Abstract:
Integrated circuits for a single-molecule nucleic-acid assay platform, and methods for making such circuits are disclosed. In one example, a method includes transferring one or more carbon nanotubes to a complementary metal-oxide semiconductor (CMOS) substrate, and forming a pair of post-processed electrodes on the substrate proximate opposing ends of the one or more carbon nanotubes.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of PCT/US13/031745, filed Mar. 14, 2013 and which claims priority to U.S. Provisional Patent Application Ser. Nos. 61/636,459, filed on Apr. 20, 2012, and 61/680,094, filed on Aug. 6, 2012, each of which is incorporated by reference herein in its entirety. 
    
    
     GRANT INFORMATION 
     This invention was made with government support under grant 0707748 awarded by the National Science Foundation, and grants HG006879 and HG006882 awarded by the National Institute of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 29, 2014, is named 070050_5387_SL.txt and is 1,344 bytes in size. 
     BACKGROUND 
     The disclosed subject matter relates to single-molecule nucleic-acid assay platforms, including techniques for making integrated circuits for single-molecule nucleic-acid assay platforms. 
     Nucleic acid assays can have many applications, including, but not limited to, gene expression studies, environmental monitoring, and infectious disease recognition. Furthermore, polymerase chain reaction (PCR) can facilitate detection and quantitation of products. However, PCR can be challenging to implement in multiplexed analyses, at least in part because primer interactions can reduce sensitivity and the repertoire of reporter systems can allow for up to 10 to 20 targets. Sample preparation can include, for example, multiple stages of thermal cycling and precise control of enzymatic conditions. 
     In contrast, DNA microarray technology can allow for extensive multiplexing, but sensitivities can be too low to allow detection without amplification. As such, DNA microarray technology can also present the similar sample preparation complexities to PCR. With direct sequencing approaches, DNA can be directly sequenced for identification, but sensitivities can be too low to allow detection without amplification, and such techniques can be unsuitable for point-of-care diagnostics. 
     Accordingly, there remains an opportunity for single-molecule nucleic-acid assay platforms that can provide improved levels of sensitivity without amplification, while also providing improved multiplexing capabilities. 
     SUMMARY 
     Systems and methods for single-molecule nucleic-acid assay platforms, including techniques for making integrated circuits for single-molecule nucleic-acid assay platforms, are disclosed herein. 
     According to one aspect of the disclosed subject matter, methods of making an integrated circuit for a single-molecule nucleic-acid assay are provided. In one example, a method of making an integrated circuit for a single-molecule nucleic-acid assay platform includes transferring one or more carbon nanotubes to a complementary metal-oxide semiconductor (CMOS) substrate, and forming a pair of post-processed electrodes on the substrate proximate opposing ends of the one or more carbon nanotubes. 
     In some embodiments, transferring the one or more carbon nanotubes can include spinning the one or more carbon nanotubes from a suspension to the substrate. Additionally or alternatively, transferring the one or more carbon nanotubes can include forming the one or more carbon nanotubes on a transfer substrate, applying a layer of polymer to the transfer substrate to adhere the one or more carbon nanotubes to the transfer substrate, and placing the layer of polymer with the one or more carbon nanotubes on the CMOS substrate. Additionally or alternatively, transferring the one or more carbon nanotubes can include placing the one or more carbon nanotubes in a suspension proximate a pair of preformed electrodes on the substrate, and applying a voltage across the pair of preformed electrodes, so that a force is applied to the one or more carbon nanotubes to urge the carbon nanotubes to be disposed across the pair of preformed electrodes. 
     In some embodiments, the substrate can include surface-exposed electrodes. Forming the pair of post-processed electrodes can include depositing titanium on a pair of the surface-exposed electrodes. Additionally or alternatively, forming the pair of post-processed electrodes can include etching away a pair of surface-exposed electrodes and replacing the pair of surface-exposed electrodes with a pair of titanium electrodes. 
     The method can further include forming one or more reference electrodes on the substrate to allow control of an electrolytic gating potential. The substrate can include one or more surface-exposed electrodes, and forming the one or more reference electrodes can include etching away the surface-exposed electrodes. The one or more surface-exposed electrodes can include aluminum, and forming the one or more reference electrodes can further include replacing the surface-exposed electrodes with gold electrodes. Forming the one or more reference electrodes can further include electroplating silver on the gold electrodes. Forming the one or more reference electrodes can further include exposing the electroplated electrodes to FeCl3 to form Ag/AgCl electrodes. Additionally or alternatively, forming the one or more reference electrodes can include depositing platinum on the one or more surface-exposed electrodes. Additionally or alternatively, forming the one or more reference electrodes can include replacing the surface-exposed electrodes with one or more platinum electrodes. 
