WO2016174525A1

WO2016174525A1 - Enhanced sensitivity in ligand binding assays performed with secondary ion mass spectrometry

Info

Publication number: WO2016174525A1
Application number: PCT/IB2016/000749
Authority: WO
Inventors: Christine HEUCLIN; Guillaume Legent
Original assignee: Biosims Technologies
Priority date: 2015-04-29
Filing date: 2016-04-29
Publication date: 2016-11-03

Abstract

The present invention relates to a method for quantitating a target molecule in at least one assay sample comprising: depositing on a biochip at least one first capture molecule; adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; adding at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface; and scanning the biochip by secondary ion mass spectrometry to measure the secondary ions that are emitted by the specific ionizable atoms. Kits comprising the nanoparticles or microparticles and methods of using the nanoparticles or microparticles are also described.

Description

ENHANCED SENSITIVITY IN LIGAND BINDING ASSAYS PERFORMED WITH SECONDARY ION MASS SPECTROMETRY

FIELD OF THE INVENTION

The present invention relates to a ligand binding assay for quantitating a target molecule in at least one assay sample comprising: depositing on a biochip at least one first capture molecule; adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; adding at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface and other various embodiments, as described herein; and scanning the biochip by secondary ion mass spectrometry to measure the secondary ions that are emitted by the specific ionizable atoms. Kits comprising the nanoparticles or microparticles, as described herein, or comprising the nanoparticles or microparticles and ionizable atoms, as described herein and methods of using the nanoparticles or microparticles, as described herein, are also disclosed.

BACKGROUND AND PRIOR ART

Nanoparticles or microparticles have many industrial applications and can be used in semiconductor technology, magnetic storage, catalysis, fabrication of electronics, medical diagnosis and medical therapies. Nanoparticles are known to be produced by gas evaporation, by sputtering, chemical reduction of metal salts, nucleation and growth, microemulsions, pulsed laser ablation, by inverse micelle techniques, by pyrolysis of organometallic compounds and by microwave plasma decomposition of organometallic compounds. The interest in nanoparticles is due to their unique chemical, optical and electronic properties arising from their small volume to large surface area ratio and the separation in the electronic energy level. It is known that nanoparticles display an optical resonance called plasmon resonance. This is due to their collective coupling of the conduction electrons in the metal sphere to the incident electromagnetic field. Thus, in the medical diagnosis a gold nanoparticle-based peptide chip prepared by forming a monolayer of gold nanoparticles onto a self-assembled monolayer constructed on a solid support and then immobilizing a peptide on the gold-based nanoparticles is disclosed in U.S. Patent 7, 951 ,572. These gold coated peptide chips are used for detecting and measuring enzyme activity using secondary ion mass spectrometry enhanced by the gold layer.

U.S. Patent application 2007/0059775 discloses water-soluble iron oxide nanoparticles, which are encapsulated in phospholipid micelles. These nanoparticles are conjugated to antibodies that are used as a contrast agent to image specific cells or proteins in a subject using fluorescence or magnetic imaging techniques. Delcourt {Applied Surface Science 252 (2006) 6582-6587) discusses matrix- enhanced secondary ion mass spectrometry using gold and silver nanoparticles on the molecular sample surface. By definition therein "matrix is any chemical added to the sample to be analyzed to increase sputtered ion yields, without any restriction concerning the nature and proportions of additives, or the structure of the resulting sample."

U.S. Patent 8,679,858 B2 describes probes which has a specific binding member for an analyte conjugated to a single linkage site on a mass dot, which mass dot is a solid lanthanide metal particle having specific features. A method to analyze these mass dots by Inductively Coupled Plasma mass spectrometry (ICP-MS) by analyzing single particles in a liquid introduction system is also described.

None of the cited prior art provides a method which can produce a multiplier coefficient to increase the sensitivity of measuring a target molecule using at least two capture molecules in a ligand binding assay. This is especially needed when secondary ion mass spectrometry is used to quantify the assay.

It is yet another object to provide a ligand binding assay that uses particles and ionizable atoms that are attached to at least one second capture molecule to generate a multiplier coefficient to quantify target molecules in a test sample. This multiplier coefficient increases the sensitivity of the biological analysis performed using the ligand binding assay of the present invention.

It is an object of the present invention to provide a ligand binding assay in which a first capture molecule is provided on a biochip and is reacted with a target molecule. Nanoparticles and microparticles are then added to the captured target molecule on the biochip. These nanoparticles and microparticles comprise specific ionizable atoms attached to at least one second capture molecule, which are attached to nanoparticles and microparticles and are allowed to react with the molecules present on the biochip. The quantity of target molecules is then measured on a secondary mass spectrometer by measuring the secondary ions that are emitted by the specific ionizable atoms (SIMS). A coefficient multiplier is obtained, which is used to in the analysis to increase the sensitivity in the ligand binding assay.

It is another object to provide a ligand binding assay that uses specific ionizable atoms that are attached to the nanoparticles and microparticles at their surface, which have said at least one second capture molecule attached to the nanoparticles or microparticles. The quantity of target molecules is measured by SIMS and a coefficient multiplier is applied in the analysis.

It is yet another object to provide a ligand binding assay that uses specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface to generate a "double" multiplier coefficient to quantify target molecules in a test sample, which is used in a multiplex assay. The quantity of target molecules is measured by SIMS and a coefficient multiplier is applied in the analysis.

It is another object to provide a ligand binding assay that uses at least two different specific ionizable atoms, which are comprised in said nanoparticles or microparticles and at least one second capture molecule, which is attached to said nanoparticles or microparticles to quantify target molecules in a test sample that provides better sensitivity due to the specific ionizable atoms being incorporated into the nanoparticles or microparticles. The quantity of target molecules is also measure by SIMS.

In yet another object the present invention provides a ligand binding assay in which the nanoparticles or microparticles comprise at least one second capture molecule attached to said nanoparticles or microparticles through a molecule comprising at least one specific ionizable atom attached to said molecule. In this aspect there is better control of the ratio of the at least second capture molecule/specific ionizable atoms during the preparation of the molecule that is attached to the nanoparticles or microparticles.

Mixtures of the various nanoparticles and microparticles and the at least one second capture molecule, as described herein, which are used in the ligand binding assay is yet another object of the invention.

It is still yet another object of the present invention to provide nanoparticles or microparticles having specific ionizable atoms as part of the particle, which have at least one second capture molecule attached thereto that can be used in a sandwich type assay to quantify target molecules in a ligand binding assay. It is another object to provide a kit containing the nanoparticles or microparticles, at least one second capture molecule and specific ionizable atoms, as described herein, for use in a secondary ion mass spectrometer assay in a ligand binding assay. These and other objects are achieved by the present invention as evidenced by the summary of the invention, the description of the preferred embodiments and the claims.

SUMMARY OF THE INVENTION

The present invention provides a ligand binding assay for quantitating a target molecule in at least one test sample comprising:

(a) depositing on a biochip at least one first capture molecule;

(b) adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule;

(c) adding at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface; and

(d) scanning the biochip by secondary ion mass spectrometry to measure the secondary ions that are emitted by the specific ionizable atoms.

The above methods provide a multiplier coefficient, which is used to in the analysis increase the sensitivity of the analysis in the ligand binding assay. In the ligand binding assay, described herein, the biochip is a silicon wafer and the target molecule can be selected from the group of a protein, a peptide, a nucleic acid, a carbohydrates, a lipid, a polysaccharide, a glycoprotein, a hormone, an antigen, an antibody, a pathogen, a toxic substance, a drug, a dye, a nutrient, an enzyme, a rheumatoid factor, a tumor marker, a microorganism, an opiate, a viral epitope, cholesterol, a heavy metal, a vitamin, an allergy, a neurodegenerative disorder, an electrolyte, a glycan, a polyamine, a fatty acid, a sugar, a chemical trace metal and mixtures thereof.

In another aspect the specific ionizable atoms attached to the nanoparticles or microparticles or attached to the at least one second capture molecule or attached to the at least one molecule attached to the nanoparticle or microparticle or being part of the at least one nanoparticle or microparticle are metals. These metals can be selected from the group of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh), iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (ln),thallium (TI), tin (Sn), lead (Pb), lanthanides, actinides, mixtures, oxides and alloys thereof.

The lanthanides include lanthanum (La), Cerium (Ce), Neodymium (Nd), Promethium (Pm), Samarium, (Sm), Europium (Eu), Gadolinium (Gd),Terbium (Tb) , Dysprosium (Dy),Holmium (Ho), Erbium (Er),Thulium (Tm),Ytterbium(Yb) and Lutetium (Lu). In another aspect the specific ionizable atoms are halogens. These halogens can be fluorine (F), chlorine (CI), bromine (Br) iodine (I) or mixtures thereof.

The nanoparticles, as described herein, are from about 5 to 500 nanometers in diameter. Nanocrystals are encompassed by the term "nanoparticles" and can also be used in the ligand binding assay of the present invention. These nanocrystals are crystalline particles with at least one dimension measuring less than 1 ,000 nm. The microparticles, as described herein, are from about 0.5 to 500 micrometers in diameter.

The first capture molecule and second capture molecules, in the ligand binding assay method as described herein, can be antibodies, antibody fragments such as Fab fragments single and chain Fv fragments (scFv), diabodies, tetrabodies, fusion antibodies, antigens, nucleic acids, aptamers, affibodies, proteins, molecular imprinted polymers, enzymes, ligands, glycans, lectins, lipids, polyamines, phages, viruses, chemicals or combinations thereof.

In one aspect in the ligand binding assay, as described herein, the target molecule reacts with the at least one first capture molecule through hybridization and the at least one second capture molecule reacts with the target molecule through hybridization. In another aspect the at least one first capture molecule is an antibody, the target molecule is an antigen that binds to the antibody and the at least one second capture molecule is also an antibody that binds to the target antigen. In another aspect a kit is provided comprising at least one first capture molecule, a biochip, nanoparticles or microparticles, specific ionizable atoms, at least one second capture molecule; reagents for reacting the target molecule with the at least one first capture molecule and at least one second capture molecule and reagents for attaching the at least one first capture molecule to the biochip and the at least one second capture molecule to the nanoparticles or microparticles and the ionizable atoms.

In another aspect a kit is provided comprising at least one first capture molecule attached to a biochip, nanoparticles or microparticles comprising specific ionizable atoms attached to the at least one second capture molecule and reagents for reacting the target molecule with the at least one first capture molecule and at least one second capture molecule.

In yet another aspect a kit is provided comprising at least one first capture molecule attached to a biochip, nanoparticles or microparticles comprising specific ionizable atoms attached to their surface and the at least one second capture molecule, which is further attached to the nanoparticles or microparticles and reagents for reacting the target molecule with the at least one first capture molecule and at least one second capture molecule.

In yet another aspect a kit is provided comprising at least one first capture molecule, attached to a biochip, specific ionizable atoms being attached to nanoparticles or microparticles at their surface and ionizable atoms attached to at least one second capture molecule, which second capture molecule is attached to said nanoparticles or microparticles and reagents for reacting the target molecule with the at least one first capture molecule and at least one second capture molecule.

In another embodiment a kit is provided comprising at least one first capture molecule attached to a biochip, nanoparticles or microparticles having at least two different specific ionizable atoms comprised in the nanoparticles or microparticles and at least one second capture molecule, which is attached to said nanoparticles or microparticles and reagents for reacting the target molecule with the at least one first capture molecule and at least one second capture molecule. In yet another aspect a kit is provided comprising nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising at least one mass tag attached to said linker and reagents for reacting the target molecule with the at least one first capture molecule and at least one second capture molecule. Mixtures of the nanoparticles and microparticles, the at least one second capture molecule and linkers, as described herein, in a kit is yet another embodiment of the invention.

