Field of the invention

The present invention relates to a method, device and compounds for detecting an analyte using Surface Enhanced Raman Spectroscopy (SERS).

Background

Development of a reliable and inexpensive continuous glucose monitoring system has been a long-standing aim of biomedical device research for the management of diabetes mellitus. Several methods have been explored with very limited success, even after years of extensive research and so far only few sensors have reached an advanced stage of development. An optical sensor for a walking diabetic could be more researched since technology already exists for making them minimally invasive through miniaturization of components. Such a device can also be used in critical care medicine for tight glycemic control, which has proven to have a dramatic improvement in recovery rate and 34% reduction in overall mortality. The current sensors are based on glucose oxidase; their performance is limited by factors, such as oxygen dependence, poor accuracy in the hypoglycemic range and consumption of glucose while making a measurement. A solution to these problems could be a sensor based on reversible binding of glucose to an artificial receptor, such as boronic acid. Ability of boronic acids to bind to glucose reversibly under physiological conditions is well documented in literature. A sensing system based on this interaction offers considerable advantages in terms of stability and ease of fabrication. A method of glucose sensing with a boronic acid functionalized dye is for example disclosed in US 5,137,883. In 1992 Yoon and Czarnik reported the first scientific study on boronic acid based glucose sensing using anthracenyl boronic acid which produced a measurable change in fluorescent intensity upon binding to glucose in solution (Yoon & Czarnik, J. Am. Chem. Soc. 1992;114:5874-5875). Since this seminal report, Shinkai and others (Kawanishi et al., J Fluorescence 2004; 14:5,499-512; Arimori et al., Tetrahedron Letters 2001, 42(27), 4553-4555; Norrild & Sotofte, Journal of the Chemical Society, Perkin Transactions 2 2002, (2), 303-311; Ma & Yang, Journal of Electroanalytical Chemistry 2005, 550(2), 348-352; Wang et al., Journal of Organic Chemistry 2005, 70(14), 5729-5732; Kanekiyo & Tao, Chemistry Letters (2005), 34(2), 196-197) have developed several abiotic sensing systems using boronic acids. All of these studies, which are based on fluorophores to which a boronic acid is appended, helped to establish the scope of this technique. Different boronic acid based glucose sensors are known in the art (International patent publication WO 2004/096817; U.S. Pat. Appl. No. 2005191761 ; U.S. Pat. Appl. No. 2005124020; Cappuccio et al., Journal of Fluorescence (2004), 14(5), 521-533). Yet, there is still need in the art for a sensor that can function efficiently under physiological conditions. The main challenges to achieve glucose sensing under physiological conditions include feasibility of transferring sensing components in minimally invasive platform such as optical fibers or implantable substrates.

Vibrational spectroscopic techniques namely infra red (IR), normal Raman and Surface Enhanced Raman (SER) have been considered for glucose detection. Since near IR and mid IR technique suffers with the limitation of competing absorption from aqueous media, Raman spectroscopic techniques have evolved as the methods of choice. Raman spectroscopy has been used as valuable tool for structural information due to the molecular vibrational finger prints available by the spectrum. However, the applications were limited under biological conditions mainly due to the poor sensitivity and the need for high laser power and complicated instrumentation. Most of these drawbacks were overcome by the development of Surface Enhanced Raman spectroscopy (SERS) where the spectral intensity is enhanced tremendously by the interaction of the analyte molecules with a nanoparticle surface of gold or silver. There are many cases where these enhancement factors are up to the level of single molecule detection (Nicholas & Ricardo, Chem. Soc. Rev., 2008, 37, 946-954). However, the detection of molecules with such extraordinary enhancement depends on the properties of the molecule-nanoparticle ensemble and is limited to certain classes of aromatic molecules. However, due to the very poor Raman cross section of glucose leading to a relatively weak SERS spectrum, the SERS based detection of glucose is still considered a challenge. Since the glucose sensing by SERS depends on the interaction of glucose with the surface of the Raman active surface, a lot of effort has been placed towards meeting with the challenge of designing methodology to bring glucose molecules close enough to the nanoparticle surface with reliable SERS response. One approach tested is to treat the silver film over the nanosphere surface with a mixture of aliphatic thiols which effectively partition glucose near the SERS active surface even in presence of biological fluids in a reproducible manner (Yonzon et al., Anal. Chem. 2004, 76, 78-85). This technique is being implemented in an implantable sensor and tested in vivo and successful sensing studies in an animal model have been reported (Stuart et al., Anal. Chem., 2006, 78, 721 1-7215; U.S. patent application No. 200901 18605; U.S. patent application Ser. No. 10/652,280; U.S. Provisional Patent Application Ser. No. 60/407,061). Other techniques included capturing glucose using well known synthetic sugar receptors (Kanayama & Kitano, Langmuir, 2000, 16 (2), 577-583). However, when these receptors were covalently anchored to the surface to follow the vibrational peaks of glucose, sensitivity of the resulting SERS sensor was not good enough for practical applications. Hence, there is still need for methods that overcome the above- detailed limitations.

Summary of the Invention

The present invention provides for methods, devices and compounds for the sensitive analyte detection using SERS.

In a first aspect, the present invention thus relates to a method for detecting an analyte using surface enhanced Raman spectroscopy (SERS), including contacting the analyte with one or more compounds selected from the group consisting of aryl boronic acid derivatives, heteroaryl boronic acid derivatives, aryl boronate derivatives and heteroaryl boronate derivatives, wherein the one or more compounds are attached to a metal substrate surface that enhances Raman scattering; and detecting a surface enhanced Raman signal from said compound. In various embodiments of this method, the surface enhanced Raman signal of the compound is correlated with the amount of the analyte.

In further embodiments, this method is suitable for the detection of the analyte in vivo or, alternatively, the analyte may be contained in a sample and the detection is in vitro.

Other embodiments of the method allow that the analyte is detected in a bodily fluid comprising said analyte. Preferably, this bodily fluid is selected from the group consisting of plasma, serum, blood, lymph, liquor and urine.

In a particular embodiment, the analyte to be detected is an a-hydroxy acid or a monosaccharide, such as glucose.

In certain embodiments of the above-detailed method, the compound is attached to the substrate surface via electrostatic, hydrophobic or covalent interactions.

In various embodiments, the compound is selected from the group consisting of phenyl boronic acid derivatives, pyridyl boronic acid derivatives and bipyridyl boronic acid derivatives and salts thereof. The compound may for example be a boronic acid substituted benzyl viologen or a 3- or 4-(purin-9-ylcarbonyl)phenyl boronic acid or salt thereof.

In further embodiments of the claimed method the compounds are covalently attached to the substrate surface via a linker. In one embodiment, the linker comprises at least one thiol group that facilitates binding to the substrate surface. In various embodiments, the linker may comprise an ethylene glycol of the formula -(0-CH ₂ -CH ₂ )„- , wherein n is an integer from 1 to 10.

In other embodiments of the invented method, the compound is either positively charged and attached to the substrate surface by electrostatic interactions or the compound is hydrophobic and attached to the substrate surface by hydrophobic interactions. In various embodiments of the claimed method, the compound can be selected from the group consisting of:

N,N'-Bis-(benzyl-3-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[J,4']bipyridinium Dibromide (J,4'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,4']bipyridinium Dibromide (J,4'-/?-BBV);

N,N'-Bis-(benzyl-3-boronic acid)-[3,3']bipyridinium Dibromide (3,3 -m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[3,3']bipyridinium Dibromide (J,3'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,3']bipyridinium Dibromide (3,3'- ?-BBV);

N,N'-Bis-(benzyl-3-boronic acid)-[4,4']bipyridinium Dibromide (¥,4'-m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[¥,4']bipyridinium Dibromide (4,4'- -BBV);

3- (6-Mercaptopurin-9-ylcarbonyl)phenylboronic acid;

4- (6-Mercaptopurin-9-ylcarbonyl)phenylboronic acid;

2-((3-Mercaptoethylcarbonyl)amino)-5-pyridyl boronic acid;

Pyrene-l-boronic acid; and

Benzothiophene-2-boronic acid;

or salts thereof. In some embodiments, the substrate used in the claimed method is a nanoparticle. The nanoparticle may be coated with, comprise or consist of a noble metal. In one embodiment, the noble metal is selected from gold and silver. In specific embodiments, the nanoparticle is a nanoparticle coated with a silver film or a citrate-stabilized gold nanoparticle.

In another aspect, the present invention relates to a biosensor for the detection of an analyte using surface-enhanced Raman spectroscopy, comprising a plurality of nanoparticles, wherein one or more compounds selected from the group consisting of aryl boronic acid derivatives, heteroaryl boronic acid derivatives, aryl boronate derivatives and heteroaryl boronate derivatives are attached to the nanoparticle surface. In one embodiment, the biosensor further comprises a substrate, wherein said nanoparticles are adherent to the substrate. Preferably, the biosensor is configured for in vivo and/or in vitro use. In one embodiment of the above biosensor, the analyte detected by the biosensor is an a-hydroxy acid or a monosaccharide, such as glucose. The biosensor may be configured for quantitative detection of glucose in a physiological concentration range.

In various embodiments, the one or more compounds of the above biosensor are attached to the substrate surface via electrostatic, hydrophobic or covalent interactions.

The one or more compounds may be selected from the group consisting of phenyl boronic acid derivatives, pyridyl boronic acid derivatives and bipyridyl boronic acid derivatives or salts thereof, such as boronic acid substituted benzyl viologens or salts thereof.

In some embodiments, the one or more compounds are covalently attached to the substrate surface via an organic linker, such as a linker which comprises at least one thiol group that facilitates binding to the substrate surface. In one embodiment, the linker comprises an ethylene glycol of the formula -(0-CH ₂ -CH ₂ ) _n -, wherein n is an integer from 1 to 10.

In other embodiments, the one or more compounds of the biosensor are either positively charged and attached to the substrate surface by electrostatic interactions or hydrophobic and attached to the substrate surface by hydrophobic interactions.

