WO2012085080A1

WO2012085080A1 - Method of detecting a proteolytic enzyme by means of gold nanoparticles- catalyzed amplification of a signal

Info

Publication number: WO2012085080A1
Application number: PCT/EP2011/073585
Authority: WO
Inventors: Leonard Jan PRINS; Paolo SCRIMIN; Renato BONOMI
Original assignee: Universita' Degli Studi Di Padova
Priority date: 2010-12-22
Filing date: 2011-12-21
Publication date: 2012-06-28
Also published as: IT1403720B1; ITPD20100388A1

Abstract

It is herein disclosed a new methodology to detect an enzyme, in particular a protease (i.e. a protein capable of hydrolyzing peptide ligands). The determination is based on the activation of colloidal gold nanoparticles with catalytic properties via enzyme hydrolysis, operated by the enzyme to be determined, of a peptide sequence acting as inhibitor for said Au nanoparticles. Following from hydrolysis, the Au nanoparticles' catalytic activity is restored by determining the production of great quantities of a "reporter" molecule via spectrophotometry. The method can be made selective with regards to the enzyme to be determined by using a specific peptide sequence. The proteases of interest for the method are subtilisin, caspases, glutamate carboxy peptidase II, granzyme and thermolysine.

Description

METHOD OF DETECTING A PROTEOLYTIC ENZYME BY MEANS OF GOLD

NANOPARTICLES- CATALYZED AMPLIFICATION OF A SIGNAL

***********

Field of the invention

The invention concerns a method of detecting a proteolytic enzyme based on a conversion reaction, catalyzed by gold (Au) nanoparticles, functionalized with positively-charged metal-ligand complexes self-assembled in a monolayer on the metal surface, with the production of a "reporter" molecule which consists of a compound which generates an instrumentally measurable signal, in particular chromogenic or fluorogenic. The method can be applied to detect protease and can be made selective by operating a suitable choice of the peptide substrate. Prior art

In enzymology the detection of an enzyme activity is essential, because it allows the study of the mechanisms at the basis of a pathology and to consequently develop new therapeutic agents for it. Among all enzymes, proteases are particularly important as the proteolytic processes are the final result of many proteins' activities [L. Hedstrom, Chem. Rev. 102, 4501 (2002); L. Tong, Chem. Rev. 102, 4609 (2002); H. Neurath, J. Cell. Biochem. 32, 35 (1986)].

Conventional methods for detecting proteases base themselves on the use of radioisotopes or fluoro- chromogenic substrates. Therefore, several approaches have been developed based on the various interactions of the enzyme's substrates and of the products thereof with a chemosensor, interactions capable to determine the production of a fluorometric or colorimetric signal such as, for example, displacement assays [B. T. Nguyen et al., Coord. Chem. Rev. 250, 31 18 (2006)] and synthetic strategies of pores on membranes [N. Sakai et al., Acc. Chem. Res. 41, 1354 (2008)].

A second strategy in developing sensitive sensors involves using nanoparticles of various types, with the exploitation of the latter's intrinsic chemical-physical properties in combination with the possibility of modifying their surface with small organic ligands [N.L. Rosi, C.A. Mirkin, Chem. Rev. 105, 1547 (2005); S.S Agasti et al., Adv. Drug. Del. Rev. 62, 316 (2010); M. De et a\., Adv. Mater. 20, 4225 (2008)]. What makes these functionalized nanoparticles of particular interest is their multivalent nature, which allows interaction with the target on multiple points and therefore with high affinity [A. Mulder et al., Org. Biomol. Chem. 2, 3409 (2004)]. On this point, extensive studies have been carried out in particular on gold monolayer protected colloids (hereinafter in short Au MPC), as they are highly stable and easy to prepare and functionalize [M.C. Daniel et al., Chem. Rev. 104, 293-346 (2004)]. The use of Au MPC is mainly based either on the induction of a cluster that causes a change of color (from red to blue) [K.H. Su et al., Nano Lett. 3, 1087-1090 (2003)] or on a displacement guided by the analyte of the fluorophore bound to the Au MPC [Sapsford, K.E. et al., Angew. Chem. Int. Ed. 45, 4562-4588 (2006); C.C. You et al., Nat. Nanotechnol. 2, 318-323 (2007)]. In particular, an example has been reported in which the action of a proteolytic enzyme induces aggregation of gold nanoparticles, and thus a change in color. [C. Guarise et al., Proc. Natl. Acad. Sci. U.S.A. 103, 3978-3982 (2006)]. Moreover, an alternative strategy was recently described, in which the analytes prevent the fluorescent extinction of a "reporter" molecule, by blocking its access to the reactive units located on a monolayer [E. Climent et al., Chem. Commun., 6531 - 6533 (2008)]. Even more recently, it was reported in the literature a sensor based on Au MPC which includes the formation of an amplification signal by the displacement of an enzyme via an analyte [O.R. Miranda et al., J. Am. Chem. Soc. 132, 5285-5289 (2010)].

The common characteristic of these methods is that the quantity of the signal generated is proportional to the quantity of substrate converted by the enzyme. The sensitivity of these assays could instead be significantly increased if the enzyme conversion of a single substrate led to the formation of a large number of reporter molecules.

