WO2013042012A1

WO2013042012A1 - Core shell conjugation of silica nanotube and polythiophene for qcm application and method for making the same

Info

Publication number: WO2013042012A1
Application number: PCT/IB2012/054745
Authority: WO
Inventors: Jean-Paul Lellouche; Natasha ESMAN; Anna Peled; Rivka BEN-ISHAY; Yaara Kapp-Barnea; Irena GRIGORIANTS
Original assignee: Bar-Ilan Research And Development Company Ltd.; Amendis Ltd.
Priority date: 2011-09-19
Filing date: 2012-09-12
Publication date: 2013-03-28

Abstract

A gold coated quartz crystal microbalance (QCM) electrode modified by a nanocomposite. The nanocomposite is based on silica nanotube covalently conjugated to polythiophene acetic acid functionalized by thiol groups in core shell morphology. Method of making the electrode and the nanocomposite are also provided.

Description

SILICA NANOTUBES

BACKGROUND OF THE INVENTION

[001] Biosensors are analytical tools that can translate a molecular recognition reaction to real-time quantitative information. The quartz crystal microbalance (QCM) is extremely sensitive and able to monitor mass variations at the nanogram level. QCM works on the basis of frequency changes of a piezoelectric gold-coated crystal, which are directly proportional to changes in masses absorbed on the electrode.

[002] One of the most straight-forward systems that could be monitored by QCM is the biotin-avidin binding event. Much effort has been devoted to develop techniques for the irreversible immobilization of biomolecules on gold surfaces, especially biotin-avidin systems (A. Dupont-Filliard, M. Billon, G. Bidan and S. Guillerez, Electroanalysis, 2004, 16, 667). Avidin has the ability to bind four biotin molecules, thus opening the possibility for more elaborate sensors in the sense of bio-macromolecules such as proteins by immobilization with antibodies, ligand etc. using protein-protein recognition and interaction. In addition, the non-covalent interaction between biotin and avidin better preserves biological molecules activity and prevents the need for additional chemical reactions and reagents.

[003] In order to functionalize such gold surfaces, functional nanomaterials can be deposited on the electrode surface. QCM electrodes have been modified using self- assembled monolayers (SAMs), (D. Samanta and A. Sarkar, Chem. Soc. Rev., 2011, 40, 2567) conducting polymer films, and carbon and indium oxide nanotubes (NTs, R. J. Chen, S. Bangsaruntip, K. A. Drouvalakis, N. Wong Shi Kam, M. Shim, Y. Li, W. Kim, P. J. Utz and H. Dai, PANS, 2003, 100, 4984.). However, only in the case of SAMs did the use of thiols allow a strong affinity between the electrode and the deposited material. In other cases they were simply deposited on the electrode from a solution. The use of functional silica NTs (SNTs) has been explored regarding this aspect.

[004] The functionalization of SNTs has been extensively explored over the last decade. These nanostructures have great potential in drug delivery (S. J. Son, J. Reichel, B. He, M. Schuchman and S. B. Lee, J. Am. Chem. Soc, 2005, 127, 7316.), biosensors (J. H. Jung, M. Park and S. Shinkai, Chem. Soc. Rev., 2010, 39, 4286) and recognition systems, DNA delivery, biocatalysis, gene delivery, MRI and more (K.-W. Hu, K.-C. Hsu and C.-S. Yeh, Biomaterials, 2010, 31, 6843). Two main approaches have been developed over the years for the synthesis of SNTs. The first one is by hard template synthesis, which utilizes anodic alumina oxide (AAO) membranes (K. Okamoto, C. J. Shook, L. Bivona, S. B. Lee and D. S. English, Nano Lett., 2004, 4, 233). The NTs are fabricated in the channels of the membrane and then liberated by either acidic or basic dissolution of the template and washing of the product. The membranes can be custom made in length and pore diameter. The second approach is self-assembly by soft template synthesis, which is based on various organogelators. Organogelators are divided into two categories according to the driving force behind their molecular aggregation: hydrogen bond-based and non-hydrogen bond-based gelators (J. H. Jung, M. Park and S. Shinkai, Chem. Soc. Rev., 2010, 39, 4286.). Examples of hydrogen bond- based gelators are amide- or urea-based cyclohexane, peptide and sugar-based derivatives that exhibit distinct helical structures in the fibrous aggregates formed in certain solvents. Examples of non-hydrogen bond-based gelators are cholesterol derivatives (J. H. Jung, Y. Ono and S. Shinkai, Langmuir, 2000, 16, 1643) which rely on π-π stacking, van der Waals forces and solvophobic properties in order to assemble and direct fiber-like nanostructure formation (J. H. Jung, M. Park and S. Shinkai, Chem. Soc. Rev., 2010, 39, 4286). This method also allows control over the dimensions of the produced SNTs. In addition, simple calcination removes the organogels yielding a clean inorganic product.

[005] In order to use SNTs for specific applications, they need to have functional groups suitable for the intended applications, such as strong affinity to gold surface or the ability to bind biological markers. A covalent strategy has been employed for linking thiols (D. R. Bae, S. J. Lee, S. W. Han, J. M. Lim, D. Kang and J. H. Jung, Chem. Mater., 2008, 20, 3809), amines, benzene rings, rhodamine B, antibodies, and various silica-based linkers, in order to obtain functional SNTs. However there has been no report regarding the covalent linkage of any functional conjugated polymer on the surface of SNTs in core- shell morphology.

[006] The core-shell morphology for inorganic-organic SNTs has not been extensively explored. There has only been one report on such a morphology using a polyaniline (PANI) shell (T. H. Kim, Y. Kim, S. J. Lee, W. S. Han and J. H. Jung, Chem. Lett., 2008, 37, 598). The interaction between bare SNTs and anilinium cations was electrostatic, followed by in situ polymerization SUMMARY OF THE INVENTION

[007] In one embodiment, this invention provides a nanocomposite comprising: (a) a polythiophene acetic acid silica nanotube (SNTs-g-Thp/PTAA) comprising: a thiol group, a carboxylic acid group, and (b) a conjugated functional polymer shell, wherein the conjugated functional polymer shell is covalently linked on the surface of the core of the polythiophene acetic acid silica nanotube in core-shell morphology.

[008] In another embodiment, this invention further provides a quartz crystal microbalance (QCM) electrode comprising a nanocomposite comprising: (a) a polythiophene acetic acid silica nanotube (SNTs-g-Thp/PTAA) comprising: a thiol group, a carboxylic acid group, and (b) a conjugated functional polymer shell, wherein the conjugated functional polymer shell is covalently linked on the surface of the core of the polythiophene acetic acid silica nanotube in a core- shell morphology. In another embodiment, the QCM electrode is a piezoelectric sensor. In another embodiment, the phrase "QCM electrode" and the phrase "piezoelectric sensor" are used interchangebly.

[009] In another embodiment, this invention further provides a method for making a nanocomposite comprising polythiophene acetic acid silica nanotubes having a core-shell morphology, comprising the steps of: grafting the surface of silica nanotubes with 2- (thiophene-2-yl)-N-(3-(triethylsilyl)propyl) (Thp-TES); dispersing the surface-grafted silica nanotubes in anhydrous CHCI₃ adding 3-thiophene-acetic acid (TAA) monomer; adding anhydrous FeCl₃ dissolved in CHC1₃ resulting in a nanocomposite dispersion; adding MeOH; recovering the resultant nanocomposite comprising polythiophene acetic acid silica nanotubes having a core-shell morphology, wherein steps (a) to (f) are carried out substantially sequentially.

[010] In another embodiment, this invention further provides a method for modifying the nanocomposite comprising polythiophene acetic acid silica nanotubes having a core- shell morphology with 1,4-diaminobutane, comprising the steps of: dispersing the resultant nanocomposite in anhydrous CHC1₃; adding Ι,Γ-carbonyldiimidazole to the dispersion; adding 1,4-diaminobutane dissolved in anhydrous CHC1₃ to the dispersion; washing the polyNH₂ modified nanocomposite with a solution comprising: water, CHC1₃, and alcohol, wherein steps (a) to (d) are carried out substantially sequentially.

[011] In another embodiment, this invention further provides a method for modifying the nanocomposite comprising polythiophene acetic acid silica nanotubes having a core- shell morphology with cysteamine, comprising the steps of: dispersing the resultant nanocomposite in THF; adding Ι,Γ-carbonyldiimidazole to the dispersion; adding cysteamine to the dispersion; washing the cysteamine-modified nanocomposite (SNT-g- Thp/PTAA(COOH)/SH) with a solution comprising: water and alcohol, wherein steps (a) to (d) are carried out substantially sequentially.

[012] In another embodiment, this invention further provides a method for derivatizing cysteamin modified nanocomposite comprising polythiophen acetic acid silica nanotubes having core-shell morphology with isoindole, comprising the steps of: dissolving SNT-g- Thp/PTAA(COOH)/SH in MeOH; adding OPA; removing unbound o-phtaldialdehyde (OPA); adding NH₄OH, wherein steps (a) to (d) are carried out substantially sequentially.