In certain embodiments of the biosensor, the nanoparticle is coated with, comprising or consisting of a noble metal. This noble metal is preferably selected from gold and silver. In a certain embodiment thereof, the nanoparticle is coated with a silver film. In another embodiment of the biosensor, the nanoparticle is a citrate-stabilized gold nanoparticle.

In a further aspect, the present invention relates to compounds of general Formula I

Formula I

wherein

A is heteroaryl and comprises at least one nitrogen atom,

L is a linker selected from the group consisting of -C(O)-, -C(0)-0-, -(CH ₂ ) _m -, - C(=CH ₂ )-, -C(0)-NH-, -NH-C(O)-, -0-, -S-, =CH- and -CH=, wherein m is an integer from 1 to 3;

L2 is a linker selected from the group consisting of a direct bond between A and SH, - C(0)-0-(CH ₂ ) _q -, -C(0)-S-(CH ₂ ) _q -, -(CH ₂ ) _p -, -C(0)-S-(0-CH ₂ -CH ₂ ) _n -, -C(0)-S -(CH ₂ - CH ₂ -0) _n -, -C(0)-S -<CH ₂ -CH ₂ -0)„-(CH ₂ ) _q - and -C(0)-S -(0-CH ₂ -CH ₂ ) _n -(CH ₂ ) _q -, wherein p is an integer from 1 to 20, n is an integer from 1 to 10 and q is an integer from 1 to 5;

B is aryl; and salts thereof. These compounds may have use as Raman receptors for monosaccharides or alpha- hydroxy acids.

In various embodiments, A is selected from the group consisting of purinyl, indolyl, pyridinyl, bipyridyl, pyrimidinyl, azaindolyl, pyrrolyl, pyrazinyl, pyridazinyl, isoindolyl, benzimidazolyl, imidazolyl and indazolyl. B is preferably selected from phenyl or naphthyl.

In some embodiments of the above compounds, L is bound to A via the at least one nitrogen atom of the heteroaryl ring.

In various embodiments, L is -C(O)-. In further embodiments, L is -C(O)-, A is purinyl, L2 is a direct bond and B is phenyl. In one embodiment, the compounds are of Formula II:

Formula II.

In other embodiments of the claimed compounds, L is -(CH ₂ ) _m -. In some embodiments, L is -(CH ₂ ) _m -, A is purinyl, L2 is a direct bond and B is phenyl.

In one embodiment, the compound is of Formula III:

Formula III.

In a still further embodiment of the compounds falling within general Formula I, A is bipyridyl, L is -(CH ₂ ) _m -, L2 is -(CH ₂ ) _P -, and B is phenyl. In one specific embodiment thereof, A is 4,4 '-bipyridyl, and L and L2 are bound to A via the nitrogen atoms of the bipyridyl moiety. embodiment, the compound is of Formula IV:

Formula IV

wherein X ^" is chloride, bromide, iodide or fluoride. In various embodiments of the above compounds of Formulae I-IV, the boronic acid substituent is in the m- or p-position of the aromatic (phenyl) ring. In further various embodiments, A is purinyl and the thiol substituent is in the 2- or 6-position of the purine ring. The compounds of the invention may be halogen salts, such as the respective bromide salts.

In various embodiments, the compounds are compounds of Formula V

Formula V

wherein X ^" is chloride, bromide, iodide or fluoride, and D is selected from the group consisting of-(CH ₂ ) _p -, -(0-CH ₂ -CH ₂ ) _n -, -(CH ₂ -CH ₂ -0) _n -, -(CH ₂ -CH ₂ -0) _n -(CH ₂ ) _m - and -(0-CH ₂ -CH ₂ ) _n -(CH2)m- ₎ wherein p is an integer from 1 to 20, n is an integer from 1 to 10 and m is an integer from 1 to 5. In one embodiment thereof, the compound is a compound of Formula VI

Formula VI

wherein X ^" is chloride, bromide, iodide or fluoride, and n is an integer from 1 to 10.

In further embodiments of the compounds of Formulae V or VI, the pyridyl rings are connected such as to form a 3,4'-, 4,3 ^? -, 3,3'- or 4,4'-bipyridyl structure. In certain embodiments, the bipyridyl benzyl boronic acid moiety is selected from the group consisting of:

N,N'-Bis-(benzyl-3 -boronic acid)-[3,4']bipyridinium Dibromide (J,4'-m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-p-BBV);

N,N'-Bis-(benzyl-3-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,3']bipyridinium Dibromide (3,3'- -BBV);

N,N'-Bis-(benzyl-3 -boronic acid)-[4,4']bipyridinium Dibromide (4,4'-w-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[4,4']bipyridinium Dibromide (^,4'-o-BBV); and

N,N'-Bis-(benzyl-4-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-p-BBV).

Further embodiments of the above compounds are the compounds, wherein the -C(O)- S-(CH ₂ -CH ₂ -0) _n -CH ₂ -SH or the -C(0)-S-D-SH group is in the m-position relative to the nitrogen atom of the pyridine ring. In one aspect of the invention, in the method according to the invention detailed above any one or more of the above-mentioned compounds are used.

In a still further aspect, the invention relates to the biosensor according to the invention, wherein the biosensor comprises one or more of the above compounds of the invention.

The present invention also relates to an optical fiber comprising a biosensor according to the invention. In another aspect, the present invention features the use of any one of the above- mentioned compounds as a monosaccharide, in particular glucose, or a-hydroxy acid receptor.

In a further aspect, the present invention relates to the use of the biosensor or the optical fiber according to the invention for the detection of an analyte. In one embodiment, the detection is in vivo. In another embodiment the detection is in vitro. In various embodiments, the analyte is a monosaccharide, such as glucose, or an a-hydroxy acid.

Brief Description of the Drawings

Figure 1 shows a schematic representation of the major components of the SERS based analyte sensing system using reporter molecules. The exemplary analyte referred to in the figure is glucose, but the claimed invention is not limited thereto. The reporter molecule can attach to the nanoparticle by electrostatic, covalent or hydrophobic interactions and upon glucose binding change the intensity or position of SERS signals. These changes can be monitored as glucose sensing event at the molecular level.

Figure 2 shows the proposed mechanism of analyte/glucose detection by SERS using a benzyl viologen as a reporter molecule and a gold nanoparticle as the SERS active surface.

Figure 3 shows UV studies of benzyl viologen boronic acids with 40 run gold nanoparticles. BBV1 has a X _max at 259.5 nm and an Au nanoparticle has a _max at 527 nm. When BBV1 and the Au nanoparticles are present together, the aggregation leads to the shift of the plasmon peak to 752 nm.

Figure 4 schematically shows the manufacturing protocol for a glucose sensing substrate (a). Figure 4 (b) shows the Emission Scanning Electron Microscopy (ESEM) characterization of the morphology of the glucose sensing substrate, and Figure 4 (c) shows SERS spectrum of BVB1 on the glucose sensing substrate. Figure 4 d) shows a Raman spectrum of BBV1 in powder form for comparison.

Figure 5 shows the response of the SERS spectrum of BBV1 upon glucose binding under physiologically relevant conditions. The actual base line is shifted from zero for clarity reasons.

Figure 6 shows the response of the four major SERS peaks of BBV1 on novel glucose sensing substrates upon glucose binding under physiological conditions.

Figure 7 shows the glucose responsiveness of the SERS peak of BBV1 under physiological conditions under different BBV1 concentrations: a) Glucose response from SERS substrate incubated with 1 μΜ BBV1 (left graph), b) Glucose response from the SERS substrate incubated with 1 mM BBV1 (right graph).

Figure 8 shows the chemical structures of benzyl viologen control compounds. The lack of glucose receptive boronic acid moieties makes them unresponsive to glucose. Figure 9 shows the chemical structures of all possible isomers of BBV1 that are synthesized and characterized with 4,4'-; 3,3'- and 4,3'- bipyridine moieties as the SERS reporting core.

Figure 10 shows the SERS spectrum of the all possible isomers of BBV1 that were synthesized to evaluate the glucose sensibility and glucose selectivity of this class of Raman reporter molecules.

Figure 11 shows the glucose response of 3,4'-o-BBV SERS spectrum peak at 1620 cm- 1.

Figure 12 shows representative examples of boronic acid derivatives that can be immobilized by hydrophobic interaction on gold nanoparticles for glucose sensing using SERS mode in solution.

Figure 13 shows the SERS spectrum of a) thionaphthene boronic acid and b) pyrene boronic acid in milliQ water after mixing with 40 nm citrate stabilized gold nanoparticles. Figure 14 shows glucose response from thionaphthene boronic acid immobilized gold nanoparticles.

Figure 15 shows the SERS spectrum of 4-(6-Mercaptopurin-9-ylcarbonyl)phenyl boronic acid (A) and 3-(6-Mercaptopurin-9-ylcarbonyl)phenyl boronic acid (B) covalently anchored via their mercapto groups on the surface on the produced SERS substrate.

Figure 16 shows the glucose response of 4-(6-Mercaptopurin-9-ylcarbonyl)phenyl boronic acid by determining the SERS intensity of the peak at 1001 cm ^"1 in a flow cell experiment where glucose concentration was systematically varied.

Figure 17 schematically shows an exemplary design of an optical fiber coupled to a biosensor according to the present invention.

Detailed Description The following detailed description refers, by way of illustration, to specific details and embodiments in which the invention may be practiced.

This invention relates to a novel method, device and compounds for detecting an analyte, specifically an analyte with poor Raman cross section, using Surface Enhanced Raman Spectroscopy (SERS).

In a first aspect, the present invention relates to a method for detecting an analyte using surface enhanced Raman spectroscopy (SERS), comprising contacting the analyte with one or more compounds selected from the group consisting of aryl boronic acid derivatives, heteroaryl boronic acid derivatives, aryl boronate derivatives and heteroaryl boronate derivatives, wherein the one or more compounds are attached to a metal substrate surface that enhances Raman scattering; and detecting a surface enhanced Raman signal from said compound.

