Microcantilever apparatus and methods for detection of enzymes

Abstract

An apparatus and a method are provided for detecting an enzyme by measuring a change in defection of a microcantilever having a substrate for the enzyme.

What is claimed is:  
     
         1 . A Method for detecting an enzyme, the method comprising: 
 depositing a coating material on a first surface of at least one microcantilever;    adding at least one substrate to the coating material, the substrate capable of interacting with the enzyme;    exposing the microcantilever with the substrate to a sample; and    measuring a deflection of the microcantilever, wherein the deflection indicates the presence of the enzyme in the sample.    
     
     
         2 . A method according to  claim 1 , wherein adding the substrate comprises adding at least one biomaterial.  
     
     
         3 . A method according to  claim 2 , wherein adding the biomaterial is adding a substance selected from the group consisting of a nucleic acid, a protein, a lipid, a hydrocarbon, and a polysaccharide.  
     
     
         4 . A method according to  claim 1 , wherein adding the substrate is adding a drug.  
     
     
         5 . A method according to  claim 1 , wherein the deflection is caused by a change in stress on the surface of the microcantilever.  
     
     
         6 . A method according to  claim 5 , wherein measuring the deflection is observing the change by a means selected from the group consisting of an optical means, an electron tunneling means, a capacitive means, a piezoelectric means, and a piezoresistive means.  
     
     
         7 . A method according to  claim 6 , wherein measuring the deflection is using the optical means.  
     
     
         8 . A method according to  claim 7 , wherein the optical means comprises a laser.  
     
     
         9 . A method according to  claim 1 , further comprising analyzing the deflection of the microcantilever as a function of a time parameter determined from the time of exposing the microcantilever to the sample.  
     
     
         10 . A method according to  claim 9 , wherein analyzing the deflection comprises using a microprocessor adapted for comparing, calculating, and storing the deflection of the microcantilever as a function of a time parameter.  
     
     
         11 . A method according to  claim 9 , wherein analyzing the deflection further comprises analyzing a parameter selected from the group of: concentration of enzyme, concentration of substrate, presence of a cofactor and presence of an inhibitor.  
     
     
         12 . A method according to  claim 1 , wherein the at least one microcantilever has a length of about 1 μm to about 500 μm, a width of about 1 μm to about 50 μm, and a thickness of about 0.1 μm to about 10 μm.  
     
     
         13 . A method according to  claim 1 , wherein depositing the coating material further comprises depositing a metal.  
     
     
         14 . A method according to  claim 13 , wherein the metal is selected from at least one of the group consisting of aluminum, copper, gold, chromium, titanium, silver, and mercury.  
     
     
         15 . A method according to  claim 14 , wherein the metal is gold.  
     
     
         16 . A method according to  claim 1 , wherein depositing the coating material further comprises depositing a plurality of metals.  
     
     
         17 . A method according to  claim 16 , wherein depositing a plurality of metals further comprises depositing a first layer of chromium and a second layer of gold.  
     
     
         18 . A method according to  claim 16 , wherein depositing a plurality of metals further comprises depositing a first layer of titanium and a second layer of gold.  
     
     
         19 . A method according to  claim 13 , wherein the metal is an amalgam or an alloy.  
     
     
         20 . A method according to  claim 1 , wherein the microcantilever has a second surface selected from the group consisting of aluminum: oxide, iridium oxide, silicon, silicon oxide, silicon nitride, tantalum pentoxide, and a plastic polymer.  
     
     
         21 . A method according to  claim 1 , further comprising prior to adding the substrate to the first surface, reacting the microcantilever with a bifunctional cross-linker, the bifunctional cross-linker capable of further reacting with the substrate.  
     
     
         22 . A method according to  claim 21 , wherein the bifunctional cross-linker is selected from the group consisting of: dithiobis(succinimido undecanoate (DSU); long chain succinimido-6-[3-(2-pyridyldithio)-propionamido] hexanoate (LCSPDP); succinimidyl-6-[3-(2-pyridyldithio)propionamido] hexanoate (SPDP); and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS).  
     
     
         23 . A method according to  claim 21 , wherein the bifunctional cross-linker is DSU.  
     