[013] In another embodiment, this invention further provides a method for derivatizing the nanocomposite comprising polythiophene acetic acid silica nanotubes having a core- shell morphology with an amine group, comprising the steps of: contacting the nanocomposite comprising polythiophene acetic acid silica nanotubes having a core-shell morphology with a solution comprising: NHS, EDC, and water; adding 1,3- diaminopropane; rinsing with water; drying under a N₂ stream; immersing in a solution comprising bis- 1 - ( 11 - { 2- [2- { 2- (2-hydroxy-ethoxy)-ethoxy } -ethoxy] -ethoxy } -undecyl) disulfide (PEG); washing in EtOH; and drying under a N₂ stream, wherein steps (a) to (g) are carried out substantially sequentially.

[014] In another embodiment, this invention further provides a method for making a quartz crystal microbalance (QCM) electrode comprising a nanocomposite, the nanocomposite comprises a polythiophene cysteamine acetic acid silica nanotube (SNT-g- Thp/PTAA/SH), comprising the steps: contacting pretreated gold QCM crystals with a dispersion comprising SNT-g-Thp/PTAA/SH nanocomposite; and drying the gold QCM crystals comprising the dispersion comprising SNT-g-Thp/PTAA/SH nanocomposite, wherein steps (a) and (b) are carried out substantially sequentially.

[015] In another embodiment, this invention further provides a method for monitoring mass variations at the nanogram level in a reaction mixture comprising contacting a quartz crystal microbalance (QCM) electrode comprising the nanocomposite of any one of claims 1-8 with a reaction mixture, thereby monitoring mass variations at the nanogram level in a reaction mixture. BRIEF DESCRIPTION OF THE DRAWINGS

[016] Figure 1. Is a scheme showing the synthetic strategy for the preparation of Thp- TES.

[017] Figure 2. Is a micrograph showing TEM (2A), HR-TEM (2B), and FE-SEM (2C) of bare SNTs.

[018] Figure 3. A scheme showing the synthetic strategy for the preparation of innovative core-shell SNT-g-Thp/PTAA NC and subsequent modifications.

[019] Figure 4. HR-TEM micrograph of core-shell morphology of the final SNT-g- Thp/PTAA NC (4A) and a graph showing a line scan analysis of SNT-g-Thp/PTAA NC (red line-Si, green line- S), inset: location of the reported line scan analysis.

[020] Figure 5. FT-IR spectra of i) bare SNTs ii) PTAA iii) SNT-g-Thp iv) SNT-g- Thp/PTAA (5 A) TGA of i) bare SNTs ii) SNT-g-Thp iii) SNT-g-Thp/PTAA (5B).

[021] Figure 6. SEM image of QCM modified electrode with SNT-g-Thp/PTAA/SH NC (6A), FT-IRRAS spectra of i) SNT-g-Thp/PTAA/SH ii) SNT-g-Thp/PTAA/SH/NH2 iii) SNT-g-Thp/PTAA/SH/Biotin (6B).

[022] Figure 7. Is a scheme showing the representation of QCM electrode modification followed by biotin-avidin interaction.

[023] Figure 8. A graph of the frequency responses of SNT-g-Thp/PTAA/SH/NH2 NC- modified electrodes during the deposition of avidin: i) 0.2 μg/μL ii) 0.65 μg/μL iii) 2.0 μg/μL (8A); merged GFP and DIC II fluorescence microscopy images of SNT-g- Thp/PTAA(COOH)/SH NC (8B).

DETAILED DESCRIPTION OF THE INVENTION

[024] The present invention provides, a QCM electrode surface modified with a novel orthogonal nanocomposite (NC). In one embodiment, this NC is the first innovative example of polythiophene acetic acid SNTs (SNTs-g-Thp/PTAA) of a core-shell morphology decorated with functional/reactive groups such as a thiol groups, where the conjugated polymer is linked onto the SNT core. In another embodiment, the NC comprises polythiophene acetic acid SNTs (SNTs-g-Thp/PTAA) in a core-shell morphology decorated with thiol groups, where the conjugated polymer is covalently linked to or onto the surface of the SNT core. In another embodiment, the functional/reactive groups such as a thiol groups are present only on the SNT and not on any other surface of a NC. In another embodiment, the functional/reactive groups such as a thiol groups are present only on the SNT and not on any other surface of a QCM electrode. In another embodiment, the functional/reactive groups such as a thiol groups are present on the SNT and on an additional QCM electrode or NC surface.

[025] In another embodiment, a quartz crystal microbalance (QCM) electrode which includes (a) a nanocomposite (MC) having a polythiophene acetic acid silica nanotube (SNTs-g-Thp/PTAA) decorated with reactive thiol and carboxylic acid groups and (b) a conjugated functional polymer shell which is covalently linked to the surface of the SNT core in core-shell morphology, are provided.

[026] According to some embodiments, during the first functionalization step, bare SNTs were grafted with a thiophene-based linker, which acts as nucleation points during the polymerization of 3-thiopheneacetic acid (TAA). In another embodiment, SNTs-g- Thp/PTAA comprises carboxylic acid functional groups on the SNT surface. In another embodiment, carboxylic acid functional groups on the SNT surface are utilized for a second step functionalization by other functional groups such as but not limited to: carboxylic acid, amines or thiols.

[027] According to some embodiments, the amount of accessible carboxylic acid groups present on the surface of the NC allows the partial use of these groups to yield an orthogonal NC, which will comprise both carboxylic acid and thiol groups on the surface. In another embodiment, a NC is an orthogonal NC. In another embodiment, an orthogonal NC comprises both carboxylic acid and thiol groups in a 1: 1 ratio.

[028] In another embodiment, the present invention provides an unexpected improvement of the state of the art by using NTs which allow high degree of control over the amount of the deposited material by varying time deposition or the concentration of the suspension. In another embodiment, after QCM electrode modification, two different functional groups are still present on the surface in a 2D pattern, which can be further utilized. In another embodiment, the current NC is used as an essential component for the covalent immobilization of a protein or a protein complex.

[029] In another embodiment, the present invention provides a device for detecting and monitoring an oscillation of an analyte in a sample, comprising a NC as described herein. In another embodiment, the present invention provides a device for detecting and monitoring an oscillation of an analyte in a sample, comprising a QCM electrode as described herein. In some embodiments, the term "sample" and the phrase "reaction mixture" are used interchangeably.

[030] In another embodiment, the current NC is used to identify and/or quantify protein- protein interactions. In another embodiment, the current NC is used to identify and/or quantify protein-carbohydrate interactions. In another embodiment, the current NC is used to identify and/or quantify protein-lipid interactions. In another embodiment, the current NC is used to identify and/or quantify protein- small molecules interactions. In another embodiment, the current NC is used to identify and/or quantify protein-organic compound/molecule interactions. In another embodiment, the current NC is used to identify and/or quantify ligand-receptor interactions. In another embodiment, the current NC is used to identify and/or quantify protein-molecule interactions. In another embodiment, the current NC is used to identify and/or quantify any interaction between two molecules.

[031] In another embodiment, the invention provides a (a) a polythiophene acetic acid silica nanotube (SNTs-g-Thp/PTAA) comprising: a thiol group, a carboxylic acid group, and (b) a conjugated functional polymer shell, the conjugated functional polymer shell is covalently linked on the surface of the core of the polythiophene acetic acid silica nanotube in core-shell morphology. In another embodiment, the invention provides a nanocomposite comprising: (a) a polythiophene acetic acid silica nanotube (SNTs-g- Thp/PTAA) comprising: a thiol group, a carboxylic acid group, and (b) a conjugated functional polymer shell, wherein the conjugated functional polymer shell is covalently linked on the surface of the core of the polythiophene acetic acid silica nanotube in core- shell morphology. In another embodiment, the nanocomposite is an orthogonal nanocomposite. In another embodiment, a thiol group, an amino group, a carboxylic acid group, or any combination thereof are present on the surface of the polythiophene acetic acid silica nanotube.

[032] In another embodiment, the thiophene groups of the nanocomposite/ silica nanotubes comprising are anchored to the silica nanotubes via a linker. In another embodiment, the thiophene groups of the nanocomposite/ silica nanotubes comprising are anchored to the silica nanotubes via a 2-(thiophen-2-yl)-N-(3- (triethylsilyl)propyl)acetamide (Thp-TES) linker.

[033] In another embodiment, the thiol group and the carboxylic acid group are present in a 1-5: 1 ratio. In another embodiment, the thiol group and the carboxylic acid group are present in a 1: 1-5 ratio.

[034] In another embodiment, the polythiophene acetic acid silica nanotube of a core- shell morphology comprises functional groups or reactive groups suitable for the intended applications, such as but not limited to: strong affinity to gold surface or the ability to bind biological markers. In another embodiment, covalent bonding is employed for linking a functional/reactive group such as but not limited to thiol, amine, benzene ring, rhodamine B2, antibody, protein, and various silica-based linkers known to one of skill in the art.

[035] In another embodiment, the invention provides that a NC is attached to a gold surface. In another embodiment, the invention provides that a NC comprises a gold surface attached thereto. In another embodiment, a gold surface is attached to a polythiophene acetic acid silica nanotube via a thiol group.

[036] In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 1 to 400 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 2 to 400 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 10 to 400 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 5 to 300 nm. In another embodiment, the invention provides that SNTs- g-Thp/PTAA comprises an inner diameter of 5 to 10 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 5 to 40 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 5 to 25 nm. In another embodiment, the invention provides that SNTs-g- Thp/PTAA comprises an inner diameter of 10 to 50 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an inner diameter of 20 to 60 nm.

