WO2016193181A1

WO2016193181A1 - Biosensors, kits and assays for measuring metalloproteinase and serine protease activities

Info

Publication number: WO2016193181A1
Application number: PCT/EP2016/062093
Authority: WO
Inventors: María Valeria GRAZÚ BONAVÍA; Mariantonietta PARRACINO; Luis UCELAY
Original assignee: Nanoimmunotech, S.L.; Lyposmol, S.L.
Priority date: 2015-05-29
Filing date: 2016-05-27
Publication date: 2016-12-08
Also published as: WO2016193181A8

Abstract

This application relates to assays for measuring metal and/or serine protease activities, such as collagenase and trypsin, in biological samples and novel biosensors for use in such assays. In particular, the present invention discloses a specific type of biosensor especially suitable for determining collagenase and/or trypsin activity and which may be visually detected via an attached colorimetric label.

Description

Biosensors, kits and assays for measuring metalloproteinase and serine protease activities. Field of the Invention

Background of the Invention

Bacterial collagenase is a metalloproteinase capable of cleaving native collagen types I, II, III, IV and V which is currently being use, among other applications, clinically to lysed adipose tissue derived from lipoaspirates for the extraction of stem cells. These stem cells are mainly later used in cell therapy medical applications.

For these specific medical applications, it is necessary to inactivate the enzyme shortly before performing the therapy in order to avoid cellular dead. In addition, it is also deemed necessary to verify that such inactivation has actually taken place and thus it is necessary to test the effectiveness of the inactivation or inhibition of the enzyme shortly before the therapeutic intervention takes place. For this purpose, using an easy, reliable and fast enzymatic assay enabling the detection of collagenase activity in a biological sample derived from i.e. a lipoaspirate, is essential.

The currently available enzymatic assays for detecting or determining collagenase activity are based on changes in physical properties, such as viscosity or turbidity, or on specific reactions of the products that are monitored using UV-Vis spectroscopy or fluorescent spectroscopy or based on the use of radio-labelled collagenase substrates. We herein describe some of these methods for detecting collagenase activity that form part of the state of the art:

A) The most basic method for determining collagenase activity is a colorimetric assay which relies on ninhidrin (2,2- dihydroxyindane-1 ,3-dione) mediated color formation. The ninhydrin assay detects the release of amino acids and peptides liberated from the breakdown of collagen. Collagen is incubated with the enzyme and the liberated peptides are measured by colorimetric ninhydrin after incubation for 5 h at 37°C. In the market there are several kits that use synthetic substrates instead of collagen (eg. Carbobenzoxy-Gly-Pro-Gly-Gly-Pro-Ala), but are also based in the use of ninhidrin for colour development. Because ninhydrin reacts with all amino acids regardless of source, an obvious problem arises when this method is used with crude enzyme preparations where the background signal may be much larger than that from cleavage of collagenase. Other disadvantage of this methodology is that in order to develop a purple color at 570nm, which is proportional to the amount of amino acids liberated, a multistep process that included the incubation of the sample at 100°C for 15-20 minutes is needed.

A second method is based on the continuous spectrophotometric measurement of the cleavage of FALGPA, which can be monitored by a decreasing absorption at 345 nm. It is not cleaved by trypsin, thermolysin or elastase. The hydrolysis follows the following reaction:

(FALGPA) collagenase FAL + Gly-Pro-Ala

Wherein FALGPA = N-(3-[2-Furyl]acryloyl)-Leu-Gly-Pro-Ala and FAL = N-(3[2-Furyl]acryloyl)- Leu

Unit Definition - One unit of Collagenase hydrolyzes 1 .0 μηηοΐβ of FALGPA per minute at 25 °C at pH 7.5 in the presence of calcium ions.

C) Lastly, we can also find methods based on the use of either radiolabeled or fluorescent labelled substrates. In the last case, the enzymatic activity of collagenase is determined by monitoring the fluorescence of specific dyes upon collagenase activity on fluorescent labelled peptides used as substrates. While both methodologies allow achieving lower detection limits, they involve the use of specialized expensive instruments.

However, none of these methods accomplish the requirements of being sensitivity enough as to allow the visual (with the naked eye) determination of at least 0,2 U/mL of active collagenase in a clinical biological sample, such as derived from a lipoaspirate, in a fast (less than 30 minutes), simple and reliable manner. Additionally, it is noted that trypsin is also used clinically to lysed adipose tissue derived from lipoaspirates for the extraction of stem cells. Therefore, there is also a need to allow the visual (with the naked eye) determination of active trypsin in a clinical biological sample, such as derived from a lipoaspirate, in a fast (less than 30 minutes), simple and reliable manner. Description of the Invention The present invention solves the above technical problem by providing a biosensor that allows for the visual (with the naked eye) determination of at least 0.2 U/mL of active collagenase in a clinical biological sample, such as a biological sample derived from a lipoaspirate, in a fast (less than 30 minutes) and reliable manner. Such biosensor is also useful for determining the presence or absence of active serine proteases such as trypsin. The aforementioned biosensor is as defined in the first aspect of the invention herein below.

A first aspect of the invention thus refers to a biosensor for detecting metal and/or serine protease activities, such as collagenase and/or trypsin activity, in an isolated biological sample, which comprises a peptide, capable of being recognized and cleavage by a metal and/or serine protease, labelled with at least one colorimetric dye, immobilized on an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL), optionally through a spacer or linker. Preferably, the metal and/or serine protease is collagenase and/or trypsin.

In a preferred embodiment of the first aspect of the invention, the peptide immobilized on the agarose support comprises, consists essentially of or consists of the following sequence: KGGPLGPPGPGG (SEQ ID NO 1 ) (from hereinafter PepX) and the metalloproteinase (from hereinafter MMP) is collagenase.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the peptide is directly bound onto the agarose support in a density equal or greater than 5 μηιοΙ of the peptide per gram of the support.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the peptide is bound to the agarose support through a spacer, wherein the agarose support comprises at least 3 μηιοΙ of the peptide per gram of the support and wherein the distance between the agarose support and the peptide is between 14 and 34.5 Angstroms. Preferably the spacer consists of polyethylene glycol (PEG) containing 4 to 45 monomers of ethylene glycol, more preferably from 20 to 25 monomers of ethylene glycol, still more preferably 23 monomers of ethylene glycol.

In another preferred embodiment of the first aspect of the invention, the peptide comprises, consists essentially of or consists of SEQ ID NO 2 (KGQGQGGKGGKGGPLGPPGPPGGCK) (from hereinafter PepXT), wherein preferably said peptide is directly bound to the agarose support in a density greater than or equal to 2.5 μηηοΙ of the peptide of SEQ ID NO 2 per gram of support; more preferably said peptide is bound to the agarose support through a spacer, in a density greater than or equal to 2.5, more preferably between 3.5 μηηοΙ and 5 μηηοΙ, of the peptide of SEQ ID NO 2 per gram of support. The biosensor of the invention comprising as its substrate peptide of SEQ ID NO 2 is capable, as shown in example 5, of detecting at least 5 U/mL of active trypsine in approximately 15 minutes at 37°C. Said biosensor is also capable of detecting the presence of active collagenase in a biological sample.

In another preferred embodiment of the first aspect of the invention or of any of its preferred embodiments, the colorimetric dye is methylene blue and the peptide is labelled with one or more colorimetric dyes.

A second aspect of the invention refers to a kit for detecting metal and/or serine protease activities, such as collagenase activity and/or trypsin activity, in an isolated biological sample, preferably for detecting the inhibition or not of metal and/or serine protease activities in a biological sample i.e derived from a lipoaspirate, which comprises the biosensor as defined in the first aspect of the invention or in any of its preferred embodiments.

In a preferred embodiment of the second aspect of the invention, the kit comprises: a. The biosensor as defined in the first aspect of the invention or in any of its preferred embodiments;

b. A positive control of metal and/or serine protease activities, such as collagenase and/or trypsin activity, and/or

c. A negative control of metal and/or serine protease activities, such as collagenase and/or trypsin activity. More preferably, the kit of the second aspect of the invention comprises the following, preferably suction-dried, products:

1 . A collagenase Substrate (PepX-MB), preferably 10 mg, for negative control (Vial

1 ) consisting of MB-SEQ ID NO 1 immobilized on an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL);

2. A collagenase Substrate (PepX-MB), preferably 10 mg, for positive control (Vial

2) consisting of MB-SEQ ID NO 1 immobilized on an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL); and 3. A collagenase Substrate (PepX-MB), preferably 10 mg, for sample (Vial 3) consisting of MB-SEQ ID NO 1 immobilized on an agarose support consisting of cross- linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL); wherein the ligand concentration contains at least 5 μηηοΙ PepX- Methylen Blue per gram of support.

