Devices and methods for target molecule characterization

Abstract

An system for recognition of a translocating polymeric target molecule includes a device having at least one constriction that is sized to permit translocation of only a single copy of the molecule. A pair of spaced apart sensing electrodes border the constriction, which may be a nanopore. The first electrode is connected to a first affinity element and the second electrode is connected to a second affinity element. Each affinity element may be connected to its corresponding electrode via one or more intermediary compounds, such as a linker molecule and/or an electrode attachment molecule. The first and second affinity elements are configured to temporarily form hydrogen bonds with first and second portions of the target molecule as the latter passes through the constriction. During translocation, the electrodes, affinity elements and first and second portions of the target molecule complete an electrical circuit and allow a measurable electrical current to pass between the first and second electrodes. The time-varying nature of this electrical current, and the specific affinity elements employed, allow one to characterize the target molecule.

1 - 63 . (canceled) 
     
     
         64 . A method of identifying a particular portion of a target molecule as the target molecule translocates through a constriction, the method comprising:
 providing an apparatus comprising a molecular recognition device, the device comprising
 a partition having a first side and a second side, and at least one constriction passing between the first and second sides, the constriction being shaped and sized to permit translocation of only a single copy of a target molecule therethough; 
 first and second sensing electrodes associated with the first side of the partition and being separated from one another by a first gap; 
 a first affinity element connected to the first electrode; 
 a second affinity element connected to the second electrode and the device being located in the apparatus such that a first chamber is located on the first side of the device and a second chamber is located on the second side of the device; 
   introducing a target molecule into the second chamber;   translocating by electrophoresis the target molecule so that it moves through the constriction; and   detecting an electrical current passing through the first electrode, the first affinity element, a first site on a portion of the target, a second site on a portion of the target molecule, the second affinity element, and the second electrode.   
     
     
         65 . The method according to  claim 64 , wherein the target molecule has a leading end and a trailing end. 
     
     
         66 . The method of  claim 65 , further comprising threading the leading end of the target molecule through the constriction and into the first chamber prior to detecting the electrical current. 
     
     
         67 . The method according to  claim 65 , further comprising affixing a first magnetic bead proximate the leading end of the target molecule to control translocation through the constriction. 
     
     
         68 . The method according to  claim 67 , further comprising affixing a second magnetic bead proximate the trailing end of the target molecule. 
     
     
         69 . The method according to  claim 64 , wherein the target molecule is DNA and the first affinity element is a guanidinium moiety. 
     
     
         70 . The method according to  claim 69 , wherein, the second affinity element comprises one of the bases A, T, C or G, or a modified version thereof, or a nucleoside analog thereof. 
     
     
         71 . The method according to  claim 64 , further comprising recording information reflective of the electrical current as a function of time; and obtaining at least one parameter reflective of an identification of the particular portion, from said recorded information. 
     
     
         72 . The method according to  claim 71 , wherein said at least one parameter comprises one from the group consisting of charge, duration of current signal, shape of current signal and decay of current. 
     
     
         73 . The method according to  claim 72 , further comprising comparing said at least one parameter with a predetermined threshold to determine whether said particular portion has been recognized. 
     
     
         74 . The method according to  claim 73 , further comprising detecting electrical current from a same portion of a predetermined number of copies of said target molecule to thereby improve recognition accuracy of said portion. 
     
     
         75 . A device for the detection of a target molecule comprising;
 a partition comprising one or more constrictions sized to permit translocation of a target molecule through the one or more constrictions;   an electrode pair on the partition in communication with the one or more constrictions;   a first affinity element and a second affinity element bound to one or both members of the electrode pair, wherein the first affinity element is capable of binding to a first site on the target molecule as it enters the one or more constrictions; and wherein the second affinity element is capable of binding to a second site on the target molecule as it enters the one or more constrictions in the presence of the first affinity element bound to the first site on the target molecule;   
       wherein the electrode pair is arranged so that binding of the target molecule by the first affinity element bound to one member of the electrode pair coupled with binding of the target molecule by the second affinity element to the other member of the electrode pair generates an electrical signal. 
     
     
         76 . The device of  claim 75 , wherein the first and second affinity elements are bound to one or both members of the electrode pair via a linker. 
     
     
         77 . The device of  claim 76 , wherein the electrodes in the electrode pair are spaced from 0.5 to 10 nm apart. 
     
     
         78 . The device of  claim 76 , wherein the target molecule is a nucleic acid. 
     
     
         79 . The device of  claim 78 , wherein the first affinity element is capable of binding to phosphate groups in the nucleic acid backbone and/or the second affinity element is capable of binding to specific bases in the nucleic acid. 
     
     
         80 . The device of  claim 79 , wherein the first affinity element comprises a guanidinium moiety, such as guanidinoethyldisulfide. 
     
     
         81 . A method for detecting a target molecule comprising;
 applying a force to translocate a target molecule though a constriction on a partition, wherein the constriction is sized to permit passage of a target molecule, wherein an electrode pair is in communication with the constriction;
 wherein a first affinity element and a second affinity element are bound to one or both members of the electrode pair; 
 wherein the first affinity element is capable of binding to a first site on the target molecule as it enters the constriction; 
 wherein the second affinity element is capable of binding to a second site on the target molecule as it enters the constriction in the presence of the first affinity element bound to the target molecule, and wherein the translocation is conducted under conditions suitable to promote binding of target molecule to the first affinity element and the second affinity element; 
   measuring an electrical signal generated by completion of an electrical circuit formed by binding of the target molecule to the first affinity element bound to one member of the electrode pair coupled with binding of the target molecule to the second affinity element to the other member of the electrode pair; and   characterizing the target molecule based on the measured electrical signal.   
     
     
         82 . The method of  claim 81 , wherein applying a force comprises applying a magnetic field or an electric field and/or an initial electrophoretic threading of the target molecule into the constriction. 
     
     
         83 . The method of  claim 81 , wherein the electrodes in the electrode pair are spaced from 0.5 to 10 nm apart. 
     
     
         84 . The method of  claim 81 , wherein the target molecule is a nucleic acid. 
     
     
         85 . The method of  claim 81 , wherein the first affinity element is capable of binding to phosphate groups in the nucleic acid backbone of the nucleic acid and/or the second affinity element is capable of binding to specific bases in the nucleic acid and/or the first and second affinity elements are bound to one or both members of the electrode pair via a linker, such as a flexible linker. 
     
     
         86 . The method of  claim 85 , wherein the first affinity element comprises a guanidinium moiety, such as guanidinoethyldisulfide. 
     
     
         87 . The method of  claim 86 , wherein the guanidinium moiety is coupled via an amide linkage. 
     
     
         88 . The method of  claim 85 , wherein the flexible linker comprises an alkane chain of two or more methylene groups.