     In some embodiments, the method can further include forming a point defect on a portion of the one or more carbon nanotubes. The method can further include coupling the one or more post-processed electrodes to a ball-grid array (BGA) package. In some embodiments, the coupling can include wirebonding. Wirebonds can be exposed on the surface of the substrate, and the method can further include covering the exposed wirebonds using dam-and-fill material. 
     According to another aspect of the disclosed subject matter, integrated circuits for a single-molecule nucleic-acid assay platform are provided. In one example, integrated circuits for a single-molecule nucleic-acid assay platform can include a CMOS substrate and an array of single-molecule field-effect transistors (smFETs) disposed on a surface of the substrate. Each smFET can include one or more carbon nanotubes and a capture probe configured to provide target specificity for a single target nucleic acid molecule proximate thereto. The integrated circuit also includes at least one pair of electrodes disposed proximate opposing ends of the one or more carbon nanotubes to electrically couple the one or more carbon nanotubes to the substrate. 
     In some embodiments, the carbon nanotube can include a point defect formed on a portion thereof. The point defect can be configured to transmit the hybridization information for the single target nucleic acid molecule. 
     In some embodiments, the CMOS substrate can have dimensions of no more than 5 mm by 5 mm. The array can include at least 12 measurement channels. Each of the measurement channels can be multiplexed to at least 500 smFETs. 
     In some embodiments, the at least one pair of electrodes can include titanium. The integrated circuit can further include one or more reference electrodes to allow control of an electrolytic gating potential. The one or more reference electrodes can include one or more Ag/AgCl electrodes or platinum electrodes. The integrated circuit can include a ball-grid array (BOA) package coupled to the at least one pair of electrodes. The integrated circuit can further include a processor coupled to the array and configured to determine a capture rate from the hybridization information. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram illustrating an exemplary embodiment of an assay platform according to the disclosed subject matter. 
         FIG. 2  is a diagram illustrating further details of the assay platform of  FIG. 1 . 
         FIGS. 3A-3D  are diagrams illustrating further details of the assay platform of  FIG. 1 . 
         FIGS. 4A-4D  are diagrams illustrating further details of the assay platform of  FIG. 1 .  FIG. 4A-4C  include illustrations of SEQ ID NOS 1-2, 1, 3, 1, and 4, respectively, in order of appearance. 
         FIG. 5  is a diagram illustrating the assay platform of  FIG. 1  configured on a form factor of a USB memory stick. 
         FIGS. 6A-6C  are diagrams illustrating exemplary techniques for transferring nanotubes onto an integrated circuit substrate. 
         FIG. 7  is a diagram illustrating an exemplary embodiment of an integrated circuit and nanotube for a single-molecule nucleic-acid assay platform. 
         FIGS. 8A-8C  are diagrams illustrating an exemplary assay platform according to the disclosed subject matter. 
         FIGS. 9A-9D  are diagrams illustrating further details of the assay platform of  FIG. 8A . 
         FIG. 10  is a diagram illustrating further details of the assay platform of  FIG. 8A . 
         FIGS. 11A-11C  are diagrams illustrating further details of the assay platform of  FIG. 8A . 
         FIGS. 12A-12D  are diagrams illustrating further details of the assay platform of  FIG. 8A . 
         FIGS. 13A-13B  are diagrams illustrating further details of the assay platform of  FIG. 8A . 
         FIGS. 14A-14B  are diagrams illustrating further details of the assay platform of  FIG. 8A . 
         FIGS. 15A-15D  are diagrams illustrating further details of the assay platform of  FIG. 8A . 
         FIG. 16  is a diagram illustrating further details of the assay platform of  FIG. 8A . 
     
    
    
     DETAILED DESCRIPTION 
     The disclosed subject matter provides systems and methods for single-molecule nucleic-acid assay platforms. Single-molecule nucleic-acid assay platforms according to the disclosed subject matter can provide improved sensitivity without amplification, while also providing improved multiplexing capabilities. Transduction can be performed label-free, which can simplify sample preparation protocols. 