Still more preferably, the kit of the second aspect of the invention comprises the following, preferably suction-dried, products:

A collagenase Substrate (PepX-MB), preferably 10 mg, for negative control (Vial 1 ) consisting of MB-SEQ ID NO 1 immobilized on an agarose support through a spacer, wherein the agarose support consist of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross- linked agarose beads of 4% in agarose (agarose 4BCL) and wherein the distance between the agarose support and the peptide is between 14 and 34.5 Angstroms. Preferably the spacer consists of polyethylene glycol (PEG) containing 4 to 45 monomers of ethylene glycol, more preferably from 20 to 25 monomers of ethylene glycol, still more preferably 23 monomers of ethylene glycol.

A collagenase Substrate (PepX-MB), preferably 10 mg, for positive control (Vial 2) consisting of MB-SEQ ID NO 1 immobilized on an agarose support through a spacer, wherein the agarose support consist of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross- linked agarose beads of 4% in agarose (agarose 4BCL) and wherein the distance between the agarose support and the peptide is between 14 and 34.5 Angstroms. Preferably the spacer consists of polyethylene glycol (PEG) containing 4 to 45 monomers of ethylene glycol; and

A collagenase Substrate (PepX-MB), preferably 10 mg, for sample (Vial 3) consisting of MB-SEQ ID NO 1 immobilized on an agarose support through a spacer, wherein the agarose support consist of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL) and wherein the distance between the agarose support and the peptide is between 14 and 34.5 Angstroms. Preferably the spacer consists of polyethylene glycol (PEG) containing 4 to 45 monomers of ethylene glycol;

Wherein the ligand concentration contains at least 3 μηηοΙ PepX- Methylen Blue per gram of support. Still more preferably, the kit of the second aspect of the invention comprises the following, preferably suction-dried, products:

1 . A collagenase and trypsine Substrate (PepXT-MB), preferably 10 mg, for negative control (Vial 1 ) consisting of MB-SEQ ID NO 2 immobilized on an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL);

2. A collagenase and trypsine Substrate (PepXT-MB), preferably 10 mg, for positive control (Vial 2) consisting of MB-SEQ ID NO 2 immobilized on an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL); and

3. A collagenase and trypsine Substrate (PepXT-MB), preferably 10 mg, for sample (Vial 3) consisting of MB-SEQ ID NO 2 immobilized on an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL); wherein the ligand concentration contains at least 2.5 μηηοΙ PepX- Methylen Blue per gram of support.

In another preferred embodiment of the second aspect of the invention or of any of its preferred embodiments, the kit further comprises reagents, recipients, pipettes, microfilter tips, centrifugal filter devices and optionally instructions for detecting metal and/or serine protease activities, such as collagenase and/or trypsin activity, in an isolated biological sample. Said instructions may preferably state the following:

Storage Conditions and Reagent Preparation: Store kit at 4°C, protected from light. Read entire protocol before performing the assay.

Assay Protocol:

Incubate vial 1 (negative control) with physiological solution only;

Incubate vial 2 (positive control) with collagenase sample before inhibition Incubate vial 3 (sample) with collagenase sample after enzyme inhibition

1 . Use 100μΙ of solution for each vial;

2. Incubate for 15 minutes at 37°C in agitation; 3. Filtrate the support with the microfilter tip and compare the colour solution with the blue scales reported in the present instructions.

A third aspect of the invention refers to a method for detecting metalloproteinase activity, preferably collagenase activity, in an isolated biological sample which comprises the following steps: a. contacting the biosensor as defined in the first aspect of the invention, or in any of its preferred embodiments, comprising as its substrate a peptide capable of being recognized and cleavage by collagenase with the biological sample; and

b. detecting cleavage of the substrate of the biosensor, wherein the level of cleavage of said substrate is correlated to the level of collagenase in said sample.

Preferably, the peptide comprises, consists essentially of or consists of sequence SEQ ID NO 1 and/or 2.

In a preferred embodiment of the third aspect of the invention, the method is for detecting metal and/or serine protease activity, preferably collagenase and/or trypsin activity, in an isolated biological sample, comprising the following steps: a. contacting the biosensor as defined in the first aspect of the invention, or in any of its preferred embodiments, comprising as its substrate a peptide capable of being recognized and cleavage by collagenase and trypsin with the biological sample; and b. detecting cleavage of the substrate of the biosensor, wherein the level of cleavage of said substrate is correlated to the level of collagenase and/or trypsin in said sample.

Preferably, the peptide comprises, consists essentially of or consists of sequence SEQ ID NO 2.

In a preferred embodiment of the third aspect of the invention or of any of its preferred embodiments, the biological sample is obtained from a lipoaspirate sample

Lastly, the present invention also refers to the use of the biosensor or the kit as described herein, for measuring metal and/or serine protease activity, such as collagenase or trypsin activity, preferably before and after inhibition during the clinical practice of stem cell extraction.

Brief Description of the figures Fig. 1 . Scheme showing the strategy designed to detect collagenase activity colorimetrically in an easy and fast way. The different cleavage sites for collagenase onto immobilized MB-PepX are also shown. Fig. 2. Plot of absorbance of remaining FALGPA versus reaction time at a fixed enzyme concentration. This figure shows how slope of the plot is different for each concentration of the substrate (FALGPA). In particular, this figure shows how by keeping the concentration of the enzyme constant (collagenase), the substrate (FALGPA), at lower concentrations, is consumed faster than at higher concentrations.

Fig. 3. Michaelis Menten Curve. This figure shows the initial rate of collagenase-catalyzed hydrolysis of FALGPA versus FALGPA concentration, including graphical determination of Km and Vmax. Fig. 4. Plot of absorbance of remaining FALGPA versus reaction time at a fixed FALGPA concentrations. This figure shows the rate of hydrolysis of the substrate using different amounts of enzyme. Only 10% of the substrate is cleaved when using collagenase solution containing 0.2 U/mL. Fig. 5. HPLC chromatograms of 1 mM (1 mg/mL) of PepX before and after treatment with 2.0 U/mL of collagenase for different time 5, 10 and 20 minutes.

Fig. 6. HPLC chromatograms of 1 mM (1 mg/mL) of PepX and after treatment with 0.5 U/mL and 2.0 U/mL of collagenase at fixed time of 15 minutes. Fig. 7. Blue intensity of the MB-PepX support functionalized with increasing applied peptide densities using carbodiimide chemistry.

Fig. 8. Characterization of the MB-PepX immobilization onto MNPs: UV-Vis spectra of input (I), supernatant (S) after chemical reaction, and washes (W1 , W2, W3).

Fig. 9. UV-Vis spectra of the supernatants of MB-PepX functionalized MNPs after incubation with 0.1 , 0.2 and 0.4 U/mL collagenase, for 15 minutes at 37°C in agitation. The supernatants of the MB-PepX functionalized MNPs were magnetically separated from the MNP suspension. Negative control refers to supernatant of MNPs not treated with collagenase. Buffer legend refers to the UV-Vis baseline correction using the buffer of incubation. Fig. 10. Photograph displays the released of MB-peptide/s, after incubation of MB-PepX- agarose with different biological samples obtained from lipoaspirates containing: 1 ) negative control (sample not containing collagenase), 2) positive control (active collagenase), 3) inhibited collagenase with serum 100%, and 4) inhibited collagenase with EDTA. MB-PepX-agarose used had a linked peptide density of 5 μηΊθΙ/g of support. The incubation was carried out for 15 minutes at 37°C.

Fig. 1 1 UV-Vis spectra of supernatants of the biological samples after incubation with MB-PepX- agarose with a linked peptide density of 5 μηΊθΙ/g of support. A) absorbance of the supernatants showing the full range of spectra between 210 and 800nm; B) absorbance spectra of supernatants showing zooming in the range between 600 and 800nm of the spectra and highlighting the absorbance peak of MB-peptide/s released in the biological samples.

Fig. 12 UV-Vis spectra of the supernatants obtained after incubation of MB-PepX activated polymeric carboxymethyl cellulose with collagenase at different concentrations (0.1 , 0.2, and 0.4 U/mL). A pale blue coloured output signal was obtained after 15 min of incubation at 37°C. A sample not containing collagenase was used as negative control.

Fig. 13 MB-peptide/s released in solution upon incubation of a buffered sample containing 0.2 U/mL with agarose support with a density of covalently linked MB-PepX of 5 μηΊθΙ/g. This blue coloured output signal was obtained after 15 min of incubation at 37°C. A sample not containing collagenase was used as negative control.

Fig. 14 UV-Vis spectra of MB-peptide/s released in solution after collagenase activity on A) MB- PepX-agarose and B) MB-AhX-PepX— agarose.

Fig. 15 Characterization of MB-PepX immobilization onto PEGylated agarose: UV-Vis spectra of input (I), supernatant (S) after chemical reaction, and washes (W1 , W2, W3,W4). The covalent immobilization of the peptide on PEG-functionalized support (1 μηηοΙ PEG/g of support) result to be around 70%.