     
         24 . A method according to  claim 1 , wherein adding the substrate is designing the microcantilever for detecting an enzyme selected from the group consisting of: a hydrolase, an oxidoreductase, a transferase, a lyase, and a ligase.  
     
     
         25 . A method according to  claim 24 , wherein the enzyme is a hydrolase.  
     
     
         26 . A method according to  claim 25 , wherein the hydrolase is a protease.  
     
     
         27 . A method according to  claim 1 , wherein the enzyme is selected from the group of consisting of: a kinase, a phosphatase, an endopeptidase, an exopeptidase, a restriction endonuclease, an exonuclease, and a polymerase.  
     
     
         28 . A method according to  claim 26 , wherein the protease is a metalloprotease or a serine protease.  
     
     
         29 . A method according to  claim 24 , wherein the transferase is selected from the group consisting of: a glycosyl transferase, a glutathione S-transferase, an acetyl transferase, and a DNA methyl transferase.  
     
     
         30 . A method according to  claim 24 , wherein the lyase is selected from the group consisting of: a polysaccharide lyase, a 3-hydroxy-3-methylglutaryl CoA lyase, an argininosuccinate lyase and an isocitrate lyase.  
     
     
         31 . A method according to  claim 24 , wherein the oxidoreductase is selected from the group consisting of: a hydroxylamine oxidoreductase, a glyphosphate oxidoreductase, a quinine oxidoreductase, an ubiquinone oxidoreductase, and a protein disulfide oxidoreductase.  
     
     
         32 . A method according to  claim 1 , wherein the sample comprises an enzyme that is substantially purified.  
     
     
         33 . A method according to  claim 1 , wherein the sample comprises a biological fluid.  
     
     
         34 . A method according to  claim 33 , wherein the biological fluid is selected from the group consisting of: a cell lysate, a culture medium, a spent medium, an animal extract, and a plant extract.  
     
     
         35 . A method according to  claim 33 , wherein the biological fluid comprises a bodily fluid from a vertebrate animal.  
     
     
         36 . A method according to  claim 35 , wherein the vertebrate is a mammal.  
     
     
         37 . A method according to  claim 36 , wherein the mammal is a human.  
     
     
         38 . A method according to  claim 35 , wherein the bodily fluid is selected from the group consisting of: blood, lymph, tissue fluid, urine, bile, sweat, synovial fluid, amniotic fluid, abdominal fluid, pericardial fluid, pleural fluid, cerebrospinal fluid, gastric juice, intestinal juice, joint cavity fluid, tears, and nasal discharge.  
     
     
         39 . A method according to  claim 1 , wherein the enzyme is associated with a medical condition in a vertebrate animal.  
     
     
         40 . A method according to  claim 39 , wherein the medical condition is a genetic defect.  
     
     
         41 . A method according to  claim 40 , wherein the medical condition is selected from the group consisting of: cystic fibrosis, Fabry disease, Gaucher disease, Tay-Sachs disease, sickle cell anemia, Lesch-Nyhan disease, Parkinsons disease, amyotrophic lateral sclerosis, Crohns disease, diabetes mellitus, mannosidosis disease, celiac disease, X-linked glomerular disease, and mucopolysaccharidosis.  
     
     
         42 . A method according  claim 39 , wherein the medical condition is a cancer.  
     
     
         43 . A method according to  claim 42 , wherein the cancer is selected from the group consisting of a cancer of the: brain, liver, pancreas, lung, prostate, and breast.  
     
     
         44 . A method according to  claim 43 , wherein the cancer is prostate cancer and the enzyme is prostate specific antigen.  
     
     
         45 . A method according to  claim 43 , wherein the cancer is breast cancer, and the enzyme is a collagenase.  
     
     
         46 . A method according to  claim 39 , wherein the medical condition is the presence of an infectious agent.  
     
     
         47 . A method according to  claim 46 , wherein the infectious agent is selected from the group consisting of: a virus, a bacterium, a fungus, a protozoan, and a helminth.  
     
