ELECTROCHEMICAL TYPE BIOSENSORS COMPRISING RESORCARENES

Abstract

The present invention relates to novel resorc[4]arenes, such as pre-organized structure linkers for the development of high performance electrochemical biosensors.

Claims

1. A compound formula (I): ##STR00009## wherein: R.sub.1 is selected from C.sub.2-6alkyl, (CH.sub.2).sub.nC(O)OX, SO.sub.3X, PO(OX).sub.2 and (CH.sub.2).sub.2OH, wherein n is an integer from 1 to 5; R.sub.2 is selected from hydrogen, [heterocycle]-C(O)OX, CH.sub.2-[heterocycle]-C(O)OX, CH.sub.2SO.sub.3X, CH.sub.2PO(OX).sub.2; preferably said [heterocycle] is selected from piperidine, pyrrolidine or piperazine; R.sub.3 is selected from a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.mSH or (CH.sub.2).sub.mS(CH.sub.2).sub.mCH.sub.3 wherein m is independently at each occurrence an integer from 5 to 11; or an aromatic group of molecular formula C.sub.6H.sub.4SH, C.sub.4H.sub.3S, C.sub.6H.sub.4N.sub.2Y; X is independently selected from positively charged atoms or groups comprising: Na.sup.+, K.sup.+, NH.sub.4.sup.+; Y is selected from negatively charged atoms or groups comprising: Cl.sup., BF.sub.4.sup..

2. The compound according to claim 1, wherein: R.sub.1 is CH.sub.2C(O)OX and/or R.sub.2 is hydrogen and/or R.sub.3 is a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.10S(CH.sub.2).sub.11CH.sub.3, preferably X is NH.sub.4.sup.+.

3. An electrode for the electrochemical detection of an analyte in a biological sample, wherein the electrode is a printed miniaturized carbon-based electrode comprising magnetic nanoparticles, said magnetic nanoparticles comprising on their surface the compound of formula (I) according claim 1.

4. The electrode for the electrochemical detection of an analyte in a biological sample according to claim 3, wherein: the printed miniaturized carbon-based electrode is a graphite, graphene, carbon nanotubes or carbon fibers electrode; and/or the magnetic nanoparticles are gold-coated magnetic nanoparticles (Au@MNPs).

5. The electrode for the electrochemical detection of an analyte in a biological sample according to claim 3, wherein the compound of formula (I) is non-covalently linked to at least one antibody able to detect to said analyte.

6. A biosensor for the electrochemical detection of an analyte in a biological sample comprising an electrode according to claim 3 as the working electrode, a reference electrode and a counter-electrode.

7. The biosensor for the electrochemical detection of an analyte in a biological sample according to claim 6, wherein the compound of formula (I) is non-covalently linked to at least one antibody able to detect atrazine (ATZ).

8. A method for the production of a biosensor for the electrochemical detection of an analyte in a biological sample according to claim 6, comprising the following steps: a) adding magnetic nanoparticles to a solution comprising the compound of formula (I), so that at least one compound of formula (I) is bound on the surface of the nanoparticles; b) adding the resulting solution on the surface of a printed miniaturized electrode and evaporating the liquid phase of the solution, preferably in the presence of a magnet located below the electrode; c) adding a solution comprising at least one antibody specific for the analyte on the surface of the electrode obtained from step (b) and incubating for sufficient time allowing the formation of a non-covalent bond among said antibody and the compound of formula (I).

9. A method for the electrochemical detection of an analyte in a biological sample comprising the following steps: a) contacting the biological sample with the biosensor according to claim 6, so that the binding of the antibody to the analyte on the working electrode results in a change in the passage of current between the working electrode and the counter electrode; b) detecting the current change generated in step a) and measuring the presence or quantity of the analyte in the sample by comparison with a calibration curve.

