Electron beam sculpting of tunneling junction for nanopore DNA sequencing

A nanodevice is provided that includes a reservoir filled with a conductive fluid and a membrane separating the reservoir. The membrane includes an electrode layer having a tunneling junction formed therein. A nanopore is formed through the membrane, and the nanopore is formed through other layers of the membrane such that the nanopore is aligned with the tunneling junction of the electrode layer. When a voltage is applied to the electrode layer, a tunneling current is generated by a base in the tunneling junction to be measured as a signature for distinguishing the base. When an organic coating is formed on an inside surface of the tunneling junction, transient bonds are formed between the electrode layer and the base.

BACKGROUND

Exemplary embodiments relate to nanodevices, and more specifically to a tunneling junction and nanopore structure in a nanodevice

Nanopore sequencing is a method for determining the order in which nucleotides occur on a strand of Deoxyribonucleic acid (DNA). A nanopore is a small hole on the order of several nanometers in internal diameter. The theory behind nanopore sequencing has to do with what occurs when the nanopore is immersed in a conducting fluid and an electric potential (voltage) is applied across the nanopore. Under these conditions, a slight electric current due to conduction of ions through the nanopore can be measured, and the amount of current is very sensitive to the size and shape of the nanopore. If single bases or strands of DNA pass (or part of the DNA molecule passes) through the nanopore, this can create a change in the magnitude of the current through the nanopore. Other electrical or optical sensors can also be put around the nanopore so that DNA bases can be differentiated while the DNA passes through the nanopore.

DNA could be driven through the nanopore by using various methods. For example, an electric field might attract the DNA towards the nanopore, and it might eventually pass through it. The scale of the nanopore means that the DNA may be forced through the hole as a long string, one base at a time, rather like thread through the eye of a needle.

BRIEF SUMMARY

According to an exemplary embodiment, a nanodevice is provided. The nanodevice includes a reservoir filled with a conductive fluid, and a membrane separating the reservoir, where the membrane includes an electrode layer having a tunneling junction formed therein. The nanodevice includes a nanopore formed through the membrane, and the nanopore is formed through other layers of the membrane such that the nanopore is aligned with the tunneling junction of the electrode layer. When a voltage is applied to the electrode layer, a tunneling current is generated by a base in the tunneling junction to be measured as a current signature for distinguishing the base. When an organic coating is formed on an inside surface of the tunneling junction, transient bonds are formed between the electrode layer and the base.

According to an exemplary embodiment, a system is provided. The system includes a nanodevice, which includes a reservoir filled with a conductive fluid. The nanodevice includes a membrane separating the reservoir, where the membrane includes an electrode layer having a tunneling junction formed therein. A nanopore is formed through the membrane, and the nanopore is formed through other layers of the membrane such that the nanopore is aligned with the tunneling junction of the electrode layer. Also, the system includes a voltage source operatively connected to the electrode layer. When a voltage is applied by the voltage source to the electrode layer, a tunneling current is generated by a base in the tunneling junction to be measured as a current signature for distinguishing the base. When an organic coating is formed on an inside surface of the tunneling junction, transient bonds are formed between the electrode layer and the base.

According to an exemplary embodiment, a nanodevice is provided. The nanodevice includes a reservoir filled with a conductive fluid, and a membrane separating the reservoir, where the membrane includes an electrode layer having a tunneling junction formed therein. The nanodevice includes a nanopore formed through the membrane, and the nanopore is formed through other layers of the membrane such that the nanopore is aligned with the tunneling junction of the electrode layer. When a voltage is applied to the electrode layer, a tunneling current is generated by a base in the tunneling junction to be measured as a current signature for distinguishing the base.

Other systems, methods, apparatus, design structures, and/or computer program products according to embodiments will be or become apparent to one with skill in the art upon review of the following drawings and detailed description. It is intended that all such additional systems, methods, apparatus, design structures, and/or computer program products be included within this description, be within the scope of the exemplary embodiments, and be protected by the accompanying claims. For a better understanding of the features, refer to the description and to the drawings.

