Abstract:
An electrically conductive nanoscale sensor includes a nanoscale pore that may be employed as a first antenna to provide precise localized measurements of the impedance-altering characteristics of a molecule such as DNA or RNA or the like passing through the pore. The use of radiofrequency measurements via a second antenna communicating with the first antenna promises high-speed analysis of long molecules (polymers).

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
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     CROSS REFERENCE TO RELATED APPLICATION 
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     BACKGROUND OF THE INVENTION 
     The present invention relates to a system for the direct sequencing of polymers such as DNA and RNA and proteins by passing the polymer through a nanoscale pore and measuring an electrical signal modulated by the polymer passing through the pore. 
     Genetic information may be encoded in a molecule of deoxyribonucleic acid (DNA) as a sequence of nucleotides: (guanine, adenine, thymine, and cytosine). Discovering the sequence of these nucleotides in DNA and other similar molecules is a foundational technology in biological studies. 
     One promising method of sequencing is “nanopore sequencing” in which a single strand of DNA, forming half of the DNA helix, is passed through a nanoscale opening in a membrane between two reservoirs. This nanopore opening may, for example be a biological pore, a solid state pore, a semiconductor pore (nano-channel) or a DNA synthesized channel held in a lipid bilayer. By a drying force, e.g. an electrical potential applied across the reservoirs, an ion flow is produced between the reservoirs pulling the strand of DNA through the nanopore. As the strand passes through the nanopore, it modulates the ion current through the nanopore as a function of the size of the nucleotide, which partially obstructs the nanopore. This fluctuation in the ion current may then be analyzed to determine the nucleotide sequence. An example system of nanopore sequencing is described in PCT patent publication WO2008102120 entitled: Lipid Bilayer Sensor System, hereby incorporated by reference. 
     The electrical signals produced by changes in ion current through a nanopore with different nucleotides are very small in amplitude and most importantly short in time span. For this reason, it can be hard to obtain reliable measurements having sufficient resolution to distinguish between different molecules in the sequence. 
     U.S. patent application Ser. No. 13/786,880, assigned to the assignee of the present application and hereby incorporated by reference, describes a method of analyzing the operation of nanopore ion channels using changes in impedance of a nanopore device measured at radio frequencies. 
     SUMMARY OF THE INVENTION 
     The present invention applies the radiofrequency measurement technique of U.S. patent Ser. No. 13/786,880 to highly sensitive analysis of molecules flowing through a nanopore by using a conductive nanopore to create a nanopore antenna. Changes in the apparent impedance of the nanopore antenna coupled to a second antenna as is coupled to the molecule reveal the passage and characteristics of molecules passing through the nanopore antenna. 
     Specifically, the present invention provides a nanochannel sensor having a first antenna structure using an electrically conductive nanoscale pore. The conductive nanoscale pore is supported to provide a passage through the membrane between the first and second membrane side and a second antenna structure is positioned to electrically couple with the nanoscale pore. The second antenna structures are adapted to communicate with a measurement circuit producing a radiofrequency signal and measuring a change in the reflected, transmitted or radiated electrical radiofrequency signal with the passage of the molecule through the first antenna structure. 
     It is thus a feature of at least one embodiment of the invention to provide an improved and high-speed method of analyzing molecules such as DNA. RNA and the like. Unlike current pore-current measurement systems, the radiofrequency measurement system of the present invention promises much higher measurement speeds. It should be noted that in current systems the diffusion speed of the molecule to be screened has to be strongly reduced. 
     The electrically conductive nanoscale pore may have an internal diameter of less than three nanometers. 
     It is thus a feature of at least one embodiment of the invention to provide a nanopore antenna that can isolate individual molecular strands for measurement. It is another feature of at least one embodiment of the invention to provide an antenna structure that is significantly affected by changes due to the molecule passing through the antenna. 
     The electrically conductive nanoscale pore may be a biomolecule formed into a pore and coated with metallic nanoparticles. In one example, the electrically conductive nanoscale pore may be constructed of DNA with metal particles attached to it. 
     It is thus a feature of at least one embodiment of the invention to provide a method of fabricating conductive structures of the necessary scale. 
     The DNA may be modified to provide side chains presenting sulfur groups and the metal particles may be gold particles 
     It is thus a feature of at least one embodiment of the invention to provide a method of fabricating metallized DNA structures suitable for constructing nanoscale antennas. 
