Patent Publication Number: US-2019170681-A1

Title: Systems and methods for electrical sensing of biomolecular targets

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of and priority to U.S. Provisional Patent Application Ser. No. 62/374,985, filed Aug. 15, 2016 and entitled “TECHNIQUES FOR ELECTRICAL SENSING OF BIOMOLECULAR TARGETS,” the entirety of which is incorporated herein by reference. 
    
    
     BACKGROUND 
     The subject matter disclosed herein relates to a detecting target molecules, such as nucleic acid molecules and, more particularly, to systems for electrical sensing of the target molecules. 
     Various methods have developed for analyzing biological samples and detecting the presence of target molecules, such as nucleic acid molecules. These methods can be used, for example, in detecting pathogens in samples. 
     Typically, detection methods use disruption techniques, such as Polymerase Chain Reaction (PCR) to extract and replicate nucleic acid molecules from a sample. PCR is a technique that allows for replicating and amplifying trace amounts of DNA fragments into quantities that are sufficient for analysis. As such, PCR can be used in a variety of applications, such as DNA sequencing and detecting DNA fragments in samples. 
     An electronic sensor for detection of specific target nucleic acid molecules can include capture probes immobilized on a sensor surface between a set of paired electrodes. An example of a system and method for detecting target nucleic acid molecules is described in U.S. Pat. No. 7,645,574, the entirety of which is herein incorporated by reference. Following PCR, amplified products or amplicons derived from targeted pathogen sequences are captured by the probes Nano-gold clusters, functionalized with a complementary sequence, are used for localized hybridization to the amplicons. Subsequently, using a short treatment with a gold developer reagent, the nano-gold clusters serve as catalytic nucleation sites for metallization, which cascades into the development of a fully conductive film. The presence of the gold film shorts the gap between the electrodes and is measured by a drop in resistance, allowing the presence of the captured amplification products to be measured. However, such sensors can be insensitive to small quantities of target molecules, resulting in false negative results or a failure to detect the target molecules. 
     SUMMARY 
     A system for detection of a target molecule includes a source terminal, a drain terminal, a gate positioned between the source terminal and the drain terminal, and a functionalized sensor surface between the source terminal and the drain terminal. The sensor surface is configured to bind target molecules and the target molecules are configured to bind functionalized nanoparticles. A sensor is coupled to the source terminal and drain terminal to monitor changes in electrical signals and detect the target molecules when changes in the electrical signals are detected. 
     In an embodiment, a system for detecting a target molecule in a sample is disclosed. The system includes a source terminal, a drain terminal, a sensor coupled to the source terminal and the drain terminal. The sensor is configured to monitor electrical signals across the source terminal and drain terminal. A gate is positioned adjacent to one of the source terminal and the drain terminal and extends partially across a gap between the source terminal and drain terminal. A sensor surface is exposed between the gate and one of the source terminal and the drain terminal. The sensor surface is a functionalized sensor surface configured to bind the target molecule. 
     A sensor surface is positioned between the source terminal and the drain terminal. The sensor surface includes a functionalized sensor surface configured to bind the target molecule. A gate is positioned adjacent to one of the source terminal and the drain terminal and extends partially across the sensor surface. 
     In another embodiment, a system for detecting a target molecule in a sample is disclosed. The system includes a source terminal, a drain terminal, and a sensor coupled to the source terminal and the drain terminal. The sensor is configured to monitor electrical signals across the source terminal and drain terminal. A gate is positioned between the source terminal and the drain terminal and a channel is positioned above the gate. The channel includes a functionalized sensor surface configured to bind the target molecule. 
     In yet another embodiment, a system for detecting a target molecule in a sample is disclosed. The system includes a substrate, a first transducer positioned on the substrate, and a second transducer positioned on the substrate. The first transducer has a signal input and the second transducer has a signal output. A sensor is coupled to the signal input to input a signal and to the signal output to measure an output signal. A delay area is positioned between the first transducer and the second transducer. The delay area has a functionalized coating configured to bind the target molecule. 
     An advantage that may be realized in the practice of some disclosed embodiments is increased sensitivity of nucleic acid sensors and improved detection of low concentrations of target materials. 