     
         48 . A method for detecting in a sample an associating substance that binds to a substrate, wherein detecting the substance involves at least one microcantilever configured to be responsive to a micro-force, the method comprising: 
 depositing a coating material on a first surface of the microcantilever;    adding at least one substrate to the coating material, the substrate capable of interaction with the substance;    exposing the microcantilever with the substrate to the sample; and    measuring a resulting free surface energy change on the surface of the microcantilever, wherein the surface energy change indicates binding to the substrate by the associating substance in the sample.    
     
     
         49 . A method according to  claim 48 , wherein the associating substance is selected from the group consisting of: a binding protein, an enzyme, a cofactor, a receptor ligand, an antibody, a polysaccharide, a lipid, a nucleic acid, and a steroid.  
     
     
         50 . A method according to  claim 48 , wherein the associating substance is an enzyme.  
     
     
         51 . A method according to  claim 50 , wherein the enzyme binds the substrate and fails to dissociate.  
     
     
         52 . A method according to  claim 51 , wherein the enzyme has no activity on the substrate.  
     
     
         53 . A method according to  claim 48 , wherein the substrate is a non-cleavable pseudosubstrate.  
     
     
         54 . A method according to  claim 1 , wherein the at least one microcantilever is a block array having a plurality of microcantilevers.  
     
     
         55 . A method according to  claim 48 , wherein the substrate is a plurality of biomaterials.  
     
     
         56 . A method according to  claim 55 , wherein the substrate comprises an inhibitor of enzymatic activity.  
     
     
         57 . A method of screening for an inhibitor of an enzyme having a substrate on a microcantilever having a coating, the method comprising: 
 adding the substrate to a first side of a first and a second microcantilever, the substrate capable of interacting with the enzyme;    exposing the first microcantilever with the substrate to a sample, the sample containing a candidate inhibitor and the enzyme; and    measuring a deflection of the first microcantilever in comparison to a deflection of a second microcantilever exposed to the enzyme in the absence of the candidate inhibitor.    
     
     
         58 . A method according to  claim 57 , wherein the first microcantilever and the second microcantilever are located in a first and second interaction well of a microfluidics device.  
     
     
         59 . A method according to  claim 58 , wherein a third microcantilever and a fourth microcantilever are located in a third and fourth interaction well, the third and fourth wells having a different concentration of enzyme than the first and second wells.  
     
     
         60 . A method according to  claim 58 , wherein a third microcantilever and a fourth microcantilever are located in a third and fourth interaction well, the third and fourth wells having different samples comprising candidate inhibitors.  
     
     
         61 . An apparatus to measure a microforce generated by an interaction between an enzyme and a biomaterial, comprising: at least one microcantilever, wherein said microcantilever has a length, a width, and a thickness; a coating material deposited on a first surface of the microcantilever; a biomaterial capable of attachment to the coating material; and at least one interaction well, wherein the microcantilever with coating material and the biomaterial is exposed to a sample, the sample comprising the enzyme.  
     
     
         62 . An apparatus according to  claim 61 , wherein the biomaterial comprises an enzymatic substrate.  
     
     
         63 . An apparatus according to  claim 61 , wherein the biomaterial comprises an enzymatic pseudosubstrate.  
     
     
         64 . An apparatus according to  claim 61 , wherein the at least one microcantilever further comprises of a block array having a plurality of microcantilevers.  
     
     
         65 . An apparatus according to  claim 61 , wherein the microcantilever length is about 1 μm to about 500 μm, the width is about 1 μm to about 50 μm, and the thickness is about 0.1 μm to about 10 μm.  
     
     
         66 . An apparatus according to  claim 64 , wherein the coating material is selected from at least one of the group consisting of copper, gold, aluminum, chromium, titanium, silver, and mercury.  
     
     
         67 . An apparatus according to  claim 66 , wherein the coating material is gold.  
     
     
         68 . An apparatus according to  claim 61 , wherein a second surface of the microcantilever is selected from the group consisting of silicon, silicon nitride, other silicon compounds, metal compounds, gallium arsenide, germanium, germanium dioxide, glass, zinc oxide, diamond, quartz, paladium and a plastic polymer.  
     
     
         69 . An apparatus according to  claim 61 , wherein the apparatus is disposable.  
     
     
         70 . An apparatus according to  claim 61 , wherein the apparatus is reusable.