Hybridization Chain Reaction Amplification Research Articles

Highly sensitive and label-free immunoassay of chloramphenicol based on poly-adenine-mediated spherical nucleic acid and hybridization chain reaction amplification

In this paper, a label-free fluorescent immunoassay for chloramphenicol was established based on a DNA-assisted signal amplification strategy. The poly-adenine mediated spherical nucleic acid was firstly employed as the substrate of antibody to construct the immunonanoprobes. Enzyme-free signal amplification techniques, hybridization chain reaction and strand displacement reaction, were utilized to develop the dual amplification strategy. The output signal was expressed through the use of G-quadruplex to achieve label free fluorescence changes. Under optimized conditions, the detection limit of this method was reduced to 0.0044 ng/L. The method exhibited a high accuracy to the detection of chloramphenicol in milk samples with a recovery rate from 94.5% to 118.1%. However, the limitations of this method probably lied in the overlong detection time and the possible interference from other compounds present in food samples. The developed label-free fluorescent immunoassay could provide an innovative view for the reliable and ultrasensitive detection of chloramphenicol, which is conducive to the food safety.

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.

An ultrasensitive dual-mode approach for AFB1 detection: Colorimetric/label-free SERS aptasensor based on a self-assembled core-shell structured Ag@Au IP6 bifunctional nanozyme

A sensitive and accurate assay of aflatoxin B1 (AFB1) is critically important to ensure food safety. This work designed a dual-mode aptasensor for the convenient and sensitive detection of AFB1 in foodstuff. The aptasensor based on a bifunctional nanozymes was prepared by combining the hybridization chain reaction (HCR) amplification and alkaline phosphatase (ALP)-induced self-assembly reaction. The Ag@Au IP6 nanozyme demonstrated better peroxidase-like activity, effectively initiating the TMB−H2O2 color reaction and facilitating the generation of the blue oxidation product oxTMB. Furthermore, Ag@Au IP6 as a surface-enhanced Raman scattering (SERS) substrate featuring a core-shell structure can notably enhance the Raman signal of oxTMB. When AFB1 existed, the trigger probe exposed and initiated HCR, leading to the introduction of ALP, consequently mediating the generation of Ag@Au IP6 nanozymes, enhancing both SERS signal and colorimetric signal in the TMB-H2O2 system. Consequently, the dual-mode aptasensor is capable of achieving SERS/colorimetric detection with no need for the modification of signal molecules and the replacement of material or reagent. In on-site detection, the visual sensing assay could achieve with collecting and transforming the colorimetric signal to RGB value by a color-assist App in smartphones. Within optimal conditions, the detection limit of dual-mode aptasensor as low as 0.58 pg/L, and with a linear range from 2 pg/L to 200 pg/L, which can meet the needs for AFB1 precise quantification and on-site detection.

Synthesis of dual models multivalent activatable aptamers based on HCR and RCA for ultrasensitive detection of Salmonella typhimurium

Aptamers have superior structural properties and have been widely used in bacterial detection methods. However, the problem of low affinity still exists in complex sample detection. In contrast, hybridization chain reaction (HCR)-based model I and rolling circle amplification (RCA)-based model II multivalent activatable aptamers (multi-Apts) can fulfill the need for low-cost, rapid, highly sensitive and high affinity detection of S. typhimurium. In our research, two models of multi-Apts were designed. First, a monovalent activatable aptamer (mono-Apt) was constructed by fluorescence resonance energy transfer (FRET) with an S. typhimurium aptamer and its complementary chain of BHQ1. Next, the DNA scaffold was obtained by HCR and RCA, and the multi-Apts were obtained by self-assembly of the mono-Apt with a DNA scaffold. In model I, when target was presented, the complementary chain BHQ1 was released due to the binding of multi-Apts to the target and was subsequently adsorbed by UIO66. Finally, a FRET-based fluorescence detection signal was obtained. In mode II, the multi-Apts bound to the target, and the complementary chain BHQ1 was released to become the trigger chain for the next round of amplification of HCR with a fluorescence detection signal. HCR and RCA based multi-Apts were able to detect S. typhimurium as low as 2 CFU mL−1 and 1 CFU mL−1 respectively. Multi-Apts amplification strategy provides a new method for early diagnosis of pathogenic microorganisms in foods.