Therefore, the main drawback of the currently known determination methods is that the amount of the signal produced, detectable via an isotope or a fluorophore or a chromophore, is linearly correlated to the product quantity deriving from the action of the enzyme on its substrate. This results in some limits to the sensitivity and speed of the measurement, because determining low enzyme concentrations requires a long time to generate a perceptible signal. A second disadvantage is that, to generate a signal detectable after the hydrolysis of a peptide ligand, it is necessary to use special enzyme substrates (isotopes, fluorophores or chromophores), and the presence of these reagents interferes with the enzyme/substrate affinity, leading to unreliable determinations.

Summary of the invention

The purpose of the invention is therefore to overcome the technical problem of the stoichiometric ratio between the quantity of substrate hydrolyzed by the enzyme and the quantity of the signal {i.e. "reporter" molecule) produced.

To remedy this limitation, a qualitative and quantitative method has been found that essentially includes a process that determines the amplification catalyzed by Au nanoparticles functionalized with positive charges, i.e. gold monolayer- protected colloids (Au MPC), of the signal to be measured.

In particular, the method to detect a proteolytic enzyme devised for the aforementioned purpose, comprises a cascade of two catalyzed processes to generate a measurable signal, in which a crucial role is held by a peptide compound having a anionic nature and having a function both as a substrate of the enzyme and as inhibitor of gold colloidal nanoparticles, functionalized with positively charged metal-ligand complexes self-assembled in a monolayer, whose catalytic activity leads to the formation of a signal consisting in an instrumentally measurable agent, for example by spectrophotometry or by fluorometry.

Therefore the subject of this invention is a method of detecting a proteolytic enzyme characterized by the use, for the detection, of a peptide compound having a anionic nature and having a function both as a substrate of the enzyme to be detected and as inhibitor of the catalytic activity of functionalized Au nanoparticles which catalyze a conversion reaction of a reagent in a product capable of generating a "reporter" molecule as an instrumentally measureable signal. The gold nanoparticles are functionalized with positively charged metal-ligand complexes self-assembled in a monolayer on the metal surface.

The method subject of the invention has shown that it is capable of fulfilling the purpose as made clearer by the following detailed description of the invention. In fact, it was possible to verify that the presence of the enzyme to be determined led to the hydrolysis of the anionic peptide compound by the enzyme itself and from this derived a catalyzed amplification in the generation of the signal. In particular, the catalyzed amplification determined the production of numerous "reporter" molecules instead of 1 , significantly increasing the assay sensitivity. This sensitivity is further improved by the fact that the method is selectively addressed to determine a specific enzyme, as the selectivity is operated by the selection of an appropriate anionic peptide compound as substrate, depending said selection on the type of enzyme to be detected. Moreover, contrary to conventional methods, it is not necessary to modify the enzyme substrate by introducing on it fluoro- or chromogenic groups suitable for generating an instrumentally measurable signal.

The proteases of interest for the method of the present invention are subtilisin, caspases, glutamate carboxy peptidase II, granzyme and thermolysine.

The invention also extends to the use of peptide compounds having a anionic nature and having a function both as a substrate of the enzyme and as inhibitor of the functionalized colloidal Au nanoparticles, and their derivatives usable for the method subject of the invention.

These and other advantages, as also other subjects of the invention, shall be made clearer by the invention's detailed description that follows, with the help of the figures attached.

Brief description of figures

Figure 1 . The figure shows a schematic representation of the functioning principle of the detection method of an enzyme subject of the invention according to an embodiment thereof: the measurable signal is the "reporter" molecule p- nitrophenol (in short PNP), starting from a peptide compound indicated in the figure as "substrate" both in the absence (A) and presence (B) of the enzyme to be detected, through functionalized positively charged Au nanoparticles (identified in the figure as Au MPC 1 ) which catalyze the reaction of trans-phosphorylation of the chromogenic agent 2-hydroxypropyl-4-nitrophenyl phosphate (in short HPNPP) and the release of the "reporter" molecule. (A) The trans-phosphorylation reaction of HPNPP is inhibited in the absence of proteases as the peptide compound, as it is anionic, forms a stable complex with the functionalized Au nanoparticles that inhibits the catalyzed hydrolysis of HPNPP. (B) The presence of a protease that can hydrolyze the peptide anionic substrate, instead, prevents the formation of the Au MPC 1 /substrate complex and allows the catalyzed trans-phosphorylation reaction of HPNPP. At the same time there is the production of the p-nitrophenol "reporter" molecule which is quantified by measuring absorbance at 405nm.

Figure 2. The figure shows the number of equivalents of p-nitrophenol (PNP) generated per catalytic unit of TACN^»Zn" in function of time. Test conditions: [TACN'Zn")] = 5.0-10^"6 M, [HPNPP]₀ = 1 .0-10^"3 M, [HEPES] = 1 .0-10^"2 M, pH = 7.0, T = 40°C. The number of equivalents of PNP per TACN*Zn" is calculated by dividing absorbance by the molar extinction coefficient of the PNP at pH 7.0 (8503 l.mo ¹.cm^"1) and the concentration of TACN*Zn" 5.0 μΜ. The final PNP concentration was 0.35 imM, corresponding to the conversion of 35% of HPNPP (1 .0 mM).

Figure 3. The figure shows a schematic representation of the protocol used for the detection method of a proteolytic enzyme according to the invention.