The compounds used in this method are Raman reporters, i.e. compounds which have a high Raman cross section and the Raman vibrational "finger print" is detectably altered, for example by a shift and/or an increase in intensity, upon the binding an analyte, such as to allow detection and quantitation of the analyte. Accordingly, the compounds can also be considered to represent reporters or receptors of the analyte, for example glucose reporters or receptors. The compounds can be stably adsorbed at a surface that enhances the Raman signal from the compound, such as a nanoparticle or other SERS active substrate surface by reversible electrostatic interaction, hydrophobic interaction or covalent anchoring. Ideally, the compound has a high Raman cross section and the capability to adsorb strongly on the surface of a metal nanoparticle in aqueous media so that it gives a fast and intense and non fluctuating SERS signal that is proportional to the concentration of the analyte, such as glucose, in bulk.

Generally, SERS using Raman reporters is advantageous over the prior art glucose oxidase based systems as it is independent of oxygen. In addition, the methods according to the invention allow the detection of analytes in lower concentration ranges, such as glucose in hypoglycemic concentration range, and thus provide a more reliable readout in the analysis of patients. By varying the amount of immobilized compound at the substrate surface, the detection range can be suitably adjusted to the desired analyte concentration range thus ensuring a linear measurement range. Compared to the methods of the prior art, a further advantage of the receptors of the present invention is that they selectively, reversibly and covalently bind to the analyte; stably enough to allow a strong SERS mediated analyte detection and reversible in order to avoid a permanent loss of analyte binding sites and analyte. As the Raman reporter selectively binds to the analyte, a more specific readout is accomplished. In addition, due to the fact that the Raman reporters are adsorbed at biocompatible substrates, such as noble metals, they can detect the desired analyte under physiological conditions, meaning that the sensing components can be integrated in a minimally invasive platform, such as optical fibers or implantable substrates. This is a clear advantage over previous approaches, e.g. those using Quantum dots, which, due to their inherent toxicity, are not suitable for implantation. In addition, the substrates of the present invention bind the Raman reporters but bind other molecules to a very low extent and thus provide a longer lasting setup, in particular in an implanted biosensor setup. As the methods of the invention do not rely on fluorescence, they overcome previous limitations of methods based on fluorescent dyes or Quantum dots, as they are not hampered by photo bleaching, broad emission profiles and peak overlapping. Moreover, the SERS-based detection methods of the invention are suitable for multiplexing, which is another advantage, both in context of sensing experiments to understand complex mechanistic pathways in biological studies and in personalized medicine.

The inventors of the present invention have surprisingly found that derivatives of aryl boronic acids, heteroaryl boronic acids, aryl boronates and heteroaryl boronates are particularly useful as Raman reporters and receptors in the SERS detection methods of the invention. Usually, in absence of analyte, these receptors exist in their trigonal neutral form. The formation of the more-acidic boronate-analyte complex shifts the acid-base equilibrium of the boronic acid towards its anionic tetrahedral "-ate" form, causing electronic and/or steric changes. This change in molecular structure causes a significant change in the orientation and distance of the molecule from the nanoparticle surface leading to a change in the SERS intensity of the molecular core.

The compounds according to the invention are aryl or heteroaryl boronic acid derivatives or aryl or heteroaryl boronate derivatives. In this connection, "aryl boronic acid" or "aryl boronate" relates to organoboranes with a carbon boron bond, wherein a boric acid or salt or ester thereof is substituted with an aryl moiety. These compounds have the general structure R-B(OH) ₂ , wherein R is the aryl moiety. In the salts or esters of such compounds the hydrogen atoms may be replaced by organic residues or suitable cations. The aryl moieties of the present invention preferably comprise 5-20 carbon atoms and can comprise 1-5 rings, which are either condensed or connected by bonds. Examples of aryl moieties according to the invention include, but are not limited to phenyl, naphthyl, pyrenyl and anthracenyl. Preferred are phenyl moieties, thus forming phenyl boronic acids. Similarly, "heteroaryl boronic acid" and "heteroaryl boronate" relates to organoboranes with a carbon boron bond, wherein a boric acid or salt or ester thereof is substituted with a heteroaryl moiety. These compounds also have the general structure R-B(OH) ₂ , wherein R is the heteroaryl moiety. In the salts or esters of such compounds the hydrogen atoms may be replaced by organic residues or suitable cations. The heteroaryl moieties of the present invention preferably comprise 5-20 carbon atoms and can comprise 1-5 rings, which are either condensed or connected by bonds, wherein at least one of the ring carbon atoms is replaced by nitrogen, oxygen or sulfur. Examples of heteroaryl moieties according to the invention include, but are not limited to purinyl, indolyl, pyridinyl, bipyridyl, pyrimidinyl, azaindolyl, pyrrolyl, pyrazinyl, pyridazinyl, isoindolyl, benzimidazolyl, imidazolyl, indazolyl, chinolinyl, isochinolinyl, acridinyl and benzothiophenyl.

The term "derivatives" in relation to the compounds of the present invention means that the aryl/heteroaryl boronic acid/boronate structure can be substituted with further substituents or groups and can thus be part of a larger molecule. When the aryl or heteroaryl group is further substituted, the substituents are one or more, preferably 1-5 groups, selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, alkylaryl, alkylheteroaryl, heteroalicyclic, alkoxy, cycloalkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, hydroxy, halo, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, nitro, silyl, sulfinyl, sulfonyl, amino, and -NR'R ² where R ¹ and R ² are independently selected from the group consisting of hydrogen, C1-C4 alkyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₄ aryl, carbonyl, acetyl, sulfonyl, amino, and trifluoromethanesulfonyl, or R ¹ and R ² , together with the nitrogen atom to which they are attached, combine to form a five-or six-membered heteroalicyclic ring. In addition, two or more of the aryl/heteroaryl boronic acid/boronate derivatives may be coupled to each other thus forming a bis(benzyl boronic acid) viologen.

"Alkyl" refers to a saturated aliphatic hydrocarbon including straight chain, or branched chain groups. Preferably, the alkyl group has 1 to 10 carbon atoms (whenever a numerical range; e.g.,"l-10", is stated herein, it means that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 10 carbon atoms). More specifically, it may be a medium size alkyl having 1 to 6 carbon atoms or a lower alkyl having 1 to 4 carbon atoms e. g., methyl, ethyl, n- propyl, isopropyl, butyl, iso-butyl, tert-butyl and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is one or more, for example one or two groups, individually selected from the group consisting of C ₃ -C _¾ cycloalkyl, C ₆ -C ₁₄ aryl, 5-10 membered heteroaryl wherein 1 to 4 ring atoms are independently selected from nitrogen, oxygen or sulfur, 5-10 membered heteroalicychc wherein 1 to 3 ring atoms are independently nitrogen, oxygen or sulfur, hydroxy, C Cjo alkoxy, C ₃ -C ₈ cycloalkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, nitro, silyl, sulfinyl, sulfonyl, amino, and -NR ^J R ² where R and R are independently selected from the group consisting of hydrogen, Ci- C ₄ alkyl, C ₃ -Cg cycloalkyl, C ₆ -CH aryl, carbonyl, acetyl, sulfonyl, amino, and trifluoromethanesulfonyl, or R ¹⁰ and R ¹¹ , together with the nitrogen atom to which they are attached, combine to form a five-or six-membered heteroalicychc ring.

A "cycloalkyl" group refers to an all-carbon monocyclic ring (i.e., rings which share an adjacent pair of carbon atoms) of 3 to 8 ring atoms wherein one of more of the rings does not have a completely conjugated pi-electron system e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like. Examples, without limitation, of cycloalkyl groups are cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, adamantane, cyclohexadiene, cycloheptane and, cycloheptatriene. A cycloalkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is one or more, for example one or two groups, individually selected from Ci-Cio alkyl, C ₃ -C ₈ cycloalkyl, C ₆ -C ₁₄ aryl, 5-10 membered heteroaryl wherein 1 to 4 ring atoms are independently selected from nitrogen, oxygen or sulfur, 5-10 membered heteroalicychc wherein 1 to 3 ring atoms are independently nitrogen, oxygen or sulfur, hydroxy, Ci-Cio alkoxy, C ₃ -C ₈ cycloalkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, C-carboxy, O- carboxy, nitro, silyl, sulfinyl, sulfonyl, amino, and -NR'R ² , with R ¹ and R ² as defined above.

An "alkenyl" group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon double bond e. g., ethenyl, propenyl, butenyl or pentenyl and their structural isomeric forms such as 1-or 2-propenyl, 1-, 2-, or 3-butenyl and the like. An "alkynyl" group refers to an alkyl group, as defined herein, consisting of at least two carbon atoms and at least one carbon-carbon triple bond e. g., acetylene, ethynyl, propynyl, butynyl, or pentynyl and their structural isomeric forms as described above. An "aryl" group, as used herein in connection with the substituent groups, refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups of 6 to 14 ring atoms and having a completely conjugated pi- electron system. Examples, without limitation, of aryl groups are phenyl, naphthenyl and anthracenyl. The aryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is one or more, for example one, two, or three substituents, independently selected from the group consisting of Q-Cio alkyl, C3-C8 cycloalkyl, C ₆ - Ci ₄ aryl, 5-10 membered heteroaryl wherein 1 to 4 ring atoms are independently selected from nitrogen, oxygen or sulfur, 5-10 membered heteroalicyclic wherein 1 to 3 ring atoms are independently nitrogen, oxygen or sulfur, hydroxy, Q-Cio alkoxy, C ₃ -Cg cycloalkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, trihalomethyl, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, nitro, silyl, sulfinyl, sulfonyl, amino, and -NR'R ² , with R ¹ and R ² as defined above.

