Microfluidic devices and methods based on massively parallel picoreactors for cell and molecular diagnostics

• US 20130244906A1
• Filed: 05/04/2012
• Published: 09/19/2013
• Est. Priority Date: 05/05/2011
• Status: Active Grant

• Current Assignee: Biopico Systems
• Original Assignee: Biopico Systems
• Inventors: Collins, John
• Granted Patent: US 9,110,026 B2
• Time in Patent Office: Days
• US Class Current: 506/12
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/185 using a liquid as fluid
• CPC Class Codes: B01L 2300/1894 Cooling means; Cryo cooling
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/0445 Natural or forced convection
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/24 Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C40B 60/08 Integrated apparatus specially adapted for both creating and screening libraries
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence

Abstract

Microfluidic devices and methods that use massively parallel anchored picoreactors as the basis for cell and molecular diagnostics (e.g., characterizing, isolation, processing, amplification) different particles, chemical compositions or biospecies (e.g., different cells, cells containing different substances, different particles, different biochemical compositions, proteins, enzymes etc.).

Claims

1. A method of forming single cell or multiple cells reactors for performing single cell PCR, multiple cells PCR or single cell or multiple cells based assays in a microfluidic chip which consists of

a. Micropillars in an arranged configuration to trap single cell or multiple cells by flow.

b. Flow of other reagents to clean the cells, to stimulate the cells, to lyse the cells, to perform biochemical reactions such as PCR, to perform cell based assay.

c. Microarray spotting and protection of reagents such as primers, fluorescence probes, other reagents in one layer of the chip which are aligned and bonded to the micropillar layer of the chip.

d. Flow of immiscible fluids around the single or multiple cells to form an encapsulated reactors array.

e. Addition of aqueous reagents in to the encapsulated rector, if necessary, is accomplished by electrical or mechanical or fluidic methods.

f. Flow of immiscible fluids to perform thermal cycling or maintain at different isothermal temperatures.

g. Performance of imaging and analysis for enumeration of positive reaction, quantification of biochemical reactions not limited to gene expression analysis, copy number variation analysis.

h. Programming of the system to perform several fluidic, thermal, imaging, robotic and statistical steps of the device using sequential and parallel processing controls for clinical diagnostics, research or pharmaceutics.

i. Preprocessing cells, tissues or blood samples using sorting techniques based on size, density or immunoagents.

2. The method according to claim 1 wherein the step of trapping single or multiple cells comprises of a set of configured micropillars with 3-10 or more elements per trapping site.

a. The trapping site can constitute two or more layers of micropillars in a concentric or other configuration to trap the cells in one area and to maintain fluids around the cells for volume metering that will provide sufficient reagents. The micropillar spacing in the inner concentric layer is larger than the micropillar spacing in the outer concentric layer to facilitate complete isolation of cells from the surrounding immiscible fluidics for cell based assay.

b. The trapping site can constitute one or more micropillars in a pointing manner at the end opposite to the fluidic inlet for smooth droplet formation during the encapsulation of the cells by immiscible fluids.

c. The micropillars based trapped site may be oriented in any angle between 0 and 180 degree compared to the direction of the flow of flow of cells or other fluids.

d. Additional pillars are included as guidance for the cells to flow into the trapping sites.

3. The method according to claim 2 wherein the guiding pillars are smaller than the trapping pillars to avoid cell clusters outside the trapping sites and the immerse the guiding pillars in the immiscible fluids. The distance between the end of the guiding pillars and the trapping sites is 2 to 6 times the size of cells to avoid formation of cell clusters between the funnel shaped configured guiding pillars and the trapping sites.

4. The method according to claim 1 wherein flow of cleaning solution, to remove weakly adhered cells or clumps near cell trapping sites, from another inlet from the direction oriented in any angle between 0 and 180 degree compared to the direction of the flow of cells.

5. The method according to claim 1 wherein different aqueous reagents are flowed to stimulate the cells and to perform different biochemical reactions for diagnostics of diseases from the cell or to carryout cell based assays.

6. The method according to claim 1 wherein a combinatorial fluids are sent and encapsulated with the cells to perform a wide range of assays or to develop drug optimization.

7. The method according to claim 1 wherein immiscible fluids for encapsulation of cells at trapping sites include vegetable oil, mineral oil, fluorocarbonated oil, oil with different composition of surfactants, paraffins or aqueous fluids such as gels that will undergo a physical transition. The encapsulation fluid can be a gel that will allow certain molecule to move within itself with a certain mobility by the application of electric field such as in electrophoresis or any variation of electrical processing of cells and molecules.

8. The method according to claim 1 wherein microarray spotting of reagents can be protected by thermal sensitive gels and are released at the time of start of the biochemical reaction within the reactor. The release can also be by pH, electrical current or any other mechanisms.

9. The method according to claim 1 wherein addition of another reagent in to the encapsulated reactor is accomplished by a slip and lock immiscible fluidic platform or robotics or by electrical means such as EWOD, liquid dielectrophoresis or electrofusion.

10. The method according to claim 9 wherein continuous droplet based mRNA purification can be accomplished by a washing step using cascaded magneto-fission and fusion of droplets

11. The method according to claim 1 wherein the flow switching of immiscible fluids from aqueous fluid is accomplished by a set of valves in the fluidic manifold.

12. The method according to claim 1 wherein thermal cycling using immiscible fluids for performing biochemical reaction such as PCR or qPCR or any variation is accomplished by a pump and a set of valves.

13. The method according to claim 1 wherein imaging of multiple reactors in the chip are carried out by fluorescence imaging or other imaging techniques to extract the positive reaction of biochemical reactions including multiplexed qRT-PCR. Further analysis not limited to copy number variation, gene expression profile is carried out using the reaction software programs.

14. The method according to claim 1 wherein all the operations of fluidic, robotic, thermal, imaging and statistical steps are performed by software control.

15. The method according to claim 1 wherein preprocessing of tissue, cells, blood samples are carried out by method no limited to magnetic, immunoassay, hydrodynamics, diffusion, inertial fluidics.

16. The method according to claim 15 wherein preprocessing of cells are performed using with or without magneto diffusion coupled cascaded diffusion channels. The output of one diffusion channel will feed into the input of the next diffusion channel. These cascaded channels can form a circle or spirals with multiple turns and interconnected fluidics channels in spirals for improved efficiency.

17. The method according to claim 16 wherein preprocessing of cells are performed by inertial fluidics devices based on high aspect ratio spiral channels with wider semi circular flow focusing regions for the isolation of larger cells such as CTC and CD4 from whole blood. Increasing or decreasing width gradient periodic pinching regions are incorporated in spiral channel inertial fluidics. Further multiple spirals can be connected together to perform sorting of cells or blood samples through inertial fluidics.

18. The method according to claim 1 wherein additional inlet and outlet channels are provided to compartmentalize the cells and gather the cells respectively. Additional inlet channels also provided to supply different combinatorial cocktails to the reactors using diffusive mixing.