Acoustically mediated fluid transfer methods and uses thereof

Abstract

Acoustic waves are used to transfer small amounts of fluid in a non-contact manner. Acoustic waves are propagated through a pool of a source fluid in such a manner that causes the ejection of a single micro-droplet from the surface of the pool. The droplet is ejected towards a target with sufficient force to provide for contact of the droplet with the target. Because the fluid is not contacted by any fluid transfer device such as a pipette, the opportunities for contamination are minimized. Methods may be employed to transfer fluids from an array of source sites to an array of target sites, thereby enabling the precise automation of a wide variety of procedures including screening and synthesis procedures commonly used in biotechnology.

1 - 57 . (canceled)  
     
     
         58 : A method for preparing an array comprised of a plurality of peptidic molecules attached to a substrate surface, the method comprising applying focused acoustic energy to each of a plurality of reservoirs each containing a peptidic molecule in a fluid, wherein the focused acoustic energy is applied in a manner effective to eject a droplet from each reservoir toward a different site on a substrate surface.  
     
     
         59 : The method of  claim 58 , wherein the focused acoustic energy is applied to each of the plurality of reservoirs by (a) acoustically coupling each reservoir in succession to an ejector that produces acoustic radiation; and (b) following each acoustic coupling step, activating the ejector to generate acoustic radiation having a focal point sufficiently near the fluid surface so as to eject a fluid droplet from the reservoir toward a designated site on the substrate surface.  
     
     
         60 : The method of  claim 58 , wherein each peptidic molecule is comprised of about two or more amino acids.  
     
     
         61 : The method of  claim 58 , wherein the fluid comprises glycerol.  
     
     
         62 : The method of  claim 58 , wherein each peptidic molecule is an oligopeptide, a polypeptide, protein, or protein fragment.  
     
     
         63 : The method of  claim 62 , wherein each peptidic molecule is selected from the group consisting of enzymes, antibodies, and antigens.  
     
     
         64 : The method of  claim 58 , wherein each peptidic molecule is different.  
     
     
         65 : The method of  claim 58 , wherein each of the ejected droplets has a diameter in the range of about 30 micrometers to about 60 micrometers.  
     
     
         66 : The method of  claim 58 , wherein each of the ejected droplets has a diameter in the range of about 120 micrometers to about 250 micrometers.  
     
     
         67 : The method of  claim 58 , wherein each of the ejected droplets has a diameter in the range of about 60 micrometers to about 500 micrometers.  
     
     
         68 : A method for preparing a peptide array comprised of a plurality of peptidic molecules attached to a substrate surface, the method comprising: 
 (a) acoustically coupling a first reservoir containing a first peptidic molecule in a first fluid to an ejector that produces acoustic radiation;    (b) activating the ejector to generate acoustic radiation having a focal point sufficiently near the surface of the first fluid so as to eject a droplet thereof toward a first designated site on the substrate surface;    (c) acoustically coupling a second reservoir containing a second peptidic molecule in a second fluid to the ejector;    (d) activating the ejector as in step (b) to eject a droplet of the second fluid from the second reservoir toward a second designated site on the substrate surface; and    (e) repeating steps (c) and (d) with additional reservoirs each containing a peptidic molecule in a fluid until a droplet has been ejected from each reservoir.    
     
     
         69 : The method of  claim 68 , wherein each fluid comprises glycerol.  
     
     
         70 : The method of  claim 68 , wherein steps (b) and (d) result in attachment of the first and second peptidic molecules, respectively, to the first and second designated sites on the substrate surface.  
     
     
         71 : The method of  claim 70 , wherein the attachment is covalent.  
     
     
         72 : The method of  claim 70 , wherein the attachment is noncovalent.  
     
     
         73 : The method of  claim 68 , wherein each peptidic molecule is comprised of two or more amino acids.  
     
     
         74 : The method of  claim 68 , wherein each fluid comprises glycerol.  
     
     
         75 : The method of  claim 68 , wherein each peptidic molecule is an oligopeptide, a polypeptide, protein, or protein fragment.  
     
     
         76 : The method of  claim 75 , wherein each peptidic molecule is selected from the group consisting of enzymes, antibodies, antigens, coagulation modulators, cytokines, endorphins, peptidyl hormones and kinins.  
     
     
         77 : The method of  claim 68 , wherein each peptidic molecule is different.  
     
     
         78 : The method of  claim 68 , wherein each of the ejected droplets has a diameter in the range of about 30 micrometers to about 60 micrometers.  
     
     
         79 : The method of  claim 68 , wherein each of the ejected droplets has a diameter in the range of about 120 micrometers to about 250 micrometers.  
     
     
         80 : The method of  claim 68 , wherein each of the ejected droplets has a diameter in the range of about 60 micrometers to about 500 micrometers.  
     