A "heteroaryl" group, as used herein in connection with the substituent groups, refers to a monocyclic or fused aromatic ring (i.e., rings which share an adjacent pair of atoms) of 5 to 10 ring atoms in which one, two, three or four ring atoms are selected from the group consisting of nitrogen, oxygen and sulfur and the rest being carbon. Examples, without limitation, of heteroaryl groups are pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1 ,2,3-triazolyl, 1 ,2,4-triazolyl, 1 ,2,3-oxadiazolyl, 1 ,2,4-oxadiazolyl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,3,4- triazinyl, 1 ,2,3-triazinyl, benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, pthalazinyl, quinoxalinyl, cinnnolinyl, napthyridinyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5, 6, 7, 8- tetra-hydroisoquinolyl, purinyl, pteridinyl, pyridinyl, pyrimidinyl, carbazolyl, xanthenyl or benzoquinolyl. The heteroaryl group may be substituted or unsubstituted. When substituted, the substituted group(s) is one or more, for example one or two substituents, independently selected from the group consisting of Ci-C ₎₀ alkyl, C ₃ -C ₈ cycloalkyl, C ₆ - C ₁₄ aryl, 5-10 membered heteroaryl wherein 1 to 4 ring atoms are independently selected from nitrogen, oxygen or sulfur, 5-10 membered heteroalicyclic wherein 1 to 3 ring atoms are independently nitrogen, oxygen or sulfur, hydroxy, Ci-C] ₀ alkoxy, C ₃ -C ₈ cycloalkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, trihalomethyl, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, nitro, silyl, sulfinyl, sulfonyl, amino,

1 2 1 2

and -NR R , with R and R as defined above. In one embodiment, if heteroaryl is pyridyl, the substituent is pyridyl to give bipyridyl. A "heteroalicyclic" group refers to a monocyclic or fused ring of 5 to 10 ring atoms containing one, two, or three heteroatoms in the ring which are selected from the group consisting of nitrogen, oxygen and -S(0)„ where n is 0-2, the remaining ring atoms being carbon. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Examples, without limitation, of heteroalicyclic groups are pyrrolidine, piperidine, piperazine, morpholine, imidazolidine, tetrahydropyridazine, tetrahydrofuran, thiomorpholine, tetrahydropyridine, and the like. The heteroalicyclic ring may be substituted or unsubstituted. When substituted, the substituted group (s) is one or more, for example one, two, or three substituents, independently selected from the group consisting of Q- Cio alkyl, C ₃ -C ₈ cycloalkyl, C ₆ -Ci ₄ aryl, 5-10 membered heteroaryl wherein 1 to 4 ring atoms are independently selected from nitrogen, oxygen or sulfur, 5-10 membered heteroalicyclic wherein 1 to 3 ring atoms are independently nitrogen, oxygen or sulfur, hydroxy, Q-Cio alkoxy, C ₃ -C ₈ cycloalkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, trihalomethyl, carbonyl, thiocarbonyl, C-carboxy, O-carboxy, nitrb, silyl, sulfinyl, sulfonyl, amino, and -NR'R ² , with R ¹ and R ² as defined above.

A "hydroxy" group refers to an -OH group.

An "alkoxy" group refers to an -O-unsubstituted alkyl and -O-substituted alkyl group, as defined herein. Examples include and are not limited to methoxy, ethoxy, propoxy, butoxy, and the like. A "cycloalkoxy" group refers to a -O-cycloalkyl group, as defined herein. One example is cyclopropyloxy.

An "aryloxy" group refers to both an -O-aryl and an -O-heteroaryl group, as defined herein. Examples include and are not limited to phenoxy, naphthyloxy, pyridyloxy, furanyloxy, and the like.

A "mercapto" group refers to a -SH group. An "alkylthio" group refers to both an S-alkyl and an -S-cycloalkyl group, as defined herein. Examples include and are not limited to methylthio, ethylthio, and the like.

An "arylthio" group refers to both an -S-aryl and an -S-heteroaryl group, as defined herein. Examples include and are not limited to phenylthio, naphthylthio, pyridylthio, furanylthio, and the like.

A "sulfinyl" group refers to a -S(0)-R' group, wherein, R' is selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, ar; yl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein.

A "sulfonyl" group refers to a -S(0) ₂ R' group wherein, R' is selected from the group consisting of hydrogen, hydroxy, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein. A "trihalomethyi" group refers to a -CX ₃ group wherein X is a halo group as defined herein e. g., trifluoromethyl, trichloromethyl, tribromomethyl, dichlorofluoromethyl, and the like.

"Carbonyl" refers to a -C(=0)-R' group, where R' is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as defined herein. Representative examples include and the not limited to acetyl, propionyl, benzoyl, formyl, cyclopropylcarbonyl, pyridinylcarbonyl, pyrrolidin-l-ylcarbonyl, and the like.

A "thiocarbonyl" group refers to a -C(=S)-R' group, with R' as defined herein.

"C-carboxy" and "carboxy" which are used interchangeably herein refer to a -C(=0)0- R" group, with R" as defined herein, e. g. -COOH, methoxycarbonyl, ethoxycarbonyl, benzyloxycarbonyl, and the like. An "O-carboxy" group refers to a -OC(=0)R' group, with R' as defined herein, e.g. methylcarbonyloxy, phenylcarbonyloxy, benzylcarbonyloxy, and the like.

An "acetyl" group refers to a -C(=0)CH ₃ group. A "carboxylic acid" group refers to a C-carboxy group in which R' is hydrogen.

A "halo" or "halogen" group refers to fluorine, chlorine, bromine or iodine.

A "cyano" group refers to a -CN group.

A "nitro" group refers to a -N0 ₂ group.

An "amino" group refers to an -NR'R ² group, wherein R ¹ and R ² are independently hydrogen or unsubstituted lower alkyl, e.g., -NH ₂ , dimethylamino, diethylamino, ethylamino, methylamino, and the like.

The term "detection", as used herein, refers to the identification of the presence or absence of an analyte and/or quantifying its amount. The analyte can be detected in a concentration range of mM, below mM, below μΜ, below nM, below pM, below fM or at a single molecule level. Surface Enhanced Raman Spectroscopy, or Surface Enhanced Raman Scattering, abbreviated SERS, is a surface sensitive technique that results in the enhancement of Raman scattering by molecules adsorbed on metal surfaces. The enhancement factor can be as much as 10 ^I4 -10 ¹⁵ , which allows the technique to be sensitive enough to detect single molecules.

Substrates suitable in the present invention comprise nanoparticles, in particular nanoparticles made of materials or coated with materials which enhance the Raman signal of a substrate-attached compound. Examples for such materials that are capable of enhancing the Raman signal of a compound attached thereto are noble metals, such as copper, silver or gold. If nanoparticles are used as a substrate, the nanoparticles may be made of plastic, ceramics, composites, glass or organic polymers and coated with the metal of choice, such as silver or gold. Alternatively, the nanoparticles may be made of a metal, for example a noble metal, such as silver or gold. Methods for the production of nanoparticles are well-known in the art and include sol-gel processes. The nanoparticles may have a medium diameter of about 1 to about 100 nm, such as 10, 20, 30, 40, 50, 60, 70, 80, or 90 nm. If gold nanoparticles are used, these may be stabilized, for example by citrate. The nanoparticles may be located on a surface and/or attached or adhered thereto. Alternatively, the surface may be an electrochemically roughened silver surface.

The compounds, i.e. the Raman receptors, which are attached to the substrate can be covalently bound, attached via electrostatic attraction or hydrophobic interaction. "Covalently bound" relates to an attachment on the surface via a covalent bond between at least one atom of the compound and at least one atom of the surface. "Electrostatic attraction" relates to attachment via salt bridges, hydrogen bonds and polar interactions, for example if the surface is charged negative and the compound bears a positive charge or vice versa. "Hydrophobic interaction" includes the interaction between uncharged and non-polar groups.

In the method of the present invention, the surface enhanced Raman signal of the compound can be correlated with the amount of the analyte in the sample. "Correlation", as used in the context of the present invention, means that the change of the SERS signal due to the binding of the analyte depends on the amount of the analyte. This correlation allows quantifying the amount of analyte. Depending on the compound used, the change of the SERS signal may be a change in intensity of certain bands and/or a shift in the peak wave numbers.

The method is suitable for the detection of an analyte in vivo, as the structures necessary for carrying out the method can be implemented on a biosensor. Alternatively, the analyte may be contained in a sample and the detection is in vitro. In any case, the method according to the invention allows detection of the analyte in a bodily fluid. This bodily fluid may be selected from the group consisting of plasma, serum, blood, lymph, liquor and urine.

The analyte to be detected according to the invented method is a monosaccharide or an a-hydroxy acid. The monosaccharide may be an aldose or ketose and can be selected from the group consisting of trioses, tetroses, pentoses, hexoses and heptoses. Exemplary monosaccharides include, but are not limited to, glucose, galactose, fructose, mannose, glyceraldehyde, dihydroxyaceton, erythrose, threose, erythrulose, ribose, arabinose, xylose, desoxyribose, ribulose, xylulose, and talose. Also encompassed by the term "monosaccharide" as used herein are sugar alcohols such as mannitol and sorbitol, sugar acids, such as glucuronic acid and galacturonic acid, or derivatives of monosaccharides, such as N-Acetyl-D-glucosamine and glucosamine. The term includes all stereoisomers, in particular the D- and the L-form and the a- and β -configuration. Exemplary monosaccharides include, but are not limited to: a-D-glucose, β-D-glucose, a-D-galactose and β-D-galactose. Preferably the monosaccharide to be detected is glucose, more preferably D-glucose, even more preferably a-D-glucose.

Also detectable according to the methods of the invention are a-hydroxy acids, including, but not limited to, glycolic acid, lactic acid, citric acid, mandelic acid, malic acid and isocitric acid. The analyte may be free in solution, either in a cell or in a bodily liquid. Alternatively, the analyte may be attached to another substance, including proteins, lipids, DNA, RNA and cells. In one embodiment, the analyte may be attached to a protein, in case the analyte is a monosaccharide thus forming a glycosylated protein. Glycosylated proteins typically comprise the following monosaccharides: N-acetyl glucosamine, mannose, fructose and galactose. By detecting these saccharides, the method of the invention also allows to detect glycosylated proteins. The glycosylated protein may be located at the outer surface of a cell or be present in a bodily fluid. Some analytes, such as monosaccharides, in particular glucose, a-hydroxy acids or glycosylated proteins, are more or less abundant in a patient with a certain disease, disorder or physical state and are thus indicators of the disease, disorder or physical state. Exemplary diseases include, but are not limited to diabetes, obesity, inflammation and cancer. Exemplary physical states include, but are not limited to hypo- and hyperglycemia. Accordingly, the methods of the invention allow monitoring an analyte which is an indicator and/or marker of these diseases/physical states in a patient. The methods allow diagnosing these diseases, disorders and physical states and establishing a prognosis. For example, the amount of glucose detected in a patient or in a patients sample may serve as an indicator of diabetes, hyper- or hypoglycemia. Alternatively, the method allows monitoring the treatment of a disease using the amount of analyte as an indicator. In patients suffering from diabetes the amount of detected analyte may indicate the need of an insulin treatment or confirm the efficiency of the insulin treatment. In patients suffering from cancer, such as prostate cancer, detection of an increased or decreased level of a specific analyte, such as a glycosylated protein, may be useful for diagnostic purpose. One analyte suitable for the diagnosis of prostate cancer includes the serum glycoprotein prostate-specific antigen.