[037] In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an outer diameter of 10 to 500 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an outer diameter of 20 to 300 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an outer diameter of 20 to 50 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an outer diameter of 50 to 150 nm. In another embodiment, the invention provides that SNTs- g-Thp/PTAA comprises an outer diameter of 50 to 100 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an outer diameter of 55 to 100 nm. In another embodiment, the invention provides that SNTs-g-Thp/PTAA comprises an outer diameter of 100 to 200 nm. In another embodiment, the invention provides that SNTs-g- Thp/PTAA comprises an outer diameter of 150 to 300 nm.

[038] In another embodiment, SNTs of the invention have an average inner diameter of 11.0 nm. In another embodiment. SNTs of the invention have an average inner diameter of 5.0-30nm. In another embodiment. SNTs of the invention have an average inner diameter of 8-25 nm. In another embodiment. SNTs of the invention have an average inner diameter of 8-12 nm. [039] In another embodiment. SNTs of the invention have an average outer diameter of 40-120 nm. In another embodiment. SNTs of the invention have an average outer diameter of 60-100 nm. In another embodiment. SNTs of the invention have an average outer diameter of 50-80 nm. In another embodiment. SNTs of the invention have an average outer diameter of 70-80 nm. In another embodiment. SNTs of the invention have an average outer diameter of 75-80 nm. In another embodiment. SNTs of the invention have an average outer diameter of 76-79 nm. In another embodiment. SNTs of the invention have an average outer diameter of 77-78 nm. In another embodiment. SNTs of the invention have an average outer diameter of 77.8 nm.

[040] In another embodiment, the invention provides a quartz crystal microbalance (QCM) electrode comprising a nanocomposite (NC) as described herein. In another embodiment, the invention provides a surface modified QCM electrode comprising orthogonal NC. In another embodiment, a QCM electrode comprises a polythiophene acetic acid SNTs (SNTs-g-Thp/PTAA) in core-shell morphology decorated with thiol groups, where the conjugated polymer is covalently linked onto the SNT core. In another embodiment, the QCM comprises a rough surface endowed by the SNT-g-Thp/PTAA/SH.

[041] In another embodiment, a QCM electrode is a biosensor. In another embodiment, a QCM electrode is an analytical tool that can translate a molecular recognition reaction to quantitative information in real time. In another embodiment, a QCM electrode is extremely sensitive for monitoring mass variations at the nanogram level. In another embodiment, a QCM electrode functions on the basis of frequency changes of the piezoelectric gold-coated crystal, which are directly proportional to the changes in the mass absorbed on the electrode. In another embodiment, a QCM electrode is used to monitor protein-protein interaction, protein-molecule interaction. In another embodiment, a QCM electrode is used to monitor a biotin-avidin binding event.

[042] In another embodiment, a molecular recognition reaction is specific binding of two or more molecules. In another embodiment, a molecular recognition reaction is a specific modification of at least one molecule. In another embodiment, a molecular recognition reaction is a specific cleavage reaction of at least one molecule.

[043] In another embodiment, a QCM electrode of the invention measures the mass and/or structural/viscoelastic changes that occur on the surface of a quartz sensor in realtime. In another embodiment, an alternating voltage is applied to the quartz sensor which starts oscillating at its resonance frequency. In another embodiment, the mass change at the sensor surface is sensed as a change in frequency (Af). In addition, the change in energy dissipation from the system (AD) when the power is shut off provides information of the viscoelastic properties of the film.

[044] In another embodiment, a QCM electrode of the invention detects cellular responses to growth factors in cells such as but not limited to cancer cells. In another embodiment, a QCM electrode of the invention provides a sensitive platform for cell- surface interactions which are monitored in real-time. In another embodiment, a QCM electrode of the invention detects protein conformational changes caused by small compounds. In another embodiment, a QCM electrode of the invention is used for protein drug target screening. In another embodiment, these assays depend on conformational changes to the tertiary structure of the proteins upon binding of small compounds. In another embodiment, a QCM electrode of the invention detects biomolecular interactions and processes. In another embodiment, a QCM electrode of the invention detects protein- protein or protein-DNA interactions. In another embodiment, a QCM electrode of the invention detects self-oligomerization or aggregation of proteins (such as oligomers formed by the Alzheimer's disease). In another embodiment, a QCM electrode of the invention detects conformational and structural changes in molecular layers.

[045] In another embodiment, QCM sensor/electrode is used for an electro acoustic method suitable for mass and viscoelastic analysis of proteins at solid/water interface. In another embodiment, a QCM sensor/electrode comprises a megahertz piezoelectric quartz crystal sandwiched between two gold electrodes. In another embodiment, the crystal is brought to resonant oscillation, and shear motions by means of A/C current between the electrodes.

[046] In another embodiment, a gold surface within a QCM sensor/electrode is substituted with an alloy, metal oxides such as tin oxide, e.g. FTO or ITO, carbon such as graphite or carbon nanotubes, electrically conducting polymers such as polyaniline, or composite materials such as organically interlinked metal nanoparticle films.

[047] In another embodiment, the QCM sensor/electrode (piezoelectric element/sensor/electrode) comprises a quartz crystal, of 0.1-0.3 mm thick. In another embodiment, the QCM sensor/electrode is characterized by high specificity and sensitivity (e.g. the order of magnitude of at least 1 ng/cm). In another embodiment, the QCM sensor/electrode is characterized by magnitude of at least 0.5 ng/cm. In another embodiment, the QCM sensor/electrode is characterized by magnitude of at least 0.1 ng/cm. In another embodiment, the QCM sensor/electrode is characterized by magnitude of at least 0.05 ng/cm. [048] In another embodiment, the QCM sensor/electrode is configured to detect binding of a molecule to the SNT layer comprising reactive groups. In another embodiment, arrays of quartz crystal resonators are fabricated on a single quartz wafer as a multichannel quartz crystal microbalance (MQCM). Mechanical oscillation of each channel is entrapped within the channel almost completely, so that the interference between the channels via the quartz crystal plate is almost negligible. In another embodiment, a mass change on each channel is quantitatively evaluated on the basis of Sauerbrey's law. Thus, each channel of a MQCM device can be used as an independent QCM. Influence from a longitudinal wave generated from another channel is also negligible compared to the influence from the wave from the monitored channel itself. In another embodiment, the simultaneous oscillation of channels is also possible. In another embodiment, the present invention provide a multichannel sensing device for contact- based, indicating and molecularly profiling of a sample such as a disease suspected tissue.

Methods of utilizing a QCM electrode

[049] In another embodiment, the phrase "physiologically active substance" includes: a polysaccharide, a carbohydrate, a protein, an antibody, a receptor, a ligand, a nucleic acid, a co-factor, an enzyme, a polypeptide, a glycoprotein, a small organic molecule, or a non- polypeptidic polymer. In another embodiment, the phrase "a test substance" includes: a polysaccharide, a carbohydrate, a protein, an antibody, a receptor, a ligand, a nucleic acid, a co-factor, an enzyme, a polypeptide, a glycoprotein, a small organic molecule, or a non- polypeptidic polymer. In another embodiment, the term "interaction" includes any biological, biochemical and chemical interaction known to one of skill in the art.

[050] In another embodiment, a method as described herein includes a detection method utilizing a QCM electrode. In another embodiment, a method as described herein includes an assay sample (a test compound) to be monitored. In another embodiment, a sample comprises an analyte. In another embodiment, a sample comprises amines, an alcohol, an aldehyde, a ketone, a carboxylic acid, a hydrocarbon, a halogenated hydrocarbon, water, or any mixture thereof. In another embodiment, a sample is gaseous, liquid or solid .

[051] In another embodiment, a detection method is used for basic scientific research, medical diagnosis, healthcare diagnosis, food quality testing, agricultural testing, security testing for explosives, toxins or harmful chemical substances, and/or for environmental monitoring .

[052] In another embodiment, piezoelectrical characterization as described herein, utilizes a piezoelectric material connected to a set of QCM electrodes ordered on one or more piezoelectrical wafer, such that when the QCM electrodes are electrified, the piezoelectric material changes its dimensions. In another embodiment, alternating voltage causes the piezoelectric material to locally vibrate. In another embodiment, the vibration frequency of the piezoelectric material depends, inter alia, on the mass of the electrodes attached thereto. In another embodiment, when each of electrodes present on the wafer binds to compounds (a test substance or a physiologically active substance) in its vicinity, the mass of the each electrode changes, and with it the vibration frequency of the adjacent area of piezoelectric wafer.