     The above embodiments are exemplary only. Other embodiments are within the scope of the disclosed subject matter. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       So that the manner in which the features of the invention can be understood, a detailed description of the invention may be had by reference to certain embodiment, some of which are illustrated in the accompanying drawings. It is to be noted, however, that the drawings illustrate only certain embodiments of this invention and are therefore not to be considered limiting of its scope, for the scope of the disclosed subject matter encompasses other embodiments as well. The drawings are not necessarily to scale, emphasis generally being placed upon illustrating the features of certain embodiments of the invention. In the drawings, like numerals are used to indicate like parts throughout the various views. 
         FIG. 1  is a perspective view of a portable diagnostic assay system operative to accept one of a plurality of disposable cartridges configured to test fluid samples of collected blood/food/biological samples; 
         FIG. 2  is an exploded perspective view of one of the disposable cartridges configured to test a blood/food/biological sample; 
         FIG. 3  is a top view of the one of the disposable cartridges illustrating a variety of assay chambers including a central assay chamber, one of which contains an assay chemical suitable to breakdown the fluid sample to detect a particular attribute of the tested fluid sample; 
         FIG. 4  is a bottom view of the disposable cartridge shown in  FIG. 3  illustrating a variety of channels operative to move at least a portion of the fluid sample from one chamber to another for the purpose of performing multiple operations on the fluid sample. 
         FIG. 5  is an illustration of an embodiment of a functionalized sensor surface; 
         FIG. 6  is an illustration of the sensor surface of  FIG. 5  having the target molecule coupled thereto; 
         FIG. 7  is an illustration of the sensor surface of  FIG. 6  with a nanoparticle coupled to the captured target molecule; 
         FIG. 8  is an illustration of the sensor surface of  FIG. 7  with a conductive film formed over the nanoparticles; 
         FIG. 9A  is a top view illustration of an embodiment of a detection system; 
         FIG. 9B  is a cross-sectional illustration of the detection system of  FIG. 9A ; 
         FIG. 10  is a flowchart illustrating an embodiment of a method of detecting a target molecule; 
         FIG. 11A  is a top view illustration of the detection system of  FIG. 9A  with catalytic nanoparticles bound to captured target molecules on the sensor surface; 
         FIG. 11B  is a cross-sectional illustration of the detection system of  FIG. 11A ; 
         FIG. 12A  is a top view illustration of the detection system of  FIG. 11A  with a conductive film formed over the catalytic nanoparticles; 
         FIG. 12B  is a cross-sectional illustration of the detection system of  FIG. 12A ; 
         FIG. 13A  is a cross-sectional illustration of an embodiment of a detection system with target molecules bound to a sensor surface; 
         FIG. 13B  is a cross-sectional illustration of the detection system of  FIG. 13A  with catalytic nanoparticles bound to the target molecules; 
         FIG. 13C  is a cross-sectional illustration of the detection system of  FIG. 13B  with a conductive film formed over the catalytic nanoparticles; 
         FIG. 14  is a flowchart illustrating another embodiment of a method of detecting a target molecule; 
         FIG. 15A  is a top view illustration of an embodiment of a detection system; 
         FIG. 15B  is a cross-sectional illustration of the detection system of  FIG. 16A  with target molecules bound to a functionalized sensor surface; 
         FIG. 15C  is a cross-sectional illustration of the detection system of  FIG. 16B  with catalytic nanoparticles bound to the target molecules; and 
         FIG. 15D  is a cross-sectional illustration of the detection system of  FIG. 16C  with a conductive film over the catalytic nanoparticles. 
     
    
    
     Corresponding reference characters indicate corresponding parts throughout several views. The examples set out herein illustrate several embodiments, but should not be construed as limiting in scope in any manner. 