A patient, as defined herein, is an animal, including mammals, in particular a human being.

The substrate with the attached Raman reporter compounds may also be integrated into cells. Hence, the method of the present invention also allows detecting an intracellular analyte. The SERS signal of the compound may be generated by a laser pointing to the cell, fluorescence microscope, a confocal microscope, or an optical fiber reaching to the cell. A confocal microscope may generate the SERS signal in a defined cell compartment. Thus, the analyte may be detected in a particular cell compartment. Deviation from the analyte level under control conditions may allow drawing conclusions about the state of the cell. Low or high glucose levels may indicate cell stress, in particular oxidative stress. Alternatively, the detected analyte may serve as an indicator of apoptosis. Cell compartments comprise the cell membrane, the cytoplasm, the ER, the Golgi apparatus, lysosomes, the nucleus, endosomes, lysosomes, mitochondria, chloroplasts and vacuoles.

In other embodiments, the method may be used in a FACS-based cell sorting assay. Cells comprising the analyte or cells with a certain level of analyte may be separated from each other into different vessels.

The transfer of substrates comprising Raman receptors into cells may be achieved by coupling to antibodies, liposome based transfection agents, coupling to or co- administration with protein transduction domains, co-administration with pore- forming agents, endocytosis, or electroporation.

In the methods of the invention, the compound, i.e. the Raman receptor, to be used in the method is attached to the substrate surface via electrostatic, hydrophobic or covalent interactions. In various embodiments of the methods of the invention, the compound is a phenyl boronic acid derivative, pyridyl boronic acid derivative or a bipyridyl boronic acid derivative. In preferred embodiments, the compound is a phenyl boronic acid derivative, i.e. a compound including a phenyl ring substituted with a boric acid group. Preferably the compound also includes a heteroaryl group coupled either directly or via a linker group to the phenyl boronic acid. In one specific embodiment, the compound is a boronic acid substituted benzyl viologen, for example a bis(phenyl boronic acid) viologen, or a purinyl phenyl boronic acid, such as a 3- or 4-(purin-9-ylcarbonyl)phenyl boronic acid. "Viologen" in this context relates to a bipyridinium core structure, wherein the pyridinium rings are coupled to each other by a 3,3'-, 3,4'- or 4,4'- bond.

In order to facilitate covalent coupling of the compound to the substrate surface, in particular a metal surface, the compounds can include a linker group. A preferred linker group is a linker comprising a thiol (-SH) group. The thiol group may facilitate covalent attachment to the metal surface by forming a covalent bond between the sulfur atom and a metal surface atom. The terms "thiol group" and "mercapto group" are used interchangeably herein and both relate to the -SH group. The linker may comprise an alkenyl or ethylene glycol chain, for example an ethylene glycol of the formula -(O- CH ₂ -CH ₂ ) _n - , wherein n is an integer from 1 to 10, or an alkenyl group of formula - (CH ₂ ) _P -, wherein p is an integer from 1 to 20.

In other embodiments of the method, the compound is positively or negatively charged and attached to the substrate surface by electrostatic interactions or the compound is hydrophobic and attached to the substrate surface by hydrophobic interactions. The compounds undergoing electrostatic interaction with the substrate may have a bipyridinium core due to its intense SERS response and the ease of functionalization by alkyl halides via quarternization to form a viologen. Boronic acid substituted benzyl viologens (See Figures 2 and 10) are particularly useful as SERS reporters for monosaccharide, in particular glucose, detection. SERS signal modulation occurs when the analyte binds to the boronic acid receptor moiety, which at pH 7.4 and in the absence of glucose exists in its trigonal neutral form. In the presence of glucose, the formation of the more-acidic glucose boronate ester shifts the acid-base equilibrium of the boronic acid towards its anionic tetrahedral "-ate" form, causing electronic and/or steric changes. These changes in the molecular structure can cause a significant change in the orientation and the distance of the molecule from the nanoparticle surface leading to a change in the SERS intensity of the molecular core. In a SERS platform for analyte, e.g. glucose, sensing under physiological conditions, such an approach leads to an added advantage over a fluorescent dye based approach or quantum dot based in terms of increased sensitivity coupled with the avoidance of problems such as photo bleaching, broad emission profiles and peak overlapping. Moreover, the SERS substrate based approach is easy to transfer into a fiber mode and allows to reduce toxicity issues due to the use of noble metal based, e.g. gold based, substrates (compared to the toxic fluorescent quantum dot based approach) that can be incorporated into a minimally invasive implantable device. The multiplexing capability of SERS provides another advantage both, in context of sensing experiments to understand complex mechanistic pathways in biological studies and personalized medicine.

The putative mechanism of SERS based monosaccharide sensing is illustrated by benzyl boronic acid viologen (BBV1) and using a SERS substrate that is fabricated with citrate stabilized gold nanoparticles (cf. Figure 2). Since the gold nanoparticle surface is negatively charged, the positively charged BBV1 can strongly adsorb on the substrate to due to electrostatic interactions. Moreover, the boronic acids groups in BBV1 can form a boronate complex with the citrate leading to the immobilization of the BBV1 molecule on the surface which provides a strong SERS signal upon laser excitation. Upon competitive binding of the analyte, e.g. glucose, to the receptor at the surface, the analyte-receptor complex can produce an increase in SERS intensity due to the steric and electronic changes that follow the binding event. The negative charge associated with the boronate complex that forms upon glucose binding to BBV1 leads to a change of the orientation of the molecule relative to the surface leading to a higher SERS signal.

Surprisingly, the inventors of the present invention have found out that BB VI, compared to the SERS active compound crystal violet, induces much smaller variations of the signal intensity on the substrate, probably due to the symmetric bis positive charge and the bis boronic acid groups that bind tightly on the surface of gold nanoparticle, leading to a more thermodynamically stable surface complex. Hence, the compounds of the present invention allow for more accurate analyte detection due to smaller signal variations. Compounds useful as Raman reports in the invented methods include, but are not limited to:

N,N'-Bis-(benzyl-3 -boronic acid)-[3,4']bipyridiniurn Dibromide (3,4'-w-BBV); N,N'-Bis-(benzyl-2-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-p-BBV);

N,N'-Bis-(benzyl-3-boronic acid)-[3,3']bipyridinium Dibromide (5,3 -w-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-q-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-/?-BBV);

N,N'-Bis-(benzyl-3-boronic acid)-[ ,4']bipyridinium Dibromide (4,4'-m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-p-BBV);

3-(6-Mercaptopurin-9-ylcarbonyl)phenylboronic acid;

4-(6-Mercaptopurin-9-ylcarbonyl)phenylboronic acid;

2-((3-Mercaptoethylcarbonyl)amino)-5-pyridyl boronic acid;

Pyrene-l-boronic acid; and

Benzothiophene-2-boronic acid. Depending on whether the above bipyridinium, pyrene and benzothiophene compounds are to be attached by covalent bonds or not, they may comprise an additional linker group as defined above, which, in case of the viologen compounds, may be attached to the bipyridinium core. In other embodiments of the above compounds, the bipyridinium compounds can be used as other salts not limited to the bromide salt, such as, for example, other halogen salts like fluoride, chloride or iodide salts.

In other embodiments of the above compounds, the purinyl compounds lack the mercapto group and are attached to the substrate by another linker group as defined above or by electrostatic or hydrophobic interactions.

In another aspect, the present invention relates to a biosensor for the detection of an analyte using surface-enhanced Raman spectroscopy, comprising a plurality of nanoparticles, wherein one or more compounds selected from the group consisting of aryl boronic acid derivatives, heteroaryl boronic acid derivatives, aryl boronate derivatives and heteroaryl boronate derivatives are attached to the nanoparticle surface. In these biosensors of the invention, the nanoparticles, the compounds or both can be as defined above in connection with the methods of the invention. Similarly, the analytes to be detected can be the same as those that have been defined above. Specifically, the analytes include monosaccharides, such as glucose, and alpha-hydroxy acids. In one specific embodiment of the biosensor, the biosensor is configured for quantitative detection of glucose in a physiological concentration range.

The biosensor can comprise a substrate to which the nanoparticles are adhered. Adherence may be achieved by any means, including covalent bonding, electrostatic or hydrophobic interactions. The attachment of the nanoparticles to the sensor surface may have the form of a coating, including a uniform coating or in form of spots.

In various embodiments, the biosensor is configured for in vivo and/or in vitro use.

In a further aspect, the present invention relates to compounds that are of use as Raman reporters for certain analytes and are thus useful in the methods of the invention. The compounds contemplated by the present invention are compounds of general Formula I

Formula I

wherein

A is heteroaryl and comprises at least one nitrogen atom,

L is a linker selected from the group consisting of -C(O)-, -C(0)-0-, -(CH ₂ ) _m -, - C(=CH ₂ , -C(0)-NH-, -NH-C(O)-, -0-, -S-, =CH- and -CH=, wherein m is an integer from 1 to 3;

L2 is a linker selected from the group consisting of a direct bond between A and SH, - C(0)-0-(CH ₂ ) _q -, -C(0)-S-(CH ₂ ) _q -, -(CH ₂ ) _p -, -C(0)-S-(0-CH ₂ -CH ₂ ) _n -, -C(0)-S -(CH ₂ - CH ₂ -0) _n -, -C(0)-S -(CH ₂ -CH ₂ -0)„-(CH ₂ ) _q - and -C(0)-S -(0-CH ₂ -CH ₂ ) _n -(CH ₂ ) _q -, wherein p is an integer from 1 to 20, n is an integer from 1 to 10 and q is an integer

B is aryl;

and salts thereof.

