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WO2015011231A1 - Procédé et système d'identification multiplex d'analytes dans des fluides - Google Patents

Procédé et système d'identification multiplex d'analytes dans des fluides Download PDF

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WO2015011231A1
WO2015011231A1 PCT/EP2014/065930 EP2014065930W WO2015011231A1 WO 2015011231 A1 WO2015011231 A1 WO 2015011231A1 EP 2014065930 W EP2014065930 W EP 2014065930W WO 2015011231 A1 WO2015011231 A1 WO 2015011231A1
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fluid
microchannel
nanoparticles
target analyte
ligand
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Ramón Ángel ÁLVAREZ PUEBLA
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FUNDACIO CENTRE TECNOLOGIC de la QUIMICA DE CATALUNYA
Institucio Catalana de Recerca i Estudis Avancats ICREA
Universitat Rovira i Virgili URV
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FUNDACIO CENTRE TECNOLOGIC de la QUIMICA DE CATALUNYA
Institucio Catalana de Recerca i Estudis Avancats ICREA
Universitat Rovira i Virgili URV
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles

Definitions

  • the present invention relates to a microfluidic method and system for the multiplex identification of target analytes, such as microorganisms, in a fluid sample.
  • the fluid sample is contacted with a set of nanoparticles which comprise at least one molecule with a high SERS signal and are functionalized with at least one ligand for each target analyte; the target analyte is identified by means of a laser-detector system recording the increase in the SERS signal due to the concentration of functionalized nanoparticles around the target analyte.
  • biodetection The ideal strategy for the clinical diagnosis and detection of biological agents (biodetection) must allow simultaneously investigating a large number of parameters, which is known as "high-throughput screening", such that a quick conclusion can be reached concerning the health conditions of a specific patient or environment.
  • tests include screening for pathogenic organisms (for example, virus, bacteria, eukaryotic pathogens, parasites, etc.) and/or specific disease markers (for example, antibodies, antigens, peptides, nucleotide sequences, etc.) in complex samples.
  • pathogenic organisms for example, virus, bacteria, eukaryotic pathogens, parasites, etc.
  • specific disease markers for example, antibodies, antigens, peptides, nucleotide sequences, etc.
  • SERS Surface enhanced Raman scattering spectroscopy
  • microfluidic systems have attracted a growing interest in the development of chemical or biological detection systems.
  • a microfluidic detection system must offer real time information about the chemical species, the biological species or about the reactions that can be present or take place in a specific fluid (liquid or gas) .
  • the coupling of these systems to detectors based on ultraviolet-visible (UV-Vis) spectroscopy, luminescence, has been successfully tested earlier.
  • UV-Vis ultraviolet-visible
  • the invention relates to a method for the identification of a plurality of target analytes present in a fluid, in a microfluidic system, which comprises:
  • each of said different nanoparticles comprises a core coated by a coating, wherein said coating comprises a Raman code and a ligand for each target analyte, under conditions allowing the interaction between said target analyte and its ligand and the formation of a target analyte-ligand complex, and
  • SERS surface enhanced Raman spectroscopy
  • the invention in another aspect, relates to a microfluidic system suitable for putting into practice said method comprising : a) a device in turn comprising a region adapted for storing a fluid sample and a transport microchannel comprising at one end an inlet for the entry of a fluid and where the other end opens into the region adapted for storing a fluid sample,
  • reading means for reading the SERS signal by surface enhanced Raman spectroscopy, or for reading the increase in said signal, in the storage region of said fluid sample excited by means of the laser emitter.
  • Figure la shows a diagram of the production of encoded plasmonic nanoparticles coated with silica and biofunctionalized with polyclonal antibodies specific to certain bacteria.
  • Figure lb schematically shows the recognition process. When the particles flow normally, in the absence of the bacterium that they selectively recognize, the SERS spectrum thereof (right panel) shows a low intensity. The presence of the bacterium concentrates the particles on the surface thereof considerably increasing the intensity of the SERS spectrum.
  • Figure lc shows a table with the agents, antibodies and Raman codes used in the study .
  • Figure 2a shows transmission electron microscopy images of the prepared encoded nanoparticles.
  • Figure 2b shows the surface plasmon resonance.
  • Figure 2c shows the spectra characteristic of each of the four prepared nanoparticles.
  • Figure 3 shows a diagram of the microfluidic device used in which optical photographs of the start where the two flows intersect one another, characterized by a low SERS signal, whereas, after the interaction of the nanoparticles with one of the sampled bacteria (Staphylococcus aureus) , with a high intensity SERS, are included.
  • the electron microscopy photographs show an S. aureus cell in the intersection, with hardly any interaction with the nanoparticles, and after the mixing channel, it is completely coated with encoded nanoparticles .
  • Figure 4 shows the experimental results of the detection.
  • Four encoded nanoparticles in the absence of bacteria, no signal (Figure 4a) .
  • Four encoded nanoparticles in the presence of S. aureus, positive signal for NBA, encoded nanoparticle assigned to this bacterium ( Figure 4b) .
  • Four encoded nanoparticles in the presence of 4 bacteria, positive signals for S. aureus (NBA) , Escherichia coli (R6G) and Pseudomonas aeruginosa (CV) Figure 4c
  • E. faecalis is not detected because no nanoparticle is assigned to that bacterium.
  • the invention relates to a method for the identification of target analytes present in a fluid, in a microfluidic system, hereinafter "method of the invention", which comprises:
  • each of said different nanoparticles comprises a core coated by a coating, wherein said coating comprises a Raman code and a ligand for each target analyte, under conditions allowing the interaction between said target analyte and its ligand and the formation of a target analyte-ligand complex, and
  • SERS surface enhanced Raman spectroscopy
  • target analyte refers to any biological entity to be detected which can be synthesized in, replicate in, or extracted from, a biological source. In its broadest sense, the term includes any substance with the capacity to bind to a ligand.
  • target analyte includes cells, microorganisms, viruses, nucleic acids, peptide nucleic acids, antigens, peptides and proteins.