[053] In another embodiment, the QCM electrode comprises an inert electrically conductive material, for example, gold. In another embodiment, the QCM electrode comprises a rough surface for increasing its surface area. In another embodiment, the QCM electrode comprises a piezoelectric material as described herein. In another embodiment, the surface or portion of the QCM electrode that specifically contacts or interacts with the subject material or sample is referred to herein as interacting layer. In some embodiments, the interacting layer within NC on each bind to marker compounds appearing in a sample such as the cytosol or on the outer surface of cells mixed with compounds that specifically bind to one kind of cells, and the results are analyzed to estimate the concentration of such cells in the subject material. In some embodiments, the interacting layer comprises compounds that specifically interacts with marker compounds appearing in the cytosol and on the outer surface of many kinds of tissues / cells, and the results are analyzed for estimating other characteristics of the subject material, for example, its viscoelasticity. Optionally, the interacting layer is made of several portions, each with other interacting moieties. Optionally, this arrangement allows characterizing concurrently several characteristics of the subject material. Optionally each of the portions is continuous. Alternatively, at least one of the portions is non-continuous and has sub portions spaced apart from one another.

[054] In another embodiment, the invention provides a method of detecting an analyte, a protein binding event, a protein cleavage event, binding between a protein and a small molecule, binding of a ligand to a receptor, a dimerization event, or the like, comprising exposing a QCM sensor according to the present invention or a sensor array of such sensors to a sample, measuring the presence or absence of an electrical signal generated by the transducer upon changes of the physical properties, wherein the presence and magnitude of the electrical signal is indicative of an "event" such as but not limited to: the presence and amount of an analyte, detecting an analyte, a protein binding event, a protein cleavage event, binding between a protein and a small molecule, binding of a ligand to a receptor, a dimerization event, or the like, in the sample.

[055] In another embodiment, a QCM electrode of the invention enables monitoring and/or measuring an interaction of a physiologically active substance with a test substance and for measuring the biological activity of the physiologically active substance in the presence of the test substance using the QCM electrode, comprising: (1) contacting at least one test substance with the QCM electrode; (2) detecting or measuring binding/ interaction (such as dimerization) of the test substance units. In another embodiment, a QCM electrode of the invention enables monitoring and/or measuring an interaction of a physiologically active substance with a test substance and for measuring the biological activity of the physiologically active substance/test substance when the physiologically active substance and the test substance are in a sample using the QCM electrode, comprising: (1) contacting the test substances, the physiologically active substance with the QCM electrode; (2) detecting or measuring binding/ interaction (such as dimerization) of the test substance and the physiologically active substance. In another embodiment, the method further includes measuring the biological activity of at least one physiologically active substance in the presence of at least one test substance, wherein, the detection and measurement of the at least one test substance interacting with at least one physiologically active substance.

[056] In another embodiment, the QCM sensor/electrode of the invention enables a method for carrying out on at least one QCM sensor/electrode the detection or measurement of a substance interacting with a physiologically active substance and the measurement of the biological activity of the above physiologically active substance.

[057] In another embodiment, the method as described herein is a screening method using the QCM sensor/electrode. In another embodiment, a test substance is characterized in that it conducts both the detection and measurement of a substance interacting with a physiologically active substance. In another embodiment, a physiologically active substance is not immobilized on a QCM sensor/electrode. In another embodiment, a physiologically active substance is immobilized on a QCM sensor/electrode. In another embodiment, a method includes the measurement of the biological activity of the above physiologically active substance .

[058] In another embodiment, the method as described herein includes a QCM sensor/electrode comprising a physiologically active substance immobilizing on the QCM sensor/electrode. In another embodiment, the method as described herein includes a QCM sensor/electrode comprising a physiologically active substance bound to reactive group on NC. In another embodiment, the method as described herein includes detection and measurement of a substance interacting with a physiologically active substance and the measurement of the biological activity of the physiologically active substance.

[059] In another embodiment, the physiologically active substance is a protein. In another embodiment, the physiologically active substance is an enzyme. In another embodiment, the method is a method for screening a test substance, which comprises: (1) a step of allowing a test substance to come into contact with a QCM electrode/sensor which comprises a physiologically active substance bound thereto (to the MC reactive/active groups), so as to detect the test substance interacting with the physiologically active substance. In another embodiment, the method further includes the step of measuring the biological activity of the physiologically active substance in the presence of the test substance.

[060] In another embodiment, binding/immobilization of a physiologically active substance and the detection of a test substance interacting with the physiologically active substance is carried out at different positions. In another embodiment, the biological activity of the physiologically active substance is indicated as any of a receptor activity level, an enzyme activity level, an antibody activity level, a metabolism activity level, a membrane potential, an ion emission level, or a gene expression level.

[061] In another embodiment, the method of the invention includes allowing a test substance to come into contact with a QCM electrode of the invention, so as to detect the test substance interacting with the physiologically active substance; and optionally (2) a step of measuring the biological activity of the above physiologically active substance in the presence of the test substance. In another embodiment, by carrying out both the detection or measurement of a substance interacting with a physiologically active substance and the measurement of the biological activity of the above physiologically active substance, it becomes possible to determine whether or not a test substance binding to a physiologically active substance has bound to the active site of the physiologically active substance, for example, thereby analyzing the effects of the test substance on the biological activity of the physiologically active substance. In another embodiment, a physiologically active substance (a protein or the like) is immobilized on the surface of a MC, and a test substance binding to the physiologically active substance can be analyzed through changes in the scattering spectrum. [062] In another embodiment, the screening method of the present invention comprises: a first step of selecting a test substance interacting with a physiologically active substance; and a second step of measuring the biological activity of the physiologically active substance in the presence of the test substance selected in the first step. In the first step, it is preferable that test substances included in the selective range of a standard value that has previously been inputted in a device be automatically selected, and that the selected test substances be automatically measured in the second step. Thereafter, in the second step, it is preferable that test substances included in the selective range of a standard value (e.g. a receptor activity level, an enzyme activity level, a metabolism activity level, a membrane potential, an ion emission level, a gene expression level, etc.), which reflects the biological activity that has previously been inputted in the device, be automatically selected .

[063] In another embodiment, provided herein a method of piezoelectrically characterizing a subject material also referred herein as a test substance or a physiologically active substance. In another embodiment, the subject material or sample comprises a body fluid, a tissue or cells adhered to or not adhered to a substrate. In some embodiments, the subject material further comprises a substrate, optionally having a form of a flat surface. In another embodiment, the subject material comprises cells pre-adhered to an inert substrate such as but not limited to a glass or plastic (such as cover glass).

[064] In another embodiment, the present invention is used as a diagnostic for a disease such as but not limited to cancer. For identifying cancer using profiles obtained in accordance with embodiments of the present invention, various characteristics may be used. In some embodiments, the acoustic profile obtained is used to estimate the viscoelastic properties of the tissue or other biophysical properties, which distinguish malignant from benign cells. In another embodiment, the profiles are used for testing a metabolic state of a tested cell or tissue. For instance, an interacting layer of metabolites that cancerous cells intend to excessively bind and insert, for example fluoro-deoxy- glucose - FDG-18, is useful for identifying such cells with profiles obtained with some embodiments of the present invention.

[065] In another embodiment, the profiles may be used for testing the biochemical features of the cellular surface. For instance, certain cancerous cells are transformed in the expression of surface macromolecules set and exhibits cancer markers on their surface. An interacting layer designed to interact with such markers may be used for obtaining profiles indicative of the malignant state of the cells. To reduce the influence of a possibly noisy environment, in some embodiments the measurement is taken against a reference, exposed to similar noise. Optionally, the reference is a piezoelectric electrode identical to the measuring sensor/electrode, but without the reactive groups as described herein. Alternatively or additionally, the reference is a QCM electrode contacting clear water or other reference material such as a healthy cell.

[066] In some embodiments, the results of the measurement, optionally together with results of a reference measurement, are displayed as graphs showing the time evolution of the frequency and/or the resistance of the sensor following the contact of the sensor to the subject material.

[067] The results of the measurements are optionally analyzed to estimate some characteristics of the subject material. Some conditions that the results may negate or confirm include cancer, psoriasis, Papilloma virus infected tissue, and other tissue infections and abnormalities.

[068] An aspect of some embodiments of the invention concerns a QCM electrode for in vivo and/or ex vivo characterization of tissue, for example, for identification of diseased tissue, utilizing molecules appearing on the surface of cells of tissue to be characterized, followed by assessment of biophysical, biochemical, and/or metabolic features or state of the cells.

[069] In another embodiment, the QCM electrode is a piezoelectric sensor comprising an interacting layer within the SNT, this interacting layer is characterized by having reactive groups. In some embodiments, the interacting layer is bound to marker molecules in the sample or tissue to be characterized.

[070] Another aspect concerns a method of characterizing tissue, ex vivo and/or in vivo. In one embodiment, the method comprises attaching a piezoelectric sensor to the tissue, obtaining by the sensor a resonant acoustic profile of the tissue, and analyzing the profile to characterize the tissue. Similarly, some embodiments provide a method of characterizing surfaces, having size comparable to the size of the sensor's electrode. Such surfaces are referred herein as large surfaces.

[071] In an exemplary embodiment, such method comprises attaching a piezoelectric sensor to the large surface, obtaining, by the sensor, a resonant acoustic profile of the large surface, and analyzing the profile to characterize the large surface. In some embodiments, attaching a sensor to a surface, tissue, or any other subject material to be characterized, comprises bringing the sensor and the subject material to a distance between them, which is small enough to allow the sensor to sense the subject material. In some embodiments, attaching a sensor to a surface, tissue, or any other subject material to be characterized, comprises contacting the sensor and the subject material. In some embodiments, the distance between a cell carrying surface and an electrode is 15 micrometers, and the cells carried by the cell carrier are 15 micrometers in diameter. In such a case, the sensor and the cells actually touch each other, although the sample-sensor distance may be defined as 15 micrometers.