The salts of the compounds of the invention include all possible salts, in particular halogen salts. If the compounds comprise quaternary nitrogen atoms, the anion may be selected from fluoride, chloride, bromide and iodide, preferably bromide.

In specific embodiments of the invention, A comprises at least 1, at least 2, 3, 4 or more nitrogen atoms. Also contemplated is that A is an heteroaryl group consisting of two or more aromatic rings that may be condensed or directly coupled to each other. In various embodiments of the invention, A is selected from the group consisting of purinyl, indolyl, pyridinyl, bipyridyl, pyrimidinyl, azaindolyl, pyrrolyl, pyrazinyl, pyridazinyl, isoindolyl, benzimidazolyl, imidazolyl, indazolyl, chinolinyl, isochinolinyl, acridinyl and benzothiophenyl. Preferably, A is selected from the group consisting of pyridinyl, bipyridinyl and purinyl. B can be any aryl group, including aryl groups consisting of 1, 2 or more rings that can either be condensed or covalently coupled. In specific embodiments, B is selected from phenyl or naphthyl, preferably phenyl.

In one embodiment of the above compounds L is carbonyl -C(O)-. In one embodiment of the compounds of the invention, wherein L is -C(O)-, A is purinyl, L2 is a direct bond and B is phenyl, thus forming mercaptopurinylcarbonyl phenyl boronic acids or salts thereof. In alternative embodiments thereof, L2 is -(CH ₂ ) _P -.

In one embodiment, the compounds are of Formula II:

Formula II.

In these compounds, the boric acid group can be in the o-, m- or p-position relative to the carbonyl group, preferably in the m- or p-position. The mercapto group can be in the 2-, 6- or 8-position of the purinyl ring, preferably in the 2- or 6-position.

In other embodiments of the compounds of the invention, L is -(CH ₂ ) _m -. In these compounds, wherein L is -(CH ₂ ) _m -, A can be purinyl, L2 can be a direct bond and B can be phenyl, thus forming mercaptopurinyl benzyl boronic acids or salts thereof. In alternative embodiments thereof, L2 is -(CH ₂ ) _P -.

In certain embodiments of these compounds, the compounds are of Formula III:

Formula III

In these compounds of Formula III, the boric acid group can be in the o-, m- or p- position relative to the carbonyl group, preferably in the m- or p-position. The mercapto group can be in the 2-, 6- or 8-position of the purinyl ring, preferably in the 2- or 6- position.

In further embodiments of the compounds of general Formula I, A is bipyridyl, L is -(CH ₂ ) _m -, L2 is -(CH ₂ ) _P -, B is phenyl. Also included are salts of these compounds, in particular halogen salts, preferably bromide salts.

Preferably, A is bipyridyl, wherein both nitrogens are in the quaternary state, L is - (CH ₂ ) _m -, L2 is -(CH ₂ ) _p -, B is phenyl and the compound is a halogen salt. More preferably, the compound is of formula IV:

Formula IV wherein X ^" is chloride, bromide, iodide or fluoride. The boric acid substituent may be in the m- or p-position of the phenyl ring.

In still further embodiments, the invention relates to compounds of Formula V:

Formula V wherein X ^" is chloride, bromide, iodide or fluoride and D is selected from the group consisting of -(CH ₂ ) _P -, -(0-CH ₂ -CH ₂ ) _n -, -iCH ₂ -CH ₂ -0) _n -, -(CH ₂ -CH ₂ -0) _n -(CH ₂ ) _m - and -(0-CH ₂ -CH ₂ ) _n -(CH ₂ ) _m -, wherein p is an integer from 1 to 20, n is an integer from 1 to 10 and m is an integer from 1 to 5.

In one embodiment thereof, the compound is a compound of Formula VI

Formula VI

wherein X ^" is chloride, bromide, iodide or fluoride and n is an integer from 1 to 10.

In the compounds of Formula V or VI, the pyridyl rings can be connected such as to form a 3,4'-, 4,3'-, 3,3 '- or 4,4'-bipyridyl structure. In these compounds, the bipyridyl benzyl boronic acid moiety may be selected from the group consisting of:

N,N'-Bis-(benzyl-3-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-m-BBV);

N,N'-Bis-(benzyl-2 -boronic acid)-[3,4']bipyridinium Dibromide (3,4'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,4']bipyridinium Dibromide (3,4'- ?-BBV);

N,N'-Bis-(benzyl-3 -boronic acid)-[3,3']bipyridinium Dibromide (3,3'-m-BBV);

N,N'-Bis-(benzyl-2-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-o-BBV);

N,N'-Bis-(benzyl-4-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-p-BBV);

N,N'-Bis-(benzyl-3-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-m-BBV);

N,N'-Bis-(benzyl-2 -boronic acid)-[ ,4']bipyridinium Dibromide (4,4'-o-BBV); and

N,N'-Bis-(benzyl-4-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-p-BBV); Further preferred embodiments of these compounds are the compounds, wherein the -C(0)-S-(CH ₂ -CH ₂ -0)„-CH ₂ -SH or the -C(0)-S-D-SH group is in the m-position relative to the nitrogen atom of the pyridine ring. All the above compounds of the invention are useful as Raman reporters in the SERS technique, in particular the SERS-based detection methods of the invention.

Therefore, the present invention also covers the above-described methods of the invention, wherein the compounds used in these methods for analyte binding and detection are those defined above, i.e. the compounds of the invention. Similarly, in various embodiments of the biosensors of the invention, the compounds used as Raman reporters are those defined above.

In a further aspect, the present invention also relates to an optical fiber comprising a biosensor as detailed-above. The optical fiber may facilitate in vivo detection of the analyte and may be used for this purpose, for example in a method for the diagnosis of a disease by means of detecting the analyte in vivo. Alternatively, the optical fiber may be used for the detection of an analyte in vitro. In any case, the analyte may be a monosaccharide, in particular glucose, or an a-hydroxy acid.

The above compounds of the invention are also contemplated for use as a monosaccharide, in particular glucose, or a-hydroxy acid receptor. The invention thus also covers methods for the detection of a monosaccharide, in particular glucose, or an α-hydroxy acid by using any of the compounds of the invention as a reporter.

Examples

Example 1 : Synthetic protocols

Synthesis of N.N'-Bis-Cbenzylboronic acidV[4,4 ^, 1bipyridinium Dibromides

General Scheme:

General Procedure: A mixture of 4,4'-bipyridine (0.5 g, 3.2 mmol) and bromomethyl- phenylboronic acid (0.47 g, 3.8 mmol) in DMF (5 ml) was heated at 80 °C for two days. On completion of the reaction, it was cooled to 0 °C and mixed with acetone to precipitate the product. The precipitate was filtered off and washed with acetone (3 x 25 ml) to remove excess DMF. The resultant solid was dried and lyophilized to afford the N,N'-Bis-(benzylboronic acid)-[4,4']bipyridinium Dibromide. Yield l.lg (85 %) In general, the yields of the addition reaction range between 80 - 90 %.

Data:

N,N'-Bis-(benzyl-4-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-para):

1H NMR (300 MHz, CDC13): δ 9.53 (d, J=6.5 Hz, 4H), 9.76 (d, J=6.5 Hz, 4H), 8.18 (s, 4H), 7.85 (d, J=7.8 Hz, 4H), 7.55 (d, J=d, J=7.8 Hz, 4H), 5.96 (s, 4H)

N,N'-Bis-(benzyl-3-boronic acid)-[4,4']bipyridinium Dibromide( 4,4'-meta):

1H NMR (300 MHz, CDC13): δ 9.50 (d, J=6.6 Hz, 4H), 8.74 (d, J=6.6 Hz, 4H), 8.19 (s, 4H), 7.94 (s, 2H), 7.85 (d, J=7.5 Hz, 2H), 7.64 (d, J=7.5 Hz, 2H), 7.44 (t, J=7.5 Hz, 2H), 5.94 (s, 4H)

N,N'-Bis-(benzyl-2-boronic acid)-[4,4']bipyridinium Dibromide (4,4'-ortho):

1H NMR (300 MHz, CDC13): δ 9.32 (d, J=6 Hz, 4H), 8.72 (d, J=6 Hz, 4H), 8.57 (s, 4H), 7.84 (d, J=6.9 Hz, 2H), 7.49-7.45 (m, 4H), 7.33 (d, J=7.2 Hz, 2H), 6.13 (s, 4H).

