Method, apparatus and flow cell for high sensitivity detection of fluorescent molecules

Abstract

Method and apparatus for high-sensitivity fluorescence detection wherein (I) a sample comprising fluorescent molecules is made to flow through a channel structure ( 1 ) including a constricted region ( 2 ) with a dimension corresponding to the size of a tightly focused laser spot and with extremely thin, transparent walls; (II) a laser beam ( 3 ) is focused inside the constricted ( 2 ) and thus exciting molecules passing through the constricted region ( 2 ); and (III) the fluorescence emitted due to excitation is detected. This enables direct determination of the concentration of a sample without use of internal or external standards. A method for the production of a flow cell for use in the method or apparatus, wherein a part of a channel structure ( 1 ) is heated until its melting point is reached, followed by pulling of the structure to lengthen the melted region and make it thinner until it has a dimension corresponding to the size of a tightly focused laser spot at the diffraction limit.

1 . An optical method for high-sensitivity detection of fluorescent molecules based on the use of a highly focused light beam and light-induced fluorescence spectroscopy characterised in that 
 (I) a sample comprising at least one fluorescent molecule is made to flow through at least one flow cell consisting of at least one channel structure ( 1 ) comprising at least one constricted region ( 2 ), said at least one constricted region ( 2 ) having a cross-section of a dimension corresponding to the size of a tightly focused light spot close to or at the diffraction limit and extremely thin, transparent walls,    (II) at least one light beam ( 3 ) is focused close to or at the diffraction limit inside said at least one constricted region ( 2 ) and thus exciting any fluorescent molecules present in the sample volume passing through said at least one constricted region ( 2 ), and    (III) the fluorescence emitted when a fluorescent molecule or a group of molecules passes through said at least one constricted region ( 2 ) and is excited is detected.    
     
     
         2 . A method according to  claim 2 , wherein said at least one light beam ( 3 ) is a laser beam.  
     
     
         3 . A method according to  claim 1  or  2 , wherein at least on single fluorescent molecule or a particle with a conservative number of fluorophores is detected.  
     
     
         4 . A method according to  claim 3 , wherein the concentration of said molecule or particle in a sample is determined directly without the use of external or internal standards.  
     
     
         5 . A method according to any one of claims  1 - 4 , wherein said at least one constricted region ( 2 ) of the flow cell is placed in a medium, such as oil or water, with a refractive index close to that of the material of said at least one constricted region.  
     
     
         6 . A method according to any one of claims  1 - 5 , wherein the channel structure ( 1 ) is a capillary.  
     
     
         7 . A method according to  claim 6 , wherein the capillary is a fused silica glass capillary.  
     
     
         8 . A method according to any one of claims  1 - 5 , wherein the channel structure ( 1 ) is a groove etched into a chip.  
     
     
         9 . A method according to any one of claims  1 - 8 , wherein said at least one constricted region ( 2 ) has an inner diameter of approximately 0.2-8 μm and an outer diameter of approximately 0.4-40 μm.  
     
     
         10 . A method according to  claim 9 , wherein said at least one constricted region ( 2 ) has an inner diameter of 1-2 μm and an outer diameter less than or equal to 5 times of the inner diameter.  
     
     
         11 . A method according-to any one of claims  1 - 10 , wherein the light source ( 4 ) is an argon ion laser.  
     
     
         12 . A method according to any one of claims  1 - 11 , wherein the wavelength of the light beam ( 3 ) used is approximately between 200 and 1500 nm.  
     
     
         13 . A method according to any one of claims  1 - 12 , wherein the detection is made through use of a highly sensitive photon detector ( 12 ), such as a single photon counting diode or a photon counting photomultiplier tube, or a highly sensitive photon counting charge coupled device, a VIM camera or a streak camera.  
     
     
         14 . A method according to any one of claims  1 - 13 , wherein detection is made at a single wavelength.  
     
     
         15 . A method according to any one of claims  1 - 14 , wherein detection is made in a multicolour format.  
     
     
         16 . A method according to any one of claims  1 - 15 , wherein detection is made in a confocal mode.  
     
     
         17 . A method according to any one of claims  1 - 16 , wherein more than one light source are used, each light source emitting light at a different wavelength.  
     
     
         18 . A method according to any one of claims  1 - 17 , wherein detection is made at more than one constricted region ( 2 ) for cross-correlation of the data.  
     
     
         19 . A method according to any one of claims  1 - 18 , wherein a two-photon or a multi-photon mode is used for the excitation of the fluorescent molecules.  
     
     
         20 . An apparatus for high-sensitivity detection of fluorescent molecules comprising at least one light source ( 4 ) and at least one fluorescence detector ( 12 ), characterised in that it further comprises at least one flow cell consisting of at least one channel structure ( 1 ) comprising at least one constricted region ( 2 ), said at least one constricted region ( 2 ) having a cross-section of a dimension corresponding to the size of a tightly focused light spot close to or at the diffraction limit and extremely thin, transparent walls, of an outer diameter no more than 10 times as large than that of said focused light spot, said at least one channel structure ( 1 ) being adapted to accommodate the sample comprising the molecule or molecules to be detected.  
     
     
         21 . An apparatus according to  claim 20 , wherein said at least one light source ( 4 ) is a laser source.  
     
