Nanopore sequencing using ratiometric impedance

Abstract

The invention relates to devices and methods for nanopore sequencing. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. The invention includes devices having sample and reference pores connecting sample, measurement and reference chambers, wherein potential measurements in each chamber is used to provide an accurate determination of current through a sample nanopore, improving nanopore sequencing.

We claim: 
     
       1. A device for determining the identity of a single molecule passing through a nanopore comprising:
 three fluidic regions comprising i) a sample fluidic region, ii) a measurement fluidic region, and iii) a reference fluidic region; 
 a substrate comprising a measurement electrode positioned to sense the potential of the measurement fluidic region; 
 a first aperture comprising a nanopore through which one or more sample molecules are transported, the first aperture connecting the sample fluidic region and the measurement fluidic region; 
 a second aperture connecting the measurement fluidic region with the reference fluidic region; and 
 a pair of drive electrodes, whereby a potential drop across the drive electrodes drives a sample molecule into the nanopore of the first aperture from the sample fluidic region, and wherein the device further comprises a sample electrode positioned to sense the potential of the sample fluidic region and a reference electrode positioned to sense the potential of the reference fluidic region, wherein the presence of the sample molecule in the nanopore of the first aperture is detected using the potential measurements at the sample electrode, the measurement electrode, and the reference electrode. 
 
     
     
       2. The device of  claim 1  wherein the sample electrode comprises the gate of a first transistor element positioned to sense the potential of the sample fluidic region, the measurement electrode comprises the gate of a second transistor element positioned to sense the potential of the measurement fluidic region, and the reference electrode comprises the gate of a third transistor element positioned to sense the potential of the reference fluidic region. 
     
     
       3. The device of  claim 2  wherein one or more of the first, second, and third transistor elements is a naked gate transistor having no metal electrode. 
     
     
       4. The device of  claim 3  wherein the gate of the one or more naked gate transistors has a thin insulating film between the gate and the fluidic region in which the transistor is positioned. 
     
     
       5. The device of  claim 2  wherein the device is fabricated in substrate is a semiconductor substrate by etching a well into the substrate, forming a semiconductor lid over the well to form a chamber, fabricating transistors onto the substrate, and etching holes through the lid into the chamber for sample and reference nanopores. 
     
     
       6. The device of  claim 5  wherein the substrate comprises silicon, silicon on insulator (SOI), or silicon on sapphire. 
     
     
       7. The device of  claim 2  wherein the device comprises sample, reference, and measurement electrodes comprise a vertical IGFET. 
     
     
       8. The device of  claim 7  wherein the vertical IGFET is produced by forming substrate is a P-doped substrate comprising an N-doped well in a P-doped substrate, filling the N-doped well filled with a P-doped semiconductor layer, forming wherein the device further comprises (i) a hole that extends through the P-doped semiconductor layer, the N-doped well, and the P-doped substrate, and fabricating (ii) electrodes onto on the top surface of the P-doped semiconductor layer and the P-doped substrate such that these become the source and drain of the IGFET. 
     
     
       9. The device of claim  2  5 wherein the transistors are formed present on the top of a the semiconductor substrate, and wherein the device comprises a fluidic structure is produced on top of the semiconductor substrate, the fluidic structure establishing sample, measurement and reference the three fluidic regions having a sample nanopore between the sample and measurement fluidic regions, and a reference nanopore between the measurement and reference regions, the first aperture, and the second aperture. 
     
     
       10. The device of  claim 1  wherein the sample molecule comprises a single stranded nucleic acid molecule. 
     
     
       11. The device of  claim 1  wherein the sample molecule comprises a nucleotide analog having a charge blockade label. 
     
     
       12. The device of  claim 1  wherein a sample potential V1, a measurement potential V2, and a reference potential V3 are measured, and whereby the presence of a sample molecule within the nanopore of the first aperture is detected using the value of (V 1 −V 2 )/(V 2 −V 3 ). 
     
     
       13. An analytical device comprising a plurality of devices of  claim 1  sharing a common substrate. 
     
     
       14. The analytical device of  claim 13  wherein the sample fluidic regions of the plurality of devices are simultaneously accessible to a fluidic sample for loading the sample fluidically connected. 
     