DETAILED DESCRIPTION

Exemplary embodiments provide an approach to make a nanometer size tunneling junction by focus electron beam cutting, and then to fine tune the junction size, by expanded electron beam techniques. Exemplary embodiments also include the integration of such tunneling junction with a nanopore for the purpose of DNA sequencing in a nanodevice.

Recently, there has been growing interest in applying nanopores as sensors for rapid analysis of biomolecules such as DNA, ribonucleic acid (RNA), protein, etc. Special emphasis has been given to applications of nanopores for DNA sequencing, as this technology is believed to hold the promise to reduce the cost of sequencing below $1000/human genome. One issue in nanopore DNA sequencing is electrically differencing individual DNA bases by leveraging this nanopore platform.

In accordance with exemplary embodiments, an approach is disclosed which uses a focused electron beam (e.g., utilizing a beam size as small as 0.4 nm) to cut a thin metal layer (shown as cut line105inFIG. 1A) to form the tunneling junction. Under a low intensity electron beam, material migration can occur, and the material migration can be used to fine tune the gap size of the tunneling junction. If the thin metal layer is on a free-standing membrane, one can also make the nanopore (shown as nanopore206,208inFIGS. 2B-2I) through the top of the membrane at the gap to create the tunneling junction right at the entrance, at the inner surface, and/or the exit of the nanopore for DNA sequencing purposes via the tunneling current.

Now turning to the figures,FIGS. 1A-1Cillustrate a schematic of a process to make a tunneling junction by focused electron beam cutting and to fine tune the junction size by expanded electron beam according to an exemplary embodiment.FIGS. 1A-1Care top views of the schematic. InFIG. 1A, a substrate101can be any electrically insulating substrate, and layer102can be any electrically conductive layer such as a metal on top of the substrate101. Voltage is applied by voltage source103between two ends of the conductive layer102and current is monitored through the ammeter104. A focused electron beam (not shown) could be as small as 0.4 nm, and the focused electron beam performs line scanning shown as line105at the center location of conductive layer102(e.g., in a vacuum). One skilled in the art understands electron beam lithography (e-beam lithography), and understands the practice of scanning a beam of electrons in a patterned fashion across a surface.

The high energy, high density electron beam can sputter/etch material on its way into the vacuum gradually. When the voltage at voltage source103is being applied, the current measured by its corresponding ammeter104serves as a feedback that the current through ammeter104will drop down to zero (0) once the conductive layer102is cut into two halves by the electron beam, as shown inFIG. 1B.FIG. 1Bshows a left half and right half of the conductive layer102. In this way, one can create a tunneling junction106without damaging the underneath substrate101. The tunneling junction106which is a nanosize gap between two electrically conductive parts corresponds to the line105previously shown inFIG. 1A.

InFIG. 1B, with an expanded (i.e., low intensity) electron beam covering area107, the (metal) material in the conductive layer102can migrate and the gap size of the tunneling junction106can be tuned; that is the tunneling junction106can be reduced or increased in size to be the tunneling junction108shown inFIG. 1C.

For example, to achieve the desired size tunneling junction (gap)108, a low intensity electron beam can be used to bombard the conductive layer102at the tunneling junction (gap)108(106inFIG. 1B); this will cause the conductive layer102material to get softer and flow under surface tension. The low intensity electron beam can be utilized to cause the conductive layer102material to flow such that the tunneling junction (gap)108is widened and/or flow such that the tunneling junction (gap)108is narrowed. As seen by the decrease in size of the tunneling junction (gap)106inFIG. 1Bto the tunneling junction (gap)108inFIG. 1C(which is not drawn to scale), material migration has caused the tunneling junction (gap)106to narrow. If the substrate101is a thin membrane, the whole tuning process can be monitored under a transmission electron microscope in real-time. Thus, one can acquire (tune) the exact size of the tunneling junction (gap)108by turning off the electron beam at the right moment. After fine tuning the tunneling junction106, the tunneling junction106is now represented as the finely tuned tunneling junction (gap)108inFIG. 1C.