     
         22 . An apparatus according to  claim 20  or  21 , further comprising a means for focusing the beam ( 3 ) from the light source ( 4 ) close to or at the diffraction limit inside said constricted region.  
     
     
         23 . An apparatus according to  claim 22 , wherein said means for focusing of the light beam ( 3 ) is a high-numerical aperture microscope objective ( 9 ).  
     
     
         24 . An apparatus according to  claim 22  or  23 , wherein a medium, such as oil or water, with a refractive index close to that of the material of said at least one constricted region ( 2 ) of the channel structure ( 1 ) is arranged between said means for focusing and said at least one constricted region ( 2 ).  
     
     
         25 . An apparatus according to any one of claims  20 - 24 , wherein the channel structure ( 1 ) is a capillary.  
     
     
         26 . An apparatus according to  claim 25 , wherein the capillary is a fused silica glass capillary.  
     
     
         27 . An apparatus according to any one of claims  20 - 24 , wherein the channel structure ( 1 ) is a groove etched into a chip.  
     
     
         28 . An apparatus according to any one of claims  20 - 27 , wherein said at least one constricted region ( 2 ) has an inner diameter of approximately 0.2-8 μm and an outer diameter of approximately 0.4-40 μm.  
     
     
         29 . A method according to  claim 28 , wherein said at east one constricted region ( 2 ) has an inner diameter of 1-2 μm and an outer diameter less than or equal to 5 times of the inner diameter.  
     
     
         30 . An apparatus according to any one of claims  20 - 29 , wherein the light source ( 4 ) is an argon ion laser.  
     
     
         31 . An apparatus according to any one of claims  20 - 30 , wherein said fluorescence detector ( 12 ) is a highly sensitive photon detector, such as a single photon counting diode or a photon counting photomultiplier tube, or a highly sensitive photon counting charge coupled device, a VIM camera or a streak camera.  
     
     
         32 . An apparatus according to any one of claims  20 - 31 , wherein the detector ( 12 ) is adapted for confocal detection.  
     
     
         33 . An apparatus according to any one of claims  20 - 32 , wherein the flow channel forms an integrated and continuos part of a flow injection analysis system or a separation system, such as a capillary electrophoresis, capillary electrochromatography, liquid chromatography, or gas chromatography system.  
     
     
         34 . A method for the production of a flow cell for use in high-sensitivity detection of fluorescent molecules characterised in that a channel structure ( 1 ) is obtained by an appropriate method, and at least one region of said channel structure ( 1 ) is then heated until the melting point of the material constituting the channel structure ( 1 ) is reached, and in that the channel structure ( 1 ) finally is pulled in order to lengthen the melted region and thus make it thinner until it has a dimension corresponding to the size of a tightly focused laser spot close to or at the diffraction limit, said material constituting the channel structure ( 1 ) being transparent or turning transparent during the heat treatment.  
     
     
         35 . A method according to  claim 34 , wherein the region ( 2 ) with a dimension corresponding to the size of a tightly focused laser spot close to or at the diffraction limit has an inner diameter of 0.2-8 μm and an outer diameter of 0.4-40 μm .  
     
     
         36 . A method according to  claim 35 , wherein the inner diameter is 1-2 μm and the outer diameter is less than or equal to 5 times of the inner diameter.  
     
     
         37 . A method according to any one of the claims claim  34 - 36 , wherein said channel structure ( 1 ) is a fused silica glass capillary.  
     
     
         38 . A method according to any one of claims  34 - 37 , wherein a butane/oxygen torch burner is used for the heating.  
     
     
         39 . A method according to any one of claims  34 - 38 , wherein a filament or a CO 2  laser is used for the heating.  
     
     
         40 . A method according to any one of claims  34 - 39 , wherein the channel structure ( 1 ) is pulled by use of electronic actuators, mechanical manipulators or weight.  
     
     
         41 . A method according to any one of claims  34 - 40 , wherein the inner surface of the channel structure ( 1 ) is modified before or after formation of the constricted region ( 2 ).  
     
     
         42 . A method according to  claim 41 , wherein the inner surface is coated with a strongly hydrophobic film.  
     
     
         43 . A method according to any one of claims  34 - 42 , wherein the channel structure ( 1 ) is packed with a suitable material, such as beads, before or after formation of the constricted region ( 2 ).  
     
     
         44 . method according to any one of claims  34 - 43 , wherein the flow cell is mounted in a holder.  
     
     
         45 . A method according to any one of claims  34 - 44 , wherein the pulled region is coated with a transparent polymeric material.  
     
     
         46 . A method according to any one of claims  34 - 45 , wherein the flow cell forms an integrated and continuous part of a separation channel.  
     
     
         47 . Use of the method according to any one of claims  1 - 19 , the apparatus according to any one of claims  20 - 33 , and/or the flow cell produced according to any one of claims  34 - 46  in combination with a microscope.  
     
     
         48 . Use according to  claim 47 , wherein said microscope is a confocal fluorescence microscope.  
     
     
         49 . Use according to  claim 48 , wherein the confocal fluorescence microscope is a scanning confocal fluorescence microscope.  
     
     
         50 . Use according to  claim 49 , wherein the scanning confocal fluorescence microscope is a multi-photon scanning confocal fluorescence microscope.