Microresonant sensors and methods of use thereof

Abstract

A sensor assembly for sensors such as microfabricated resonant sensors is disclosed. The disclosed assembly provides improved performance of the sensors by providing a thermally insensitive environment and short pathways for signals to travel to processing components. Further, the assembly provide modular construction for the sensors and housing modules, thereby allowing replacement of the sensors at a lower cost. The assembly includes a sensor module including a sensor formed on a conductive substrate with a cavity formed on one surface. The substrate has conductive vias extending from the cavity to a second surface of the substrate. A housing assembly accommodates the sensor and includes a rigid housing, preferably made from a ceramic. An electronic component, such as an amplifier, is mounted on the rigid housing. The electronic component electrically engages the vias substantially at the second surface of the substrate. The electronic component receive signals from the sensor through the vias. The signals are then processed through an amplifier and a digital signal processor using a modified periodogram.

We claim:  
     
         1 . A method of monitoring density in a fluid, comprising: 
 placing said fluid into contact with a micromechanical sensor assembly comprising a membrane having an upper surface facing toward said fluid and a lower surface facing away from said fluid, wherein said membrane vibrates in response to electrical current; and    monitoring a vibrational frequency of said membrane, wherein said vibrational frequency is related to density of a volume of said fluid at or near said membrane upper surface.    
     
     
         2 . The method of  claim 1 , wherein said fluid is an aqueous liquid environment comprising cells, and the density of said liquid environment changes due to a change in the number of cells in said liquid.  
     
     
         3 . The method of  claim 2 , wherein said cells increase in number due to division of said cells.  
     
     
         4 . The method of  claim 2 , wherein said cells are suspended in said liquid environment.  
     
     
         5 . The method of  claim 2 , wherein said cells are directly or indirectly affixed to said membrane upper surface.  
     
     
         6 . The method of  claim 5 , wherein said liquid environment comprises a hydrogel layer within the sensed volume of said micromechanical sensor assembly, and said cells are indirectly affixed to said membrane upper surface through said hydrogel.  
     
     
         7 . The method of  claim 1 , wherein said micromechanical sensor assembly is contained within a sensor array comprising a plurality of discretely addressable micromechanical sensor assembly sites.  
     
     
         8 . The method of  claim 1 , wherein said fluid is a gas.  
     
     
         9 . The method of  claim 1  wherein the fluid contains dense organic or inorganic particles and the sensor is used to distinguish the mass or density of said particles.  
     
     
         10 . The method of  claim 9 , wherein said particles comprise heavy metals.  
     
     
         11 . The method in  claim 7 , wherein said density measurement is correlated to mobility of cells or cell processes across the surface of the sensor array  
     
     
         12 . The method of  claim 2 , wherein said cells are bacteria.  
     
     
         13 . The method of  claim 2 , wherein said cells are mammalian cells.  
     
     
         14 . The method of  claim 13 , wherein said mammalian cells are lymphocytes or hybridomas.  
     
     
         15 . The method of  claim 2 , wherein said cells are cells are yeast cells.  
     
     
         16 . The method of  claim 15 , wherein said yeast cells display antibody or antibody fragments, or protein libraries or protein fragment libraries.  
     
     
         17 . The method of  claim 1 , wherein said fluid is a liquid environment comprising virus or phage particles, and the density of said liquid changes due to a change in the number of virus or phage particles in said liquid.  
     
     
         18 . The method of  claim 17  in which said virus or phage particles display antibodies or protein libraries on a particle surface.  
     
     
         19 . The method of  claim 1 , wherein said fluid is a liquid environment comprising molecules that bind to a receptor within the sensed volume of said membrane upper surface, and wherein the density of said liquid changes due to further addition of mass or density to the bound molecules.  
     
     
         20 . The method of  claim 19 , wherein addition of mass or density to the bound molecules comprises the use of an enzymatic reaction selected from the group consisting of strand displacement nucleic acid amplification, polymerase chain reaction nucleic acid amplification, isothermal nucleic acid amplification, or rolling circle nucleic acid amplification.  
     
     
         21 . The method of  claim 19 , wherein addition of mass or density to the specifically bound molecules comprises the use of one or more second binding partners for the bound molecules.  
     
     
         22 . The method of  claim 21 , wherein said one or more second binding partners comprise particles conjugated thereto.  
     
     
         23 . The method of  claim 7 , wherein said aqueous liquid environment is an electrophoretic gel within the sensed volume of an array of sensors, and said sensor array monitors flow of molecules through said electrophoretic gel.  
     
     
         24 . The method of  claim 1 , wherein said fluid is a liquid environment comprising molecules that bind to a second molecule within the sensed volume of said membrane upper surface, and wherein the method further comprises identification of molecules bound to one or more sensor sites by mass spectometry.  
     