Figure 4. The figure shows the results obtained with the detection assay of Subtilisin A described in example 1 : a) absorbance at 405 nm measured 30 minutes after adding Au MPC/TACN^»Zn" and HPNPP to a solution of substrate peptide Ac-Asp-Asp-Asp-OH (SEQ ID NO. B) incubated with Subtilisin A at different concentrations for 1 hour in the absence (■) and in the presence (□) of an inhibitor of the enzyme phenylmethylsulphonyl fluoride (PMFS) (H₂O buffered at pH 7.5 ([HEPES]=10 mM), T = 25 °C); b) the difference in absorbance at 405 nm between Ac-Asp-Asp-Asp-OH (SEQ ID NO. B) incubated for 1 hour with different concentrations of Subtilisin A and the reference sample containing Ac-Asp-Asp- Asp-OH (SEQ ID NO. B) in function of the assay time; c) the linearity of the response compared with the enzyme concentrations in a nanomolar system and the positive effect with longer assay times (■ : 20 minutes,□ : 20 hours) on the strength of the output signal (= PNP measurement).

Figure 5. The figure shows the results obtained with the detection assay of Caspase 1 described in example 2: the increase in absorbance at 400 nm in function of time following addition of Au MPC/TACN^»Zn" and HPNPP to a solution of substrate peptide Ac-YVADD-OH (SEQ ID NO. 1 ) and Caspase 1 incubated for 4 days at 37 °C in the absence (I) and presence (II) of the enzyme Ac-YVAD-CMK (SEQ ID NO. 15) inhibitor. Test conditions: [Ac-YVADD-OH] = 5.0-10^"6 M, caspase 1 = 0.1 AU, [TACN-Zn"] = 5.0-10^"6 M, [HPNPP] = 1 .0-10^"3 M, [HEPES] = 1 .0-10^"2 M, pH = 7.2, T = 40°C.

Detailed invention description

Definitions and abbreviations

Unless otherwise defined, all scientific and technical terms used in all parts of the invention description have the meanings that one skilled in the art of the invention field would commonly understand. In particular, an expert in the field would know that the terms "nanoparticles" and "colloids" are to be considered equivalent.

Au MPC is herein used as abbreviation for "Au nanoparticles functionalized with positively charged ligands self-assembled in a monolayer" and for "Au colloids protected monolayer of organic molecules" and equivalent expressions. Such expressions herein used are to be considered equivalent and as their extended meaning for the purposes of describing this invention.

Preferably, the gold functionalized nanoparticles (Au MPC) usable for the implementation of the method subject of the invention are those known and described in F. Manea et al., Angew.Chem.lnt.Ed. 43, 6165 (2004). In particular, the Au MPC are the gold nanoparticles functionalized on the surface with the complex between 1 ,4,7-triazacyclononane (TACN) and ion Zn". Therefore, the gold functionalized nanoparticles can be indicated herein also as Au MPC/TACN'Zn"-.

"Reporter" molecule or "measurable signal" are equivalent terms, by which it is intended to indicate the product deriving from the conversion reaction, catalyzed by Au nanoparticles functionalized with positively charged ligands self-assembled in a monolayer, of a chromogenic or fluorogenic reagent, substrate of the Au MPC themselves. The conversion generates a chromophore or fluorophore compound, the so-called "reporter molecule", instrumentally measurable with methods known to an expert of the field. By way of a non-limiting example, in a preferred embodiment of the invention, said conversion reaction is the trans-phosphorylation of the chromogenic agent 2-hydroxypropyl-4-nitrophenyl phosphate (which is therefore the Au MPC's substrate) with generation of the chromophore agent p- nitrophenol (which is the "reporter molecule") measurable with absorbance at 405 nm. The abbreviations reported below refer to the catalyzed conversion reaction or to the affinity of the Au MPC compared with the anionic peptide substrate/inhibitor: Acat = rate constant of the catalyzed conversion by Au MPC of a chromogenic/fluorogenic agent.

(uncat = rate constant of the conversion of a chromogenic/fluorogenic agent in the absence of Au MPC.

K_M = dissociation constant between Au MPC and the chromogenic/fluorogenic agent or the anionic peptide.

Description

The essential and key technical feature of the method of detecting a protease according to the invention is the use of an anionic peptide that can act simultaneously as enzyme substrate, and therefore can be hydrolyzed by it specifically, and as inhibitor of the catalytic activity of Au nanoparticles functionalized with positively charged ligands self-assembled in a monolayer on the metal surface. The use of a peptide with these characteristics determines a cascade process of two catalytic events with the generation of a measurable two- way signal depending on whether the enzyme is present or absent in the sample to be analyzed:

A. in the absence of the enzyme, the substrate peptide is not hydrolyzed and therefore, as it has an anionic nature, with the positively charged Au nanoparticles it forms a complex that inhibits the activity thereof in the catalysis of the reagent conversion for the generation of a measurable signal, consisting in particular of a chromophore of fluorophore agent;

B. in the presence of a protease able to hydrolyze the anionic peptide substrate, on the contrary, there is no formation of the Au MPC/peptide substrate complex, thus allowing the catalyzed conversion of the reagent with generation of the measurable signal in terms of the concentration of a chromophore or fluorophore compound.

This process is laid out in detail in figure 1 with reference to a specific example of catalyzed conversion reaction, preferred for the purposes of this invention, of a chromogenic agent, such as the trans-phosphorylation reaction of 2- hydroxypropyl-4-nitrophenyl phosphate (HPNPP) and its conversion into p-nitro- phenol (PNP).

