Hybridization Chain Reaction Amplification Strategy Research Articles

Hepar-on-a-sensor-platform with hybridization chain reaction amplification strategy to intuitively monitor the hepatoxicity of natural compounds.

The irrational use of natural compounds in the treatment of diseases can lead to serious side effects, especially hepatoxicity, and its toxic effects are usually cumulative and imperceptible. Therefore, an accurate sensing platform is urgently needed to monitor the hepatotoxicity of natural compounds. Here, we deposited a thermo-responsive alginate-RGD/Pluronic hydrogel to construct an in vitro three-dimensional(3D) hepar-platform, and a thorough validation was adopted to evaluate the bioprinted hepatic constructs. The engineered hepar-platform was then employed to access its biological response toward Emodin (EM) and Triptolide (TP), two typical hepatotoxic natural compounds. Subsequently, we integrated it with a robust fluorescent sensor based on hybridization chain reaction amplification strategy (HCR) to monitor the early hepatotoxic biomarker - glutathione-S-transferase-alpha (GST-α) secreted by this 3D constructs. Our study was the first attempt to construct an accurate hepar-on-a-sensor platform that could effectively detect GST-α for monitoring the hepatoxic effects of natural compounds. The limit of detection of the platform was 0.3 ng ml-1 and the accuracy of this platform was verified by enzyme linked immunosorbent assay. Furthermore, the variation of GST-α induced by EM and TP was consistent with hepatotoxicity studies, thus providing an important application value for evaluating the hepatotoxicity of natural compounds. STATEMENT OF SIGNIFICANCE: 1. We deposited a thermo-responsive alginate-RGD/Pluronic hydrogel to construct an in vitro three-dimensional(3D) hepar-platform, and elucidated the essential reasons why hybrid bioinks more suitable for 3D extrusion from biomaterials itself. Also, a thorough validation associated with a series of important proteins and genes involved in liver cell metabolism was adopted to evaluate the bioprinted hepatic constructs accurately 2. Glutathione-S-transferase-alpha is a soluble trace biomarker for acute hepatotoxic injury, the hepatotoxic effects of natural compounds on the secretion of GST-α has not been reported to date. We integrated our 3D hepar-platform with recognition molecules-aptamers and HCR amplification strategy to monitor the variation of GST-α, aiming at developing a robust and stable fluorescent biosensing platform to monitor the hepatoxicity of natural compounds.

A new ratiometric electrochemical aptasensor for the sensitive detection of carcinoembryonic antigen based on exonuclease I-assisted target recycling and hybridization chain reaction amplification strategy

In this work, a novel ratiometric electrochemical aptasensor was designed on the basis of dual-amplification mechanism by using exonuclease I (Exo I)-assisted target recycling and hybridization chain reaction (HCR). The thiol-modified capture DNA (Cp-DNA) was fixed on the surface of the gold electrode (AuE) modified with gold nanoparticles (AuNPs) through Au-S bonds. The ferrocene (Fc)-labeled DNA (Fc-DNA) hybridized with Cp-DNA to form Fc-DNA/Cp-DNA duplex. The presence of carcinoembryonic antigen (CEA) led to the dissociation of Fc-DNA from Fc-DNA/Cp-DNA duplex and initiated Exo Ⅰ-assisted target recycling to digest Fc-DNA, so numerous Fc left away from the electrode surface and resulting in the “signal off” of Fc. Moreover, Cp-DNA was exposed to bind trigger DNA and initiate HCR with the presence of H1 and H2 probes, resulting in the intercalating of numerous methylene blue (MB) molecules and the “signal-on” of MB. This dual signal strategy significantly amplified the decrease of Fc signal and the increase of MB signal, achieving a linear range of 1 pg mL−1 to 100 ng mL−1 with a detection limit of 0.479 pg mL−1 (S/N = 3). Besides, the developed aptasensor exhibited high specificity, good stability and reproducibility. More importantly, the aptasensor had been applied in detection of CEA in diluted human serum and displayed satisfactory anti-interference property. By changing the corresponding aptamer sequences, the designed strategy can be employed to detect other targets, which holds promising application in clinical diagnosis.