  • the term "cell” refers to the smallest unit maintaining the fundamental properties of life, and comprises prokaryotic cells, eukaryotic cells and mesokaryotic cells, such as cells from unicellular organisms and multicellular organisms.
  • prokaryotic cells such as cells from unicellular organisms and multicellular organisms.
  • eukaryotic cells such as cells from unicellular organisms and multicellular organisms.
  • Non-limiting illustrative examples of said cells include tumor cells, tumor cell precursors, blood cells, fetal cells, stem cells and microorganisms or viruses infected cells.
  • microorganism includes very small or microscopic organisms which can be unicellular or multicellular organisms.
  • the concept of microorganism lacks any taxonomic or phylogenetic implication since it encompasses unicellular organisms not related to one another, such as prokaryotes (for example, bacteria (including mycoplasmas) and archaebacteria) , eukaryotes (for example, protozoa, fungi, algae, microscopic plants (green algae) and microscopic animals (for example, rotifera and planarias) .
  • prokaryotes for example, bacteria (including mycoplasmas) and archaebacteria
  • eukaryotes for example, protozoa, fungi, algae, microscopic plants (green algae)
  • microscopic animals for example, rotifera and planarias
  • Non- limiting illustrative examples of said microorganisms include pathogenic bacteria and non-pathogenic bacteria of interest, such as for example, Bacillus, Vibrio, for example, V. cholerae, Escherichia, for example enterotoxigenic E. coli, Shigella , for example, S. dysenteriae, Salmonella , for example, S. typhi, Mycobacterium for example M. tuberculosis, M. leprae, Clostridium, for example, C. botulinum, C. tetani, C. difficile, C. perfringens; Corynebacterium, for example, C. diphtheriae; Streptococcus, for example, S.
  • pathogenic bacteria and non-pathogenic bacteria of interest such as for example, Bacillus, Vibrio, for example, V. cholerae, Escherichia, for example enterotoxigenic E. coli, Shigella , for example, S. dysenteriae
  • said cell is a eukaryotic cell.
  • said cell is a bacterium, such as a eubacterium or a proteobacterium .
  • virus includes infectious agents having a small size and simple composition which can only multiply in animal, plant or bacteria cells in which they live. They are made of a nucleic acid (DNA or RNA) and of a protein layer.
  • viruses which can be detected by means of the present invention include orthomyxovirus (for example, flu virus, etc.), paramyxovirus (for example, respiratory syncytial virus, mumps virus, measles virus, etc.), adenovirus, rhinovirus, coronavirus, reovirus, togavirus (for example, rubella virus, etc.), parvovirus, smallpox virus, vaccinia virus, enterovirus (for example, poliovirus, etc.), hepatitis virus (including hepatitis A, B, C virus, etc.), herpesvirus (for example, herpes simplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr
  • nucleic acid refers to polymers formed by the repetition of nucleotides bound by means of phosphodiester bonds.
  • nucleic acids store the genetic information of living organisms.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • This term includes viroids, transposons, retrotransposons and plasmids or vectors .
  • the term "viroid” refers to the infectious particles which are smaller than any of the known viruses. These particles are made up only of an extremely small circular RNA and lack virus coating protein. Even though only viroids causing certain diseases in plants are known today it is possible that certain diseases in mammals may be caused by viroids. This is also the case with the other two types of infectious nucleic acid molecules, the transposons and the retrotransposons.
  • the term "transposon” refers to a DNA sequence which is capable of being inserted into a new location in the genome. Transposons have a typically linear structure, contain only one or a few genes and have special sequence structures (inverted repetitions) at their ends.
  • retrotransposon refers to a transposon which is mobilized through an RNA form; the DNA element is transcribed into RNA and then transcribed, by means of reverse transcription, into DNA which is inserted into a new site in the genome.
  • plasmid refers to an autonomous, self-replicating, extrachromosomal circular DNA.
  • peptide nucleic acid refers to a nucleic acid in which the phosphate- (deoxy) ribose skeleton has been substituted with 2- (N-aminoethyl ) glycine, bound by a conventional peptide bond.
  • the purine and pyrimidine bases are bound to the skeleton by the carbonyl carbon.
  • the term "antigen" includes any substance which triggers the formation of antibodies and can cause an immune response; i.e., it includes any self and foreign substance which can be recognized by the adaptative immune system.
  • the antigens are usually peptides, proteins or polysaccharides, and include, by way of illustration, parts of bacteria (e.g., capsule, cell wall, flagella, fimbriae, and toxins), viruses, parasites, etc.
  • peptide and protein are virtually synonyms and refer to molecules having polymerized amino acids due to the formation of peptide bonds.
  • peptide is normally used in relatively short polymerized amino acid chains whereas the term “protein” is used to refer to larger molecules and/or molecules having a specific activity. These terms include prions, antibodies, hormones, cytokines, etc.
  • the term "prion" refers to an aberrant form of an amyloid protein which is usually harmless, found in mammals and birds.
  • the normal form of the protein is located on the surface of brain cells.
  • antibody refers to gamma-globulin type glycoproteins.
  • the typical antibody is made up of basic structural units, each of which with two large heavy chains and two smaller light chains, forming monomers (with one unit) , dimers (with two units) or pentamers (with five units), for example.
  • monomers with one unit
  • dimers with two units
  • pentamers with five units
  • the term “antibody” includes natural antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, etc. Likewise, the term “antibody” refers both to complete antibodies and to fragments thereof maintaining the capacity to bind to the corresponding antigen, as well as to recombinant antibodies, such as for example, single-chain Fv fragments (scFv) , diabodies, single-domain antibodies (VH) , nanobodies (VHH) , etc.
  • scFv single-chain Fv fragments
  • VH single-domain antibodies
  • VHH nanobodies
  • the target analytes to be identified according to the method of the invention are present in a fluid or a continuous medium formed by a substance having a weak attractive force between its molecules.
  • a fluid is a group of particles which remain bound to one another by weak cohesive forces and/or by the walls of a recipient.
