Hybridization Chain Reaction Signal Amplification Research Articles

A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

A Highly Sensitive Electrochemical Aptasensor for Kanamycin: Leveraging RecJf Exonuclease-Assisted Target Recycling and Hybridization Chain Reaction Signal Amplification

Enzyme-free and label-free detection of HER2 in human serum based on Ag+-released hybridization chain reaction signal amplification

The overexpression of human epidermal growth factor receptor 2 (HER2) has been linked to aggressive breast cancer and worse clinical outcomes. However, accurately measuring HER2 remains challenging, due to its low serum content. Herein, we developed a novel method for HER2 detection in serum by the combination of aptamer with C-Ag+-C structure, Ag+-released hybridization chain reaction (Ag+-HCR) and coprecipitation of Ag+ and gold nanoparticles (AuNPs) in the solution including Eosin Y (EY) and AgI colloids. Following the aptamer bound to HER2, signal amplification occurred through the release of numerous Ag+ by Ag+-HCR. This process subsequently induced AuNPs aggregation on the surface of AgI colloids, resulting in reduced fluorescence intensity of EY in the supernatant. The reduced value of EY fluorescence intensity in the supernatant was proportional to the level of HER2. Under optimized reaction conditions, a wide detection range was obtained between 0.09 and 9000 pg/mL with a limit of detection of 11.7 fg/mL in diluted human serum, and 96 serum samples in a 96-well plate could be measured simultaneously by a microtiter plate reader within 2 h. Moreover, the quantitative results of HER2 were consistent with those obtained using the enzyme-linked immunosorbent assay kit (r = 0.9995). Therefore, leveraging the delicate design of Ag+-HCR signal amplification, this enzyme-free, label-free, and effective method has demonstrated to be capable of detecting HER2 in serum due to its high sensitivity. Remarkably, this method may be extended to detect other cancer-related proteins and genes by replacing the corresponding target sequences.

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.

Artificial nanochannels for highly selective detection of miRNA based on the HCR signal amplification

Artificial solid-state nanochannels have nanoscale spatial confinement and unique ion transport properties that have been widely used in biosensing, nanofluidic devices, and nucleic acid sequencing. However, most of the nanochannel sensors for miRNA detection are grounded on the complementary pairing of bases, lacking signal amplification, which limits its application in many fields. Herein, we proposed a signal amplification strategy for miRNA-182 detection based on introducing hybridization chain reaction (HCR) in artificial nanochannels to improve the detection sensitivity. In this method, the capture DNA (cDNA) was anchored in the nanochannel, and when the target miRNA appeared, the hairpin probes H1, H2, and initiator were introduced to trigger the HCR reaction to form long nucleic acid double strands, which significantly enhanced the negative charge and affected ion transport in nanochannel. The nanochannel sensor performed excellent selectivity to the target miRNA-182, and it can realize the detection of miRNA-182 with an ultra-low concentration of 1.65 aM, which can be further applied to the detection of miRNA-182 in actual biological samples. Moreover, the nanochannel system was proved by using the AND logic gate that cDNA, initiator, H1, H2, and miRNA-182 are indispensable for successfully triggering HCR. This work provides a simple and efficient signal amplification method for miRNA detection, which considerably expands the application of nanochannel sensors and holds great prospects in biomedical and clinical detection.

Multiplex, Quantitative, High-Resolution Imaging of Protein:Protein Complexes via Hybridization Chain Reaction.

Signal amplification based on the mechanism of hybridization chain reaction (HCR) facilitates spatial exploration of gene regulatory networks by enabling multiplex, quantitative, high-resolution imaging of RNA and protein targets. Here, we extend these capabilities to the imaging of protein:protein complexes, using proximity-dependent cooperative probes to conditionally generate a single amplified signal if and only if two target proteins are colocalized within the sample. HCR probes and amplifiers combine to provide automatic background suppression throughout the protocol, ensuring that even if reagents bind nonspecifically in the sample, they will not generate amplified background. We demonstrate protein:protein imaging with a high signal-to-background ratio in human cells, mouse proT cells, and highly autofluorescent formalin-fixed paraffin-embedded (FFPE) human breast tissue sections. Further, we demonstrate multiplex imaging of three different protein:protein complexes simultaneously and validate that HCR enables accurate and precise relative quantitation of protein:protein complexes with subcellular resolution in an anatomical context. Moreover, we establish a unified framework for simultaneous multiplex, quantitative, high-resolution imaging of RNA, protein, and protein:protein targets, with one-step, isothermal, enzyme-free HCR signal amplification performed for all target classes simultaneously.

Ratiometric electrochemical biosensor based on hybridization chain reaction signal amplification for sensitive microRNA-155 detection.