10. An electrode for the electrochemical detection of an analyte in a biological sample, wherein the electrode is a printed miniaturized carbon-based electrode comprising gold-coated nanoparticles, said nanoparticles comprising on their surface a compound of formula (II): ##STR00010## wherein R.sub.1 is selected from hydrogen, alkyl, (CH.sub.2).sub.nC(O)OX, SO.sub.3X, PO(OX).sub.2, (CH.sub.2).sub.2OH, wherein n is an integer from 1 to 5; R.sub.2 is selected from hydrogen, [heterocycle]-C(O)OX, CH.sub.2-[heterocycle]-C(O)OX, CH.sub.2SO.sub.3X, CH.sub.2PO(OX).sub.2; preferably said [heterocycle] is piperidine, pyrrolidine or piperazine; R.sub.3 is selected from a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.mSH or (CH.sub.2).sub.mS(CH.sub.2).sub.mCH.sub.3, wherein m is independently at each occurrence an integer from 5 to 11; or an aromatic group of molecular formula C.sub.6H.sub.4SH, C.sub.4H.sub.3S, C.sub.6H.sub.4N.sub.2Y; X is independently selected from positively charged atoms or groups comprising: Na.sup.+, K.sup.+, NH.sub.4.sup.+; Y is selected from negatively charged atoms or groups comprising: Cl.sup., BF.sub.4.sup.. wherein said compound of formula (II) is non-covalently bound to at least one antibody able to detect said analyte.

Description

[0012] The present invention will now be illustrated with non-limiting examples with reference to the following figures.

[0013] FIG. 1. Electrode current intensity variations following deposition of Ab-ATZ at different concentrations.

[0014] FIG. 2. Histogram relative to the values of i (A) due to the different ATZ incubation times (1 ng/ml).

[0015] FIG. 3. ATZ adsorption isotherm and sensor calibration straight line in the range 0.05-1 ng/mL.

[0016] FIG. 4. DPV signals relative to the interaction between the immunosensor and ATZ standard solutions.

[0017] FIG. 5. Comparing AbATZ adsorption isotherms relative to oriented immobilization (in black) and randomization (in white).

DETAILED DESCRIPTION OF THE INVENTION

[0018] The authors of the present invention have developed a resorc[4]arene linker, able to direct the Abs, with the advantage of increasing the quantity immobilized while maintaining the ability to bind specifically with the Ags, allowing to increase the sensitivity of the measuring device. In order to obtain a good oriented immobilization of the Abs and, in the same way, extend the use of artificial receptors to screen printed gold electrodes (SPE), thus allowing the miniaturization of the measuring device, the compounds object of the present patent application, represented for example by the compound identified with the acronym RW, have been designed and synthesized.

[0019] The linker compounds of the invention are macrocyclic systems belonging to the resorcarene family. These cyclic oligomers, derived from resorcinol, are among the classes of compounds most used in supramolecular chemistry as host species for molecular recognition of host species. The resorcarenes have a unique three-dimensional structure, characterized by a large central cavity, which can be chemically modified in the upper and lower rims with different functional groups. In the present invention, the rational design of resorc[4]arene linkers, such as for example the RW compound, has provided for: [0020] I) the introduction of polar groups in the upper rim, with the aim of favouring interaction with specific amino acid residues of the Fc portion of the antibody in the end-on configuration; [0021] II) the functionalization of the lower rim with alkyl chains containing thioether groups, functional to the anchoring on the surface of gold electrodes through the formation of the SAM; [0022] III) the introduction of negatively charged groups, capable of favouring solubility in aqueous, non-toxic and biocompatible solvent, in order to avoid degradation of SPEs.

[0023] In particular, the RW linker, thus designed and synthesized, turned out to be able to bind the Fc portion of different Abs by non-covalent interactions, e.g., dipole-dipole and electrostatic, allowing the possible regeneration of the functionalized surface after the measurement of the Ag-Ab interaction. One of the main advantages in using the RW compound for the development of high-performance immunosensors consists in combining the correct immobilization of the antibody with the possibility of developing electrochemical sensors.

[0024] In one embodiment the present invention concern compounds of formula (I), such as artificial linkers capable of directing site-specific immobilization of antibodies according to an end-on orientation, wherein:

##STR00001## [0025] R.sub.1 is selected from C.sub.2-6alkyl, (CH.sub.2).sub.nC(O)OX (wherein 1n5), SO.sub.3X, PO(OX).sub.2, (CH.sub.2).sub.2OH; [0026] wherein X are positively charged atoms or groups comprising: Na.sup.+, K.sup.+, NH.sub.4+; [0027] R.sub.2 is selected from hydrogen, [heterocycle]-C(O)OX, CH.sub.2-[heterocycle]-C(O)OX, CH.sub.2SO.sub.3X, CH.sub.2PO(OX).sub.2, wherein X are positively charged atoms or groups such as: Na.sup.+, K.sup.+, NH.sub.4.sup.+; preferably said heterocycle is piperidine, pyrrolidine or piperazine; [0028] R.sub.3 is selected from [0029] a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.mSH (5m11) or (CH.sub.2).sub.mS(CH.sub.2).sub.mCH.sub.3 (5m11), or [0030] an aromatic group of molecular formula C.sub.6H.sub.4SH, C.sub.4H.sub.3S or C.sub.6H.sub.4N.sub.2Y wherein Y are negatively charged atoms or groups comprising: Cl.sup., BF.sub.4.sup..