UIO66 low background signal and fluorescence synergism strategy for highly sensitive detection of Salmonella typhimurium

Successful construction of a detection method for Salmonella typhimurium (S. typhimurium) based on the synergy of hybridization chain reaction (HCR) and fluorescence was realized in this paper. First, the aptamer modified with the quenching group Black Hole Quencher-1 acid (BHQ1) was immobilized on the magnetic beads in combination with the complementary chain of the aptamer modified with 6-carboxyfluorescein (6-FAM). Second, S. typhimurium and cDNA-6-FAM immobilized on magnetic beads competitively bound to the aptamer. Finally, the cDNA-6-FAM was released after magnetic separation acted as a promoter to trigger HCR amplification when the target presented. The fluorescence signal could be significantly improved by the combination of green SYBR Green I (SGI) and HCR long double-stranded DNA and the fluorescent synergy of 6-FAM and SGI. Because of the separation of target and its aptamer, the trigger strand was abstracted by magnetic separation. There was no HCR to generate long double-stranded DNA, and the fluorescence of excess hairpin/SGI could be adsorbed through UIO66 so that only a very low background signal was detected. This fluorescent sensor was capable of monitoring S. typhimurium in the range of 10–3.2 × 107 CFU mL−1 with a limit of detection as low as 1.5 CFU mL−1. Because of the excellent properties of the aptasensor and the validity of SGI fluorescence synergy, this HCR enzyme-free amplification strategy could be generalized to other areas.

Boosting the sensitivity for tau protein detection with a bifunctional nanoenzyme signal amplifier

The trace amounts of tau protein in the blood are considered a significant biomarker in the early diagnosis of Alzheimer's disease. Herein, an electrochemical biosensor has been developed that utilizes a bifunctional nanoenzyme signal amplifier (Nanosa), which incorporates hybridization chain reaction (HCR) amplification and peroxide-mimicking nanoenzymes enhancement to enable ultrasensitive detection of tau. The Nanosa utilizes a peroxide-active MoS2/Au substrate on which DNA aptamer and hairpin probes are fixed as signal amplifiers capable of triggering HCR. The Fc molecules were initially aggregated at the electrode interface through HCR amplification, and were tested by adding an electrolyte solution containing hydrogen peroxide (H2O2). Under the catalysis of a peroxide nanoenzyme, the electrochemical signal of Fc molecules was further enhanced. The bifunctional Nanosa can not only achieve the pre-enrichment of Fc molecules at the electrode interface but also catalyze the enhancement of its electrochemical signal. The well-designed and optimized poly adenine tail not only as an electrode coupling agent but also as an excellent antifouling coating. The ingenious design of the Nanosa-based biosensing strategy enables the ultrasensitive analysis of tau with a broad linear range from 0.1 pg/mL to 100 ng/mL and a lower LOD of 33.4 fg/mL. Meanwhile, the outstanding analytical performance of this Nanosa-based biosensing strategy enables its successful application in determining tau levels in real plasma samples, and showed good agreement with results obtained using a commercial ELISA kit.

Exploring T7 RNA polymerase-assisted CRISPR/Cas13a amplification for the detection of BNP via electrochemiluminescence sensing platform

Brain natriuretic peptide (BNP) is considered to be an important biomarker of heart failure (HF) attracting attention. However, its low concentration and short half-life in blood lead to a low-sensitivity detection of BNP, which is a challenge that has to be overcome. In this work, we propose a highly specific, highly sensitive T7 RNA polymerase-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system to detect BNP via an electrochemiluminescence (ECL) sensing platform and incorporate exonuclease III (Exo III)-hairpin and dumbbell-shaped hybridization chain reaction (HCR) technologies. In this detection scheme, the ECL sensing platform possesses low background signal and high sensitivity. Firstly, the T7 promoter-initiated T7 RNA polymerase acts as a signal amplification technique to generate large amounts of RNAs that can activate CRISPR/Cas13a activity. Secondly, CRISPR/Cas13a is able to trans-cleave the surrounding trigger strand to produce DNA1. Thirdly, DNA1 is involved in the co-amplification reaction of Exo III and hairpin DNA, which subsequently triggers a dumbbell-shaped HCR technology. Eventually, a large number of Ru (II) molecules are inserted into the interstitial space of the dumbbell-shaped HCR to generate a strong ECL signal. The CRISPR/Cas13a possesses outstanding specificity for a single base and increased sensitivity. The tightly conformed dumbbell-shaped HCR provides higher sensitivity than the traditional linear HCR amplification technique. Ultimately, the clever combination of several amplification reactions enables the limit of detection (LOD) as low as 3.2 fg/mL. It showed promise for clinical sample testing, with recovery rates ranging from 98.4% to 103% in 5% human serum samples. This detection method offered a valuable tool for early HF detection, emphasizing the synergy of amplification strategies and specificity conferred by CRISPR/Cas13a technology.