     The nanoscale pore may include one or more conductive side antenna structures extending perpendicular to the pore axis. 
     It is thus a feature of at least one embodiment of the invention to significantly increase the coupling to the nanoscale antenna by increasing its extent with respect to a wavelength of an electromagnetic signal developed perpendicular to the pore axis. 
     The nanoscale pore can be made of semiconductor material, which functions as an antenna via regulation of the semiconductor doping. 
     It is thus a feature of at least one embodiment of the invention to permit the use of a variety of nanoscale pore materials. 
     The second antenna structure may provide a gap along a third axis across which an electromagnetic field is generated by the measurement circuit and the nanoscale pore may be oriented so that the second axis is aligned with the third axis. 
     It is thus a feature of at least one embodiment of the invention to significantly increase the coupling of the nanoscale antenna by adjusting its orientation with respect to a developed electromagnetic signal by the second antenna. 
     The nanochannel sensor may further include measurement circuitry providing a radiofrequency signal source and measuring a change in the reflected, transmitted or radiated electrical radiofrequency signal of the second antenna due to the passage of molecules through the nanoscale pore of the first antenna. 
     It is thus a feature of at least one embodiment of the invention to provide an integrated system for measuring the property of molecules passing through the nanopore antenna. 
     The measurement circuitry may monitor a change in the resonant frequency of the coupled first and second antenna system. 
     It is thus a feature of at least one embodiment of the invention to provide measurement that may be accomplished at high speed commensurate with radiofrequency signals. 
     The electrically insulating membrane may be a lipid bilayer. 
     It is thus a feature of at least one embodiment of the invention to provide a fluid-resistant surface that may be readily integrated with a biomolecule nanopore. 
     These particular features and advantages may apply to only some embodiments falling within the claims and thus do not define the scope of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a simplified perspective view of a nanochannel sensor of the present invention providing a conductive nanopore suspended across a gap in an interrogation antenna and separating two chambers to receive a biomolecule passing between the chambers for sequencing; 
         FIG. 2  is a plot of reflectance versus frequency for the system of  FIG. 1  showing variations in the measurements associated with a metallized and non-metallized nanopores with and without the passage of DNA therethrough; 
         FIG. 3  a simplified diagram of the metallization of the nanopore formed from DNA; 
         FIG. 4  is an elevational cross-section of a nanopore and interrogation antenna system used for nanopore measurement; and 
         FIG. 5  is a perspective top view of the interrogation antenna system with a fragmentary detail showing the nanopore. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     Referring now to  FIG. 1 , a nanochannel sensor  10  may provide for an electrically conductive nanopore  12  supported on a membrane  14 , the latter separating a first chamber  16  on the first side of the membrane  14  from a second chamber  18  on a second side of the membrane. The chambers may hold, for example, a fluid such as a saline solution in which biomolecules to be analyzed may be suspended. 
     As supported, the nanopore  12  extends generally perpendicularly through the membrane  14  to provide a through-channel  20  through the membrane  14  along a transfer axis  22  generally perpendicular to the upper face of the membrane  14  and suitable for passing a single strand of a biomolecule  24  such as DNA, RNA, peptides, proteins or the like. The membrane  14  may, for example, be a lipid bilayer of the type known in the art and similar to that forming a cellular membrane or may be a solid state nanomembrane into which the nanopore- 12  can be inserted.]. 
     In one embodiment, a generally cylindrical torus  25  of the nanopore  12  surrounding the through-channel  20  is attached to electrically conductive laterally extending arms  26 , for example, extending in opposite directions on opposite sides of the cylindrical torus  25  in directions generally perpendicular to the transfer axis  22 . 
     The arms  26  may lie closely proximate to the surface of the membrane  14  and may stabilize the nanopore  12  with respect to the membrane  14 . The arms  26  may extend along a second axis  28  generally perpendicular to axis  22  and may lie within a gap  30  between secondary antenna lobes  32  and  34  of a driving antenna  35 , the lobes  32  and  34  also lying along axis  28  positioned just outside the periphery of the membrane  14 . In this way, each one of the arms  26  points respectively to a different lobe  32  and  34 . The invention is not limited to this orientation however but also accommodates some misalignment. The arms  26  may also extend into the torus  25  but not into the through channel  20 . 
