Hybridization Chain Reaction System Research Articles

Target and acidic tumor microenvironment-induced in situ HCR for bispecific tumor cell imaging and therapy

The tumor microenvironment is characterized by an acidic extracellular milieu generated through the anaerobic glycolysis of tumor cells. Leveraging this characteristic, we suggest employing an in situ hybridization chain reaction (HCR) activated by both target recognition and the acidic microenvironment for bispecific tumor cell imaging and therapy. By integrating the recognition capability of aptamers, the pH responsiveness of i-motifs, and the signal amplification of HCR, we have developed a pH-induced HCR (pH-HCR) system. This system consists of three DNA probes: pH-Apt, HP1, and HP2. pH-Apt is composed of the Apt-T-B and Imotif sequences (sequences that can form an i-motif structure). Apt-T-B includes two segments: an aptamer sequence for MUC1 recognition and a trigger sequence blocked by the Imotif sequence for HCR. Under acidic conditions, upon binding to MUC1, the Imotif sequence forms an i-motif structure, detaching from pH-Apt and exposing the trigger sequence. This initiation of HCR, in conjunction with HP1 and HP2, amplifies fluorescent signals, achieving the bispecific imaging of tumor cells. Simultaneously, doxorubicin (Dox) can be loaded onto probes and released under acidic conditions, resulting in a 47% cytotoxic efficiency against tumor cells. pH-HCR enables successful bispecific imaging and cell killing induced by MUC1 in the acidic microenvironment of SW620 cells.

Tumor-associated miRNAs activated HCR-DNAzyme theranostic nanosystem to trigger innate- and adaptive-immune responses for long-term immunotherapy

Targeted manipulation of cytoplasmic double-strand (dsDNA) generation in tumors to activate cGAS-STING signaling remains challenging in antitumor immunity. Herein, we proposed an endogenous tumor-associated microRNAs-triggered theranostic nanosystem for codelivery of Hybridization Chain Reaction (HCR) and DNAzyme systems to achieve robust long-term antitumor immunity based on MnO2-adjuvant nanoparticles. The MnO2 was decomposed into Mn2+ ions in the endosome and thus released HCR and DNAzyme systems. Endogenous miR-21-triggered HCR for dsDNA generation, thereby resulting in cGAS-STING signaling activation. Meanwhile, the DNAzyme system was unlocked and catalyzed respectively via endogenous miR-146a and cofactor Mn2+ ions for gene silencing of programmed cell death ligand 1 (PD-L1) to achieve immune-checkpoint blockade (ICB). The combination of innate immunity based on HCR system and adaptive immune-responses mediated by DNAzyme system acquired robust antitumor immunity and long-term immune effect on distal tumors and pulmonary metastasis in vivo. In summary, this endogenously-responsive nanosystem with self-supplied cytoplasmic dsDNA presents strong antitumor immune activation and provides a long-term immunotherapeutic potential for cancer therapy.

Development of a hybridization chain reaction-powered lab-on-fiber device for ultrafast point-of-care testing of circulating tuor DNA in whole blood

Circulating tumor DNA (ctDNA) demonstrates great promise in the guidance of prognostication, diagnosis, and surveillance of cancers, which highlights the need for rapid and sensitive point-of-care testing (POCT) technologies. Hybridization chain reaction (HCR)-based optical biosensors provide excellent solutions due to their prominent features. However, the requirement of a sophisticated and expensive optical readout device, relatively long detection time, and heating hold back their scalability and clinical applications. Here, an innovative HCR-powered lab-on-fiber device (HCR-LOFD) was developed for rapid on-site detection of ctDNA with high sensitivity, specificity, and reproducibility. A LOFD with a compact all-fiber optical structure was constructed for the fluorescence detection of the HCR system. Combining HCR, fluorescence energy resonant transfer, and the evanescent wave fluorescence principle, HCR-LOFD achieved the quantitative detection of KRAS G12D, the 12th amino acid from glycine (Gly) mutated aspartate (Asp) and the most common mutation of KARS, in 5 min at room temperature based on end-point detection mode or real-time fluorescence detection mode. This new assay platform was also successfully applied for the direct detection of KRAS G12D in whole blood with simple dilution. The application of HCR-LOFD not only greatly simplifies the complexity of optical readout devices and improves their scalability but also potentially serves as a sample-to-answer solution for the detection of biomarkers in limited medical resource regions.