     
         25 . The method of  claim 1 , wherein said fluid is a liquid environment comprising molecules that bind to one or more nucleic acid molecules within the sensed volume of said membrane upper surface, and wherein the density of said liquid changes due to binding of nucleic acid binding proteins to said one or more nucleic acid molecules.  
     
     
         26 . The method of  claim 25 , wherein said nucleic acid binding proteins are transcription factors or nucleic acid replication molecules.  
     
     
         27 . A method of screening a library of test molecules for the ability to bind to a population of cells, comprising: 
 contacting an array comprising a plurality of discretely addressable micromechanical sensor assembly sites with said population of cells, wherein each site comprises a membrane having an upper surface facing toward an aqueous liquid environment comprising said population of cells and a lower surface facing away from said aqueous liquid environment, wherein said membrane vibrates in response to electrical current, and wherein each of said plurality of micromechanical sensor assembly sites comprise one or more test molecules from said library directly or indirectly affixed to said membrane upper surface; and    determining whether a change in vibrational frequency of said membrane occurs at each of said plurality of micromechanical sensor assembly sites, wherein said vibrational frequency at each individual micromechanical sensor assembly site is related to binding of one or more cells to said one or more test molecules affixed at said individual micromechanical sensor assembly site.    
     
     
         28 . The method of  claim 27 , wherein said membrane upper surface comprises a hydrogel layer, and said one or more test molecules are indirectly affixed to said membrane upper surface through said hydrogel.  
     
     
         29 . The method of  claim 27 , wherein each member of said library of molecules is independently selected from the group consisting of small molecules, prodrugs, polypeptides, antibodies, antibody fragments, single-chain variable region fragments, polynucleotides, oligonucleotides, oligonucleotide analogs, oligosaccharides, polysaccharides, cyclic polypeptides, peptidomimetics, and aptamers.  
     
     
         30 . A method of screening a library of test molecules for the ability to inhibit binding of a ligand to a cell surface molecule, comprising: 
 contacting an array comprising a plurality of discretely addressable micromechanical sensor assembly sites with a population of cells expressing said cell surface molecule and one or more test molecules, wherein each site comprises a membrane having an upper surface facing toward an aqueous liquid environment comprising said population of cells and a lower surface facing away from said aqueous liquid environment, wherein said membrane vibrates in response to electrical current, and wherein each of said plurality of micromechanical sensor assembly sites comprise said ligand directly or indirectly affixed to said membrane upper surface, and wherein each discretely addressable micromechanical sensor assembly site is contacted with different test molecules; and    determining whether a change in vibrational frequency of said membrane occurs at each of said plurality of micromechanical sensor assembly sites, wherein said vibrational frequency at each individual micromechanical sensor assembly site is related to binding of one or more cells to said ligand affixed at said individual micromechanical sensor assembly site.    
     
     
         30 . A method of sorting cells in a cell population according to the expression of a plurality of cell surface molecules on said cells, comprising: 
 contacting a sensor array with one or more test molecules that bind one or more cell surface molecules on one or more cells in the cell population, wherein said sensor array comprises 
 (i) a plurality of discretely addressable micromechanical sensor assembly sites, each site comprising a micromechanical sensor assembly comprising a membrane having an upper surface facing toward an aqueous environment and a lower surface facing away from said fluid, wherein said membrane vibrates in response to electrical current, wherein a vibrational frequency of said membrane is related to density of a volume of said fluid at or near said membrane upper surface, and  
 (ii) one or more cells from said cell population bound to an immobilized receptor for a cell surface molecule, thereby immobilizing said one or more cells within the sensed volume of each of said plurality of discretely addressable micromechanical sensor assembly sites; and  
   monitoring a change in vibrational frequency of said membrane at each site resulting from one or more test molecules binding to said one or more cells within the sensed volume at each site.    
     
     
         31 . The method of  claim 30 , wherein a change in vibrational frequency at a site results from a change in a physiologic state of cell(s) bound at said site causing a change in size or density of said cell(s).  
     
     
         32 . The method of  claim 30 , wherein said method measures chemosensitivity of cells in said cell population to drugs, biological agents, pathogens, or environmental toxins.  
     
     
         33 . The method of  claim 30 , wherein said one or more cells are immobilized within the sensed volume of each of said plurality of discretely addressable micromechanical sensor assembly sites via binding to an integrin, selectin, cadherin, N-CAM, or I-CAM protein.  
     
     
         34 . The method of  claim 30 , wherein said one or more cells are immobilized within the sensed volume of each of said plurality of discretely addressable micromechanical sensor assembly sites via binding to an extracellular matrix protein.  
     
     
         35 . The method of  claim 30 , said method measures inotropic or chronotropic changes in cells in said cell population.