Synthesis of N,N'-Bis-(benzylboronic acid)-[3.4 ^, l ^M P ^y ridinium Dibromides General Procedure: ) ₂

Step-1 :

To a solution of 3-bromopyridine (7.7 g, 0.049 mol) in dioxane (80 ml) pyridine 4- boronic acid (5 g, 0.04 mol) was added followed by the addition of a degassed solution of sodium carbonate (6 ml, 2 M solution) under inert atmosphere. Triphenyl phosphine (2.66 g, 0.01 mol) and Pd(OAc)2 (0.46 g, 2.03 mmol) were added to the reaction mixture and heated to 80 °C for 12 h. The solution was concentrated, mixed with water and extracted using ethyl acetate (2 x 100 ml). The organic layer was washed with water and brine solution, dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude material was recrystallized using an ethyl acetate - hexane mixture to afford 3,4'-bipyridine. Yield 3 g (47 %)

Step-2:

A mixture of 3,4'-bipyridine (1 g, 6.4 mmol) and bromomethylphenylboronic acid (1.37 g, 0.13 mmol) in DMF (10 ml) was heated to 80 °C for two days. On completion of the reaction, the solution was cooled to 0 °C and mixed with acetone to precipitate the product. The precipitate was filtered off and washed with acetone (3 x 50 ml) to remove excess DMF. The resultant solid was dried and lyophilized to afford the N,N'-Bis- (benzyl-boronic acid)-[3,4']bipyridinium Dibromide. Yield 2.2 g (81 %)

In general, the yields of the addition reaction range between 80 - 90 %

Data:

N,N'-Bis-(benzyl-4-boronic acid)-[3,4']bipyridinium Dibromide (3 ,4 '-para):

1H NMR (300 MHz, CDC13): δ 10.14 (s, 1H), 9.55 (d, J=6.3 Hz, 2H), 9.37 (d, J=6 Hz, 1H), 9.23 (d, J=8.1 Hz, 1H), 8.77-8.75 (m, 2H), 8.44-8.39 (m, 1H), 8.17 (s, 4H), 7.87- 7.82 (m, 4H), 7.60- 7.54 (m, 4H), 6.01 (s, 2H), 5.96 (s, 2H) N,N'-Bis-(benzyl-3-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-meta):

1H NMR (300 MHz, CDC13): δ 10.07 (s, 1H), 9.52 (d, J=6.9 Hz, 2H), 9.34 (d, J=6 Hz, 1H), 9.22 (d, J=8.1 Hz, 1H), 8.74 (d, 3=6.9 Hz, 2H), 8.44-8.39 (m, 1H), 8.19 (s, 2H), 8.16 (s, 2H), 7.94 (s, 2H), 7.86-7.82 (m, 2H), 7.63-7.60 (m, 2H), 7.47-7.39 (m, 2H), 5.98 (s, 2H), 5.95 (s, 2H)

N,N'-Bis-(benzyl-2-boronic acid)-[3,4']bipyridinium Dibromide (3,4'-ortho):

1H NMR (300 MHz, CDC13): δ 9.31 (s, 1H), 9.21 (d, J=8.1 Hz, 2H), 9.1 1 (d, J=6.3 Hz, 1H), 8.74 (d, J=6.6 Hz, 1H), 8.70-8.53 (m, 6H), 8.39-8.34 (m, lH), 7.86-7.81 (m, 2H), 7.52-7.32 (m, 6H), 6.16 (s, 2H), 6.14 (s, 2H)

Synthesis of N,N'-Bis-(benzylboronic acidV[3.3'1bipyridinium Dibromides

Step-1 :

To a solution of 3-bromopyridine (5 g, 0.032 mol) in dioxane (50 ml) pyridine 3- boronic acid (5.43 g, 0.044 mol) was added, followed by the addition of a degassed solution of sodium carbonate (6.7 g, 2M solution) under inert atmosphere. Triphenyl phosphine (2.48 g, 0.009 mol) and Pd(OAc)2 (0.7 g, 0.003 mol) were added to the reaction mixture and heated to 80 °C for 12 h. The reaction mix was concentrated; water was added and extracted using ethyl acetate (2 x 100 ml). The organic layer was washed with water and brine solution, dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude material was recrystallized using an ethyl acetate - hexane mixture to afford 3,3'-bipyridine. Yield 2 g (41 %)

Step-2: A mixture of 3,3'-bipyridine (0.5 g, 3.2 mmol) and bromomethylphenylboronic acid (0.47 g, 3.8 mmol) in DMF (20 ml) was heated to 80 °C for two days. On the completion of the reaction, the mixture was cooled to 0 °C and mixed with acetone to precipitate the product. The precipitate was filtered off ahd washed with acetone (5 x 25 ml) to remove excess of DMF. The resultant solid was dried and lyophilized to afford the N,N'-Bis-(benzylboronic acid)-[3,3']bipyridinium Dibromide. Yield 1.2 g. (87 %). In general, the yields of the addition reaction range between 80 - 90 %

Data:

N,N'-Bis-(benzyl-4-boronic acid)-[3,3']bipyridinium Dibromide (3,3 '-para):

1H NMR (300 MHz, CDC13): δ 9.99 (s, 2H), 9.33 (d, J=6 Hz, 2H), 9.11 (d, J=8.1 Hz, 2H), 8.45- 8.40 (m, 2H), 8.17 (s, 4H), 7.84 (d, J=7.6 Hz, 4H), 7.57 (d, J=7.6 Hz, 4H), 5.97 (s, 4H)

N,N'-Bis-(benzyl-3-boronic acid)-[3,3']bipyridinium Dibromide(3,3'-meta):

1H NMR (300 MHz, CDC13): δ 10.03 (s, 2H), 9.33 (d, J=6 Hz, 2H), 9.14 (d, J=6.6 Hz, 2H), 8.46- 8.41 (m, 2H), 8.16 (br s, 4 H), 7.84 (d, J=7.5 Hz, 2H), 7.68 (d, J=7.8 Hz, 2H), 7.45-7.40 (m, 2H), 5.98 (s, 4H) N,N'-Bis-(benzyl-2-boronic acid)-[3,3']bipyridinium Dibromide (3,3'-ortho): 1H NMR (300 MHz, CDC13): δ 9.8 (s, 2H), 9.08 (d, J=6.9 Hz, 4H), 8.57 (br s, 4H), 8.38 (t, J=6.9 Hz, 2H), 7.84 (d, J=7.8 Hz, 2H), 7.51-7.42 (m, 4H), 7.31 (d, J=7.2 Hz, 2H), 6.15 (s, 4H).

Example 2: Nanoparticle work: General protocols

All the glass wares were rinsed in aqua regia (nitro hydrochloric acid) and washed thoroughly with water and ethanol and oven dried before using them. The sodium citrate and gold chloride were obtained from Sigma Aldrich. The gold nanoparticles were prepared by the citrate reduction of HAuCl ₄ . The UV spectral experiments were carried using a Hitachi-9820 spectrophotometer. The TEM measurements were carried out using a JEOL 2010 transmission electron microscope. Example 3: Synthesis of gold nanoparticles

Gold nanoparticles were prepared according to the reported protocols with the following changes. 25mg of hydrogen tetrachloroaurate (III) hydrate powder was added to 200ml of distilled water and the mixture was brought to rolling boil on a hot-plate with vigorous stirring. Afterwards, about 34.2 mg of sodium citrate dehydrate powder dissolved in 3ml of distilled water were rapidly added to the stirred tetrachloroaurate solution. The mixture was further boiled for 10 minutes, during which the solution exhibited several color changes starting with yellowish then purplish and finally ruby- red. The final solution was stored at 4°C until needed.

Example 4: Substrate fabrication using chemisorption of gold nanoparticles

Microscope glass slides were cut into squares ( =4cm x 1cm) and were immersed in freshly prepared Piranha solution for 1 hour. The slides were then rinsed several times with distilled water and finally with ethanol three times and blow dried using a stream of Argon gas. These slides were then treated with a 1% solution of 3- Mercaptopropyltrimethoxy-silane solution (3MP-TMS) in hexane for 2 hours in a mechanical shaker (180 rpm) at room temperature. The soaked glass squares were incubated in a sealed glass bottle containing 1% 3MP-TMS solution. The glass slides were then washed several times with anhydrous ethanol and water respectively and dried under a stream of Argon gas. Each of the resulting 3MP-TMS treated glass slides was then put into an individual scintillation vial and treated with 10ml of Au nanoparticle solution for 24 hours to obtain the SERS substrate. These substrates were then removed from the nanoparticle solution and washed with water and stored in a dry box until use. Field Emission Scanning Electron Microscopy (FESEM) Measurements of each batch of the substrates were carried out in scanning electron microscope (JEOL, SEM6340F) to examine the surface morphology. Example 5: SERS instrumentation and measurement

Raman and SERS measurements were carried out in a Renishaw In Via Raman (UK) microscope system with an excitation laser at 633 nm. The laser intensity was set to 100% laser power. The laser beam focused on the sample, after passing through the objective lens, had a power of about 6.2 mW. The Raman system was connected to a Leica microscope, wherein the laser light was coupled through a 50 X 0.75 N.A objective lens, which was used to excite the sample and also to collect the returning Raman signal. The detector to collect Raman signals was a Peltier cooled CCD. The WiRE 3.0 software package (provided with the Renishaw system) was used for instrument control and data acquisition. A 1800 1/mm grating was chosen for the spectral measurement with a resolution of about 1 cm ^-1 . Before each set of measurements the system was calibrated with a silicon standard (520cm ^_1 ).

Example 6: SERS glucose reporters

A bipyridinium compound (N,N'-Bis-(benzyl-3-boronic acid)-[4,4']bipyridinium Dibromide (¥,4'-/w-BBV; BBV1)) was chosen as a reporting moiety for glucose sensing due to its intense SERS signal and ease of chemical functionalization by alkyl halides via quarternization of the nitrogen atoms to form viologens. Initial studies to evaluate the interaction of BBV1 with gold nanoparticles were carried out using UV visible spectroscopy and Transmission electron microscopy. Figure 3 shows the UV spectrum of gold nanoparticles in the presence and absence of BBV1. The BBV1 absorption peak was around 280 nra (Figure 3) and did not interfere with the gold absorption at 520 nm. Upon the addition of BBV 1 to the gold nanoparticle solution, an additional peak appeared around 800 nm. Upon further successive addition, the intensity at the 520 nm peak decreased and the additional peak underwent a red shift, indicating that BBV1 caused the aggregation of the gold nanoparticles. A 2D substrate array was prepared, consisting of approximately 40 nm gold nanoparticles on a modified glass support. The glass surface was modified by thiol groups (Figure 4) and chemisorption of the nanoparticles carried out using citrate stabilized gold nanoparticles (C-Au) synthesized by the Turkevich method (Kimling et al. J. Phys. Chem. B 2006, 110, 15700-15707). The C-Au substrate was incubated with 1 μΜ of BBV1 in pH=7.4 PBS and produced a surface layer of adsorbed BBVl molecules as evidenced by the stable SERS spectrum of BBVl (Figure 4). Whereas 1 μΜ BBVl alone did not produce a significant Raman signal, upon incubation of BBVl with the C-Au substrate, instantly a strong SERS signal was detected (Figure 4c) that correlates with the Raman spectra of BBVl in powder form (Figure 4d).