[0031] As used herein, the terms heterocycle or saturated heterocycle, used interchangeably, mean a 4-7 term saturated cyclic compound comprising at least one heteroatom; examples of heterocycles are, for example, azetidine, pyrrolidine, piperidine, piperazine, morpholine, thiomorpholine, azacycloheptane; preferably the saturated heterocycle is piperidine, pyrrolidine or piperazine, preferably linked via the nitrogen atom, directly or via a methylene bridge, to the aryls of the compound of general formula (I) or of general formula (II), as will be described below.

[0032] As used herein, (CH.sub.2).sub.nC(O)OX indicates an aliphatic chain comprising n methylene units (CH.sub.2), functionalized at one end with a carboxyl group C(O)OX, wherein X is the positively charged counterion balancing the negative charge of the carboxylate, and bonded at the other end to the reference scaffold. Thus in the present invention, (CH.sub.2).sub.nC(O)OX indicates for example CH.sub.2C(O)OX, CH.sub.2CH.sub.2C(O)OX, CH.sub.2CH.sub.2CH.sub.2C(O)OX etc., wherein X is the positively charged counterion balancing the negative charge of the carboxylate. Similarly, (CH.sub.2).sub.mSH indicates an aliphatic chain comprising m methylene units (CH.sub.2), functionalized at one end with an SH group and linked at the other end to the reference scaffold; (CH.sub.2).sub.2OH indicates an aliphatic chain comprising two methylene units (CH.sub.2), functionalized at one end with an OH group and linked at the other end to the reference scaffold. The terms CH.sub.2-[heterocycle]-C(O)OX, CH.sub.2SO.sub.3X, CH.sub.2PO(OX).sub.2 indicate methylene groups linked to the reference scaffold and to the indicated functional group.

[0033] A preferred embodiment of the invention subject-matter of the present description concerns a compound of general formula (I) wherein: R.sub.1 is (CH.sub.2).sub.nC(O)OX, preferably CH.sub.2C(O)OX, and/or R.sub.2 is hydrogen and/or R.sub.3 is a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.10S(CH.sub.2).sub.11CH.sub.3; in a further preferred embodiment X is NH.sub.4.sup.+; in a further preferred embodiment R.sub.1 is CH.sub.2C(O)OX, R.sub.2 is hydrogen, R.sub.3 is a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.10S(CH.sub.2).sub.11CH.sub.3 and X is NH.sub.4.sup.+.

[0034] In a further embodiment, the invention concerns an electrode for the electrochemical detection of an analyte in a biological sample, wherein the electrode is a printed miniaturized carbon-based electrode comprising magnetic nanoparticles, preferably coated with gold, said nanoparticles comprising on their surface the compound of formula (I) as defined above; preferably said printed miniaturized carbon-based electrode is a graphite, graphene, carbon nanotubes or carbon fibers electrode; in a further preferred embodiment the compound of formula (I) is non-covalently linked to at least one antibody able to detect to said analyte.

[0035] Gold-coated magnetic nanoparticles (Au@MNPs) are currently used primarily for the bioseparation and the development of electrochemical and optical sensors, for the preparation of contrast agents for magnetic resonance imaging, or for the targeted delivery of drugs. The gold-coated magnetic nanoparticles used herein are commercially available or can be obtained by procedures described in the literature (Chem. Commun., 2016, 52, 7528-7540).

[0036] In a further embodiment, the invention concerns a biosensor for the electrochemical detection of an analyte in a biological sample comprising the electrode as above defined as a working electrode, a reference electrode and a counter electrode.

