Devices and processes for analysing individual cells

Abstract

A device for individually analysing cells of interest, comprising (a) a channel for receiving the contents of a cell of interest, wherein the channel has an input end and an output end, and (b) a cell trapping site in proximity to the input end of the channel, wherein (i) the input end of the channel is adapted such that an intact cell of interest cannot enter the channel; and (ii) the channel contains one or more analytical components for analysing the contents of the cell of interest. In use, a cell is applied to the device, where it is trapped by the cell trapping means. The cell cannot enter the channel intact, but its contents can be released in situ to enter the channel's input end. The contents can then move down the channel, towards the output end, and they encounter the immobilised reagents, thereby permitting analysis of the cell contents.

1 - 68 . (canceled) 
     
     
         69 . A device for individually analysing cells of interest, comprising:
 a channel for receiving the contents of a cell of interest, wherein the channel has an input end and an output end; and   a cell trapping site in proximity to the input end of the channel,   
       wherein:
 the input end of the channel is adapted such that an intact cell of interest cannot enter the channel; 
 the channel contains one or more analytical component(s) arranged in discrete patches for analysing the contents of the cell of interest; 
 the contents of the cell of interest can be moved along the channel, in a direction from the input end towards the output end; and 
 the device includes one or more feature selected from the group consisting of (a), (b) and (c); 
 
       (a) the channel has a rectangular cross-sectional shape and a height <50 μm; 
       (b) each of said patches has an area of less than 10 −6  m 2 ; and/or 
       (c) said patches are arranged such that they occupy the full width of the channel. 
     
     
         70 . The device of  claim 69 , wherein the device has: a plurality of said channels, each of which is for receiving the contents of an individual cell of interest; and a cell trapping site in proximity to the input end of each channel. 
     
     
         71 . The device of  claim 70 , wherein the channels are substantially identical to each other such that, during use, cells in different channels are separately subjected to substantially the same treatment and analysis as each other. 
     
     
         72 . The device of  claim 71 , wherein each channel contains a sequence of analytical components along the channel, and wherein the sequence of analytical components in one channel is the same as in another channel. 
     
     
         73 . The device of  claim 69 , wherein the channels are substantially parallel to each other. 
     
     
         74 . The device of  claim 73 , wherein the channels are arranged next to each other within a single plane. 
     
     
         75 . The device of  claim 69 , wherein the channel(s) have a substantially constant cross-sectional area and/or a substantially constant cross-sectional shape. 
     
     
         76 . The device of  claim 69 , wherein the channel(s) have a rectangular cross-sectional shape. 
     
     
         77 . The device of  claim 69 , wherein the channel(s) have a height <50 μm. 
     
     
         78 . The device of  claim 69 , wherein the channel(s) are closed except in the direction of flow from input end to output end. 
     
     
         79 . The device of  claim 69 , wherein the cell trapping site(s) is/are in the form of a tapered inlet before the input end of a channel. 
     
     
         80 . The device of  claim 79 , wherein a cell can be moved into the tapered inlet by the use of electrokinesis. 
     
     
         81 . The device of  claim 69 , wherein a channel includes an expansion chamber either (i) between its cell trapping site and its input end or (ii) immediately downstream of its input end. 
     
     
         82 . The device of  claim 69 , wherein a channel comprises a polydimethylsiloxane wall. 
     
     
         83 . The device of  claim 69 , wherein a channel comprises a glass wall. 
     
     
         84 . The device of  claim 83 , wherein a channel is formed from polydimethylsiloxane mounted on a flat glass support. 
     
     
         85 . The device of  claim 69 , wherein the analytical component(s) are immobilised binding reagents. 
     
     
         86 . The device of  claim 85 , wherein the analytical component(s) are covalently immobilised. 
     
     
         87 . The device of  claim 85 , wherein the binding reagents include nucleic acids for hybridisation. 
     
     
         88 . The device of  claim 87 , wherein the nucleic acids can retain mRNA transcripts. 
     
     
         89 . The device of  claim 87 , wherein the nucleic acids are DNA. 
     
     
         90 . The device of  claim 87 , wherein the nucleic acids are <200 nt. 
     
     
         91 . The device of  claim 85 , wherein the binding reagents include immobilised proteins. 
     
     
         92 . The device of  claim 85 , wherein the binding reagents include immobilised antibodies. 
     
     
         93 . The device of  claim 85 , wherein the analytical reagent(s) are immobilised along only one side of a channel. 
     
     
         94 . The device of  claim 85 , comprising at least 100 different analytical reagents per channel. 
     
     
         95 . The device of  claim 85 , wherein different immobilised binding reagents are arranged in discrete patches. 
     
     
         96 . The device of  claim 95 , wherein each patch of immobilised reagent has an area of less than 10 −8  m 2 . 
     
     
         97 . The device of  claim 95 , wherein the centre-to-centre separation of adjacent patches is less than 10 −3  m. 
     
     
         98 . The device of  claim 95 , wherein the patches have a rectangular shape. 
     
     
         99 . The device of  claim 95 , wherein the patches are arranged singly in series along a channel's length from input end to output end. 
     
     
         100 . The device of  claim 95 , wherein the patches occupy the full width of a channel. 
     
     
         101 . The device of  claim 69 , comprising a plurality of channels and a plurality of immobilised analysis reagents, wherein the channels intersect lines of immobilised analysis reagents. 
     
     
         102 . The device of  claim 101 , wherein the channels are straight and are substantially parallel to each other. 
     
     
         103 . The device of  claim 101 , wherein the lines of immobilised analysis reagents are straight and are substantially parallel to each other. 
     