  • the term "fluid" encompasses liquids and gases.
  • Non-limiting illustrative examples of fluids include solutions, suspensions, etc., in which the target analyte can be dissolved or dispersed in a suitable medium, such as an aqueous medium, an organic medium, or aqueous-organic medium.
  • said fluid comprises a biological fluid (e.g., blood, urine, exudates, tears, etc.), natural water, waste water, airflows, etc .
  • a fluid sample comprising a plurality of target analytes is contacted, in a microfluidic system, with a set of different nanoparticles, wherein each of said nanoparticles comprises a core coated by a coating, wherein said coating comprises a Raman code and a ligand for each target analyte.
  • nanoparticles is used for designating colloidal systems of sphere, rod, polyhedron shapes, etc., or similar shapes, having a size less than 1 micrometer ( ⁇ ) , individually or forming organized structures (dimers, trimers, tetramers, etc.), dispersed in a fluid (aqueous solution) .
  • the nanoparticles suitable for putting the invention into practice have a size less than 1 ⁇ , generally comprised between 1 and 999 nanometers (nm) , typically between 5 and 500 nm, preferably between 10 and 150 nm, approximately.
  • the shape of the nanoparticles can vary greatly; advantageously, said nanoparticles will adopt any optically efficient shape such as spheres, rods, stars, cubes, polyhedra or any another variant as well as complex associations of several particles; in a particular embodiment, the shape of the nanoparticles for putting the invention into practice is spherical, or it has the shape of a rod, cube, octahedron, decahedron, star, etc.
  • the core of the nanoparticles comprises one or more particles of any suitable material known in the art such as, for example, any metals and doped semiconductors that can sustain Raman signal amplification are suitable for use in the present invention .
  • the core of said nanoparticles can be prepared with a material capable of generating high electric or electromagnetic fields at the particle surface by means of the interaction thereof with a light beam, such as materials which generate surface plasmon resonances or "whispering gallery modes" (Mie Resonances) excited by means of monochromatic light beams, for example, lasers, LEDs, OLEDs, lamps with filters, etc. (J Phys Chem Lett 3 (2012) 857-866) .
  • a material capable of generating high electric or electromagnetic fields at the particle surface by means of the interaction thereof with a light beam such as materials which generate surface plasmon resonances or "whispering gallery modes" (Mie Resonances) excited by means of monochromatic light beams, for example, lasers, LEDs, OLEDs, lamps with filters, etc.
  • a material capable of generating high electric or electromagnetic fields at the particle surface by means of the interaction thereof with a light beam such as materials which generate surface plasmon resonances or "whispering gallery modes" (Mie
  • Plasmonic materials comprising metals such as gold, silver, copper, aluminum, rhodium, ruthenium, indium, alkaline metals, alkaline-earth metals, the alloys of these metals, the alloys of these metals with other metals, etc. (Laser & Photonics Reviews 4 (6) . doi : 10.1002 /lpor .200900055 ) ; as well as semiconductors in which plasmons are generated by inducing vacancies in the crystalline structure, for example, CuSe, CuSe or CuTe (JACS 135 (2013) 7098-7101; Adv. Mater. 2013, 25, 3264- 3294) ; and
  • Materials with a high refractive index capable of sustaining Mie resonances such as silicon, etc.
  • the plasmonic materials provide the electromagnetic field necessary for enhancing the SERS signal of the Raman molecule.
  • the plasmonic material comprises, but is not limited to gold, silver, or copper, aluminium, or alloys thereof, or a combination thereof.
  • the metal may be gold, silver or copper .
  • the material of the core is forming particles, typically nanoparticles (nanospheres, nanorods, nanotriangles , nanostars or other nanogeometries ) .
  • nanoparticles nanospheres, nanorods, nanotriangles , nanostars or other nanogeometries
  • Methods of preparing metallic nanoparticles are well-known to those of skill in the art (Lee, Meisel J. Phys. Chem., (1982) 86, 3391-3395; Baker et al. Baker et al. Anal. Bioanal. Chem., (2005) 382, 1751- 1770.2005), and are not further described herein.
  • the nanoparticles of plasmonic material or material having a high refractive index capable of sustaining Mie resonances may be of a suitable size and type.
  • the average particle size i.e., diameter
  • the average size of said nanoparticles may be in the range of about 1 to 100 nm; for example, the average size of said nanoparticles may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm, or any amount therebetween, or any range defined by the values just recited.
  • the nanoparticle core is coated by a coating comprising a Raman code (i.e., at least one Raman active reporter molecule) and a ligand specific for a target analyte.
  • a Raman code i.e., at least one Raman active reporter molecule
  • a ligand specific for a target analyte i.e., at least one Raman active reporter molecule
  • the coating of the nanoparticles has several objectives: (i) to increase nanoparticle colloidal stability in biological or other natural fluids; (ii) to prevent plasmonic couplings between different nanoparticles, which can give rise to the generation of hot-spots with the subsequent uncontrolled increase in Raman signal; (iii) to allow the efficient functionalization of the nanoparticles with the suitable ligands; and (iv) to avoid the leaching of the Raman active reporter molecule or molecules (i.e., the Raman code) .
  • the coating can vary greatly; by illustrative, the coating may be formed of any suitable material known in the art; for example, and not wishing to be limiting in any manner, it may comprise silica, or a suitable metal oxide, or one or more than one polymer, or block copolymer, etc.
  • said coating comprises a metal oxide, for example, silicon dioxide (Si0 2 ) , titanium dioxide (Ti0 2 ) , etc.; a polymer, for example, polyethylene glycol (PEG) , sulfonated polystyrene (SPS) , polyether ether ketone (PEEK) , polyvinylpyrrolidone (PVP) , polystyrene (PS) , polyacrylic acid (PAA) , poly (methyl methacrylate) ( PMMA) , etc .