Although this is a preferred embodiment of the invention, the conversion reaction that is exploited for the production of a measurable signal, catalyzed Au MPC conversion, can also use other substrates and generate other "reporter molecules", by adequately modifying the leaving group that generates the "reporter molecule" on a base structure that can function as substrate of the Au MPC.

Therefore, the chromogenic or fluorogenic reagent, substrate of the conversion reaction catalyzed by the Au nanoparticles, can be a 2-hydroxypropyl-(R-) phosphate compound, wherein R is a group that becomes a chromo- or fluorophore compound when cut from the substrate. Some alternatives to the HPNPP chromogenic reagent can be, for example, 2-hydroxypropyl-(2,4- dinitrophenyl) phosphate, 2-hydroxypropyl-(2,4-dinitro-7-sulfonate-naftyl) phosphate, 2-hydroxypropyl-(3,6,8-trisulfonate pyrene) phosphate, reagents that generate respectively the "reporter" molecules whose structure formula is reported below.

As previously reported the preferred Au nanoparticles usable as catalyst for the conversion reaction of a chromophore or a fluorophore molecule are known and described in [F. Manea et al., Angew.Chem.lnt.Ed. 43, 6165 (2004)].

In brief, the characteristics that these Au MPC nanoparticles must possess for the purposes of this invention are as follows:

- a size preferably comprised from 1 .5 to 20 nm;

- positive charges resulting from the complex between 1 ,4,7- triazacyclononane (TACN) and ion Zn", whose structure formula is reported below self-assembled in a monolayer on the metal surface

- a charge density of at least 1 positive charge per thiol and preferably between 2 and 3 positive charges per thiol.

The use of these Au nanoparticles in the catalysis of the hydrolysis of HPNPP into PNP is known [F. Manea et al., ref. cit] and consolidated, and this makes it particularly advantageous for its reliability and ease of implementation. Catalysis is obtained from the cooperative action of two TACN^»ZN" complexes located on the surface of the monolayer. As is common knowledge, these Au nanoparticles have a saturation behavior similar to that of an enzyme with values equal to for k_ca = 6.7 x 10^"3 s^"1 and for K_M = 0.31 imM at pH = 7.5 in H₂O. Moreover, for analytical purposes, the use of these Au nanoparticles and the HPNPP's trans- phosphorylation reaction for the generation of a measurable signal present the additional benefit that at the assay conditions there is virtually no competitive reaction, because k_unca ior the non-catalyzed reaction of HPNPP is around 10^"7 s^"1 in the same assay conditions and that the reaction can easily be monitored visually, by measuring the absorbance of p-nitrophenol produced at 405 nm.

For another aspect the use of these Au MPC is particularly advantageous, because they can have a high number of positive charges due to the TACN^»Zn" complex positioned on the metal surface and, as a consequence, they have a great affinity for negatively charged molecules. Turnover experiments, described in the following experimental part, carried out for the setting up of the method subject of the invention, have highlighted that these Au nanoparticles actually have a primary role in the amplification of the signal, as the tests showed that the system is capable of generating at least 70 PNP molecules per TACN*Zn" complex (fig. 2).

Considering the fact that the method according to the invention is based on a cascade of two catalyzed processes regulated by the interactions of enzyme/substrate peptide and substrate peptide or its product deriving from the enzyme hydrolysis with Au MPC, it is necessary to take into due consideration also some requirements relative to usable peptides. Indeed, it is the substrate peptide that is a key player in inducing or inhibiting the two catalyzed processes, the first of the hydrolysis catalyzed by the enzyme and the second catalyzed by the Au nanoparticles.

In particular, as this peptide has a function of both enzyme substrate and inhibitor of the Au nanoparticles, it must have an adequate amino acid sequence and at the same time a suitable number of negative charges.

Therefore, the peptides useful for these purposes can be peptides represented by general formula (I)

RNH-[AA - AA]„[AA - AA]_rCOOH

(I)

and derivatives thereof

wherein:

R is an acetyl group;

[AA - AA], (/^' = an integer comprised from 1 to 4) defines an affinity domain for Au nanoparticles with a positively charged monolayer comprising an amino acid or an amino acid sequence wherein the amino acid or the amino acids, equal or different one from the other, are selected from aspartic acid or glutamic acid;

[AA - AA]_n {n = an integer comprised from 1 to 8) defines the selectivity domain for the enzyme to be detected comprising an amino acid or an amino acid sequence wherein the amino acid or the amino acids, equal or different one from the other, are independently selected from both of L and

D series proteinogenic amino acids.

The derivatives of the peptides of general formula (i) are known to an artisan skilled in the art. The amino acids in the sequence [AA - AA]_/ imay be glutamic acid or aspartic acid or non-natural equivalents thereof, whereas no particular restrictions occur to amino acids in the sequence [AA - AA]_n.

Within these peptides of general formula (I), a substantial role is played by the [AA - AA]_n portion that defines the selectivity domain as this is specifically designed in order that said peptides can be the specific substrates of the enzyme to be detected, whilst the [AA - AA], portion, defining the affinity for Au nanoparticles, is significant to obtain a suitable charge difference between the substrate peptide and the product derived from its enzyme hydrolysis.