Fig. 16 UV-Vis spectra of the supernatants obtained after 15 minutes incubation at 37°C of MB- PepX-PEG- agarose with 0.1 , 0.2, and 0.4 U/mL of collagenase. Fig. 17 Photograph of released MB-peptide/s after incubation of buffered samples containing different amounts of collagenase with MB-PepX-PEG-agarose. The MB-peptide-support used had a linked peptide density of 3 μηΊθΙ/g of support. The incubation was carried out for 15 minutes at 37°C. Fig. 18 a) Visual blue scale of increasing concentrations of PepX-MB; b) Calibration curve to correlate Absorbance at 660 nm with the amount (μΜ) of the released MB-peptide/s. Fig. 19 Photograph of released MB-peptide/s after of incubation of collagenase samples inhibited by EDTA. Inhibited samples containing different amounts of collagenase were incubated with MB-PepX-agarose with a linked peptide density of 3 μηΊθΙ/g of support. The incubation was carried out for 15 minutes at 37°C. Fig. 20 a) UV-Vis spectra of MB-PepTX at different concentration, solubilized in 10mM MES, pH6; b) Calibration curve to correlate Absorbance at 660 nm with the amount (μΜ) of the released MB-peptide/s.

Fig. 21 Visual blue scale of increasing concentrations of PepXT-MB in the range between 0 and 100μΜ. The visual LOD is achieved when at least 10 μΜ of PepXT-MB is hydrolized.

Fig. 22 Trypsin activity on QD-substrate at different concentrations in the range between 1 and 100 U/mL. The enzymatic activity has been measured continuously monitoring, for 30 minutes, the emission of fluorescence at 515 nm of the proteolyzed QD-substrate, upon excitation at 495nm.

Fig. 23 Characterization of the MB-PepXT directly linked onto carboxylated-agarose. UV-Vis spectra of input (I), supernatants (S) after chemical reaction, and washes (W1 , W2, W3, W4), of MB-PepXT at different applied densities of a) 3.5μη-ιοΙΛ} of agarose; b) δμηιοΙ^ of agarose; c) 7. δμηιοΙ^ of agarose.

Fig. 24 Characterization of the trypsin activity onto MB-PepXT directly linked onto carboxylated agarose. UV-Vis spectra of supernatants obtained after incubation of 5 and 50 U/mL of trypsin samples with supports functionalized with different densities of linked peptide: a) 2.5μη"ΐοΙΛ} of agarose; b)3^mol/g of agarose; c) δμη-ιοΙΛ^ of agarose.

Fig. 25 Calibration curve to correlate Absorbance at 660 nm with the amount (μΜ) of the released MB-peptide/s proteolysis upon 5U/ml_ of trypsin activity. Fig. 26 Calibration curve to correlate Absorbance at 660 nm with the amount (μΜ) of the released MB-peptide/s lysed upon 50U/ml_ of trypsin activity. Fig. 27 Visual blue scale of increasing concentrations of soluble MB-PepXT in the range between 0 and 100μΜ. The figure indicates that the absorbance values of the released MB- peptide upon collagenase activity is higher than the set LOD of 10 μΜ. Fig. 28 Characterization of MB-PepXT immobilization onto PEGylated agarose: UV-Vis spectra of Input, supernatants after chemical reaction, and washes. The covalent immobilization of the peptide on the PEG (1 μηιοΙ PEG/g) activated support results to be around 90%.

Fig. 29 Comparison between of trypsin activity onto MB-PepXT-PEG-agarose and MB-PepXT- agarose. UV-Vis spectra supernatant after incubation for 15 minutes at 37°C with: A) 5 U/mL and B) 50 U/mL of trypsin.

Fig. 30 Calibration curve to correlate Absorbance at 660 nm with the amount (μΜ) of the released MB-peptide/s hydrolysed upon 0.1 U/mL of collagenase activity.

Definitions

In the context of the present invention, "U/mL" refers to units of hydrolysed FALGPA per minute at 25 °C at pH 7.5 in the presence of calcium ions. In the context of the present invention, proteolytic enzymes are enzymes that perform hydrolysis of the peptide bonds that link amino acids together in a polypeptide chain. Show evidence of their evolutionary relationship by their similar tertiary structures, by the order of

catalytic residues in their sequences, or by common sequence motifs around the catalytic residues.

In the context of the present invention metal or metallo proteases or proteinases are understood as a family of proteolitic enzymes whose catalytic mechanism involves a metal.

In the context of the present invention, serine proteases are a family of proteolitic enzymes that have a common catalytic mechanism in which serine serves as the nucleophilic amino acid at the active site.

In the context of the present invention, the term "active collagenase" is understood as collagenase molecules with a quaternary structure that allow them to catalyse the cleavage of the bond between an often neutral amino acid (X) and glycine in the sequence Pro-X-Gly-Pro, which is found with high frequency in collagen. In the context of the present invention, the term "agarose beads" is understood as microspheres of agarose gels. Thus, they are composed of an inert polysaccharide polymer made up of the repeating unit of agarobiose which is a disaccharide made up of D-ga lactose and 3,6-anhydro-L- galactopyranose.

In tte context of the present invention, the term„crosslinked agarose beads" is understood as agarose beads that have been chemical crosslinked providing to the microspheres higher chemical, mechanical and thermal stability. In the context of the present invention, the term "collagenase" is understood as zinc containing endopeptidase capable of digesting native, undenatured collagen under physiological conditions of pH and temperature.

In the context of the present invention, the term "active trypsin" is understood as trypsin molecules with a quaternary structure that allow them to catalyse the cleavage of peptides on the C-terminal side of lysine and arginine amino acid residues except when either is bound to a C-terminal proline. If an acidic residue is on either side of the cleavage site, the rate of hydrolysis has been shown to be slower. In the context of the present invention, the term "trypsin" is understood as a protease including a serine residue at the active site (serin protease) with a substrate specificity based upon positively charged lysine and arginine side chains.

In the context of the present invention, the term "colorimetric dye" is understood as a molecule that possesses color as it has a visible light absorbance maximum at a particular wavelength and transmits or reflects others. Preferably, the colorimetric dye has an absorbance maximum which differs from heme proteins, characterized by the presence of Soret band in range of wavelengths between 405 and 430nm, depending of the oxidation state of the prosthetic group.

In the context of the present invention, the term "PEG" is understood as a synthetic polyether composed of repeating ethylene glycol units. In the context of the present invention, we use "heterofunctional PEG" to create the spacer, wherein "heterofunctional PEG" is understood as a PEG derivative activated at each terminus with a different functional group. Preferably containing a carboxyl and a primary amine group.

In the context of the present invention, the term "spacer" is understood as a molecule used to keep at a given distance the peptide and the surface of agarose^'s beads. Detailed Description of the Invention

The present invention provides agarose supports functionalized with labeled synthetic peptides (substrates) that preferably mimic collagen's structure. These agarose supports functionalized with labeled synthetic peptides that preferably mimic collagen^'s structure, result in a biosensor that allows for the visual (with the naked eye) determination of at least 0.2 U/mL of active collagenase in a clinical biological sample in a fast, understood as less than 30 minutes, preferably less than 20 minutes, more preferably less than about 15 minutes, simple and reliable manner. The biosensor of the present invention performs the above said determination based on the amount of label molecule released in solution upon collagenase activity (cleavage of the substrate) on the substrate (synthetic peptide), providing a different color scale that is proportional to the capability of collagenase to hydrolyze the peptide substrate attached to the support, giving the possibility of a visual and quantitative detection of its presence (see figure 1 ). During the development of the biosensor a number of synthetic peptides were tested. For this purpose, a number of enzymatic assays were performed (see example 1 ) from which the inventors selected two sequences that mimic collagen's structure and that are remarkably useful for determining collagenase activity in a biological sample, namely SEQ ID NO 1 (KGGPLGPPGPGG) and SEQ ID NO 2 (KGQGQGGKGGKGGPLGPPGPPGGCK). Nevertheless, the present invention can also be carried out by using other substrates such as, but not limited to: GPLGMRG (SEQ ID NO: 3), GPINLHG (SEQ ID NO: 4), GPSELKG (SEQ ID NO: 5), PHPFRG (SEQ ID NO: 6), GPHPFRG (SEQ ID NO: 7), GPSGIHV (SEQ ID NO: 8), VTPYNMRG (SEQ ID NO: 9), GPLQFRG (SEQ ID NO: 10), GPKGMRG (SEQ ID NO: 1 1 ), GPYGMRA (SEQ ID NO: 12), GPKGITS (SEQ ID NO: 13), GPRPFRG (SEQ ID NO: 14), GPLSISG (SEQ ID NO: 15), GPMSYNG (SEQ ID NO: 16), GPLSIQD (SEQ ID NO: 17), GPSGIHL (SEQ ID NO: 18), GPVNLHG (SEQ ID NO: 19), PSGIHL (SEQ ID NO: 20), GPFGLKG (SEQ ID NO: 21 ), GPHPMRG (SEQ ID NO: 22), GPLQMRG (SEQ ID NO: 23), DEGPMGLKC(Me)YLG (SEQ ID NO: 24), GPVNLHGR (SEQ ID NO: 25), VC(Me)PKGITSXVFR (SEQ ID NO: 26), SYPSGIHLC(Me)LQR (SEQ ID NO: 27), GPLGLHG (SEQ ID NO: 28), GPLGFRG (SEQ ID NO: 29), GPLGFRV (SEQ ID NO: 30), GPLPFHV (SEQ ID NO: 31 ), GPSPFHV (SEQ ID NO: 32), GPSPLHG (SEQ ID NO: 33), GPVNFRV (SEQ ID NO: 34), GPAPFRG (SEQ ID NO: 35), GPAPFRV (SEQ ID NO: 36), GPAPLHG (SEQ ID NO: 37), GPLPFRG (SEQ ID NO: 38), GPLPFRV (SEQ ID NO: 39), GPSPFRG (SEQ ID NO: 40), GPAPLHV (SEQ ID NO: 41 ), GPLGLHV (SEQ ID NO: 42), and GPLPLHG (SEQ ID NO: 43) and N-[3-(2-furyl)acryloyl)]-Leu-Gly-Pro-Ala (FALGPA).