The kits, as described herein, comprise specific ionizable atoms attached to the nanoparticles or microparticles or attached to the at least one second capture molecule or attached to a molecule or being part of the at least one nanoparticle or microparticle which are metals selected from the group of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb),tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh), iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (ln),thallium (Tl), tin (Sn), lead (Pb), lanthanides, as described herein, actinides, mixtures, oxides and alloys thereof.

The lanthanides include lanthanum (La), Cerium (Ce), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd),Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium(Yb) and Lutetium (Lu).

In another aspect the specific ionizable atoms are halogens. These halogens can be fluorine (F), chlorine (CI), bromine (Br) iodine (I) or mixtures thereof.

Nanoparticles or microparticles comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle, said at least one nanoparticle or microparticle comprising specific ionizable atoms being attached to said at least one nanoparticle or microparticle at their surface or specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or specific ionizable atoms being a part of the at least one nanoparticle or microparticle and mixtures thereof for use in secondary ion mass spectroscopy to quantify target molecules in a ligand binding assay is another aspect of the invention. Nanoparticles or microparticles comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle, said at least one nanoparticle or microparticle comprising specific ionizable atoms being attached to said at least one nanoparticle or microparticle at their surface or specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or specific ionizable atoms being a part of the at least one nanoparticle or microparticle and mixtures thereof for use in measuring the secondary ions emitted after destructive ionization in a ligand binding assay. The secondary ions can be measured with a secondary ion mass spectrometer. Nanoparticles or microparticles comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle, said at least one nanoparticle or microparticle comprising specific ionizable atoms being attached to said at least one nanoparticle or microparticle at their surface or specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or specific ionizable atoms being a part of the at least one nanoparticle or microparticle and mixtures thereof, as described herein, comprise ionizable atoms, which are metals. These metals can be selected from the group of of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh),iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (In), thallium (TI), tin (Sn), lead (Pb), lanthanides, actinides, mixtures, oxides and alloys thereof.

The lanthanides include lanthanum (La), Cerium (Ce), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb) and Lutetium (Lu).

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a diagrammatic representation of one embodiment of the method using an embodiment of the nanoparticles and microparticles as described herein. The capture molecules (antibodies) are attached to the biochip. Two different capture molecules are used on separate areas of the biochip to provide a 2-plex ligand binding assay. The 2 corresponding target molecules are symbolized by small circles and triangles. The nanoparticle or microparticle is symbolized by the large circle to which is attached the secondary capture molecule. The nanoparticle or microparticle is made up of Iron. The Iron is ionized through a primary ion beam. The emitted secondary ions are then measured.

Fig. 2 is a diagrammatic representation of another embodiment of a nanoparticle or microparticle to which is attached through molecules such as chemical linkers at least one second capture molecule and one DOTA (chelating agent) complexing lanthanide ions (Thulium - Tm). The large circle symbolizes the nanoparticles or microparticles to which are attached other linkers and chelating agents complexing lanthanide ions. The secondary capture molecule is attached to the nanoparticle or microparticle through a molecule that is a chemical linker. The nanoparticle or microparticle might feature other linkers (represented as up and down lines) without secondary capture molecules, to improve the particle solubility.

Fig. 3 is a diagrammatic representation of yet another embodiment of a nanoparticle or microparticle comprising two different specific ionizable atoms and at least one second capture molecule attached to the nanoparticles or microparticles through a molecule which is a chemical linker. The large circle symbolizes the nanoparticles or microparticles to which are attached at least one second capture molecule and at least a part of the nanoparticle or microparticle is made up of Ag and Au. The at least secondary capture molecule is attached to the nanoparticle or microparticle through a molecule that is a chemical linker. The nanoparticle or microparticle might feature other linkers without secondary capture molecules, to improve the particle solubility.

Fig. 4 is a diagrammatic representation of a nanoparticle or microparticle comprising linkers which are attached to the nanoparticle or microparticle and second capture molecules is yet another embodiment encompassed by the invention. The second capture molecules have attached thereto ionizable Lanthanide (Ytterbium - Yb) atoms. The large circle symbolizes the nanoparticles or microparticles to which are attached several linkers. The nanoparticle or microparticle might feature linkers without secondary capture molecules and without Lanthanides, to improve the particle solubility.

Fig. 5 is a diagrammatic representation of yet another embodiment of a nanoparticle or microparticle comprising second capture molecules attached to the nanoparticle or microparticle through linkers. The molecule attached to the nanoparticle or microparticle are linkers that have Lanthanide (Erbium - Er) ionizable atoms attached thereto. The large circle symbolizes the nanoparticles or microparticles to which are attached several linkers. The secondary capture molecules are attached to the nanoparticle or microparticle through the linkers that contain the complexed lanthanides. The nanoparticle or microparticle might feature linkers without secondary capture molecules and without lanthanides, to improve the particle solubility.

Fig. 6 is a schematic representation of an array showing the disposition of the different spots on the array.

Fig. 7 is a schematic representation of the hybridization procedure used in Example 13.

Fig. 8 shows the disposition of the different samples on the silicon wafer in Example

13.

Fig. 9 shows the disposition of the different samples on the silicon wafer in Example

13. Fig. 10 is a standard curves for the two target molecules ; left side of the curve is model HRP biotin ; right side of the curve is antibody against IL6 labeled with NHS-LC- LC biotin or NHS-PEG12 biotin obtained from the results in Example 13.

Fig. 11 shows the disposition of different spots of the array in Example 14.

Fig. 12 shows the disposition of the different samples on the silicon wafer in Example 15.

Fig. 13 is a schematic representation of the hybridization procedure used in Example

15. Fig. 14 shows the disposition of the different samples on the silicon wafer in Example

15.

Fig. 15 shows a procedure for normalization after SIMS analysis to obtain quantification of the entire spots on the array in Example 15. Fig. 16 is a calibration curve of the different target molecules. The x-axis is the quantity of target molecules, while the y-axis is the normalized SIMS signal based on the data obtained in Example 15.

Fig. 17 is a Table showing the disposition of different spots on the array in Example

16. Fig. 18 is a schematic showing the distribution of different samples on the silicon wafer in Example 17.

Fig. 19 is a schematic representation of the hybridization procedure used in in Example 17.

Fig. 20 shows the difference in signals between two concentrations of target IL6 in Example 17.

Fig. 21 is a calibration curve of the different target molecules. The x-axis is the quantity of target molecules, while the y-axis is the normalized SIMS signal, including different CV concentrations based on the data obtained in Example 17.

Fig. 22 is a Table showing the different concentrations of CV based on Example 17. Fig. 23 is a Table showing the disposition of different spots on the array based on Example 18.

Fig. 24 is a schematic representation of the hybridization procedure used in Example

19.

Fig. 25 shows the distribution of different samples on the silicon wafer based on Example 19. Fig. 26 shows the distribution of different samples on the silicon wafer based on Example 19.

Fig. 27 is a calibration curve of the different target molecules. The x-axis is the quantity of target molecules, while the y-axis is the normalized SIMS signal, including different CV concentrations based on the data obtained in Example 19.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term "test sample," as described herein, means any sample that is fixed as a point of examination. The test sample contains the target molecules that are to be quantified. In this regard the test sample can be derived from a histological sample, a cytological sample, a biological sample or an environmental sample. Examples of histological samples include, but are not limited to, tissues, tumors, organs, bone or skin. The test sample can be a lysate of a tissue, a lysate of a tumor, a lysate of an organ or a lysate of skin. Examples of cytological samples include, but are not limited to, cells, cell lysates or cell culture. Examples of biological samples include, but are not limited to, blood, serum, plasma, cerebrospinal fluid, seminal fluid, amniotic fluid, bile, gastric juices, saliva, tears, sweat, sputum, semen, peritoneal fluid, pericardial fluid, urine, feces, hair, nail clippings and the like. Air, water, food, mining waste, pulp and paper waste or general manufacturing waste and the like are examples of environmental samples. The samples can be obtained from any animal and especially mammals or any environmental sample.

Examples of target molecules that can be quantified include proteins, peptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, antigens, antibodies, pathogens such as viruses, bacterium, or fungus, toxic substances, drugs, dyes, nutrients, enzymes, rheumatoid factor, tumor markers, microorganisms, drugs, opiates, viral epitopes, cholesterol, heavy metals, vitamins, allergies, neurodegenerative disorders, electrolytes, glycans, polyamines, fatty acids, sugars, allergens, chemicals trace metals, organic contaminants, mercury, dioxin, emergent chemicals, polychlorinates biphenyls and the like.

As used herein "biochips" refers to microarrays of molecules arranged on a substrate that permits testing to be performed at the same time in the ligand binding assay. The biochips can contain a defined set of capture molecules that are immobilized at high density in a geometric pattern on the surface.

"Measuring" means quantitating the amount of target molecule present in the sample in the ligand binding assay by measuring the secondary ions or equivalent ions that are emitted. This measurement can be performed using a secondary ion mass spectrometer or similar spectroscopic techniques. Examples of secondary mass spectrometers or similar spectroscopic techniques include nano-Secondary Ion Mass Spectrometer (nano-SIMS), Time of Flight Secondary Ion Mass Spectrometer (TOF- SIMS), Time of Flight Matrix-Assisted Laser Desorption/lonization (Maldi-Tof), electron spray ionization mass spectrometry (ESIMS), Ion Mobility Spectrometer (IMS), Secondary Ion Mass Spectrometer (SIMS), Matrix-Assisted laser Desorption/lonization (MALDI), Surface Enhanced Laser Desorption/lonization (SELDI), Surface-Assisted Laser Desorption/lonization (SALDI), Auger Electron Spectroscopy (AES), Energy- Dispersive X-ray Spectroscopy (EDS or EDX), X-ray Photoelectron Spectroscopy (XPS) and Dynamic Secondary Ion Mass Spectrometer (D-SIMS). In one aspect Dynamic Secondary Ion Mass Spectrometer (D-SIMS) is used.

The "multiplier coefficient" as described herein is a numerical quantity in front of a variable, which is the multiplier, that is an outcome of the methods described herein to increase the number of ionizable atom measurements and hence increase the sensitivity of the biological analysis of the at least one target sample. This multiplier coefficient increases the sensitivity of the biological analysis performed using the ligand binding assay of the present invention.

As used herein the term "capture molecule" refer to molecules such as ligands, receptors, aptamers, DNA segments, nucleic acids, affibodies, proteins, molecular imprinted polymers, enzymes, antigens, antibodies, glycans, lectins, lipids, polyamines, phages, viruses, chemicals, combinations thereof and the like which are used to specifically bind the target molecules in the test sample. The at least one first capture molecule and the at least one second capture molecule may be in the same category of molecules or may be in a different category. For example, the at least one first capture molecule may be an antibody and the at least one second capture molecule may also be an antibody. The at least one first capture molecule may be an antibody and the at least one second capture molecule may be an enzyme.

The term "specific ionizable atoms" means, as used herein, an atom acquiring a negative or positive charge by gaining or losing electrons, which atom is particularly fitted to a use or purpose.

By "attached" when referring to the ionizable atoms on the at least one second capture molecule is meant that the ionizable atoms are joined to the at least one second capture molecule. In one aspect the ionizable atoms are joined via a covalent bond or can be joined through a molecule such as a linker. In another aspect the ionizable atoms can be joined via non-covalent interactions such as the strong interation (Kd=10^~ ¹⁵M) known between some vitamins and proteins. The joining of the ionizable atoms to the at least one second capture molecule does not greatly affect the binding activity of this capture molecule.

