Devices and methods for sample analysis

Abstract

Methods, devices, and systems for analyte analysis using a nanopore are disclosed. The methods, devices, and systems utilize a first and a second binding member that each specifically bind to an analyte in a biological sample. The method further includes detecting and/or counting a cleavable tag attached to the second binding member and correlating the presence and/or the number of tags to presence and/or concentration of the analyte. Certain aspects of the methods do not involve a tag, rather the second binding member may be directly detected/quantitated. The detecting and/or counting may be performed by translocating the tag/second binding member through a nanopore. Devices and systems that are programmed to carry out the disclosed methods are also provided.

What is claimed is: 
     
         1 . A method for measuring or detecting an analyte present in a biological sample, the method comprising:
 contacting the sample with a first binding member, wherein the first binding member is immobilized on a solid support and wherein the first binding member specifically binds to the analyte;   contacting the analyte with a second binding member, wherein the second binding member specifically binds to the analyte;   removing second binding member not bound to the analyte bound to the first binding member;   disassociating a portion of the second binding member thereof bound to the analyte;   translocating the portion of the second binding member thereof through a nanopore; and   detecting the portion of the second binding member thereof translocating through the nanopore.   
     
     
         2 . The method of  claim 1 , wherein the nanopore is disposed in a device comprising:
 a microfluidics module; and   a nanopore module in fluid communication with the microfluidics module and comprising the nanopore,   wherein the microfluidics module is configured to introduce a fluid droplet to the nanopore.   
     
     
         3 . The method of  claim 2 , wherein the nanopore is formed in situ within the device. 
     
     
         4 . The method of  claim 1 , wherein the second binding member comprises a cleavable tag or an aptamer. 
     
     
         5 . The method of  claim 4 , wherein the second binding member comprises the cleavable tag, and wherein the cleavable tag is selected from the group consisting of an anionic polymer, a cationic polymer, a dendrimer, and a nanoparticle. 
     
     
         6 . The method of  claim 5 , wherein the cleavable tag is negatively charged and the translocating comprises applying a positive potential across the nanopore thereby translocating the cleavable tag through the nanopore. 
     
     
         7 . The method of  claim 5 , wherein the cleavable tag is positively charged and the translocating comprises applying a negative potential across the nanopore thereby translocating the cleavable tag through the nanopore. 
     
     
         8 . The method of  claim 5 , wherein each cleavable tag translocating through the nanopore is a translocation event and the method further comprises measuring the translocation events to measure an amount of analyte present in the sample, wherein the amount of analyte present in the sample is determined by:
 counting a number of translocation events during a set period of time and correlating the number of translocation events to a control;   measuring an amount of time for a set number of translocation events to occur and correlating to a control; or   determining an average time between translocation events and correlating to a control, wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction.   
     
     
         9 . The method of  claim 8 , wherein the reference standard is determined by:
 measuring the number of translocation events for the control during a predetermined period of time;   measuring the amount of time for a predetermined number of translocation events to occur compared to the control; or   measuring the average time between translocation events to occur for the control.   
     
     
         10 . The method of  claim 8 , wherein measuring the translocation events comprises observing a change in current induced by an interaction of the cleavable tag with the nanopore. 
     
     
         11 . The method of  claim 4 , wherein second binding member comprises the aptamer, and wherein the aptamer is a DNA aptamer or an RNA aptamer. 
     
     
         12 . The method of  claim 11 , further comprising detecting the aptamer by single molecule counting. 
     
     
         13 . The method of  claim 11 , wherein each aptamer translocating through the nanopore is a translocation event and the method further comprises measuring the translocation events to measure an amount of analyte present in the sample, wherein the amount of analyte in the sample is determined by at least one of:
 counting a number of translocation events during a set period of time and correlating the number of translocation events to a control;   measuring an amount of time for a set number of translocation events to occur and correlating to a control; or   measuring an average time between translocation events to occur and correlating to a control,   wherein the control is a reference standard comprising a calibration curve, standard addition, or digital polymerase chain reaction.   
     
     
         14 . The method of  claim 13 , wherein the reference standard is determined by:
 measuring the number of translocation events for the control during a predetermined period of time;   measuring the amount of time for a predetermined number of translocation events to occur compared to the control; or   measuring the average time between translocation events to occur for the control.   
     
     
         15 . An integrated digital microfluidics nanopore device comprising:
 a microfluidics module comprising an array of electrodes and a first substrate and a second substrate, wherein the first substrate is separated from the second substrate by a gap;   a nanopore module in fluid communication with the microfluidics module and comprising at least one nanopore,   wherein the microfluidics module is configured to introduce a fluid droplet to the at least one nanopore,   and wherein at least one of contacting a sample with a first binding member, contacting an analyte with a second binding member, removing second binding member not bound to the analyte bound to the first binding member, and disassociating a portion of the second binding member thereof is performed in the gap between the first and second substrates.   
     
     
         16 . The device of  claim 15 , wherein the microfluidics module is further configured to transport the fluid droplet to a first transfer position in the array of electrodes, and wherein the first transfer position is proximate an interface between the microfluidics module and the nanopore module;
 the nanopore module comprising:
 a first capillary channel, 
 a second capillary channel intersecting the first capillary channel at an intersection region, and 
 a nanopore disposed in a nanopore layer disposed between the first and second capillary channels proximate the intersection region, 
   wherein at least the first capillary channel extends to the interface and adjacent the first transfer position and is positioned to receive the fluid droplet disposed at the first transfer position.   
     
     
         17 . The device of  claim 16 , wherein the first capillary channel comprises a first pair of electrodes and the second capillary channel comprises a second pair of electrodes,
 wherein the first pair of electrodes is disposed in the first capillary channel and flank the nanopore in the nanopore layer and wherein the second pair of electrodes is disposed in the second capillary channel and flank the nanopore in the nanopore layer.   
     
     
         18 . The device of  claim 16 , wherein the second capillary channel extends between a vent or a reservoir proximate one or both ends of the second capillary channel. 
     
     
         19 . The device of  claim 18 , wherein the second capillary channel is joined to a first reservoir at a first end and a second reservoir at the opposing end. 
     
     
         20 . The device of  claim 19 , wherein at least one of the first reservoir and the second reservoir comprises the fluid droplet to be positioned within the second capillary channel proximate the intersection region and configured to facilitate operation of the nanopore layer to drive current through the nanopore disposed in the nanopore layer.