In order to test the biosensor of the present invention, SEQ ID NO 1 labelled with methylene blue (MB) was used to functionalize an agarose solid support, in particular an agarose support consisting of cross-linked agarose beads, preferably cross-linked agarose beads of from 2% to 10% in agarose, more preferably cross-linked agarose beads of 4% in agarose (agarose 4BCL), by using an applied density of pepX (SEQ ID N01 ) of at least 0.05 μη-iol/g. More particularly, MB-SEQ ID NO 1 was directly covalently immobilized through its only free primary amine to carboxyl or aldehyde groups introduced onto the agarose beads surface via two different chemistries (carbodiimide and Schiff Base bond reduction) (See example 6) and using different applied peptide densities. An example of the intensity of the blue color of the functionalized agarose supports obtained by using carbodiimide for the activation of the support, at different peptide densities, is shown in figure 7. In addition, as shown in example 2, a biosensor consisting of SEQ ID NO 1 labelled with methylene blue (MB) onto an agarose solid support, in particular an agarose support consisting of cross-linked agarose beads, by using a density of at least 5 μηιοΙ pepX(SEQ ID N01 )/g, was capable of determining the presence of collagenase activity in a visual (with the naked eye) assay with a final catalytic unit of collagenase of 0.2U/ml_ which corresponded to 0.02mg/ml_ of the enzyme in less than 15 minutes at 37°C.

It is further noted, that in order to develop the aforesaid biosensor, the authors of the present invention tested different types of support materials such as hydrogels of carboxymethyl cellulose, magnetic and gold nanoparticles and agarose supports. However, surprisingly determining the required sensitivity (of at least 0.2 U/mL of active collagenase) in less than 30 minutes, preferably less than 20 or 15 minutes, was only achieved by using agarose as a solid support (see examples 2 and 3).

In addition, it is also noted that hydrolysis of the immobilized substrate depends on the time and temperature of reaction. It is a mandatory aspect of the present invention to be capable of performing the above mentioned determination in less than 30 minutes, more preferably less than 20 minutes and still more preferably about 15 minutes or less than 15 minutes. Suitable temperatures for this purpose are preferably from 20°C to 40°C degrees, more preferably from 25°C to 38°C degrees, still more preferably room temperature or 37°C. In a preferred embodiment, it is noted that by using MB-SEQ ID NO 1 directly and covalently immobilized onto an agarose support at a temperature of 37C and at a linked peptide density of at least 5 μηηοΙ pepX(SEQ ID N01 )/g, the peptide hydrolysis after only 15 minutes of enzymatic reaction at 37°C revealed a clearly visible blue color in solution by using a concentration of collagenase of about 0.2 U/mL. In fact, upon collagenase activity the modified solid support releases the labeled peptide in solution as it is shown in figure 13.

In addition, SEQ ID NO 1 labelled with methylene blue (MB) was used to functionalize an agarose solid support by using different applied densities of the peptide using carbodiimide chemistry. The absorbance values of the supernatants and the relative concentration of the MB- peptide/s released in solution upon incubation of 5 U/mL (0,5 mg/mL) of collagenase with the biosensor is summarized in the table below. Table 1. Amount of Methylene Blue (MB)-peptide/s released from functionalized MB-PepX-agarose with different densities of linked peptide/g of support. The molar absorption coefficient of MB at 660 nm used was 0,0227 μΜ^" αη^"1.

From the results obtained it is possible to conclude that a minimum density of peptide on the support is needed (> 5 μηηοΙ PepX/g), when a biosensor consisting of SEQ ID NO 1 labelled with methylene blue (MB) is directly bound onto an agarose solid support is used, in order to detect the presence of at least 0.2 U/mL of active collagenase at 37°C in less than 30 minutes, preferably in about or less than 15 minutes. It was also concluded that immobilizing the peptide via carbodiimide chemistry onto agarose support was better than using Schiff base bond reduction chemistry and that the use of a spacer between the peptide and the support surface reduces the minimal amount of the peptide that is necessary in order to detect the target collagenase concentration in about 15 min at 37° C (see example 4).

In addition, it has also been demonstrated that by using a biosensor consisting of SEQ ID NO 1 labelled with methylene blue (MB) directly bound onto an agarose solid support, in particular an agarose support consisting of cross-linked agarose beads, by using a density of at least 5 μηιοΙ PepX (SEQ ID N01 )/g, the amount of hydrolyzed labeled peptide in solution is correlated with the enzyme concentration (see figure 17). In fact, collagenase activity is not affected by difference in buffer composition or pH (such as 10mM MES pH 6 and 50 mM Tricine, 10mM CaCI₂ and 450 mM NaCI pH 7.5) Additionally, the amount of hydrolyzed peptide, upon collagenase cleavage, was quantified, resulting to be within the range of linear relation between the absorbance and the concentration of the labelled peptide used in solution to obtain the standard blue scale (see figure 18).

Lastly, a biosensor consisting of SEQ ID NO 1 labelled with methylene blue (MB) directly bound onto an agarose solid support, by using a density of at least 5 μηιοΙ pepX (SEQ ID N01 )/g, was also tested in presence of several collagenase inhibitors such as ethylenediaminetetraacetic acid (EDTA). The results show that when collagenase is inhibited there is not blue color released in solution (see figure 19). Therefore, the present invention provides, among other embodiments, novel biosensors for determining the presence of active metal proteases or serine proteases such as collagenase or trypsin in a biological sample. Such biosensors comprise peptides comprising sequences that are recognized and cleaved by collagenase and/or trypsin. These substrates are labelled with a colorimetric dye such as MB. It is noted that the present invention is not restricted to the determination of active collagenase enzyme but, as shown in example 5, the biosensors of the present invention are also capable of determining the presence of at least 5 U/mL of active trypsin in a biological sample, therefore the present biosensors are useful for all types of serine proteases. In order to carry-out the present invention, peptides are described herein that demonstrate increased selectivity and/or specificity for collagenase and/or trypsin over peptide substrates of the prior art, and may visually (with the naked eye) detect these enzymes at low concentrations in a biological sample. "Specificity" in the context of this application means the fractional turnover of a substrate per unit of time (units s-'M-1 ), or in other words, how quickly a unit of an enzyme converts a substrate.

"Selectivity" is the ratio of specificity of two enzymes for a particular substrate, or in other words, the speed of a first enzyme divided by the speed of a second enzyme against the same substrate.

In general, peptide substrates having the selectivities and specificities described herein have been enumerated above or might comprise a consensus sequence of the formula P4-P3-P2-PI- ΡΓ-Ρ2'-Ρ3'-Ρ4', wherein a typical sequence comprises: (a) a glycine residue at position P4; (b) a proline residue at position P3; (c) an amino acid residue at position P2 that is selected from the group consisting of leucine, serine, valine, alanine, methionine, histidine, arginine, lysine, tyrosine and isoleucine; (d) an amino acid residue at position P1 that is selected from the group consisting of glycine, proline, serine, asparagine, glutamine and glutamate; (e) an amino acid residue at position PV that is selected from the group consisting of leucine, phenylalanine, methionine, isoleucine, tyrosine and methionine; an amino acid residue at position P2' that is selected from the group consisting of histidine, arginine, serine, asparagine, glutamine, lysine and threonine; (g) an amino acid residue at position P3' that is selected from the group consisting of glycine, valine, alanine, serine, leucine and aspartate; and (h) a natural or synthetic amino acid residue at the P4'position that can attach to a detectable label. The detectable label could also be attached to an aminoacid at the amino-terminal end of the peptide sequence. This aminoacid could be linked directly by its C-terminal side directly to the peptide sequence containing one or more enzyme cleavege sites. Another design implies its covalent linking trough a peptide spacer containing a variable number of aminoacids. It will also be possible to include internal amino acids capable of attaching to a detectable label. It should be understood that "attached" encompasses both "incorporated" labels (whereby a labeled amino acid is incorporated into the peptide sequence during synthesis) and "conjugated" labels (which are attached to peptide following synthesis). It may also be possible to replace a side chain of an amino acid, thereby changing the nature of the labelled amino acid in the process of the labelling reaction.

