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

Biophotonics as a highly interdisciplinary frontier often requires the assistance of optical agents to control the light pathways in cells, tissues and living organisms for specific biomedical applications. Organic semiconducting materials (OSMs) composed of π-conjugated building blocks as the optically active components have recently emerged as a promising category of biophotonic agents. OSMs possess common features including excellent optical properties, good photostability and biologically benign composition. This review summarizes the recent progress in the development of OSMs based on small-molecule fluorophores, aggregation-induced emission (AIE) dyes and semiconducting oligomer/polymer nanoparticles (SONs/SPNs) for advanced biophotonic applications. OSMs have been exploited as imaging agents to transduce biomolecular interactions into second near-infrared fluorescence, chemiluminescence, afterglow or photoacoustic signals, enabling deep-tissue ultrasensitive imaging of biological tissues, disease biomarkers and physiological indexes. By fine-tuning the molecular structures, OSMs can also convert light energy into cytotoxic free radicals or heat, allowing for effective cancer phototherapy. Due to their instant light response and efficient light-harvesting properties, precise regulation of biological activities using OSMs as remote transducers has been demonstrated for protein ion channels, gene transcription and protein activation. In addition to highlighting OSMs as a multifunctional platform for a wide range of biomedical applications, current challenges and perspectives of OSMs in biophotonics are discussed.

Development of organic semiconducting materials for deep-tissue optical imaging, phototherapy and photoactivation

Rolling circle amplification (RCA) is an isothermal enzymatic process where a short DNA or RNA primer is amplified to form a long single stranded DNA or RNA using a circular DNA template and special DNA or RNA polymerases. The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. The power, simplicity, and versatility of the DNA amplification technique have made it an attractive tool for biomedical research and nanobiotechnology. Traditionally, RCA has been used to develop sensitive diagnostic methods for a variety of targets including nucleic acids (DNA, RNA), small molecules, proteins, and cells. RCA has also attracted significant attention in the field of nanotechnology and nanobiotechnology. The RCA-produced long, single-stranded DNA with repeating units has been used as template for the periodic assembly of nanospecies. Moreover, since RCA products can be tailor-designed by manipulating the circular template, RCA has been employed to generate complex DNA nanostructures such as DNA origami, nanotubes, nanoribbons and DNA based metamaterials. These functional RCA based nanotechnologies have been utilized for biodetection, drug delivery, designing bioelectronic circuits and bioseparation. In this review, we introduce the fundamental engineering principles used to design RCA nanotechnologies, discuss recently developed RCA-based diagnostics and bioanalytical tools, and summarize the use of RCA to construct multivalent molecular scaffolds and nanostructures for applications in biology, diagnostics and therapeutics.

Rolling circle amplification: A versatile tool for chemical biology, materials science and medicine

https://dr.ntu.edu.sg/bitstream/10356/88820/1/Recent%20progress%20on%20semiconducting%20polymer%20nanoparticles%20for%20molecular%20imaging%20and%20cancer%20phototherapy.pdf

Recent progress on semiconducting polymer nanoparticles for molecular imaging and cancer phototherapy

ConspectusBiosensors for highly sensitive, selective, and rapid quantification of specific biomolecules make great contributions to biomedical research, especially molecular diagnostics. However, conventional methods for biomolecular assays often suffer from insufficient sensitivity and poor specificity. In some case (e.g., early disease diagnostics), the concentration of target biomolecules is too low to be detected by these routine approaches, and cumbersome procedures are needed to improve the detection sensitivity. Therefore, there is an urgent need for rapid and ultrasensitive analytical tools. In this respect, single-molecule fluorescence approaches may well satisfy the requirement and hold promising potential for the development of ultrasensitive biosensors.Encouragingly, owing to the advances in single-molecule microscopy and spectroscopy over past decades, the detection of single fluorescent molecule comes true, greatly boosting the development of highly sensitive biosensors. By in vitro/in vivo ...

Fluorescent Biosensors Based on Single-Molecule Counting

Supramolecular structures composed of padlock probes and primers were used to perform rolling circle amplification (RCA) which was achieved by metal ion (Hg2+ or Ag+) induced DNA ligase activity. In the presence of Hg2+ (or Ag+), the specific and strong interaction between thymidine–thymidine and Hg2+ (or cytosine–cytosine and Ag+) at the terminal of the padlock probe enabled the circularization of the padlock probe with primer in the aid of DNA ligase. An RCA process was then accomplished by DNA polymerase/dNTPs. The RCA product containing multiple tandem repeats could hybridize with a large number of molecular beacons (reporter), resulting in an enhanced fluorescence signal. This proposed single-input YES gate enabled the sensitive and selective detection of Hg2+ (or Ag+). Additionally, based on the principle of DNA hybridization and displacement, a NOT logic gate was constructed by designing a double-stranded fluorescence probe as reporter. Significantly, this assay was further applied to the construction of a complete set of two-input molecular-scale logic gates and three advanced logic devices.

Metal ions triggered ligase activity for rolling circle amplification and its application in molecular logic gate operations

Sortase A (Srt A) is a key enzyme that catalyzes the covalent anchoring of surface proteins to the bacterial cell wall and plays a dominant role in the late stage of Gram-positive bacterial infections. Achieving activity detection of Srt A and inhibitors screening is important for the treatment of Gram-positive bacterial infections and novel antibacterial drugs research. Here, an electrochemical sensor pattern is proposed. Black phosphorus (BP) is a novel graphite-like two-dimensional material with excellent electrochemical properties. However, the extremely poor environmental stability limits its practical application. The surface modification of BP using graphene oxide (GO) significantly improves the stability, and obtained partially reduced graphite oxide functionalized BP composites combine both electrical conductivity and stability. In this paper, we develop an electrochemical biosensor based on partially reduced graphite oxide functionalized BP /gold nanoparticles (p-rGO-BP/AuNPs) nanocomposites for detecting Srt A activity. The assay utilizes two peptide chains, P1 (CLPETG) and P2 (GGGGG), which can be linked by the presence of Srt A. At the same time, the process is monitored by differential pulse voltammetry (DPV). The sensor showed good selectivity and stability with a linear dynamic range of 1 pM-100 nM and a detection limit (LOD) of 0.65 pM. In addition, the method was successfully used to detect Srt A in complex samples and estimate the half-inhibitory concentration (IC50) of Srt A inhibitors (curcumin, dihydromyricetin and rutin). 

An electrochemical biosensor based on graphene intercalated functionalized black phosphorus/gold nanoparticles nanocomposites for the detection of bacterial enzyme

A sensitive biosensor based on carbon nanohorn/rhodamine B for toxicity detection of polystyrene microplastics and typical pollutants

A chemiluminescence imaging array for the detection of cancer cells by dual-aptamer recognition and bio-bar-code nanoprobe-based rolling circle amplification

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Metal ions triggered ligase activity for rolling circle amplification and its application in molecular logic gate operations

An electrochemical biosensor based on graphene intercalated functionalized black phosphorus/gold nanoparticles nanocomposites for the detection of bacterial enzyme

A sensitive biosensor based on carbon nanohorn/rhodamine B for toxicity detection of polystyrene microplastics and typical pollutants

A chemiluminescence imaging array for the detection of cancer cells by dual-aptamer recognition and bio-bar-code nanoprobe-based rolling circle amplification