     The antenna lobes  32  and  34  may be driven electrically by measuring circuit  36  providing a radiofrequency signal source  38  and measuring device  40  that together operate to generate a radiofrequency field across antenna lobes  32  and  34  and to measure the effective impedance of a system comprising the driving antenna  35  coupled to the nanopore  12  as influenced by the passage of biomolecules  24  through the through-channel  20 . The measuring device  40  measures a change in the reflected, transmitted or radiated electrical radiofrequency signal from the nanopore  12  via the antenna lobes  32  and  34  with the passage of the molecule through the nanopore  12 . In this regard, nanopore  12  forms a driven nanopore antenna structure coupled to the driving antenna  35  of lobes  32  and  34 . The arms  26  improve the coupling between the nanopore  12  and the driving antenna  35  of lobes  32  and  34 . 
     Biomolecules  24  may be encouraged through the through-channel  20  from chamber  16  to chamber  18  by a number of different means including, for example, a slight electrical bias between chambers  16  and  18  provided by DC biasing source  39  such as may act upon ions and charged molecules under test (DNA) within fluids of the chambers  16  and  18 . 
     Referring to  FIG. 2 , theoretical evidence indicates that the use of an electrically conductive nanopore  12  substantially increases the quality (Q) factor of a resonant circuit created by the system of the driving antenna  35  and the nanopore  12 . A plot of an attenuation of the radiofrequency signal from the radiofrequency signal source  38  versus frequency of that radiofrequency source  38  shows its natural resonance in the form of a negative-going “trough”  41  that is sharper and larger with the metallized nanopore  12  as compared to a resonant trough  42  for a nanopore that is not electrically conductive. Similar troughs  44  for the conductive nanopore  46  and for a nonconductive nanopore both with DNA passing through the nanopores are also shown indicating the effect of the presence of a biomolecule within the nanopore through-channel  20 . Measurement of the nanopore  12  may analyze the biomolecule through any one or more of frequency change of these peaks and/or the amplitude change in these peaks and/or phase change of the signal or the like known in the art. 
     Referring now to  FIG. 3 , in one embodiment, the cylindrical torus  25  of the nanopore  12  may be fabricated of DNA using DNA-prototyping, for example, as described in Synthetic Lipid Membrane Channels Formed by Designed DNA Nanostructures, Martin Langecker et al., Science 338, 932 (2012); DOI: 10.1126/science.1225624 hereby incorporated by reference. An average diameter of the through-channel  20  of the nanopore  12  will be such as to limit the passage through the through-channel  20  of a single biomolecule of interest and will typically be less then three nanometers or preferably less than two nanometers in diameter. The height of the cylindrical torus  25 , in one embodiment, as measured along the transfer axis  22  may be at least five nanometers and typically 10 to 20 nanometers and an outer diameter of the cylindrical torus  25  may be greater than five nanometers and typically 5 to 20 nanometers. The arms  26  are constructed of DNA strands attached to the cylindrical body as discussed above. The arms  26  will generally extend by an amount no less than five nanometers from the transfer axis  22 . 
     Electrical conductivity of the structure of the cylindrical body and arms  26  is provided by replacing side chains of the DNA molecule of the cylindrical body and arms  26  with sulfur groups  48  which will link to gold nanoparticles  50  applied to the nanopore  12 . 
     Alternatively the cylindrical torus  25  may be a wild-type or genetically engineered nanopores such as α-hemolysin or a semiconductor nanotubes such as carbon nanotubes or colloidally grown nanotubes. 
     Referring now to  FIG. 4 , the membrane  14  may be supported at its periphery on an opening  52  in an insulating planar support  55  such as a glass or quartz plate with the antenna lobes  32  and  34  being conductive traces deposited on the upper surface of the planar support  55  closely adjacent to the edges of the opening  52 . The membrane  14  may be attached to the opening  52  by painting or by applying vesicles of lipid bilayers 
     The conductive material of the lobes  32  and  34  may be a variety of different metals including aluminum, copper, silver, gold and the like or conductive metal compounds such as silver chloride. 
     An electrically insulating layer  54  may cover the lobes  34  and  32  against electrical contact with the liquid in chamber  16 . The electrically insulating layer  54  may, for example, be a thin coating of Parylene-C commercially available from a number of suppliers A similar insulating layer  56  may be applied on the lower surface of the planar support  55 . 