In this work, a sensitive ratiometric electrochemical biosensor for microRNA-155 (miRNA-155) detection is reported based on a hybridization chain reaction amplifying the electrochemical signal. The biosensor was fabricated using Au NPs as a modified material to assemble capture DNA labeled with ferrocene (Fc) molecules, and a DNA probe labeled with methylene blue (MB) was employed for the signal probe. In the presence of target miRNA-155, it can be dual hybridized with capture and signal probe, especially with signal probe to continuously produce long concatemers containing lots of MB molecules. The electrochemical signal of Fc was used for the internal signal, and the signal from MB was used as an indicator signal. As the concentration of miRNA-155 was altered, the internal reference signal of Fc remained constant, and only the indicator signal changed in a sensitive way. The change in the ratio (IMB/IFc) between the indicator signal of MB and internal reference signal of Fc can be used to monitor the concentration of miRNA-155. Under optimal conditions, the prepared ratiometric biosensor could detect miRNA-155 within a wide linear range from 100 fM to 100 nM with low detection limit of 33 fM (at S/N = 3). Moreover, the biosensor was evaluated with human serum samples, and satisfactory recoveries were obtained, indicating that the ratiometric biosensor can be applied to clinical sample analysis.

Electrochemical Detection of Tumor Cell-Derived Exosomes Based on Cyclic Enzyme Scission and Hybridization Chain Reaction Dual-Signal Amplification

Tumor cell-derived exosomes are considered a potential source of cancer biomarkers. Here, we developed an electrochemical sensing platform for the rapid and simple detection of exosomes, using the CCRF-CEM exosome as a model. The platform utilizes cyclic nicking enzyme cleavage and a hybridization chain reaction (HCR) for dual-signal amplification. A hairpin aptamer probe (HAP) containing an aptamer was designed for the assay. The specific binding between the aptamer and PTK7, present on the exosome surface, causes a conformational change in the HAP. This facilitates hybridization between the HAP and the linker DNA, which subsequently triggers cyclic cleavage of the nicking endonuclease towards the linker DNA. Therefore, exosome detection is transformed into DNA detection. By combining this approach with HCR signal amplification, we achieved high-sensitivity electrochemical detection of CCRF-CEM exosomes, down to 1.1 × 104 particles/mL. Importantly, this assay effectively detected tumor exosomes in complex biological fluids, demonstrating the potential for clinical diagnosis.

Target-triggered enzyme-free amplification for highly efficient AND-gated bioimaging in living cells.

Rapid, simultaneous, and sensitive detection of biomolecules has important application prospects in disease diagnosis and biomedical research. However, because the content of intracellular endogenous target biomolecules is usually very low, traditional detection methods can't be used for effective detection and imaging, and to enhance the detection sensitivity, signal amplification strategies are frequently required. The hybridization chain reaction (HCR) has been used to detect many disease biomarkers because of its simple operation, good reproducibility, and no enzyme involvement. Although HCR signal amplification methods have been employed to detect and image intracellular biomolecules, there are still false positive signals. Therefore, a target-triggered enzyme-free amplification system (GHCR system) was developed, as a fluorescent AND-gated sensing platform for intracellular target probing. The false positive signals can be well avoided and the accuracy of detection and imaging can be improved by using the design of the AND gate. Two cancer markers, GSH and miR-1246, were used as two orthogonal inputs for the AND gated probe. The AND-gated probe only works when GSH and miR-1246 are the inputs at the same time, and FRET signals can be the output. In addition to the use of AND-gated imaging, FRET-based high-precision ratiometric fluorescence imaging was employed. FRET-based ratiometric fluorescent probes have a higher ability to resist interference from the intracellular environment, they can avoid false positive signals well, and they are expected to have good specificity. Due to the advantages of HCR, AND-gated, and FRET fluorescent probes, the GHCR system exhibited highly efficient AND-gated FRET bioimaging for intracellular endogenous miRNAs with a lower detection limit of 18 pM, which benefits the applications of ratiometric intracellular biosensing and bioimaging and offers a novel concept for advancing the diagnosis and therapeutic strategies in the field of cancer.

The helper oligonucleotides enable detection of folded single-stranded DNA by lateral flow immunoassay after HCR signal amplification

A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3′-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures.The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure.In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.