A fluorescence aptasensor based on hybridization chain reaction for simultaneous detection of T-2 toxins and zearalenone1

It is extremely necessary to establish a rapid and high-throughput method to detect mycotoxins in food, because grains and cereals are greatly vulnerable to mycotoxins before and after harvest. In this study, we developed a portable aptasensor based on streptavidin magnetic microspheres (MMPs) and hybridization chain reaction (HCR) to simultaneously detect T-2 toxin and zearalenone (ZEN) in corn and oat flour. The MMPs compete with the aptamer for binding, which releases more H0 and triggers HCR with the H1 intermediate modified using 6-FAM and BHQ-1 and the unmodified H2. Subsequently, placing the HCR system corresponding to T-2 and ZEN in a constant-temperature fluorescence detector resulted in well-recovered fluorescence of the HCR products. T-2 and ZEN exhibited good fluorescence response in the dynamic range of 0.001–10 ng mL−1 and 0.01–100 ng mL−1 with detection limits of 0.1 pg mL−1 and 1.2 pg mL−1, respectively. In addition, this strategy achieved the selective detection of T-2 and ZEN in the spiked corn and oat flour samples. The results are also in good agreement with those obtained using commercial ELISA kits. This developed aptasensor with the characteristics of simple operation and portability has the application potential of establishing sensitive and portable field detection of various mycotoxins.

Ultrasensitive hairpin mediated upconversion fluorescence biosensor for Staphylococcus aureus detection in foods and waters exploiting g–C3N4–assisted catalysis

A novel g-C3N4 nanosheets (g-C3N4 NSs)-assisted upconversion fluorescent aptasensor was proposed for Staphylococcus aureus (S. aureus) detection by adopting hybridization chain reaction (HCR) as a sensitizer. Two hairpin (H1 and H2) structured DNA probes were engineered predicated on the partial complementary sequence (cDNA) of S. aureus aptamer and modified on the exterior of the upconversion nanoparticles (UCNPs), respectively. The presence of S. aureus initiated the HCR system and activated H1 and H2 probes to form a double-helix away from the g-C3N4 NSs vicinity. This led to the decrease in peroxidase-like activity (PA) of the g-C3N4 NSs and corresponding fluorescence recovery proportional to the concentration of S. aureus (10-106 cfu mL−1). The method was applied to real food samples with acceptable recoveries (91.1–101.6%) and further validated by traditional plate counting method (p > 0.05).

Fluidic Membrane Accelerating the Kinetics of Photoactivatable Hybridization Chain Reaction for Accurate Imaging of Tumor-Derived Exosomes.

Slow intermolecular collisions and "always active" responses compromise the amplification efficiency and response accuracy of nonenzymatic hybridization chain reaction (HCR). In this study, a photoactivatable membrane-oriented HCR (MOHCR) system was rationally designed by binding a photocleavable initiator probe onto a target protein and then anchoring cholesterol-modified hairpin-structure fuel probes. When irradiated, the bound initiator probe was photoactivated and initiated self-assembly to generate activatable and amplified imaging. In a proof-of-concept assay, breast-cancer-derived exosomes were imaged based on the surface protein epithelial cell adhesion molecule (EpCAM). Photoactivatable responses provided precise spatiotemporal control of the MOHCR, and fluidic membranes enabled accelerated reaction kinetics. Our MOHCR system demonstrated high efficiency and accuracy in differentiating between plasma samples from breast cancer patients and healthy donors.