Enzyme-free sensitive SERS biosensor for the detection of thalassemia-associated microRNA-210 using a cascade dual-signal amplification strategy

Backgroundβ-thalassemia is a blood disorder caused by autosomal mutations. Gene modulation therapy to activate the γ-globin gene to induce fetal hemoglobin (HbF) synthesis has become a new option for the treatment of β-thalassemia. MicroRNA-210 (miR-210) contributes to studying the mechanism regulating γ-globin gene expression and is a potential biomarker for rapid β-thalassemia screening. Traditional miRNA detection methods perform well but necessitate complex and time-consuming miRNA sample processing. Therefore, the development of a sensitive, accurate, and simple miRNA level monitoring method is essential. ResultsWe have developed a non-enzymatic surface-enhanced Raman scattering (SERS) biosensor utilizing a signal cascade amplification of catalytic hairpin assembly reaction (CHA) and proximity hybridization-induced hybridization chain reaction (HCR). Au@Ag NPs were used as the SERS substrate, and methylene blue (MB)- modified DNA hairpins were used as the SERS tags. The SERS assay involved two stages: implementing the CHA-HCR cascade signal amplification strategy and conducting SERS measurements on the resulting product. The HCR was started by the products of target-triggered CHA, which formed lengthy nicked double-stranded DNA (dsDNA) on the Au@Ag NPs surface to which numerous SERS tags were attached, leading to a significant increase in the SERS signal intensity. High specificity and sensitivity for miR-210 detection was achieved by monitoring MB SERS intensity changes. The suggested SERS biosensor has a low detection limit of 5.13 fM and is capable of detecting miR-210 at concentration between 10 fM and 1.0 nM. SignificanceThe biosensor can detect miR-210 levels in the erythrocytes of β-thalassemia patients, enabling rapid screening for β-thalassemia and suggesting a novel approach for investigating the regulation mechanism of miR-210 on γ-globin gene expression. In the meantime, this innovative technique has the potential to detect additional miRNAs and to become an important tool for the early diagnosis of diseases and for biomedical research.

Hybridization chain reaction assisted multicolor immunosensor for sensitively detection of human chorionic gonadotropin

The level of human chorionic gonadotropin (HCG) is an important indicator for early pregnancy, pregnancy-related diseases trophoblastic diseases and even cancer diagnosis. Therefore, sensitive detection of HCG has crucial significance in clinical, especially in gynaecology and obstetrics. Herein, a hybridization chain reaction (HCR) assisted multicolor immunosensor have been developed for HCG analysis. The proposed method introduced HCR after the immunoreaction between antibody and HCG protein, and produced long double strand DNA (dsDNA) that contain biotin sites. The streptavidin-horseradish peroxidase was linked on the dsDNA by the interaction between biotin and streptavidin, and can further mediated gold nanobipyramids (Au NBPs) etching. The localized surface plasmon resonance absorption peaks of Au NBPs blue shift and accompanied a vivid color change after etching effect. Based on this color change, HCG could be qualitative and semi-quantitative detected. Because of the introduction of HCR and enzyme amplification technique, the proposed method exhibited high sensitivity with a linear range of 0.1–2000 pg/mL and limit of detection (LOD) of 0.1 pg/mL. Finally, the proposed immunosensor was used to detect clinical serum samples. The results show there are no significant differences between clinical results and the test results by this method, indicating the practicability of the proposed method.

NAD+ mediated photoelectrochemical biosensor for histone deacetylase Sirt1 detection based on CuO-BiVO4-AgNCs heterojunction and hybridization chain reaction amplification