A novel electrochemical immunosensor for dibutyl phthalate based on Au@Pt/PEI-rGO and DNA hybridization chain reaction signal amplification strategy

Herein, a sensitive immunosensor for the rapid detection of Dibutyl phthalate (DBP) small molecules was developed. Its principle is based on Au@Pt/Polyethyleneimine (PEI)-rGO and hybridization chain reaction signal amplification system. First, Au@Pt/PEI-rGO modified electrode is used as a signal amplification platform and the antibody is fixed. In the presence of target DBP, it competes with DBP-bovine serum albumin-S0 for binding antibody. Finally, the DNA concatemer carrying a large number of signal molecules methylene blue combines with its S0 to produce a reduced electrochemical signal. Under the most reasonable conditions, this immunosensor showed a good linear range from1 pg mL−1 to 0.1 μg mL−1with a low detection limit of 0.276 pg mL−1. It has been verified that this immunosensor has good selectivity, reproducibility and stability. The designed biosensing solution will open up a new path for DBP monitoring and other small molecule targets in the food field.

Ultrasensitive detection of tumor-derived small extracellular vesicles based on nonlinear hybridization chain reaction fluorescence signal amplification and immunomagnetic separation.

Small extracellular vesicles (sEVs) have attracted wide attention as a promising tumor biomarker. However, sensitive and selective detection of sEVs is challenging due to the low levels of sEVs in the early stage of cancers. Herein, a novel fluorescent sensor was developed for the detection of sEVs with high sensitivity and selectivity based on nonlinear hybridization chain reaction (nHCR) signal amplification and immunomagnetic separation. Firstly, sEVs were captured and enriched by CD63 antibody conjugated magnetic beads via antibody-antigen reactions. Then, cholesterol-modified DNA probes were anchored spontaneously on lipid membranes of sEVs through efficient hydrophobic interactions between the cholesterol moiety and the phospholipid bilayer of sEVs. The simultaneous recognition of the transmembrane protein and the phospholipid bilayer structure of the sEVs could effectively eliminate interferences from free proteins. The sticky ends of the cholesterol-modified DNA probes acted as the initiator to trigger nHCR to form a hyperbranched network of DNA structure that could recruit more fluorescent signal molecules for signal amplification. Under the optimal conditions, the nHCR-based strategy showed high sensitivity for the detection of sEVs with a limit of detection of 80 particles per μL. In addition, the as-constructed method was successfully applied for the analysis of clinical samples. It provides a sensitive and selective platform for the isolation and detection of sEVs in the early diagnosis of cancers.

Hybridization chain reaction enables a unified approach to multiplexed, quantitative, high-resolution immunohistochemistry and in situ hybridization.

ABSTRACTRNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.

Dual CHA-mediated high-efficient formation of a tripedal DNA walker for constructing a novel proteinase-free dual-mode biosensing strategy

DNA walkers have been recognized as a type of powerful signal amplification tool for biosensors, but how to adopt a proper strategy to increase their amplification efficiency is still highly desirable. Herein we design a dual-catalytic hairpin assembly (CHA)-mediated strategy for the high-efficient formation of a tripedal Mg2+-dependent DNAzyme (MNAzyme)-DNA walker, and thus develop a novel proteinase-free dual-mode biosensing method for the kanamycin (Kana) antibiotic assay. The first CHA is initiated by a target-biorecognition reaction, which can produce the DNA walker and also induce the target recycling. The second CHA is initiated by a special base sequence designed as a one-half substrate of the MNAzyme. Upon the first CHA-triggered DNA walking at a magnetic bead (MB) track, this “pseudo-target” sequence can be released to induce another CHA-cycle for the formation of the same DNA walker. Meanwhile, the other one-half substrate strand exposed on the MB surface will trigger the quantitative hybridization chain reaction (HCR)-assembly of a G-quadruplex DNAzyme (G-DNAzyme)-enriched double-stranded DNA polymer. So the enzymatic reaction of G-DNAzymes enabled the convenient colorimetric and photoelectrochemical dual-mode signal transduction of the method. Due to the dual-CHA facilitation to the tripedal and three-dimensional DNA walking and synergetic signal amplification of HCR, this method exhibits very low detection limits of 9.4 and 0.55 fg mL−1, respectively. In combination with its wide linear range, automated manipulation, and excellent selectivity, repeatability and reliability, the proposed method is expected to be used for the convenient semiquantitative screening and accurate determination of possible antibiotic residues in complicated matrices.

Ultrasensitive zero-background photoelectrochemical biosensor for analysis of organophosphorus pesticide based on in situ formation of DNA-templated Ag2S photoactive materials.