Indeed, so that such process can be determined efficiently, the following requirements relative to the substrate peptide/Au MPC inhibitor must be fulfilled:

- the substrate peptide and the product deriving from its hydrolysis catalyzed by the presence of the enzyme to be detected must have different affinities for the Au nanoparticles used in the second catalysis and, in particular, as the interaction is electrostatic, this implies that the enzyme must modify the substrate's charge by diminishing it; and

- the peptide substrate concentration to be used in the assay must be such that the substrate and the product deriving from the enzyme hydrolysis have the greatest difference in inhibition power. In any case, the concentration regimen is indicatively determined by the number of negative charges present on the substrate according to the criterion:

negative charges on the peptide usable concentration regimen

substrate

1 > 1 x10^"3 M

2 5x10^"3 M - 1 x10^"3 M

3 5x10^"6 M - 1 x10^"4 M

4 5x10^"7 M - 1 x10^"5 M.

Therefore, the concentrations of the peptides can vary from 1 x10^"3 M to 5x10^"7 M and preferably the concentration is 5x10^"6 M. For the method according to the invention, the putative peptide substrates of proteolytic enzymes and inhibitors of Au MPC are as follows:

Enzyme substrate Deotides/inhibitors (SEQ. ID NO.)

Glutamate carboxy peptidase II Ac-DE-OH (SEQ. ID NO. A)

Subtilisin Ac-DDD-OH (SEQ. ID NO. B)

Caspase 1 Ac-YVADD-OH (SEQ. ID NO. 1 )

Caspase 2 Ac-VDVADDD-OH (SEQ. ID NO. 2)

Caspase 3 Ac-DEVDDD-OH (SEQ. ID NO. 3)

Caspase 4 Ac-LEVDDD-OH (SEQ. ID NO. 4)

Caspase 5 Ac-WEHDDD-OH (SEQ. ID NO. 5)

Caspase 6 Ac-VEIDDD-OH (SEQ. ID NO. 6)

Caspase 8 Ac-IETDDD-OH (SEQ. ID NO. 7)

Caspase 9 Ac-LEHDDD-OH (SEQ. ID NO. 8)

Caspase 10 Ac-AEVDDD-OH (SEQ. ID NO. 9)

Caspase 12 Ac-ATADDD-OH (SEQ. ID NO. 10)

Caspase 13 Ac-LEEDDD-OH (SEQ. ID NO. 1 1 )

Thermolysine Ac-XXXDDD-OH (X = any AA) (SEQ. ID

NO. 12)

Granzyme Ac-IEPDDD-OH (SEQ. ID NO. 13).

Taking into account what has been mentioned above, as an example we provide, in table 1 , the conditions under which the requirements indicated above are fulfilled if the enzyme to be determined is subtilisin A, glutamate carboxy peptidase II or caspase 1 respectively.

Table 1 : Substrate peptides and products for different enzymes. For every enzyme the concentration of the peptide substrate is provided at which the difference in the inhibiting power of the substrate and products is at maximum level

Glutamate A/-acetyl-L- A/-acetyl-L- 5.0 x 10^"° carboxy aspartyl-L- aspartate (-2) and

peptidase II glutamate (-3) glutamate (-1 )

(SEQ. ID NO. A)

4.0x10^"5

9.0x10^"7

Caspase 1 Ac-YVADD (-3) Ac-YVAD (-2) 5.0 x 10^"°

(SEQ. ID NO. 1 ) (SEQ. ID NO. 14)

4.0x10^"6 1 x10^"4

The detail of inhibition tests carried out for the set up and assessment of the method of the present invention are reported in the following experimental part. Detection of a proteolytic enzyme according to the method subject of the invention comprises at least the following steps:

- adding to a sample containing the proteolytic enzyme to be detected in an aqueous solution or suspension buffered at pH between 7.0 and 8.0, a peptide compound of general formula (I) having a function both as substrate of the enzyme and as inhibitor of Au nanoparticles, functionalized with positively charged ligands self-assembled in a monolayer;

- adding after incubation for at least 20 min. to the mixture previously obtained the functionalized Au-nanoparticles and a reagent compound substrate thereof for the conversion reaction;

- instrumental^ detecting the generation of the "reporter" molecule starting from the addition of functionalized Au-nanoparticles and its substrate;

- calculating the enzyme concentration on the basis of the "reporter" molecule concentration on the basis of a calibration curve obtained with a series of known titre samples of enzyme.

The protocol to execute the method is laid out in figure 3. Below a preferred embodiment is reported in detail.

The assay according to the method of detecting a proteolytic enzyme of the invention foresees the following experimental conditions. An aqueous solution of a general formula (I) peptide is added, in a quantity comprised between 0.25 mg and 5 mg and preferably of 2.5 mg at a concentration between 5.0 x 10^"7 and 1 .0 x 10^"3 M, and preferably of 5.0 x 10^"6 M, to an aqueous enzyme solution buffered with HEPES (10x10^"3 M) at pH between 7.0 and 8.0 (preferably 7.5) with an unknown enzyme concentration. The mixture is incubated at a temperature between 25 and 40 °C for a period of between 20 minutes and 24 hours (preferably 30 minutes). After this incubation, the Au MPC/TACN^»Zn" nanoparticles and HPNPP are added to reach a final concentration of TACN^»Zn" of 5.0 x 10^"6 M and of 1 .0 x 10^"3 M respectively. Absorbance is measured at a wavelength of 405 nm after an incubation time that varies between 20 minutes and 24 hours. From the value of measured absorbance the concentration of PNP is measured by means of one the following equations (1 a or 1 b):

[PNP] = A₄₀₅ nm /13135 (at pH 7.5) (1 a)

[PNP] = A₄₀₅ nm /8503 (at pH 7.0) (1 b) where the value of 13135 corresponds to the molar extinction coefficient of the PNP at pH of 7.5 and the value of 8503 corresponds to the molar extinction coefficient of PNP at a pH of 7.0.