     DETAILED DESCRIPTION 
     A disposable cartridge is described for use in a portable/automated assay system such as that described in commonly-owned, co-pending U.S. patent application Ser. No. 15/157,584 filed May 18, 2016 entitled “Method and System for Sample Preparation” which is hereby included by reference in its entirety. While the principal utility for the disposable cartridge includes DNA testing, the disposable cartridge may be used to detect any of a variety of diseases which may be found in either a blood, food or biological detecting hepatitis, autoimmune deficiency syndrome (AIDS/HIV), diabetes, leukemia, graves, lupus, multiple myeloma, etc., just naming a small fraction of the various blood borne diseases that the portable/automated assay system may be configured to detect. Food diagnostic cartridges may be used to detect  salmonella, e - coli, staphylococcus aureus  or dysentery. Diagnostic cartridges may also be used to test samples from insects and specimen. For example, blood diagnostic cartridges may be dedicated cartridges useful for animals to detect diseases such as malaria, encephalitis and the west nile virus, to name but a few. 
     More specifically, and referring to  FIGS. 1 and 2 , a portable assay system  10  receives any one of a variety of disposable assay cartridges  20 , each selectively configured for detecting a particular attribute of a fluid sample, each attribute potentially providing a marker for a blood, food or biological (animal borne) disease. The portable assay system  10  includes one or more linear and rotary actuators operative to move fluids into, and out of, various compartments or chambers of the disposable assay cartridge  20  for the purpose of identifying or detecting a fluid attribute. More specifically, a signal processor  14 , i.e., a PC board, controls a rotary actuator (not shown) of the portable assay system  10  so as to align one of a variety of ports  18 P, disposed about a cylindrical rotor  18 , with a syringe barrel  22 B of a stationary cartridge body  22 . The processor  14  controls a linear actuator  24 , to displace a plunger shaft  26  so as to develop pressure i.e., positive or negative (vacuum) in the syringe barrel  22 . That is, the plunger shaft  26  displaces an elastomer plunger  28  within the syringe  22  to move and or admix fluids contained in one or more of the chambers  30 ,  32 . 
     The disposable cartridge  20  provides an automated process for preparing the fluid sample for analysis and/or performing the fluid sample analysis. The sample preparation process allows for disruption of cells, sizing of DNA and RNA, and concentration/clean-up of the material for analysis. More specifically, the sample preparation process of the instant disclosure prepares fragments of DNA and RNA in a size range of between about 100 and 10,000 base pairs. The chambers can be used to deliver the reagents necessary for end-repair and kinase treatment. Enzymes may be stored dry and rehydrated in the disposable cartridge  20 , or added to the disposable cartridge  20 , just prior to use. The implementation of a rotary actuator allows for a single plunger  26 ,  28  to draw and dispense fluid samples without the need for a complex system of valves to open and close at various times. This greatly reduces potential for leaks and failure of the device compared to conventional systems. Finally, it will also be appreciated that the system greatly diminishes the potential for human error. 
     In  FIGS. 3 and 4 , the cylindrical rotor  18  includes a central chamber  30  and a plurality of assay chambers  32 ,  34  surrounded, and separated by, one or more radial or circumferential walls. In the described embodiment, the central chamber  30  receives the fluid sample while the surrounding chambers  32 ,  34  contain a premeasured assay chemical or reagent for the purpose of detecting an attribute of the fluid sample. The chemical or reagents may be initially dry and rehydrated immediately prior to conducting a test. Some of the chambers  32 ,  34  may be open to allow the introduction of an assay chemical while an assay procedure is underway or in-process. The chambers  30 ,  32 ,  34  are disposed in fluid communication, i.e., from one of the ports  18 P to one of the chambers  30 ,  32 ,  34 , by channels  40 ,  42  molded along a bottom panel  44 , i.e., along underside surface of the rotor  18 . For example, a first port  18 P, corresponding to aperture  42 , may be in fluid communication with the central chamber  30 , via aperture  50 . 
       FIG. 5  illustrates an embodiment of a sensor surface  70 . The sensor surface  70  includes a plurality of capture probes  72  in the form of a functionalized oxide surface allowing attachment and immobilization of capture probe molecules  72  on the sensor surface  70 . The capture probes  72  are designed to capture or bind target molecules  74  ( FIG. 6 ) by interaction between complementary sequences. The target molecules  74  can be collected from a biological sample. The biological sample could be any suitable type of material, such as blood, mucous, and skin, among others. 