A SERS/electrochemical dual-signal readout immunosensor using highly-ordered Au/Ag bimetallic cavity array as the substrate for simultaneous detection of three β-adrenergic agonists

A surface-enhanced Raman scattering (SERS)/electrochemical dual-signal readout immunosensor was developed for simultaneous detection of β-adrenergic agonists salbutamol (SAL), ractopamine (RAC) and phenylethanolamine A (PA). The highly-ordered gold/silver bimetallic cavity array (BMCA) was prepared by electrodepositing Au/Ag nanoparticles to the interstice of highly ordered close-packed polystyrene templates. After electrochemical and SERS characterization, the BMCA was used as the substrate for constructing SERS/electrochemical dual-signal readout immunosensor. 3,3′,5,5′-tetramethylbenzidine (TMB), methylene blue (MB) and Nile blue (NB) were selected as the dual-signal reporters, and hybridization chain reaction (HCR) was used as the signal amplifier. The immunoprobe was prepared by absorption of the antibody (Ab) and constructing HCR system embedded with electro/SERS reporter on Au nanoparticles (NPs). After competitive immuno-reaction between coating antigen and analyte for limited Ab on immunoprobe, the SERS/electrochemical dual-signals on BMCA were measured for quantitatively detecting SAL, RAC and PA simultaneously. SAL, RAC and PA were detected in concentration range of 1 pg mL−1 to 100 ng mL−1 with LOD of 0.8, 0.4, and 1.3 pg mL−1, respectively. The applicability of the proposed immunosensor in spiked pork liver samples was verified by the recovery of 95.0%–108.5% with RSD of 6.9%–10.7%. It was proven that the immunosensor was able to detect multiple β-adrenergic agonists with high sensitivity, specificity, accuracy and precision. The immunosensor can be used as a platform for the determination of other small molecular compounds in biological, food and environmental analytical fields.

Construction of a Stimuli-Responsive DNAzyme-Braked DNA Nanomachine for the Amplified Imaging of miRNAs in Living Cells and Mice

Construction of a Stimuli-Responsive DNAzyme-Braked DNA Nanomachine for the Amplified Imaging of miRNAs in Living Cells and Mice

Cationic Copolymer-Augmented DNA Hybridization Chain Reaction.

Various DNA assembly techniques and structures have emerged with the continuous progress of DNA nanotechnology. DNA hybridization chain reaction (HCR) is a representative example owing to isothermal and enzyme-free features. However, HCR is time consuming and is inhibited by nucleases present in biological samples. Herein, we demonstrated that a cationic copolymer, poly(l-lysine)-graft-dextran (PLL-g-Dex), significantly facilitated HCR and increased its initiator sensitivity by 40-fold. PLL-g-Dex promoted the generation of HCR products with high molecular weight by accelerating the initiation and the subsequent growth steps of HCR. Moreover, PLL-g-Dex protected the HCR system from nucleases, permitting HCR in the presence of serum components. Addition of PLL-g-Dex is a universal and efficient strategy that does not require optimization of the reactor setup or DNA sequences, thus laying a solid foundation for the wider application of HCR.

Determination of Endonuclease Activity by an Enzyme-Free Fluorescent Biosensor Using the Hybridization Chain Reaction (HCR)

A fluorescent labeling system for the determination of endonuclease activity using the hybridization chain reaction (HCR) is reported. Two hairpin probes, H1 and H2, were well-designed in which two split cleavage sequences of endonuclease EcoRI were respectively integrated into H1 and H2. Black Hole Quencher 1 (BHQ1) and carboxyfluorescein (FAM) were labeled at the 5′ and 3′ ends of H1, respectively. Once trigger DNA was introduced, HCR between H1 and H2 was triggered to form long DNA nanowires containing the intact cleavage sequences of EcoRI. At the same time, the BHQ1 and FAM were close together and the fluorescence signal was quenched. In the presence of EcoRI, the cleavage sequences were recognized by EcoRI and long DNA nanowires were cleaved to short double-stranded DNA (dsDNA) polymers. The FAM was far from BHQ1, resulting in the recovery of fluorescence signal. Under the optimized conditions, the fluorescence intensity was linear with EcoRI concentration over the range from 0.001 to 0.2 U μL−1 and the detection limit is 1.2 × 10−4 U µL−1. In addition, this strategy is versatile and may be used to determine other endonucleases activity by simply changing the specific cleavage sites in HCR system.