By "attached" when referring to the nanoparticles or microparticles is meant that the ionizable atoms and/or the at least one second capture molecule are fixed to the nanoparticles or microparticles at their surface either directly by, for example, covalent bonds or through a molecule such as a linker or through a non-covalent interaction. This fixing does not affect the binding activity of the at least one second capture molecule.

The term "molecule" when referring to the specific ionizable atoms being attached to the molecule, which is attached to the at least one nanoparticle or microparticle as used herein means a molecule that includes the specific ionizable atoms as a part thereof. In this aspect the molecule can be a "linker," which can be used to connect the at least one first capture molecule to the biochip and/or the ionizable atoms and/or the at least one second capture molecule to the nanoparticles or microparticles.

"Nanoparticles," as used herein, refers to particles between 1 and 100 nanometers in size and are small objects that behave as a whole unit with respect to their transport and properties. Included in this definition of nanoparticles are nanocrystals, which are crystalline particles with at least one dimension measuring less than 1 ,000 nm. Thus, nanoparticles includes and encompasses nanocrystals.

As used herein, "microparticles" are particles between 0.1 and 100 microns in size and are small objects that behave as a whole unit with respect to their transport and properties.

"Metallic," when referring to nanoparticles or microparticles or ionizable atoms attached to the at least one second capture molecule means that these particles are fabricated using the metals selected from the group of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh),iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (In), thallium (TI), tin (Sn), lead (Pb), lanthanides, as described herein, actinides, mixtures, oxides, and alloys thereof and mixtures thereof.

As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of reaction assays, such delivery systems include the biochip and/or supporting materials (e.g., buffers, written instructions for performing the assay) from one location to another. For example, kits include one or more enclosures (e.g., boxes) containing the biochip and/or relevant reaction reagents. As used herein, the term "fragmented kit" refers to delivery systems comprising two or more separate containers that each contain a subportion of the total kit components. The containers may be delivered to the intended recipient together or separately. For example, a first container may contain the biochip for use in an assay, while a second container contains relevant reaction agents.

As used herein, the terminology "ligand binding assay" means an assay that measures at least one analyte by capturing the analyte on a solid surface using a capture molecule.

By "consisting essentially of means that the method, as described herein, can have additional steps or less steps in the procedure that does not affect quantitating the target molecule. When consisting essentially of refers to the kits, it means that the kits may have additional reagents or less reagents that do not affect the quantitation of the target molecule.

In one aspect, the present invention relates to a ligand binding assay for quantitating a target molecule in at least one assay sample comprising: depositing on a biochip at least one first capture molecule;adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; adding at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the

microparticles and nanoparticles at their surface; and scanning the biochip by secondary ion mass spectrometry to measure the secondary ions that are emitted by the specific ionizable atoms. More specifically the present invention provides a ligand binding assay for quantitating a target molecule in at least one test sample. This ligand binding assay comprises (a) depositing on a biochip at least one first capture molecule; (b) adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; (c) adding nanoparticles or microparticles comprising specific ionizable atoms attached to at least one second capture molecule that reacts with the target molecule; and (d) measuring by secondary ion mass spectrometry the secondary ions that are emitted.

In another aspect the present invention relates to a ligand binding assay for quantitating a target molecule in at least one test sample, this ligand binding assay method comprises (a) depositing on a biochip at least one first capture molecule;(b) adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; (c) adding nanoparticles or microparticles comprising specific ionizable atoms at their surface and at least one second capture molecule that is attached to the nanoparticles or microparticles said at least one second capture molecule reacts with the target molecule; and (d) measuring by secondary ion mass spectrometry the secondary ions that are emitted.

In yet another aspect the present invention relates to a method for quantitating a target molecule in at least one test sample in a ligand binding assay this method comprises (a) depositing on a biochip at least one first capture molecule;(b) adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; (c) adding nanoparticles or microparticles comprising specific ionizable atoms at their surface and at least one second capture molecule comprising specific ionizable atoms attached thereto that is further attached to the nanoparticles or microparticles said at least one second capture molecule reacts with the target molecule; and (d) measuring by secondary ion mass spectrometry the secondary ions that are emitted.

The present invention also relates to a method for quantitating a target molecule in at least one test sample in a ligand binding assay this method comprises (a) depositing on a biochip at least one first capture molecule;(b) adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; (c) adding at least two different specific ionizable atoms which are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles said at least one second capture molecule reacts with the target molecule; and (d) measuring by secondary ion mass spectrometry the secondary ions that are emitted.

Two different specific ionizable atoms can be formulated as the nanoparticles or microparticles themselves such as gold and silver can be used to synthesize the nanoparticles or microparticles or can be synthesized with specific ionizable atom such as gold and a linker such as DOTA complexed with a lanthanide.

Thus, 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacteic acid-gadolinium (DOTA- Ln),diethylenetriaminepentaacetic acid- gadolinium (DTPA-Ln), triethylenetetramine- gadolinium (TETA-Ln) and 1 ,4,7-triazacyclononane-1 ,4,7-trisacetic acid-gadolinium (NOTA-Ln) can also be used as the linker in all of the formulations as described herein. In yet another aspect the present invention provides a method for quantitating a target molecule in at least one test sample in a ligand binding assay this method comprises (a) depositing on a biochip at least one first capture molecule;(b) adding at least one target molecule and allowing the target molecule to react with said at least one first capture molecule; (c) adding nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker; and (d) measuring by secondary ion mass spectrometry the secondary ions that are emitted.

The methods, disclosed herein, can also use mixtures of the nanoparticles and microparticles, as described herein, in their various embodiments having the specific ionizable atoms, the at least one second capture molecule and the linkers.

The above methods provide a multiplier coefficient, which increases the sensitivity of the target quantitation as disclosed in the ligand binding assays of the present invention. The at least one test sample that contains the target molecules to be quantified can be derived from a histological sample, a cytological sample, a biological sample or an environmental sample. Examples of histological samples include, but are not limited to, tissues, tumors, organs, bone or skin. The test sample can be a lysate of a tissue, a lysate of a tumor, a lysate of an organ or a lysate of skin. Examples of cytological samples include, but are not limited to, cells, cell lysates or cell culture. Examples of biological samples include, but are not limited to, blood, serum, plasma, cerebrospinal fluid, seminal fluid, amniotic fluid, bile, gastric juices, saliva, tears, sweat, sputum, semen, peritoneal fluid, pericardial fluid, urine, feces, hair, nail clippings and the like. Air, water, food, mining waste, pulp and paper waste or general manufacturing waste and the like are examples of environmental samples. The samples can be obtained from any animal and especially mammals or any environmental sample.

The at least one first capture molecule can be antibodies, antibody fragments such as Fab fragments, single chain Fv fragments (scFv), diabodies, tetrabodies, fusion antibodies, nucleic acids, aptamers, affibodies, proteins, antigens, molecular imprinted polymers, enzymes, ligands, glycans, lectins, lipids, polyamines, phages, viruses, chemicals, DNA segments, receptors or combinations thereof.

Likewise the at least one second capture molecule can also be antibodies, antibody fragments such as Fab fragments single and chain Fv fragments (scFv), diabodies, tetrabodies, fusion antibodies, fragments of antibodies, nucleic acids, aptamers, affibodies, proteins, antigens, molecular imprinted polymers, enzymes, ligands, glycans, lectins, lipids, polyamines, phages, viruses, chemicals, DNA segments, receptors or combinations thereof.

Both the at least one first capture molecule and the at least one second capture molecule bind to the target molecule in some manner. This binding has to be to an extent that the target molecule is not washed away after hybridization so that a correct measurable result can be obtained.

The biochip that can be utilized can be any collection of microarrays arranged on a substrate that permits testing to be performed at the same time. The biochip can be a solid planar surface or alternatively it can be a solid porous surface. It can also be in the form of a bead. The substrate can be fabricated from a material of plastic, silicon, fused quartz, soda lime glass, ceramics, pyrex, metals such as aluminum, titanium, stainless steel, glassy carbon and polymeric materials such as polystyrene and polycarbonate. Thus, the biochip can be in the form of glass slides, polyacrylamide gel pads, nitrocellulose membranes, beads, silicon slides or microtiter plates to which a capture molecule can be bound. In one aspect of the invention the biochip is a silicon wafer.

The biochip can have a rigid or semi-rigid surface. At least one surface of the substrate can be essentially flat in one embodiment. The substrate or solid substrate can in the shape of a rectangle, a square, a circle, a triangle, an oval, a pentagon, an octagon or a hexagon. The size and shape of the substrate may vary depending on the size of the sample chamber in the surface mass spectrometer or similar spectroscopic techniques used and its application. For instance, they can range from 1 cm x 2 cm, 2.5 cm x 2.5 cm, 2 cm x 3 cm, 6 cm x 12 cm or 6 mm x 6 mm for slides. In another embodiment, to physically separate the capture molecules the biochip can have wells, raised regions, etched trenches and the like.

Prior to use the biochip can be washed using, for example, organic solvents, methylene chloride, dimethylformamide (DMF), ethyl alcohol or the like. The solvents will depend on the material of the substrate chosen and are well known in the art. The surface of the biochip can be functionalized to permit the conjugation thereto of the at least one first capture molecules. The at least one first capture molecules are spotted on the biochip using, for example, a microarrayer. The attachment of the at least one spot(s) containing the at least one first capture molecules can be by any means known in the art such as by nonspecific and noncovalent immobilization, nonspecific and covalent immobilization, specific and noncovalent immobilization or specific and covalent immobilization.

Nonspecific and noncovalent immobilization relies on physical adsorption of proteins or molecules to substrates. As a general rule, essentially all proteins will adsorb to essentially all surfaces. Physical adsorption can be to biochips made of nitrocellulose, polystyrenes or silanized glass. The at least one first capture molecules can be adsorbed to the surface of the biochip by applying a drop of buffer containing these molecules to the surface of the substrate. In this embodiment the capture molecules are attached to the substrate through non covalent bonds. Nonspecific and covalent immobilization requires substrates that have functional groups that can be used to covalently link the molecule to the biochip to avoid the exchange of immobilized proteins, and can be applied to low-molecular-weight molecules including peptides. Thus, the functionalized moiety to which the at least one first capture molecules are attached to the biochip include, but are not limited to, amino groups, aldehyde groups, carboxyl groups, sulfhydryl groups, phenyl groups, benzyl groups, hydroxyl groups, carbonyl groups, imide groups, thiol groups, phosphate groups, epoxy silane groups and the like. In one aspect expoxy silane groups are used to functionalize the biochip.

Examples of functionalized moieties containing functional groups that can be used to immobilize the capture molecules include glass slides modified with N- hydroxysuccinimide (NHS) esters or with aldehydes, cyano, amino/bifunctional N- hydroxysuccinmde, mercapto or expoxy functional groups that are covalently bonded to amino groups, hydroxyl groups or thiol groups. In one embodiment the substrate is functionalized with epoxysilane. Specific and noncovalent immobilization includes the use of substrates that present molecular groups that can selectively interact with a tag on the protein. Attachment of biotin-tagged ligands to substrates that are modified with a layer of streptavidin is the most common example of a specific but noncovalent strategy for immobilizing molecules as described by Yam et al J. Colloid. Interf Sci 296:1 18- 130(2006).