[0037] In a preferred embodiment, the biosensor for the electrochemical detection of an analyte in a biological sample comprises: [0038] a printed miniaturized carbon-based electrode comprising magnetic nanoparticles; preferably said electrode is a graphite, graphene, carbon nanotubes or carbon fibers electrode; [0039] the compound of formula (I) as defined above non-covalently linked with at least one antibody capable of recognizing the analyte.

[0040] In a further preferred embodiment, the magnetic nanoparticles are gold-coated magnetic nanoparticles modified on the surface with the compound of general formula (I).

[0041] In a further preferred embodiment, in the biosensor for the electrochemical detection of an analyte in a biological sample, the compound of formula (I) non-covalently binds at least one antibody capable of recognizing atrazine (ATZ).

[0042] In a further embodiment, the present invention comprises a method for the production of the biosensor as defined above for the electrochemical detection of an analyte in a biological sample comprising the following steps: [0043] (a) adding magnetic nanoparticles preferably coated with gold to a solution comprising the compound of formula (I), such that at least one compound of formula (I) is bound on the surface of the nanoparticles; [0044] (b) depositing the resulting solution on the surface of a printed miniaturized carbon-based electrode and evaporating the liquid phase of the solution, preferably in the presence of a magnet located below the electrode; [0045] (c) adding to the electrode surface a solution comprising at least one antibody specific for an analyte obtained at the end of step (b) and incubating for sufficient time for the formation of a non-covalent bond with the compound of formula (I).

[0046] Preferably, in step (a) the compound of formula (I) is used in aqueous solution and in a concentration comprised between 1 M and 4 mM; again preferably, in step (c) the antibody is used in a concentration comprised between 0.1 and 100 g/ml.

[0047] In a further embodiment, the present invention concerns a method for the electrochemical determination of an analyte in a biological sample comprising the following steps: [0048] a) contacting the biological sample with the biosensor as defined above, so that the antibody present on the working electrode binds the analyte resulting in a change in the passage of current between the working electrode and the counter electrode; [0049] b) detecting the current change generated in step a); [0050] c) measuring the presence and/or the quantity of the analyte in the sample by comparison with a calibration curve.

[0051] In a further embodiment, the invention concerns an electrode for the electrochemical detection of an analyte in a biological sample, wherein the electrode is a printed miniaturized carbon-based electrode comprising magnetic nanoparticles, preferably coated with gold, said nanoparticles comprising on their surface the compound of formula (II):

##STR00002## [0052] Wherein [0053] R.sub.1 is selected from hydrogen, alkyl, (CH.sub.2).sub.nC(O)OX (1n5), SO.sub.3X, PO(OX).sub.2, (CH.sub.2).sub.2OH, wherein X are positively charged atoms or groups comprising: Na.sup.+, K.sup.+, NH.sub.4.sup.+; [0054] R.sub.2 is selected from hydrogen, [heterocycle]-C(O)OX, CH.sub.2-[heterocycle]-C(O)OX, CH.sub.2SO.sub.3X, CH.sub.2PO(OX).sub.2 wherein X are positively charged atoms or groups such as: Na.sup.+, K.sup.+, NH.sub.4.sup.+; preferably the heterocycle is piperidine, pyrrolidine or piperazine; [0055] R.sub.3 is selected from [0056] a saturated linear aliphatic group containing sulfur of molecular formula (CH.sub.2).sub.mSH (5m11) or (CH.sub.2).sub.mS(CH.sub.2).sub.mCH.sub.3 (5m11); [0057] an aromatic group of molecular formula C.sub.6H.sub.4SH, C.sub.4H.sub.3S, C.sub.6H.sub.4N.sub.2Y wherein Y are negatively charged atoms or groups, preferably Cl.sup., BF.sub.4.sup.; [0058] wherein said compound of formula (II) non-covalently binds at least one antibody capable of recognizing said analyte.

[0059] The invention concerns the use of said electrode as a working electrode in a biosensor for the electrochemical detection of an analyte in a biological sample, said biosensor further comprising a reference electrode and a counter electrode.

[0060] Overall, the data of the invention in question propose a new artificial linker, such as in particular the RW compound, capable of directing the site-specific immobilization of antibodies according to an end-on orientation, and emphasize the effectiveness of the compound as a new potential linker capable of increasing the performance of electrochemical immunosensors.

Examples

[0061] The following examples are reported for illustrative purposes only and are not intended to limit the scope of the present invention. Variations and modifications of any of the embodiments described herein, which are obvious to a person skilled in the art, are encompassed by the scope of the appended claims.