Methods of making NCs/SNTs

[072] In another embodiment, a first functionalization step includes grafting bare SNTs with a thiophene-based linker. In another embodiment, such thiophene-based linker species act as nucleation points during the polymerization of TAA. In another embodiment, SNTs- g-Thp/PTAA have carboxylic acid functional groups on the surface. In another embodiment, carboxylic acid functional groups on the surface are further used for an additional functionalization step. In another embodiment, an additional functionalization step utilized functional groups such as but not limited to amines or thiols. In another embodiment, the accessible amount of carboxylic acid groups present on the surface of the NC allows the partial use of these groups to yield an orthogonal NC, which comprises both carboxylic acid and thiol on the surface.

[073] In another embodiment, the invention provides a method for making a nanocomposite comprising polythiophene acetic acid silica nanotubes having a core-shell morphology, comprising the following sequential steps of: grafting the surface of silica nanotubes with 2-(thiophen-2-yl)-N-(3-(triethylsilyl)propyl) (Thp-TES); dispersing the surface grafted silica nanotubes in CHCI₃ or anhydrous CHCI₃; adding 3-thiophene-acetic acid (TAA) monomer; adding anhydrous FeCl₃ dissolved in CHC1₃ - resulting in a nanocomposite dispersion; adding MeOH; recovering the resultant nanocomposite comprising polythiophene acetic acid silica nanotubes having a core- shell morphology. In another embodiment, recovering includes drying. In another embodiment, drying comprises drying in a vacuum using a rotary evaporator.

[074] In another embodiment, the invention further includes modifying the nanocomposite comprising polythiophene acetic acid silica nanotubes having core-shell morphology with 1,4-diamino-butane, comprising the following sequential steps of: dispersing nanocomposite and anhydrous CHC1₃; adding Ι,Γ-carbonyldiimidazole to the dispersion; adding 1,4-diaminobutane dissolved in anhydrous CHC1₃ to the dispersion; and washing the polyNH₂ modified nanocomposite with a solution comprising: water, CHC1₃, and alcohol. [075] In another embodiment, alcohol is methanol. In another embodiment, alcohol is ethanol. In another embodiment, ethanol is substituted with methanol or a mixture of ethanol-methanol. In another embodiment, ethanol is absolute ethanol. In another embodiment, methanol is absolute methanol. In another embodiment, water is distilled water. In another embodiment, water is double distilled water.

[076] In another embodiment, the invention further includes modifying the nanocomposite comprising polythiophene acetic acid silica nanotubes having core-shell morphology with cysteamin, comprising the following sequential steps of: dispersing the resultant nanocomposite in THF; adding Ι,Γ-carbonyldiimidazole to the dispersion; adding cysteamine to the dispersion; washing the cysteamine-modified nanocomposite (SNT-g- Thp/PTAA(COOH)/SH) with a solution comprising: water and alcohol.

[077] In another embodiment, the invention further includes derivatizing cysteamin modified nanocomposite comprising polythiophene acetic acid silica nanotubes having core-shell morphology with isoindole, comprising the following sequential steps of: dissolving the SNT-g-Thp/PTAA(COOH)/SH in alcohol; adding o-phtaldialdehyde (OPA); removing unbound OPA; adding NH₄OH.

[078] In another embodiment, the invention further includes derivatizing the nanocomposite comprising polythiophene acetic acid silica nanotubes having a core-shell morphology with an amine group, comprising the following sequential steps of: contacting the nanocomposite comprising polythiophen acetic acid silica nanotubes having a core- shell morphology with a solution comprising: NHS, EDC and water; adding 1,3- diaminopropane; rinsing with water; drying under a N₂ stream; immersing in a solution comprising bis- 1 - ( 11 - { 2- [2- { 2- (2-hydroxy-ethoxy)-ethoxy } -ethoxy] -ethoxy } -undecyl) disulfide (PEG); washing in alcohol; and drying under a N₂ stream.

[079] In another embodiment, derivatizing includes the introduction of a functional group. In another embodiment, derivatizing includes the introduction of a reactive group. In another embodiment, derivatizing includes the introduction of an amine group. In another embodiment, derivatizing includes the introduction of a thiol group. In another embodiment, derivatizing includes the introduction of a carboxyl group. In another embodiment, derivatizing includes the introduction to the surface of a SNT. In another embodiment, derivatizing includes the introduction to the surface of a nanocomposite.

[080] In another embodiment, the invention further includes a method of making a quartz crystal microbalance (QCM) electrode comprising a nanocomposite, wherein the nanocomposite comprises a polythiophene cysteamine acetic acid silica nanotube (SNT-g- Thp/PTAA/SH), comprising the following sequential steps of: (a) contacting pretreated gold QCM crystals with a dispersion comprising SNT-g-Thp/PTAA/SH nanocomposite; and (b) drying the gold QCM crystals comprising the dispersion comprising SNT-g- Thp/PTAA/SH nanocomposite.

[081] In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises an additional step (c) comprising rinsing the resulting product of step (b) with an alcohol.

[082] In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) and (b) at least 2 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) and (b) at least 2 to 15 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) and (b) at least 2 to 10 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) and (b) at least 8 to 12 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) and (b) at least 10 times.

[083] In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) through (c) at least 2 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) through (c) at least 2 to 15 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) through (c) at least 2 to 10 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) through (c) at least 8 to 12 times. In another embodiment, a method of making a quartz crystal microbalance (QCM) electrode further comprises sequentially repeating steps (a) through (c) at least 10 times.

[084] In another embodiment, dispersion comprises an alcohol. In another embodiment, dispersion comprises EtOH. In another embodiment, dispersion comprises MeOH. In another embodiment, drying is evaporating an alcohol. In another embodiment, drying is evaporating EtOH. In another embodiment, drying is evaporating MeOH.

[085] In another embodiment, the invention further includes derivatizing a QCM electrode surface or the surface of a polythiophene acetic acid silica nanotubes having core-shell morphology with an amine/biotine group, comprising the following sequential steps of: treating QCM modified electrode substrates or a polythiophene-acetic acid silica nanotubes having a core-shell morphology with NHS and EDC in water; adding 1,3- diaminopropane; rinsing with water; drying under a stream of N₂; immersing in a solution of bis- 1-(1 l-{2-[2-{2-(2-hydroxy-ethoxy)-ethoxy}-ethoxy]-ethoxy}-undecyl) disulfide (PEG); washing with alcohol; and drying under a stream of N₂. In another embodiment, the resulting amino-terminal groups are reacted with sulfosuccinimidyl biotin via an ester link and the biotin modified QCM electrode surface or the surface of a polythiophen acetic acid silica nanotubes having core- shell morphology is rinsed with water and dried under a stream of N₂.

[086] In another embodiment, the term "comprise" includes the term "consist" or is replaceable by the term "consist".

EXAMPLES

Chemicals and Materials

[087] Lyophilized avidin (Av), phosphate buffer saline (PBS), sulfosuccinimidyl biotin, 1,3-diaminopropane, N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N'- ethylcarbodiimide hydrochloride (EDC), biotin 3-sulfo-Nhydroxysuccinimide ester sodium salt (sulfosuccinimidyl biotin), cysteamine, ophtaldialdehyde (OPA) were purchased from Sigma- Aldrich. Bis-l-(l l-{2-[2-{2- (2-hydroxy-ethoxy)-ethoxy } -ethoxy] -ethoxy } - undecyl) disulfide was purchased from ProChimia Surfaces. Ethanol (AR, Frutarom) was used as received. Water was doubly distilled (ddH₂0). The inert gas used was household nitrogen.

[088] All the experimental details related to the fabrication of (i) dodecyltriethylammonium bromide (DTEAB), of (ii) N-dodecanoyl-L-histidine (DHis), and of (iii) SNTs appear in (S. Lei, J. Zhang, J.-R. Wang and J.-B. Huang, Langmuir, 2010, 26, 4288.).