An example of specific and covalent immobilization is the immobilization domain of a fusion protein was made to interact with an irreversible inhibitor, leading to a selective and covalent attachment of the protein to the substrate such as the binding of a serine esterase cutinase to a self-assembled monolayer presenting a phophonate ligand as described by Hodneland et al, PNAS vol. 99 No. 8 5048-5052 (2002).

Processes for attachment of the at least one first capture molecules to the biochip that can be used include expressed protein ligation as described in Camarero et al, J. Am Chem Soc 2004:126(45)14730-1 , click chemistry as described by Sun et al Bioconjugate Chem (2006) 17 52-57 or trans-splicing as described by Kwon et al Angew Chem Int Ed 2006:45(1 1 )1726-9.

The biochip can also be layered with a polymer or different polymers such as polyallylamine, polyethylene terephthalate, polyethylene glycol, polyacrylamide, nitrocellulose or agarose. If the substrate is a polymer or layered with a polymer it can also be coated with a layer of carbon or diamond-like carbon by spattering, chemical vapor deposition (CVD) or physical vapor deposition (PVD) to ensure covalent bonds with the polymer.

Besides functionalizing the biochip the at least one first capture molecules can be functionalized with the functional groups, as described herein, for their attachment to the biochip. Linkers can also be used to attach the functionalizing group on the substrate or capture molecules or signal control molecules. The linkers can be short peptide sequences or bifunctional chemical linkers.

Peptide linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another.

Bifunctional chemical linkers, include, for example, 3-maleimidobenzoic acid N- hydroxyysuccinimide ester (MBS), N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP), [N-Succinimidyl)-S-acetylthioacetate] (SATA), [(N-succinimidiyloxy carbonyl)1 -methyl-1 - (2-pyridyldithio) toluene] (SMPT), 2-iminothiolane (2-IT), (Sulfosuccinimidyl N-[3- (Acetylthio)-3-methylbutyryl)-beta-alanine]) (sulfoNHS-ATMBA), thioimidates such as AMPT and M-CDPT and 2-[N-chlorocarbonyl)-N-methylamino]1 -ethyl 2-pyridyldisulfide can be used. The at least one first capture molecules, whether functionalized or not, are then spotted on the biochip using, for example, a microarrayer. The spots can be any geometric shape such as square, rectangular, triangular hexagonal, oval, octagonal or hexagonal. The size of the spot may vary depending on the assay being performed and the substrate or solid substrate used. The size of the spot can range from 1 μηη to 4.0 mm. In another embodiment the spots can range from 5.0 μηη to 500 μηη or 10 μηη to 200 μηι or 50 μηι to 100 μπι.

A typical biochip, as described herein, can have between 1 to 1 ,000,000 spots. In another embodiment the number of spots can range from 4 to 5,000 spots. In yet another embodiment the number of spots can range from 4 to 500. Thus the term "at least one" encompasses one spot, but can also encompass 400 spots or 1 ,000 spots or 8,000 spots. The term at least three spots can encompass three spots or 450 spots or 80,000 spots. In one aspect there can be 10 to 900,000 spots.

In the methods, as described herein, the target molecule can be selected from the group of a protein, a peptide, a nucleic acid, a carbohydrates, a lipid, a polysaccharide, a glycoprotein, a hormone, an antigen, a pathogen, a toxic substance, a drug, a dye, a nutrient, an enzyme, a rheumatoid factor, a tumor marker, a microorganism, an opiate, a viral epitope, cholesterol, a heavy metal, a vitamin, an allergy, a neurodegenerative disorder, an electrolyte, a glycan, a polyamine, a fatty acid, a sugar, a chemical trace metal and mixtures thereof.

In one aspect the at least one first capture molecule is an antibody and the target molecule is an antigen and the at least one second capture molecule that is attached to the nanoparticle or microparticle is an antibody. In this aspect a type of sandwich assay is carried out. The specific ionizable atoms used in the ligand binding assay described herein can be metals selected from the group of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh),iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (ln),thallium (Tl), tin (Sn), lead (Pb), lanthanides, as described herein, actinides, mixtures, oxides and alloys thereof. In another aspect the ionizable ion used in the method is iron(Fe).

In another aspect specific ionizable atoms used in the ligand binding assay, as described herein, can be halogens such as fluorine (F), chlorine (CI), bromine (Br), iodine (I) or mixtures thereof. In one aspect a sole type of specific ionizable atoms such as iron (Fe) is used to fabricate the nanoparticles or microparticles and to attach the specific ionizable atoms to the at least one second capture molecule, which is attached to the nanoparticles or microparticles or both the nanoparticles or microparticles have the specific ionizable atoms attached at their surface and the at least second capture molecule is also attached to the nanoparticles or microparticles. In this aspect the same specific ionizable atom is used to analyze different target molecules on the same biochip at the same time.

In another aspect more than one different specific ionizable atom is used in the method described herein. For example, the nanoparticles or microparticles can have gold (Au) attached thereto at their surface and the at least one second capture molecule can have silver attached thereto, the at least one second capture molecule being attached to the gold nanoparticles or microparticles. In a different embodiment, the nanoparticles or microparticles can be made of iron (Fe) and the at least one second capture molecule can have silver attached thereto. In yet another embodiment both the nanoparticles or the microparticles can be made of gold and have attached at their surface silver and the at least one second capture molecule, which is attached to the nanoparticles or microparticles can have iron attached thereto. In this aspect different ionizable atoms can be used to analyze different targets on the same biochip at the same time. The specific ionizable atoms are used to fabricate the nanoparticles or microparticles using methods known in the art such as by gas evaporation, by sputtering, chemical reduction of metal salts, nucleation and growth, gaz phase growth by laser pyrolysis, microemulsions, pulsed laser ablation, by inverse micelle techniques, by pyrolysis of organometallic compounds, by microwave plasma decomposition of organometallic compounds or as described herein.

In one aspect the nanoparticles or microparticles can be fabricated using any natural or synthetic polymers such as cellulose, chitosan, polyethylene glycol (PEG), polylactic acid (PLA), polystyrene, neoprene, polyvinylchloride(PVC), polyvinylpyrrolidone (PVP), polypropylene and the like. The nanoparticles or microparticles can be made via suspension and dispersion-precipitation polymerization or emulsion polymerization methods. These methods are known in the art.

In another aspect, once made the nanoparticles or microparticles can have additional ionizable atoms attached to their surface. This attachment can be by any method known in the art as long as the ionizable atoms do not become prematurely detached in the method as described herein. These methods include adding functional groups to the nanoparticles or microparticles such as thiol, carboxylic acid, amine, methoxy or PEG and grafting the ionizable atoms thereto. Diazonium functionalized nanoparticles or microparticles can also be used to which are covalently bound on their surfaces the ionizable atoms using the procedure of Joselevich et al Langmuir (2008) 24(20) pgs. 1 171 1 -1 1717.

The nanoparticles can range from about 5 to about 500 nanometers in diameter and the microparticles can range from about 0.5 to about 500 micrometers in diameter. The nanocrystals are crystalline particles with at least one dimension measuring less than 1 ,000 nm and are encompassed by the term "nanoparticles. "The size of the nanoparticle or microparticles depends upon the type of assay being performed. It also can vary depending upon what is attached to the nanoparticles or microparticles and the sensitivity wanting to be achieved. For example, when more ionizable atoms have to be attached to increase sensitivity either alone or being attached to the at least one second capture molecule a larger particle may be required. This size parameter can be manipulated according to the assay undertaken and what is well known within the art.

The choice of the types and sizes of nanoparticles or microparticles that are chosen for use in the methods of the present invention are based on many factors such as the density of these nanoparticles or microparticles in the aqueous medium, the size of these nanoparticles or microparticles, the ionizable atoms that are used to fabricate them, the composition of the target sample and their ability for these nanoparticles or microparticles to be efficiently washed on the biochip and remain thereon without being washed off. The nanoparticles or microparticles can be fabricated from ionizable atoms from a single source such as Fe or can have mixtures of ionizable atoms such as a combination of silver (Ag) and gold (Au). The nanoparticles or microparticles, as described herein, can be fabricated with between dozens and millions of ionizable atoms, depending upon the application and sensitivity needed in the assay. For instance, at least between 50 and 5,000,000 ionizable atoms or between at least 300 and 300,000 ionizable atoms or between at least 1 ,000 and 1 ,000,000 ionizable atoms can be used. These ionizable atoms used in the ligand binding assays, as described herein, provide a multiplier coefficient, which increases the sensitivity of the biological analysis disclosed in the ligand binding assays of the present invention. In another aspect the target molecule reacts with the at least one first capture molecule through hybridization and the at least one second capture molecule reacts with the target molecule through hybridization. Thus, the at least one first capture molecule is attached to a biochip, the target molecule is added and the biochip is then washed to rid the biochip of uncaptured target molecules. The nanoparticles or microparticles containing the specific ionizable atoms attached to the at least one second capture molecule, which is further attached to the nanoparticles or microparticles or said specific ionizable atoms being attached to said nanoparticles or microparticles at their surface, which have said at least one second capture molecule attached to the nanoparticles or microparticles or said specific ionizable atoms being attached to said nanoparticles or said microparticles at their surface and said specific ionizable atoms also being attached to said at least one second capture molecule, which is further attached to said nanoparticles or microparticles or at least two different specific ionizable atoms are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles or nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and mixtures thereof is then added to the biochip and allowed to react for a certain period of time with the target molecule. This reaction time will depend upon the type of target molecule being quantitated, the amount of target molecule introduced onto the biochip and other reaction conditions such as temperature. The reaction time can thus vary between 5 minutes and 4 hours, 10 minutes to 2 hours or 20 minutes to 90 minutes or 30 minutes to 1 hour.

After the reaction period, the surface of the specimen is sputtered with a focused primary ion beam. Primary beam species useful in the present invention include Cs+, 02+, O, Ar+, and Ga+ at energies between 1 and 30 keV. Primary ions are sputtered with sample atoms to various depths, which depends upon the analysis. For instance, the depths may vary between a few nanometers to 1 micrometer or more. This depends on whether surface analysis is being performed or an analysis that requires deeper depths. Thus depths may vary between 1 to 10 nm or 10 nm to 1 mm or between 100 nm to 5 mm. The depth in which the primary ions are sputtered depends also upon where the specific ionizable atoms are located on the nanoparticles or microparticles, the size of the particle, the type of specific ionizable atoms and the like that are used in the multiplex assay method.

The time for sputtering may vary depending on the size of the nanoparticle or microparticle, the area to which the specific ionizable ions are attached; i.e., on the surface or inside the particle, the size of the raster dimension, the instrument utilized, as well as the sensitivity to be achieved. Sputtering rate is generally determined by the mass, energy, and angle of incidence of the bombarding ions, temperature and the current density of the sputtering ion beam, which is the beam current that impinges the crater area divided by the rastered area. Thus the sputtering time can be altered. If higher sensitivity is to be achieved, when the sensitivity objective is far above the signal that is permitted due to the multiplier coefficient, sputtering time can be reduced, thus permitting the acquisition of more data within a given time frame.

The SIMS ionization efficiency is called ion yield, defined as the fraction of sputtered atoms that become ionized. Ion yields vary over many orders of magnitude for the various elements and the chemistry of the sputtered surface. For instance some atoms like iodine are easily ionizable and result in about 20% ionization yield, while for others like Thulium the ionization yield is about 1 %. In the case of Thulium it is estimated that 100 atoms are needed to generate a single count on the average. The secondary ions emitted from the nanoparticles or microparticles are collected and analyzed by counting. The secondary ions differ depending on the type of specific ionizable atoms used to fabricate the nanoparticles or microparticles or the specific ionizable atoms that are attached to the surface or the nanoparticles or microparticles or the specific ionizable atoms attached to the at least one secondary capture probe. The mass/charge ratios of these secondary ions are then measured and counted with a mass spectrometer.