     In an embodiment, a sample including the target molecules  74  is mixed with a solution containing magnetic nanoparticles (not shown) and the target molecules  74  and magnetic nanoparticles hybridize. Using a magnet (not shown), the hybridized target molecules are attracted to the functionalized sensor surface  70  for binding to the capture probe molecules  72 . 
     After the target molecules  74  are bound to or captured by the capture probe molecules  72  ( FIG. 6 ), a catalytic nanoparticle  76 , such as a gold catalyst reagent, is directly hybridized to the captured or bound target molecules  74 , as illustrated in  FIG. 7 . Any suitable method of hybridizing the catalytic nanoparticles  76  and target molecules  74  can be used. In an embodiment, the catalytic nanoparticles  76  are in the form of catalyst clusters. In an embodiment, a single catalyst cluster  76  binds to each captured target molecule  74 . In another embodiment, and depending on the length of the target molecule  74 , a plurality of catalyst clusters  76  bind to each captured target molecule  74 . 
     Following hybridization of the captured target molecules  74  with the catalytic nanoparticles  76 , metallization of the catalytic nanoparticles  76 , which serve as catalytic nucleation sites, can be performed to form a conductive film  78  ( FIG. 8 ) that can be used to detect the target molecules  74 . In an example, the catalytic nanoparticles  76  are gold clusters and a gold developer reagent is applied to the catalyst clusters to cascade into the development of the conductive film  78 , which in this example is a gold film. 
       FIGS. 9A-9B  illustrate an embodiment of a detection system  80  for detecting a target molecule  76 . The system includes a well  82  on which are formed a source terminal  84  and a drain terminal  86 . A sensor  88  is coupled to the source terminal  84  and drain terminal  86 . The sensor  88  includes a microcontroller  90  for applying an electrical signal to the source terminal  84  and for measuring resistance across the source terminal  84  and drain terminal  86 . A functionalized sensor surface  92  is positioned between the source terminal  84  and drain terminal  86  and separated from the source terminal  84  and drain terminal  86  by spacers  94 . Similar to the sensor surface  70  described above ( FIG. 5 ), the functionalized sensor surface  92  includes a plurality of capture probes (not shown) in the form of a functionalized oxide surface allowing attachment and immobilization of capture probe molecules on the sensor surface  92 . In an embodiment, the spacers  94  can be formed of an insulating material, such as silicon dioxide, silicon oxynitride, or a high-K dielectric material. 
     A gate  96  is separated from the source terminal  84  by the spacer  94  and extends partially across a gap between the source terminal  84  and drain terminal  86  toward the drain terminal  86 , exposing the functionalized sensor surface  92 . Alternatively, the gate  96  can be separated from the drain terminal  86  by the spacer  94  and extend toward the source terminal  84 . The gate  96  can be formed from a metal material or from a polysilicon (polycrystalline silicon). In an embodiment, the system  80  is in the form of an incomplete metal oxide semiconductor field effect transistor (MOSFET) in which the gate  97  is incomplete, exposing a portion of the gate dielectric. In one embodiment, the gate  96  is formed as a complete gate extending across the gap and a portion of the gate  96  is removed. In another embodiment, the gate  96  is formed as a partial gate. 
       FIG. 10  illustrates an embodiment of a method  96  of detecting a target molecule with a detection system, such as the detection system  80  ( FIGS. 9A-9B ). At block  98 , target molecules  108  are hybridized or bound to the functionalized sensor surface  92 , as illustrated in  FIGS. 11A-11B . In particular, the target molecules  108  are bound to capture molecules on the functionalized sensor surface  92 . 
     At block  100  of the method  96  ( FIG. 10 ), catalytic nanoparticles  110  are hybridized or bound to the captured target molecules  108 . The hybridization of the catalytic nanoparticles  110  creates more “gate” material, extending the gate  96  across the sensor surface  92 . 