Synthetic strategy for the preparation of Thp-TES

[089] 3-thiopheneacetic acid (TAA) (1.42 g, 10.00 mmol) and Ι,Γ-carbonyldiimidazole (CD I) (1.96 g, 12.00 mmol) were dissolved in dry THF (40.00 mL) in a two neck-round bottom flask equipped with a drying tube. After stirring at room temperature (RT) for 2 hours (h), 3-aminopropyltriethoxysilane (APTES 2.30 mL, 10.00 mmol) was added to the reaction mixture, which was stirred at RT overnight. At completion of the reaction (TLC checking), the medium was concentrated in vacuum and purified by flash chromatography on silica gel (eluent: diethyl ether/n-hexane = 85: 15) affording pure ThpTES (1.62 g, 4.7 mmol) in a 47 % yield. Yellow oil; TLC: Rf = 0.34 (eluent: diethyl ether/n-hexane: 85/15); vmax (KBr easy diff)/cm-l 3292 (NH), 2975 and 2886 (CH stretching), 2930 (CH2 stretching), 1648 and 1440 (Thiophene), 1553 (C=0 amide), 1390 and 1193 (C=C), 1271 (C-O), 764 (CH2 bending), 1102 (Si-O-Si), 957 (Si-OH); δΗ 1H NMR (300 MHz; DMSO- d6) 7.97 (1H, br t, NH), 7.42 (1H, m, Ar-H), 7.21 (1H, m, Ar-H), 7.01 (1H, m, Ar-H), 3.72 (6H, q, J = 6.9 Hz, 0-CH2-CH3), 3.06-3.00 (2H, m, NH-CH2-CH2), 1.50-1.43 (2H, m, CH2-CH2-CH2), 1.16 (9H, t, J = 6.9 Hz, 0-CH2-CH3), 0.55-0.50 (2H, m, CH2-Si); 5C ¹³C NMR (75 MHz, DMSO-d6, TMS) 169.49 (C), 136.34 (C), 128.57 (CH), 125.49 (CH), 121.90 (CH), 57.66 (CH2), 41.34 (CH2), 37.13 (CH2), 22.68 (CH2), 18.16 (CH3), 7.29 (CH2); HRMS (DCI+CH4) m/z calcd for C13H22N03SSi (MH-EtOH)+ 300.4692 found 300.1090. The synthesis scheme of 2-(thiophen-2-yl)-N-(3-(triethylsilyl)propyl) acetamide (Thp-TES) is provide in Fig. 1.

Surface grafting of SNTs with Thp-TES

[090] SNTs (30.0 mg) were suspended in 5 mL of EtOH by sonication for 20 minutes. A freshly prepared solution of Thp-TES (30 mg, 2.5 mL EtOH absolute, 0.2 w/v) was added to the solution. The total volume of the reaction was adjusted to 15 mL by adding 7.5 mL of ddH₂0 to the reaction mixture. The resultant reaction flask was immersed into a preheated oil bath and stirred for 24h at 500C. The modified SNTs were washed with EtOH (5x30mL) using centrifugation (10 min, 10,000 rpm, lOoC), and dried under vacuum using a rotary evaporator.

Preparation of SNTs-g-Thp/PTAA NCs with core-shell morphology

[091] The modified SNTs-g-Thp (40.00 mg) were dispersed in 10.00 mL of anhydrous CHC13 by sonication (30 min, bath sonicator). To the resulting dispersion, TAA monomer (142.20 mg, 1.00 mmol) was added and the mixture was stirred for 30 min at RT. Thus, the oxidant anhydrous FeC13 (405.50 mg, 2.50 mmol) was dissolved in anhydrous CHC13 (5.00 mL) and the oxidant solution was added to the SNT-g-Thp/TAA dispersion. During the stirring process (3h), the color of the mixture changed from white to a dark black. To the resulting NC dispersion, 30.00 mL of absolute MeOH were added resulting in a color change of the suspension from black to orange. The resulting NC dispersion was stirred for 20h at RT. The NC was washed with MeOH (4x25 mL) using centrifugation (15 min, 10,000 rpm, OoC) and dried in vacuum using a rotary evaporator.

Modification of SNTs-g-Thp/PTAA NCs by 1,4-diaminobutane

[092] The previous SNT-g-Thp/PTAA core-shell NC (5.0 mg) was dispersed in anhydrous CHC13 (2.0 mL) by sonication (30 min, bath sonicator). CDI (5.0 mg, 0.03 mmol) was added to the resulting dispersion and mixed for 2h at RT. Next, 1,4- diaminobutane (2.63 mg, 3.0 μί, 0.03 mmol) was dissolved in anhydrous CHC13 (1.0 mL) and added to the NC dispersion for 24h at RT and stirring. The modified polyNH2 NC was then washed with ddH₂0 (3 x 2 mL), CHC13 (3 x 2 mL) and EtOH (1 x 2 mL) using centrifugation (10 min, 10,000 rpm, 00C). The amount of amine for this novel SNT-g- Thp/PTAA/NH2 NC was measured using an analytical sensitive UV spectroscopic Kaiser test (E. Kaiser, R. L. Colescott, C. D. Bossinger and P. I. Cook, Anal.l Biochem., 1970, 34, 595.).

Modification of SNTs-g-Thp/PTAA NCs by cysteamine

[093] The former SNT-g-Thp/PTAA core-shell NC (30.0 mg) was dispersed in THF (lOmL) by sonication (30 min, bath sonicator). CDI (1.21 mg, 0.0075 mmol) was added to the resulting dispersion and mixed for 2h at RT. Cytsteamine (0.6 mg, 0.0075 mmol) was then added to the NC dispersion for 24h at RT under stirring. The modified NC was then washed with ddH₂0 (3 x 2 mL) and EtOH (2 x 2 mL) using centrifugation (10 min, 10,000 rpm, 00C) yielding SNT-g-Thp/PTAA(COOH)/SH NC.

Isoindole derivatization

[094] The former SNT-g-Thp/PTAA(COOH)/SH NC (2.0 mg) was dissolved in 500 of MeOH by sonication (30 min, bath sonicator). A stock solution of OPA with a concentration of ImM in MeOH was prepared. 100 μΐ_^ of the OPA solution were added to the NC solution, and the reaction was continued for 20 min at RT. The resulting NC dispersion was washed by MeOH (3 x 500 μί) to remove unbound OPA molecules, after which 100 of NH40H (28-30%) were added to the NC solution and allowed to react for 10 min at RT. A control experiment was performed using starting bare non-functional SNTs using the same procedure. Fluorescent images were recorded with a Zeiss Axio Imaging Zl fluorescent microscope using a GFP filter.

Biosensor preparation and QCM measurements

Preparation of gold surfaces

[095] Gold QCM crystals were cleaned by immersion in a freshly prepared hot piranha solution (1:3 H₂0₂:H₂S0₄) for 3 minutes followed by rinsing with dd H₂0, and drying under a stream of N₂.

Self-assembly of SNT- g-Thp/PTAA/SH NC on gold QCM electrodes

[096] The pretreated gold QCM crystals were directly used for the adsorption of SNT-g- Thp/PTAA/SH NC. The gold substrate was positioned horizontally on a hard surface. A drop of 7.0 μΐ, of a SNT-g-Thp/PTAA/SH NC dispersion (1.5mg NC/mL EtOH) was carefully placed on the surface of the electrode and the EtOH suspension spread to cover the full area of the gold electrode substrate. After EtOH evaporation, the substrate was rinsed using pure EtOH and dried in RT. The deposition was repeated 10 times.

Biotine-derivatized QCM electrode surfaces

[097] To obtain an outer amine functionality, QCM modified electrode substrates were initially treated with a solution of NHS (11.5 mg) and EDC (62 mg) in ddH₂0 (3.75 mL), and followed by the addition of 1,3-diaminopropane (61.65 μί) overnight. The substrate was rinsed with ddH₂0 and dried under a stream of N₂. To prevent non-specific bio- adsorption during the subsequent reaction, the corresponding substrates were immersed in a solution of bis-l-(l l-{2-[2-{2-(2-hydroxy-ethoxy)-ethoxy}-ethoxy]-ethoxy}-undecyl) disulfide (PEG) (100.0 μg/mL, 3.75 mL per electrode) followed by 10 min washing in EtOH and drying under a stream of N₂. The terminal amino groups were subsequently able to react with sulfosuccinimidyl biotin (0.000375 mM, 1 mL) in an aqueous solution via an ester link. The modified substrates were removed from the solution and rinsed several times with ddH₂0, then dried under a stream of N₂.

Avidin affinity measurement

[098] The gold modified sensing electrode was assembled into the crystal holder and connected to the QCM 200 crystal oscillator. A volume of 400.0 of a PBS buffer (pH 7.4) was spread on the biotin modified QCM electrode until stabilization of the resonance frequency was reached. The binding interaction of avidin to the biotin-modified electrode was investigated at various avidin concentrations of 0.2, 0.65, and 2.0 μg/μL PBS.

Characterization

[099] QCM. Changes in resonance frequency from adsorption processes onto the sensor electrode were monitored by a SRS QCM 200 sensor (Stanford Research Systems, Sunnyvale USA) system. AT-cut quartz crystals with gold electrodes and a fundamental oscillating frequency of 5 MHz (Stanford Research Systems, Inc.) were used as sensing substrates. The sampling period and the resolution of the frequency counter were 1 sec. and 0.1 Hz respectively. All the measurements were done at room temperature. The QCM electrode (active sensing area) surface is 1.26 cm (manufacturer' s specifications.

[0100] Fourier-Transform Infrared Reflection-Absorption Spectroscopy (FT-IRRAS). FT-IRRAS spectra were recorded on a Bruker Tensor spectrometer equipped with a grazing angle specular reflectance accessory Pike 80Spec. A reference spectrum was taken on a clean gold sample. For all spectra, 100 scans were collected.

EXAMPLE 1: Characterization of bare SNTs [0101] The as prepared SNTs morphology was characterized by FE-SEM and TEM (Fig. 2). These NTs have well defined average inner and outer diameters of 11.0 and 77.8 nm respectively. In addition, HR-TEM revealed an ordered pattern of fringes indicated by the arrows. XRD revealed a low resolution peak due to the hollow structure of SNTs at29=2.80 (ESI, SI).