       FIG. 1  is a diagram illustrating an exemplary nanoscale single-molecule field effect transistor (smFET)  100  according to the disclosed subject matter. A smFET  100 , which can be utilized to perform transduction, can be configured as a carbon nanotube device  110  disposed on a substrate  115 , which can be a silicon substrate and can have a silicon oxide layer disposed thereon. A capture probe  160  (as shown in  FIGS. 3B and 3D ), for example and as embodied herein single-stranded DNA, can be immobilized onto the nanotube  110 , and can be coupled to source electrode  120   a  and drain electrode  120   b , each of which are embodied herein as titanium electrodes, and can be disposed at opposing ends of the nanotube  110 . To form source and drain electrodes  120   a ,  120   b , a metal, such as titanium, can be deposited onto the substrate  120 . Additionally, source and drain electrodes  120   a ,  120   b  can be passivated with a photoresist or an e-beam resist. The smFET  100  can be configured to detect hybridization with a single target molecule at a relatively high signal-to-noise ratio, for example 3 or more, without labeling. As such, background or non-specific adsorption can be reduced. 
     The smFET  100  assay platform can operate differently from traditional ensemble assays. For example, and as embodied herein, rather than measuring the hybridization behavior of an ensemble, as in a traditional microarrays, the smFET  100  assay platform can measure the time between capture events. Capture rates can thus be diffusion limited and concentration dependent. As such, low levels of detection, as low as one molecule, can be performed, which can be affected at least in part by diffusion time of the target to the sensor site. With reference to  FIG. 1 , the diffusion time can be reduced with a bias applied between the nanotube  110  and a reference electrode  125 , for example and embodied herein as an Ag/AgCl electrode or platinum electrode, in the electrolyte  130  that surrounds it, and the bias can be referred to as a liquid gate bias V lg .  FIG. 2  is a diagram illustrating the estimated capture rate for 33-mer ssDNA, with a 300 mV bias applied, as a function of target concentration. Furthermore, the capture rate can be independent of the size of the target DNA being captured. Additionally or alternatively, and as described further below, the carrier density in the nanotube  110  can be controlled through a global back gate V bg  applied through the substrate  115 . 
     Nanotube and nanowire field-effect sensors can be utilized as biosensors. In some implementations, an electrolyte buffer with mobile ions can be used to gate the transistor. The sensing mechanism can be attributed at least in part to changes in the Schottky barrier at the contacts and electrostatic doping of the nanotube channel due to adsorption of biomolecules. 
     As embodied herein, introducing a defect onto the nanotube  110  surface can provide smFET  100  sensors with localized charge sensitivity and improved gain. Such defects can, in turn, be used to covalently bind molecules at the scattering site. The resulting smFET  100  device can have improved sensitivity and detect the binding of a single molecule, due at least in part to Coulomb interactions between the molecule and the defect which modulates scattering in the 1D channel. The charge sensitivity can be screened by counterions and can be localized to the region of the defect. The single-point defects can be electrochemically created in a controllable manner, as described in further herein. Such defect-dominated conductance in nanotubes can produce measurements of DNA hybridization kinetics with suitable high signal-to-noise ratio (SNR) and bandwidth to measure single-molecule kinetics and thermodynamics through a label-free smFET approach. 
     The point-functionalized defect areas on the smFETs  100 , for example and as embodied herein, can be less than two nanometers in diameter. Scanning gate microscopy can be used to image the sensitivity of the nanotube  110  before and after the oxidation by using a biased tip (−5V) of an atomic force microscope. Before oxidation, electron transport in the nanotube can be sensitive both to the Schottky barrier of the lower electrode, and also to other points along the channel. After oxidation, the sensitivity can be localized to a region proximate the middle of the channel. A short chemical reaction with a strong oxidizer (for example potassium permanganate or the like) can convert the defect functional group to a carboxylic acid, to which a probe molecule can be covalently attached. 