A magnetic separation-assisted cascade hybridization chain reaction amplification strategy for sensitive detection of flap endonuclease 1

Sensitive and accurate detection of flap endonuclease 1 (FEN1) is essential to understand its roles in DNA replication and repair and explore its functions in tumor diagnosis and prognosis. Here, we designed a magnetic separation-assisted cascade hybridization chain reaction (HCR) amplification strategy for enzyme-free and sensitive detection of FEN1. Firstly, the forked dsDNAs contained the recognition site (5′ overhanging DNA flap) of FEN1 were modified on magbeads surface to form magnetic recognition probes. By the recognition and catalytic cleavage of FEN1, the 5′ flaps were removed into solution and then purified by magnetic separation. Then, the obtained flaps served as triggers to activate the linear-assembly of hairpin probes H1 and H2 (primary HCR), forming nicked double helices with split fragments. Finally, the fragments acted as new triggers to activate the branched-assembly of hairpin probes H3 and H4 (secondary HCR), causing fluorophore/quencher labeled on H3 far away and fluorescence recovery. Benefiting from the purification of triggers by magnetic separation and the signal amplification effect of cascade HCR, this strategy achieved a low detection limit of 3.6 × 10−5 U μL−1 for FEN1, and enabled the assay of it in actual samples. The results demonstrate that our strategy will provide a potent approach for sensitive detection of FEN1 in early tumor diagnosis and prognosis.

Lateral flow biosensor for universal detection of various targets based on hybridization chain reaction amplification strategy with pregnancy test strip

Abstract Pregnancy test strip is currently one of the most mature and widely used biomedical test products. In this study, based on its simplicity and rapidity, the detecting target-human chorionic gonadotropin (hCG) is coupled with different hybridization probes, while the aptamer is linked with the initiator used for hybridization chain reaction (HCR), which can construct a signal amplification platform based on HCR. The method converts the detection of other biochemical molecules into the detection of hCG signal, and combines the magnetic separation technology to realize the detection of various targets with pregnancy test strip. The rapid quantitative detection can be achieved with smartphone App and portable detection device. Under the optimal experiment conditions, the method can realize rapid and sensitive detection of adenosine triphosphate, thrombin, carcinoembryonic antigen and DNA fragment in human serum, and the visual detection limits can reach 100 pM, 0.2 pM, 5 ng/mL and 5 pM, respectively. The design has low cost, simple operation, and good sensitivity and selectivity for target detection. Based on this design, the detection of multiple analytes can be implemented with different aptamer sequences, thereby broadening the application of commercial pregnancy test strip in the biomedical testing field.

Electrochemical detection of microRNA-21 based on a Au nanoparticle functionalized g-C3N4 nanosheet nanohybrid as a sensing platform and a hybridization chain reaction amplification strategy.

Here, a sensitive sandwich-type electrochemical biosensor for microRNA-21 detection was reported. It was based on the use of a Au NP functionalized graphite-like carbon nitride nanosheet (g-C3N4 NS) nanohybrid (Au NPs-g-C3N4 NS) as a sensing platform and DNA concatemers containing methylene blue (MB) as a signal probe. The signal probe was prepared by using two different single strand DNAs with a complementary sequence (one of them labeled with MB at the 3' end) to form long concatemers via continuous hybridization chain reaction (HCR); thus numerous MB signal molecules were loaded on long concatemers. The biosensor was fabricated following the next step: a thiolated hairpin probe (HP) was first immobilized on the surface of the glassy carbon electrode (GCE) modified with a Au NPs-g-C3N4 NS nanohybrid. After it was blocked with MCH, the modified electrode was sequentially hybridized with microRNA-21 and a signal probe, respectively. As a result, a sandwich structure of HP-microRNA-signal probe covered the surface of the modified electrode. Differential pulse voltammetry (DPV) was employed to measure the sensing signal in phosphate buffered solution (0.10 M PBS, pH 7.4). The experimental conditions were optimized such as the hybridization time and the amount of g-C3N4 NS. The proposed biosensor exhibited a wide linear response range (1.0 fM to 500 nM) and a low limit of detection (0.33 fM; at S/N = 3) under the optimal conditions. Meanwhile, the biosensor could discriminate single base mismatched microRNA-21, indicating that the biosensor possessed high selectivity.