Smart Hairpins@MnO2 Nanosystem Enables Target-Triggered Enzyme-Free Exponential Amplification for Ultrasensitive Imaging of Intracellular MicroRNAs in Living Cells.

Sensitive and specific imaging of microRNA (miRNA) in living cells is of great value for disease diagnosis and monitoring. Hybridization chain reaction (HCR) and DNAzyme-based methods have been considered as powerful tools for miRNA detection, with low efficient intracellular delivery and limited amplification efficiency. Herein, we propose a Hairpins@MnO2 nanosystem for intracellular enzyme-free exponential amplification for miRNA imaging. The enzyme-free exponential amplification is based on the synergistic cross-activation between HCR and DNAzymes. The MnO2 nanosheets were employed as the carrier of three kinds of hairpin DNA probes and further provided appropriate Mn2+ as DNAzyme cofactors in the living cell. Upon entering cells and in the presence of highly expressed glutathione (GSH) in tumors, MnO2 is reduced to release Mn2+ and the three kinds of hairpin DNA probes. In the presence of target miRNA, the released hairpin DNA H1 and H2 probes self-assemble via HCR into the wire-shaped active Mn2+-based DNAzymes which further catalyze the cleavage of H3 to generate numerous new triggers to reversely stimulate HCR amplifiers, thus offering tremendously amplified Förster resonance energy transfer readout. The method has a detection limit of 33 fM, which is 2.4 × 104 times lower than that of the traditional HCR system. The developed method also has a high specificity; even miRNAs with a single base difference can be distinguished. Live cell imaging experiments confirmed that this Hairpins@MnO2 nanosystem allows accurate differentiation of miRNA expression of cancer cells and normal cells. The method holds great potential in biological research of nucleic acids.

Construction of a Homogeneous Enzyme-Free Autocatalytic Nucleic Acid Machinery for High-Performance Intracellular Imaging of MicroRNA

Construction of a Homogeneous Enzyme-Free Autocatalytic Nucleic Acid Machinery for High-Performance Intracellular Imaging of MicroRNA

Specific and sensitive detection of CircRNA based on netlike hybridization chain reaction

Circular RNA (circRNA), as a new class of biomarker, plays an important role in the occurrence and development of cancer. However, the limitations of detection methods in recent years have severely restricted the related research of circRNA. Here, we have developed an effective circRNA detection method based on the thermostatic netlike hybridization chain reaction (HCR). It combines reverse transcription-rolling circle amplification (RT-RCA) with well-designed netlike HCR to achieve dual selection and dual signal amplification, which can eliminate the interference of linear isomers. This two-dimensional netlike HCR is composed of an ingeniously designed trigger chain and two hairpin fuel probes, which can generate a stable network structure with RT-RCA products containing multiple sets of repeats at a constant temperature, thereby producing enhanced fluorescent signals. Systematic studies reveal that the optimized netlike HCR system has higher detection efficiency for DNA strands containing multiple sets of repetitive sequences, can detect circRNA as low as 0.1 pM, and has excellent selectivity. By using human tumor cell lines and tissues, it has been verified that the netlike HCR-based method can accurately detect specific circRNA in real biological samples without RNase R enrichment, which provides a simple and useful platform for detecting low-abundance circRNA. Furthermore, the proposed strategy is also a potential method for detecting some genes containing repetitive sequences, such as telomere DNA, centromere DNA and ribosomal DNA (rDNA).

Study on the Functionalization and Signaling Efficiency of the Hybridization Chain Reaction Using Traditional and Single Molecular Characterizations.