Peptides having longer sequences than the consensus sequence shown above are also encompassed as illustrated herein. It may also be possible to substitute the various amino acids listed through-out the present specification with other natural, unnatural and synthetic amino acids having similar properties. For instance, and in a preferred embodiment for detecting collagenase III, the inventors envision that appropriate amino acids at position P2 may be aliphatic, hydrophobic and/or positively charged. Similarly, appropriate amino acid residues for position P1 may be aliphatic and/or hydrogen bonding.

Methods of attaching colorimetric labels to amino acids and other materials, and methods of using labels to detect and monitor enzyme reactions and conversion of substrates, are well known in the art. For instance, The Handbook of Fluorescent Probes and Research Products published and frequently updated by Molecular Probes, Inc. provide a comprehensive overview of appropriate labels for particular methodologies, including compounds for labelling peptides.

The biosensors described herein may be used in methods for detecting active metal or serine protease enzymes in biological samples. For instance, a method for detecting the presence of collagenase I in a sample may comprise (a) contacting the sample with at least one biosensor as described herein, and (b) detecting cleavage of said substrate, wherein the level of cleavage of said substrate is correlated to the level of active metalloproteinase enzyme in said sample. Some embodiments included herein comprise adding at least one further biosensor for one or more competing metal or serine proteases at step (a). A "further" biosensor may comprise a natural further substrate for the competing metal or serine protease or an internal peptide sequence of said natural substrate. "Natural" substrates refer to substrates for metal or serine protease enzymes encountered in nature, such as type 1 collagen, type II collagen, fibronectin, and gelatin, for instance. Such "further" substrates will also be attached to a detectable colorimetric label according to the methods described herein.

The methods reported herein are applicable to measuring activities of metal or serine protease in any biological sample, preferably in a biological sample obtained from lipoaspirates. For example, the methods reported herein could be used to measure the purity, stability or remaining activity of MMP enzyme preparations, for instance after a period in storage in order to monitor any enzyme breakdown. The substrates reported herein may be attached to the solid agarose supports using any suitable linker, ligand or chemical attachment means as long as the distance between the solid support and the substrate or peptide is between 14 and 34.5 Angstroms, or alternatively may be covalently linked directly on the solid support.

Also envisioned are multiple enzyme/multiple substrate embodiments which include methods of determining the concentrations of multiple active enzymes in a sample, comprising (a) contacting the sample with one or more biosensors of the invention comprising multiple substrate reagents; (b) determining the reaction rate for each individual substrate/enzyme interaction; and (c) comparing the determined reaction rates to standard reaction rates based on known quantities of each enzyme to calculate the amount of each enzyme present in said sample. Such methods are applicable to any mixed sample of enzymes, or any non-enzyme system, i. e. catalyzed reactions, for which specific substrates, ligands, etc. are available for measuring the activities of sample components. More specifically, such multiple enzyme/multiple substrate methods comprise: a) dividing the sample into a number of portions, the number of portions equaling the number of substrate biosensors used; (b) contacting the sample with multiple substrate reagents, each sample portion contacting one substrate reagent in a separate vessel (well); (c) determining the reaction rate for each sample portion exposed to its substrate reagent; (d) contacting known quantities of each enzyme with multiple substrate reagents, each substrate reagent contacting each enzyme in a separate vessel (well); (e) determining the reaction rate for each enzyme exposed to each individual substrate reagent; (f) formulating a set of simultaneous equations relating the reaction rates and concentrations of each enzyme to the reaction rate of the sample portion; each equation relating the reaction rates for one of the substrate reagents; and (g) solving the set of simultaneous equations from (f) to calculate the concentration of each tested enzyme present in said sample. The multiple enzyme/multiple substrate embodiments may be performed with any number of substrate reagents and enzymes.

Lastly, the present invention provides, as a preferred embodiment, the novel biosensors or kits described herein for detecting inhibition of metal or serine proteases. Commonly inhibitors that could had been used might comprise chelating compounds (such as EDTA, 1 ,10- phenanthroline, etc), classical lock and key inhibitors also known as transition state analogues, proteinaceous inhibitors, alkylating, acylating, phophonylating and sulfonylating agents. All references and patents cited in this application are incorporated by reference in their entirety. The following examples are merely illustrations of the many embodiments covered by the present invention, and should not be construed as limited the claims in any way. Those of skill in the art upon reading the exemplary embodiments will immediately envision many similar assays and methods which should also be considered as a part of the invention.

Examples

Example 1. Comparison of enzymatic activity using peptide N-(3-[2-uryl1acryloyl)-Leu-Glv-Pro- Ala (FALGPA) and KGGPLGPPGPGG (PepX) as substrates.

In these examples the following was determined:

• Enzymatic activity of C. Histoliticum collagenase using N-(3-[2-Furyl]acryloyl)-Leu-Gly- Pro-Ala (FALGPA): spectrophotometric assay monitoring the decrease of absorbance at 345nm.

· Determination of the enzyme kinetic constants using FALGPA as substrate

• Enzymatic assay of Collagenase using as substrate peptide KGGPLGPPGPGG (PepX), at different concentrations of collagenase and substrate: activity monitored with the modification of elution pattern of the hydrolyzed peptide in HPLC chromatogram. a. Quantification of enzymatic activity of collagenase using peptide N-(3-[2- furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA). Determination of the enzyme kintetic constants (Km and Vmax) The enzymatic activity was monitored using different concentrations of the substrate (FALGPA) in order to calculate the kinetic parameters of collagenase. The assay was carried out in a final volume of 200μΙ, measuring the decrease of absorbance at 345nm of N-(3-[2-Furyl]acryloyl)- Leu-Gly-Pro-Ala in a concentration range between 0.3125 mM and 5mM in a 50mM buffer tricine containing 10mM CaCI₂ and 400mM NaCI at pH 7.5.

The slope of the plot is different for each concentration of the substrate. In fact, if we keep the concentration of the enzyme constant, the substrate, at lower concentrations, is consumed faster than at higher concentrations as shown in figure 2.

Afterwards, the rate of the enzymatic activity was calculated for each concentration of the substrate and the Michaelis-Menten plot was constructed in order to determine the K_M and Vmax of reaction as shown in figure 3. The Vmax represents the maximum rate achieved by the system, at saturating substrate concentrations, and the Km is the substrate concentration at which the reaction rate is half of Vmax. In this case the Vmax is 0.04 OD/min, (18.9μη"ΐοΙ/η"ΐίη) and the corresponding Km is 0.5 mM of FALGPA. b. Enzyme detection limit using the peptide FALGPA

Once the Km was calculated as shown above, the limit of detection of collagenase using a fixed amount of FALGPA (1 mM) was determined.

The enzymatic assay was performed by using different concentrations of the enzyme in a final volume of 250μΙ, measuring the decreased of absorbance at 345nm of 1 mM N-(3-[2- Furyl]acryloyl)-Leu-Gly-Pro-Ala, in a 96 wells plate:

• 200μΙ of 1 mM FALGPA in 50mM buffer tricine containing 10mM CaCI₂ and 400mM NaCI pH 7.5;

• 50 μΙ of Collagenase at the respective concentrations of 0.025mg/mL, 0.05mg/mL, and 0.1 mg/mL.

The activity was measured continuously for 5 min (see figure 4) by using the following equation.

\(( ^^ in Test -^ m?i±n¾lank) / W ol tot*dilution factor )l J

Units/mL=^ ^m

(Coeff Ext Mol*Vol enzym)

Coefficient Extinction Molar 0.53 Mem

Total volume= 0.25mL

Dilution factor= 5

Volume of enzyme= 0.050mL

The enzymatic activity of each collagenase sample, is reported in the table below (table 2) Table 2.

Around 20% of the substrate was hydrolyzed after 5 minutes by using 0.1 mg/mL of collagenase. Lower concentrations of the enzyme, such as 0.05 or 0.025 mg/mL, hydrolyzed less than 10% of the substrate after 5 minutes of reaction. At any rate, the three concentrations of the enzyme were detected by using this substrate. c. Quantification of enzymatic activity using the peptide: KGGPLGPPGPGG (PepX)

Collagenase activity was also tested by using SEQ ID NO 1 (KGGPLGPPGPGG) (PepX) (MW=990.13) (PepX). This larger peptide has three sites of cleavage between glycine and proline residues. For the quantification of the enzymatic activity, the assay was performed in a volume of 250μΙ as follows:

100μΙ of 2.5 mg/mL PepX for a final concentration of 1 mg/mL.

