaching (UV light bleaches visible dyes to a greater extent than visible light). Blue-shifted reference dyes that can be used in embodiments of the present invention (depending on, for example, the wavelength width of the excitation filter and 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 systems.

Various other agents may be coupled to the dextran to improve its conformation and/or the hydrophilic-hydrophobic balance, generally enhancing hydrophilicity. Exemplary reagents include cyclic anhydrides (e.g., phthalic anhydride), derivatives of tartaric anhydride (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 stability of the assay by improving the solubility of the ligand. This has shown even better stability than the fluorogenic ligand. This 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 fiducial ("where it absorbs more"). This also enables further dose response optimization.

It has been found that when using ligands labeled with fluorophores only (e.g., AF647-100dex (d)), increasing the (AF647-100dex (d)) concentration in the assay results in a higher baseline (REF) signal (risk of REF saturation) and greater wash-over into the Assay (ASY) channel, thereby reducing the sensor Dose Response (DR). Due to the above problems, the (dex) concentration and degree of labelling (DOL) of pure AF647 ligand are limited. High DOL HMCV1-dex had solubility problems, and AF647-dex appeared to be more soluble in either Tris or water. Addition of HCMV1 to AF647 ligand reduced the REF signal and spill wave without reducing quenching capacity. The addition of AF647 to HMCV1 ligand increases solubility. Thus, particular embodiments of the present invention provide for the addition of AF647 to HMCV 1-dextran, thereby forming an AF647-HMCV1-Dex ligand with improved solubility, thereby improving assay stability. AF647 may be replaced with AF 700. The combination ligand as well as only the HMCV1-Dex ligand can be slightly succinylated to further improve assay stability.

In another aspect of the invention, a method for preparing a multi-labeled/combinatorial 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 stain dextran simultaneously. In one case, 10X HMCV1-SE and 10X AF647-SE was added to 100Dex (d). This is usually followed by purification, dialysis or by a small Gel Permeation Chromatography (GPC) column.

The degree of labelling (DOL) of individual dyes on Multiply Labelled Dextran (MLD) was determined by uv-vis spectroscopy. The DOL of the two dyes was varied to obtain the best point of engagement. FromSpectra generated by these two dyes on dextranIs the spectrum of the individual dyesAndlinear combinations with the corresponding dye concentration (Dyex). The dye concentration was determined by solving all λ of the following equation in the three recorded spectra (using HMCV1 and AF647 as examples).

ε_HMCV1(λ_max)＝42000M^-1cm^-1

ε_AF647(λ_max)＝270000M^-1cm^-1

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

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

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

the relative DR loss is used as a key parameter to assess sensor quality. Historically, this term is the loss of DR, so the negative development in DR results in a positive loss, and thus is a (-1) multiplication in the "slope" equation. Baseline drift was evaluated, but the baseline drift drifted to a much lesser degree. In an exemplary experiment, as shown in fig. 13-17, single labeled dextran showed relative DR losses between 3% and 6% per day, while multi-labeled dextran surprisingly drifted only between 0.5% and 2.5%.

Blue shift reference dyes

Embodiments of the present invention include a set of different fluorescent dyes (e.g., a reference fluorophore and a measurement fluorophore) that are selected for use together due to their wavelength characteristics. The reference dye in (intensity) fluorescence measurements is needed in order to track changes in the experimental setup, such as light source fluctuations, optical path changes (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. When a fluorophore is excited using light of a lower wavelength with more energy than necessary, the risk of an electronic transition in the fluorescent molecule from the ground electronic state (S0) to the second excited state (S2) instead of the first excited electronic state (S1) increases. The molecules at S2 are much more likely to decompose than the same molecules at S1, so excitation to S2 achieves photobleaching at a faster rate than 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 the conventional red-shifted reference fluorophores that can exhibit photolabile due to low wavelength excitation, certain embodiments of the present invention instead use reference fluorophores that are 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 baseline fluorescenceStability of the bolus, a key characteristic in providing accurate assay measurements. In one or more embodiments, competitive glucose binding affinity assays include the use of an assay fluorophore (Alexa)647) Labeled glucose receptor/lectin (e.g., mannan-binding lectin) and use of a reference fluorophore (e.g., Alexa594) And a quencher dye (e.g., hexamethoxy crystal violet-1), wherein the reference fluorophore is blue-shifted relative to the assay fluorophore.

As is known in the art, the spectrum and available light source determine the choice of fluorophore and the design of the optical setup when such a system is employed. There are a variety of semiconductor Light Sources (LEDs) that can be used with embodiments of the present invention, such as those found in the MIGHTEX SYSTEMS LED wavelength combination. In addition, in embodiments of the present invention, a continuous light source (white light source with laser characteristics) may be filtered to select a particular wavelength range of 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 effect of the measurement fluorophore "red" tail spill, and must be separated by approximately 50nm to avoid the reference fluorophore "blue" tail spill to the measurement fluorophore and thus reduce the dose response. For a blue-shifted reference, the fluorophore must typically be blue-shifted by about 50nm to avoid a reference assay "blue" tail-over to the reference fluorophore and thus desensitize the reference to the assay fluorophore fluorescence level. In this case, the reference fluorophore concentration can be reduced to avoid a decrease in dose response due to a reference "red" tail spill into the measured fluorophore emission. For most fluorophore pairs, it is often difficult to separate the fluorophores by more than 100nm and still be able to excite most red-shifted fluorophores simultaneously with blue-shifted fluorophores. One way to circumvent this problem is to use two different light sources for excitation, but this adds complexity to the optical system, as the output of these 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 Mw in kDa of the glucan;

y is an ion exchange chromatography (IEX) fraction of amino-dextran;

z is "r" representing recombinant MBL, "p" representing plasma MBL, "UHP" representing ultra-high purity MBL;

succ represents the case where dextran is treated with a molar excess of succinic anhydride of "c". If succ is not indicated, the dextran is stained with only one or more dyes; and is

(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 of Dye2, and "Xc" is the molar excess of succinic anhydride used. DOL is defined as the number of dyes per unit of dextran, i.e. a dimensionless number.

An illustrative example is as follows:

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

the conjugate was a 100kDa dextran IEX peak-d labeled with HMCV1 and AF647 and having DOLs of 5.2 and 3.9, respectively.

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

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

AF647-rMBL(0.51)

The conjugate was recombinant MBL labeled with AF647 and having a DOL of 0.51.

The assays are described according to the same nomenclature as MBL and ref-conjugates (where necessary) and the concentrations of the individual conjugates are set forth in brackets (PP; DD; RR), where PP is the MBL (rMBL) concentration in μ M, DD is the ligand dextran concentration in μ M (Dex), and RR is the baseline dextran concentration in μ M.

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