EXAMPLE 2: Characterization of bare SNTs-g-Thp

[0102] Figure 3 demonstrates the synthetic strategy toward the preparation of core-shell SNTs-g-Thp/PTAA NC. Surface grafting of the SNTs was performed using the oxidizable thiophene-based Thp-TES linker. This allowed the anchoring of thiophene groups onto the surface of bare SNTs in a 1st step. In the 2nd step, the TAA monomer was oxidatively polymerized in the presence of the grafted SNTs to afford corresponding targeted NCs of controlled core-shell morphology.

[0103] In order to confirm the successful modification of bare SNTs by the Thp-TES linker and PTAA, X-ray photoelectron spectroscopy (XPS), which is a highly surface specific technique with a probing depth of a few nanometers (5-10 nm), was applied to compare the elemental compositions of the surface on bare SNTs, modified SNT-g-Thp, and of the final functional SNT-g-Thp/PTAA NC (Table 1). [0104] Table 1: XPS elemental compositions of SNTs, SNT-g-Thp, and SNT-g- Thp/PTAA

XFS s (atomic : con< ;e ratic :ui. %}

>>isiiipi

N C S Si S/Si

SNTs 39.25 60.75 0

SNT- -Hip 1 76 79.25 1.15 1 " S4 0.064

8NT- -T!i?¾^;PTA A - 91.69 7.21 1.10 6.554

[0105] As can be seen from Table 1, bare SNTs contain only C and Si elements. The SNT-g-Thp however, contains both N and S elements in addition to the C and Si. These additional elements originate from the Thp-TES linker, which confirms the successful surface modification of the bare SNTs. EXAMPLE 3: Characterization of SNTs-g-Thp/PTAA core-shell NC

[0106] According to XPS (Table 1), it was found that the final SNTs-g-Thp/PTAA NC does not contain a N element. This can be explained by the fact that XPS has a maximal probing depth of up to 10 nm, while the polymeric shell has been measured to be 20 nm thick in average. Because of this, it is unlikely to detect the N element in the final functional NC. In addition, the increase in the S/Si ratio from 0.064 for the intermediate product SNT-g-Thp to 6.554 for the final product SNT-g-Thp/PTAA NC, further suggests the deposition of the PTAA shell around SNT-g-Thp leading to the targeted core-shell morphology of the final NC.

[0107] HR-TEM was also used to confirm the core-shell morphology of the final SNT-g- Thp/PTAA NC (Fig. 4). As can be seen from the corresponding HR-TEM image, an ordered pattern of fringes can be observed similar to the one observed for bare SNTs. However, one may also observe an amorphous polymeric shell with an average thickness of 20 nm.

[0108] HR-TEM line scan analysis (Fig. 4(b)) was performed based on two elements, Si and S. It should be noticed that the inorganic SNT core is the only source for Si, while only the organic PTAA shell contains the S element. Accordingly, a high amount of S was detected at the NC edges, indicative of the presence of the deposited polymer, while a high amount of Si was detected at the NC center. Both observations fully validate the as- designed core- shell morphology of the corresponding NC.

[0109] For further investigation into the organic content of SNT-g-Thp/PTAA NC, TGA and EA were also performed. TGA curves are presented in Fig. 3(b). For bare SNTs, there is a slight decrease in weight -7% at lOOOC, which can be attributed to H₂0 (dehydration process). Indeed, EA showed that only 0.1% out of 1.53% of the organic content originates from C. The incomplete hydrolysis of TEOS ethoxy groups might account for the presence of C. In contrast, both intermediate SNT-g-Thp and final SNT-g-Thp/PTAA NC disclose additional weight lost at ~ 3250C of 7.5% and 45.76% (not considering the first weight loss) respectively. This indicates the significant increase observed in the NC organic content after each step of modification. Moreover, EA fully correlates to this data, and after each modification step a significant increase in organic content is observed (ESI, Table SI).

[0110] NC functionality was been also checked using FT-IR. FT-IR spectra of PTAA, bare SNTs, SNT-g-Thp, and final SNT-g-Thp/PTAA are presented in Fig. 5(a). All the corresponding spectra of the various NTs have characteristic peaks of (i) Si-O-Si at 800 and 1100 cm^" ascribed to symmetric and non-symmetric stretching vibrations respectively, of (ii) Si-OH group stretching vibrations appearing at 958 cm-1, and of (iii) O-Si-0 vibrations from bridge bending modes at 460 cm^"1. Both 1600 and 1420 cm^"1 peaks correspond to symmetric and asymmetric thiophene ring stretching, respectively. These peaks can be observed in both SNT-g-Thp and SNT-g-Thp/PTAA composites. In addition, for the SNT-g-Thp NC, the peak appearing at 1545 cm-1 characterizes the amide bond present in the Thp-TES linker. Moreover, the strong C=0 stretching peak appearing at 1715 cm^"1 and the broad O-H stretching peak at 2700-3600 cm-1 are also characteristic of both PTAA and SNT-g-Thp/PTAA.

EXAMPLE 4: Post-polymerization chemical modifications of the core-shell SNT-g- Thp/PTAA NC

Modification by 1,4-diaminobutane

[0111] In order to prepare functional SNTs, and since the TAA monomer possesses a carboxylic group, it was of prime importance to further chemically modify the SNT-g- Thp/PTAA NC. Using a simple amidation reaction with 1,4-diaminobutane, a novel polyaminated SNT-g-Thp/PTAA/NH₂ NC was prepared. The quantification of NH₂ groups present on the surface of the NC after the modification was done using a well known sensitive UV analytical spectroscopic Kaiser test. In this way, the amount of carboxylic acid groups accessible for chemical modifications was measured. According to EA, 27.53% of organic matter for this NC is assigned to C, meaning that there are 3.821 mmol of COOH/g of SNT-g-Thp/PTAA NC. Kaiser test revealed a value of 0.5 mmol of free primary NH₂/g of SNT-g-Thp/PTAA/ NH₂ NC. This data means that 13.08% of the carboxylic acid groups originating from PTAA are accessible for chemical modification. Modification by cysteamine

[0112] Another more attractive partial (half amount of accessible COOH groups-0.25 mmol/g of SNT-g-Thp/PTAA NC) chemical modification of the core-shell SNT-g- Thp/PTAA NC was performed using cysteamine36 in order to produce a bi-functional SNT-g-Thp/PTAA(COOH)/SH NC, enabling QCM Au electrode functionalization. EA showed a slight, but detectable, increase of 0.5 weight % in both amounts of N and S elements. This data makes sense due to the very small amount of cysteamine (0.0075 mmol) that has been used for SNT-g-Thp/PTAA NC modification. On the other hand, the O weight % values decreased by 2.21%, since only half of the carboxylic acid groups were modified to thiols. To confirm the presence of multiple thiol groups, they were transformed into isoindoles species of which fluorescence was readily detected by fluorescence microscopy (Fig. 5(b)). It can be clearly seen from the merged images between DIC II and GFP filters that the fluorescence originates only from the chemically modified SNT-g-Thp/PTAA/SH NC. A control experiment performed with bare SNTs did not afford any detectable fluorescence signal.

EXAMPLE 5: Biotin-avidin interaction detection using QCM sensing SNT-g- Thp/PTAA/SH NC modified electrode

[0113] In order to optimize the coverage of the electrodes surface, several concentrations (0.5, 1, 1.5 mg/mL EtOH) and different number of deposition cycles of SNT-g- Thp/PTAA/SH NC were examined. The optimal result was received after 10 cycles using a concentration of 1.5 mg/mL of the NC (Fig 6(a)). Next, QCM analysis showed the ability of the SNT-g-Thp/PTAA/SH NC to bind onto the QCM Au electrode after 10 min and reached an equilibrium after 30 min from a suspension of 1.5mg NC/mL. (ESI, S2). This is due to the strong interactions between the Au electrode surface and SH groups present on the NC. According to the Sauerbrey equation, QCM measurements allow the calculation of the change in mass Am on the quartz crystal surface by measuring the change in vibration frequency, where Af is the observed residual frequency change (Hz), Am is the change in mass per unit area ^g/cm2), and Cf is the crystal sensitivity factor (56.6 Hz μg-lcm-2). Accordingly, the electrode surface coverage and the total weight of bound SNT-g- Thp/PTAA/SH NC have been measured to be 1300.0 ng cm-2 and 1650.0 ng respectively. Af = -CfAm

[0114] Next, the SNT-g-Thp/PTAA/SH NC modified electrode was tested for the sensing of biotin-avidin interactions as a biological model system. The remaining COOH groups on the surface of SNT-g-Thp/PTAA/SH NC were modified to amines, yielding SNT-g- Thp/PTAA/SH/NH2 NC (Fig. 7). Due to amine modification, a sulfo-succinimidyl biotin could be covalently attached to the SNT-g-Thp/PTAA/SH/NH2 NC, which was confirmed by FT-IRRAS (Fig. 6). As evidenced by FT-IR measurement (Fig. 4b-ii) the peak appearing at 1715 cm-1 corresponding to C=0 stretching of carboxylic acid decreased in intensity due to amine modification. In addition, a new peak can be observed at 3360 cm-1, which can be ascribed to N-H stretching. Following the biotin binding (Fig. 6b-iii) an appearance of typical peak at 1650 cm-1 provides evidence to successful covalent binding event. [0115] Various concentrations of an avidin suspension in PBS were introduced to the modified electrode, and were assessed by QCM analysis (Fig 8(a)). As can be seen from Fig 5(a), the reaction was almost immediate, and equilibrium was reached after 50 sec. As the avidin concentration increased, the electrode frequency decreased. Table 2 shows the corresponding amounts of anchored avidin when interacting with the biotin-functionalized modified electrode. These values are much higher in comparison to those measured for a monolayer - 367 ng cm^" , and for those measured for conjugated polymer films - 534 ng cm^" (these are the maximum values for both monolayers and film.