  • PEG polyethylene glycol
  • SPS sulfonated polystyrene
  • PEEK polyether ether ketone
  • PVP polyvinylpyrrolidone
  • PS polystyrene
  • PAA polyacrylic acid
  • PMMA poly (methyl methacrylate)
  • the coating comprises silica (glass) or other suitable material.
  • the coating in general, and silica, in particular, provides the core (which is surrounded/ coated by the coating) with mechanical and chemical stability, prevents the core from exterior reactions, and renders the core amenable to use in many solvents without disrupting the SERS response. Further, the coating prevents other analytes from entering SERS hot sites to displace the signal of the Raman active reporter molecules. Additionally, the coating enables attachment of suitable ligands (e.g., biomolecules ) .
  • This core and coating structure is well- known for the skilled person in the art. Methods for preparing a coating comprising silica are also well-known to those of skill in the art.
  • the thickness of the coating may vary. By illustrative, and without wishing to be limiting in any manner, the thickness of the coating may be applied in a controlled manner over the core- Raman active reporter molecules.
  • the thickness of the coating once complete, may be about 1 nm and 100 nm, or any value there between; for example, the silica coating may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm thick, or any value therebetween. In a specific, non-limiting example, the thickness of the silica coating may be about 10 to 30 nm.
  • the ligand is the actual coating of the nanoparticle .
  • the Raman code comprises one or more molecules having a high SERS signal (i.e., one or more Raman active reporter molecules) .
  • Said Raman active reporter molecules may be adsorbed onto the core or onto the nanoparticles that constitute the core or otherwise aggregated with said core or core nanoparticles.
  • the Raman active reporter molecule may comprise at least one organic compound; the organic compound may comprise, normally, at least one isothiocyanate, thiol, or amine group, or multiple sulfur atoms, and/or multiple nitrogen atoms.
  • Non- limiting illustrative examples of said molecules having a high SERS signal include organic dyes, for example, rhodamines, indole derivatives, azo dyes, porphyrins, perylenes, acridines, anthraquinones , arylmethanes , indamines, oxazones, oxazines, indophenols, thiazines, xanthens, fluorenes, etc.; organometallic compounds, for example, porphyrins, phthalocyanines , organic lacquers coordinated with transition metals, etc.; aromatic compounds, for example, thiolated, aminated or nitrated derivatives of the benzene, n
  • the Raman active reporter molecule may be, but is not limited to, rhodamine 6G, tetramethyl-rhodamine-5-isothiocyanate, X- rhodamine-5- (and-6) -isothiocyanate, or 3,3'- diethylthiadicarbo- cyanine iodine, or a combination thereof.
  • the Raman active reporter molecule is a rhodamine, such as, for example, rhodamine 6G (R6G) .
  • the nanoparticles used in putting the method of the invention into practice generate surface electromagnetic fields capable of enhancing the Raman signal of the codifying molecule.
  • the nanoparticles of plasmonic material can have any shape but have a size less than the wavelength of the incident light beam.
  • those fields can be generated by means of association of those nanoparticles in which hot-spots are generated, from uncontrolled aggregates to controlled nanoparticle dimers, trimers, tetramers, etc. (Angew Chem Int Ed 51 (2012) 12688-12693) .
  • this effect is achieved by means of using spherical materials with a high refractive index, for example, silicon of 100 to 1,000 nm. 1.2.4 Ligand
  • the coating of the nanoparticles further comprises a ligand for a target analyte; said ligand is preferably specific for said target analyte.
  • ligand refers to a compound which is used for probing the presence of the target analyte.
  • the term includes any substance with the capacity to bind to a target analyte, and particularly any molecule capable of giving rise to selective interactions with the target analyte.
  • the person skilled in the art will understand that the chemical nature of the ligand will depend on the chemical nature of the target analyte.
  • Ligands, preferably selective ligands, for a wide variety of target analytes are known or can be easily found using known techniques. By way of non-limiting illustration:
  • the ligand when the target analyte is a protein, can be a protein (for example, an antibody or a fragment thereof ( Fabs , etc . ) ) ;
  • the ligand when the target analyte is an enzyme, can be a substrate or an inhibitor of said enzyme;
  • the ligand when the target analyte is a nucleic acid-binding protein or a nucleic acid, the ligand can be an aptamer, such as a peptide aptamer or a DNA or RNA aptamer; and
  • the ligand can be a nucleic acid, such as a probe, etc.
  • the ligand is an antibody, or a fragment thereof, recognizing the target analyte.
  • the characteristics of the antibodies have already been defined above .
  • the ligand is a substrate or an inhibitor of an enzyme.
  • the ligand is an aptamer.
  • aptamer includes single-chain oligonucleotides with a length usually comprised between 70 and 100 nucleotides, including a fixed sequence or primer in each end of the aptamer for amplification by means of polymerase chain reaction (PCR) , capable of specifically recognizing several types of target molecules with a high affinity by means of a three-dimensional folding of its chain.
  • PCR polymerase chain reaction
  • oligonucleotides have a central region of variable size and with a random sequence with a length comprised between 30 and 60 nucleotides, and two flanking regions with known sequence.
  • molecular targets can be small molecules, such as proteins, nucleic acids, etc.
  • aptamers based on peptide chains comprising a variable peptide loop attached to the ends of a protein support, which increases the affinity of the peptide aptamer to levels comparable to antibodies (nanomolar range) .
  • the ligand is a molecule giving rise to a selective interaction with the target analyte; i.e., the ligand has the capacity to distinguish between several target analytes present in the fluid sample.
  • selective interaction include the antigen-antibody interactions, enzyme-substrate interactions, key-lock interactions, etc.
  • the binding of the ligand to the target analyte is specific, i.e., the ligand unambiguously identifies the target analyte in the presence of other target analytes which can be expected to be present in the fluid sample.
  • specific binding is understood as the ligand binding to the target analyte with sufficient specificity so as to distinguish between the target analyte and other components or contaminants of the test sample. The binding must be strong enough for the ligand to remain bound to the target analyte under the test conditions.
