JPWO2022117547A5

JPWO2022117547A5 -

Info

Publication number: JPWO2022117547A5
Application number: JP2023530741A
Authority: JP
Publication date: 2024-08-27
Anticipated expiration: 2041-11-30

Claims

1. A system for classifying particles in a liquid medium, comprising:
a cartridge comprising at least one microfluidic sorting unit [9] ;
wherein the unit is
(i) at least one sample supply conduit [1] for supplying a sample fluid to a sorting junction point [2] ;
The sample fluid comprises a mixture of positive and negative particles to be sorted;
(ii) at least one microfluidic sorted positive particle conduit [4] for removing fluid containing positive particles from said sorting junction [2] ;
(iii) at least one microfluidic waste conduit [5] for removing waste fluid containing negative particles from said sorting junction [2] ;
and (iv) at least one extrusion fluid conduit [3] for supplying an extrusion fluid to said sorting junction point [2].
With
said sorting unit [9] being characterised in that at least four microfluidic conduits (i), (ii), (iii) and (iv) meet at said sorting junction point [2];
the cartridge further comprises at least one detection zone for detecting positive particles in the sample supply conduit (i) upstream of the sorting junction;
and an apparatus for controlling fluid flow through the at least one microfluidic sorting unit and temporarily reducing a flow rate of particles in the sample supply conduit [1] in response to detecting positive particles in the supply conduit upstream of the sorting junction [2] ,
The system wherein the particles are double emulsion droplets.

2. The system of claim 1,
The at least one microfluidic sorting unit [9] comprises:
(v) a spacer fluid conduit [11] and a spacer fluid junction [10] arranged in the sample supply conduit [1] upstream of the sorting junction point [2] for supplying a spacer fluid into the sample fluid containing the particles to be sorted.
Further equipped with
The system specifically comprises at least two separate detection zones;
an upstream detection zone [12] is disposed upstream of said spacer fluid junction [10] for detecting particles before they reach said spacer fluid junction [10];
and
a downstream detection zone [13] located downstream of the spacer fluid inlet for detecting positive particles before they enter the sorting junction [2];
and
The downstream detection zone is specifically designed to allow the device to detect whether a correct classification has been made.
system.

A system according to any one of claims 1 to 2 ,
The cartridge comprises a set of wells or containers for fluids capable of accommodating the total volume of fluids required to accomplish the classification, including the sample fluid, the waste fluid, and the classified positive particle fluid.

A system according to any one of claims 2 to 3 ,
means for triggering a sorting event by applying air pressure to a spacer fluid inlet [18] in response to detecting a positive particle upstream of the sorting junction [2] ;
and/or
means for temporarily reducing the flow rate of particles in the sample supply conduit [1] for a period of one second or less after detecting a positive particle upstream of the sorting junction;
system.

A system according to any one of claims 1 to 4 ,
A system in which both the extrusion fluid and the spacer fluid are water-based fluids.

A system according to any one of claims 1 to 5 ,
A system comprising means for applying a variable negative pressure to said sorted positive particle conduit [4] and to said waste conduit [5].

A system according to any one of claims 1 to 6 ,
the cartridge comprises two or more sorting lanes;
and the two or more sorting lanes are arranged in pairs ,
The at least one detection zone specifically comprises at least two separate particle supply conduit portions of a sorting lane pair,
and
Each pair of sorting lanes specifically comprises an upstream detection zone [12] and a downstream detection zone [13].
system.

8. A system according to claim 7 , comprising:
the instrument detects signals from two separate droplet sample supply conduits of a classification lane pair at the upstream detection zone;
and wherein each of the two sample supply conduits includes a detection loop, and the two detection loops are of different lengths.

A system according to any one of claims 7 to 8 ,
A system in which the two or more sortation lanes are pneumatically connected to the same pressure source.

A system according to any one of claims 1 to 9 ,
The device is designed to fit into the cartridge and to control and drive the fluid through the cartridge, as well as a system designed to fit into, control and drive the fluid through a cartridge made to produce single emulsion droplets and a cartridge made to produce double emulsion droplets.