BackgroundHistone deacetylate Sirt1 has been involved in many important biological processes and is closely related to the occurrence and development of many diseases. Therefore, the accurate detection of Sirt1 is of great significance for the diagnosis and treatment of diseases caused by Sirt1 and the development of related drugs. ResultsIn this work, a photoelectrochemical biosensor was developed for Sirt1 detection based on the NAD + mediated Sirt1 recognition and E. Coli DNA ligase activity. CuO-BiVO4p-n heterojunction was employed as the photoactive material, rolling circle amplification (RCA), hybridization chain reaction (HCR) and AgNCs were used as triple signal amplifications. As a bifunctional cofactor, NAD+ played a crucial role for Sirt1 detection, where the peptide deacetylation catalyzed by Sirt1 consumed NAD+, and the decreased amount of NAD + inhibited the activity of E. Coli DNA ligase, leading to the failure on RCA reaction, and improving the HCR reaction. Finally, AgNCs were generated using C-rich DNA as carrier. The surface plasmon effect of AgNCs and its heterojunction with CuO and BiVO4 accelerated the transfer rate of photogenerated carriers and improved the photocurrent signal. When the detection range was 0.001–200 nM, the detection limit of the biosensor was 0.76 pM (S/N = 3). SignificanceThe applicability of the method was evaluated by studying the effects of known inhibitors nicotinamide and environmental pollutant halogenated carbazole on Sirt1 enzyme activity. The results showed that this method can be used as a new platform for screening Sirt1 enzyme inhibitors, and also provided a new biomarker for evaluating the ecotoxicological effects of environmental pollutants.

Hybrid chain reaction and selective recognition-based homogeneous dual-fluorescence analysis of circulating tumor cells in clinical ovarian cancer samples

BackgroundOncological analysis is important in tumor diagnosis. We constructed a dual-fluorescence and binary visual analysis system for circulating tumor cells (CTCs) using the folate receptor as a biomarker, combined with hybridization chain reaction and nanomaterial amplification. This strategy integrates terminal protection, selective recognition properties of N-methyl mesoporphyrin IX and CdTe quantum dots for Cu2+ and double-stranded templated copper nanoparticles, and inkjet printing technology. ResultsIn fluorescence mode, folate receptor and A2780 ovarian cancer cells were specifically detected with a limit of detection of 0.1 fg mL−1, and 10 cells mL−1 were observed. The detection limits of both the color and distance reading modes were comparable to those obtained in fluorescence mode. The applicability of the method for quantifying CTCs was validated using 27 (6 negative and 21 positive) clinical ovarian cancer samples; the results agreed with those of both the clinical folate receptor-polymerase chain reaction kit and radiological and pathological results. SignificanceThis dual-fluorescence and binary visual CTCs detection method provides multiple options for clinical tumor liquid biopsy.

Low-Background Signal-On Homogeneous Electrochemiluminescence Biosensor for Hepatitis B Virus Detection Based on the Regulation of the Length of DNA Modified on the Nanoparticles by CRISPR/Cas12a and Hybridization Chain Reaction.

In this work, combined with the high amplification efficiency of hybridization chain reaction (HCR), high specificity of the CRISPR/Cas12a system, and convenience of the homogeneous electrochemiluminescence (ECL) assay based on the regulation of negative charge on the reporting probes, a sensitive ECL biosensor for hepatitis B virus DNA (chosen as a model target) had been developed. The initiator chain trigger DNA that can induce HCR amplification is modified on the surface of ruthenium bipyridine-doped silica nanoparticles (Ru@SiO2 NPs) first, and large amounts of negative charges modified on the particles were achieved through the HCR amplification reaction. The efficiency of the nanoparticles reaching the negatively charged working electrode can be regulated and realize the change of the ECL signal. In addition, long DNA on the surface of the luminescent body may prevent the coreactant from entering the pore to react with ruthenium bipyridine. These factors combine to produce a low-background system. The presence of the target can activate the CRISPR/Cas12a system and make trigger DNA disappear from the nanoparticle surface, and strong ECL can be detected. The sensor does not require a complex electrode modification; therefore, it has better reproducibility. Additionally, due to dual signal amplification, the sensor has a high sensitivity. In the range of 10 fM to 10 nM, the ECL intensity exhibits a strong linear relationship with the logarithm of the target concentration, and the detection limit is 7.41 fM. This sensor has shown high accuracy in detecting clinical samples, which holds significant potential for application in clinical testing.