     Referring now to  FIG. 5 , the lobes  34  and  32  may provide a bowtie antenna in which electrode lobe  34  connects to measuring circuit  36  by a conductor  58  passing along an upper surface of the insulating planar support  55  along axis  28  as flanked on either side by a ground plane conductor  60 . As conductor  58  approaches the nanopore  12 , it forms lobe  34  and the ground plane conductor  60  diverges from axis  28  (for example, by opposite angles of 45 degrees) to a point past the lobe  32 . The ground plane conductor  60  then converges to rejoin on the left and right side of axis  28  to attach to the end of lobes  32  removed from the nanopore  12  to provide a “bowtie” type antenna. 
     The concept of measuring the electrical properties of the nanopore  12  should be understood to include both direct and indirect measurements and to consider radiofrequency energy that is reflected, transmitted or radiated by the nanopore  12 . 
     It will be appreciated that additional side arms  26  may be added to the cylindrical torus  25  so as to promote increased coupling with the electromagnetic field developed between the lobes  32  and  34  of the driving antenna  35 . 
     The following documents describing previous work by the inventors and are hereby incorporated by reference: 
     Radio Frequency Tank Circuit for Probing Planar Lipid Bilayer Formation’, A. Bhat, H. Qin, J. Rodriguez, H. C. Shin, H. Shin, D. Kreft, J. Park, E. Stava, M. Yu, and, R. H. Blick, Soft Nanoscience Letters 3, 87-92 (2013); DOI: 10.4236/snl.2013.34016 
     ‘Rapid fabrication and piezoelectric tuning of micro- and nanopores in single crystal quartz’, Eric Stava, Minrui Yu, Hyun Cheol Shin, Hyuncheol Shin, Dustin Kreft, and Robert H. Blick, Lab Chip 13, 156-160 (2013): DOI: 10.1039/C2LC40925A. http://pubs.rsc.org/ doi: 10.1039/C2LC40925A 
     ‘Mechanical actuation of ion channels using a piezoelectric planar patch clamp system’, E. Stava, M. Yu, H. C. Shin, H. Shin, J. Rodriguez, and R. H. Blick, Lab Chip 12, 80-87 (2012), DOI: 10.1039/C1LC20636B; Advance Article—inside cover title (http://pubs.rsc.org/en/content/articlelanding/2012/lc/c1lc90128a); DOI: 10.1039/C1LC90128A. 
     ‘Radio Frequency Response of Single Pores and Channels’, H. S. Kim, S. Ramachandran, E. Stava, D. W. van der Weide, and R. H. Blick, New Journal of Physics 13, 093033 (2011); http://iopscience.iop.org/1367-2630/13/9/093033/; DOI: 10.1088/1367-2630/13/9/093033. 
     ‘Direct Microwave Transmission Measurement on Single α-HL pores’, S. Ramachandran, D. W. van der Weide, and R. H. Blick, Applied Physics Letters 99, 093105 (2011); DOI.org/10.1063/1.3626586. 
     The term “electrically conductive” applied to the nanopore means an electrical conductivity suitable for generating measurable antenna currents and typically exceeding that of unmodified DNA. It is expected that the production of an electrically conductive nanopores suitable for this purpose will require the introduction of foreign conductive materials such as metals to a DNA structure or construction of the nanopore of a conventionally conductive material. 
     Certain terminology is used herein for purposes of reference only, and thus is not intended to be limiting. For example, terms such as “upper,” “lower,” “above,” and “below” refer to directions in the drawings to which reference is made. Terms such as “front,” “back,” “rear,” “bottom,” “side,” “left” and “right” describe the orientation of portions of the component within a consistent but arbitrary frame of reference which is made clear by reference to the text and the associated drawings describing the component under discussion. Such terminology may include the words specifically mentioned above, derivatives thereof, and words of similar import. Similarly, the terms “first,” “second” and other such numerical terms referring to structures do not imply a sequence or order unless clearly indicated by the context. 
     When introducing elements or features of the present disclosure and the exemplary embodiments, the articles “a,” “an,” “the” and “said” are intended to mean that there are one or more of such elements or features. The terms “comprising,” “including” and “having” are intended to be inclusive and mean that there may be additional elements or features other than those specifically noted. It is further to be understood that the method steps, processes, and operations described herein are not to be construed as necessarily requiring their performance in the particular order discussed or illustrated, unless specifically identified as an order of performance. It is also to be understood that additional or alternative steps may be employed. 
     All of the publications described herein including patents and non-patent publications are hereby incorporated herein by reference in their entireties.