Rational Drawing and Synthesis of the RW Compound

Chemicals, Reagents and Methods of Analysis

[0062] All reagents and solvents are commercially available and have been used without further purifications.

[0063] Silica gel (230-400 mesh) was used for purification by flash column chromatography. All reactions were monitored by thin layer chromatography (TLC) and F254 fluorescence silica gel plates (Sigma-Aldrich 99569) were used. The melting points were determined with a Buchi Melting Point B-454. The 1H and 13C NMR spectra were recorded with a Bruker 400 Ultra Shield instrument (400 MHz for .sup.1H NMR and 100 MHz for .sup.13C NMR), using tetramethylsilane (TMS) as a standard. Chemical shifts are reported in parts per million (ppm). Multiplicities were reported as follows: singlet (s), doublet (d), triplet (t) and multiplet (m). Mass spectrometry was performed with the Thermo Finnigan LXQ linear ion trap mass spectrometer, equipped with electrospray ionization (ESI). High-resolution mass spectra (HRMS) were recorded with a Bruker BioApex Fourier transform ion cyclotron resonance (FT-ICR).

Synthesis Procedures

[0064] Synthesis of compound 2: 10-undecen-1-ol (MERCK 203-971-0) (41.11 mmol, 7 g) was added to a solution of pyridinium chlorochromate (MERCK 247-595-5) (61.66 mmol, 13.3 g) and celite (MERCK 272-489-0) (3 g) in dichloromethane (DCM) (250 ml). The reaction, which takes on a dark colouring, was allowed to stir at room temperature for 1.5 hours. Subsequently, the reaction was filtered over gooch, using as eluent mixture a hexane:ethyl acetate (AcOEt) solution in 9:1 ratio. The filtrate was concentrated under reduced pressure and the compound 2 was obtained in yield of 80%. [Corey E. J. and Suggs J. W. 1975]

##STR00003##

[0065] Oil (80% yield). .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm)=9.76 (s, 1H, RCHO), 5.87-5.73 (m, 1H, RCHCH.sub.2), 4.98 (d, J=17.1 Hz, 1H, RCHCH.sub.2), 4.92 (d, J=10.2 Hz, 1H, RCHCH.sub.2), 2.07-1.98 (m, 2H, RCH.sub.2CHO), 1.68-1.54 (m, 2H, RCH.sub.2CHCH.sub.2), 1.43-1.21 (m, 12H, CH.sub.2). .sup.13C-NMR (CDCl.sub.3, 100 MHz): (ppm)=202.98, 139.15, 114.17, 43.91, 33.77, 29.29, 29.25, 29.14, 29.03, 28.88, 22.07.

[0066] ESI-HRMS (positive) m/z: [M+Na].sup.+ calculated for C.sub.11H.sub.20ONa 191.29; it was found: 191.29

[0067] Synthesis of compound 3: resorcinol (MERCK 203-585-2) (38 mmol, 4.18 g), previously crushed with mortar and pestle, was added to a solution of ethanol (EtOH) (16.25 ml) and hydrochloric acid (37% HCl) (5.41 ml). After 30 minutes, the solution takes on a white colouring, and undecylenic aldehyde 2 (38 mmol, 6.40 g.) previously solubilized in EtOH (10.58 ml) is added. Subsequently, the reaction was allowed to stir and reflux for 24 hours at 70 C. Subsequently, the reaction was brought to room temperature and concentrated under reduced pressure. The residue was purified by flash chromatography column using DCM:Methanol (MeOH) in 95:5 ratio as eluent mixture. The compound 3 was obtained in yield of 70%. [Thoden van Velzen E. U. et al. 1995]

##STR00004##

[0068] Brown powder (70% yield); p.f. 2900.5 C. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm)=9.60 (br s, 8H, ArOH), 7.21 (s, 4H, ArHext.), 6.11 (s, 4H, ArHint.), 5.88-5.74 (m, 4H, RCHCH.sub.2), 4.99 (dd, J=17.1, 1.4 Hz, 4H, RCHCH.sub.2), 4.95 (d, J=10.1 Hz, 4H, RCHCH.sub.2), 4.30 (pseudo t, J=7.2 Hz, 4H, ArCHAr), 2.44-2.09 (m, 8H, RCH.sub.2CHAr.sub.2), 2.12-1.95 (m, 8H, RCH.sub.2CHCH.sub.2), 1.45-1.20 (m, 48H, CH.sub.2). .sup.13C NMR (CDCl.sub.3, 100 MHz): (ppm)=139.20, 114.15, 33.85, 29.71, 29.52, 29.17, 28.99, 27.99.