Dandelion-like CuO microspheres decorated with Au nanoparticle modified biosensor for Hg2+ detection using a T-Hg2+-T triggered hybridization chain reaction amplification strategy

We fabricate a novel electrochemical biosensor based on the specific thymine-Hg2+-thymine (T-Hg2+-T) base pair for the highly sensitive detection of mercury ions (Hg2+) and utilize toluidine blue (TB) as a redox indicator that is combined with a hybridization chain reaction (HCR) for signal amplification. The dandelion-like CuO (D-CuO) microspheres that were assembled using Au nanoparticles were first introduced as support materials, which produced more active sites for the thiolated probe (P1) combination. Then, the presence of Hg2+ induced P1 to hybridize with the other oligonucleotide (P2) through Hg2+-mediated T-Hg2+-T complexes. In addition, the partial sequence of P2 acted as an initiator sequence, which led the two hairpin DNA (H1 and H2) strands to collectively form the extended double-strand DNA through the HCR process on the electrode surface. TB was employed to interact with the double strands and produce an efficient electrochemical signal. The proposed strategy combined the amplification of the HCR and the inherent redox activity of TB and utilized D-CuO/Au composites, which exhibited high sensitivity for Hg2+ determination. Under the optimum conditions, the proposed biosensor showed a prominent response for Hg2+, including a linear range from 1 pM to 100 nM and a detection limit of 0.2 pM (S/N = 3). Moreover, the new biosensor proved its potential application for trace Hg2+ determination in environmental water samples.

Bioinspired Photonic Barcodes with Graphene Oxide Encapsulation for Multiplexed MicroRNA Quantification.

Multiplexed microRNA (miRNA) quantification has a demonstrated value in clinical diagnosis. In this paper, novel mussel-inspired photonic crystal (PhC) barcodes with graphene oxide (GO) encapsulation for multiplexed miRNA detection are presented. Using the excellent adhesion capability of polydopamine, the dispersed GO particles can be immobilized on the surfaces of the PhC barcodes to form an additional functional layer. The GO-decorated PhC barcodes have constant characteristic reflection peaks because the GO immobilization process not only maintains their periodic microstructure but also enhances their stability and anti-incoherent light-scattering capability. The immobilized GO particles are shown to enable high-sensitivity miRNA screening on the surface of the PhC barcodes by integration with a hybridization chain reaction amplification strategy. Because the PhC barcodes have stable encoding reflection peaks, multiplexed low-abundance miRNA quantification can also be achieved rapidly, accurately, and reproducibly by employing different GO-decorated PhC barcodes. These features should make GO-encapsulated PhC barcodes ideal for many practical applications.

Framework Nucleic Acid-Mediated Pull-Down MicroRNA Detection with Hybridization Chain Reaction Amplification.

Gastric cancer remains a disease of high mortality worldwide due to its poor prognosis. Previous studies have shown that microRNAs (miRNAs) are effective biomarkers for early diagnosis of gastric cancer. To realize sensitive detection of related miRNAs for improved early diagnosis, classification, and survival prognosis of gastric cancer, herein we developed a framework nucleic acid (FNA)-mediated microarray for quantitative analysis of multiple miRNAs. By rationally designing FNA with different sizes, we systematically modulated the surface density and lateral interactions of DNA probes, which provides an effective means for programmable tailoring of the hybridization efficiency and kinetics of the biosensing interface. We found that the hybridization efficiency was increased along with the size of the FNA and was optimum for FNA-17. In combination with the hybridization chain reaction amplification strategy, this established FNA microarray can serve as an ultrasensitive and selective analytical platform for simultaneous multiplexed detection of miRNA (e.g., FNA-miR-652, FNA-miR-627, and FNA-miR-629) biomarkers in gastric cancer.