  • the diassociation constant of the target analyte and the ligand will be equal to or less than about 10 ⁇ 4 M -1 , preferably equal to or less than 10 ⁇ 6 M -1 , more preferably equal to or less than 10 ⁇ 8 M -1 , still more preferably equal to or less than 10 ⁇ 10 M -1 .
  • said ligand Due to the nature of the ligand, some of its characteristics, for example, its molecular weight, can vary within a wide range; nevertheless, in a particular embodiment, said ligand has a molecular weight of at least 250 Dalton (Da) , typically of at least 500 Da, usually of at least 1, 000 Da, preferably of at least 5, 000 Da, more preferably of at least 100,000 Da and, still more preferably of at least 5,000,000 Da.
  • Da Dalton
  • the nanoparticles suitable for putting the method of the invention into practice can be obtained by conventional methods known by the persons skilled in the art.
  • the production of the nanoparticles for putting the invention into practice is carried out by means of synthetic methods ranging from “top-down” to "bottom-up” type methods including chemical methods comprising, for example, metal salt reduction, overgrowth, light ablation, or physical methods, for example, physical or chemical vapor evaporation, sputtering, milling, etc.
  • the plasmonic particles are encoded with molecules with Raman activity or which can develop Raman activity including organic systems, inorganic systems, organometallic systems, etc., with or without functionalization .
  • the Raman code can be added before or after the coating with inorganic oxides and/or polymers or organic macromolecules such as proteins, sugars or lipids.
  • the coating could be avoided if the ligand is used as coating.
  • the ligand can be stabilized on the surface of the nanoparticles by means of suitable chemical techniques, for example, depending on the functional groups eventually present on the surface of the nanoparticles and on the ligand, such as, for example, creation of bonds by means of chemical synthesis or self-assembly, or by means of physical phenomena based on electrostatic or hydrophobic interactions.
  • said nanoparticles comprise a gold core obtained by chemical reduction with sodium citrate; said gold core stabilized with PEG is encoded with the Raman code by means of adding one or more dyes (molecules with a high SERS signal) and it is then coated with tetraethyl orthosilicate (TEOS) under controlled conditions. Finally, the gold nanoparticles coated with TEOS and containing the Raman code are functionalized with the suitable ligand, such as a selective ligand or a ligand specific for a target analyte.
  • the suitable ligand such as a selective ligand or a ligand specific for a target analyte.
  • a bifunctional reagent such as a carbodiimide for example, which binds at one end to the nanoparticle and, at the other end to the ligand, for example, an antibody.
  • the invention contemplates the possibility of a target analyte having one or more binding, interaction or recognition sites (generally, for simplicity, "binding site") for binding to, interacting with or recognizing one and the same ligand or different ligands.
  • binding site generally, for simplicity, "binding site”
  • the target analyte has a single ligand binding site; in that case, the nanoparticles will include the ligand corresponding to the target analyte; this can be the case of a protein or peptide (target analyte) , for example, having a single epitope and an antibody recognizing said protein or peptide, or the case of a nucleic acid (target analyte) having a single target nucleotide sequence and a probe hybridizing with said target nucleotide sequence.
  • a protein or peptide for example, having a single epitope and an antibody recognizing said protein or peptide
  • a nucleic acid target analyte
  • the target analyte has two (or more) binding sites, wherein in each of said binding sites the same ligand or a different ligand can bind.
  • the target analyte can have several repetitions of one and the same motif (e.g., epitope) which is recognized by the same ligand and to which the ligand binds; in this case, the nanoparticles will include the ligand corresponding to the target analyte; and in the second case, the target analyte has two (or more) different ligand binding sites wherein a different ligand can bind to each binding site, for example, a cell (target analyte) can have different proteins (e.g., the different PI and P2 proteins) that can be recognized by different antibodies (e.g., the Acl and Ac2 antibodies recognizing the PI and P2 proteins, respectively) , and, in that case, the nanoparticles can be functionalized with the 2 Acl and Ac2 antibodies, or there may be nanoparticles functionalized with Acl and other nanoparticles functionalized with Ac2 ; in this last case, the nanoparticles functionalized with different ligand
  • the target analyte has one or more different enzymatic activities (e.g., lactonase and acylase, etc.), in which case, the nanoparticles could be functionalized with the different substrates for said enzymatic activities or nanoparticles functionalized with a substrate for a specific enzymatic activity and other nanoparticles functionalized with another substrate for a different enzymatic activity can be provided; in this last case, if desired, the nanoparticles functionalized with different ligands (substrates) can have the same Raman code;
  • enzymatic activities e.g., lactonase and acylase, etc.
  • the target analyte has one or more nucleic acid-binding proteins
  • the nanoparticles could be functionalized with the different aptamers which bind to said nucleic acid-binding proteins or nanoparticles functionalized with an aptamer which binds to a nucleic acid-binding protein and other nanoparticles functionalized with another different aptamer which binds to another nucleic acid-binding protein can be provided; in this last case, if desired, the nanoparticles functionalized with different ligands (aptamers) can have the same Raman code; or
  • the target analyte is a nucleic acid which may have several repetitions of one and the same nucleotide sequence to which one and the same probe binds
  • the nanoparticles will include the probe corresponding to the target analyte (target nucleotide sequence) , or said nucleic acid can have two (or more) different ligand (probe) binding sites wherein a different probe can bind in each target nucleotide sequence; in that case, the nanoparticles can be functionalized with the same probe, or some nanoparticles can be functionalized with a specific probe and other nanoparticles can be functionalized with another different probe, wherein each of said probes binds to a different target nucleotide sequence; in this last case, the nanoparticles functionalized with different ligands can have the same Raman code, if desired.
  • Step a) of the method of the invention comprises contacting a fluid sample comprising a plurality of target analytes with a set of different nanoparticles, wherein each of said different nanoparticles comprises a core coated by a coating, wherein said coating comprises a Raman code and a ligand for each target analyte.
  • a fluid sample comprising a plurality of target analytes with a set of different nanoparticles
  • each of said different nanoparticles comprises a core coated by a coating, wherein said coating comprises a Raman code and a ligand for each target analyte.