FIGS. 2A-2Fillustrate a schematic of the integration of the tunneling junction108with a nanopore in accordance with an exemplary embodiment.FIGS. 2A,2B,2D, and2E (including4A and4B) are a cross-sectional view of the schematic, andFIGS. 2C and 2F(including4C) are top views of the schematic. InFIG. 2A, the substrate201can be any substrate, such as Si (silicon). Layers202and203are electrically insulating films, such as Si3N4(compound of silicon and nitrogen). The insulating layer203serves as an etching mask for etching thorough the substrate201via either dry or wet etching, and the etching stops on insulating layer202. In this way, part of the insulating layer202will be a free-standing membrane. Conductive layer204(corresponding to conductive layer102inFIG. 1) is an electrically conductive layer, and tunneling junction205(corresponding to tunneling junction/gap108inFIG. 1) is the tunneling junction made in the free-standing membrane part of conductive layer204using the method described inFIG. 1. The tunneling junction205will be visible under a transmission electron microscope, and a nanometer size pore (nanopore)206can be made through the tunneling junction205and the underneath insulating layer202, as shown inFIG. 2B. In this way, the tunneling junction205is integrated with the nanopore206. As seen inFIG. 2B, the nanopore206is a hole through the insulating layer202while the tunneling junction205is a gap in the conductive layer (metal)204.

FIG. 2Cshows a top view of the schematic inFIG. 2B. As seen in the top view ofFIG. 2C, the tunneling junction205(corresponding to tunneling junction/gap108inFIG. 1) is only between the conductive layer (metal)204(corresponding to conductive layer102), and the tunneling junction205splits the conductive layer204into a left half and a right half. The nanopore206is formed through the tunneling junction205and goes through the substrate201.

In order to work with an electrically conductive solution, an insulating (cap) layer207(also called the passivation layer which may be a layer of oxide and/or silicon nitride) is deposited on the conductive layer204, as shown inFIG. 2D(e.g., right after the tunneling junction205is made). The tunneling junction205will be visible under a transmission electron microscope and a nanometer size pore (nanopore)208can be made through the tunneling junction205and the underneath insulating layer202, as shown inFIG. 2E. In this way, the tunneling junction205is embedded in the nanopore208. The nanopore206may now be considered part of the nanopore208. Via windows209and210are opened through the insulating layer207down to the conductive layer204, for electrically accessing the two sides of the tunneling junction205. The windows209and210will be used as electrodes/connections for connecting, e.g., a wire to the left and right halves of the conductive layer204.

FIG. 2Fillustrates the top view ofFIG. 2E. InFIG. 2F, the conductive layer204(shown as an outline with a dotted line) is buried underneath the insulation (passivation) layer207with windows209and210of the conductive layer204exposed. Although not visible inFIG. 2F, the nanopore208goes through the insulating layer202and the insulation (passivation) layer207.

FIGS. 4A,4B, and4C illustrate a variation ofFIGS. 2A-2Fin which the nanopore208and tunneling junction are made in the same electron beam cutting process and have the same shape in accordance with an exemplary embodiment. An insulating (cap) layer207(also called the passivation layer which may be a layer of oxide and/or silicon nitride) is deposited on the conductive layer204, as shown inFIG. 4A. A focused electron beam is used to cut through all layers207,204, and202at the freestanding membrane part and to cut conductive layer204into two halves, as shown inFIG. 4B. In this way, the tunneling junction205and the nanopore208have exactly the same shape. Via windows209and210are opened through the insulating layer207down to the conductive layer204, for electrically accessing the two sides of the tunneling junction205. The windows209and210will be used as electrodes/connections for connecting, e.g., a wire to the left and right halves of the conductive layer204.

