Universal molecular processor for precision medicine

Patent metadata

Field	Value
Publication number	US-10830757-B2
Application number	US-201916513947-A
Country	US
Kind code	B2
Filing date	Jul 17, 2019
Priority date	Mar 23, 2015
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

The present invention is directed to methods comprising a device that comprises a biomolecular processor and one or more nanotubes. Each biomolecular processor comprises a bioreactor chamber defined by a solid substrate, a plurality of spaced support structures within said bioreactor chamber and attached to the solid substrate, one or more nanotubes defined by the solid substrate and fluidically coupled to the bioreactor chamber and one or more capture molecules immobilized to some or all of said plurality of spaced support structures, said one or more capture molecules suitable to bind to a portion of a target nucleic acid molecule in a sample. The nanotubes have a passage extending between an input end proximate to the bioreactor chamber and an output end distal to the bioreactor chamber, and comprises one or more nanopores within the passage with each nanopore having a reduced diameter relative to the passage.

First claim

What is claimed:

1. A method for detecting a presence of a target nucleic acid molecule in a sample, said method comprising:
providing a device comprising:
a biomolecular processor, each biomolecular processor comprising:
a bioreactor chamber defined by a solid substrate;
a plurality of spaced support structures within said bioreactor chamber and attached to the solid substrate;
one or more capture molecules immobilized to some or all of said plurality of spaced support structures, said one or more capture molecules suitable to bind to a portion of a target nucleic acid molecule in a sample;
one or more nanotubes defined by the solid substrate and fluidically coupled to the bioreactor chamber; each of said one or more nanotubes having a passage extending between an input end proximate to said bioreactor chamber and an output end distal to said bioreactor chamber and comprising one or more nanopores within the passage with each nanopore having a reduced diameter relative to the passage;
electrodes positioned at locations upstream of said bioreactor chamber and downstream of said one or more nanotubes;
a voltage source electrically coupled to said electrodes to establish a voltage gradient between a location upstream of said bioreactor chamber and downstream of said one or more nanotubes causing molecules to pass from said bioreactor chamber through said one or more nanotubes to the output end; and
a detector positioned to measure changes in current levels across the one or more nanopores as molecules pass through said one or more nanotubes;

feeding a sample comprising a target nucleic acid molecule into said biomolecular processor under conditions effective for the target nucleic acid molecule to bind to the capture molecules and to be immobilized to the spaced support structures;
subjecting the immobilized target nucleic acid molecule or immobilized extension products thereof to a ligase detection reaction to produce ligation products hybridized to the immobilized target nucleic acid molecules or immobilized extension products thereof;
denaturing the ligation products from the immobilized target nucleic acid molecules, or immobilized extension products thereof, to release the ligation products from the spaced support structures;
passing the ligation products through the one or more nanotubes;
detecting, with said detector, identifying signatures of ligation products passing through the one or more nanotubes; and
identifying the presence of the target nucleic acid molecule in the sample, differing from other nucleic acid molecules in the sample, based on said detecting.

2. The method of claim 1 , wherein the device further comprises:
one or more units for sample preparation upstream of said biomolecular processor and one or more nanotubes, wherein the one or more units comprise:
a separator unit defined by the solid substrate and upstream of said biomolecular processor and one or more nanotubes, said separator unit comprising a separation chamber including solid surfaces defining channels between them with cell specific capture agents attached to the solid surfaces, an inlet to the chamber, and an outlet from the chamber,
said method further comprising:
passing the sample through said separator unit prior to said feeding the sample into said biomolecular processor.

3. The method of claim 1 , wherein the device further comprises:
one or more units for sample preparation upstream of said biomolecular processor and one or more nanotubes, wherein the one or more units comprise:
a sensor unit defined by the solid substrate and upstream of said biomolecular processor and one or more nanotubes, said sensor unit comprising:
an inlet;
an outlet; and

a cell counter positioned to count cells passing from the inlet to the outlet of said sensor unit,
said method further comprising:
passing the sample through said sensor unit prior to said feeding the sample into said biomolecular processor.

4. The method of claim 1 , wherein the device further comprises:
one or more units for sample preparation upstream of said biomolecular processor and one or more nanotubes, wherein the one or more units comprise:
an extractor unit defined by the solid substrate and upstream of said biomolecular processor and one or more nanotubes, said extractor unit comprising solid supports and passages between them, wherein the solid supports are provided with a material suitable to immobilize nucleic acids, or exosomes,

said method further comprising:
passing the sample through said extractor unit prior to said feeding the sample into said biomolecular processor.

5. The method of claim 1 , wherein the device further comprises:
one or more units for sample preparation upstream of said biomolecular processor and one or more nanotubes, wherein the one or more units comprise:
a longitudinally-extending plasma isolation unit defined by the solid substrate and upstream of said biomolecular processor and one or more nanotubes, said longitudinally-extending plasma isolation unit comprising:
an entrance passage;
a discharge passage which is wider and shallower than the entrance passage;
a transition passage connecting the entrance passage and the discharge passage, said transition passage becoming wider and shallower as the transition passages progresses from the entrance passage to the discharge passage;
primary side channels extending laterally away from the entrance passage, wherein a separator, positioned between the entrance passage and each primary side channel, is sized to permit plasma, but not cells, to pass from the entrance passage to the primary side channels; and
secondary side channels extending laterally away from the discharge passage, wherein a separator, positioned between the discharge passage and each secondary side channel, is sized to permit plasma, but not cells, to pass from the entrance passage to the secondary side channels,

said method further comprising:
passing the sample through said plasma isolation unit prior to said feeding the sample into said biomolecular processor.

6. The method of claim 1 , wherein the one or more units comprises:
a separator unit defined by the solid substrate, said separator unit comprising:
a separation chamber including solid surfaces defining channels between them with cell specific capture agents attached to the solid surfaces;
an inlet to the chamber; and
an outlet from the chamber;

a longitudinally-extending plasma isolation unit defined by the solid substrate and upstream of said biomolecular processor and one or more nanotubes, said longitudinally-extending plasma isolation unit comprising:
an entrance passage;
a discharge passage which is wider and shallower than the entrance passage;
a transition passage connecting the entrance passage and the discharge passage, said transition passage becoming wider and shallower as the transition passages progresses from the entrance passage to the discharge passage;
primary side channels extending laterally away from the entrance passage, wherein a separator, positioned between the entrance passage and each primary side channel, is sized to permit plasma, but not cells, to pass from the entrance passage to the primary side channels; and
secondary side channels extending laterally away from the discharge passage, wherein a separator, positioned between the discharge passage and each secondary side channel, is sized to permit plasma, but not cells, to pass from the entrance passage to the secondary side channels;

a first extractor unit defined by the solid substrate and fluidically coupled to said longitudinally extending plasma isolation unit, said first extractor unit comprising solid supports and passages between them, wherein the solid supports are provided with a m

Assignees

• Univ North Carolina Chapel Hill
• Univ Cornell
• Univ Louisiana State

Inventors

• Soper Steven A
• Barany Francis
• Park Sunggook
• Murphy Michael
• Mckinney Collin J
• Efcavitch John William
• Hupert Mateusz

Classifications

• B01L2300/0896

  Nanoscaled · CPC title

• B01L2200/0647

  Handling flowable solids, e.g. microscopic beads, cells, particles · CPC title

• C12M1/40

  Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• B01L3/502761

  specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads or physically stretching molecules · CPC title