Microfluidic Aliquoting For Single-Cell Isolation

Abstract

According to the invention, generally, a microfluidic aliquoting (MA) chip, adapted to fit in a Petri dish, has a center well (inlet) connected by branched channels to a plurality of side wells (outlets). The chip comes in various types, including a bMA Chip T1, bMA Chip T2, bMA Chip T3, and an rMA Chip. The branched channel improvement provides for a greater distance between neighboring channels and a decreased density near the center well. Design improvements including an injection mold design for an insert and a base and a multiplex hole punch allow for rapid fabrication of the MA chip.

What is claimed is: 
     
         1 . A microfluidic aliquot (MA) chip for performing single-cell isolation, comprising:
 a chip having a radius, a center, a top surface, a bottom surface, an outer edge and a thickness;   a single center inlet well disposed substantially at the center of the chip, extending into and accessible from the top surface of the chip;   a plurality of side outlet wells disposed in an annular outer portion of the chip, extending into and accessible from the top surface of the chip; and   a plurality of channels having multiple segments that extend into the bottom surface of the chip and extending from the center inlet well to the side outlet wells in fluid communication with the center inlet well and the side outlet wells, configured to maintain uniform distribution of a liquid from the center inlet well to the side outlet wells.   
     
     
         2 . The MA chip of  claim 1 , wherein the multiple segments comprise:
 a plurality of first segments that form an inner layer, a plurality of last segments that form an outer layer, and a plurality of segments between the first segments and the last segments form a middle layer;   wherein the side outlet wells and the multiple segments are configured to uniformly distribute liquid and a plurality of cells when the liquid and cells are placed in the MA chip;   wherein the first segment is connected to the center inlet well in a radial pattern; and   wherein each segment after the first segment is divided from a prior segment in a radial pattern.   
     
     
         3 . The MA chip of  claim 2 , wherein a plurality of segments in the middle layer form a curved portion generally in the shape of a bend, whereby one of the first segments is joined to the bend of a segment in the middle layer. 
     
     
         4 . The MA chip of  claim 1 , wherein the multiple segments comprise:
 a plurality of first segments that extend from the center inlet well to a plurality of second segments;   wherein an end of a first segment branches at a 90 degree angle into two opposite directions that each lead into second segments; and   wherein subsequent branches of segments branch at 90 degree angles.   
     
     
         5 . The MA chip of  claim 4 , wherein the chip comprises:
 a sheet of flexible or semi-rigid material selected from a group consisting of polydimethylsiloxane (PDMS), PMMA (poly(methyl methacrylate)), PS (polystyrene), and PC (polycarbonate);   wherein the sheet has a top surface and a bottom surface, that correspond to the top and bottom surfaces of the chip, respectively.   
     
     
         6 . The MA chip of  claim 4 , wherein μm-scale markings are disposed on an inside of the side outlet wells and mm-scale markings are disposed on an outside of a first segment of a branched channel along four cardinal directions. 
     
     
         7 . The MA chip of  claim 4 , wherein mm-scale markings are disposed on an outside of the side outlet wells. 
     
     
         8 . The MA chip of  claim 4 , wherein the chip comprises:
 a center inlet well having a diameter of approximately 3 mm and a volume of 4 μl;   a plurality of side outlet wells having a diameter of approximately 1.5 mm and a volume of 1 μl;   a plurality of branched channels having a width of approximately 50 μm;   wherein the chip is sized and shaped to fit within a Petri dish of approximately 8.5 cm; and   wherein the chip is sized and shaped to fit within a Petri dish of approximately 10 cm.   
     
     
         9 . The MA chip of  claim 1 , wherein the chip comprises:
 a top layer having a center inlet well and a first plurality of channels;   a bottom layer having a well array and a second plurality of channels; and   wherein the second plurality of channels on the bottom layer are shorter and wider than the first plurality of channels on the top layer.   
     
     
         10 . The MA chip of  claim 1 , wherein the chip comprises:
 a top layer having a center inlet well and a well array; and   a bottom layer having an alignment mark array and a plurality of channels that extend outwards radially from a center of the bottom layer.   
     
     
         11 . The MA chip of  claim 12 , wherein the chip comprises:
 a well array having a diameter of 1-2 mm;   a center inlet well having a diameter of 2-4 mm;   a plurality of radial channels having a width of 0.03-0.1 mm and a length of 2-4 mm; and   an alignment mark array having a diameter of 0.5-1.5 mm.   
     
     
         12 . The MA chip of  claim 1 , wherein the chip comprises:
 a top layer having an adhesive side;   a bottom layer having a well array and a channel array; and   wherein the adhesive side of the top layer adheres to the bottom layer.   
     
     
         13 . The MA chip of  claim 14 , wherein the chip comprises a plurality of wells, each well labeled with an alphabetic letter. 
     
     
         14 . The MA chip of  claim 14 , wherein the channel array comprises:
 a first plurality of channels;   a second plurality of channels;   wherein the second plurality of channels is shorter and narrower than the first plurality of channels;   wherein wells are symmetrically distributed on both sides of the first plurality of channels; and   wherein the first plurality of channels are configured to distribute liquid into the second plurality of channels, and the second plurality of channels is configured to distribute liquid into the outlet wells.   
     
     
         15 . The MA chip of  claim 14 , wherein the chip comprises:
 a membrane on the top layer having a thickness of 0.03-0.3 mm;   a plurality of wells having a diameter of 0.5-5 mm and a height of 1-10 mm; and   wherein the chip has a length of 127.8±5 mm, a width of 85.5±3 mm, and a height of 1-10 mm.   
     
     
         16 . The MA chip of  claim 1 , wherein the center inlet well is configured to receive and distribute liquid through the multiple segments and into the wells. 
     
     
         17 . A method for making an MA chip, comprising:
 forming a pair of metal injection molds using a Computer Numerical Control (CNC) machine tool;   inserting a first metal injection mold into an injection molding machine;   injecting thermoplastic into the first metal injection mold to produce an insert;   inserting a second metal injection mold into the injection molding machine;   inserting thermoplastic into the second metal injection mold to produce a base; and   inserting a plurality of rivets on the base into a plurality of rivet through holes on the insert, thereby sealing the insert into the base.   
     
     
         18 . A method for creating holes in an MA chip, comprising:
 inserting an MA chip between a top and a bottom of an enclosure;   aligning the MA chip directly above a mold in the enclosure;   inserting a plurality of pins attached to a bottom of a pin head into a plurality of through holes on the top of the enclosure; and   pressing a top of the pin head so that the MA chip is pushed into the mold.   
     
     
         19 . A method for mass production of MA chips, comprising:
 conducting photolithography to produce a silicon mold;   injecting Polydimethylsiloxane (PDMS) into the silicon mold;   heating the silicon mold containing the PDMS to produce a first layer of an MA chip;   using a laser to cut a well array into a plastic material to produce a second layer of an MA chip; and   placing the first layer on a top of the second layer aligned directly below the first layer.