Table 2 miRNA amplification and detection methods for POC diagnostics

Isothermal amplification-based assays

Real-time fluorescence detection of exponential amplification reaction (EXPAR) products. This reaction is used to amplify short oligonucleotides by a combination of polymerase strand extension and single-strand nicking.

let‐7 miRNA family (let-7a–i)

0.1 zmol (100 copies) per 10 µL reaction

0.1 zmol to 1.0  pmol

Synthetic miRNA

≤30 min

[52]

One-step loop-mediated isothermal amplification (LAMP).

let‐7 miRNA family (let-7a–e)

1.0 amol per 10 µL reaction

1.0 amol to 1.0 pmol

Synthetic miRNA

≤90 min

[53]

Assay based on a target-primed branched rolling-circle amplification (BRCA) reaction and fluorescence quantification.

let‐7 miRNA family (let-7a–c)

10 fM

0.05–0.5 pM

Spiked synthetic miRNA in total human lung RNA

≤90 min

[56]

Lateral flow assay-based systems

DNA-gold nanoparticle (DNA-GNP)-based lateral flow nucleic acid biosensor for visual detection of miRNA. Particularly, the sandwich-type hybridization reactions among GNP-labeled DNA probe, miRNA, and biotin-modified DNA probes on the lateral flow device enables the visual detection of miRNA based on the accumulation of GNPs on the test line of the biosensor.

miRNA-21

5.0 amol per 50 µL reaction

5.0–100 amol

Synthetic miRNA

~70 min

[60]

Enzyme-amplified lateral flow biosensor based on an enzyme-based dual-labeled nanoprobe. Particularly, the GNPs surface was functionalized with detection probe and horseradish peroxidase (HRP). The capture DNA probes are immobilized on the test zone of the lateral flow biosensor. In miRNA positive samples, the enzyme-based dual-labeled nanoprobes are captured by forming the “sandwich structure” in the test zone of the lateral flow biosensor, enabling visualization of the detected miRNA.

miRNA-244

7.5 pM

7.5 pM–75 nM

Synthetic miRNA and miRNA-224 in A549 cell lysate

≤30 min

[61]

pH-responsive method based on the byproduction of H⁺, a significant change of the pH in the system during the miRNA amplification based RCA technique.

miRNA-21

9.3 fM

20 fM to 20 pM

Synthetic miRNA, a tumor-associated miRNA, spiked serum samples and cells

≤70 min

[62]

Oligonucleotide-templated reaction (OTR)

The miRNA of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction between chemically engineered peptide nucleic acid hybridization probes.

miRNA-141, miRNA-375, miRNA-132

60.77 nM

50 nM to 1 µM

miRNA in human blood samples

Not reported

[64]

Nanobead-based systems

miRNA quantification based on the enzymatic processing of DNA probes immobilized on PEGylated GNPs. The fluorescence of FAM-labeled DNA probes is initially quenched via nanomaterial surface energy transfer by the proximity to the gold surface.

miRNA-203, miRNA-21

0.2–0.3  fmol or 5–8 pM

5–300 pM

Cancer related miRNA-203 and miRNA-21 in samples of extracted total RNA from cell cultures

5 h

[66]

DNA functionalized Fe₃O₄@Ag core-shell magnetic NPs for miRNA capture and DSN signal amplification to be detected via surface-enhanced Raman spectroscopy (SERS) spectroscopy.

miRNA-let7b

0.3 fM

1 fM–1 nM

Synthetic miRNA

Not reported

[67]

Electrochemical-based systems

Oligonucleotide capture probes on the surface of indium tin oxide electrodes hybridize to target miRNA specifically. After forming a DNA/miRNA duplex through hybridization, isoniazid-capped OsO₂ nanoparticles are introduced to form a chemical ligation between tag miRNA and OsO₂ nanoparticles. Consequently, this electrocatalytic system generates a measurable current that enables to detect miRNA.

let‐7 miRNA family (let-7b, 7c), miRNA-106, miRNA 139

80 fM

80–200 pM

miRNA expression analysis of HeLa cells

~60 min

[70]

Amperometric magnetobiosensors involving RNA-binding viral protein p19 enable to quantify miRNA.

miRNA-21

0.4 fmol in 10 µL of sample

0.14–10 nM

Synthetic miRNA and endogenous miR-21 in total RNA extracted from cancer cells and human breast-tumor specimens.

~2 h

[71]

Amperometric magnetobiosensors involving use of magnetic beads (MBs) modified with a specific DNA-RNA antibody as for the determination of miRNAs.

miRNA-211, miRNA-205, miRNA-223, miRNA-155

2.4 pM

8.2–250 pM

Synthetic miRNA and miRNA in total RNA extracted from metastatic cancer cell lines and human tumor tissues.

2 h

[72]

miRNA detection based on hybridization protection against nuclease S1 digestion.

miRNA-319a

1.8 pM

5–1000 pM

Synthetic miRNA

Not reported

[74]

Microfluidic chip-based

The power-free microfluidic device driven by degassed poly-di-methyl-siloxane (PDMS) was applied for target miRNA detection by sandwich hybridization taking advantage of the coaxial stacking effect.

miRNA-21

0.62 nM

Not reported

Synthetic miRNA

20 min

[78]

The immobilization of miRNA capture probe onto the glass surface, and microchannels convey the sample to the immobilized probes initiating the miRNA hybridization resulting in the amplified signal by laminar flow-assisted dendritic amplification.

miRNA-21

0.5 pM

Not reported

Synthetic miRNA

20 min

[79]

A fully integrated fluorescence reader into a microfluidic device for successful screening and detection of breast cancer by blood test using miRNA beacon probe, which was designed to include multiple miRNA to detect multiple miRNA levels.

miRNA-21

Not reported

Not reported

Blood samples

30 min

[81]