  • the fluid sample comprising a plurality of target analytes and the set of different nanoparticles are contacted in a recipient independently fed by a first feed comprising said sample fluid and by a second feed comprising the set of nanoparticles.
  • said recipient wherein both first and second feeds are fed in the same recipient, acting as a premixture container, is placed within the microfluidic system.
  • Said fluid sample comprising a plurality of target analytes is contacted with said set of different nanoparticles under conditions allowing the interaction between the target analyte and its ligand and the formation of a target analyte-ligand complex (agglutination) .
  • Said conditions depend on the nature of the target analyte and on its ligand. The person skilled in the art knows or can readily identify the conditions allowing said interaction.
  • said conditions include those allowing the formation of an immunocomplex between the protein (target analyte) and the antibody (ligand) ; when the target analyte is an enzyme and the ligand is a substrate of said enzyme, said conditions include those allowing the enzyme (target analyte) to act on the substrate (ligand) ; when the target analyte is a nucleic acid- binding protein and the ligand is an aptamer, said conditions include those allowing the interaction (binding) between the nucleic acid-binding protein (target analyte) and the aptamer (ligand) ; when the target analyte is a nucleic acid and the ligand is a probe, said conditions include those allowing the interaction (hybridization) between the target sequence of the nucleic acid (target analyte) and the probe (ligand); etc.
  • Step b) of the method of the invention comprises subjecting the resulting mixture of step a) to surface enhanced Raman spectroscopy (SERS) and analyzing the spectra associated with the target analyte-ligand complexes formed, and thereafter identifying the target analytes present in said fluid.
  • SERS surface enhanced Raman spectroscopy
  • the analysis of the obtained Raman spectrum is carried out by deconvoluting the spectrum, i.e., dividing the spectrum into the individual contributions associated with each Raman code used .
  • the present invention allows the multiplex identification and, if desired, the count of target analytes, for example, microorganisms, in liquid or gas fluids.
  • the invention enables real time multiplex biodetection and diagnosis of target analytes present in a fluid.
  • each of said nanoparticles comprises a core coated by a coating, wherein said coating comprises a Raman code and a ligand for each target analyte, and contacting said set of nanoparticles with a fluid sample comprising a plurality of target analytes under conditions allowing the interaction between the target analyte and its ligand and the formation of a target analyte-ligand complex; said interaction enables identifying the target analyte or analytes by means of a laser-detector system recording the increase in the SERS signal due to the concentration of functionalized nanoparticles around the target analytes.
  • the method of the invention allows the simultaneous detection (multiplex) of a plurality of target analytes present in a fluid, for example, microorganisms (pathogenic or non-pathogenic microorganisms), cells (normal or tumor cells), etc., in a biological or environmental fluid sample .
  • a fluid for example, microorganisms (pathogenic or non-pathogenic microorganisms), cells (normal or tumor cells), etc.
  • the method of the invention involves some relevant features, such as, for example:
  • Raman-coded nanoparticles i.e., a plasmonic nanoparticle, coded with a Raman active reporter molecule and having high SERS activity and coated with a suitable coating
  • the coating provides several technical effects such as: (i) increases nanoparticle colloidal stability; (ii) prevents plasmonic couplings between different nanoparticles, which can give rise to the generation of hot-spots with the subsequent uncontrolled increase in Raman signal; (iii) allows the efficient functionalization of the nanoparticles with the suitable ligands; and (iv) maintains the Raman active reporter molecule or molecules ;
  • the nanoparticles are of the type "label-free", i.e., the positives are due to an increase in the Raman signal due to the aggregation of nanoparticles with the same ligand and Raman code induced by the presence of the target analyte;
  • the target analyte is a microorganism, for example, a virus, a bacteria, etc., or a cell, such as an eukaryotic cell, since the nanoparticles are smaller than those microorganisms and cells and, normally, those microorganisms and cells have more than one surface receptor for the ligand present in the nanoparticles, said microorganisms and cells can accumulate said nanoparticles on their surface; and c) the use of a microfluidic system for which the set of Raman-coded nanoparticles and sample are forced to pass through what allows:
  • the sample moves through a conveyor microchannel and positives are duly counted; since the nanoparticles are single, homogeneus and are protected by a coating, they do not couple their fields and, therefore, the increase in the signal is due to the increase in the number of nanoparticles in the studied area (which is limited by the flow rate) ; and further
  • the sample is analyzed in order to obtain the target analyte concentration (i.e., it is a quantitative method) .
  • plasmonic nanoparticles encoded with different Raman codes, coated with silica and biofunctionalized with polyclonal antibodies specific to the bacteria under study are produced; the obtained nanoparticles are recognized by the corresponding bacteria what increases the intensity of the SERS spectrum.
  • plasmonic nanoparticles are coated with silica, and then encoded with a Raman code, the surface is functionalized with an amino- silane [NH 2 -R-Si (OR) 3 ] type reagent and biofunctionalized with a polyclonal antibody recognizing a specific bacterium by means of using a bifunctional coupling agent, such as a carbodiimide , for example, EDC [ethyl-3- (3-dimethylaminopropyl) carbodiimide] .
  • a bifunctional coupling agent such as a carbodiimide , for example, EDC [ethyl-3- (3-dimethylaminopropyl) carbodiimide] .
  • plasmonic nanoparticles encoded with NBA Renishaw code or Raman active reporter molecule
  • silica and biofunctionalized with anti-Staphylococcus aureus polyclonal antibodies recognizing the presence of S. aureus in a fluid sample containing said bacterium;
  • plasmonic nanoparticles encoded with R6G (Raman code) , coated with silica and biofunctionalized with anti- Escherichia coli polyclonal antibodies, recognizing the presence of E. coli in a fluid sample containing said bacterium;
  • plasmonic nanoparticles encoded with CV (Raman code) , coated with silica and biofunctionalized with anti- Pseudomonas aeruginosa polyclonal antibodies, recognizing the presence of P. aeruginosa in a fluid sample containing said bacterium.
