System and apparatus for sequential processing of analytes

• US 20090143244A1
• Filed: 10/12/2007
• Published: 06/04/2009
• Est. Priority Date: 05/23/1997
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Solexa Incorporated (Illumina Incorporated)
• Inventors: Corcoran, Kevin, Golda, George, Pallas, Michael C., Bridgham, John, Brenner, Sydney
• Granted Patent: US 8,361,713 B2
• Time in Patent Office: Days
• US Class Current: 506/12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2400/0403 specific forces
• CPC Class Codes: B01L 2400/0475 specific mechanical means and fluid pressure
• CPC Class Codes: B01L 2400/0622 distribution valves, valves having multiple inlets and/or outlets, e.g. metering valves, multi-way valves
• CPC Class Codes: B01L 2400/0644 rotary valves
• 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: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 15/1456 : without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 15/1484 : microstructural devices
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/0015 : in solids
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00

Abstract

An apparatus and system are provided for simultaneously analyzing a plurality of analytes anchored to microparticles. Microparticles each having a uniform population of a single kind of analyte attached are disposed as a substantially immobilized planar array inside of a flow chamber where steps of an analytical process are carried out by delivering a sequence of processing reagents to the microparticles by a fluidic system under microprocessor control. In response to such process steps, an optical signal is generated at the surface of each microparticle which is characteristic of the interaction between the analyte carried by the microparticle and the delivered processing reagent. The plurality of analytes are simultaneously analyzed by collecting and recording images of the optical signals generated by all the microparticles in the planar array. A key feature of the invention is the correlation of the sequence of optical signals generated by each microparticle in the planar array during the analytical process.

Claims

1. A closely packed planar array of microparticles,

wherein the number of microparticles per unit area of a planar array is at least eighty percent of the number of microparticles in a hexagonal array of equal area,

and wherein each microparticle carries a polynucleotide analyte and is marked with a fluorescently labelled processing reagent.

2. The array of claim 1, wherein the diameter of each of the microparticles is between about 0.1 μ

m and 100 μ

m.

3. The array of claim 1, wherein each microparticle is marked with a different polynucleotide analyte.

4. The array of claim 3, wherein said different polynucleotide analytes comprise a cDNA library.

5. The array of claim 1, wherein the planar array of microparticles is disposed in a flow chamber.

6. A fluidics system for sequentially delivering reagents to a population of analytes anchored to microparticles, said fluidics system comprising

a flow chamber for disposing said microparticles in a planar array such that the movement of the microparticles is restricted,

a computer controlled valve,

a series of reagent reservoirs,

and computer controlled means for moving said reagents from a selected reservoir into said flow chamber via said valve,

wherein said flow chamber is operationally associated with said fluidics system and with a detection system effective to visualize said microparticles, such that operation of said fluidics system and detection system is under computer control.

7. The fluidics system of claim 6, where said means for moving said reagents comprises gravity feed or pressure feed.

8. The fluidics system of claim 7, wherein said pressure feed is created via a peristaltic pump or syringe pump.

9. The fluidics system of claim 6, wherein said computer control operates to execute multiple cycles of (i) process steps, comprising said moving of reagents, and (ii) microparticle visualization by said detection system.

10. A method for simultaneously analysing the nucleotide sequences of a plurality of different-sequence polynucleotides, wherein

polynucleotides of different sequences are anchored to separate microparticles,

greater than 80% of the microparticles carry multiple copies of a single polynucleotide sequence, and the number of microparticles having an anchored polynucleotide is at least 100000,

and said method comprises

repeating processing cycles which comprise (i) exposing said microparticles and said polynucleotide sequences to fluorescently labelled reagents and (ii) detecting the fluorescent labels of said reagents.

11. The method according to claim 10, wherein the microparticles are substantially immobilized in a closely packed planar array in a flow chamber.

12. The method according to claim 10, wherein the plurality of different sequence polynucleotides comprises a cDNA library.

13. An apparatus for monitoring the interactions of fluorescent processing reagents with polynucleotide analytes on the surfaces of microparticles, said apparatus comprising

a computer controlled stage comprising a flow cell in which said microparticles are immobilized in a closely packed planar array,

an autofocus system operatively associated with said computer controlled stage, effective to optimize the focus of said array of microparticles,

an illumination system, effective to produce detectable fluorescent signals from said fluorescent reagents,

a motorised filter wheel comprising a plurality of band pass filters suitable for the detection of said signals, and

a CCD camera effective to record said signals.

14. The apparatus of claim 13, wherein said interactions of said fluorescent processing reagents with said polynucleotide analytes are dependent on the sequence of said polynucleotide analytes, and said fluorescent signals correspond to the identity of nucleotides in said polynucleotides.

15. The apparatus of claim 14, wherein said polynucleotide analytes comprise a cDNA library.

16. A method for tracking and analysis of multiple polynucleotide analytes through a series of processing steps, wherein said polynucleotide analytes are anchored to a plurality of microparticles, the method comprising:

(i) providing said plurality of microparticles, held in a closely packed planar array within a flow chamber,

(ii) delivering fluorescently labelled processing reagents to the flow chamber, such that said multiple polynucleotide analytes are contacted with the processing reagents to produce microparticles having fluorescent labels,

(iii) illuminating said plurality of microparticles, thereby producing fluorescent signals from the microparticles,

(iv) detecting and recording said fluorescent signals, and

(v) treating the microparticles to remove said fluorescent labels; and

repeating cycles of processing steps (ii)-(v) to generate a series of images of fluorescent signals from the microparticles.

17. The method of claim 16, further comprising the step of tracking the positions of said plurality of microparticles within the array between said cycles of processing steps.

18. The method of claim 17, wherein the average distance between centers of adjacent microparticles in the closely packed planar array is less than two microparticle diameters.

19. The method of claim 17, wherein the number of microparticles per unit area of the closely packed planar array is at least eighty percent of the number of microparticles in a hexagonal array of equal area.