A hybridization chain reaction amplification strategy for fluorescence imaging of human telomerase activity in living cells

A hybridized chain reaction (HCR)-based biosensing method has been developed for the imaging detection of intracellular telomerase activity. The telomerase-targeting responder-transmitter DNA complex (HPT) consisting of telomerase primer sequence (HP) and a HCR initiator (trigger) is transfected into cell plasma. In the presence of telomerase, HPT can be recognized and extended, producing plenty of triggers which initiate HCR amplification reaction. Finally, a long nicked dsDNA with a lot of outstretched single chains was formed by hybridizing with Q of the reporter complex, generating an enhanced fluorescence signal. The developed biosensing approach can be used for the detection of telomerase activity in cell lysate with the detection limit of 578 cells/100 μl. In addition, this strategy has been successfully applied not only for the sensitive and specific imaging of telomerase activity in living cells but also for comparing of telomerase activity among different cell lines. Therefore, the method might become a potential alternative tool for telomerase-related cancer diagnosis and therapy in medical research and early clinical diagnosis.

Aptamer based electrochemical assay for protein kinase activity by coupling hybridization chain reaction

The present work reported a simple, lable-free and sensitive electrochemical method for the detection of protein kinase A (PKA) activity. This method was based on the specific recognition of aptamer and the aptamer-induced hybridization chain reaction (HCR) amplification strategy. The aptasensor was constructed by immobilizing capture probe on a gold electrode via an Au–S bond. When adenosine triphosphate (ATP) aptamer was introduced, its one terminus hybridized with capture probe and the other hybridized with the complementary region of an auxiliary probe, which other region triggered HCR between two hairpin DNA (H1 and H2) to form a long DNA concatamer. At last a large number of electroactive methyle blue (MB) molecules were assembled on the dsDNA concatamer, which generated a significantly amplified electrochemical signal. In the presence of ATP, the HCR would not be performed because the aptamer specifically bond to ATP and the electrochemical response would decrease. However, when ATP and PKA coexisted, the electrochemical response would recovery because that ATP had been translated into ADP by PKA. So the activity of PKA could be effectively monitored according to the change of electrochemical signal. Based on the HCR amplification strategy, the aptasensor showed a wide linear range (4 − 4 ×105 U L−1) and a low detection limit (1.5 U L−1) for the detection of PKA. Furthermore, the method was applied to study the inhibitory effect of H-89 on PKA activity. The developed aptasensor was also used to the analysis of drug-induced PKA activity in cell lysates, indicating the potential application of the developed method in the fields of clinical diagnostics and discovery of new targeted drugs.

A DNA nanostructured aptasensor for the sensitive electrochemical detection of HepG2 cells based on multibranched hybridization chain reaction amplification strategy

Sensitive detection of cancer cells is beneficial to the early diagnosis of cancer and individual treatment. In the present study, a DNA nanostructured aptasensor was used for the sensitive electrochemical detection of human liver hepatocellular carcinoma cells (HepG2) based on multibranched hybridization chain reaction amplification strategy. We established a well-designed platform by immobilizing DNA tetrahedron, a three-dimensional DNA nanostructure, on the gold electrode to capture HepG2 cells more specifically and efficiently. Meanwhile, functional hybrid nanoprobes consisted of MIL-101@AuNPs (Au nanoparticles), numerous hemin/G-quadruplex DNAzyme from multibranched hybridization chain reaction, and natural horseradish peroxidase (HRP) was designed. The hybrid nanoprobes possessed the functions of specific discernment and enzymatic signal amplification simultaneously. With the help of nanoprobes, HepG2 cells were recognized and captured to form a DNA tetrahedron-cell-nanoprobe sandwich-like structure on the electrode surface. The lower detection limit of this established cytosensor is 5 cells per ml. Moreover, it delivered a broad detection range from 102 to 107 cells per ml. The results revealed that the as-proposed cytosensor may be utilized as a powerful tool for early diagnosis of cancer in the future.

