Microfluidic apparatus, systems, and methods for performing molecular reactions

Abstract

Disclosed herein are methods, apparatuses, and systems for performing nucleic acid sequencing reactions and molecular binding reactions in a microfluidic channel. The methods, apparatuses, and systems can include a restriction barrier to restrict movement of a particle to which a nucleic acid is attached. Furthermore, the methods, apparatuses, and systems can include hydrodynamic focusing of a delivery flow. In addition, the methods, apparatuses, and systems can reduce non-specific interaction with a surface of the microfluidic channel by providing a protective flow between the surface and a delivery flow.

1 . A method to detect a nucleotide, comprising:
 a) restraining movement of a nucleic acid molecule attached to a single particle using a restriction barrier located within a first channel;   b) contacting the nucleic acid molecule with an exonuclease to release the nucleotide, wherein the nucleotide is a terminal nucleotide; and   c) identifying the released nucleotide by associating the released nucleotide with a surface enhanced Raman spectroscopy-active surface, irradiating the released nucleotide with a detection laser beam and measuring Raman emission from the irradiated nucleotide, thereby detecting the nucleotide.   
     
     
         2 . The method of  claim 1 , wherein the restriction barrier comprises a plurality of walls. 
     
     
         3 . The method of  claim 2 , wherein the restriction barrier comprises a first angled wall and a second angled wall positioned relative to the first angled wall to capture the single particle having the surface with the attached nucleic acid molecule. 
     
     
         4 . The method of  claim 1 , wherein a gradient force optical trap captures the single particle downstream of the restriction barrier, transports the single particle upstream of the restriction barrier, and release the single particle. 
     
     
         5 . The method of  claim 1 , wherein a gradient force optical trap captures the single particle downstream of the detection laser beam, the detection laser beam and the restriction barrier are moved downstream of the captured single particle, and the single particle is released. 
     
     
         6 . A method to determine a nucleotide sequence of a nucleic acid molecule, comprising:
 a) restraining movement of a single particle using a restriction barrier located within a first channel, wherein the nucleic acid molecule is attached to the single particle;   b) contacting the nucleic acid molecule with an exonuclease to release a terminal nucleotide; and   c) identifying a first released nucleotide and a second released nucleotide by irradiating the first released nucleotide and then the second released nucleotide with light from a detection light source, by associating the first released nucleotide and the second released nucleotide with a surface enhanced Raman spectroscopy-active surface, and measuring Raman emission from the irradiated first released nucleotide and then from the second released nucleotide, thereby determining a nucleotide sequence of the nucleic acid.   
     
     
         7 . The method of  claim 6 , wherein the restriction barrier comprises a plurality of walls. 
     
     
         8 . The method of  claim 7 , wherein the restriction barrier comprises a first angled wall and a second angled wall positioned relative to the first angled wall to capture the single particle having the surface with the attached nucleic acid molecule. 
     
     
         9 . The method of  claim 6 , wherein a gradient force optical trap captures the single particle downstream of the light from the detection light source, transports the single particle upstream of the restriction barrier, and releases the single particle. 
     
     
         10 - 45 . (canceled) 
     
     
         46 . The method of  claim 3 , wherein the first angled wall and the second angled wall form a first opening at least 1 micron in width or diameter and a second opening less than 10 microns in width or diameter, wherein the first opening has a greater width or diameter than the second opening. 
     
     
         47 . The method of  claim 4 , wherein the gradient force optical trap is operable as optical tweezers. 
     
     
         48 . The method of  claim 1 , wherein the particle has a diameter between 0.1 and 20 microns. 
     
     
         49 . The method of  claim 6 , wherein there is a gap between a wall of the channel and the restriction barrier. 
     
     
         50 . The method of  claim 6 , wherein a portion of a flow path in optical communication with the detection light source is coated with silver, gold, platinum, copper or aluminum. 
     
     
         51 . The method of  claim 1 , wherein a single nucleotide is attached to a single particle. 
     
     
         52 . The method of  claim 1 , further comprising sequentially detecting a plurality of nucleotides.