Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research Thus far, many signal amplification strategies have been developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA) In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence in situ hybridization (FISH) imaging, live cell imaging, and targeted drug delivery In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples Finally, the review provides insight into the challenges and future perspectives of HCR

Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine

ConspectusMicroRNAs (miRNAs) are a class of small noncoding RNAs that act as pivotal post-transcriptional regulators of gene expression, thus involving in many fundamental cellular processes such as cell proliferation, migration, and canceration. The detection of miRNAs has attracted significant interest, as abnormal miRNA expression is identified to contribute to serious human diseases such as cancers. Particularly, miRNAs in peripheral blood have recently been recognized as important biomarkers potential for liquid biopsy. Furthermore, as miRNAs are expressed heterogeneously in different cells, investigations into single-cell miRNA expression will be of great value for resolving miRNA-mediated regulatory circuits and the complexity and heterogeneity of miRNA-related diseases. Thus, the development of miRNA detection methods, especially for complex clinic samples and single cells is in great demand. In this Account, we will present recent progress in the design and application of isothermal amplification...

Isothermal Amplification for MicroRNA Detection: From the Test Tube to the Cell

Owing to its important physiological functions, especially as molecular biomarkers of diseases, RNA is an important focus of biomedicine and biochemical sensing. Signal amplification detection has been put forward because of the need for accurate identification of RNA at low expression levels, which is significant for the early diagnosis and therapy of malignant diseases. However, conventional amplification methods for RNA analysis depend on the use of enzymes, fixation of cells, and thermal cycling, which confine their performance to cell lysates or dead cells, thus the imaging of RNA in living cells remained until recently little explored. In recent years, the advance of isothermal amplification of nucleic acids has opened paths for meeting this need in living cells. This minireview tracks the development of in situ amplification assays for RNAs in living cells, and highlights the potential challenges facing this field, aiming to improve the development of in vivo isothermal amplification as well as usher in new frontiers in this fertile research area.

In Situ Amplification‐Based Imaging of RNA in Living Cells

Modern optical detection technology plays a critical role in current clinical detection due to its high sensitivity and accuracy However, higher requirements such as extremely high detection sensitivity have been put forward due to the clinical needs for the early finding and diagnosing of malignant tumors which are significant for tumor therapy The technology of isothermal amplification with nucleic acids opens up avenues for meeting this requirement Recent reports have shown that a nucleic acid amplification-assisted modern optical sensing interface has achieved satisfactory sensitivity and accuracy, high speed and specificity Compared with isothermal amplification technology designed to work completely in a solution system, solid biosensing interfaces demonstrated better performances in stability and sensitivity due to their ease of separation from the reaction mixture and the better signal transduction on these optical nano-biosensing interfaces Also the flexibility and designability during the construction of these nano-biosensing interfaces provided a promising research topic for the ultrasensitive detection of cancer diseases In this review, we describe the construction of the burgeoning number of optical nano-biosensing interfaces assisted by a nucleic acid amplification strategy, and provide insightful views on: (1) approaches to the smart fabrication of an optical nano-biosensing interface, (2) biosensing mechanisms via the nucleic acid amplification method, (3) the newest strategies and future perspectives

Optical nano-biosensing interface via nucleic acid amplification strategy: construction and application

DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems We also discuss the outstanding challenges in the field and outline potential solutions

DNA-Based Nanostructures for Live-Cell Analysis.

We herein report a novel finding that nucleic acid molecular aggregates (NAMAs) self-assembled on graphene oxide nanoplates (GONPs) as a result of DNA rolling circle amplification (RCA) and a functionalized triple-helix probe (THP) in single cells. The functionalized THP containing the aptamer region for target recognition and the trigger DNA region for RCA was firstly used to activate RCA for miRNA imaging in single cells. Interestingly, NAMAs with the fluorescent labels were hybridized by both the RCA products and FAM-DNA, and could partly self-assemble on GONPs; meanwhile, NAMAs could extend from the GONPs, which led to the quenched fluorescence being renewed. Significantly, the NAMAs were successfully applied for low-abundance miRNA detection and imaging in single cells. The self-assembled NAMAs could generate prominent and agminated fluorescence-bright spots in single cancer cells, which will effectively drive cell imaging into a new era.

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Self-assembly of nucleic acid molecular aggregates catalyzed by a triple-helix probe for miRNA detection and single cell imaging

Orderly nucleic acid aggregates (ONAAs) self-assembled on mesoporous silica nanoparticles (MSNs) with positively charged aminopropyl groups (PC) were firstly developed. Interestingly, a novel electrostatic DNA self-assembly could realize hybridization chain reaction (HCR) on the surface of PCMSNs in single cells. Significantly, a non-destructive amplification strategy based on ONAAs-PCMSNs was successfully developed for miRNA detection and in situ imaging by the prominent and agminated fluorescence-bright spots in living cells.

Orderly nucleic acid aggregates by electrostatic self-assembly in single cells for miRNA detection and visualizing.

Here, we propose a strategy for unique nuclear-shell biopolymers initiated by telomere elongation for telomerase activity detection and precise drug delivery to individual cancer cells. Telomerase-triggered DNA rolling-circle amplification (RCA) is used to assemble nuclear-shell biopolymers with signal molecules for selective cancer cell recognition and efficient drug delivery to targeted individual cells. This strategy not only should allow the creation of clustered 5-carboxyfluorescein (FAM)-fluorescence spots in response to human-telomerase activity in individual cancer cells but also could efficiently deliver drugs to reduce the undesired death of healthy cells. These findings offer new opportunities to improve the efficacy of cancer cell imaging and therapy.

Nuclear-Shell Biopolymers Initiated by Telomere Elongation for Individual Cancer Cell Imaging and Drug Delivery

A novel core-shell DNA self-assembly catalyzed by thiol-disulfide exchange reactions was proposed, which could realize GSH-initiated hybridization chain reaction (HCR) for signal amplification and molecules gathering. Significantly, these self-assembled products via electrostatic interaction could accumulate into prominent and clustered fluorescence-bright spots in single cancer cells for reduced glutathione monitoring, which will effectively drive cell monitoring into a new era.

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Zhen Zhang

Papers

Self-assembly of nucleic acid molecular aggregates catalyzed by a triple-helix probe for miRNA detection and single cell imaging

Orderly nucleic acid aggregates by electrostatic self-assembly in single cells for miRNA detection and visualizing.

Nuclear-Shell Biopolymers Initiated by Telomere Elongation for Individual Cancer Cell Imaging and Drug Delivery

Core-shell self-assembly triggered via a thiol-disulfide exchange reaction for reduced glutathione detection and single cells monitoring.

Ultrasensitive SERS assay of lysozyme using a novel and unique four-way helical junction molecule probe for signal amplification