A microfluidics chemiluminescence immunosensor based on orientation of antibody for HIV-1 p24 antigen detection

The HIV-1 capsid protein, p24 antigen, is a recognizable early diagnostic biomarker for human immune deficiency virus (HIV) infection. However, the existing methods address the limitations of high cost, cumbersome operations, long analysis time, and insufficient sensitivity and selectivity. Herein, an antibody oriented microfluidic chemiluminescence biosensor based on multiple signal amplification strategy was developed for rapid and ultrasensitive detection of HIV-1 p24 antigen. The microfluidic device contained an upstream snake-shape mixing channel and a downstream ship-shape microcavity with micropillar arrays for sufficient mixing of CL reagents, efficient capture of p24 antigen, double antibody sandwich reaction, hybridization chain reaction (HCR) amplification and CL detection. The multiple signal amplification was realized by antibody orientation, multiple HCRs on antibody and initiator chain modified gold nanoparticles (Ab2-AuNPs-I), and HRP and AuNPs synergistic catalyzed CL reaction. Using the platform, the detection of target p24 is converted to the detection of nucleic acid with a limit detection of 0.212 pg mL−1 and excellent specificity within 2 h of total analysis time, which is better or comparable to previously reported biosensors. Moreover, the biosensor exhibited an excellent recovery rate ranging from 92.7% to 102.3%. Therefore, the proposed method holds great potential in clinical diagnosis.

PH-Stimulated Self-Locked DNA Nanostructure for the Effective Discrimination of Cancer Cells and Simultaneous Detection and Imaging of Endogenous Dual-MicroRNAs.

In this study, a pH-stimulated self-locked DNA nanostructure (SLDN) was developed to efficiently distinguish cancer cells from other cells for the simultaneous detection and imaging of endogenous dual-microRNAs (miRNAs). Impressively, the SLDN was specifically unlocked in the acidic environment of cancer cells to form unlocked-SLDN to disengage the i-motif sequence with a labeled fluorophore for the recovery of a fluorescence signal, resulting in the differentiation of cancer cells from normal cells. Meanwhile, unlocked-SLDN could combine and recognize the targets miRNA-21 and miRNA-155 simultaneously to trigger the hybridization chain reaction (HCR) amplification for sensitive dual-miRNA detection, with detection limits of 1.46 pM for miRNA-21 and 0.72 pM for miRNA-155. Significantly, compared with the current miRNA imaging strategy based on the traditional DNA nanostructure, the strategy proposed here remarkably eliminates the interference of normal cells to achieve high-resolution colocation imaging of miRNAs in tumor cells with an ultralow background signal. This work provided a specific differentiation method for tumor cells to materialize sensitive biomarker detection and distinguishable high-definition live-cell imaging for precise cancer diagnosis and multifactor research of tumor progression.

Enhanced imaging of protein-specific palmitoylation with HCR-based cis-membrane multi-FRET

Palmitoylation plays an important role in modulating protein trafficking, stability, and activity. The major predicament in protein palmitoylation study is the lack of specific and sensitive tools to visualize protein-specific palmitoylation. Although FRET approach was explored by metabolically labeled palmitic acid and antibody recognized target protein. The trans-membrane strategy suffers from low FRET efficiency due to the donor and acceptor located at different sides of membrane. Herein, we proposed a cis-membrane multi–fluorescence resonance energy transfer (multi–FRET) for amplified visualization of specific palmitoylated proteins through metabolic labeling and targeted recognition. The azido-palmitic acid (azido-PA) was metabolically incorporated into cellular palmitoylated proteins, followed by reacting with dibenzylcylooctyne-modified Cy5 (DBCO-Cy5) through copper-free click chemistry. The protein probe was attached to targeted protein by specific peptide recognition, which initiates a hybridization chain reaction (HCR) amplification process. The cis-membrane labeling method enables effective intramolecular donor-acceptor distance and allow to increase FRET efficiency. Simultaneously, HCR amplification triggered multi–FRET phenomenon with significantly improved FRET efficiency. With the superiority, this strategy has achieved the enhanced FRET imaging of palmitoylated PD-L1 and visualizing the palmitoylation changes of on PD-L1 under drug treatment. Furthermore, the established method successfully amplified visualization of PD-L1 palmitoylation in vivo and mice tumor slice. We envision the approach would provide a useful platform to investigate the effects of palmitoylation on the protein structure and function.