100μΙ of 0.5 mg/mL Collagenase for a final concentration of 0.2 mg/mL (2 U/mL)

50μΙ of 50mM buffer tricine containing 10mM CaCI₂ and 400mM NaCI pH 7.5

The reaction was carried out at 37°C for different times, in particular respectively for 5, 10, 20 minutes. The reaction was stopped by adding 50μΙ of 1 M EDTA in order to remove the Ca²⁺ ion (molar ratio 20X) and stop the enzymatic activity.

The collagenase activity was observed by monitoring the variation of the elution of PepX peak using a C8 HPLC column and H₂0-TFA 0.1 %- AcCN 90%/H2O 10%-TFA 0.1 % as the mobile phase, using a linear gradient of 30 min till 50% of AcCN90%H2O10%-TFA 0.1 %.

In figure 5 the chromatograms of the peptide alone and after different times of incubation with collagenase is displayed.

The PepX not hydrolyzed was eluted after 7 minutes and the area of the peak was 3.19. When the peptide was incubated with collagenase new peaks corresponding to small fragments of the PepX appeared at different elution times. After 5 minutes of collagenase treatment the area of PepX peak was reduced (2.12), envisaging that already after 5 minutes 32% of the peptide was is cleaved. After 10 minutes of reaction, the peptide was 100% hydrolyzed.

d. Enzyme detection limit using peptide PepX After studying time dependence test of the collagenase's proteolytic activity on PepX, the effect of enzyme concentration in the assay was tested.

PepX at a final concentration of 1 mM (1 mg/mL), solubilized in 50mM buffer tricine containing 10mM CaCI₂ and 400mM NaCI pH 7.5, was tested at different concentrations of the enzyme in a range between 0.050 mg/mL (0.5 U/mL) and 0.2 mg/mL (2.0 U/mL).. The reaction was carried out in 50mM buffer tricine containing 10mM CaCI₂ and 400mM NaCI pH 7.5 at 37C for 20 minutes. The assay was performed in a final volume of 250μΙ.Τϊιβ reaction was stopped by adding 50μΙ of 1 M EDTA in order to remove the Ca₂ ⁺ ion (molar ratio 20X). In order to remove the enzyme before HPLC injection, 300μΙ of each sample were filtrated by using the centrifugal filter Amicon Ultra, with a cut off of 30K. The samples were centrifuged for 15 min at 13400 rpm. Collagenase activity was observed by monitoring the variation of the elution of PepX using a C8 HPLC column with a mobile phase H₂0-TFA 0.1 %- AcCN90%-H2O10%-TFA 0.1 %.; the sample was eluted with a linear gradient of 15 m, from 0% till 25% of AcCN90%-H2O10%-TFA 0.1 %.

Collagenase treatment produced an alteration of the HPLC elution time of the PepX, as well as the peak area, which is directly related with the concentration of PepX and the concentration of the enzyme.

The chromatogram of 1 mM (1 mg/mL) of PepX treated with 0.5 U/mL of collagenase differs from the chromatogram obtained by using the untreated PepX, as shown in figure 6.

Even lowering 4 times the enzyme concentration a 75% of hydrolysis is achieved. These results clearly point out to the possibility of reaching a lower limit of enzyme activity detection using PepX as substrate than by using the shortest peptide.

In conclusion, this example demonstrates that C. Histoliticum collagenase is capable of hydrolyzing both substrates, FALGPA and PepX, being the enzymatic activity higher for PepX than for FALGPA.

Example 2. Determination of collagenase activity using MB-PepX directly and covalently immobilized onto an agarose support For this assay we tried the determination of collagenase activity of biological samples obtained from lipoaspirates using MB-PepX directly covalently immobilized via carbodiimide chemistry onto a carboxy-modified agarose support. The procedure carried out was as follows:

500μΙ of an isolated lipoaspirate biological sample was centrifuged for 5 min at 700 g. The centrifugation step produced 3 layers: The upper layer corresponded to the liquid fat; the second layer to the fat containing the SVF (stromal vascular fraction) and the third layer to the red cells.

An aliquot of 200μΙ obtained from the second layer was treated with 200μΙ of collagenase from C. Histoliticum, previously solubilized in 20 mL of saline buffer (1 vial). The sample was incubated for 30 minutes at 37°C by using a gentle agitation.

After incubating the sample it was centrifuged for 5 minutes at 800 g to obtain a supernant. Then the supernatant containing the exogenous collagenase, was treated with two dilutions of patient's serum, respectively 200μΙ (hereafter named as 100 %) and 100μΙ (hereafter named as 50 %) of serum, and then a saline buffer was added in order to obtain a final volume of 1 .6 mL. After this enzyme inhibition step, the sample was again centrifuged in order to separate the cellular part from the supernatant containing the inhibited enzyme. The final catalytic unit of collagenase to be detected in this assay was 0.2U/mL which correspond to 0.02mg/mL of enzyme.

As controls we prepared two additional samples, one without collagenase (negative control), and another with collagenase not inhibited (positive control).

For testing the enzymatic activity, 10mg of MB-PepX directly bound to agarose support with a density of linked peptide of 5 μηΊθΙ/g of support was used for each of the following 5 samples:

Biological sample without collagenase (NC) (tissue protease endogenous activity)

Samplel : collagenase from C. Histoliticum in real sample (positive control: active enzyme)

Sample 2: 100% serum/collagenase from C. Histoliticum in real sample (serum inhibited sample 1 )

Sample 3: 50% serum/collagenase from C. Histoliticum in real sample (serum inhibited sample 2)

Sample 4: 0,1 M EDTA/collagenase from C. Histoliticum in real sample (positive control of inhibition) 10ΟμΙ of each sample was used for the enzymatic assay, which was carried out for 15 minutes at 37°C in agitation. After the incubation period, the solid supports were filtrated and the supernatants were analyzed as it is displayed in figure 10. In addition, the relative UV-Vis spectra of each sample was measured and displayed as shown in figure 1 1 . From the results obtained it is possible to conclude that the sample in absence of active collagenase fails to show a visually detectable blue color; however, the supernatant of the sample containing the non-inhibited enzyme shows a visually detectable blue color due to the MB released in solution, corresponding to amaximum of absorption at 662nm in the Vis spectra. These results lead to the conclusion that basal endogenous collagenase fails to cause the hydrolysis of the immobilized substrate. In contrast, the hydrolysis of support linked peptide and therefore the release of MB in solution is only due to the catalytic activity of the exogenous C. Histoliticum added for tissue treatment. In addition, the intensity of the blue colour decreases when the enzyme is in presence of collagenase activity inhibitors (e.i. patient^'s serum, EDTA), this fact becomes clear when comparing samples 2 or 3 with the negative control (sample no treated with collagenase, sample 4), indicating that the inhibited enzyme is not able to hydrolyzed the MB labelled peptide immobilized on solid support.

Example 3. Immobilization of the PepX-MB in different types of solid supports.

PepX-MB peptide (as substrate) was immobilized in different solid supports as follows: · Immobilization of PepX-MB onto carboxylated magnetic nanoparticles (200 nm) and onto carboxy-pegylated AuNPs (30 nm);

• Immobilization of PepX-MB onto carboxymethyl cellulose (hydrogel). a. Immobilization of PepX onto magnetic nanoparticles

The immobilization of 9μg of PepX-MB onto 40mg of MNP of 200 nm of diameter, functionalized with -COOH groups, was tested at different conditions, such as at 10mM MES pH6 and at 20mM HEPES pH8 450 mM NaCI, using the EDC/NHS chemistry and a conjugation ratio between the peptide and the nanoparticles of 5:1.

After mixing all these components, the reaction was carried-out for a period of 2 hours at room temperature. The MNPs were then washed and the supernatants of the reaction characterized using UV-Vis spectroscopy as it is shown in figure 8. MB-PepX functionalized MNPs were then incubated with collagenase at 0.01 ,0.02 and 0,03 mg/mL (0.1 U/mL, 0.2U/ml_ and 0.3 U/mL) for 15 minutes at 37°C in agitation. Afterwards, MB- PepX functionalized MNPs were magnetically separated from the supernatant, and this was analyzed by using UV-Vis spectroscopy. The spectra of the supernatants showed a low absorbance at 660nm as it is showed in figure 9.

The experiment was repeated by using MB-PepX immobilized on pegylated-AuNPs of 30nm. Again, the immobilization of the peptide in this case was also ineffective. b. Immobilization of PepX-MB onto carboxymethyl cellulose

As shown in figure 12, collagenase activity on activated polymeric carboxymethyl cellulose with PepX-MB produced a low intensity of color in solution. This could be due to the high level of crosslinking within the active groups of the hydrogel and the peptide that induces steric hindrance and blocks the proteolytic activity of the enzyme.