As an important enzyme-free amplifier, the hybridization chain reaction (HCR) uses an ssDNA to trigger cycled displacement interactions between substrate hairpins and finally form elongated dsDNA concatamer mixtures. In many cases, to provide a signal probe or advanced function, additional oligonucleotides (named hairpin tails) have to be extended upon classic HCR hairpin substrates, but by doing so the HCR assembly efficiency and signal-to-noise ratio (SNR) may get seriously reduced. In this Article, a rational and general model that may guide the study on HCR functionalization and signaling efficiency is provided. We rationally design a four-hairpin model HCR system (4H-HCR) in which one or more hairpin substrates are appended with additional tails as a signaling probe. After HCR assembly, two adjacent tails are supposedly integrating into a full G-quadruplex structure to provide the evidence or signal for the assembly. A systematic study has been applied to reveal the relationship between the "tail-design" with assembly efficiency and SNR. A clear design rule-set guiding the optimized assembly and signal has been provided for traditional electrophoresis and G-quadruplex-enhanced fluorescence signal. Importantly, solid-state nanopore single molecular detection has been innovatively introduced and recommended as an "antirisk" and "mutual benefit" readout to traditional G-quadruplex signaling. Nanopore detection can provide a clear signal distinguished before and after the HCR reaction, especially when the traditional G-quadruplex-enhanced signal only provides low SNR. The G-quadruplex, in turn, may enhance the nanopore signal amplitude via increasing the diameter of the HCR products.

Construction of an Enzyme-Free Initiator-Replicated Hybridization Chain Reaction Circuit for Amplified Methyltransferase Evaluation and Inhibitor Assay.

The enzyme-free nucleic acid amplification circuit, for example, hybridization chain reaction (HCR), has paved a broad avenue for evaluating various enzyme-involved biotransformations, including DNA methyltransferases (MTases). The nonenzymatic MTase-sensing platform has supplemented a versatile toolbox for monitoring aberrant methylation in intricate biological samples, yet their amplification efficiency is always constrained by the initiator-depletion paradigm. Herein, the autonomously initiator-replicated HCR (IR-HCR) was developed as a versatile amplification system for detecting MTase with ∼100-fold sensitivity of the conventional HCR system. The initiator I-triggered HCR leads the assembly of a tandem DNAzyme concatemer that cleaves its substrate. This leads to the cyclic replication of a new initiator I for reversely motivating the initial HCR circuit, resulting in a dramatic Förster resonance energy transfer (FRET) readout. Without M.SssI MTase, hairpin HM can be recognized and digested by restriction endonuclease HpaII to release initiator I for stimulating a high FRET signal. While the M.SssI-methylated HM prohibits the HpaII-mediated cleavage of HM, the caged initiator I fails to trigger the IR-HCR circuit. Based on a systematic investigation, the IR-HCR circuit readily achieves selective and sensitive analysis of M.SssI MTase and its inhibitors. As a general MTase-sensing platform, the IR-HCR principle was further applied to analyze another MTase (Dam) by redesigning HM with the Dam recognition sequence. Overall, the versatile homogeneous MTase sensing platform was achieved via an efficient and robust initiator replication amplification circuit and may have enormous potential for early disease diagnosis.

A minimal hybridization chain reaction (HCR) system using peptide nucleic acids.

The HCR represents a powerful tool for amplification in DNA-based circuitry and sensing applications, yet requires the use of long DNA sequences to grant hairpin metastability. Here we describe a minimal HCR system based on peptide nucleic acids (PNAs). A system comprising a 5-mer stem and 5-mer loop/toehold hairpins was found to be suitable to achieve rapid amplification. These hairpins were shown to yield >10-fold amplification in 2 h and be suitable for the detection of a cancer biomarker on live cells. The use of γ-peg-modified PNA was found to be beneficial.

An amplification strategy for detecting HER2 with a quasi-targeted proteomics approach coupled with aptamer-triggered hybridization chain reaction

