Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

Isothermal Amplification of Nucleic Acids

Colorimetric detection of target analytes with high specificity and sensitivity is of fundamental importance to clinical and personalized point-of-care diagnostics. Because of their extraordinary optical properties, plasmonic nanomaterials have been introduced into colorimetric sensing systems, which provide significantly improved sensitivity in various biosensing applications. Here we review the recent progress on these plasmonic nanoparticles-based colorimetric nanosensors for ultrasensitive molecular diagnostics. According to their different colorimetric signal generation mechanisms, these plasmonic nanosensors are classified into two categories: (1) interparticle distance-dependent colorimetric assay based on target-induced forming cross-linking assembly/aggregate of plasmonic nanoparticles; and (2) size/morphology-dependent colorimetric assay by target-controlled growth/etching of the plasmonic nanoparticles. The sensing fundamentals and cutting-edge applications will be provided for each of them, pa...

Plasmon-Based Colorimetric Nanosensors for Ultrasensitive Molecular Diagnostics.

Surface plasmon resonance (SPR) has been intensively studied and widely employed for light trapping and absorption enhancement. In the mid-infrared and terahertz (THz) regime, graphene supports the tunable SPR via manipulating its Fermi energy and enhances light-matter interaction at the selective wavelength. In this work, periodic arrays of graphene rings are proposed to introduce tunable light trapping with good angle polarization tolerance and enhance the absorption in the light-absorbing materials nearby to more than one order. Moreover, the design principle here could be set as a template to achieve multi-band plasmonic absorption enhancement by introducing more graphene concentric rings into each unit cell. This work not only opens up new ways of employing graphene SPR, but also leads to practical applications in high-performance simultaneous multi-color photodetection with high efficiency and tunable spectral selectivity.

Tunable light trapping and absorption enhancement with graphene ring arrays

Surface plasmon resonance (SPR) has been intensively studied and widely employed for light trapping and absorption enhancement. In the mid-infrared and terahertz (THz) regime, graphene supports tunable SPR via manipulating its Fermi energy and enhances light–matter interaction at the selected wavelength. Most previous studies have concentrated on the absorption enhancement in graphene itself while little attention has been paid to trapping light and enhancing the light absorption in other light-absorbing materials with graphene SPR. In this work, periodic arrays of graphene rings are proposed to introduce tunable light trapping with good angle polarization tolerance and enhance the absorption in the surrounding light-absorbing materials by more than one order of magnitude. Moreover, the design principle here could be set as a template to achieve multi-band plasmonic absorption enhancement by introducing more graphene concentric rings into each unit cell. This work not only opens up new ways of employing graphene SPR, but also leads to practical applications in high-performance simultaneous multi-color photodetection with high efficiency and tunable spectral selectivity.

A pure DNA hydrogel with stable catalytic ability produced by one-step rolling circle amplification.

A methylation-blocked cascade amplification strategy for label-free colorimetric detection of DNA methyltransferase activity

The invention discloses a cascading isothermal amplification based miRNA fluorescence detection kit and a miRNA fluorescence detection method. The target miRNA can specifically recognize and form a stable three-way crossing structure with a ternary probe through complementary hybridization of nucleic acids; a ternary primer in the three-way crossing structure performs strand displacement amplification (SDA) along a ternary template and generates a number of SDA template products; the SDA template product can open a difunctional hairpin probe to trigger nickase to perform signal amplification reaction finally; and a cyclophorase digestion molecular beacon can generate cascading amplified fluorescence signals. The kit can detect miRNA low to 100fM; and the detection method has superhigh specificity, can well distinguish highly homologous sequences at different mispairing positions and solves the difficult problem of false positive result caused by poor specificity in the existing detection method.

Cascading isothermal amplification based miRNA fluorescence detection kit and miRNA fluorescence detection method

MicroRNAs (miRNAs) have been considered as promising biomarkers for cellular events and disease diagnosis. However, current methods for miRNA detection often require sophisticated instruments that may limit the applications in a resource-constrained setting. Herein, we developed a plasmonic biosensor for the colorimetric detection of miRNA with high specificity based on hairpin capture probe-coated magnetic beads (MBs) and catalase (CAT)/streptavidin (SA)-functionalized SiO2 nanoparticles. One end of the hairpin capture probe was labeled with a biotin group which was located close to the surface of MBs and failed to bind to CAT/SA-SiO2 nanoparticles in the absence of target. However, the toehold-mediated strand displacement between target miRNA and hairpin capture probe stretched the biotin group far away from the surface of MBs, enabling the capture and separation of CAT/SA-SiO2 nanoparticles. CAT can rapidly consume hydrogen peroxide (H2O2) which was then used to mediate the growth of gold nanoparticles (AuNPs). A blue aggregated AuNP solution and a red non-aggregated AuNP solution were obtained under low and high concentrations of H2O2, respectively. A single SiO2 nanoparticle was decorated with a large number of CAT molecules to offer an amplified colorimetric signal. As low as 5 amol miRNA was detected with the naked eye. The biosensor can also distinguish target miRNA from homologous sequences even with single nucleotide variation due to the long stem-containing hairpin capture probe. Furthermore, the practical application in real samples was demonstrated by detecting the small RNA samples extracted from cancer cells. Thus, we expect this simple biosensor may work as a routine tool for on-site detection of miRNA in resource-limited settings for clinical early diagnosis.

Catalase-functionalized SiO2 nanoparticles mediate growth of gold nanoparticles for plasmonic biosensing of attomolar microRNA with the naked eye

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A methylation-blocked cascade amplification strategy for label-free colorimetric detection of DNA methyltransferase activity

Cascading isothermal amplification based miRNA fluorescence detection kit and miRNA fluorescence detection method

Catalase-functionalized SiO2 nanoparticles mediate growth of gold nanoparticles for plasmonic biosensing of attomolar microRNA with the naked eye