The specific ionizable atoms attached to the at least one second capture molecule, which is attached to the nanoparticle or microparticle or the specific ionizable atoms that are attached to the nanoparticles or microparticles at their surface and at least one second capture molecule attached to the nanoparticles or microparticles or the specific ionizable atoms are attached to both of the nanoparticles or microparticles at their surface and the at least one second capture molecule having specific ionizable atoms attached thereto and that is also attached to the nanoparticles or microparticles or at least two different specific ionizable atoms are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles or nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and mixtures thereof can be joined using techniques known in the art such as by bioconjugation. Bioconjugation involves the covalent binding of specific ionizable atoms to the at least one second capture molecule or the specific ionizable atoms that are attached to the nanoparticles or microparticles at their surface or the specific ionizable atoms are attached to both of the nanoparticles or microparticles at their surface and the at least one second capture molecule that is also attached to the nanoparticles or microparticles or nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and mixtures thereof via functional groups. Functional groups on the at least one second capture molecule may include primary amine reactive groups, carboxyl reactive groups, sulfhydryl reactive groups, carbonyl reactive groups and the like.

In another aspect the specific ionizable atoms or linkers can be attached to the at least one second capture molecule or the nanoparticles or microparticles via lysine residues via amine-reactive succinimidyl esters such as an NHS-ester, isocyanates or isothiocyanates. The ionizable atoms or linkers can also be attached to the at least one second capture protein or the nanoparticles or microparticles through a cysteine residue via a thiolate nucleophile which will react with soft electrophiles such as maleimides and iodoacetamides. Tyrosine residues on the at least second capture molecule can be modified through electrophilic substitution reactions and it is selective for the aromatic carbon adjacent to the phenolic hydroxyl group as described in Stephanopoulos, et al (201 1 ) Nature Chemical Biology 7 (12):876-884.

N-terminal and C- terminal reactions to biocongate the specific ionizable atoms to the at least one second capture protein or the linkers is yet another aspect of the invention for attachment. For example, the oxidation of the N-terminal serine and threonine residues can generate N-terminal aldehydes, which can undergo further bioorthogonal reactions. Condensation of the N-terminal cysteine with aldehyde generates thiazolidone that can be further reacted. Pyridoxal phosphate can be used to yield a N-terminal aldehyde on N-terminal glycine and aspartic acid. An example of C- terminal modification is the use of native chemical ligation, which is the coupling between a C-terminal thioester and a N-terminal cysteine. The specific ionizable atoms can be connected to at least one second capture protein or the specific ionizable atoms that are attached to the nanoparticles or microparticles at their surface or the specific ionizable atoms are attached to both of the nanoparticles or microparticles at their surface and the at least one second capture molecule that is also attached to the nanoparticles or microparticles or at least two different specific ionizable atoms are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles or nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and mixtures thereof through a linker such as a substituted aliphatic carbon chain or a polymer chain. Examples of polymers chain include polyethylene glycol, polylactic acid, chitosan and the like, as well as the other linkers described herein.

In one aspect the linker can be a S-R1 -R2-R3 moiety in which R1 is (CH₂-CH₂- 0)„; R2 is (CH₂-CH₂)m and R3 is an amide having the structure of C=0-NH or NH-C=0 and n is an integer from 2 to 3 and m is an integer from 1 to 2.

A kit is also provided comprising nanoparticles or microparticles comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface and mixtures thereof; and reagents for the ligand binding assay. In another aspect a kit comprising nanoparticles or microparticles comprising the specific ionizable atoms attached to the at least second capture antibody or the ionizable atoms that are attached to the nanoparticles or microparticles at their surface and the at least one second capture molecule that is also attached to the nanoparticles or microparticles or the specific ionizable atoms are attached to both of the nanoparticles or microparticles at their surface and the at least one second capture molecule that is also attached to the nanoparticles or microparticles or at least two different specific ionizable atoms are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles or nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and mixtures thereof, as described herein, and reagents for reacting with the target molecule by secondary ion spectrometry is provided. Such reagents may include hybridization buffers, diluents, wash solutions, NP-40, 20 x SSC, IgG and the like. The content of the kit will depend on the assay that is being performed.

The kit can further comprise at least one first capture molecule and reagents to attach the at least one first capture molecule to the biochip. Alternatively the kit contains biochips that have the at least one first capture molecule already attached to the biochip. This attachment is described herein.

The kit may additionally contain at least one second capture molecule that is affixed to the nanoparticle or microparticle having or not having specific ionizable atoms attached to this capture molecule. In the case where there is no specific ionizable atoms attached to the at least one second capture molecule, the specific ionizable atoms are attached to the nanoparticle or microparticle at their surface. In another embodiment the kit may contain specific ionizable atoms, which are attached to the at least one second capture molecule which are attached to the nanoparticle or microparticle and the nanoparticles and microparticles also have specific ionizable atoms attached thereto at their surface. In another aspect the kit may contain at least two different specific ionizable atoms that are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles and reagents for reacting with the target molecule.

Nanoparticles or microparticles are also provided in a kit comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and reagents for reacting with the target molecule.

The kit, as described herein, that contains nanoparticles or microparticles comprising the ionizable atoms as described herein, or the ionizable atoms attached to the at least second capture antibody, as described herein, are metals selected from the group of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb),tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh), iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (ln),thallium (TI), tin (Sn), lead (Pb), lanthanides, as described herein, actinides, mixtures, oxides, and alloys thereof. In another aspect the ionizable ion used in the method is iron(Fe).

The nanoparticles or microparticles can be fabricated with ionizable atoms that are made of halogens such as fluorine (F), chlorine (CI), bromine (Br), Iodine (I) or mixtures thereof.

Nanoparticles or microparticles comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanopartides or micropartides or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the micropartides and nanopartides at their surface and mixtures thereof for use in secondary ion mass spectroscopy to quantify target molecules in a ligand binding assay is also an embodiment of the invention.

In another aspect of the invention, nanopartides or micropartides comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanopartides or micropartides or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the micropartides and nanopartides at their surface mixtures thereof for measuring the secondary ions emitted after destructive ionization in a ligand binding assay.

Nanopartides or micropartides comprising specific ionizable atoms attached to at least one second capture molecule, which is attached to said nanopartides or micropartides or at least one second capture molecule which is attached to a nanoparticle or microparticle, said nanoparticle or microparticle having specific ionizable atoms attached thereto at their surface or nanopartides or micropartides having specific ionizable atoms attached thereto at their surface and also attached to these particles is at least one second capture molecule to which is also attached specific ionizable atoms or at least two different specific ionizable atoms are comprised in said nanoparticles or microparticles and at least one second capture molecule is attached to said nanoparticles or microparticles or nanoparticles or microparticles comprising at least one second capture molecule attached to said nanoparticles or microparticles through a linker comprising specific ionizable atoms attached to said linker and mixtures thereof for use in secondary ion mass spectroscopy to quantify target molecules in a ligand binding assay is provided. These specific ionizable atoms provide a multiplier coefficient, which increases the sensitivity of the biological analysis disclosed in the ligand binding assays of the present invention. In another aspect, nanoparticles or microparticles, as described herein, and at least one second capture molecule, as described herein, are used in a method for measuring the secondary ions emitted after destructive ionization in a ligand binding assay. The secondary ions are measured with a secondary ion mass spectrometer.

In the above two applications; i.e., for quantifying target molecules or for measuring the secondary ions emitted after destructive ionization, the nanoparticles or microparticles are ionizable atoms or ionizable atoms attached to the at least second capture molecule are metals selected from the group of lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) , francium (Fr), berrylium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), radium (Ra), yttrium (Y), lanthanum (La), actinium (Ac), titanium (Ti), zirconium (Zr), hafnium (Hf), vanadium (V), niobium (Nb), tantalum (Ta), chromium (Cr), molybdenum(Mo), tungsten (W), manganese (Mn), technetium (Tc), rhenium (Re), iron (Fe), ruthenium (Ru), osmium (Os), cobalt (Co), rhodium (Rh),iridium (Ir), nickel (Ni), palladium (Pd), platinum (Pt), copper (Cu), silver (Ag), gold (Au), zinc (Zn), cadmium (Cd), aluminum (Al), indium (In), thallium (TI), tin (Sn), lead (Pb), lanthanides, as described herein, actinides, mixtures, oxides, and alloys thereof and mixtures thereof. In another aspect the specific ionizable ion used in the method is iron(Fe).

The nanoparticles or microparticles can be fabricated with ionizable atoms that are made of halogens such as fluorine (F), chlorine (CI), bromine (Br), Iodine (I) or mixtures thereof. In order to further illustrate the present invention and advantages thereof the following specific examples are given it being understood that the same are intended only as illustrative and in nowise limitative.

EXAMPLES

Example 1 -Synthesis of metal nanoparticles

The synthesis of nanoparticles was performed by following the procedure of Panigrahi et al, Journal of Nanoparticle Research 6 41 1 -414 (2004). More specifically, the reagents used are chloroautic acid (HAuCU), silver nitrate (AgN0₃), chloroplatininc acid (H₂PtCI₆) and paladous chloride (PdCI₂). Reagent grade fructose, glucose and sucrose are used and purchased from a chemical company. Ultrapure water is used in the synthesis.

A series of solution are prepared by dissolving 0.2 g of sugar in 3.9 ml of ultrapure water and then 100 μΙ (10^~2 M) of the corresponding metal salt solutions is added so that the total volume is 4 ml. Fructose, glucose and sucrose are used individually as reducing agents for the synthesis of each metal nanoparticle. The concentration of metal salts is 2.5 x 10^"4M in the final solution. HAuCI , AgN0₃, H₂PtCI₆ and PdCI₂ are used the preparation of Au³⁺, Ag⁺, Pt²⁺ and Pd²⁺ ionized nanoparticles. The solution is then heated in a hot water bath at around 70°C to 75 °C and the heating is continued until the solution evaporated to dryness, which takes around 2 hours. 4 ml of water is then added and the solution is then sonicated for 30 minutes.

Example 2-Functionalizing the metallic nanoparticle by covalent bonding to the antibody

An aqueous solution of IL-1 at 5 mg/ml is allowed to react with 0.77 mg of N- succinimidyl S-acetylthioacetate at room temperature for 1 hour. The resulting conjugate of IL-1 -acetylthioacetate is purified on a gel filtration desalting column using a protein dye kit as an indicator of the antibody-containing fractions. The purified IL-1 - acetylthioacetate is then reacted with 0.216 mg of p-isothiocyanatobenzyl- diethylenetriaminepentaacetic acid at 4°C overnight and the pH is adjusted to 8 using NaHC03. The product is purified by passing over a desalting column to remove excess p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid to yield diethylenetriaminepentaacetic acid-IL-1 -acetylthioacetate (DTPA-IL-1 -ATA).

The same procedure is used to functionalize IL-6, IL-10, IL-17 and TNF antibodies. Example 3- Conjugation of the Antibodies to the gold nanoparticle

An aqueous solution of DTPA-IL-1 -ATA at 20 μg/ml is treated with hydroxylamine (50 μΙ) at room temperature for 2 hours to expose the free SH groups. After passing through a desalting column the resulting DTPA-IL-1 -SH is added to an aqueous solution of the gold nanoparticles as prepared in Example 1 at a concentration of 7.3 x 10¹⁰ particles/ml) to a final antibody concentration of 5 μg/ml. The suspension is stirred for 1 hour at room temperature. Polyethylene glycol-SH is added to the antibody-coated Au nanoparticles to a final concentration of 0.2 mg/ml and the mixture is reacted for an additional hour. DTPA-IL-1 is centrifuged for 5 minutes at 8,000 x rpm and the resulting is washed twice with deionized water. The presence of free IL-1 is examined using a protein dye. No free IL-1 is detected after the second wash. The Au-NP-IL-1 are resuspended in 0.1 mmol/L PBS and stored at 4°C for further use.

The other metallic nanoparticles are prepared in the same manner.

Example 4- TEM analysis A transmission electron microscopic analysis of the Au-NP is undertaken using a

JEOL TEM at an accelerating voltage of 80kV. The average nanodiameter and thickness of the shell is determined by measuring 50 individual AuNP particles. The particle size is also determined using dynamic light scattering at a scatter angle of 90°C. UV-visible spectroscopy of the particles is recorded on the spectrometer and the concentration of gold atoms is analyzed via inductively coupled plasma mass spectroscopy. Example 5-Preparation of ionizable atoms with an antibody IL1

The procedure in U.S. Patent 5,460,785 is followed. More specifically, IL-1 is subjected to a reaction with 2mM stannous ions during a 21 hour incubation. The antibody is used at a concentration of 5 mg/ml and in a buffer composed of 10 mM tartrate and 40 mM phthalate, pH 5.5. The reduction step is followed by buffer exchange through Sephadex G-25. The antibody solution is then subjected to adjustment to 1.25mM stannous tartrate by adding 10 mM tartrate/ 40 mM phthalate buffer, pH 5.5

IL-1 is prepared in the same manner except no stannous ions are added in the antibody reaction step. The IL-1 is then added to Cu²⁺, mixed and incubated at room temperature for up to 60 minutes. The mixture can then be analyzed by Sephadex G-25 to reveal the amount of

Cu²⁺ incorporated into IL-1 .

The same procedure is used for IL-6, IL-10, IL-17 and TNF antibodies.

Example 6- Test of the Quantification of the Target Molecules in a Biological

Sample using Au⁺³ nanoparticles

A. Materials: The materials used in this example are the following:

(1 ) One biochip fabricated as follows: (a) Silicon wafers (18 mm x 18 mm) (Siltronix) functionalized with epoxysilane coating (Sigma). There are 16 different zones (4x4) on the wafer with a size of 4 mm x4 mm. These zones are called wells. (b) Capture molecules: antibodies for the following target molecules: a. Anti IL1 alpha (R&D Systems MAB200)

b. Anti-IL6 (R&D Systems MAB206)

c. Anti-IL-10 (R&D Systems MAB2172)

d. Anti-IL 17 (R&D Systems MAB1248)

e. Anti-TNF (R&D Systems MAB610)

(c) Protein Printing Buffer 2X, Nexterion^®

(d) BSA fraction V (Sigma Aldrich)

(e) PBS buffer

The biochips are formulated in the following manner:

In 384 well plate, the different antibodies were diluted in protein printing buffer 2X as follows: o Well A1 : 10μΙ_ of antibody anti IL1 at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

o Well A2 : 10μΙ_ of antibody anti IL6 at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

o Well A3 : 10μΙ_ of antibody anti IL10 at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

o Well A4 : 10μΙ_ of antibody anti IL17 at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

o Well A5 : 10μΙ_ of antibody anti TNF at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X The well plate and the silicon wafers are placed in a SciFLEXArrayer (Scienion).

The arrayer, after being programmed appropriately, fabricates the arrays of capture molecules in each well on the biochip.

The biochip with printed arrays is stored at 4°C overnight and then stored under vacuum at room temperature.

3.) The target molecules used for calibration curve are the following recombinant molecules:

a. IL1 alpha (R&D Systems 200-LA-002)

b. IL6 (R&D Systems 206-IL-010)

c. IL-10 (R&D Systems 217-IL-005)

d. IL 17 (R&D Systems 317-ILB-005)

e. TNF alpha (R&D Systems 210-TA-005) The hybridization of targets is carried out as follows. The wells are first blocked with a solution containing 1 % BSA in a 1X PBS buffer. The different wells of the silicon wafer are incubated for 2H with different samples containing various quantities of target molecules. These molecules will interact with the capture molecules.

Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 0 pg/mL (10)

Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 0.1 pg/ml_ (12)

Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 1 pg/mL (14)

Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 5 pg/ml_ (16)

Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 25 pg/mL (18) f. Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 50 pg/mL (20)

g. Recombinant Target molecules (IL1 , IL6, IL10 IL17 & TNF) at 100 pg/mL (22)

h. to p. the biological samples.

3) Second capture molecules:

Gold nanoparticles set forth in Example 3 are conjugated to the following antibodies: a Anti -IL1 alpha (R & D Systems AF-200-NA)

b Anti -IL6 (R & D Systems AF-206-NA)

c Anti -IL10 (R & D Systems AF-217-NA)

d Anti -IL17 (R & D Systems AF-317-NA)

e Anti -TNF alpha (R & D Systems AF-210-NA)

All of the wells are washed with 1X PBS and 0.1 % Tween. The wells are hybridized for 2H with a mix of the following Au-NP-IL or Au-Np-TNF conjugated antibodies :

- Anti IL1 alpha 2μΙ_ - Αηίί-ΙΙ_6 2μΙ_

- Anti-IL-10 2μί - Anti-IL 17 2μΙ_

- Anti-TNF alpha 2μΙ_

The silicon wafer (all of the wells) is washed with 1X PBS and 0.1 % Tween, followed by washing with PBS 1X and then washed with HPLC grade distilled H₂0.

The silicon wafer is dried under air flow and stored under vacuum at 4°C. SIMS analysis is carried out as follows. The biochip is installed in the SIMS analyser, put under high vacuum and all the wells position are programmed to be analysed.

For each well, all the surface is analysed by D-SIMS. The detector is tuned on the mass 197 (gold).This results in a matrix of 300 columns (number of pixels in width) and 300 lines (number of pixels in height). For each position (x,y) the value corresponds to the quantity of the specific secondary ion detected by the analyser.

Measurements of the signal on each spot of the well is undertaken. All the data are normalized and formulated in an excel table.

A calibration curve of the target molecules is then formulated using the different calibration samples to establish a calibration curve of the different target molecules (x- axis quantity of target molecules and y-axis normalized SIMS signal).

The signal of the different target molecules of the biological sample are plotted on the curve, the concentration value resulting in that corresponding to the concentration in the sample.

Example 7- Conjugation of Antibodies and of Lanthanides to nanoparticles

1 ,4,7,10-tetraazacyclododecane (DOTA) chelating agents are conjugated to IMH2- polyethylene glycol-SH via an amidification reaction yielding to DOTA-polyethylene glycol-SH. Thulium salts (lanthanides) are added to the DOTA- polyethylene glycol-SH leading to a DOTA-Thulium-Polyethylene glycol-SH.

An aqueous solution of DTPA-IL-1 -ATA at 20 μg/ml is treated with hydroxylamine (50 μΙ) at room temperature for 2 hours to expose the free SH groups. After passing through a desalting column the resulting DTPA-IL-1 -SH is added to an aqueous solution of the gold nanoparticles as prepared in Example 1 at a concentration of 7.3 x 10¹⁰ particles/ml) to a final antibody concentration of 5 μg/ml. The suspension is stirred for 1 hour at room temperature. Polyethylene glycol-SH and DOTA-Thulium-Polyethylene glycol-SH (1 :1 molar ratio) are added to the antibody-coated Au nanoparticles to a final concentration of 0.2 mg/ml and the mixture is reacted for an additional hour.

DTPA-IL-1 is centrifuged for 5 minutes at 8,000 x rpm and the resulting is washed twice with deionized water. The presence of free IL-1 is examined using a protein dye. No free IL-1 is detected after the second wash. The Tm-NP-IL-1 are resuspended in 0.1 mmol/L PBS and stored at 4°C for further use.

A schematic diagram of the nanoparticle having the ionizable atoms and second capture molecule is shown in Figure 2.

Example 8- lonizable Nanoparticles having at least one Second Capture Molecule and at Least one Specific lonizable Atom attached to the Exterior through a Linker

Lanthanide conjugated nanoparticles are used in this example and are synthesized as set forth in Example 7. The size of this nanoparticle is 100 nm in diameter. The biochip has the first capture molecule attached thereto through epoxysilane groups that are attached to the biochip. The assay is performed using SIMS as set forth in Example 6 with the mass detector set on the lanthanide mass and on the gold mass to normalize the signal. A schematic diagram of the nanoparticle having the ionizable atoms and second capture molecule is shown in Figure 2. This embodiment permits the control of the at least one capture agents and the ionizable atoms in the preparation of the nanoparticles and the control of the ratio of the ionizable atoms/Au at the core of the nanoparticles.

Example 9-lonizable Nanoparticles having the at least one Second Capture molecules attached to the exterior through a linker and specific ionizable atoms forming part of the Nanoparticle

Silver nanoparticles are used in this example and are synthesized as set forth in Example 1 except that gold atoms are also added to the solutions. The size of the particles is 20 nm and 3 nanoparticles are fixed to each second capture molecule.

The biochip has the first capture molecule attached thereto through epoxysilane groups that are attached to the biochip. The assay is performed using SIMS as set forth in Example 6 with the mass detector set on the silver mass and on the gold mass. A diagram of the nanoparticle having the ionizable atoms incorporated therein and second capture molecule is shown in Figure 3.

This embodiment has the advantage due to the ionizable atoms being in the nanoparticle itself which makes the method more sensitive and the possibility of constructing a sole nanoparticle with many different second capture molecules (20 nm particles) or constructing different types of nanoparticles with different ionizable atoms and a sole second capture molecule.

Example 10-lonizable Nanoparticles having the second capture molecules attached to the exterior through a linker and the specific ionizable atoms attached to the second capture molecule Gold nanoparticles are used in this example and is synthesized as set forth in

Example 1. The size of this nanoparticle is 100 nm in diameter. Linkers of S(CH₂)603NH are attached to the nanoparticles via the sulfur moiety. The second capture molecule is attached to the linker through the amine group. In this embodiment the molecule containing lanthanide is attached to the second capture molecule. The biochip has the first capture molecule attached thereto through epoxysilane groups that are attached to the biochip. The assay is performed using SIMS as set forth in Example 6 with the mass detector set on the lanthanide mass and on the gold mass to normalize the signal. A diagram of the nanoparticle having the ionizable atoms incorporated therein and second capture molecule is shown in Figure 4. The advantage of this embodiment is that the ionizable atoms and at least one second capture agent can be formulated prior to fixing it to the nanopartide. There is also control of the ratio of the second capture molecule to the nanopartide.

Example 11 -Ionizable Nanoparticles having the Second Capture molecules attached to the exterior through a Linker and the specific ionizable atoms attached to the Linker

Gold nanoparticles are used in this example and is synthesized as set forth in Example 1. The size of this nanopartide is 100 nm in diameter. Linkers having a sulfur group at one end and an amide group at the other end which is attached to the second capture molecule. This attachment is performed through a thiol or amine reactive group. The lanthanide ionizable atoms are complexed with the linker.

The biochip has the first capture molecule attached thereto through epoxysilane groups that are attached to the biochip. The assay is performed using SIMS as set forth in Example 6. 6 with the mass detector set on the lanthanide mass and on the gold mass to normalize the signal. A diagram of the nanopartide having the ionizable atoms incorporated therein and second capture molecule is shown in Figure 5.