FIG. 4Cillustrates the top view ofFIG. 4B. InFIG. 4C, the conductive layer204(shown as a dotted line) is buried underneath the insulating (passivation) layer207with windows209and210of the conductive layer204exposed. Although not visible inFIG. 4C, the nanopore208goes through the insulating layer202and the insulating (passivation) layer207.

FIGS. 3A and 3Billustrate a schematic (system) of a tunneling junction (e.g., tunneling junction106,108, and205) and nanopore device300for DNA sequencing according to an exemplary embodiment.FIGS. 3A and 3Bshow a cross-sectional view of the tunneling junction and nanopore device300.

InFIGS. 3A and 3B, elements301-310are the same as elements201-210respectively. However,FIG. 3Bincludes an organic coating as discussed herein. The tunneling junction and nanopore device300partitions two reservoirs311and312. Electrically conductive solution313fills the two reservoirs311and312as well as the nanopore308. A negatively charged DNA314(with each base illustrated as base315) can be driven into the nanopore308by a voltage of the voltage source318applied between the two reservoirs311and312via two electrodes316and317, respectively. Voltage of the voltage source319is applied between the two sides (at left window309and right window310) of the tunneling junction305, and a baseline tunneling current is monitored at ammeter320. The baseline tunneling current may be stored in memory15of a computer600(shown inFIG. 6) for further use as discussed herein. As DNA bases315pass through the tunneling junction305(which is the gap in the conductive (metal) layer304), each of the DNA bases315can be indentified by its respective tunneling current signal at the ammeter320.

For example, voltage source318is turned on to drive the DNA314into the tunneling junction305which is the gap separating the conductive layer304into two halves. When, e.g., a base315ais in the tunneling junction305, voltage source319is turned on (while voltage source318is turned off) to measure the tunneling current of the base315a. For instance, with voltage applied by voltage source319, current flows through window309(acting as an electrode) of conductive layer304, through the conductive layer304, into the conductive solution (liquid)313, into the DNA base315a(which produces the tunneling current signature), out through the conductive solution313, into the right side of the conductive layer304, out through the window310(acting as an electrode), and into the ammeter320for measurement. The ammeter320may be implemented by and/or integrated in the computer600(test equipment) for measuring the baseline tunneling current and tunneling current generated by the DNA base315a. A software application605of the computer600is configured to measure, display, plot/graph, analyze, and/or record the measured tunneling current for each DNA base315that is tested. In the example above, the software application605(and/or a user utilizing the software application605) can compare the baseline tunneling current measured with no DNA base315in the tunneling junction305to the tunneling current corresponding to each DNA base315(at a time) that is measured in tunneling junction305. In the example, the tunneling current (signal) for the DNA base315ais compared against the baseline tunneling current by the software application605(or a user utilizing the software application605). The tunneling current (signature) for the DNA base315amay have particular characteristics that are different from the baseline tunneling current measured by the ammeter320, and the tunneling current (signatures) for the DNA base315acan be utilized to identify and/or differentiate the DNA base315afrom other DNA bases315on the DNA314.

For example, the measured tunneling current signature for DNA base315amay have a positive pulse, a negative pulse, a higher or lower current (magnitude), an inverse relationship, a rising or falling plot, a particular frequency, and/or any other difference from the baseline tunneling current that can be determined by the software application605(and/or a user viewing the display45of the two different plots). This unique tunneling current signature can be utilized (by the software application605) to distinguish the DNA base315afrom other DNA bases315. Note that, the tunneling current measured at ammeter320between electrode layers does not require any electrical wiring between the left and right parts (which will be shown as electrodes304aand304binFIG. 3B) of the conductive (electrode) layers304as electrons simply move from one electrode to the other in a quantum mechanical way. For example, there will be a baseline tunneling current when DNA base315ais away (e.g., with distance much longer than the wavelength of an electron) from the tunneling junction305. When DNA base315ais close (e.g., within the distance of the wavelength of an electron) to the tunneling junction305, the tunneling path of the electron will be rerouted to tunnel from the left part of the conductive (electrode) layer304to the DNA base315aand then to the right part of the conductive (electrode) layer304. In this way, the tunneling current (electrons) through the DNA base315awill create a current signature (such as an increase of tunneling current, typically in the order of tens of pA (picoamperes)) added onto the baseline tunneling current trace. The tunneling current across DNA bases is dependent on the electronic and chemical structure of the DNA bases; thus, a different DNA base will generate a different tunneling current signature. If the difference between the tunneling current signatures of different bases is small or stochastic, repeating measurements on the same DNA base can be done; a histogram of the amplitudes of the tunneling current signatures can be fit and the statistical data will provide enough resolution to differentiate DNA bases.

FIG. 3Butilizes the approach discussed forFIG. 3Aexcept that the conductive layer304is coated with organic coating325aand325b, which can form transient bonds321aand321b(such as a hydrogen bond (i.e., transient bonds321) with the DNA base315). InFIG. 3B, these transient bonds321formed by the organic coating325aand325bwill fix the orientation of the DNA base315and the relative distance of the DNA base315to the conductive layer304, for improving the tunneling current signal measured by ammeter320and for better identifying DNA bases315. If the organic coating325aand325band/or transient bonds321aand321bare electrically conductive, they will help to shrink the tunneling gap size and enhance the tunneling current signatures too. Also, the transient bonds321aand321bby the organic coating325aand325bhold the DNA314in place against thermal motion when measuring the tunneling current of the base315. The forces of thermal motion may cause the DNA314to move, but the transient bonds321aand321bfix the base315in the tunneling junction305against the DNA movement caused by thermal motion.

In one implementation, the organic coating325aand325bconsists of bifunctional small molecules which at one end form covalent bonds with conductive layer304, and at the other end (of the organic coating325a/b) which is exposed in the nanopore308, the organic coating325aand325bconsists of functionalities which can form strong hydrogen bonds with DNA and/or can protonate nucleotides to form acid base interactions. If the conductive layer304is made of metals such as gold, palladium, platinum etc., the first functionality which bonds to the conductive layer304can be chosen as thiols, isocyanides, and/or diazonium salts. If the conductive layer304is made of titanium nitrides or indium tin oxide (ITO), the covalent bonding functionality is chosen from phosphonic acid, hydroxamic acid, and/or resorcinol functionality. The small bifunctional molecules are designed in such a way that any charge formation due to interaction with DNA can easily be transferred to the conductive layer304and therefore a pi-conjugated moiety (e.g., benzene, diphenyl, etc.) are sandwiched between two functionalities. The second functionality is a group which can form a strong hydrogen bond with DNA. Examples of such groups include but are not limited to alcohols, carboxylic acids, carboxamides, sulfonamides, and/or sulfonic acids. Other groups which can be used to form interactions with DNA are individual self-assembled nucleotides. For example, adenine monophosphonic acid, guanine monophosphonic acid, etc., can be self-assembled on titanium nitride electrodes or mercapto thymine or mercapto cytosine self-assembles on metal electrodes such as gold and/or platinum.FIG. 5illustrates examples of molecules for self-assembly inside nanopores according to exemplary embodiments. The molecules may be utilized as the organic coating325aand325b.

Referring toFIG. 3B, as discussed above, the voltage source318is applied to move the DNA314into the nanopore308. When voltage of the voltage source319is applied (and the voltage source318is turned off), current flows through left electrode304a, into the organic coating325a, into the transient bond321a(which acts as or can be thought of as a wire), into the DNA base315a(producing the tunneling current), out through the transient bond321b, out through the organic coating325b, out through the right electrode304b, and into the ammeter320to measure the tunneling current of the DNA base315a. The ammeter320may be integrated with the computer600, and the computer600can display on display45the tunneling current of the DNA base315aversus the baseline tunneling current measured when no base315is in the tunneling junction305.

FIG. 7illustrates a method700according to exemplary embodiments, and reference can be made toFIGS. 1,2, and3.

At operation705, a tunneling junction108,205,305is made by electron beam sculpting (cutting or size-tuning). Using a low intensity electron beam, the tunneling junction108,205,305can be widened by causing the material (metal) of the conductive layer102,204,304to migrate away from the tunneling junction gap, thus making the gap wider; similarly, using a low intensity electron beam spread across area107inFIG. 1, the tunneling junction108,205,305can be narrowed to cause the material of the conductive layer102,204,304to flow toward (into) the tunneling junction gap thus make the gap smaller.

At operation710, the tunneling junction108,205is integrated with a nanopore208as shown inFIGS. 2B-2F. The integrated (combined) tunneling junction205and nanopore208form a hole through multiple layers207,204, and202as shown inFIG. 2E. The distinction between the tunneling junction205and the nanopore208can be seen inFIG. 2F. This distinction is carried through to the tunneling junction305shown inFIG. 3in which the tunneling junction305is the gap between the conductive layer304(i.e., separating the conductive layer304into two halves) but not layers307,302,301, and303. In one implementation, the tunneling junction108,205is formed prior to forming the nanopore208(and/or nanopore206).

At operation715, the nanopore208partitions two conductive ionic buffer reservoirs312and313, and the DNA314is electrically loaded into the nanopore308and the tunneling junction305. The tunneling junction305is between the left half304aand right half304bof the conductive layer304. The left and right halves304aand304bserve as electrodes for accessing the tunneling junction305(and the base315therein) by the voltage source319to measure the tunneling current with ammeter320.

At operation720, the DNA bases315are differentiated using the tunneling current of each individual base315(measured by ammeter320) with and/or without organic coating325aand325bon the inside surface of the tunneling junction305. The computer600can measure, analyze, differentiate, display, and record/store (in memory15) the different tunneling currents measured for the different bases315of the DNA314. The tunneling current measurements of the bases315with the organic coating325aand325bcausing the transient bonds321aand321bwould be different from the tunneling currents measurements of the same bases315without the organic coating325aand325band without the transient bonds. For example, the tunneling current measured for base315awith the organic coating325aand325b(causing transient bonds321aand321b) may have a greater magnitude than without the organic coating325aand325b.

Now turning toFIG. 6,FIG. 6illustrates a block diagram of the computer600having various software and hardware elements for implementing exemplary embodiments. The computer600may be utilized in conjunction with any elements discussed herein.

The diagram depicts the computer600which may be any type of computing device and/or test equipment (including ammeters, voltage sources, connectors, etc.). The computer600may include and/or be coupled to memory15, a communication interface40, display45, user interfaces50, processors60, and software605. The communication interface40comprises hardware and software for communicating over a network and connecting (via cables, plugs, wires, electrodes, etc.) to the nanodevices discussed herein. Also, the communication interface40comprises hardware and software for communicating with, operatively connecting to, reading, and controlling voltage sources, ammeters, tunneling currents, etc., as discussed herein. The user interfaces50may include, e.g., a track ball, mouse, pointing device, keyboard, touch screen, etc, for interacting with the computer600, such as inputting information, making selections, independently controlling different voltages sources, and/or displaying, viewing and recording tunneling current signatures for each base, etc.

The computer600includes memory15which may be a computer readable storage medium. One or more applications such as the software application605(e.g., a software tool) may reside on or be coupled to the memory15, and the software application605comprises logic and software components to operate and function in accordance with exemplary embodiments in the form of computer executable instructions. The software application605may include a graphical user interface (GUI) which the user can view and interact with according to exemplary embodiments.