       FIGS. 3A-3D  are diagrams illustrating carbon nanotube FET time-domain measurements showing the two-level response to single-molecule hybridizations. As shown in  FIG. 3B , capture probe  160 , which can be terminated with an amine group and a three-carbon linker at the 5′ end, can be covalently attached to the carboxyl defect  140  on the nanotube  100  through a standard coupling reaction using sulfo-N-hydroxysuccinimide (sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). As shown in  FIG. 3A , the carbon nanotube  100 , which was measured in 1×PBS buffer solution with a 10-mer oligonucleotide probe attached at the 5′ end (NH2-5′-GTGAGTTGTT-3′) (SEQ ID NO: 1), has a conductance with a single state dominated by flicker noise. In  FIG. 3D , complementary ssDNA molecules were added to the solution, and the conductance exhibited two level fluctuations, as shown in  FIG. 3C . Examining the fluctuations as a function of temperature can illustrate the high state in the conductance correlated to the defect with probe only and the low state correlated to a defect with duplex DNA. The negatively charged target DNA can modulate a tunnel barrier at the defect site through electrostatic interaction or scattering and can thereby reduce its conductance. Further details of exemplary single-molecule detection using nanotubes are shown and described in International Application No. PCT/US2012/020955, filed on Jan. 11, 2012, which is incorporated by reference herein in its entirety. 
     The effect of Debye length screening of the region around the point defect of the nanotube can be useful for the assay applications of the smFET  100  devices. In one example, measurements were performed with a 10-mer probe, covalently attached at the 5′ end to the nanotube, and hybridized to targets of varying length such that the distance was varied between the 3′ end of the target and the nanotube. As such, the probe lengths were comparable to the persistence length of ssDNA.  FIGS. 4A-4D  illustrate the results of the measurements.  FIGS. 4A-4C  are histograms illustrating the two-level conductance fluctuations that show binding and melting of targets of different length at a temperature of 17° C., which can be lower than the melting temperature. The resistance amplitude that resulted from these different target lengths is shown in  FIG. 4D  as a function of distance of the 3′-end of the target to the nanotube for a fixed buffer (embodied herein as 1×PBS). The results can be represented by simple Debye length screening 
               ∝     e   ⁢     x     λ   d           ,         
where λ d  represents the Debye length. Similar Debye-length trends can be shown by varying the buffer concentration. As such, the interactions of the biomolecular system can be controlled with the charge-sensitive region of the smFET  100  device through proximity and buffer strength.
 
     According to another aspect of the disclosed subject matter, an integrated circuit for a single-molecule nucleic-acid assay platform is provided. With reference to  FIGS. 5-7 , in an exemplary embodiment, the assay platform  200  can include one or more of smFETs  100  disposed on a complementary metal-oxide semiconductor (CMOS) integrated circuit  210 . Platform  200  can allow for the development of an assay device  100  configured on a form factor of a USB memory stick, as shown for example in  FIG. 5 . The integrated circuit  210  can be configured, for example and without limitation, with dimensions from 1-mm to 10-mm on each side, and as embodied herein as a 5-mm-by-5-mm integrated circuit chip fabricated in an IBM 0.13-μm CMOS technology. Integrated circuit  210  can include, for example and without limitation, 10 to 100 measurement channels each multiplexed to 100 to 1000 smFET devices per channel, and as embodied herein having 12 measurement channels each multiplexed to 500 smFET devices per channel. Platform  200 , in addition to improved integration, can allow for improved fabrication of these nanotube devices. 
     Various techniques can be utilized to transfer one or more nanotubes  110  to a substrate of a CMOS integrated circuit  210 .  FIG. 6A  illustrates an exemplary technique for transferring nanotubes  110  to a substrate of CMOS integrated circuit  210 . The technique of  FIG. 6A  involves spinning on the carbon nanotubes from a suspension. Alternatively, as illustrated in  FIG. 6B , nanotubes  110  can be transferred by growing carbon nanotubes  110  on solid substrates and then spin on a polymer  205  (for example, embodied herein as polymethyl methacrylate) that can lift off in strong bases (for example, potassium hydroxide or sodium hydroxide) along with the nanotube  110 . The thin film polymer  205  can then be placed on top of the CMOS substrate  210 , along with the nanotubes  110 . As such, the density of nanotubes  110  can be adjusted through the growth recipe. Furthermore, using quartz substrates can improve density and alignment of the nanotube  110  arrays, which can be suitable for biosensors.  FIG. 6C  illustrates another exemplary technique for transferring nanotubes  110  using dielectrophoresis. In this technique, by applying an AC voltage between electrodes  215   a ,  215   b , a force can be applied to nanotubes  110  proportional to the gradient of electric fields. The CMOS chip  210  can aid in applying the field and stop the field when a nanotube  110  has bridged the electrodes. In each of the above transfer techniques, an active CMOS chip  210  with a dense electrode array, for example having 550 or more electrode pairs per mm 2 , can be used to locate where a nanotube bridges two contacts, and in this manner can guide the location of the nanotubes  110 . 