Example 4. Use of spacers in the biosensors of the invention.

In the present invention we have tested the functionalization of agarose solid supports by using a substrate containing a spacer between the aminoacidic sequence and the MB (hereafter named as MB-AhX-PepX). The extinction coefficient of the peptide was calculated and resulted to be 5,40^*10⁴M^"1cm^"1. The higher extinction coefficient of this peptide, compared with the calculated coefficient of the PepX (5,19^*10⁴ M^*cm^"1), resulted in a darker blue intensity.

The peptide was directly immobilized covalently onto carboxylated- agarose support. After proving the effectiveness of the peptide immobilization, the enzymatic assay was carried out using three different concentrations of collagenase. Unfortunately the catalytic activity of the enzyme could not be detected as shown in figure 14. The intensity of the absorbance of MB released in solution upon collagenase activity on MB-PepX-support is 7 times higher than the intensity of the Absorbance of MB released upon enzymatic activity on the MB-AhX-PepX- support.

Therefore, although the spacer between the peptide sequence and the dye improved the molar extinction coefficient of the dye, it introduced a steric hindrance which blocked the enzyme activity.

We also tested the carboxy-modified agarose functionalization with a bi-functional PEG (MW 1000 Da) containing an amino and a carboxyl group at each terminus. The amino group allows its covalent attachment via carbodiimide (EDO) chemistry to carboxylated-agarose support. Once the PEG was covalently bound to the agarose support, its COOH groups were activated via EDC chemistry in order to link the MB-PepX peptide via its only primary amino group. The pre-activation of carboxylated-agarose with PEG improve the accessibility of the bound peptide reducing steric hindrance, making the collagenase able to better reached and cleaved the labelled peptide.

The agarose PEGylation was carried out using different densities of PEG (in a range between 0.5 to 10 μηΊθΙ/g of agarose). As a density of δμηηοΙ^ of MB-PepX was the optimal in terms of output signal/cost when attaching the peptide directly to the support, the same amount of peptide was offered to different obtained PEGylated agarose supports. The covalent immobilization of the peptide on the different PEG activated supports results to be 70%, as it is shown by the UV-Vis spectrain figure 15. However, the best PEG density in terms of enzyme activity was 1 μηηοΙ of PEG/g of agarose.

In order to improve the yield of peptide immobilization, and avoid losing unbound peptide when using a density of PEG of 1 μηηοΙ of PEG/g of agarose as spacer, it was possible to lower the amount of applied MB-PepX to the PEG-modified support, from 5 to 3 μηΊθΙ/g of labelled peptide. By using this latter amount of applied peptide nearly 100% of the peptide was covalently attached to de surface. Collagenase proteolysis of immobilized MB-peptide, using different concentration of collagenase (0,01 mg/mL; 0,02 mg/mL and 0,04 mg/mL corresponding to 0.1 U/mL, 0.2 U/mL, 0.4 U/mL), induced the release of MB in solution. The relative absorbance of the supernatants were measured and shown in figure 16. The release of MB-peptide/s in solution, upon collagenase activity, was improved almost 30 % compared with the enzymatic activity on the MB-Pep support in which the labeled peptide was bound directly to the support at a density of 5 μηΊθΙ/g of MB-PepX.

Example 5. Proof of concept of the trypsin activity on the peptide functionalized solid support of the invention. a. Peptide design

We designed a peptide labelled with methylene blue capable of be hydrolysed by trypsin. The MB-KGQGQGGKGGKGGPLGPPGPPGGCK (PepXT) has four possible sites of cleavage for trypsin. In addition, it also contains three possible cleavage sites for collagenase.

This synthetic peptide constitutes a magnificent substrate to determine the presence of trypsin and collagenase activity in a biological sample, since it offers cleavage sites compatible for both proteolytic enzymes. b. UV-VIS Characterization of PepXT

Peptide PepXT labelled with methylene Blue after solubilisation in 10mM MES pH was characterized by using UV-VIS spectroscopy. In particular, absorbance in the range between 210 and 850 nm was measured at different concentrations of the peptide, in particular, in the range between 0 and 100μΜ. The results are shown in figure 20

The calculated extinction coefficient of the Methylene blue labelled PepTX was 1 ,87^*10⁺⁴ M^" ^1*cm^"1, which was lower from coefficient for MB-PepX (5.19^*10⁺⁴M^*cm^"1).

The blue colour of the peptide solution is clearly visible for concentrations higher than 10μΜ of the peptide as shown in figure 21. It is noted that lower concentrations of released MB-peptide/s could be detected by using a spectrophotometer. c. Characterization of the trypsin activity and LOD detection of a commercially available detection kit based on fluorescence.

Trypsin from bovine pancreas from Sigma (T8003-500mg; #SLBK4769V) 10.000 U/mg, was solubilized in 1 ml_ of buffer 50mM tricine, 10mM CaCI₂, 0.45mM NaCI pH7. The enzyme had been opportunely diluted at O-l pg/mL (1 U/ml_), 1 g/mL (10U/ml_), and 10 g/mL (100U/ml_) and tested with the EnzChek assay kit (LifeTechnologies). In this kit, the protease substrate is heavily labelled with fluorescein (QD-substrate), but the fluorescence was quenched. The substrate emitted fluorescence at 515 nm upon excitation at 495nm, upon protease activity such as in the case of the trypsin, as well as for the collagenase. The increase of fluorescence was proportional to proteolytic activity.

The sensitivity of the kit for trypsin detection was determined monitoring for 30 minutes the protease activity on QD-substrate at increasing applied enzymatic catalytic units in the range between 1 U/mL to 100U/ml_ (figure 22). Trypsin solutions containing less than 1 U/mL could not be detected using this commercial kit based on fluorescence detection. d. Functionalization of agarose with the designed peptide

The peptide was conjugate through the NH₂ group of the COOH-terminal lysine to the agarose support. We studied the immobilization of the selected peptide: Directly onto carboxylated agarose and through the use of a bifunctional PEG spacer, and

At different densities of applied peptide per gram of solid support For this purpose, the peptide was directly linked onto carboxylated- activated agarose using different densities of applied peptide per gram of support: in particular 3.5, 5 and 7.5 μηιοΙ of peptide/g of agarose. The peptide immobilization onto carboxylated-agarose was carried out by using EDC chemistry. After the immobilization reaction, the peptide functionalized-agarose was extensively washed with 10mM MES pH6 containing 0.5M NaCI. The high ionic strength helped eliminating the non- covalent immobilized peptide. After four washes, the possible remaining reactive carboxylic groups were blocked with 0.5M TRIS pH 8.5.

The peptide immobilization was characterized by using UV-Vis Spectroscopy resulting in a final density value of the immobilized peptide of: 2.5, 3.5 and 5.0 μηιοΙ of peptide/g of agarose (figure 23). e. Trypsin activity on Methylene blue labelled peptide immobilized directly onto carboxylated-Agarose

Determination of the presence of active trypsin in a sample was tested by using methylene blue labelled PepXT peptides immobilized on solid agarose supports by using the three densities of PepXT above mentioned. Considering that the activity of 1 U/ml_ of Trypsin (corresponding to O.^g/mL of enzyme), resulted in a low activity by using the EnzChek assay kit, it was decided to test enzymatic units higher than 1 U/mL, such as 5U/ml_ (O^g/mL) and 50 U/mL ^g/mL).

Aliquots of 10mg of peptide-functionalized agarose were incubated with a trypsin solution in agitation for 15 minutes at 37°C, in a final volume of 100μΙ_. After incubation the solutions were separated from the solid supports by filtration, and the supernatant was characterized by using UV-Vis spectroscopy.

The results are indicated in figure 24, in which the determination of the trypsin activity using the directly functionalized peptide-support with different peptide densities is shown. The graphs display a higher absorbance at 660nm for all densities of peptide-support using 50U/ml_ of trypsin than the absorbance shown by using 5U/ml_. Comparing the absorbance at 660 nm of the MB-peptide hydrolyzed, using the two concentrations of trypsin, with the absorbance of the PepTX in solution, it is possible to calculate the amount of peptide proteolyzed, In this sense, a sample containing 5U/ml_ of trypsin is able to hydrolyze respectively 5 μΜ, 10 μΜ, and 15 μΜ of peptide from peptide- agarose functionalized with an linked density of peptide of 2.5, 3.5, and 5.0 μηΊθΙ/g as shown in figure 25.

When using a solid support functionalized with 3.5 and 5.0 μηΊθΙ/g of agarose, it is possible to detect the presence of 5 U/mL of trypsin visually. As the amount of peptide released is higher than the LOD previously fixed for visual detection (10 μΜ).