A cartridge comprising at least one microfluidic sorting unit [9] according to any one of claims 1 to 10 ,
The unit comprises:
(i) at least one sample supply conduit [1] for supplying a sample fluid to a sorting junction point [2] ;
The sample fluid comprises a mixture of positive and negative particles to be sorted;
(ii) at least one microfluidic sorted positive particle conduit [4] for removing fluid containing positive particles from said sorting junction [2] ;
(iii) at least one microfluidic waste conduit [5] for removing waste fluid containing negative particles from said sorting junction [2] ;
and (iv) at least one extrusion fluid conduit [3] for supplying an extrusion fluid to said sorting junction point [2].
With
said sorting unit [5] being characterised in that at least four microfluidic conduits (i), (ii), (iii) and (iv) meet at said sorting junction point [2];
the cartridge further comprises at least one detection zone for detecting positive particles in the sample supply conduit (i) upstream of the sorting junction;
The cartridge wherein the particles are double emulsion droplets.

A system as claimed in claims 1 to 10 ,
said instrument comprises an optical head forming an integral unit comprising an optical system forming two linear detection zones, i.e. said upstream and said downstream detection zones;
The optical head further comprises the necessary optics and detectors for the detection of positive particles ;
The instrument is specifically adapted to detect positive particles by laser-induced fluorescence signals,
The instrument is specifically adapted to collect the fluorescent signals emanating from positive particles in both the upstream and downstream detection zones through a single lens system;
and
The instrument may include an automatic alignment system that aligns the cartridge with the optical system;
the alignment system includes three actuators that move the optical head in the XY plane to align the optical system with the cartridge;
system.

A system as claimed in any one of claims 1 to 10 and 12 ,
the instrument controls the flow of fluid through the at least one microfluidic sorting unit by a pneumatic system operating under both variable positive pressure and variable negative pressure ;
The pneumatic system specifically comprises a set of directional control valves capable of supplying high or low positive pressure to the microfluidic system, and another set of directional control valves capable of supplying variable negative or ambient pressure to the microfluidic system.
system.

A method for classifying particles comprising the use of a system according to any one of claims 1 to 10 and 12 to 13 ,
the particles are double emulsion droplets;
The method comprises the steps of:
i. providing a sample fluid containing particles;
ii. Providing a microfluidic cartridge according to claim 11 , comprising:
a supply well or vessel comprising a volume of extrusion fluid;
a supply well or vessel comprising a volume of spacer fluid;
and a supply well or vessel containing a volume of said particle sample fluid;
iii. Inserting the cartridge into the device;
iv. initiating said classification on said system;
and v. transferring the sorted positive particles into a suitable container after the sorting is complete;
Including;
A method wherein particle flow rate is temporarily reduced in response to a positive signal being detected by said instrument at said upstream detection zone [12] of said cartridge.

1. An in vitro method for enriching one or more target nucleic acid molecules from a sample of mixed nucleic acid molecules, comprising the steps of:
i. providing a liquid sample of mixed nucleic acid molecules comprising at least one or more specific target nucleic acid molecules and at least one reagent for specifically detecting at least one of said target nucleic acid molecules;
ii. forming an emulsion from the liquid sample, the emulsion comprising a plurality of double emulsion droplets, each of the emulsion droplets comprising a mixed nucleic acid molecule;
iii. Incubating the emulsion droplets to obtain a specific detectable reaction in the droplets containing at least one specific target nucleic acid molecule;
iv. loading the reacted droplets into a system for sorting droplets as described in any one of claims 1 to 10 and 12 to 13 ;
v. sorting the microdroplets using the system;
vi. collecting the sorted droplets in a suitable container and combining said sorted droplets;
and vii. subjecting the combined and selected droplets from step vi to a general amplification procedure;
The method includes:

A kit of parts for carrying out any of the methods according to any one of claims 14 to 15 , comprising:
a) at least one cartridge according to claim 11 ,
b) at least one gasket for fitting said at least one cartridge to an implement of a system for sorting particles, in particular to an implement of a system according to any one of claims 1 to 10 or 12 to 13 ;
The particles are double emulsion droplets;
and c) at least one vial of buffer fluid in an amount sufficient to perform the number of sorts provided by said at least one cartridge;
A kit comprising: