Abstract:
A method of analyzing molecules using a nanopore array including a plurality of cells included on a chip is disclosed. Nanopores are caused to be formed in at least a portion of the plurality of the cells. A first physical measurement of the nanopores is evaluated. It is determined whether to cause the molecules to interact with the nanopores. At least a portion of the nanopores is caused to interact with the molecules. A second physical measurement of the nanopores that indicates a property of the molecules is evaluated. It is determined whether to cause the nanopores to be reformed so that the cells may be reused to interact with additional molecules.

Description:
CROSS REFERENCE TO OTHER APPLICATIONS 
       [0001]    This application is a continuation of co-pending U.S. patent application No. Ser. No. 13/759,701 entitled NANOPORE ARRAYS filed Feb. 5, 2013 which is incorporated herein by reference for all purposes. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Advances in micro-miniaturization within the semiconductor industry in recent years have enabled biotechnologists to begin packing their traditionally bulky sensing tools into smaller and smaller form factors, onto so-called biochips. It would be desirable to develop techniques for biochips that make them more robust, efficient, and cost-effective. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0003]    Various embodiments of the invention are disclosed in the following detailed description and the accompanying drawings. 
           [0004]      FIG. 1  is a block diagram illustrating an embodiment of a system  100  for analyzing molecules using nanopore devices. 
           [0005]      FIG. 2  is a block diagram illustrating an embodiment for applying a voltage stimulus to a cell in nanopore array  102 . 
           [0006]      FIG. 3  is a diagram illustrating an embodiment of a nanopore device  300  within a cell of nanopore array  102 . 
           [0007]      FIG. 4A  is a diagram illustrating that nanopore device  300  is in a state in which a lipid bilayer has not yet been formed. 
           [0008]      FIG. 4B  is a diagram illustrating that nanopore device  300  is in a state in which a lipid bilayer  302  has been formed. 
           [0009]      FIG. 4C  is a diagram illustrating that nanopore device  300  is in a state in which a nanopore structure  308  with a nanopore  310  has been inserted into lipid bilayer  302 . 
           [0010]      FIG. 5  is a flow diagram illustrating an embodiment of a process  500  for analyzing molecules using nanopore devices. 
       
    
    
     DETAILED DESCRIPTION 
       [0011]    The invention can be implemented in numerous ways, including as a process; an apparatus; a system; a composition of matter; a computer program product embodied on a computer readable storage medium; and/or a processor, such as a processor configured to execute instructions stored on and/or provided by a memory coupled to the processor. In this specification, these implementations, or any other form that the invention may take, may be referred to as techniques. In general, the order of the steps of disclosed processes may be altered within the scope of the invention. Unless stated otherwise, a component such as a processor or a memory described as being configured to perform a task may be implemented as a general component that is temporarily configured to perform the task at a given time or a specific component that is manufactured to perform the task. As used herein, the term ‘processor’ refers to one or more devices, circuits, and/or processing cores configured to process data, such as computer program instructions. 
         [0012]    In various embodiments, the techniques described herein are implemented in a variety of systems or forms. In some embodiments, the techniques are implemented in hardware as an application-specific integrated circuit (ASIC) or a field-programmable gate array (FPGA). In some embodiments, a processor (e.g., an embedded one such as an ARM core) is used where the processor is provided or loaded with instructions to perform the techniques described herein. In some embodiments, the technique is implemented as a computer program product which is embodied in a computer readable storage medium and comprises computer instructions. 
         [0013]    A detailed description of one or more embodiments of the invention is provided below along with accompanying figures that illustrate the principles of the invention. The invention is described in connection with such embodiments, but the invention is not limited to any embodiment. The scope of the invention is limited only by the claims and the invention encompasses numerous alternatives, modifications and equivalents. Numerous specific details are set forth in the following description in order to provide a thorough understanding of the invention. These details are provided for the purpose of example and the invention may be practiced according to the claims without some or all of these specific details. For the purpose of clarity, technical material that is known in the technical fields related to the invention has not been described in detail so that the invention is not unnecessarily obscured. 
         [0014]    Advances in micro-miniaturization within the semiconductor industry in recent years have enabled biotechnologists to begin packing their traditionally bulky sensing tools into smaller and smaller form factors, onto so-called biochips. These chips are essentially miniaturized laboratories that can perform hundreds or thousands of simultaneous biochemical reactions. Biochips enable researchers to quickly screen large numbers of biological analytes for a variety of purposes, from disease diagnosis to detection of bioterrorism agents. 
         [0015]    Typically, a biochip includes a large array of cells. For example, a biochip for nucleotide sequencing may contain thousands or millions of single cells in an array. Each cell includes a molecular complex composed of monomers that make up an oligomeric nanopore. Each cell may further include a single strand of DNA, and anything bound to that single strand of DNA. The nanopore is a small hole in an electrically insulating membrane that can be used as a single-molecule detector. A nanopore may be formed using a biological material, such as α-hemolysin or MspA. A nanopore may be formed using a solid-state material, such as a semiconductor material. When a small voltage is applied across a molecular complex containing a nanopore, an ionic current through the molecular complex can be measured to provide information about the structure of a molecule transiting the molecular complex. In a single cell of the array, an electrical circuit may be used for controlling the electrical stimulus applied across a lipid bilayer which contains a nanopore, and for detecting and analyzing the electrical patterns, or signatures, of a molecule passing through the nanopore. 
         [0016]      FIG. 1  is a block diagram illustrating an embodiment of a system  100  for analyzing molecules using nanopore devices. System  100  includes a nanopore array  102 , a master controller  104 , a temperature controller  106 , a fluidic system  108 , a storage device  110  for storing extracted results, and a memory  112 . In some embodiments, some of the modules may be combined together as a single module, and some of the modules may be optional. In some embodiments, the cells of nanopore array  102  and the nanopore devices within the cells are individually controllable and individually addressable by other modules of system  100 , including by master controller  104 , temperature controller  106 , and fluidic system  108 . In some embodiments, performance data or other data corresponding to each of the cells may be sent from nanopore array  102  to other modules in system  100 . Control, address, performance, or other data signals may be communicated between nanopore array  102  and other modules in system  100  via signal lines  114 ,  116 , and  118 A, respectively. 
         [0017]    In some embodiments, the cells of nanopore array  102  and the nanopore devices within the cells are individually controllable and individually addressable by master controller  104 . This allows master controller  104  to control each of the cells or each group of cells in nanopore array  102  such that the particular cell or particular group of cells performs different functions or transits through different states independently, without affecting the functioning or progress of other cells or other groups of cells in nanopore array  102 . In one example, a mal-functioning cell in nanopore array  102  may be put in a state (e.g., disabled state) by master controller  104  such that the mal-functioning cell does not affect the functioning of other cells in nanopore array  102 . For example, if a lipid bilayer fails to form in a particular cell, the cell may be disabled such that no electrical stimulus is applied to the cell; otherwise, the cell may draw a large current, which may affect the performance of other cells in nanopore array  102 . 
         [0018]    In another example, master controller  104  may send control signals to nanopore array  102  such that different stimuli are applied to different cells or groups of cells. For example, a first stimulus (e.g., a voltage) is applied to a first group of cells and a second stimulus is applied to a second group of cells at time t l . The first stimulus may be a stimulus corresponding to a particular state of a cell, and the second stimulus may be a stimulus corresponding to a different state of a cell. The stimulus that is applied to the first group of cells may vary over time, as the first group of cells transits from one state to another.  FIG. 2  is a block diagram illustrating an embodiment for applying a voltage stimulus to a cell in nanopore array  102 . As shown in  FIG. 2 , control signals from master controller  104  may be used as input to a multiplexer  202  to select one of two voltages that can be applied to a cell in nanopore array  102 . 
         [0019]    In some embodiments, performance or other data corresponding to each of the cells may be received by master controller  104 . By monitoring the performance or other data of the cells, master controller  104  may determine any state transitions of the cells. The state information of the cells may be stored in memory  112  by master controller  104 . In addition, if the overall performance of nanopore array  102  falls below a certain threshold, master controller  104  may reset and re-initialize nanopore array  102  such that any processes running on nanopore array  102  may be terminated or restarted again. In some embodiments, nanopore array  102  may also be reused multiple times. For example, nanopore array  102  may be used for analyzing different types of samples during different runs. In another example, nanopore array  102  may be reused for analyzing a single type of samples over multiple runs. In some embodiments, nanopore array  102  may be reused after the contents in nanopore array  102  have been flushed out or rinsed out by master controller  104  and fluidic system  108 . 
         [0020]    In some embodiments, the cells of nanopore array  102  are individually controllable and individually addressable by temperature controller  106  via signal line  116 . Temperature or other data corresponding to a cell may be received by temperature controller  106  via signal line  116 . Depending on the state or condition of a particular cell or a group of cells, different temperature stimuli may be applied to the cell or group of cells by temperature controller  106 . In some embodiments, temperature controller  106  receives state information of the cells via signal line  120  and applies the appropriate temperature stimuli to the cells in nanopore array  102  at least in part based on the state information. In some embodiments, temperature controller  106  receives control signal via signal line  120  from master controller  104 , and then temperature controller  106  applies the appropriate temperature stimuli to the cells in nanopore array  102  based on the received control signal. 
         [0021]    In some embodiments, the cells of nanopore array  102  are individually controllable and individually addressable by fluidic system  108 . The control and address information is communicated between nanopore array  102  and fluidic system  108  via signal lines  118 A. Different contents may be delivered in and out of the individual cells of nanopore array  102  via channels  118 B. The contents may be any fluids or reagents that are used for the operations within the cells of nanopore array  102 , including saline solution for rinsing, samples to be analyzed by nanopore array  102 , lipid bilayer forming reagent, nanopore forming reagent, gas catalyst, and the like. The contents delivered out of nanopore array  102  may be any molecules that are extracted from the samples that have been analyzed by nanopore array  102 , and the extracted molecules may be further delivered to a storage device  110  by fluidic system  108 . The contents may be in any form, including liquid or gas. Depending on the state or condition of a particular cell or a group of cells, different fluids may be delivered to or from the cell or group of cells by fluidic system  108 . In some embodiments, fluidic system  108  receives state information of the cells via signal line  122  and delivers the appropriate fluid to or from the cells in nanopore array  102  at least in part based on the state information. In some embodiments, fluidic system  108  receives control signal via signal line  122  from master controller  104 , and then fluidic system  108  delivers the appropriate fluid to or from the cells in nanopore array  102  based on the received control signal. In some embodiments, nanopore array  102  may be reused after the contents in nanopore array  102  have been flushed out or rinsed out by master controller  104  and fluidic system  108 . 
         [0022]    Nanopore array  102  includes a large array of cells. Each cell includes a nanopore device for analyzing and characterizing molecules. Within a nanopore device, a lipid bilayer is formed, and a nanopore structure is then formed on the lipid bilayer. The nanopore structure has a nanopore that is large enough for enclosing at least a portion of a molecule that is being analyzed or passing at least a portion of the molecule between the two sides of the lipid bilayer. The nanopore device also includes a sample chamber for holding a solution of the analyzed molecules. The solution may be provided over the lipid bilayer for introducing the analyzed molecules for characterization. The nanopore device further includes means for providing electrical stimulus, sensing electrical characteristics, detecting and processing signal of the nanopore device. 
         [0023]      FIG. 3  is a diagram illustrating an embodiment of a nanopore device  300  within a cell of nanopore array  102 . Nanopore device  300  includes a lipid bilayer  302  formed on a lipid bilayer compatible surface  304  of a conductive solid substrate  306 . Lipid bilayer compatible surface  304  may be isolated by lipid bilayer incompatible surfaces  305 , and conductive solid substrate  306  may be electrically isolated by insulating materials  307 . Lipid bilayer  302  may be surrounded by an amorphous lipid  303  formed on lipid bilayer incompatible surfaces  305 . 
         [0024]    In some embodiments, lipid bilayer  302  is embedded with a single nanopore structure  308  having a nanopore  310  large enough for passing at least a portion of a molecule  312  being characterized and/or small ions (e.g., Na + , K + , Ca 2+ , Cl − ) between the two sides of lipid bilayer  302 . A layer of water molecules  314  (also referred to as an aqueous film  314 ) may be adsorbed on lipid bilayer compatible surface  304  and sandwiched between lipid bilayer  302  and lipid bilayer compatible surface  304 . Aqueous film  314  adsorbed on the hydrophilic lipid bilayer compatible surface  304  may promote the ordering of lipid molecules and facilitate the formation of lipid bilayer  302  on lipid bilayer compatible surface  304 . 
         [0025]    A sample chamber  316  may be provided over lipid bilayer  302  for introducing a sample for characterization. The sample may be a solution of molecule  312  that is being characterized. The solution may be an aqueous solution containing electrolytes and buffered to an optimum ion concentration and maintained at an optimum pH to keep nanopore  310  open. In some embodiments, sample chamber  316  receives the sample from fluidic system  108 . The sample may also be flushed out of nanopore device  300  by fluidic system  108  after the characterization of the sample has been performed. Sample chamber  316  may also be rinsed with saline solution by fluidic system  108  such that nanopore device  300  may be reused again. 
         [0026]    Nanopore device  300  includes a pair of electrodes  318  (including a negative node  318   a  and a positive node  318   b ) coupled to a variable voltage source  320  for providing electrical stimulus (e.g., voltage bias) across the lipid bilayer  302  and for sensing the electrical characteristics of the lipid bilayer  302  (e.g., resistance, capacitance, and ionic current flow). The surface of the negative positive electrode  318   b  is or forms a part of the lipid bilayer compatible surface  304 . The conductive solid substrate  306  may be coupled to or forms a part of one of the electrodes  318 . Nanopore device  300  may also include an electrical circuit  322  for controlling electrical stimulation and for processing the signal detected. In some embodiments, the variable voltage source  320  is included as a part of the electrical circuit  322 . The electrical circuitry  322  may include amplifiers, integrators, noise filters, feedback control logic, and/or various other components. In some embodiments, the electrical circuitry  322  may be an integrated electrical circuitry integrated within a silicon substrate  328  and may be further coupled to a computer processor  324  coupled to a memory  326 . For example, computer processor  324  may be a portion of master controller  104 , and memory  326  may be memory  112  that is coupled to master controller  104 . Master controller  104  may control the various components of nanopore device  300  via electrical circuit  322 . Master controller  104  may also receive data collected by nanopore device  300  via electrical circuit  322 . 
         [0027]    The lipid bilayer compatible surface  304  can be formed from various materials that are suitable for ion transduction and gas formation to facilitate lipid bilayer formation. In some embodiments, conductive or semi-conductive hydrophilic materials as opposed to insulating hydrophilic materials are preferred because they may allow better detection of a change in the lipid bilayer electrical characteristics. Example materials include Ag—AgCl, Ag—Au alloy, Ag—Pt alloy, or doped silicon or other semiconductor materials. 
         [0028]    The lipid bilayer incompatible surface  305  can be formed from various materials that are not suitable for lipid bilayer formation and they are typically hydrophobic. In some embodiments, a non-conductive hydrophobic material is preferred, since it electrically insulates the lipid bilayer regions in addition to separating the lipid bilayer regions from each other. Example lipid bilayer incompatible materials include silicon nitride (e.g., Si 3 N 4 ) and Teflon. 
         [0029]    In one particular example, nanopore device  300  of  FIG. 3  is a alpha hemolysin (αHL) nanopore device having a single αHL protein embedded in a diphytanoylphosphatidylcholine (DPhPC) lipid bilayer  302  formed over a lipid bilayer compatible silver-gold alloy surface  304  coated on a copper material  306 . The lipid bilayer compatible silver-gold alloy surface  304  is isolated by lipid bilayer incompatible silicon nitride surfaces  305 , and the copper material  306  is electrically insulated by silicon nitride materials  307 . The copper  306  is coupled to electrical circuitry  322  that is integrated in a silicon substrate  328 . A silver-silver chloride electrode placed on-chip or extending down from a cover plate contacts an aqueous solution containing dsDNA molecules. 
         [0030]    The αHL nanopore is an assembly of seven individual peptides. The entrance or vestible of the αHL nanopore is approximately 26 Å in diameter, which is wide enough to accommodate a portion of a dsDNA molecule. From the vestible, the αHL nanopore first widens and then narrows to a barrel having a diameter of approximately 15 Å, which is wide enough to allow a single ssDNA molecule to pass through but not wide enough to allow a dsDNA molecule to pass through. At a given time, approximately 1-20 DNA bases can occupy the barrel of the αHL nanopore. 
         [0031]    In addition to DPhPC, the lipid bilayer of the nanopore device can be assembled from various other suitable amphiphilic materials, selected based on various considerations, such as the type of nanopore used, the type of molecule being characterized, and various physical, chemical and/or electrical characteristics of the lipid bilayer formed, such as stability and permeability, resistance, and capacitance of the lipid bilayer formed. Example amphiphilic materials include various phospholipids such as palmitoyl-oleoyl-phosphatidyl-choline (POPC) and dioleoyl-phosphatidyl-methylester (DOPME), diphytanoylphosphatidylcholine (DPhPC) dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, and sphingomyelin. 
         [0032]    In addition to the αHL nanopore shown above, the nanopore may be one of various other types of nanopores; examples include γ-hemolysin, leukocidin, melittin, and various other naturally occurring, modified natural, and synthetic nanopores. A suitable nanopore may be selected based on various characteristics of the analyte molecule, such as the size of the analyte molecule in relation to the pore size of the nanopore. For example, the αHL nanopore is a nanopore that has a restrictive pore size of approximately 15 Å. It is suitable for analyzing DNA molecules since it allows a single strand DNA (ssDNA) to pass through while restricting a double strand DNA (dsDNA). 
         [0033]      FIGS. 4A-4C  illustrate three different states of nanopore device  300 .  FIG. 4A  is a diagram illustrating that nanopore device  300  is in a state in which a lipid bilayer has not yet been formed.  FIG. 4B  is a diagram illustrating that nanopore device  300  is in a state in which a lipid bilayer  302  has been formed.  FIG. 4C  is a diagram illustrating that nanopore device  300  is in a state in which a nanopore structure  308  with a nanopore  310  has been inserted into lipid bilayer  302 . 
         [0034]      FIG. 5  is a flow diagram illustrating an embodiment of a process  500  for analyzing molecules using nanopore devices. In some embodiments, process  500  is a process that is performed by system  100  of  FIG. 1 . 
         [0035]    At  502 , various functionalities of system  100  are verified. In some embodiments, master controller  104  may send test signals to the modules of system  100 , including nanopore array  102 , temperature controller  106 , and fluidic system  108 . In response, each module may perform verification steps at the module. For example, nanopore array  102  may measure the current flowing in a particular nanopore device. After the verification steps are performed at the modules, each of the modules may send a response back to master controller  104  for verification purposes. Depending on the responses received from the various modules, master controller  104  may determine whether further verifications are needed. In some embodiments, the verification results may be stored in a log file. In some embodiments, if master controller  104  has detected any errors, then an alarm may be triggered or process  500  may be terminated. 
         [0036]    In some embodiments, verification of the different modules may be performed at different levels, and the levels may be configurable. For example, master controller  104  may verify the functionalities of nanopore array  102  at the printed circuit board level or at the semiconductor chip level. In some embodiments, master controller  104  may verify the functionalities of a group of cells. If the number of cells within the group that are functioning properly falls below a certain threshold, then master controller  104  may determine that the group of cells is mal-functioning and that the group of cells should be disabled. 
         [0037]    At  504 , lipid bilayers are assembled. In some embodiments, master controller  104  may cause fluidic system  108  to deliver a lipid forming reagent to the cells of nanopore array  102 . The lipid forming reagent is then deposited on lipid bilayer compatible surface  304  within a cell. As discussed above, the lipid bilayer may be formed using different materials, including different amphiphilic materials. Depending on the type of lipid bilayers to be formed, master controller  104  may cause different stimuli (e.g., electrical, temperature, chemical, or gas) to be applied to the cells to facilitate the assembling of the lipid bilayers. 
         [0038]    At  506 , it is determined whether the lipid bilayers are properly formed. Depending on the type of lipid bilayers to be formed, different physical or electrical property measurements (e.g., resistance, current, or capacitance measurements) may be made at the cells and then sent to master controller  104  via signal lines  114  for determining whether lipid bilayers are properly assembled. In some embodiments, steps  504  and  506  are repeated until master controller  104  has determined that lipid bilayers have been properly assembled in a minimum number of cells in nanopore array  102 . In some embodiments, if the number of cells with lipid bilayers properly assembled falls below a certain threshold after a fixed period of time, master controller  104  may terminate process  500 . In addition, an alarm may be triggered or an error message may be written to the log file. In some embodiments, if the number of cells with lipid bilayers properly assembled is above a certain threshold, master controller  104  may cause system  100  to proceed to step  508 . 
         [0039]    At  508 , nanopore structures with nanopores are inserted. In some embodiments, master controller  104  may cause fluidic system  108  to deliver a nanopore forming reagent (e.g., a solution containing α-hemolysin) to the cells of nanopore array  102 . Master controller  104  may cause different stimuli (e.g., electrical, temperature, chemical, or gas) to be applied to the cells to facilitate the insertion of the nanopore structures into the lipid bilayers. 
         [0040]    At  510 , it is determined whether the nanopore structures are properly formed. Depending on the type of nanopores to be formed, different measurements (e.g., resistance, current, or capacitance measurements) may be made at the cells and then sent to master controller  104  via signal lines  114  for determining whether nanopores are properly inserted. In some embodiments, steps  508  and  510  are repeated until master controller  104  has determined that nanopores have been properly inserted in a minimum number of cells in nanopore array  102 . In some embodiments, if the number of cells with nanopores properly inserted falls below a certain threshold after a fixed period of time, master controller  104  may terminate process  500 . In addition, an alarm may be triggered or an error message may be written to the log file. In some embodiments, if the number of cells with nanopores properly inserted is above a certain threshold, master controller  104  may cause system  100  to proceed to step  512 . 
         [0041]    At  512 , samples are analyzed using the nanopores in nanopore array  102 . In some embodiments, master controller  104  may cause fluidic system  108  to deliver samples to the sample chambers  316  in nanopore array  102 . Depending on different factors, including the type of samples that are being analyzed and the type of nanopores formed, master controller  104  may cause different stimuli (e.g., electrical, temperature, chemical, or gas) to be applied to the cells to facilitate the manipulating, detecting, correlating, characterizing, analyzing and/or sequencing of molecules in the nanopores. Different measurements (e.g., resistance, current, or capacitance measurements) may be made at the cells and then sent to master controller  104  via signal lines  114 . Master controller  104  may use the received measurements to detect, correlate, determine, characterize, sequence and/or discriminate various structural and chemical features of a molecule as the molecule stays inside the nanopore, traverses through the nanopore, or interacts with the nanopore. 
         [0042]    At  514 , nanopore array is reset and re-initialized for repeated uses. In some embodiments, nanopore array  102  may be reused multiple times. For example, nanopore array  102  may be used for analyzing different types of samples during different runs. In another example, nanopore array  102  may be reused for analyzing a single type of samples over multiple runs. New nanopores may be reformed in nanopore array  102  such that nanopore array  102  may be reused. New nanopores may be reformed in nanopore array  102  after the contents (e.g., lipid bilayers with nanopores inserted, lipid bilayers without nanopores inserted, and samples) in nanopore array  102  have been flushed out or rinsed out (e.g., using saline solution) by master controller  104  and fluidic system  108 . 
         [0043]    In some embodiments, master controller  104  may detect and determine whether there are any molecules or other contents of interest remaining in the cells of nanopore array  102 . Master controller  104  and fluidic system  108  may selectively rinse out the contents (e.g., lipid bilayers) within cells in which no molecules or other contents of interest are found. The molecules or other contents of interest in the remaining cells may be retrieved. In one example, the molecules may be retrieved manually. In another example, master controller  104  and fluidic system  108  may deliver the molecules or other contents of interest to storage device  110  before the remaining contents are rinsed out. After  514 , nanopore array  102  is ready for repeated uses again, and process  500  may be restarted at  502 . In some embodiments, step  514  is performed before a nanopore array  102  is used for the first time. For example, nanopore array  102  is rinsed with saline solution before the functionalities of system  100  is checked at  502 . 
         [0044]    Although the foregoing embodiments have been described in some detail for purposes of clarity of understanding, the invention is not limited to the details provided. There are many alternative ways of implementing the invention. The disclosed embodiments are illustrative and not restrictive.