Human epidermal growth factor receptor 2 (HER2)-positive is a particularly aggressive type of the breast cancer. Because of the evidence has revealed that accurate HER2 status detection is crucial for prognosis and treatment strategy selection, great effort has been taken to develop assays for sensitive and accurate quantification of HER2. However, nonspecific amplification effect of most current assays limits the quantification accuracy of low abundance HER2. In the present work, we developed an LC-MS/MS-based quasi-targeted proteomics strategy coupled with hybridization chain reaction (HCR) for amplification of the HER2 protein signal. In the described strategy, the aptamer triggered the HCR system to undergo a cascade of hybridization events, with the two locked hairpins conjugated to the substrate peptide to form aptamer–HCR peptide probes. The membrane protein HER2 was recognized by probe and the signal was to be converted and then amplified into the mass response of the reporter peptide, which could be quantified using LC-MS/MS. The signal intensity was approximately five fold greater than that without signal amplification. Finally, the developed assay was applied for the quantitative analysis of HER2 in breast cell lines and monitor the dynamic change of HER2 in drug induced HER2 negative cells. The result demonstrated that combination of HCR signal amplification and mass spectrometry provides a novel approach for simple, accurate, and quantitative monitoring of low abundance protein.

Branched hybridization chain reaction\u2014using highly dimensional DNA nanostructures for label-free, reagent-less, multiplexed molecular diagnostics

The specific and multiplexed detection of DNA underpins many analytical methods, including the detection of microorganisms that are important in the medical, veterinary, and environmental sciences. To achieve such measurements generally requires enzyme-mediated amplification of the low concentrations of the target nucleic acid sequences present, together with the precise control of temperature, as well as the use of enzyme-compatible reagents. This inevitably leads to compromises between analytical performance and the complexity of the assay. The hybridization chain reaction (HCR) provides an attractive alternative, as a route to enzyme-free DNA amplification. To date, the linear nucleic acid products, produced during amplification, have not enabled the development of efficient multiplexing strategies, nor the use of label-free analysis. Here, we show that by designing new DNA nanoconstructs, we are able, for the first time, to increase the molecular dimensionality of HCR products, creating highly branched amplification products, which can be readily detected on label-free sensors. To show that this new, branching HCR system offers a route for enzyme-free, label-free DNA detection, we demonstrate the multiplexed detection of a target sequence (as the initiator) in whole blood. In the future, this technology will enable rapid point-of-care multiplexed clinical analysis or in-the-field environmental monitoring.

Amplified Split Aptamer Sensor Delivered Using Block Copolymer Nanoparticles for Small Molecule Imaging in Living Cells.

We develop a novel amplified split aptamer sensor for highly sensitive detection and imaging of small molecules in living cells by using cationic block copolymer nanoparticles (BCNs) with entrapped fluorescent conjugated polymer as a delivery agent. The design of a split aptamer as the initiator of hybridization chain reaction (HCR) affords the possibility of enhancing the signal-to-background ratio and thus allows high-contrast imaging for small molecules with relatively weak interactions with their aptamers. The novel design of using fluorescent cationic BCNs as the nanocarrier enables efficient and self-tracking transfection of DNA probes. Results reveal that BCNs exhibit high fluorescence brightness allowing direct tracking of the delivery location. The developed amplified split aptamer sensor is shown to have high sensitivity and selectivity for in vitro quantitative detection of adenosine triphosphate (ATP) with a detection limit of 30 nM. Live cell studies show that the sensor provides a "signal on" approach for specific, high-contrast imaging of ATP. The DNA sensor based HCR system may provide a new generally applicable platform for detection and imaging of low-abundance biomarkers.

Toehold integrated molecular beacon system for a versatile non-enzymatic application.

A molecular beacon (MB) is an oligonucleotide hybridization probe with a hairpin-shaped structure that leads to specific and instantaneous nucleic acid hybridization, enabling a variety of applications. However, integration of additional module sequences interferes with the performance of MBs and increases the complexity of sequence design. Herein, we develop and characterize a toehold integrated molecular beacon (ToMB) strategy for nucleic acid hybridization, where the reaction rate can be flexibly regulated by a target-induced MB conformational switch. Using this basic mechanism, the ToMB is capable of identifying nucleic acids with high specificity and a wider linearity range compared with the conventional molecular beacon system. We further applied the ToMB to the construction of a hybridization chain reaction system and a basic OR logic gate VJHto explore its programmability and versatility. Our results strongly suggest that the novel ToMB can act as a powerful nano-module to construct universal and multifunctional biosensors or molecular computations. Graphical abstract Molecular beacon is employed as a flexible and switchable spacer to control the toehold-mediated strand displacement reaction.