An electrochemical aptasensor for exosomes based on strand displacement amplification and hybridization chain reaction amplification

Exosomes are lipid bilayer vesicles, relating to the happening and progress of cancer. However, exploring reliable, convenient and sensitive exosomes detection methods in early cancer diagnosis is still challenging. Here, an electrochemical aptasensor based on both signal amplifications of strand displacement amplification (SDA) and hybridization chain reaction (HCR) was developed to sensitively detect tumor exosomes. Firstly, exosomes were recognized by CD63 aptamer, and converted into DNA concentration by chain replacement reaction and magnetic separation. A produced DNA fragment (P) was obtained by SDA，then P was captured at the modified gold electrode and amplified using HCR. Abundant horseradish peroxidase (HRP) attached to DNA concatemers and catalyzed the oxidation of o-phenylenediamine (OPD). The reduction current of 2,3-diaminopenazine (DAP) was measured and the exosomes were quantized. Under the optimal conditions, the DPV response showed a great linear relationship with exosomes concentration range of 44 ∼ 8.8 × 106 particle/μL. The limit of detection was 18 particle/μL. Experimental results show that this is an alternative method to detect exosomes and other tumor markers.

Hybridization chain reaction cascaded amplification platform for sensitive detection of pathogen

Rapid, sensitive, and accurate identification of pathogens is vital for preventing and controlling fish disease, reducing economic losses in aquaculture, and interrupting the spread of food-borne diseases in human populations. Herein, we proposed a hybridization chain reaction (HCR) cascaded dual-signal amplification platform for the ultrasensitive and specific detection of pathogenic microorganisms. A couple of specific primers for target bacterial 16S rRNAs were used to obtain amplified target single-stranded DNAs (AT-ssDNA). Then, AT-ssDNA initiated HCR amplification along with the opening of fluorophore (FAM) and a quencher (BHQ1) labeled hairpin reporter probe (H1), and the FAM fluorescence signal recovered. The proposed strategy could achieve a detection limit down to 0.31 CFU/mL for Staphylococcus aureus (S. aureus), 0.49 CFU/mL for Escherichia coli (E. coli) in buffer, and a linear range from 1 to 1 × 106 CFU/mL for S. aureus, 1 to 1 × 107 CFU/mL for E. coli. Furthermore, this platform enabled sensitive and precise detection of pathogenic microorganisms in complex samples such as fish blood and different organ tissues (large intestine, gallbladder, heart, liver, ren, gill, skin), which shows great potential in disease prevention and control in aquatic products.

Colorimetric aptasensor based on spherical nucleic acid-induced hybridization chain reaction for sensitive detection of exosomes.

Exosomes are one of the most promising biomarkers for tumor diagnosis and prognosis. Therefore, the development of convenient and sensitive exosome sensing strategies is of great significance. Herein, we integrated aptamer-based spherical nucleic acids (SNAs) and hybridization chain reaction (HCR) into a colorimetric aptasensor platform and applied it to the detection of exosomes. In this design, the CD63-specific aptamer pre-immobilized on the microplate was used to capture target exosomes, while the SNAs conjugated with nucleolin-specific aptamer and trigger probe H1 were designed for amplifying signal. In the presence of target exosomes, the SNAs can be attached to the microplate by the bridge effect of exosomes, resulting in the trigger of HCR. This process is accompanied by the formation of abundant G-quadruplex/hemin DNAzyme, enabling the visual quantitative analysis of exosomes. Featured with the dual amplification of SNAs and HCR, the proposed aptasensor achieved a considerable detection limit of 50 particles/μL. The practicability of this method was further verified by testing the different clinical samples. Given the ability of the aptasensor to visually detect exosomes in scenarios lacking instruments and resources, we believe that the aptasensor can be serve as a potential on-site test for liquid biopsy.

Highly sensitive plasmonic biosensor for hepatitis B virus DNA based on the surface etching of the active helical gold nanorods

As one of the most prominent global public health problems, which seriously threats public health security and human health, hepatitis B virus (HBV) infection is the main cause of liver cirrhosis and cancer. Herein, an innovative and sensitive biosensor for HBV-DNA detection was developed based on the surface etching of the helical gold nanorods (HGNRs) at single-particle level under a dark-field microscope (DFM). In this work, the unique HGNRs with high surface activity were functionalized as the optical probe, which were etched to the smooth gold nanorods and even to gold nanospheres, resulting in a distinct color and light scattering change of single particle with a more sensitive result of HBV-DNA detection than the common gold nanorods. Furthermore, the hybridization chain reaction amplification and DFM technique enabled the proposed strategy with a high sensitivity, offering the limit of detection (LOD) as low as 30.15 fM (3σ/k) and realizing sensitive detection of HBV-DNA from 0.05 to 10 pM. Moreover, the proposed strategy supplied the good specificity, which was capable of distinguishing mismatch sequence and other viral nucleic acids. In addition, this strategy was universal and provided a new idea for early diagnosis of other viral nucleic acids, such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV).

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development.

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development.