[0069] ESI-HRMS (positive) m/z: [M+Na].sup.+ calculated forC.sub.68H.sub.96O.sub.8Na 1063. 69974; it was found 1063.70010.

[0070] Synthesis of compound 4: potassium carbonate (K.sub.2CO.sub.3) (19.2 mmol, 2.65 g) and methylbromoacetate (BrCH.sub.2COOCH.sub.3) (9.6 mmol, 1.47 g) (ratio of starting substrate to reagents 1:20:10) were added to a solution of resorcarene 3 (0.96 mmol, 1 g) in acetonitrile (ACN) (131.5 ml). The reaction was allowed to stir and reflux for 24 hours at 82 C. The solution was then evaporated under reduced pressure to remove ACN in excess and dissolved in DCM. The obtained organic phase was washed once with a 1 N HCl solution (70 ml) and twice with a saturated sodium chloride (NaCl) solution (140 mL). Finally, it was dehydrated with anhydrous sodium sulfate (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The residue was allowed to stir 12 hours in MeOH, the precipitate was vacuum filtered obtaining the compound 4 in yield of 77%.

##STR00005##

[0071] White powder (77% yield) p.f. 2680.5 C. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm)=6.60 (s, 4H, ArHext.), 6.20 (s, 4H, ArHint.), 5.86-5.71 (m, 4H, RCHCH.sub.2), 4.96 (d, J=17.1 Hz, 4H, RCHCH.sub.2), 4.90 (dd, J=10.2, 0.9 Hz, 4H, RCHCH.sub.2), 4.58 (t, J=7.4 Hz, 4H, ArCHAr), 4.21

[0072] (s, 16H, ArOCH.sub.2CO), 3.68 (s, 24H, CH.sub.3OCOR), 1.99-1.88 (m, 8H, RCH.sub.2CHCH.sub.2), 1.82-1.70 (m, 8H, RCH.sub.2CHAr.sub.2), 1.34-1.10 (m, 48H, CH.sub.2). .sup.13C NMR (CDCl.sub.3, 100 MHz): (ppm)=169.94, 154.60, 139.35, 128.62, 126.68, 114.20, 100.87, 67.26, 52.05, 35.81, 34.63, 33.96, 30.08, 29.82, 29.80, 29.36, 29.11, 28.17.

[0073] ESI-HRMS (positive) m/z: [M+Na].sup.+ calculated for C.sub.92H.sub.128O.sub.24Na 1640.9537; it was found 1640.8669.

[0074] Synthesis of compound 5: 1-dodecanthiol (14.09 mmol, 2.85 g) and 9-borabicyclo[3.3.1.]nonane (BBN) (24.75 mmol, 3 g) were added at 0 C. to a solution of resorcarene 4 (0.618 mmol, 1 g) in tetrahydrofuran (THF) (53.74 ml). The reaction was allowed to stir for 12 hours at room temperature. Subsequently, the solvent was evaporated under reduced pressure and the residue was purified by crystallization in MeOH. The compound 5 was obtained in yield of 82%. [Thoden van Velzen E. U. et al. 1995]

##STR00006##

[0075] White powder (82% yield); p.f. 2900.5 C. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm)=6.59 (s, 4H, ArHext.), 6.20 (s, 4H, ArHint.), 4.57 (t, J=7.4 Hz, 4H, ArCHAr), 4.27 (s, 16H, ArOCH.sub.2CO), 3.75 (s, 24H, CH.sub.3OCOR), 2.53-2.39 (m, 16H, CH.sub.2SCH.sub.2), 1.92-1.75 (m, 8H, CH.sub.2), 1.67-1.46 (m, 16H, CH.sub.2), 1.39-1.19 (m, 128H, CH.sub.2), 0.87 (t, J=6.7 Hz, 12H, CH.sub.3). .sup.13C NMR (CDCl.sub.3, 100 MHz): (ppm)=169.94, 154.61, 128.62, 126.68, 100.89, 67.27, 52.05, 35.82, 34.63, 32.35, 32.05, 30.11, 29.96, 29.94, 29.91, 29.88, 29.83, 29.80, 29.77, 29.76, 29.69, 29.53, 29.49, 29.43, 29.21, 29.13, 28.20, 22.82, 14.26.