In this embodiment there is better control of the ratio of the second capture agent to the ionizable atoms in the preparation of the linker, as well as the final nanopartide. Example 12: Determination of the Limit of Detection with the nano particles

1 - Materials used in Example 12:

Silicon wafers (19 mm x 19 mm) (Siltronix) functionalized with epoxysilane coating (Sigma). There are 16 different zones (4x4) on the wafer with a size of 4 mm x 4 mm. These zones are called wells.

- Target molecules:

o antibodies labelled with biotin with LC LC linker

o antibodies labelled with biotin with PEG12 linker o HRP Biotinylated (Pierce)

Dilution molecule: antibody unlabeled

o Non-specific control (AbD Serotec HCA096): non relevant antibody Protein Printing Buffer 2X, Nexterion

- BSA fraction V (Sigma Aldrich)

- PBS buffer - building of the arrays:

1 . In a 384 well plate, the different antibodies were diluted in protein printing buffer 2X.

o Well A1 : 10μΙ_ of LC-LC biotin labelled non-specific control at 37.5 μg/mL + 10μΙ_ of protein printing buffer 2X

o Well A2: 10μΙ_ of LC-LC biotin labelled non-specific control at 3.75 μg/mL + 10μί of protein printing buffer 2X

o Well A3: 10μΙ of LC-LC biotin labelled non-specific control at 375 ng/mL + 10μί of protein printing buffer 2X

o Well A4: 10μΙ of LC-LC biotin labelled non-specific control at 37.5 ng/mL + 10μί of protein printing buffer 2X

o Well A5: 10μί of LC-LC biotin labelled non-specific control at 3.75 ng/mL + 10μί of protein printing buffer 2X

o Well A6: 10μΙ of LC-LC biotin labelled non-specific control at 375 pg/mL + 10μί of protein printing buffer 2X

o Well B1 : 10μί of PEG12-biotin labelled non-specific control at 37.5 μg/mL + 10μί of protein printing buffer 2X

o Well B2: 10μί of PEG12-biotin labelled non-specific control at 3.75 μg/mL + 10μί of protein printing buffer 2X

o Well B3: 10μί of PEG12-biotin labelled non-specific control at 375 ng/mL + 10μί of protein printing buffer 2X

o Well B4: 10μί of PEG12-biotin labelled non-specific control at 37.5 ng/mL + 10μί of protein printing buffer 2X o Well B5: 10μΙ_ of PEG12-biotin labelled non-specific control at 3.75 ng/mL + 10μΙ_ of protein printing buffer 2X

o Well B6: 10μΙ_ of PEG12-biotin labelled non-specific control at 375 pg/mL + 10μΙ_ of protein printing buffer 2X

o Well C1 : 10μΙ_ of HRP biotin labelled 10 μg/mL + 10μΙ_ of protein printing buffer 2X

o Well C2: 10μί of HRP biotin labelled 1 μg/mL + 10μΙ_ of protein printing buffer 2X

o Well C3: 10μΙ_ of HRP biotin labelled 100 ng/mL + 10μΙ_ of protein printing buffer 2X

o Well C4: 10μΙ_ of HRP biotin labelled 10 ng/mL + 10μΙ_ of protein printing buffer 2X

o Well C5: 10μί of HRP biotin labelled 1 ng/mL + 10μΙ_ of protein printing buffer 2X

o Well C6: 10μΙ_ of HRP biotin labelled 100 pg/mL + 10μΙ_ of protein printing buffer 2X

The well plate and the silicon wafers were placed in a Sciflex Arrayer (Scienion)

The arrayer built the arrays following Figure 6.

The printed arrays were stocked at 4°C overnight and then stocked under vacuum condition at room temperature.

Example 13: Test of quantification of the target molecules with a microbeads detection

1 -Material used in Example 13:

- One Array built in example 12.

- Detection molecules: o Polymer Tb core nanobeads of 30 nm coated with streptavidin

- BSA fraction V (Sigma Aldrich)

- PBS buffer - Hybridization:

1. The wells were blocked with a solution containing BSA 1 % in a PBS buffer 1X

2. The wells were hybridized with polymer nanobeads.

3. The silicon wafer was washed with PBS 1X

4. The silicon wafer was washed with H₂0

5. The silicon wafer was dried under air flow and stocked under vacuum at 4°C. The hybridization was carried out according to the schematic drawing in Figure 7. - SIMS analysis:

1. The wafer was installed in the SIMS analyzer, put under high vacuum and all the wells position were programmed to be analyzed.

2. For each well, all the surface was analyzed by D-SIMS. The detector was tuned on the mass 159

3. The result was a matrix of 100 columns (number of pixels in width) and 100 lines (number of pixels in height). For each position (x,y) the value corresponded to the quantity of the secondary ions generated by the ionization of the iron and detected by the analyzer.

4. Measurements of the signal on each spot of the well.

The distribution of the samples on the silica wafer is shown in Figures 8 and 9. Intra well signal control curve: the value of the spots signal control molecule were extracted to determine a signal control curve (Quantity of label on the x-axis / Signal on the y-axis).

Inter well comparison: the signal control curves were correlated with each other and normalized: All the different wells can be compared with each other independently of the SIMS performance during the analysis.

All the data were normalized and showed in an excel table.

Figure 10 shows the standard curves for the two target models. The left curve is the model HRP biotin. The right curve is the antibody against IL6 labeled with NHS-LC-LC biotin or NHS-PEG12 biotin.

Calibration curve of the target molecules: use of the different calibration samples (see 2) to establish a calibration curve of the target molecules (x-axis quantity of target molecules and y-axis normalized SIMS signal)

Example 14: realization of a bio array containing signal control molecules and capture molecules 1 - Materials used in Example 14:

- Silicon wafers (19 mm x 19 mm) (Siltronix) functionalized with epoxysilane coating (Sigma). There are 16 different zones (4x4) on the wafer with a size of 4 mm x 4 mm. These zones are called wells.

- Capture molecules: antibodies for the following target molecule:

o Anti-IL6 (Ebioscience BMS 130)

- Signal control molecule: Antibody labelled with Chelated Thulium

o Non-specific control (AbD Serotec HCA096): non relevant antibody

- Dilution molecule: antibody unlabelled

o Non-specific control (AbD Serotec HCA096): non relevant antibody

- Protein Printing Buffer 2X, Nexterion

- BSA fraction V (Sigma Aldrich) PBS buffer

2- building of the arrays:

1. In a 384 well plate, the different antibodies were diluted in protein printing buffer 2X.

o Well A1 : 10μΙ_ of labelled non-specific control at 1 mg/mL + 10μΙ_ of protein printing buffer 2X

o Well A2: 5μΙ_ of non-specific control at 1 mg/mL + 5μΙ_ of labelled nonspecific control at 1 mg/mL + 10μί of protein printing buffer 2X o Well A3: 7.5μί of non-specific control at 1 mg/mL + 2.5μί of labelled nonspecific control at 1 mg/mL + 10μί of protein printing buffer 2X o Well A4: 10μί of antibody non-specific control at 1 mg/mL + 10μί of protein printing buffer 2X

o Well A5: 10μί of antibody anti IL6 at 1 mg/mL + 10μί of protein printing buffer 2X

2. The well plate and the silicon wafers were placed in a Sciflex Arrayer

(Scienion)

3. The arrayer built the arrays following Figure 6.

Figure 1 1 shows the disposition of the different spots on the array.

4. The printed arrays were stocked at 4°C overnight and then stocked under vacuum condition at room temperature.

Example 15: Test of quantification of the target molecules with a Iron microbeads detection

1 -Materials used in Example 15:

- One Array built in the example 14. - Recombinant Molecules: Target molecules used for calibration curve

o IL6 (R&D Systems 206-IL-010)

- Detection molecules:

o antibodies labelled with biotin at 1 μg/mL Anti-IL6 (R&D Systems BAF-206). o Microbeads of magnetic Iron Oxide coated with streptavidin of 1 μηη of diameter.

- BSA fraction V (Sigma Aldrich)

- PBS buffer ybridization:

1. The wells were blocked with a solution containing BSA 1 % in a PBS buffer 1X

2. The different wells of the wafer were incubated with 10μΙ_ of different samples containing a various quantity of target molecules.

a. Recombinant Target molecule IL6 at 0 fg/mL

b. Recombinant Target molecule IL6 at 10 fg/mL

c. Recombinant Target molecule IL6 at 100 fg/mL

d. Recombinant Target molecule IL6 at 1000 fg/mL

Figure 12 shows the distribution of the different samples on the silicon wafer.

3. All the wells were washed with PBS 1X Tween 0.1 %

4. The wells were firstly hybridized with the detection molecules Anti-IL6

5. The silicon wafer (all the wells) was washed with PBS 1X Tween 0.1 %

6. The wells were secondly hybridized with the Iron beads.

7. The silicon wafer was washed with PBS 1X

8. The silicon wafer was washed with H₂O

9. The silicon wafer was dried under air flow and stocked under vacuum at 4°C.

The hybridization was carried out according to the schematic drawing in Figure 13. -SIMS analysis:

2. For each well, all the surface was analyzed by D-SIMS. The detector was tuned on the mass 56

4. Measurements of the signal on each spot of the well was undertaken.

Figure 14 shows the distribution of the different samples on the silicon wafer.

5. Intra well signal control curve: the value of the spots signal control molecule were extracted to determine a signal control curve (Quantity of label on the x-axis / Signal on the y-axis).

6. Inter well comparison: the signal control curves were correlated with each other and normalized: All the different wells can be compared with each other independently of the SIMS performance during the analysis.

The procedure of normalization after the SIMS analysis to obtain a quantification of the entire spots on the array is shown in Figure 15.

7. All the data were normalized and showed in an excel table.

8. Calibration curve of the target molecules: use of the different calibration samples (see 2) to establish a calibration curve of the target molecules IL6 (x-axis quantity of target molecules and y-axis normalized SIMS signal). Figure 16 shows a calibration curve of the different target molecules with Terbium nanobeads; x-axis quantity of target molecules and y-axis normalized SIMS signal.

Example 16: Test of quantification of the target molecules with a Terbium

nanobeads detection

1 - Material used in Example 16:

- Silicon wafers (19 mm x 19 mm) (Siltronix) functionalized with epoxysilane

coating (Sigma). There are 16 different zones (4x4) on the wafer with a size of 4 mm x 4 mm. These zones are called wells.

- Capture molecules: antibodies for the following target molecule:

o Anti-IL6 (Ebioscience BMS 130)

- Signal control molecule: Antibody labelled with Chelated Thulium

o Non-specific control (AbD Serotec HCA096): non relevant antibody

- Dilution molecule: antibody unlabelled

o Non-specific control (AbD Serotec HCA096): non relevant antibody

- Protein Printing Buffer 2X, Nexterion

- BSA fraction V (Sigma Aldrich)

- PBS buffer

2- building of the arrays

o Well A1 : 10μΙ_ of labelled non-specific control at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

o Well A2: 5μΙ_ of non-specific control at 1 mg/ml_ + 5μΙ_ of labelled nonspecific control at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X o Well A3: 7.5μΙ_ of non-specific control at 1 mg/mL + 2.5μΙ_ of labelled nonspecific control at 1 mg/mL + 10μΙ_ of protein printing buffer 2X o Well A4: 10μΙ_ of antibody non-specific control at 1 mg/mL + 10μί of protein printing buffer 2X

2. The well plate and the silicon wafers were placed in a Sciflex Arrayer

(Scienion)

3. The arrayer built the arrays following Figure 6.

Figure 17 is a table showing the disposition of the different spots of the array.