     
         104 . The device of  claim 101 , wherein the lines of immobilised analysis reagents run substantially orthogonal to the channels. 
     
     
         105 . The device of  claim 69 , including a delivery line in communication with the cell trapping site(s). 
     
     
         106 . The device of  claim 105 , wherein the delivery line runs perpendicular to the channel(s). 
     
     
         107 . The device of  claim 105 , wherein the delivery line runs parallel to the channel(s). 
     
     
         108 . The device of  claim 69 , including a reagent supply line in communication with the cell trapping site(s). 
     
     
         109 . The device of  claim 105 , wherein the delivery line is taller than the channels. 
     
     
         110 . The device of  claim 69 , including an exhaust in communication with the output end(s) of the analysis channel(s). 
     
     
         111 . The device of  claim 69 , including a pump for moving liquids through the channel(s). 
     
     
         112 . The device of  claim 69 , including one or more electrodes, or connectors for attachment of electrodes. 
     
     
         113 . The device of  claim 112 , wherein the electrodes can be used to generate an electrical potential along a channel. 
     
     
         114 . The device of  claim 69 , including a light source. 
     
     
         115 . The device of  claim 69 , including a camera. 
     
     
         116 . A process for analysing an individual cell of interest, comprising the steps of: trapping the cell in proximity to the input end of a channel that has an input end and an output end, the input end being adapted such that the cell of interest cannot enter the channel intact; releasing the cell's contents such that they enter the input end of the channel; allowing the released contents to move from the input end towards the output end, such that they interact with one or more analytical component(s) within the channel, thereby permitting analysis of the contents,
 said device comprising:
 a channel for receiving the contents of a cell of interest, wherein the channel has an input end and an output end; and 
 a cell trapping site in proximity to the input end of the channel, 
   wherein:
 the input end of the channel is adapted such that an intact cell of interest cannot enter the channel; 
 the channel contains one or more analytical component(s) arranged in discrete patches for analysing the contents of the cell of interest; 
 the contents of the cell of interest can be moved along the channel, in a direction from the input end towards the output end; and 
 the device includes one or more feature selected from the group consisting of (a), (b) and (c); 
   (a) the channel has a rectangular cross-sectional shape and a height <50 μm;   (b) each of said patches has an area of less than 10 −6  m 2 ; and/or   (c) said patches are arranged such that they occupy the full width of the channel.   
     
     
         117 . The process of  claim 116 , wherein the contents of a cell are moved along the channel by electrokinesis. 
     
     
         118 . The process of  claim 116 , wherein the contents of a cell are moved along the channel by pumping. 
     
     
         119 . The process of  claim 116 , wherein the analytical component(s) can capture mRNA from a cell by nucleic acid hybridisation. 
     
     
         120 . The process of  claim 119 , wherein mRNA captured by analytical component(s) are reverse transcribed. 
     
     
         121 . The process of  claim 120 , wherein analysis comprises the steps of (i) permitting the mRNA to hybridise to immobilised nucleic acids in a channel, such that the mRNA has a single stranded overhang in the hybrid; (ii) extending the immobilised nucleic acid in the hybrid using the single stranded overhang as a template, wherein the extension reaction incorporates a detectable label into the immobilised nucleic acid. 
     
     
         122 . The process of  claim 121 , wherein analysis further comprises: (iii) melting the hybrid and allowing the free nucleic acid to re-anneal to an immobilised nucleic acid, to form a new hybrid in which the free nucleic acid has a single stranded overhang; and (iv) repeating step (ii) at least n times, where n is an integer >1, provided that where n>1 then step (iii) is performed after at least the first n−1 repeats of step (ii). 
     
     
         123 . The process of  claim 119 , wherein a DNA/mRNA hybrid is detected using an apparatus that can identify single fluorophores. 
     
     
         124 . The process of  claim 119 , wherein at least 80% of the mRNA targets within a cell are captured for analysis. 
     
     
         125 . A process for making the device of  claim 69 , wherein the channels are formed within a polymeric material. 
     
     
         126 . The process of  claim 125 , wherein the polymeric material is photo-polymerisable. 
     
     
         127 . The process of  claim 125 , wherein the channels are formed by casting or injection molding of the polymeric material. 
     
     
         128 . The process of  claim 125 , wherein the polymeric material is PDMS. 
     
     
         129 . The process of  claim 125 , wherein the analytical component(s) is/are immobilised nucleic acids for hybridisation. 
     
     
         130 . The process of  claim 129 , wherein the nucleic acids are attached to a surface of the device using an in situ synthesis method. 
     
     
         131 . The process of  claim 129 , wherein the nucleic acids are synthesised before being attached to a surface of the device. 
     
     
         132 . The process of  claim 131 , wherein the nucleic acids are applied to a surface of the device by forming a contact between a reaction surface on a reaction substrate and an open microfluidic channel on a microfluidic channel substrate; (b) introducing a reagent into the microfluidic channel such that the reagent contacts the reaction surface along a contact line formed by the contact between the reaction surface and the open microfluidic channel; and (c) separating the reaction surface and the microfluidic channel, leaving the reagent immobilised along the contact line on the reaction surface. 
     
     
         133 . The process of  claim 131 , wherein the nucleic acids are applied to a surface of the device by deprotecting a region on the surface of a substrate to expose a reactive group, and applying pre-synthesised nucleic acids to the substrate to allow them to bind to the exposed reactive groups. 
     
     
         134 . The process of  claim 133 , wherein the deprotection is photo-deprotection. 
     
     
         135 . The process of  claim 133 , wherein the deprotection is electrochemical deprotection. 
     
     
         136 . The device of  claim 108 , wherein the reagent supply line is taller than the channels.