Using 50U/ml_ of Trypsin, the amount of hydrolyzed peptide is higher than when using 5 U/mL of enzyme, respectively 17 μΜ, 20 μΜ, and 22 μΜ of peptide for 2.5, 3.5 and 5.0 linked peptide/g of activated supports as shown in figure 26.

50 U/mL of trypsin, are able to hydrolyze the same amount of peptide on the support activated with 3.5 and 5.0 μηηοΙ of linked peptide/g of MB-PepXT activated agarose, indicating that 22μΜ of peptide is the limit of proteolysis, clearly visible in the blue scale shown in figure 27, and higher that the set LOD.

For cell trypsinization usually a trypsin solution in a concentration range between 0.05% and 2.5% is used. As trypsin used has about 10.000U/mg, following the suggested dilutions, the lowest amount of trypsin, used in the protocols of cell trypsinization should be of 500U/mL. Solid supports functionalized with an applied peptide density of 3.5 and 5.0 μη-iol/g of agarose, enable the visual detection of 5U/mL of trypsin, which is much lower than the amount often used for cell trypsinization. f. Immobilization of the labelled peptide onto carboxylated-Agarose- using PEG as a spacer. Carboxylated-agarose, modified with COOH-PEG-NH₂ (1 .000 Da) as a spacer, at a density of 1 μη-iol/g of support, was used.

The peptide was linked to the PEGylated-agarose applying 5 μηιοΙ of peptide/g of agarose. Using EDC chemistry we carried out the peptide covalently immobilization to the carboxylic groups of the support. After the immobilization reaction, the peptide activated solid support was extensively washed with 10mM MES pH6 containing 0.5M NaCI. The high ionic strength helped eliminating the non- covalent immobilized peptide. After the washes, the potential remaining carboxylated reactive groups were blocked with 0.5M TRIS pH8.5.

The percentage of peptide immobilization was calculated to be about around 90%, thus, resulting in a final density of the immobilized peptide on the support of about 4.5 μηηοΙ of peptide/g of agarose (figure 28). g. Trypsin activity on Methylen blue labeled peptide onto PEGylated-agarose supports

For this experiment, determination of the presence of trypsin activity was tested by the use of the obtained MB-PepXT-PEG-agarose. Figure 29 indicates that PEG modification of the carboxylated agarose support fails to improve the colour output signal after incubation with buffered samples with 5 and 50 U/mL. h. Detection of collagenase using the support optimized for trypsin detection (PepXT- agarose).

Methylen blue labeled PepXT directly linked onto Agarose support (functionalized with three densities of linked PepXT, 2.5, 3.5, and 5.0 μηΊθΙ/g of agarose) was used to test the activity of 0.1 U/mL collagenase. As in the case of trypsin, only when using supports functionalized with a linked peptide density of 3.5, and 5 μηΊθΙ/g of agarose, it is possible to achieve a visual detection (12,5 μΜ of MB- peptide/s released).

Collagenase activity could be detected visually using the KIT optimized for Trypsin detection (PepXT as substrate). Even though, for equal units of collagenase, the amount of MB-peptide/s released using PepX as substrate is 60% higher. Thus, the KIT optimized for Collagenase detection (PepX as substrate) improves visual detection of the enzyme (see Table below)

Table 3. Comparison of the visual output signal of MB for collagenase detection obtained using the supports optimized for Trypsin (PepXT-agarose) and collagenase (PepX-PEG-agarose) detection. i. Detection of Trypsin using a support optimized for collagenase detection (PepX-PEG- agarose)

The trypsin activity was tested using a support optimized for collagenase detection. The lowest concentration of trypsin (5U/ml_) was tested by using the MB-labeled PepX-onto PEG Agarose activated support functionalized with a linked density of peptide of 3 μηΊθΙ/g agarose.

The comparison of the absorbance at 660nm of MB upon enzymatic activities is reported in the following Table:

Table 4. Comparison of the visual output signal of MB for trypsin detection obtained using the supports optimized for trypsin (PepXT-agarose) and collagenase (PepX-PEG-agarose) detection.

A visual detection of 5 or 50 U/mL of trypsin is not feasible using the KIT optimized for Collagenase detection (PepX as substrate).

In conclusion, in this example the following was demonstrated:

1 . MB-PepTX is well immobilized on a carboxylated-agarose support at the three applied densities used, respectively 3.5, 5 and 7.5 μη-iol/g of agarose.

2. The introduction of a linker or spacer consisting of PEG in the biosensor of MB-PepTX immobilized onto the carboxylated-agarose support, fails to improve the enzymatic activity in comparison to the use of a biosensor without the linker or spacer.

3. Hydrolysis of peptide MB-PepXT from the support correlates to the trypsin concentration.

4. Biosensor using PepXT as substrate are capable in about 15 min at 37°C, to visually detect low amounts of trypsin (5 U/mL)

5. Biosensor using PepXT as substrate are capable in about 15 min at 37°C, to visually detect low amounts of collagenase (0.1 U/mL).

Example 6.Synthesis of carboxylated-agarose. For the preparation of support agarose functionalized with carboxylic groups, the agarose was first modified with epoxide groups. More in details 10g of Agarose 4BCL has been slowly resuspended in a solution prepared as follow 3.3 g of NaOH have been solubilized into 50ml_ of cold Mili-Q (MQ) water, followed by the addition of 0.2 of NaBH₄ and 16 mL of acetone.

The resuspended agarose 4BCL was then incubated overnight (=16 hours) in agitation with 1 1 mL epicloridine, gradually added to the suspension. The agarose was washed with abundant MQ water.

The epoxy activated agarose was then functionalized with iminodiacetic (IDA) acid for introducing abundant carboxylic groups. The IDA activation has been carried out incubating overnight the epoxy agarose with 0.5M IDA adjusted at pH 1 1.

Quantification of the amount of COOH introduced per g of agarose: As the IDA groups introduced in the agarose are able to chelate divalent metals, chelation of cooper ions could be used to quantify the number of carboxyl groups introduced per gram of agarose.

Firstly, a calibration plot with CuS0₄ solutions in EDTA 0,5 M at different concentrations was measured in the spectrophotometer. These solutions present a maximum absorption around 733 nm (Figure 1 ).

The support Ag-IDA was incubated with an excess of a solution 30 mg/mL of CuS0₄. After 1 hour, the supernatant was recovered and the support washed twice with 10 mM MES pH 6. When the Agarose-IDA was properly washed, and all the Cu²⁺ cations removed from the supernatant, the support was incubated with a solution of EDTA 0.5 M. The supernatant from this incubation was recovered and its absorbance was measured spectrophotometrically. (Table 5)

From the absorbance value, we calculate the total amount of Cu⁺² moles chelate by per gram of supportsupport. Each mole of CuS04 reacts with two moles of carboxylic groups, hence the final COOH content of the support was found to be around 80 μηιοΐββ COOH per gram agarose.

Table 5: Functional COOH groups content of Ag-IDA per gram of support.

Claims

1 . A biosensor for detecting collagenase activity, in an isolated biological sample, which comprises a peptide consisting of SEQ ID NO 1 labelled with at least one colorimetric dye, immobilized on an agarose support consisting of cross-linked agarose beads, optionally through a spacer or linker.

2. The biosensor according to claim 1 , wherein the peptide is directly bound to the agarose support and wherein the agarose support comprises at least 5 μηιοΙ of linked peptide of

SEQ ID NO 1 per gram of support.

3. The biosensor according to claim 1 , wherein the peptide is bound to the agarose support through a spacer in a density of at least 3 μηηοΙ of the peptide of SEQ ID NO 1 per gram of support, and wherein the distance between the agarose support and the peptide is between 14 and 34.5 Angstroms, preferably the spacer consists of polyethylene glycol (PEG) containing 4 to 45 monomers of ethylene glycol (PEG).

4. The biosensor of any of the precedent claims, wherein the colorimetric dye is methylene blue.

5. A kit for detecting collagenase activity in an isolated biological sample, which comprises the biosensor as defined in any of claims 1 -4.

6. The kit of claim 5, wherein said kit comprises:

a. The biosensor as defined in any of claims 1 to 4;

b. A positive control of the collagenase activity; and/or

c. A negative control of the collagenase activity.

7. The kit of claim 5 or 6, which further comprises reagents, recipients, pipettes, microfilter tips or centrifugal filtration devices and optionally instructions for detecting collagenase activity in an isolated biological sample.

8. A method for detecting collagenase activity in an isolated biological sample which comprises the following steps: a. contacting the biosensor as defined in any of claims 1 to 4 with a biological sample suspected of having collagenase activity; and b. Visually (with the naked eye) detecting cleavage of the substrate of the biosensor, wherein the level of cleavage of said substrate is correlated to the level of active collagenase, in said sample.

Use of the biosensor according to any of claims 1 to 4 or the kit according to any of claims 5 to 7 for measuring collagenase activity, preferably before and after inhibition during the clinical practice of stem cell extraction.