In case of the enzyme is measured on the concentration of PNP, the concentration thereof can thus be calculated according to the equation (2):

[enzyme] = (A₄₀₅ nm, 64soo _s-3.75x10^"2)/4.74x10^"2 (μΜ) (2) obtained from a calibration curve using a series of known titre enzyme samples.

EXPERIMENTAL PART

Turnover experiments

HPNPP was added to an aqueous solution ([HEPES] = 10 mM, pH =7.0, T = 40^QC) of Au MPC/TACN^»Zn" (5.0 μΜ with reference to the concentration of TACN and

5.0 μΜ with reference of Zn(NO3)2, so that the final concentration is 1 .0 mM.

Absorbance at 405 nm was measured for a period of 7 days. The final PNP concentration was calculated with the equation 1 b

[PNP] = A₄₀₅nm/8503 (M)

where the value of 8503 corresponds to the molar extinction coefficient of the PNP at pH 7.0. The number of PNP molecules produced per TACN*Zn" complex (70) was calculated by dividing the final PNP concentration (0.35 imM) by the concentration of TACN*Zn".

The results obtained are listed in the aforementioned figure 2. The figure shows the number of PNP molecules produced per TACN*Zn" complex in function of time.

Inhibition experiments

The inhibition experiments for setting up the method were carried out on Subtilisin A, Glutamate carboxy peptidase II and Caspase 1 .

A quantity of peptide inhibitor of these proteases was added to an aqueous solution ([HEPES] = 10 mM, pH =7.0, T = 40 ^QC) of Au MPC/TACN'Zn" (5.0 μΜ with reference to the concentration of TACN and 5.0 μΜ with reference to the concentration of ZnNO₃) covering the concentration interval as indicated in Table 2 for the various inhibitors. After 5 minutes, HPNPP (1 .0 mM) was added and absorbance at 405 nm was measured for 30 min. Initial rate is given by the slope of the absorbance curve against time. The inhibition constant as indicated in the previously reported table 1 is defined as the concentration of inhibitor necessary to halve initial rate in the absence of inhibitor.

Table 2. Interval of concentration for the various inhibitors

The results obtained are listed in the above table 1 . Optimal concentration for the assay has been defined as the concentration at which the inhibitory power between the substrate and the product is at its maximum. For all pairs of peptides Ac-DDD-OH/Ac-DD-OH, Ac-YVADD-OH/Ac-YVAD-OH and Ac-DE-OH/Ac-D-OH this concentration is 5.0x10^"6 M.

For illustrative and non-limiting purposes, below we report some examples of detections of proteases, such as the aspecific proteases Subtilisin A and caspase 1 , from which it is possible to infer both the ease and versatility of applying the method according to the invention, and the benefits obtainable therewith.

EXAMPLES

Example 1: assay of detecting Subtilisin

Method

All commercial products (including the enzyme) were purchased from Sigma Aldrich. Peptide Ac-DDD-OH (SEQ ID NO. B) was synthesized according to the solid phase peptide synthesis (Wang resin) using Fmoc chemistry.

The assay of detecting the enzyme was carried out at pH 7.5 ([HEPES]=10 imM) in two different experiments: (A) in a fixed concentration of Subtilisin (42 μΜ) or (B) with different concentrations of Subtilisin (66nm to 42 μΜ). An inhibition experiment was also carried out (C).

(A) . An aqueous solution of the Ac-DDD-OH substrate peptide (925 μΙ_, 10.7 μΜ) was incubated with 10 μΙ_ of an aqueous solution of Subtilisin A buffered at pH 7.5 with HEPES (10 imM) at a concentration of 42 μΜ. The solution was maintained at 25 °C for 20 minutes, after which Au MPC/TACN^»Zn" and HPNPP were added to have, respectively, the concentrations of 5x10^"6 μΜ (in reference to TACN*Zn"), 5x10^"6 μΜ and 1 .0 imM The test tube was kept thermostatically at 40 °C for 30 minutes, during which the formation of the p-nitro-phenol PNP product is followed by measuring absorbance at 405 nm.

(B) . Peptide Ac-Asp-Asp-Asp-OH (SEQ ID NO. B) is incubated with Subtilisin A in concentrations between 66 nM and 42μΜ. After 60 minutes, Au MPC/TACN^»Zn"

(TACN^»Zn" = 5 μΜ) and HPNPP (1 imM) are added to the mixture and we begin measuring absorbance at 405 nm for 30 minutes. (C). The inhibition experiment with control phenyl-methylsulfonyl fluoride (PMFS) was carried out similarly to what is described above, adding 175 ng of phenyl-methylsulfonyl fluoride (PMFS) inhibitor (1 μΜ) to the solution of the enzyme 30 minutes before adding the substrate tripeptide of SEQ ID NO. B.