     
         81 : The method of either  claim 58  or  claim 68 , wherein the substrate surface is activated as a result of pretreatment with an activating agent.  
     
     
         82 : The method of either  claim 58  or  claim 68 , wherein the substrate surface is comprised of a porous material.  
     
     
         83 : The method of  claim 82 , wherein the porous material is a permeable material.  
     
     
         84 : A peptide array comprised of a plurality of peptidic molecules each attached through an optional linking moiety to a different site on a substrate surface, wherein each of the peptidic molecules is deposited onto the substrate surface using focused acoustic energy.  
     
     
         85 : A peptide array comprised of a plurality of peptidic molecules each attached through an optional linking moiety to a different site on a substrate surface, wherein substantially none of the peptidic molecules exhibits signs of shear stress and substantially all of the peptidic molecules are intact.  
     
     
         86 : The peptide array of  claim 84  or  claim 85 , wherein the attachment of the peptidic molecules to the substrate surface is covalent.  
     
     
         87 : The peptide array of  claim 84  or  claim 85 , wherein the attachment of the peptidic molecules to the substrate surface is noncovalent.  
     
     
         88 : The peptide array of  claim 84  or  claim 85 , wherein each peptidic molecule is different.  
     
     
         89 : The peptide array of  claim 84  or  claim 85 , wherein each peptidic molecule is comprised of two or more amino acids.  
     
     
         90 : The peptide array of  claim 84  or  claim 85 , wherein each peptidic molecule is an oligopeptide, a polypeptide, protein, or protein fragment.  
     
     
         91 : The peptide array of  claim 90 , wherein each peptidic molecule is selected from the group consisting of enzymes, antibodies, antigens, coagulation modulators, cytokines, endorphins, peptidyl hormones and kinins.  
     
     
         92 : The peptide array of  claim 84  or  claim 85 , wherein the substrate surface is comprised of a porous material.  
     
     
         93 : The peptide array of  claim 92 , wherein the porous material is a permeable material.  
     
     
         94 : The peptide array of  claim 84  or  claim 85 , wherein more than a thousand peptidic molecules are present on the substrate surface.  
     
     
         95 : The peptide array of  claim 84  or  claim 85 , wherein more than a thousand peptidic molecules are present on the substrate surface, said substrate comprising a microscope slide.  
     
     
         96 : The peptide array of  claim 84  or  claim 85 , wherein thousands of peptidic molecules are present on the substrate surface.  
     
     
         97 : The peptide array of  claim 84  or  claim 85 , wherein thousands of peptidic molecules are present on the substrate surface, said substrate comprising a microscope slide.  
     
     
         98 : The peptide array of  claim 84  or  claim 85 , wherein many thousands of peptidic molecules are present on the substrate surface.  
     
     
         99 : The peptide array of  claim 84  or  claim 85 , wherein many thousands of peptidic molecules are present on the substrate surface, said substrate comprising a microscope slide.  
     
     
         100 : The peptide array of  claim 84  or  claim 85 , wherein at least one of the peptidic molecules on the substrate surface is in a lipidic material.  
     
     
         101 : The peptide array of  claim 100 , wherein the lipidic material is a phospholipid.  
     
     
         102 : The peptide array of  claim 100 , wherein the lipidic material is a liposome.  
     
     
         103 : The peptide array of  claim 100 , wherein at least one of the peptidic molecules on the substrate surface is in an aqueous fluid.  
     
     
         104 : A peptide array comprised of a plurality of peptidic molecules each attached through an optional linking moiety to a different site on a porous substrate surface, wherein each of the peptidic molecules is deposited onto the substrate surface using focused acoustic energy.  
     
     
         105 : A peptide array comprised of a plurality of peptidic molecules each attached through an optional linking moiety to a different site on a porous substrate surface, wherein said plurality comprises more than a thousand peptidic molecules.  
     
     
         106 : The peptide array of  claim 104  or  claim 105 , wherein said porous substrate surface comprises a microscope slide.  
     
     
         107 : The peptide array of  claim 104  or  claim 105 , wherein said plurality comprises thousands of peptidic molecules.  
     
     
         108 : The peptide array of  claim 104  or  claim 105 , wherein said plurality comprises thousands of peptidic molecules and said porous substrate surface comprises a microscope slide.  
     
     
         109 : The peptide array of  claim 104  or  claim 105 , wherein said plurality comprises many thousands of peptidic molecules.  
     
     
         110 : The peptide array of  claim 104  or  claim 105 , wherein said plurality comprises many thousands of peptidic molecules and said porous substrate surface comprises a microscope slide.  
     
     
         111 : The peptide array of  claim 104  or  claim 105 , wherein the porous substrate surface is a surface of a nonporous substrate.  
     