  • nanoplasmonic particles coated with silica but not encoded with Raman code or biofunctionalized
  • plasmonic nanoparticles encoded with TB Raman code
  • silica and biofunctionalized with bovine serum albumin (BSA) are produced.
  • Figure 2a shows how the transmission electron microscopy images of the plasmonic nanoparticles encoded with Raman code, coated with silica and biofunctionalized obtained as mentioned above would be.
  • the spectra characteristic of each of the four prepared nanoparticles [plasmonic nanoparticles encoded with NBA, coated with silica and biofunctionalized with anti-S. aureus polyclonal antibodies, plasmonic nanoparticles encoded with R6G, coated with silica and biofunctionalized with anti-£. coli polyclonal antibodies, plasmonic nanoparticles encoded with CV, coated with silica and biofunctionalized with anti-P. aeruginosa polyclonal antibodies, and plasmonic nanoparticles encoded with TB, coated with silica and biofunctionalized with BSA] are shown in Figure 2c, whereas the surface plasmon resonance is shown in Figure 2b.
  • the SERS signal when the bacterium (e.g., S. aureus) has not interacted with the corresponding nanoparticles (e.g., plasmonic nanoparticles encoded with NBA, coated with silica and biofunctionalized with anti-S. aureus polyclonal antibodies), the SERS signal is very low; however, when the plasmonic nanoparticles encoded with NBA, coated with silica and biofunctionalized with anti-S. aureus polyclonal antibodies have interacted with the bacterium S. aureus then the SERS signal has a high intensity.
  • the plasmonic nanoparticles encoded with NBA, coated with silica and biofunctionalized with anti-S. aureus polyclonal antibodies have interacted with the bacterium S. aureus then the SERS signal has a high intensity.
  • a positive signal is observed for NBA when the plasmonic nanoparticles encoded with NBA, coated with silica and biofunctionalized with anti-S. aureus polyclonal antibodies, obtained as described above, are contacted with a fluid sample containing S. aureus ( Figure 4b) ; and positive signals are observed for CV, NBA and R6G when the plasmonic nanoparticles encoded with CV, coated with silica and biofunctionalized with anti-P. aeruginosa polyclonal antibodies, the plasmonic nanoparticles encoded with NBA, coated with silica and biofunctionalized with anti-S. aureus polyclonal antibodies, and the plasmonic nanoparticles encoded with R6G, coated with silica and biofunctionalized with anti-£.
  • coli polyclonal antibodies are contacted with a fluid sample containing P. aeruginosa , S. aureus and E. coli ( Figure 4b) ; however, E. faecalis is not detected because no nanoparticle is assigned to that bacterium. It is worth highlighting that there is no signal for TB, nanoparticle functionalized with BSA, in any case and it therefore does not react with any bacterium .
  • the method of the invention can be carried out in any suitable microfluidic system comprising the means suitable for performing it. Nevertheless, in a particular embodiment the method of the invention is carried out in a microfluidic system comprising :
  • a device in turn comprising a region adapted for storing a fluid sample and a transport microchannel , comprising at one end an inlet for the entry of a fluid and where the other end opens into the region adapted for storing a fluid sample, b) a laser emitter adapted for striking on the storage region of the sample of said fluid;
  • reading means for reading the SERS signal by surface enhanced Raman spectroscopy, or for reading the increase in said signal, in the storage region of said fluid sample excited by means of the laser emitter.
  • the fluid sample is delivered to the region adapted for storing the fluid sample
  • microfluidic device and system are additional aspects of the present invention.
  • microchannel refers to a channel the width of which is comprised in the range of 500 nm to 10 mm.
  • the microchannels having a width close to the upper limit of the preceding range, particularly those having a width of more than a millimeter, are sometimes called “millichannels”. It will therefore be understood that millichannels having a width comprised in the range of 500 nm to 10 mm are included in the definition of microchannel according to the invention.
  • a "microfluidic device” according to the invention is the device including microchannels.
  • the region adapted for storing the fluid sample is envisaged for storing the sample during the detection time, until the acquisition of the corresponding spectrum Raman. Said region must be suitable to allow the laser beam to strike on the sample, preferably due to having at least one area which is transparent to the laser radiation used.
  • the device comprises a single inlet envisaged for introducing the mixture formed by the fluid sample to be analyzed and the nanoparticles into the microchannel .
  • the device has two inlets, one of them being envisaged for introducing the fluid sample to be analyzed and the other for introducing a solution containing the SERS-encoded nanoparticles and functionalized with the ligands, preferably selective or specific ligands, for each target analyte.
  • Each inlet is connected with the transport microchannel by means of corresponding channels.
  • the system of channels with two inlets preferably has a substantially Y or T shape .
  • the device can have more than two inlets envisaged for introducing samples of different origin.
  • each inlet is connected with the transport microchannel by means of corresponding channels.
  • the device of the invention is manufactured from PDMS, glass or any other polymer or organic and inorganic material.
  • the device of the invention comprises :
  • a first microchannel for introducing the fluid sample and comprising at one end a first input port
  • the ends of the first microchannel and of the second microchannel opposite the ends having the input port converge in the inlet of the transport microchannel; and for contacting the fluid sample with the nanoparticles the fluid sample is introduced in the input port of the first microchannel and the second fluid, comprising the medium (excipient) with the nanoparticles, is introduced in the input port of the second microchannel, delivering both fluids to the inlet of the transport microchannel; and delivering both through said transport microchannel to the region adapted for storing a fluid sample.
  • the device comprises passive drive means for driving the fluid through its channels, particularly, incorporating on at least part of the inner surface of each channel, a hydrophilic material which allows moving the fluid from the inlet by capillarity.
  • the device comprises active drive means for driving the fluid through its channels, such as for example, piston pumps or peristaltic pumps, peristaltic pumps being particularly preferred in auto-sampling systems.
  • the thickness and height of the channels of the device can be selected depending on the application and on the agents to be studied.