     After transfer of nanotubes  110  to the substrate of the CMOS integrated circuit  210 , additional lithography and metallization can be performed to deposit a metal, such as titanium, to form electrodes  220   a ,  220   b  connecting the nanotubes  110  to the integrated circuit  210 , as shown for example in  FIG. 7 . The metal can be deposited onto exposed aluminum electrodes on substrate  215 , or alternatively, the exposed aluminum electrodes can be etched away and replaced with titanium. Additionally, a further set of surface-exposed aluminum electrodes can be etched away and replaced with gold. The gold electrodes can be used to create one or more integrated reference electrodes  225  to allow control of the electrolytic potential gating the nanotube sensors. Integrated circuit  210  can be wirebonded to a ball-grid array (BGA) package. A dam-and-fill doughnut epoxy encapsulation (for example, using Hysol FP4451 dam and FP4650 fill) can cover the exposed gold wirebonds, which can leave the die surface exposed. The reference electrodes  225  can be formed, for example and without limitation, by electroplating silver with subsequent chlorination with FeCl3. The reference electrodes can thus be configured as Ag/AgCl electrodes, which can have a relatively low current level therethrough, and thus can operate for days continuously before being exhausted. Chlorination of the Ag/AgCl electrodes can be repeated multiple times before the silver electrode can become exhausted and can need to be re-electroplated. Additionally or alternatively, platinum can be deposited to create the reference electrodes  225  to allow control of the electrolytic potential gating the nanotube  110 . Platinum can be deposited onto the set of surface-exposed aluminum electrodes, or the set of surface-exposed aluminum electrodes can be etched away and replaced with platinum. Additionally or alternatively, one or more external reference electrodes  125  can be utilized to allow control of the electrolytic gating potential, as described herein. 
     Integration of nanotubes  110  with CMOS chips  210  can allow multiple devices to be integrated on the same measurement substrate and can allow reduction in the parasitic capacitance associated with assay measurements. As such, measurement bandwidth can be increased while reducing amplifier noise. Furthermore, the integrated circuit  210  CMOS substrates can also be automated to quickly probe devices and select those with suitable performance. 
     Concentrations of target analytes can be determined by the mean time between capture events, as shown for example in  FIG. 2 . Concentrations as low as 100 fM can be detectable within approximately 8 hours. The assays can be performed at a temperature such that after capture, there is suitable thermal energy, which can be based at least in part on the size of the target and the buffer concentration, for the captured molecule to escape again and allow for a new capture event. After a capture event, the molecule can again be recaptured. The detailed kinetics of these multiple capture events can provide additional information to identify single-nucleotide polymorphisms (SNPs) and other partial mismatches. By contrast, in certain microarrays, concentration and mismatch affinity can not be independently determined. 
     Capture probe  160  can be immobilized or programmed on a site-specific basis, for example and as embodied herein through either robotic spotting or through electrically programmed immobilization. With robotic spotting, drops of capture probe  160  can be placed over each selected site. In this manner, sensor density can have a pitch as low as approximately 150-μm. With electrically programmed immobilization, individual probe sites can be electrically selected such that only those selected sites can bind the probe. In this manner, sensor density can be increased without limit over that obtained with robotic spotting. 
     Example 
     In one example, chemical vapor deposition was used to grow carbon nanotubes  310  approximately 1.4-1.6 nm in diameter on silicon wafers  315  with a thin (about 300 nm) grown silicon oxide layer. Photolithography was used to pattern titanium electrodes on top of the grown nanotubes to make source and drain contacts separated by about 2.5 μm. From a single carbon nanotube  310 , 20-30 individual devices can be created, which can then be wirebonded and epoxy encapsulated to perform the following biological assay techniques. An exemplary smFET assay platform  300  configuration is shown in  FIG. 8A , for purpose of illustration. A small bias can be applied between the source and drain electrodes  320   a ,  320   b , embodied herein as titanium electrodes, on the order of 100 mV, and a current can be measured through the individual carbon nanotube  310 , as shown for example in the scanning electron microscope image of  FIG. 8B . 
     The carrier density in the nanotube  310  can be controlled through a global back gate V bg . The carbon nanotube  310  can also be gated through the liquid electrolyte  330 , for example by controlling the electrolyte potential with an Ag/AgCl or platinum pseudo reference electrode  325 . Mobile ions in the electrolyte  330  can cause an electric field at the nanotube liquid interface and induce carriers in the nanotube channel. The amount by which the potential drops can be given by the Debye length, as described further herein, which, as embodied herein, can be about 0.7 nm for a liquid electrolyte  340  buffer of 1×PBS at a pH of 7.4. Due at least in part to the larger capacitance (C lg /C BG ˜10), the semiconducting nanotube  310  can be turned off at smaller voltages with the electrolyte gate V lg  than with the back gate V bg , as shown for example in  FIG. 8C , which illustrates the current through the back gate (bottom) and liquid gate (top). 
     One-dimensional conductors, such as single-walled carbon nanotubes, can act as high-gain field-effect sensors, in which the conductance can vary strongly with local charge density. As an example of ensemble sensing using the assay platform  300  to sense bovine serum albumin (BSA) is provided.  FIG. 9A  is an atomic force microscope (AFM) image showing carbon nanotube  310  of assay platform  300 . The nanotube  310  was immersed in 50 nM BSA in 1×PBS solution for 1 hour, which is shown in the AFM image of  FIG. 9B . As shown in  FIG. 9B , the BSA proteins tend to stick preferentially to the carbon nanotube  310 . The device  300  was measured before and after this exposure using the back gate in air and the adsorbed protein cause a doping of the channel, resulting in a threshold shift of −3V, as shown in  FIG. 9C . A shift in the same direction was also observed in device  300  under the aqueous buffer. For purpose of comparison, as shown in  FIG. 9D , the Ag/AgCl electrolyte gate V lg  response was shifted by around −120 mV when exposed to a 1 μM BSA buffer, which is also illustrated in real time in the inset of  FIG. 9D . In this manner, the device  300  was held under a constant bias and the conductance was observed. The device  300  shows no response when only the electrolyte solution is applied, but a drop in conductance is measured when the device  300  is immersed in the BSA solution. As such, carbon nanotube  310  in assay device  300  can be used for label-free detection of biomolecules. 
     Sensitivity of device  300  can be improved by creating a point defect  340  in the carbon nanotube  310 , which can reduce the region of charge sensitivity to the region around the defect. The defect can be created, as embodied herein, by conductance-controlled electrochemical etching of the tube in 1 M sulfuric acid using a platinum electrode, followed by a 30 to 45 s exposure to 6.5 mM potassium permanganate to create a defect with a carboxylic acid functional group. Conductance-based oxidation of the carbon nanotube  310  is illustrated in  FIG. 10 . As shown in  FIG. 10 , as the electrolytic gate voltage applied to the platinum electrode was changed below a threshold voltage (around −1V), the conductance exhibits a slow decrease, followed by an abrupt jump. A Labview program detected this jump and immediately ramped up the platinum voltage in order to avoid further oxidation. The potential was held constant, and the device was immersed in 6.5 mM potassium permanganate to create the carboxylic acid functional group. 
     Scanning gate microscopy (SGM), which used the biased tip of an atomic force microscope as a localized gate, was used to characterize the location of the defect  340 .  FIG. 11A  shows an SGM image of the defect overlaid with a topography image of nanotube  310 . The Schottky barriers at the contacts (shown in  FIG. 11A  as the darker areas) can be seen when the tip is biased with a −5 V bias. With the presence of point defect  340 , the sensitivity can be localized to a small region around the center of the nanotube, as shown in  FIG. 11B . Localization can be further illustrated by a coupling reaction with gold-labeled streptavidin. A gold nanoparticle can be found around the region of highest sensitivity, as shown in  FIG. 11C , indicating that the defect can be both localized and chemically reactive. 
     In one example, device  300  can be used to measure DNA hybridization. A 10-mer DNA probe with an amine group at the 5′ end (NH2-5′-GTGAGTTGTT-3′) (SEQ ID NO: 1) was attached to the point defect  340  in the 310 nanotube by an amine to carboxylic acid coupling reaction assisted by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and sulpho-N-hydroxysuccinimide (sulpho-NHS). The device  300  was rinsed with deionized water, and the conductance was monitored in phosphate buffered saline buffer (1×PBS).  FIG. 12A  illustrates real time conductance data obtained with the DNA probe  360  only, with a corresponding histogram of the data illustrated in  FIG. 12B . As shown in  FIG. 12A , no particular features in the conductance are observed and the device  300  exhibits flicker (1/f) noise. The histogram of the conductance shows the conductance data fit to a single Gaussian pattern, as shown in  FIG. 12B . 