     
       15. An analytical device comprising 1,000 to 100,000 devices of  claim 1 . 
     
     
       16. The device of claim 1 further comprising a polymerase enzyme attached proximal to the nanopore of the first aperture whereby when a nucleotide analog comprising a phosphate linked blockade label is associated with the polymerase enzyme, the blockade label at least partially blocks the pore such that the association between the nucleotide analog and the polymerase enzyme is detected. 
     
     
       17. A nucleic acid sequencing analytical device comprising a plurality of devices on a single substrate, each device comprising:
 three fluidic regions comprising i) a sample fluidic region, ii) a measurement fluidic region, and iii) a reference fluidic region;   a measurement electrode positioned to sense the potential of the measurement fluidic region;   a first aperture comprising a nanopore through which one or more sample molecules are transported, the first aperture connecting the sample fluidic region and the measurement fluidic region;   a second aperture connecting the measurement fluidic region with the reference fluidic region;   a polymerase enzyme-template nucleic acid complex attached proximal to the first aperture;   a sequencing reaction mixture in the sample fluidic region comprising a plurality of types of labeled nucleotide analogs, each having a different type of blockade label; and   a pair of drive electrodes, whereby a potential drop across the drive electrodes drives a blockade label into the nanopore of the first aperture from the sample fluidic region, and wherein the device further comprises a sample electrode positioned to sense the potential of the sample fluidic region and a reference electrode positioned to sense the potential of the reference fluidic region, wherein the presence of the blockade in the nanopore of the first aperture is detected using the potential measurements at the sample electrode, the measurement electrode, and the reference electrode.   
     
     
       18. The analytical device of claim 17 wherein the plurality of devices comprises greater than 1000 devices. 
     
     
       19. A method of fabricating the device of claim 2, wherein the substrate is a semiconductor substrate, the method comprising:
 etching a well into the semiconductor substrate;   forming a semiconductor lid over the well to form a chamber;   fabricating transistors onto the substrate; and   etching holes through the lid into the chamber for sample and reference nanopores.   
     
     
       20. A method of fabricating the device of claim 2, wherein the substrate is a semiconductor substrate, the method comprising:
 forming the transistors on the top of the semiconductor substrate;   producing a fluidic structure on top of the semiconductor substrate establishing the sample, measurement and reference fluidic regions;   wherein the fluidic structure comprises the first and second aperture.   
     
     
       21. A method of fabricating the device of claim 7, wherein the substrate is a P-doped substrate, the method comprising:
 forming an N-doped well in the P-doped substrate;   filling the N-doped well with a P-doped semiconductor layer;   forming a hole that extends through the P-doped semiconductor layer, the N-doped well, and the P-doped substrate; and   fabricating electrodes onto the top surface of the P-doped semiconductor layer and the P-doped substrate such that these become the source and drain of the IGFET.   
     
     
       22. A method of determining the identity of a single molecule, the method comprising:
 contacting the sample fluidic region of the device of claim 1 with a sample comprising a single molecule;   providing a potential drop across the drive electrodes to drive the single molecule into the nanopore of the first aperture from the sample fluidic region;   detecting the presence of the single molecule in the nanopore of the first aperture by measuring the potential at the sample electrode, the measurement electrode, and the reference electrode; and   determining the identity of the single molecule based on the potential measurements.   
     
     
       23. The method of claim 22 wherein the single molecule comprises a single stranded nucleic acid molecule. 
     
     
       24. The method of claim 22 wherein a sample potential V1, a measurement potential V2, and a reference potential V3 are measured, and whereby the presence of the single molecule within the nanopore of the first aperture is detected using the value of (V1−V2)/(V2−V3). 
     
     
       25. The method of claim 22 wherein the single molecule comprises a blockade label. 
     
     
       26. The method of claim 25 wherein the blockade label is a label that is released from a labeled nucleotide analog when the nucleotide analog is incorporated into a template nucleic acid, releasing the blockade label. 
     
     
       27. The method of claim 26 wherein the potential measurements comprise measurements of current blockage over time.