     At block  102 , a conductive film  112  can be formed by applying a reagent to the catalytic nanoparticles  110 . For example, a bath can be applied to the catalytic nanoparticles to initiate development of the conductive film  112 . In an embodiment, the catalytic nanoparticles  110  are gold clusters that act as nucleation sites for the development of a gold film. The conductive film  112  can further increase the development of the gate material, bridging the gap between the gate  96  and the drain terminal  86  or source terminal  84  and completing the MOSFET. In an embodiment, development of the conductive film  112  is optional. 
     At block  104  ( FIG. 10 ) the sensor  88  ( FIG. 9A ) monitors transistor output characteristics to determine (block  106 ) the presence of target molecules  108 . For example, if a change in voltage is detected, target molecules, catalytic nanoparticles, and, optionally, a conductive film have been deposited on the sensor surface  92 , bridging the gap between the gate  96  and the source terminal  84  or the drain terminal  86 , thus indicating the presence of the target molecules. By contrast, if no change in voltage is detected, the gap between the gate  96  and the source terminal  84  or drain terminal  86  has not been bridged, indicating that no target molecules  108  are present. While monitoring has been described in the context of voltage, it is to be understood that other characteristics of electrical signals can be monitored in order to detect the presence of target molecules. Various techniques, such as drain biasing, can be applied for partial metallization or higher sensitivity situations. 
       FIGS. 13A-13C  illustrate another embodiment of a detection system  114 . As illustrated in  FIG. 13A , the detection system  114  is in the form of a thin film transistor (TFT)  115  coupled to a sensor  130 . A source terminal  116  and a drain terminal  118  are formed on a body  120 . The body  120  can be formed of any suitable material, such as a glass or silicon. A gate  122  is positioned between the source terminal  116  and the drain terminal  118  and spacers  124  are interposed between the gate  122  and the source terminal  116  and drain terminal  118  to prevent direct physical contact. An exposed channel  126  is positioned above the gate  122 . The channel  126  can be formed of Silicon, indium, gallium, zinc, and oxygen (IGZO) semiconductor, or any other suitable semiconductor. The channel  126  includes a functionalized sensor surface  128 , similar to the functionalized sensor surface  92  described above with respect to  FIGS. 9A-9B . A sensor  130 , having a microcontroller  132 , is coupled to the source terminal  116  and the drain terminal  118  to monitor the state of the transistor, such as monitoring voltage or conductivity. 
     As described above with respect to  FIG. 10 , the detection system  114  can be used to detect the presence of target molecules. In this embodiment, the target molecules  134  are bound to the functionalized sensor surface  128  ( FIG. 13A ). As illustrated by  FIG. 13B , catalytic nanoparticles  136  are hybridized to the bound target molecules  134 . The hybridization of the catalytic nanoparticles  136  produces a second or top gate above the channel  126  and gate  122 . Optionally, the catalytic nanoparticles can serve as nucleation sites for the development of a conductive film  138  ( FIG. 13C ) to further develop the top gate. 
     The development of the top gate serves to change the conductive state of the transistor  115 . By monitoring the state of the transistor  115  via the sensor  130 , the presence of the target molecules  134  can be detected. For example, if changes in the conductive state of the transistor  115  are detected, the presence of the target molecules  134  is detected. If there are not changes in the conductive state of the transistor  115 , the top gate has not developed and no target molecules  134  are detected. 
       FIG. 14  illustrates another method  140  for detecting a target molecule. In an example, the method  140  can be used to detect a target molecule with a detection system, such as the detection system  154  illustrated by  FIGS. 15A-15D . The detection system  154  includes a substrate  156  on which a first transducer  158  and a second transducer  160  are positioned. A signal is input  162  at the first transducer  158  and a signal is output  164  at the second transducer  160 . A sensor  166 , having a microcontroller  168  is coupled to the signal input  162  and the signal output  164  to input the signal and measure or monitor the output signal  164 . In an embodiment, the sensor  166  is an acoustic wave mass sensor. 
     A functionalized delay area  170  is positioned on the substrate  156  between the first transducer  158  and the second transducer  160 . In an embodiment, the surface  172  of the delay area  170  is functionalized with a bio-specific coating  173  ( FIG. 15B ), such as nucleic acid primers or proteins. 
