Exchange-induced remnant magnetization for label-free detection of dna, micro-rna, and dna/rna-binding biomarkers

Abstract

A method of using an exchange-induced remnant magnetization (EXIRM) technique for label free detection of short strands of nucleotides and cancer biomarkers, such as DNA and microRNA strands, DNA/RNA-binding biomarkers, and cancer-specific antigens, with high sensitivity, high specificity, and broad dynamic range. The method may provide a label-free approach aimed to facilitate high reliability, and to require a minimum amount of biochemical reagents.

What is claimed is: 
     
         1 . A method of detecting nucleotide sequences, comprising
 a) immobilizing a first nucleotide single strand on a surface;   b) adding a second nucleotide single strand to the first nucleotide single strand to form a hybridized double strand, wherein said second strand comprises:
 a first magnetic particle; and 
 a nucleotide sequence that is less than 100% complementary to the first nucleotide single stand, and comprises at least a first mismatched base; 
   c) measuring a first magnetic signal value for said hybridized double strand;   d) incubating a third nucleotide strand with said hybridized double strand; wherein said third strand is complementary to said first strand, and wherein said incubating forms an exchange product;   e) measuring a second magnetic signal value for the exchange product of step d after applying a weak mechanical force to remove nonspecifically bound magnetic particles; and   f) quantifying the amount of said third nucleotide strand from the difference in magnetic signal values measured in step c and step e   
     
     
         2 . The method of  claim 1 , wherein said first nucleotide single strand is derivatized. 
     
     
         3 . The method of  claim 1 , wherein said first nucleotide strand is immobilized to said surface through a S—Au covalent bond or by a streptavidin-biotin covalent bond. 
     
     
         4 . The method of  claim 1 , wherein said magnetic particle is attached to said second nucleotide strand by a streptavidin-biotin covalent bond. 
     
     
         5 . The method of  claim 1 , wherein said magnetic particle is about 1 nm to about 10 μm in size. 
     
     
         6 . The method of  claim 1  wherein said magnetic particle is about 3 μm in size. 
     
     
         7 . The method of  claim 1 , wherein said measuring comprises an atomic magnetometer. 
     
     
         8 . The method of  claim 1 , wherein said first and said second magnetic signal comprise magnetic moment measurements. 
     
     
         9 . The method of  claim 8 , wherein step f comprises measuring the change in magnetic signal (ΔB). 
     
     
         10 . The method of  claim 9 , further comprising calculating the molar concentration of said third nucleotide strand, wherein said concentration is linearly related to ΔB. 
     
     
         11 . The method of  claim 1 , wherein said quantifying further comprises calculating the number of free magnetic particle labels, wherein the number of said free magnetic particles corresponds to the number of exchange product molecules. 
     
     
         12 . The method of  claim 1 , wherein said weak mechanical force is supplied by a shaker, centrifuge, or sonicator. 
     
     
         13 . The method of  claim 1 , wherein said hybridized strand is in a liquid environment, a cell lysate, blood plasma, or urine. 
     
     
         14 . The method of  claim 1 , wherein said first nucleotide strand is a RNA or a DNA sequence of about 1-100 nucleotides. 
     
     
         15 . The method of  claim 1  wherein said third nucleotide strand is a DNA or microRNA sequence of about 1 to about 100 nucleotides. 
     
     
         16 . 
     
     
         17 . The method of  claim 1 , wherein said exchange product is thermodynamically more stable than said hybridized double strand. 
     
     
         18 . A method of simultaneously detecting an array of heterologous nucleotide sequences; the method comprising:
 coating a sample well comprising an array of compartments; wherein the surface of said compartments are alternatively:   a) coated with a hybridized nucleotide double strand; and   b) uncoated; wherein said uncoated compartment produces no magnetic signal; and each said coated compartment comprises a heterologous hybridized double strand sequence;   c) measuring magnetic signals for each compartment;   d) incubating said array with a sample comprising free target nucleotide sequences, and forming exchange products;   d) measuring magnetic signals for each compartment comprising exchange products;   e) calculating the difference in said signals from step c and d; and   f) quantifying and identifying said target sequence based on the change in signal calculated in e.   
     
     
         19 . The method of  claim 17 , wherein said measuring said magnetic signal from said sample array is by: a scanning single sensor, scanning the sample well, a two-dimensional sensor array for simultaneous detection or combinations thereof. 
     
     
         20 . An exchange induced remnant magnetization method to detect cancer biomarkers, the method comprising:
 (a) immobilizing a first sequence on a surface; wherein said first sequence comprises N bases;   (b) adding a second sequence to said first sequence, wherein said second sequence comprises N complementary bases or less; and wherein said second sequence hybridizes to said first sequence forming a hybridized double strand; and   (c) incubating said hybridized double strand with a biomarker; and wherein said biomarker exchanges with said second strand to form an exchange product; wherein said exchange product is thermodynamically more stable than said hybridized double strand.