[0076] ESI-HRMS (positive) m/z: [M+H].sup.+ calculated for C.sub.140H.sub.232O.sub.24S.sub.4 2425.58109; it was found [M+Na].sup.+ 2447.56295.

[0077] Synthesis of compound 6: Resorcarene 5 (0.124 mmol, 300 mg) was solubilized in THF (17.3 ml) and subsequently treated with an aqueous solution of 2M potassium hydroxide (KOH) (7.44 ml) for 4 hours at room temperature. The reaction was then acidified with 2M HCl and concentrated under reduced pressure. The obtained residue was washed with water and dried at 80 C. under vacuum. The compound 6 was obtained in yield of 93%. [Hua B. et al. 2016]

##STR00007##

[0078] White powder (yield 93%); p.f. 2700.5 C. .sup.1H NMR (CDCl.sub.3:CD.sub.3OD=98:2, 400 MHz): (ppm)=6.64 (s, 4H, ArHext.), 6.16 (s, 4H, ArHint.), 4.54 (t, J=7.1 Hz, 4H, ArCEAr), 4.48-3.98 (m, 16H, ArOCH.sub.2CO), 2.54-2.34 (m, 16H, CH.sub.2), 1.78 (br s, 8H, CH.sub.2), 1.56-1.46 (m, 16H, CH.sub.2), 1.37-1.10 (m, 128H, CH.sub.2), 0.83 (t, J=6.6 Hz, 12H, CH.sub.3). .sup.13C NMR (CDCl.sub.3:CD.sub.3OD (98:2), 100 MHz): (ppm)=170.12, 154.45, 128.52, 126.59, 100.84, 67.14, 35.56, 34.61, 32.23, 31.95, 30.01, 29.84, 29.81, 29.77, 29.73, 29.70, 29.67, 29.65, 29.58, 29.42, 29.38, 29.31, 29.09, 29.01, 28.06, 22.72, 14.12. ESI-HRMS (negative) m/z: [M2H].sup.2 calculated for C.sub.132H.sub.216O.sub.24S.sub.4 1155.72094; it was found 1155.72196.

[0079] Synthesis of compound 7: Resorcarene 6 (0.043 mmol, 100 mg) was treated with 25-28% ammonium hydroxide (NH.sub.4OH) solution at room temperature for 24 hours. Subsequently, the solution was concentrated under reduced pressure and the compound 7 was obtained in a quantitative yield. [Hua B. et al. 2016]

##STR00008##

Pink Powder (Quantitative Yield)

Modification of Magnetic Nanoparticles Decorated with Gold (Au@MNPs)

[0080] The RW compound was used to modify the surface of gold-decorated magnetic nanoparticles (Au@MNPs), allowing the realization of functionalized nanoparticles (RW/Au@MNPs). To this end, the Au@MNPs, once washed with water, were incubated in a rotating stirrer away from light, with a RW compound solution. Different concentrations of RW compound in water were evaluated, in a range comprised between 4 mM and 1.8 M, (Ha, Solovyov, and Katz 2009) evaluating the stability thereof over time. The optimal concentration of RW compound was found to be 100 M, and was chosen for realizing the immunosensor. The graphite screen printed (SPE) electrodes were used as electrochemical transducers for the realization of the immunosensor, depositing on the surface of the graphite working electrode, 20 L of the RW/Au@MNPs solution. A magnet was used to prevent the loss of material during the washing processes that characterize the measurements. The system thus realized was then evaluated in the antibody loading capacity for atrazine (Ab-ATZ). Immobilization was achieved by depositing the antibody solution at different concentrations on the electrode surface. Subsequently, the surface was washed with phosphate buffer (PBS) and a 0.1 mg/mL albumin solution (BSA) was used to deactivate the RW compound molecules that did not react with the specific antibody, preventing the occurrence of any non-specific interactions in the incubation step with the antigen. The electrochemical characterization of the various phases involved in the realization of the immunosensor was carried out by differential pulse voltammetry (DPV) using as redox probe the iron-ferricyanide pair [Fe(CN).sup.6].sup.3/4, an electrochemically active substance which is discharged on the surface of the electrode at a certain applied potential giving rise to a current signal. [Ramnani P. et al. 2016] Any modification made to the sensor surface, hindering the diffusion of the redox probe towards the electrode surface, results in the detected current intensity being lowered which is proportional to the quantity of substance that interacted on the electrode surface. [Ha J. M. et al. 2009; Ramnani P. et al. 2016]