     Returning to  FIG. 14 , at block  142 , an initial output signal is measured as a baseline reference point. At block  144 , target molecules  174  are hybridized or bound to the bio-specific coating  173  ( FIG. 15B ). For example, a sample containing the target molecules  174  can be introduced to the delay area  170  and allowed to interact with the coating  173 . The sample can be rinsed to leave only the bound target molecules  174 . 
     At block  146  ( FIG. 14 ), functionalized nanoparticles  176  are hybridized to the bound target molecules  174  ( FIG. 15C ). In an example, the functionalized nanoparticles  176  are catalytic nanoparticles, such as gold clusters, as discussed above. Optionally, the functionalized nanoparticles can act as nucleation sites for the development of a film  178  ( FIG. 15D ) to increase the mass of the delay area  170 . 
     At block  148  ( FIG. 14 ) the acoustic wave mass sensor  166  is initiated to measure the output signal. At block  150 , the sensor  166  determines if the measured output signal exceeds the baseline reference point to detect, at block  152 , the presence of target molecules. When the measured output signal does exceed the baseline reference point, the target molecules are detected. When the measured output signal does not exceed the baseline reference point, the target molecules are not detected. 
     While the detection systems  80 ,  114 ,  154  have been discussed in terms of independent detection systems, it is to be under stood that the detection systems  80 ,  114 ,  154  can be incorporated in other systems, such as the portable assay system  10  or disposable assay cartridge  20  described above with respect to  FIGS. 1-4 . 
     Possible advantages of the above described method include improved sensitivity of target molecule detection and improved detection of small quantities of target molecules. 
     While the present invention has been particularly shown and described with reference to certain exemplary embodiments, it will be understood by one skilled in the art that various changes in detail may be effected therein without departing from the spirit and scope of the invention that can be supported by the written description and drawings. Further, where exemplary embodiments are described with reference to a certain number of elements it will be understood that the exemplary embodiments can be practiced utilizing either less than or more than the certain number of elements. 
     The patentable scope of the invention is defined by the claims, and may include other examples that occur to those skilled in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differ from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims. 
     To the extent that the claims recite the phrase “at least one of” in reference to a plurality of elements, this recitation is intended to mean at least one or more of the listed elements, and is not limited to at least one of each element. For example, “at least one of an element A, element B, and element C,” is intended to indicate element A alone, or element B alone, or element C alone, or any combination thereof. “At least one of element A, element B, and element C” is not intended to be limited to at least one of an element A, at least one of an element B, and at least one of an element C. 
     PARTS LIST 
     
         
           10  portable assay system 
           14  processor 
           18  rotor 
           18 P port 
           20  disposable assay cartridge 
           22  cartridge body 
           22 B syringe barrel 
           24  linear actuator 
           26  plunger shaft 
           28  elastomeric plunger 
           30  central chamber 
           32  assay chamber 
           34  assay chamber 
           40  channel 
           42  channel 
           44  bottom panel 
           50  aperture 
           70  functionalized sensor surface 
           72  capture molecule 
           74  target molecule 
           76  catalytic nanoparticle 
           78  conductive film 
           80  detection system 
           82  well 
           84  source terminal 
           86  drain terminal 
           88  sensor 
           90  microcontroller 
           92  functionalized sensor surface 
           94  spacer 
           96  gate 
           97  method 
           98 - 106  method blocks 
           108  target molecule 
           110  catalytic nanoparticles 
           112  conductive film 
           114  detection system 
           115  thick film transistor 
           116  source terminal 
           118  drain terminal 
           120  body 
           122  gate 
           124  spacers 
           126  channel 
           128  functionalized sensor surface 
           130  sensor 
           132  microcontroller 
           134  target molecule 
           136  catalytic nanoparticles 
           138  conductive film 
           140  method 
           142 - 152  method blocks 
           154  detection system 
           156  substrate 
           158  first transducer 
           160  second transducer 
           162  signal input 
           164  signal output 
           166  sensor 
           168  microcontroller 
           170  delay area 
           172  delay area surface 
           173  coating 
           174  target molecules 
           176  functionalized nanoparticles 
           178  film