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

• US 10,640,813 B2
• Filed: 10/25/2018
• Issued: 05/05/2020
• Est. Priority Date: 04/10/2013
• Status: Active Grant

• Current Assignee: University of Houston System
• Original Assignee: University of Houston System
• Inventors: Xu, Shoujun, Yao, Li, Wang, Yuhong, Hu, Qiongzheng, Yang, Haopeng
• Primary Examiner(s): Bertagna, Angela M.
• Application Number:
  US16/170,208
• Publication Number:
  US 20190048403A1
• Time in Patent Office: 558 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads

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.

Claims

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 comprises at least one base that does not form a complementary base pair with said first nucleotide single strand;

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 and randomizing the magnetic dipoles of nonspecifically bound magnetic particles, wherein said measuring occurs in the absence of removing said nonspecifically bound magnetic particles from said surface; and

f) quantifying the amount of said third nucleotide strand from the difference in magnetic signal values measured in step c and step e, wherein the difference in the magnetic signal values comprises a decrease in magnetic signal values due to the randomizing of the magnetic dipoles of the nonspecifically bound magnetic particles.

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 non-covalent bond.

4. The method of claim 1, wherein said first magnetic particle is about 1 nm to about 10 μ

m in size.

5. The method of claim 1, wherein said first and said second magnetic signal comprise magnetic moment measurements.

6. The method of claim 1, wherein said weak mechanical force is supplied by a shaker, centrifuge, or sonicator.

7. The method of claim 1, wherein said hybridized double strand is in a liquid environment, a cell lysate, blood plasma, or urine.

8. 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 less than N complementary bases and a magnetic particle; and

wherein said second sequence hybridizes to said first sequence forming a hybridized double strand;

(c) measuring a first magnetic signal value for said hybridized double strand;

(d) incubating said hybridized double strand with a cancer biomarker nucleic acid;

wherein said cancer biomarker nucleic acid exchanges with said second strand to form an exchange product;

wherein said exchange product is thermodynamically more stable than said hybridized double strand;

(e) measuring a second magnetic signal value for the exchange product of step (d) after applying a weak mechanical force and randomizing the magnetic dipoles of nonspecifically bound magnetic particle, wherein said measuring occurs in the absence of removing said nonspecifically bound magnetic particles from said surface; and

f) quantifying the amount of said cancer biomarker nucleic acid from the difference in magnetic signal values measured in step (c) and step (e) wherein the difference in the magnetic signal values comprises a decrease in magnetic signal values due to the randomizing of the magnetic dipoles of the nonspecifically bound magnetic particles.