Example 17: Test of quantification of the target molecules with a nanobeads detection

1 - Material used in Example 17:

- One Array built in example 16.

- Recombinant Molecules: Target molecules used for calibration curve

o IL6 (R&D Systems 206-IL-010)

- Detection molecules:

o antibodies labelled with biotin at 1 μg/mL Anti-IL6 (R&D Systems BAF-206) o Polymer Tb core nanobeads of 30 nm coated with streptavidin

- BSA fraction V (Sigma Aldrich)

- PBS buffer

2- Hybridization:

1. The wells were blocked with a solution containing BSA 1 % in a PBS buffer 1X 2. The different wells of the wafer were incubated with 10μΙ_ of different samples containing a various quantity of target molecules.

a. Recombinant Target molecule IL6 at 0 fg/mL

b. Recombinant Target molecule IL6 at 10 fg/mL

c. Recombinant Target molecule IL6 at 100 fg/mL

d. Recombinant Target molecule IL6 at 1000 fg/mL

Figure 18 shows the distribution of the different samples on the silicon wafer.

3. All the wells were washed with PBS 1X Tween 0.1 %

4. The wells were firstly hybridized with the detection molecules Anti-IL6

5. The silicon wafer (all the wells) was washed with PBS 1X Tween 0.1 %

6. The wells were secondly hybridized with the terbium nanobeads.

7. The silicon wafer was washed with PBS 1X

8. The silicon wafer was washed with H₂0

9. The silicon wafer was dried under air flow and stocked under vacuum at 4°C.

The hybridization was carried out according to the schematic drawing in Figure 19. - SIMS analysis:

4. Measurements of the signal on each spot of the well was undertaken. Figure 20 shows the difference of signal between two concentrations of target IL6.

7. All the data were normalized and showed in an excel table.

8. Calibration curve of the target molecules: use of the different calibration samples (see 2) to establish a calibration curve of the target molecules IL6 (x-axis quantity of target molecules and y-axis normalized SIMS signal).

Figure 21 is a calibration curve of the different target molecules; x-axis quantity of target molecules and y-axis normalized SIMS signal including CV for the different concentrations.

Figure 22 is a Table showing the different concentrations of CV.

Example 18: Test of quantification of the target molecules with a nanobeads detection

1 - Material used in Example 18:

- Silicon wafers (19 mm x 19 mm) (Siltronix) functionalized with epoxysilane

coating (Sigma). There are 16 different zones (4x4) on the wafer with a size of 4 mm x 4 mm. These zones are called wells. - Capture molecules: antibodies for the following target molecule:

o Polyclonal Anti-IL6 (R&D AF 206)

- Signal control molecule: Antibody labelled with Chelated Thulium

o Non-specific control (AbD Serotec HCA096): non relevant antibody

- Dilution molecule: antibody unlabelled

o Non-specific control (AbD Serotec HCA096): non relevant antibody

- Protein Printing Buffer 2X, Nexterion

- BSA fraction V (Sigma Aldrich)

- PBS buffer - building of the arrays:

o Well A2: 5μΙ_ of non-specific control at 1 mg/ml_ + 5μΙ_ of labelled nonspecific control at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X o Well A3: 7.5μΙ_ of non-specific control at 1 mg/ml_ + 2.5μΙ_ of labelled nonspecific control at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X o Well A4: 10μΙ_ of antibody non-specific control at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

o Well A5: 10μΙ_ of antibody anti IL6 at 1 mg/ml_ + 10μΙ_ of protein printing buffer 2X

2. The well plate and the silicon wafers were placed in a Sciflex Arrayer

(Scienion)

3. The arrayer built the arrays following Figure 6.

Figure 23 is a Table showing the distribution of different spots on the array. 4. The printed arrays were stocked at 4°C overnight and then stocked under vacuum condition at room temperature.

Example 19: Test of quantification of the target molecules with a nanobeads detection

1 - Materials used in Eaxmple 19:

- One Array built in the Example 18.

- Recombinant Molecules: Target molecules used for calibration curve

o IL6 (R&D Systems 206-IL-010)

- Detection molecules:

o Monoclonal antibodies labelled with biotin (Pierce NHS-PEG4-Biotin) at

I pg/mL Anti-IL6 (R&D Systems MAB-206)

o Polymer Tb core nanobeads of 30 nm coated with streptavidin

- BSA fraction V (Sigma Aldrich)

- PBS buffer

2- Hybridization:

1. Microtubes were prepared with 100 μΙ_ of different concentration of recombinant IL6 diluted in PBS 1 % BSA:

a. Recombinant Target molecule IL6 at 0 fg/mL

b. Recombinant Target molecule IL6 at 10 fg/mL

c. Recombinant Target molecule IL6 at 100 fg/mL

d. Recombinant Target molecule IL6 at 1 pg/mL

e. Recombinant Target molecule IL6 at 10 pg/mL

f. Recombinant Target molecule IL6 at 100 pg/mL

2. Each tube received 100μΙ_ of solution containing 2μg/ml of anti IL6 monoclonal antibodies labeled with biotin.

3. The tubes were incubated 1 hour at 20°C under agitation

4. Each tube received 100μΙ_ of solution containing 10μg/ml Streptavidin nanobeads 5. The tubes were incubated 1 hour at 20°C under agitation

6. The tubes were centrifuged 10 minutes at 1500G, the supernatant was discarded and 50μΙ_ of the precipitate were kept on dry ice

7. The wells were blocked with a solution containing BSA 1 % in a PBS buffer 1X

8. The different wells of the wafer were incubated with 50μΙ_ of the different

precipitates (step 6) during 4 hours at 20°C under agitation.

9. All the wells were washed with PBS 1X Tween 0.1 %

10. The silicon wafer was washed with PBS 1X

1 1 .The silicon wafer was washed with H2O

12. The silicon wafer was dried under air flow and stocked under vacuum at 4°C.

The hybridization was carried out according to the schematic drawing in Figure 24. - SIMS analysis:

4. Measurements of the signal on each spot of the well was undertaken.

Figures 25 and 26 show the distribution of the different samples on the silicon wafer. Intra well signal control curve: the value of the spots signal control molecule were extracted to determine a signal control curve (Quantity of label on the x-axis / Signal on the y-axis).

All the data were normalized and showed in an excel table.

Calibration curve of the target molecules: use of the different calibration samples to establish a calibration curve of the target molecules IL6 (x-axis quantity of target molecules and y-axis normalized SIMS signal).

Figure 27 is a calibration curve of the different target molecules; x-axis quantity of target molecules and y-axis normalized SIMS signal including CV for the different concentrations.

While the invention has been described in terms of various preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions and changes may be made without departing from the scope thereof. Accordingly, it is intended that the scope of the present invention be limited by the scope of the claims, including equivalents thereof.

Claims

CLAIMS What is claimed is:

1. A ligand binding assay for quantitating a target molecule in at least one assay

sample comprising:

(a) depositing on a biochip at least one first capture molecule;

2. The ligand binding assay according to Claim 1 , wherein said multiplex assay method provides a multiplier coefficient, which increases the sensitivity of the biological analysis in the multiplex assay method.

3. The ligand binding assay according to Claim 1 or Claim 2, wherein the biochip is a silicon wafer.

4. The ligand binding assay according to any one of Claims 1 to 3, wherein said target molecule selected from the group of a protein, a peptide, a nucleic acid, a carbohydrates, a lipid, a polysaccharide, a glycoprotein, a hormone, an antigen, an antibody, a pathogen, a toxic substance, a drug, a dye, a nutrient, an enzyme, a rheumatoid factor, a tumor marker, a microorganism, an opiate, a viral epitope, cholesterol, a heavy metal, a vitamin, an allergy, a neurodegenerative disorder, an electrolyte, a glycan, a polyamine, a fatty acid, a sugar, a chemical trace metal and mixtures thereof.

5. The ligand binding assay according to any one of Claims 1 to 4, wherein the ionizable atoms are selected from the group of Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Sr, Ba, Ra, Y, La, Ac, Ti, Zr, Hf, V, Nb, Ta. Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, Al, In, TI, Sn, Pb, lanthanides, actinides, halogens, mixtures, oxides and alloys thereof.

6. The ligand binding assay according to any one of Claims 1 to 5, wherein the nanoparticles are from about 5 to 500 nanometers in diameter and the microparticles are from about 0.5 to 500 micrometers in diameter.

7. The ligand binding assay according to any one of Claims 1 to 6, wherein said at least one first capture molecule and at least one second capture molecule are antibodies, antibody fragments, antigens, diabodies, tetrabodies, nucleic acids, aptamers, affibodies, proteins, molecular imprinted polymers, enzymes, ligands, glycans, lectins, lipids, polyamines, phages, viruses, chemicals or combinations thereof.

8. The ligand binding assay according to any one of Claims 1 to 7, wherein the target molecule reacts with the first capture molecule through hybridization and the second capture molecule reacts with the target molecule through hybridization.

9. A kit comprising: nanoparticles or microparticles comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface and mixtures thereof; and reagents for the ligand binding assay.

10. The kit according to Claim 10, further comprising a biochip.

1 1 . The kit according to Claim 10, further comprising a first capture molecule.

12. The kit according to Claim 10, wherein the biochip has spotted thereon a first capture molecule.

13. The kit according to Claim 9, wherein said reagents for the multiplex assay are hybridization buffers, diluents and wash solutions.

14. The kit according to any one of Claims 9 to 13, wherein the ionizable atoms are selected from the group of Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Sr, Ba, Ra, Y, La, Ac,

Ti, Zr, Hf, V, Nb, Ta. Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, Al, In, TI, Sn, Pb, lanthanides, actinides, halogens, mixtures, oxides and alloys thereof.

15. Nanoparticles or micropartides comprising at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or micropartides or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the micropartides and nanoparticles at their surface and mixtures thereof for use in secondary ion mass spectroscopy to quantify target molecules in a ligand binding assay.

16. Nanoparticles or micropartides comprising adding at least one second capture molecule that reacts with the target molecule, this at least one second capture molecule being attached to at least one nanoparticle or microparticle wherein specific ionizable atoms are part of the at least one nanoparticle or microparticle or at least one second capture molecule being attached to at least one nanoparticle or microparticle comprising specific ionizable atoms being attached at their surface or to said at least one nanoparticle or microparticle having specific ionizable atoms being part of a molecule attached to the at least one nanoparticle or microparticle at their surface or at least one second capture molecule having specific ionizable atoms attached thereto wherein said at least one second capture molecule is attached to nanoparticles or microparticles or specific ionizable atoms that are attached to at least one second capture molecule and specific ionizable atoms that are also attached to the microparticles and nanoparticles at their surface and mixtures thereof for measuring the secondary ions emitted after destructive ionization in a ligand binding assay.

17. The nanoparticles or microparticles according to any one of Claims 15 to 16, wherein the ionizable atoms are selected from the group of Li, Na, K, Rb, Cs, Fr, Be, Mg, Ca, Sr, Ba, Ra, Y, La, Ac, Ti, Zr, Hf, V, Nb, Ta. Cr, Mo, W, Mn, Tc, Re, Fe, Ru, Os, Co, Rh, Ir, Ni, Pd, Pt, Cu, Ag, Au, Zn, Cd, Al, In, TI, Sn, Pb, lanthanides, actinides, halogens, mixtures, oxides and alloys thereof.