Results

The dependence of absorbance at 405 nm measured at the initial enzyme concentration demonstrates that the method is capable of detecting enzyme activity (fig. 4a). Indeed, no enzyme activity was observed in the case when Subtilisin A was pre-treated with the PMFS (phenyl-methylsulfonyl fluoride) inhibitor. The fact that absorbance plateaus at Subtilisin concentrations greater than 20 μΜ indicates that substrate Ac-Asp-Asp-Asp-OH was hydrolyzed at its maximum degree.

The advantage of the signal's catalyzed amplification is evident when the incubation time for the production of p-nitrophenol via Au MPC/TACN*Zn" is increased (fig. 4b). As a consequence of the catalytic production of p-nitrophenol, the absolute difference in absorbance with and without the enzyme presents increases in function of time. As a result, even small quantities of substrate hydrolyzed by an enzyme solution 66 nM in 60 minutes is sufficient to generate a perceptible difference of approximately 0.04 absorbance units after 20 hours of incubation with HPNPP. The linearity of the absorbance curve against low enzyme concentrations (fig. 4c) indicates that the protocol is usable and reliable also for a quantitative determination of the enzyme concentration in a nanomolar interval. The maximum difference in absorbance observed (0.12 AU; fig. 4b) corresponds to a difference of 15 μΜ in the concentration of p-nitrophenol.

Example 2: assay of detecting Caspase 1

Method

All commercial products (including the enzyme) were purchased from Sigma Aldrich. Peptide Ac-YVADD-OH (SEQ ID NO. 1 ) was synthesized according to the peptidic synthesis on solid phase (Wang resin) using Fmoc chemicals.

The assay of detecting the enzyme was carried out at pH 7.2 ([HEPES]=10 imM). A solution of substrate pentapeptide Ac-YVADD-OH (5.0 μΙ_, 1 .0 imM) was incubated with 1 .0 μΙ_ of a Caspase 1 enzyme solution (a recombinant human enzyme expressed in E.coli; 1 00 AU have been regenerated with 1 00 μΙ_ of buffering solution 0.5M HEPES) at a concentration of 0.1 ΑΙΙ/μΙ_. The solution was maintained at 37 °C for 4 days, after which Au MPC/TACN^»Zn" and HPNPP were added to have, respectively, the final concentrations of 5x1 0^"6 μΜ (in reference to TACN*Zn"), 5x1 0^"6 μΜ and 1 .0 mM The test tube was kept thermostatically at 40 °C for 48 hours, during which the formation of the p-nitro-phenol product is followed by measuring absorbance at 405 nm.

The control inhibition experiment with Ac-YVAD-chloromethyl ketone (chloromethyl ketone = CMK) (SEQ ID NO. 1 5) was carried out in a similar way to what is described above, adding 1 51 μg of inhibitor with Ac-YVAD-CMK (0.28 mM) to the enzyme solution 40 minutes before adding the substrate peptide Ac-YVADD-OH. Caspase 1 (0.1 active units) was incubated with substrate Ac-YVADD-OH (5 μΜ) for 3 days at 40 °C and at pH 7.0 after having added Au MPC/TACN'Zn" (TACN^»Zn" = 5 μΜ) and HPNPP (1 mM). The same experiment was also carried out in presence of inhibitor Ac-YVAD-CMK (CMK: chloromethyl ketone).

Results

The purpose of the example is to illustrate how the method subject to the invention could be made selective by choosing a suitable peptide substrate depending on the type of enzyme to be detected and/or determine quantitatively. Caspase 1 is paradigmatic in this sense as it is currently measured with a colorimetric assay based on substrate Ac-YVAD-pNA (p-NA= p-nitroaniline) (SEQ ID NO. 1 6) because this enzyme hydrolyzes the ligand after aspartic acid, releasing p- nitroaniline). For the purposes of this determination, we synthesized the substrate Ac-YVADD-OH containing more aspartic acid after the cut site which increases the affinity of said substrate for Au MPC.

The initial rate of production of the p-nitrophenol was significantly higher when the enzyme was present (Avi_nit,meas = 0.30, Avi_nit,_exp = 0.42, based on inhibition experiments) and it was consistent with the expected rates on the basis of the inhibition curves.

Similarly to what was observed for the Subtilisin A example reported previously, the increase in absorbance was measured in an extended time interval, determining a difference in absorbance between the two samples of 0.76 units after 24 hours (fig. 5). This corresponds to a concentration difference of p- nitrophenol of 90 μΜ. Under the same operating conditions, the conventional colorimetric assay, which uses 5 μΜ of Ac-YVAD-pNA, allows a maximum concentration difference of 5 μΜ p-nitroaniline (data not shown). The difference corresponds to a signal amplification factor of 18 and it illustrates the advantages of the method of the invention.

Claims

1 . A method of detecting a proteolytic enzyme characterized by the use, for the detection, of a peptide compound represented by the general formula (I)

RNH-[AA - AA]„[AA - AA]_rCOOH

(I)

and derivatives thereof

wherein:

R is an acetyl group;

[AA - AA]_/ (/^' = an integer comprised from 1 to 4) defines an affinity domain for Au nanoparticles with a positively charged monolayer consisting of an amino acid or an amino acid sequence wherein the amino acid or the amino acids, equal or different one from the other, are selected from aspartic acid or glutamic acid;

[AA - AA]_n {n = an integer comprised between 1 and 8) defines the selectivity domain for the enzyme to be detected consisting of an amino acid or an amino acid sequence wherein the amino acid or the amino acids, equal or different one from the other, are independently selected from both of L and D series proteinogenic amino acids,

having a function both as a substrate of the enzyme to be detected and as inhibitor of gold nanoparticles, functionalized with positively charged metal-ligand complexes self-assembled in a monolayer on the metal surface, which catalyze a conversion reaction of a reagent capable of generating a "reporter" molecule as an instrumentally measurable signal.