Immobilization of Ab-ATZ on RW/Au@AuMNPs

[0081] The electrodes were previously modified by deposition of Au@MNPs functionalized with a 100 M solution of RW compound. In order to trace back the optimal concentration of antibody to be immobilized, a loading curve was constructed by observing the decrease in the current signal (I) resulting after incubation of increasing quantities of antibody in the concentration range; 0.1-100 g/ml (FIG. 1) in PB buffer (phosphate buffer) pH 7.4. The measurements were performed by differential pulse voltammetry in the presence of a solution 1.1 mM Fe(CN).sub.6.sup.3/4, 100 mM KCl, in the potential range [0.4-+0.6].

[0082] The antibody concentration chosen for realizing the immunosensor was equal to 20 g/ml.

Optimization of ATZ Incubation

[0083] In order to improve the interaction of Ab.sub.ATZ with ATZ, different incubation times of an ATZ solution (1 ng/ml) on the surface of the realized immunosensor, in a range between 15 and 50 min, were evaluated by measuring the lowering of the intensity of current (FIG. 2).

[0084] The measurements were carried out in [Fe(CN).sub.6].sup.3/4 in 1.1 mM deionized water (R=18 mOhm) in the range of potentials [0.4-+0.6].

ATZ Calibration

[0085] For immunosensor characterization, various concentrations of Atrazine were evaluated in a range comprised between 0.05 ng/mL and 10 ng/ml, with an incubation time of 30 minutes.

[0086] At the end of the process, the excess unbound antigen was removed by rinsing with 20 mM dilution phosphate buffer (PBS) pH 7.4 (FIGS. 3 and 4).

[0087] The measurements were carried out in [Fe(CN).sub.6].sup.3/4 in 1.1 mM deionized water (R=18 mOhm) in the range of potentials [0.4-+0.6].

[0088] The sensor obtained showed a sensitivity equal to 5.79 mL*A/ng, a limit of detection (LOD) of 0.015 ng/mL and a linear dynamic range (linear range) 0.05-1 ng/mL, better analytical performance when compared to a comparison sensor obtained by chemical immobilization (Tab.1).

TABLE-US-00001 TABLE 1 Analytical performance obtained with RW-oriented Ab immobilization and random immobilization. Linear Sensitivity Analytical Range (mL/ng)* LOD Performances (ng/mL) A (ng/mL) Resorcarene 0.05-1 5.7876 0.015 Immobilization Random 0.3-2 2.2215 0.035 immobilization

Description of the Random Immobilization Method

[0089] The graphite electrodes (SPE) were previously modified by depositing mercapto propionic acid (MPA)-functionalized Au@MNPs derived from incubating 10 L of Au@MNPs in a 230 mM MPA solution in PBS pH7.4.

[0090] The MPA carboxyl groups were subsequently activated by treatment with 1:1 EDC/NHS at 4 mM concentration for 15 min. Once the excess reagent was rinsed with MES buffer pH 5.4, the surface was incubated with a 20 g/mL solution of Anti-Atrazine Antibody (AbATZ) for 30 minutes and subsequently rinsed by PBS buffer. The electrode is then rinsed in PBS and incubated for 20 minutes with 1 M aqueous ethanolamine solution to deactivate the activated sites that have not reacted with AbATZ.

[0091] The measurements were carried out in FeCN.sub.6.sup.3/41.1 mM 100 mM KCl cell in deionized water (R=18 mOhm) in the range of potentials [0.4; +0.6]V using a graphite counter electrode and a calomel reference electrode (SCE). The electrochemical experiments were carried out using the Palmsens potentiostat.

[0092] As shown in FIG. 5, the curve concerning the oriented immobilization of the AbATZ thanks to the presence of the RT linkerwith the same concentration usedis higher than the random chemical immobilization, to the advantage of the subsequent Ab-Ag interaction phase.

BIBLIOGRAPHY

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