# US 11,111,528 B2 · Method and apparatus for ultrasensitive quantification of microRNA using an atomic force microscope and a hybrid binding domain

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- Number: US 11,111,528 B2
- Document ID: US11111528B2
- Status: Granted
- Kind: B2
- Application number: 15/412,025
- Filed: 2017-01-22
- Published: 2017-08-17
- Granted: 2021-09-07
- Earliest claimed: 2015-11-23
- Assignees: Posco; Postech Academy-Industry Foundation
- Applicants: Posco; Postech Academy-Industry Foundation
- Inventors: Joon Won Park; Joung Hun Kim; Young Kyu Kim; Hyun Seo Koo; Yoon Hee Lee
- Web page: https://patentgrep.com/patent/US11111528B2

## CPC

- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- G01Q 60/38: Probes, their manufacture, or their related instrumentation, e.g. holders
- G01Q 60/42: Functionalisation
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01Q: SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]

## Abstract

The present invention provides an apparatus and a method for detecting the presence of and/or determining the amount of a label-free microRNA using an atomic force microscope. The method is extremely selective and/or ultrasensitive. In particular, the present invention provides a cantilever comprising a probe that selectively binds to a double strand of DNA/RNA hybrid complex. The probe comprises a hybrid binding domain (HBD) or a variant thereof.

## Claims

### Claim 1

An atomic force microscope comprising a cantilever, wherein said cantilever comprises a probing tip and a probe that is immobilized on said probing tip, wherein said probe comprises a hybrid binding domain (HBD) or a variant thereof, wherein said variant of HBD is elected from the group consisting of:
an HBD linked to a glutathione S-transferase (GST);
an HDB linked to a histidine-tag; and
a biotinylated HBD,
and wherein said HBD is capable of binding to a minor groove of a DNA/RNA hybrid duplex.

### Claim 2 (depends on 1)

The atomic force microscope of claim 1, wherein said HBD comprises an amino terminal (N-terminal) domain of a human ribonuclease 1 (RNase I).

### Claim 3 (depends on 1)

The atomic force microscope of claim 1, wherein said HBD comprises an amino acid sequence of SEQ ID NO: 1.

### Claim 4 (depends on 1)

The atomic force microscope of claim 1, wherein said probe comprises the variant of the HBD.

### Claim 5 (depends on 4)

The atomic force microscope of claim 4, wherein in said variant of the HBD is said HBD linked to said GST, wherein said GST is immobilized on said probing tip and is attached to the N-terminal of said HBD.

### Claim 6 (depends on 1)

The atomic force microscope of claim 1, wherein said probe is capable of complementarily binding to a target microRNA (miRNA).

### Claim 7 (depends on 1)

The atomic force microscope of claim 1, wherein said probe is capable of binding to a 2′—OH group of two consecutive bases of an RNA strand; three phosphodeoxyribose units of a DNA strand, or a combination thereof.

### Claim 8 (depends on 1)

A method for detecting the presence of a target microRNA in a sample without labeling or amplifying, said method comprising:
(a) contacting a substrate comprising a probe DNA that is immobilized on the surface of said substrate with a sample under conditions sufficient to form a DNA/RNA hybrid complex when a target microRNA (miRNA) is present in the sample and
(b) detecting a presence of said DNA/RNA hybrid complex using the atomic force microscope of claim 1.

### Claim 9 (depends on 8)

The method of claim 8, wherein said probe DNA comprises a nucleotide sequence that is complementary to said target miRNA.

### Claim 10 (depends on 8)

The method of claim 8 further comprising the step of determining the amount of said target miRNA in said sample.

### Claim 11 (depends on 10)

The method of claim 10, wherein said step of determining the amount of said target miRNA in said sample comprises:
determining the number of said DNA/RNA hybrid complexes detected per spot area of said substrate and
calculating a total number of said target miRNA in said sample using Equation 1:
TmiRNA=(N×(S/U))/(E/100) (Eq. 1),
wherein
TmiRNA=total number of said target miRNA;
N=the number of said target miRNA per unit area of spot;
S=total spot area within said substrate;
U=unit area of spot; and
E=% capture efficiency of miRNA by said probe.

### Claim 12 (depends on 8)

The method of claim 8, wherein said step of detecting the presence of said DNA/RNA hybrid complex comprises (i) determining a site where the adhesion force is observed at four consecutive pixels of 8 nm pixel size or (ii) determining three consecutive pixels where an adhesion force is observed at three consecutive pixels of 10 nm pixel size.

### Claim 13 (depends on 8)

The method of claim 8, wherein the sample is a fluid sample, and wherein the concentration of the target miRNA in the sample is 5×10−20 to 2×10−13 M.

### Claim 14 (depends on 8)

The method of claim 8, wherein a size of a probe DNA spot is calculated by an equation selected from the group consisting of:
SD(μm)=([M]×1019/5)0.5 (Eq. 2) and
SD(μm)=([unit]/10)0.5 (Eq. 3),
wherein
SD is Probe DNA spot diameter;
[M] is miRNA concentration; and
[unit] is miRNA number.

### Claim 15 (depends on 8)

The method of claim 8, wherein the substrate is selected from the group consisting of glass, metal, plastic, silicon, silicate, ceramic, a semiconductor, synthetic organic metal, a synthetic semiconductor, an alloy, and a combination thereof.

### Claim 16 (depends on 8)

The method of claim 8, wherein a site of the HBD binding to the DNA/RNA hybrid complex comprises:
Y3, K33, and K34 of the HBD bound with the DNA strand of the DNA/RNA hybrid complex;
W17 and F32 of the HBD bound with the RNA strand of the DNA/RNA hybrid complex; and
a combination thereof.

### Claim 17 (depends on 8)

The method of claim 8, wherein said sample is obtained from a single cell.