     
         112 : The peptide array of  claim 104  or  claim 105 , wherein the porous substrate surface is a surface of a porous substrate.  
     
     
         113 : The peptide array of  claim 104  or  claim 105 , wherein the porous substrate surface is a permeable surface.  
     
     
         114 : The peptide array of  claim 113 , wherein the permeable surface is a surface of a permeable substrate.  
     
     
         115 : A device for acoustically ejecting a droplet of fluid from each of a plurality of fluid reservoirs, comprising: 
 a plurality of reservoirs each adapted to contain a fluid;    an acoustic ejector comprising an acoustic radiation generator for generating acoustic radiation and a focusing means for focusing the acoustic radiation generated; and    a means for positioning the acoustic ejector in acoustic coupling relationship to each of the reservoirs.    
     
     
         116 : The device of  claim 115 , comprised of a single acoustic ejector.  
     
     
         117 : The device of  claim 115 , wherein each of the reservoirs is removable from the device.  
     
     
         118 : The device of  claim 115 , wherein the reservoirs comprise individual wells in a micro-titer or well plate.  
     
     
         119 : The device of  claim 118 , wherein the distance between the centers of any two adjacent reservoirs is about 2.25 mm.  
     
     
         120 : The device of  claim 115 , wherein the reservoirs are substantially acoustically indistinguishable.  
     
     
         121 : The device of  claim 115 , wherein the device comprises 1536 reservoirs.  
     
     
         122 : The device of  claim 115 , wherein the device comprises more than one thousand reservoirs.  
     
     
         123 : The device of  claim 115 , wherein the device comprises thousands of reservoirs.  
     
     
         124 : The device of  claim 115 , wherein at least one reservoir has a volume of no more than about 10 μL of fluid.  
     
     
         125 : The device of  claim 115 , wherein each reservoir contains a fluid.  
     
     
         126 : The device of  claim 125 , wherein the fluid in each reservoir contains a biomolecule.  
     
     
         127 : The device of  claim 126 , wherein the biomolecule in each reservoir is different.  
     
     
         128 : The device of  claim 115 , wherein at least one of the reservoirs contains a nonaqueous fluid.  
     
     
         129 : The device of  claim 128 , wherein the nonaqueous fluid comprises an organic solvent.  
     
     
         130 : The device of  claim 128 , wherein the nonaqueous fluid comprises a non-biological fluid.  
     
     
         131 : The device of  claim 126 , wherein the biomolecule is selected from nucleotidic, peptidic, monomeric, oligomeric, and polymeric biomolecules.  
     
     
         132 : The device of  claim 115 , wherein the ejector positioning means is adapted to eject droplets from each reservoir in a predetermined sequence.  
     
     
         133 : The device of  claim 115 , further comprising a substrate positioning means for positioning the substrate surface with respect to the ejector.  
     
     
         134 : The device of  claim 133 , further comprising a substrate in which individual sites on the substrate form an array.  
     
     
         135 : The device of  claim 115 , wherein the acoustic coupling relationship comprises positioning the ejector such that the acoustic radiation is generated and focused external to the reservoirs.  
     
     
         136 : The device of  claim 135 , wherein acoustic coupling relationship between the ejector and the fluid in each reservoir is established by providing an acoustically conductive medium between the ejector and the reservoir.  
     
     
         137 : The device of  claim 115 , wherein acoustic coupling between the ejector and the fluid in each reservoir is established at a predetermined distance between the ejector and each reservoir.  
     
     
         138 : A method for generating an array of chemical entities on the surface of a substrate, the method comprising applying focused acoustic energy to each of a plurality of reservoirs each containing a chemical entity in a fluid, wherein the focused acoustic energy is applied using a single ejector comprised of an acoustic radiation generator and a focusing means, in a manner effective to eject a droplet from each reservoir toward a site on the surface of the substrate such that the chemical entity in each droplet attaches thereto.  
     
     
         139 : The method of  claim 138 , wherein the focused acoustic energy is applied to each of the plurality of reservoirs by (a) acoustically coupling each reservoir in succession to the acoustic ejector, and (b) following each acoustic coupling step, activating the ejector to generate acoustic radiation having a focal point sufficiently near the fluid surface so as to eject a fluid droplet from the reservoir toward a site on the substrate surface.  
     
     
         140 : The method of  claim 139 , wherein each of the ejected droplets has a diameter of less than about 120 micrometers.  
     
     
         141 : The method of  claim 139 , wherein each of the ejected droplets has a diameter in the range of about 30 micrometers to about 60 micrometers.  
     
     
         142 : The method of  claim 139 , wherein each of the ejected droplets has a diameter in the range of about 120 micrometers to about 250 micrometers.  
     
     
         143 : The method of  claim 139 , wherein each of the ejected droplets has a diameter in the range of about 60 micrometers to about 500 micrometers.  
     