[0116] In this study however, one could still increase the amount of deposited NC or the amount of COOH present on the surface of the NC, and thus increase the final amount of anchored avidin. This result could be attributed to the surface roughness endowed by the SNT-g-Thp/PTAA/SH NC. The surface area of the NC-modified electrode is significantly larger than its strict geometric surface area. In this sensing system several non specific binding (NSB) interactions need to be examined. First of all, the NSB of avidin alone onto a bare gold electrode was examined; with an observed frequency change was of 50 Hz. Second, examination of the NSB to the SNT-g-Thp/PTAA/SH/NH₂ NC itself was performed, since proteins in general exhibit a high degree of NSB onto NTs. This fact could be attributed to both hydrophilic and hydrophobic interactions between the NTs and the protein as reported previously. Indeed, the electrode was modified with SNT-g- Thp/PTAA/SH/NH₂ without biotin, an extremely high change in frequency - 425 Hz was recorded. This high value might suggest that both specific binding and NSB took place. Since much lower values were obtained once the biotin was introduced, this means that the NSB was eliminated. Prior to introducing the biotin, avidin could interact with the NC surface at any location. Once the biotin was introduced, avidin binding was almost immediate, which is evident from the rapid decrease in frequency. Thus, the avidin itself blocks the rest of the SNT-g-Thp/PTAA/SH/NH₂ NC surface, preventing NSB of other avidin molecules.

[0117] Table 2. Frequency and mass changes for avidin deposition

1 1.40 2473

^■ 1 ?(} 3003

^ 1 ^ % [0118] In another embodiment, a new type of functionalized orthogonal SNT-g- Thp/PTAA/SH NC is provided. By a method of surface modification a functional polymer shell was covalently linked onto a SNT core. The presence of the functional shell was verified by a combination of FE-SEM, HR-TEM, TGA, EA, XPS and FT-IR. Moreover, this organic shell was utilized for further chemical modifications towards the preparation of multi-functional NCs that possess quantified amounts of COOH and SH groups. Such orthogonal NC was used for the surface modification of a QCM electrode for sensing detection capability. Based on the NC multi-functionality, it could be used as a sensor for biological markers. In addition, surface roughness of the polymer contributed to signal enhancement. This concept has been fully validated for the well-known biotin-avidin system. Both specific and non-specific binding phenomena were intensively examined. It was found that an NSB phenomenon was prevented by the specific binding itself, without the need for any additional electrode modification. This makes this overall sensing process simple to run and effective regarding detection capability.

Claims

CLAIMS What is claimed is:

1. A nanocomposite comprising: (a) a polythiophene acetic acid silica nanotube (SNTs-g-Thp/PTAA) comprising: a thiol group, a carboxylic acid group, and (b) a conjugated functional polymer shell, said conjugated functional polymer shell is covalently linked on the surface of the core of said polythiophene acetic acid silica nanotube in core- shell morphology.

2. The nanocomposite of claim 1, wherein said nanocomposite is an orthogonal nanocomposite.

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3. The nanocomposite of claim 1, wherein said thiol group, said carboxylic acid group, or both are present on said polythiophene acetic acid silica nanotubes surface.

4. The nanocomposite of claim 1, wherein said thiol group and said carboxylic acid group are present in a 1: 1 ratio.

5. The nanocomposite of claim 1, further comprising a gold surface attached to said polythiophene acetic acid silica nanotubes via said thiol group.

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6. The nanocomposite of claim 1, wherein thiophene groups are anchored to the silica nanotubes via a 2-(thiophen-2-yl)-N-(3-(triethylsilyl)propyl)acetamide (Thp-TES) linker.

7. The nanocomposite of claim 1, wherein said SNTs-g-Thp/PTAA comprises an inner diameter of 5 to 25 nm.

8. The nanocomposite of claim 1, wherein said SNTs-g-Thp/PTAA comprises an outer diameter of 55 to 100 nm.

9. A quartz crystal microbalance (QCM) electrode comprising the nanocomposite of any one of claims 1-8.

10. A method making a nanocomposite comprising polythiophen acetic acid silica nanotubes having core-shell morphology, comprising the steps of:

(a) grafting the surface of silica nanotubes with 2-(thiophen-2-yl)-N-(3- (triethylsilyl)propyl) (Thp-TES);

(b) dispersing the surface grafted silica nanotubes in anhydrous CHCl_3;

(c) adding 3-Thiopheneacetic acid (TAA) monomer;

(d) adding anhydrous FeCl₃ dissolved in CHC1₃ resulting in a nanocomposite dispersion;

(e) adding MeOH; and

(f) recovering the resultant nanocomposite comprising polythiophen acetic acid silica nanotubes having core-shell morphology, wherein steps (a) to (f) are carried out substantially sequentially.

11. The method of claim 10, wherein said recovering comprises drying in a vacuum using a rotary evaporator.

12. The method of claim 10, further comprising modifying said nanocomposite comprising polythiophen acetic acid silica nanotubes having core- shell morphology with 1,4-diaminobutane, comprising the steps of:

(a) dispersing said the resultant nanocomposite in anhydrous CHC1₃;

(b) adding Ι,Γ-carbonyldiimidazole to the dispersion;

(c) adding 1,4-diaminobutane dissolved in anhydrous CHC1₃ to the dispersion; and

(d) washing the polyNH₂ modified nanocomposite with a solution comprising: water, CHC1₃, and alcohol, wherein steps (a) to (d) are carried out substantially sequentially.

The method of claim 12, wherein said alcohol is ethanol.

14. The method of claim 10, further comprising modifying said nanocomposite comprising polythiophen acetic acid silica nanotubes having core- shell morphology with cysteamin, comprising the steps of:

(a) dispersing said resultant nanocomposite in THF;

(b) adding Ι,Γ-carbonyldiimidazole to the dispersion;

(c) adding cysteamine to the dispersion; and

(d) washing the cysteamin modified nanocomposite (SNT-g-Thp/PTAA(COOH)/SH) with a solution comprising: water and alcohol, wherein steps (a) to (d) are carried out substantially sequentially.

The method of claim 14, wherein said alcohol is ethanol.

16. The method of claim 14, further comprising derivatizing cysteamin modified nanocomposite comprising polythiophene acetic acid silica nanotubes having core- shell morphology with isoindole, comprising the steps of:

(a) dissolving said SNT-g-Thp/PTAA(COOH)/SH in MeOH;

(b) adding OPA;

(c) removing unbound o-phtaldialdehyde (OPA); and

(d) adding NH₄OH, wherein steps (a) to (d) are carried out substantially sequentially.

17. The method of claim 10, further comprising derivatizing said nanocomposite comprising polythiophen acetic acid silica nanotubes having core- shell morphology with an amine group, comprising the steps of:

(a) contacting said nanocomposite comprising polythiophen acetic acid silica nanotubes having core-shell morphology with a solution comprising: NHS, EDC and water;

(b) adding 1,3-diaminopropane;

(c) rinsing with water;

(d) drying under a N₂ stream. (e) immersing in a solution comprising bis-l-(l l-{2-[2-{2-(2-hydroxy-ethoxy)- ethoxy}-ethoxy]-ethoxy}-undecyl) disulfide (PEG);

(f) washing in EtOH; and

(g) drying under a N₂ stream, wherein steps (a) to (g) are carried out substantially sequentially

18. The method of claim 17, wherein said amine group is a surface functional amine group.

19. A method of making a quartz crystal microbalance (QCM) electrode comprising a nanocomposite, said nanocomposite comprises a polythiophene cysteamine acetic acid silica nanotube (SNT-g-Thp/PTAA/SH), comprising the steps:

(a) contacting pretreated gold QCM crystals with a dispersion comprising the SNT-g- Thp/PTAA/SH nanocomposite; and

(b) drying the gold QCM crystals comprising said dispersion comprising the SNT-g- Thp/PTAA/SH nanocomposite, wherein steps (a) and (b) are carried out substantially sequentially

20. The method of claim 19, wherein said dispersion comprises EtOH.

21. The method of claim 20, wherein said drying is evaporating EtOH.

22. The method of claim 19, further comprising step (c) comprising rinsing the resulting product of step (b) with EtOH.

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23. The method of claim 19, further comprising sequentially repeating steps (a) and (b) at least 2 times.

24. The method of claim 22, further comprising sequentially repeating steps (a) through (c) at least 2 times.

25. A method of for monitoring mass variations at the nanogram level in a reaction mixture comprising contacting a quartz crystal microbalance (QCM) electrode comprising the nanocomposite of any one of claims 1-8 with said reaction mixture, thereby monitoring mass variations at the nanogram level in a reaction mixture.

26. The method of claim 25, wherein said reaction mixture is a biological sample comprising cells.