It was found that the detection of an SERS signal from the substrate required the treatment of the gold nanoparticles with a BBV 1 solution of an at least nanomolar concentration. Further experiments were performed which showed that the glucose binding to the sensor does not produce any considerable wave number shift in the SERS spectrum but rather produces an increase in the peak intensity. The SERS spectrum shows strong vibrational bands that can be followed for glucose sensing applications.

Since 2D nanoparticle array types of SERS substrates such as C-Au substrate were known to exhibit large point to point variations in signal intensity, optimizations of the array fabrication were carried out to reduce the variation to 5% of the average SERS intensity for a known strongly Raman active molecule, such as crystal violet on a given batch of substrate. It was noted that, compared to crystal violet, BBVl induced much smaller variations of the signal intensity on the substrate, probably due to the symmetric bis positive charge and the bis boronic acid groups that bind tightly on the surface of gold nanoparticle, leading to a more thermodynamically stable surface complex. Example 7: Glucose Detection

The glucose sensing experiments on the produced SERS substrate were carried out, first by incubating the substrate with ΙμΜ BBVl for 12 hours, followed by 30 minute incubation with glucose in a physiological range of concentrations (0-25 mM) in pH=7.4 PBS. The SERS intensity from these substrates was recorded for 10 seconds using 633 nm at a maximum laser power of 6.2 mW. A large number of glucose sensing experiments were carried out and the spectral data from a typical experiment under physiological conditions are shown in Figure 5. The BBVl SERS spectrum shows an increase in intensity that is proportional to the increase in glucose concentration. All the peaks of the BBVl SERS spectrum show a varying degree of sensitivity to glucose concentrations. The most representative peaks at six different wave numbers were followed for glucose response and demonstrated the possibility to apply the detection in a multiplexing or ratiometric approach. The glucose sensing profiles for the four major peaks are shown in Figure 6. Example 8: Optimization for low glucose levels

The glucose sensing data from Figure 6 indicates that the used configuration the SERS system was more sensitive to the higher end of the physiologically relevant concentration, i.e. 10-25 mM. In order to change the sensitivity to lower glucose concentrations, the amount of BBVl adsorbed to the nanoparticle surface was increased. Then the measurements were carried out again and the obtained data showed that the sensitivity of the assay was shifted to the lower end of the tested glucose concentrations. Figure 7 depicts the glucose sensing experiment, wherein the 1620 cm ^"1 peak intensity in the SERS spectrum was followed at different BBVl concentrations (left graph: 1 μΜ BBVl 12 h; right graph: 1 mM BBVl). The obtained results clearly demonstrate the advantages of this two component (receptor molecule-nanoparticle) approach that allows tuning the sensitivity of the SERS assay to the desired range.

Example 9: Mechanism of glucose detection

In order to investigate the mechanism of glucose sensing, bipyridine salts that lack the boronic acid moiety (4,4'-BV; 3,4'-BV and 3,3 '-BV; Figure 8) were synthesized and subjected to glucose sensing conditions. As expected, there was no glucose response when any of these compounds was used for the SERS-based glucose detection. The SERS spectrum was not significantly changed upon a systematic change in glucose concentration.

Example 10: BBV compounds for the detection of glucose

All possible isomers of BBVl were synthesized to evaluate the glucose sensibility and glucose selectivity of this class of Raman reporter molecules. Figure 9 shows all 9 possible isomers of bipyridinium boronic acids. All of these isomers showed an intense SERS spectrum (Figure 10) and are thus suitable for glucose detection and glucose selectivity experiments. The isomer 3,4'-o-BBV on a SERS substrate had excellent selectivity and dynamic range for glucose and was therefore tested in further glucose sensing experiments.

Example 11 : 3,4'-o-BBV for the detection of glucose

3,4'-o-BBV was used for glucose sensing experiments on the produced SERS substrate, first by incubating the substrate with 1 μΜ 3,4'-o-BBV for 12 hours, followed by 10 minute incubation with glucose in the concentration range of 0-100 mM in pH=7.4 PBS in flow cell configuration. The SERS intensity from this substrate was recorded for 10 seconds using 633 nm at a maximum laser power of 0.62 mW. Figure 11 shows the glucose response of 3,4'-o-BBV SERS spectrum peak at 1620 cm-1.

Example 12: Hydrophobic SERS glucose sensors

The experiments were focused on high cross section aromatic boronic acids which can bind with gold nanoparticles via hydrophobic interactions. The molecules bound to the nanoparticle surface (nanoparticle-glucose reporter complex) can be further stabilized by thiolated PEG/small molecule encapsulation to allow their use in biosensing. Upon glucose binding to the receptor, a boronate ester is formed which leads to a change in the SERS spectrum of the nanoparticle-reporter complex that can be monitored spectroscopically. Two representative examples of these molecules that allow the SERS-based glucose detection are shown in Figure 12. Compounds of this class are insoluble in aqueous media. However, suitable experimental conditions allowing their immobilization on citrate stabilized gold nanoparticles were identified. These conditions comprise the incubation of a solution of the compound with dimethyl sulfoxide (DMSO). After incubation, the reporter attached nanoparticles were centrifuged to remove the excess reporter and were functionalized with PEG or mercapto propionic acid to impart buffer stability. Interestingly, the mercapto propionic acid stabilized particles showed an efficient immobilization of the two compounds pyrene-l-boronic acid and thionaphthene boronic acid. The SERS spectra of the compounds are shown in Figure 13. The glucose response of the SERS spectrum (peak wave number shift) from the mercapto propionic acid stabilized gold nanoparticles, which were adsorbed with thionaphthene boronic acid is shown in Figure 14. This is a representative example for glucose detection with compounds of this category. The experiment clearly demonstrates that also by hydrophobic interactions the glucose reporter can be immobilized on a gold nanoparticle surface and can be utilized for glucose sensing.

Example 13: Covalentlv bound SERS glucose sensors

4-(6-Mercaptopurin-9-ylearbonyl)phenyl boronic acid and 3-(6-Mercaptopurin-9- ylcarbonyl)phenyl boronic acid were covalently anchored via their mercapto groups on the surface on the produced SERS substrate by incubating the substrate with 1 μπι of a solution of 4-(6-Mercaptopurin-9-ylcarbonyl)phenyl boronic acid or 3-(6- Mercaptopurin-9-ylcarbonyl)phenyl boronic acid in ethanol overnight. Thereafter the substrate was washed with ethanol several times to remove the non-covalently bound molecules and used in glucose sensing in a flow cell configuration as in the experiment of 3,4'-o-BBV. The SERS spectrum of these compounds is shown in Figure 15 (A: 4- (6-Mercaptopurin-9-ylcarbonyl)phenyl boronic acid; B: 3-(6-Mercaptopurin-9- ylcarbonyl)phenyl boronic acid) and the glucose response of the SERS intensity of the peak at 1001 cm-1 of 4-(6-Mercaptopurin-9-ylcarbonyl)phenyl boronic acid in a flow cell experiment where glucose concentration was systematically varied is shown in Figure 16.

Example 14: Fiber optic probe

An exemplary design of a fiber optic probe according to the invention is schematically shown in Figure 17. Such a device may be used in in vitro diagnostics or in a minimal invasive in vivo setup.

Example 15: SERS glucose sensing

Six pieces of SERS substrates were incubated with ImM BBV1 solution overnight. Afterwards, the substrates were removed from the BBV1 solution and rinsed one time with 10 mL of deionized water. The substrates were then removed from the glucose solution and placed on glass a glass microscopic slide with a cover glass. The SERS spectra of all the substrates were recorded using a 633 nm laser, as described above, with 10 second acquisition time. For each substrate the SERS spectrum was recorded at 8 different points, collecting three different accumulations at each point. Each of the BBV1 treated and SERS analyzed substrates was then immersed in 2ml glucose solutions of the following concentrations 0, 5mM, lOmM, 15mM, 20mM and 25mM in PBS for 30 minutes. The substrates were then removed from the glucose solution and placed on a glass microscopic slide with a cover glass. The SERS spectra from these substrates were recorded at eight different points, wherein for each point an average of three spectra was calculated.

Example 16: Synthesis of N SP-Bis-fbenzylboronic acid ^" )-f4.4 ^, 1 bipyridinium (polyethylene glycol thioalkyl thioester Dibromides The general scheme:

Step a:

To a solution of 3-bromo nicotinic acid ethyl ester in p-dioxane is added pyridine 3- boronic acid followed by the addition of degassed solution of sodium carbonate under inert atmosphere. Triphenyl phosphine and Pd(OAc)2 are added to the reaction mixture and heated at 100 °C for 12 h. The reaction mixture is concentrated, combined with water and extracted using ethyl acetate (2 x 100 ml). The organic layer is washed with water and brine solution, dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude material is recrystallized using ethyl acetate -hexane mixture to afford ethyl 5-pyridin-3-ylpyridine-3-carboxylate.

Step b:

5-pyridin-3-ylpyridine-3-carboxylate is dissolved in THF. To the solution concentrated HC1 is added and the solution boiled under reflux.

Step c:

To the solution of Step b thionyl chloride is added and the solution further boiled under reflux.

Step d:

For the formation of the thioester derivative, the compound of step c is mixed with the corresponding bis-thiol and under addition of Et3N in methylenchoride as solvent the reaction is stirred for 12 hrs. Step e:

The corresponding N,N'-Bis-(benzylboronic acid)-[4,4'] bipyridinium (poly)ethylene glycol thioalkyl thioester Dibromide is obtained by mixing the compound of step d and bromomethylphenylboronic acid in DMF (20 ml). The mixture was heated at 80 °C for two days. On completion of the reaction, it was cooled down to 0 °C and added to acetone resulting in the formation of a precipitate. The precipitate was filtered off and washed with further acetone (5 x 25 ml) to remove excess DMF. The resultant solid was dried and lyophilized to afford the desired N,N'-Bis-(benzylboronic acid)-[4,4'] bipyridinium (poly)ethylene glycol thioalkyl thioester Dibromide.

While the invention has been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is thus indicated by the appended claims and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced.

All documents cited or referred to are herein incorporated by reference in their entirety.