Ultrasensitive electrochemical biosensor for silver ion based on magnetic nanoparticles labeling with hybridization chain reaction amplification strategy

Silver ion (Ag+) is a highly toxic heavy metal ion to aquatic organisms and accumulates in the human body via the food chain. Therefore, fast and accurate detection of Ag+ in water and food resources has become a critical issue within the scope of human health. Herein, we developed an ultrasensitive electrochemical biosensor for detection of Ag+ based on magnetic Fe3O4@gold core-shell nanoparticles (Fe3O4@Au NPs) labeling with hybridization chain reaction (HCR) amplification strategy. In this sensing strategy, the magnetic Fe3O4@Au NPs were selected for labeling with HCR product and enrichment on the surface of magnetic gold electrode. Thiolated-oligonucleotide (S1) was firstly immobilized on the surface of Fe3O4@Au NPs through Au–S chemical bond. In the presence of Ag+, cytosine-rich DNA oligonucleotide S2 hybridized with S1 to form an intramolecular duplex, in which Ag+ can selectively bind to cytosine–cytosine mismatches forming C–Ag+–C complex. The exposed stem of the C–Ag+–C complex opened two alternating ferrocene-labeled DNA hairpins (H1 and H2) in turn and triggered HCR to form a supersandwich DNA structure on the surface of Fe3O4@Au NPs. The HCR products modified Fe3O4@Au NPs were brought to the surface of magnetic gold electrode for direct electrochemical measurements. The proposed strategy led to a low detection limit of 0.5fM and a wide dynamic range of 1fM–100pM for target Ag+. The developed biosensor was highly selective and its practical applicability in tap water and lake water samples was also investigated with a satisfactory result.

Gold nanoparticles labeling with hybridization chain reaction amplification strategy for the sensitive detection of HepG2 cells by inductively coupled plasma mass spectrometry

Sensitive detection of circulating tumor cells (CTCs) is of great significance in the early detection of cancer and cancer metastasis. This work reported an efficient, specific, and sensitive immunoassay protocol for detection of tumor cells by using inductively coupled plasma mass spectrometry (ICP-MS) with gold nanoparticles (AuNPs) labeling and hybridization chain reaction (HCR) amplification. In the established approach, antibodies against epithelial cell adhesion molecule (anti-EpCAM) conjugated magnetic beads (MBs) were used for selective capture of tumor cells from peripheral blood, aptamer was applied for the recognition of captured tumor cells, and AuNPs labeled DNA concatamer was used as the signal probe for tumor cell labeling and ICP-MS detection. Due to the dual amplification effect of AuNPs and HCR, the limit of detection of this ICP-MS based method for HepG2 cells was as low as 15 cells, and the linear range was 40–8000 cells with the relative standard deviation for seven replicate detections of 200 HepG2 cells was 8.7%. Furthermore, the applicability of the method for the analysis of peripheral blood samples was demonstrated by the spiking tests. The established method was highly specific and sensitive for the detection of HepG2 cells, and has a good application potential in clinical diagnosis.

Enzyme-free electrochemical detection of microRNA-21 using immobilized hairpin probes and a target-triggered hybridization chain reaction amplification strategy

We describe a sensitive enzyme-free bioassay for the determination of microRNA-21. It is based on a combination of target-triggered hybridization chain reaction, tagging with CdTe quantum dots (QDs), and anodic stripping voltammetry. Firstly, a thiolated capture hairpin probe SH-HP1 was immobilized on the surface of a gold electrode. HP1 unfolds in the presence of microRNA-21. If hairpin probe 2 (HP2) is present, a HP1-HP2 complex will be formed which possesses an exposed stem of HP2, and microRNA is released in parallel. The released microRNA-21 triggers a hybridization chain reaction and this leads to form an exposed DNA segment of HP2 and cycle use microRNA-21. With the aid of assistant DNA A1 and A2, the exposed DNA segment of HP2 progressed to a long double strand. The strand is rich in CdTe QDs with the help of QDs-A1. Then, the attached QDs were dissolved with HNO3 to give dissolved Cd(II) ions. Finally, the corresponding electrochemical current response of Cd(II) is monitored by anodic stripping voltammetry and used to quantify the concentration of microRNA-21. More microRNA-21 participated in this reaction increases the number of CdTe QDs, which results in increased electrochemical current. Thus, an ultrasensitive detection of microRNA-21 is accomplished by anodic stripping voltammetry. This gene assay displays a detection limit as low as 33 aM. It can discriminate between complementary DNA sequence and single-base mismatched DNA, indicating its high specificity.