     The device  300  was then exposed to a 1 μM complementary DNA concentration  350  at 28° C.  FIG. 12C  illustrates real time conductance data obtained with the complementary DNA concentration  350  added, with a corresponding histogram of the data illustrated in  FIG. 12D . As shown in  FIG. 12C , the device  300  exhibits two-level fluctuations, which can also be seen in the corresponding histogram in  FIG. 12D . In this manner, a model can be shown in which the low-conductance state represents device  300  with DNA in the duplex form and the high-conductance state with only DNA probe  360  attached. 
     In addition to the two-level fluctuations, there was a small decrease in the overall conductance level with the addition of target DNA  350 , which can be attributed at least in part to non-specific adsorption to the sidewall of the nanotube  310 . From the model, a DNA melting curve (shown in  FIG. 13A ) can be extracted by taking the ratio of the areas under the Gaussian fits to the histograms in  FIG. 12D . The melting point measured with the nanotube conductance on a single DNA molecule was slightly lower (29.4° C.) than the measurements in solution by UV-Vis spectroscopy (36.2° C.). The fit can be represented by the Langmuir isotherm K=a/(1−a)C, where C can represent the concentration of target DNA together with the thermodynamic relationship −RT ln(K)=ΔH°−TΔS°. The kinetics of both the high and low states were also determined, as shown in the Arrhenius plot in  FIG. 13B . 
     Random telegraph noise can be a challenge in nanoscale CMOS devices. The assay devices according to the disclosed subject matter, which can be sealed to nanometer dimensions, can be affected by trapping phenomena at the silicon-dielectric interface in one or more traps. Certain data analysis techniques, including time lag plots (TLP), which are two dimensional histograms of the conductance at consecutive data points, can be utilized to analyze random telegraph noise.  FIG. 14A  is a diagram illustrating the single state in the TLP with DNA probe  360  only.  FIG. 14B  illustrates the two-states in the TLP after adding complementary DNA target  350 . 
     Having the channel of device  300  exposed to the environment can be advantageous at least in part because molecules can be directly tethered to it, which can improve sensitivity of the device  300 . However, small fluctuating changes in the environment can affect the electronic transport. The underlying noise of device  300  can be examined.  FIGS. 15A-15B  illustrate the noise power spectral density of the carbon nanotube  310  current as a function of bias both for the back gate configuration ( FIG. 15A ) and the electrolyte gate ( FIG. 15B ).  FIGS. 15C-15D  illustrate the corresponding flicker noise for the back gate configuration ( FIG. 15C ) and the electrolyte gate ( FIG. 15D ).  FIGS. 15A-15D  illustrate that, for each configuration, the noise included mostly flicker or 1/f noise, and the noise amplitude was similar. 
     Two exemplary models can be utilized to represent the flicker noise in carbon nanotube  310 . The first exemplary model can be the empirical Hooge model, which can suggest that noise can be caused by independent scattering events of charge carriers, and as such can scale inversely with the number of charge carriers in the nanotube  310 . The second exemplary model can be the charge-noise model, which can suggest that noise can be attributed to random fluctuations of charge in the environment of the channel. The flicker noise can be represented by the charge-noise model as:
 
 S   l ∝1 /C   g   2 ( dI/dV   g ) 2 ,  (1)
 
where S can represent the noise due to fluctuations of local environmental charge, C g  can represent the capacitive coupling between the gate and the carbon nanotube  310 , dI can represent the change in current through the nanotube  310  and dV g  can represent the change in gating potential of the nanotube  310 . As shown in  FIG. 16 , the noise spectrum and fitted to the above charge-noise model using the gate sweeps in  FIGS. 15A-15B . One difference between the back gate and the liquid gate can be the gate capacitance. As such, the liquid itself can be considered to not contribute significant noise, which can be beneficial for biosensor applications in aqueous environments. The majority of the noise can be due at least in part to both traps at the interface between the nanotube  310  device and the dielectric substrate on which the nanotube  310  device is supported and noise from diffusion or absorption/desorption of biomolecules near the point defect  340 .
 
     The foregoing merely illustrates the principles of the disclosed subject matter. Various modifications and alterations to the described embodiments will be apparent to those skilled in the art in view of the teachings herein. It will be appreciated that those skilled in the art will be able to devise numerous modifications which, although not explicitly described herein, embody its principles and are thus within its spirit and scope.