2. The method according to claim 1 , wherein the gold functionalized nanoparticles have:

a size comprised from 1 .5 to 20 nm;

positive charges resulting from the complex between 1 ,4,7- triazacyclononane (TACN) and ion Zn";

a charge density of at least 1 positive charge per thiol and up to 3 positive charges per thiol.

3. The method according to claim 1 , wherein the conversion reaction is a conversion reaction of 2-hydroxypropyl-(R-)phosphate, wherein R represents a leaving group generating a "reporter" molecule.

4. The method according to claim 3, wherein R is selected from 2- hydroxypropyl-4-nitrophenyl phosphate, 2-hydroxypropyl-(2,4-dinitrophenyl) phosphate, 2-hydroxypropyl-(2,4-dinitro-7-sulfonate-naftyl) phosphate, 2- hydroxypropyl-(3,6,8-trisulfonate pyrene) phosphate.

5. The method according to claim 3, wherein the conversion reaction is the trans-phosphorylation reaction of 2-hydroxypropyl-4-nitrophenyl phosphate yielding p-nitrophenol as a "reporter molecule".

6. The method according to one of the claims from 1 to 5, wherein the peptide compound is selected from the peptides having SEQ. ID NO. A, B and 1 to 13.

7. The method according to claim 1 comprising at least the steps of:

- adding to a sample containing the proteolytic enzyme to be detected in an aqueous solution or suspension buffered at pH between 7.0 and 8.0, a peptide compound represented by the general formula (I) and having a function both as substrate of the enzyme to be detected and as inhibitor of gold nanoparticles, functionalized with positively charged ligands self- assembled in a monolayer on the metal surface, which catalyze a conversion reaction of a reagent capable of generating a "reporter" molecule as an instrumentally measurable signal;

- adding after incubation for at least 20 min. to the mixture previously obtained the functionalized gold-nanoparticles and a reagent compound substrate thereof for the conversion reaction;

- instrumentally determining the generation of the "reporter" molecule starting from the addition of functionalized gold-nanoparticles and its substrate;

- calculating the enzyme concentration on the basis of the "reporter" molecule concentration.

8. The method according to claim 7, wherein the gold functionalized nanoparticles have:

a size comprised from 1 .5 to 20 nm; positive charges resulting from the complex between 1 ,4,7- triazacyclononane (TACN) and ion Zn";

9. The method according to claim 7, wherein the conversion reaction is a conversion reaction of 2-hydroxypropyl-(R-)phosphate, wherein R represents a leaving group generating a "reporter" molecule.

10. The method according to claim 9, wherein R is selected from 2- hydroxypropyl-4-nitrophenyl phosphate, 2-hydroxypropyl-(2,4-dinitrophenyl) phosphate, 2-hydroxypropyl-(2,4-dinitro-7-sulfonate-naftyl) phosphate, 2- hydroxypropyl-(3,6,8-trisulfonate pyrene) phosphate.

1 1 . The method according to claim 9, wherein the conversion reaction is the trans-phosphorylation reaction of 2-hydroxypropyl-4-nitrophenyl phosphate yielding the p-nitrophenol "reporter molecule".

12. The method according to claim 7, wherein the peptide compound is added at the concentrations from 5.0 x 10^"7 to 1 .0 x 10^"3 M.

13. The method according to claim 7, wherein gold functionalized nanoparticles are added up to the final concentration of 5 x 10^"6 M and substrate thereof is added up to the final concentration of 1 x 10^"3 M.

14. The method according to claim 1 1 , wherein the concentration of the

"reporter" molecule is calculated on the basis of equation:

[PNP] = A₄₀₅ nm /13135 (pH 7.5)

[PNP] = A₄₀₅ nm /8503 (a pH 7.0)

where the value of 13135 corresponds to the molar extinction coefficient of the PNP at pH of 7.5 and the value of 8503 corresponds to the molar extinction coefficient of PNP at a pH of 7.0.

15. The method according to claim 1 1 , wherein the enzyme concentration is calculated on the equation:

[enzyme] = (A₄₀₅ nm, 64800 s-3.75x10^"2)/4.74x10^"2 (μΜ) obtained from a calibration curve using a series of known titre enzyme samples.

16. The method according to one of the claims from 7 to 15, wherein the peptide compound is selected from the peptides having SEQ. ID NO A, B and 1 to 13.

17. Use of peptide compounds representable by the general formula (I)

RNH-[AA - AA]„[AA - AA]_rCOOH

(I)

and derivatives thereof

wherein:

R is an acetyl group;

[AA - AA]_/ (/^' = an integer comprised from 1 to 4) defines an affinity domain for gold nanoparticles with a positively charged monolayer consisting of an amino acid or an amino acid sequence wherein the amino acid or the amino acids, equal or different one from the other, are selected from aspartic acid or glutamic acid;

for a method of detecting a proteolytic enzyme.

18. Use of peptide compounds according to claim 17 selected from peptides having SEQ. ID NO. A, B and 1 to 13.