     
         144 : The method of  claim 139 , wherein each of the ejected droplets has a diameter in the range of about 500 micrometers to about 1,000 micrometers.  
     
     
         145 : The method of  claim 139 , wherein each of the ejected droplets has a diameter in the range of about 1 micrometer to about 10,000 micrometers.  
     
     
         146 : The method of  claim 139 , wherein each chemical entity is a molecule selected from a biomolecule, a nucleotide or an oligonucleotide, a peptidic and a saccharidic molecule, and an oligosaccharide.  
     
     
         147 : A method for preparing an array of chemical entities attached to the surface of a substrate, the method comprising: 
 (a) acoustically coupling a first reservoir containing a first chemical entity in a first fluid to an ejector that produces acoustic radiation;    (b) activating the ejector to generate acoustic radiation having a focal point sufficiently near the surface of the first fluid so as to eject a droplet thereof toward a first site on the substrate surface;    (c) acoustically coupling a second reservoir containing a second chemical entity in a second fluid to the ejector;    (d) activating the ejector as in step (b) to eject a droplet of the second fluid from the second reservoir toward a second site on the substrate surface; and    (e) repeating steps (c) and (d) with additional reservoirs each containing a chemical entity in a fluid until a droplet has been ejected from each reservoir, wherein steps (b) and (d) result in attachment of the chemical entity in each droplet to the surface of the substrate.    
     
     
         148 : The method of  claim 147 , wherein the target surface is comprised of a porous permeable material.  
     
     
         149 : The method of  claim 147 , wherein the target surface is comprised of a nonporous material.  
     
     
         150 : The method of  claim 147 , wherein the array includes more than one thousand array elements on the substrate surface.  
     
     
         151 : The method of  claim 147 , wherein the array includes thousands of array elements on the substrate surface.  
     
     
         152 : The method of  claim 147 , wherein the array includes more than one thousand array elements on the substrate surface, said substrate comprising a microscope slide.  
     
     
         153 : The method of  claim 147 , wherein the array includes thousands of array elements on the substrate surface, said substrate comprising a microscope slide.  
     
     
         154 : The method of  claim 147 , wherein at least two ejected droplets are deposited at the same designated site on the substrate surface.  
     
     
         155 : The method of  claim 154 , wherein each of the at least two ejected droplets contains a biomolecule capable of covalent or noncovalent binding to another biomolecule.  
     
     
         156 : The method of  claim 147 , further comprising, prior to step (a), employing acoustic ejection in order to fill the first reservoir with the first fluid.  
     
     
         157 : The method of  claim 147 , further comprising, prior to step (a): 
 (a-1) acoustically coupling the ejector to a modifier reservoir containing a surface modification fluid; and    (a-2) activating the ejector to generate a modifier ejection acoustic wave having a focal point near the surface of the surface modification fluid in order to eject at least one droplet of the surface modification fluid toward the substrate surface for deposition thereon at the first designated site.    
     
     
         158 : The method of  claim 157 , wherein, steps (a-1) and (a-2) are repeated to deposit a droplet of the surface modification fluid at all designated sites.  
     
     
         159 : The method of  claim 158 , wherein the surface modification fluid increases the surface energy of the substrate surface with respect to each of the ejected fluids.  
     
     
         160 : The method of  claim 158 , wherein the surface modification fluid decreases the surface energy of the substrate surface with respect to each of the ejected fluids.  
     
     
         161 : The method of  claim 147 , further comprising, before each ejector activation step, acoustically measuring the fluid level in the reservoir in acoustically coupled relationship with the ejector, and using the measurements to adjust the focus of the acoustic radiation needed in each activation step.  
     
     
         162 : The method of  claim 161 , further comprising, before each ejector activation step, determining the position of the fluid surface in each reservoir in relation to the acoustic radiation generator, and using the determination to adjust the focused acoustic radiation required to ensure consistency in droplet trajectory.  
     
     
         163 : A biomolecular array comprising an array of a plurality of biomolecules on a substrate surface divided into a plurality of discrete surface sites, each site containing one biomolecule attached to the substrate surface in a localized region within the site, wherein the biomolecules are deposited onto the substrate surface in droplets having diameters in the range of about 1 micrometer to about 10,000 micrometers.  
     
     
         164 : The biomolecular array of  claim 163  wherein the array is formed by acoustic deposition of droplets.  
     
     
         165 : A biomolecular array comprising an array of a plurality of biomolecules on a substrate surface divided into a plurality of discrete surface sites, each site containing one biomolecule attached to the substrate surface in a localized region within the site, wherein more than one thousand biomolecules are deposited onto the substrate surface, said substrate comprising a microscope slide.  
     
     
         166 : The biomolecular array of  claim 165 , wherein thousands of biomolecules are deposited onto the substrate surface.