  • all the channels included in the device are microchannels, i.e., they have a width of between 500 nanometers and 10 millimeters. More preferably, the width and the height of the channels are comprised in the range of 1 ⁇ to 5 mm.
  • said storage region where the reading of the SERS signal or of the increase thereof is carried out comprises a narrowing which reduces its section such that only the passage and observation of a target analyte with the nanoparticle agglomeration is allowed.
  • a two-dimensional detector (2D-CCD) is located in the storage region where the reading of the SERS signal is carried out whereby the target analytes with the nanoparticle agglomeration are simultaneously but individually analyzed.
  • the light beam strikes on the width of the channel in the form of a line, each pixel of the 2D-CCD camera acts as an individual detector recording a single signal.
  • the system of channels of the microfluidic device of the invention can be manufactured by means of various techniques among which nanoimprint lithography, electron beam lithography, ion beam lithography or laser lithography are included, or these lithographies can be used for obtaining the mold with which circuits are imprinted by means of soft lithography techniques.
  • the channels are obtained, they are sealed with a window transparent to the laser detection beam by means of materials such as quartz, glass, inorganic oxides transparent to the laser beam or polymers transparent to the laser beam.
  • the system of the invention comprises, in addition to the microfluidic device, a laser emitter and a Raman detection system, preferably including a diffraction grating and a detector (CCD or other) integrated or not integrated in the device .
  • the system of the invention additionally comprises processing means configured for interpreting the results, such as a system for transmitting data to a computer with deconvolution software.
  • processing means configured for interpreting the results, such as a system for transmitting data to a computer with deconvolution software.
  • the software can be programmed for indicating positive or negative events depending on the concentration of target analyte determined.
  • the system of the invention allows automatic auto-sampling devices in which different samples are arranged in vials in an automatic injection carousel and are injected into the microfluidic device at programmed intervals.
  • a microfluidic device was manufactured from poly (dimethylsiloxane) (PDMS) /glass by means of conventional soft lithography methods. Briefly, the device was initially designed with AutoCad 2007 (Autodesk) and a dark field mask was imprinted (Circuit-graphics) .
  • the light sensitive material SU-8 2025 (Micro-Chem) was applied by means of rotation (spin-coating) on a silicon wafer of 76.2 mm in diameter (Compart Technology Ltd.) at 500 rpm for 5 seconds and then in a ramp at 1,000 rpm with an acceleration of 300 rpm s -1 for 33 seconds. The final thickness of the light sensitive layer was measured by means of profilometry on the end master mold (Dektak 150) .
  • the wafer was then precooked for 3 minutes at 65°C, then for 9 minutes at 95°C, and finally for 3 minutes at 65°C.
  • the mask was exposed to UV light through the mask in a mask aligner (MJB4, Suss Microtech) .
  • a mask aligner MJB4, Suss Microtech
  • the master mold was intensely cooked in an oven for 1 minute at 170°C.
  • a mixture of PDMS (Sylgard 184) and cross-linking agent (ratio of 10:01, w/w) was poured into the master mold, it was degassified for 30 minutes and then cured overnight at 75°C.
  • the curing device was cut and removed from the master mold and holes were made for the tubes with a biopsy needle.
  • the device After the treatment with air plasma for 30 seconds, the device was attached to a glass slide for the purpose of sealing the microfluidic device. The device was cooked at 90°C for 30 minutes to make the sealing permanent. Finally, the microfluidic channels were treated with Aquapel (Pittsburg, USA) , a fluorosilane available on the market, followed by washing the channels with fluorinated oil.
  • Aquapel Pittsburg, USA
  • the mixture was left to react for 2 hours, and in this step, the casing was left to grow after the controlled addition of TEOS (tetraethyl orthosilicate ) . Centrifugation is recommended before each addition of TEOS to remove the free silica cores.
  • TEOS tetraethyl orthosilicate
  • the Raman probes were encapsulated by simply adding the dye (diluted in ethanol) dropwise under gentle stirring to the gold colloids stabilized with PEG immediately before the addition of TEOS.
  • the final concentration of added dye was 6 mM for each dye .
  • the encoded nanoparticles were treated with the coupling agent (3-aminopropyl) trimetoxysilane (APS) by means of stirring at 60-70°C for 90 minutes.
  • the particles were centrifuged at 2, 500 rpm for 15 minutes and washed with ethanol and phosphate buffered saline solution (PBS) .
  • Carbodiimide chemistry was used for conjugating the primary amines APS with the carboxyl groups of the dodecanedioic acid, providing-COOH groups to the surface of the nanoparticles.
  • SERS experiments were conducted in a micro-Renishaw Invia Reflex system.
  • the spectograph uses a high resolution grid (1,200 grooves mm -1 ) with additional band-pass filter optics, a confocal microscope and a 2D-CCD camera.
  • a laser excitation energy of 785 nm (diode) was used for all the in-line measurements.
  • the in-line measurements were made in a confocal microscope in the backscattering geometry using an objective of 20 times magnification (20x) .

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Abstract

La présente invention concerne un procédé d'identification d'une pluralité d'analytes cibles présents dans un fluide, le procédé comprenant : (a) le contact d'un échantillon dudit fluide comprenant une pluralité d'analytes cibles avec un ensemble de nanoparticles différentes, chacune desdites nanoparticles comprenant un noyau revêtu d'un revêtement, ledit revêtement comprenant un code Raman et un ligand pour chaque analyte cible, dans des conditions permettant une interaction entre ledit analyte cible et son ligand et la formation d'un complexe analyte cible/ligand, et (b) la soumission du mélange résultant à une spectroscopie Raman exaltée de surface (SERS), et l'analyse des spectres Raman associés aux complexes analyte cible/ligand formés, puis l'identification des analytes cibles présents dans ledit fluide.
PCT/EP2014/065930 2013-07-25 2014-07-24 Procédé et système d'identification multiplex d'analytes dans des fluides Ceased WO2015011231A1 (fr)

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