Herein, a novel signal-on photoelectrochemical (PEC) biosensor with nearly zero background noise (ZBN) was first fabricated to determine the presence of organophosphorus pesticide based on in situ formation of DNA-templated Ag2S photoactive materials, accompanied by hybridization chain reaction (HCR) signal amplification. The capture probe (S1) on the gold nanoparticle-modified electrode can hybridize with the aptamer molecule to generate a simple PEC biosensor. In the presence of a target molecule, the aptamer molecule is released on the double-stranded DNA (dsDNA)-modified PEC biosensor. Meanwhile, the capture probe remains on the electrode and can open the DNA hairpins (H1, H2) which are rich in cytosine, to trigger the HCR reaction. The rich "C" strands are uncovered after formation of a long dsDNA polymer strand, which can assemble multiple silver ions (Ag+) by means of by C-Ag+-C chelation. Then, a large number of Ag2S can be generated by challenging with S2- solution, producing a satisfactory photocurrent signal. The photoactive material is formed in situ, which eliminates the laborious operation. Moreover, the signal can be highly amplified with nearly zero background noise and HCR signal amplification. Under optimal conditions, the ZBN aptasensor exhibited high sensitivity and selectivity, with a low detection limit of 2pgmL-1 for malathion. Importantly, the sensing platform can also be applied to determine the presence of malathion in real samples. In this assay, a novel signal-on photoelectrochemical biosensor with nearly zero background noise was first fabricated to determine the presence of organophosphorus pesticide based on in situ formation of DNA-templated Ag2S photoactive materials, accompanied by hybridization chain reaction signal amplification.

Graphene Oxide Biosensors Based on Hybridization Chain Reaction Signal Amplification for Detecting Biomarkers of Radiation-Resistant Nasopharyngeal Carcinoma and Imaging in Living Cells.

Since microRNA-205 (miRNA-205) is a predictive biomarker for antiradiation of nasopharyngeal carcinoma (NPC), quantitative detection of miRNA-205 is important for developing personalized strategies for the treatment of NPC. In this investigation, based on the graphene oxide (GO) sensor and hybridization chain reaction (HCR) for fluorescence signal amplification, a highly sensitive and selective detection method for miRNA-205 was designed. A target-recycling mechanism is employed, where a single miRNA-205 target triggers the signal amplification of many DNA signal probes. The biosensor shows the ability to analyze miRNA-205 in solution, and it can detect miRNA-205 at concentrations as low as 311.96 pM. Furthermore, the method is specific in that it distinguishes between a target miRNA and a sequence with single-, double-, and three-base mismatches, as well as other miRNAs. Considering its simplicity and superior sensitivity, it was also verified in 1‰ serum with a detection limit of 111.65 pM. Importantly, the method successfully demonstrated that miRNA-205 could be imaged in living cells, which provided the possibility of localizing target molecules in live cell imaging applications. This method has great clinical application potential in the determination of miRNA-205, a biomarker for radiation-resistant NPC.

Three kinds of porphyrin dots as near-infrared electrochemiluminescence luminophores: Facile synthesis and biosensing

Three kinds of porphyrin dots, including TCPP dots, ZnTCPP dots and TCPP-based metal organic framework (pMOF) dots, have been subtly synthesized for the first time by a universal straightforward exfoliation of bulk porphyrin aggregates or pMOF. To explore their ECL application, a novel label free “signal-on” ECL biosensor for ultrasensitive detection of Pb 2+ was developed based on TCPP dots as DNA intercalators and DNAzyme-based hybridization chain reaction signal amplification. • Three kinds of porphyrin dots are subtly prepared by a simple and universal exfoliation. • The novel porphyrin dots show excellent annihilation ECL activity. • They all exhibit stable and robust near-infrared co-reactant ECL performance. • A label free ECL biosensor is established for trace Pb 2+ detection based on TCPP dots. • The porphyrin dots as ECL emitters give a new proof of concept. Exploring novel near-infrared (NIR) luminophore with highly efficient and benign is strongly anticipated for electrochemiluminescence (ECL) evolution. Here, three kinds of porphyrin dots including 5,10,15,20-tetrakis (4-carboxyphenyl) porphyrin (TCPP) dots, ZnTCPP dots and TCPP-based metal organic framework (pMOF) dots have been subtly synthesized for the first time by a universal straightforward exfoliation of bulk porphyrin aggregates or pMOF. These as-designed porphyrin dots perform excellent annihilation ECL activity, also exhibit stable and robust cathodic or anodic NIR ECL properties with different coreactants (potassium peroxydisulfate, hydrogen peroxide or tri-n-propylamine) in aqueous phase. Especially, the ECL efficiency of three kinds of porphyrin dots in S 2 O 8 2− cathodic system are 56, 44, 39-fold higher than that of the Ru(bpy) 3 2+ standard, respectively. As a new type of ECL luminophores, TCPP dots coupled with a lead ion-dependent DNAzyme and hybridization chain reaction cycle amplification are used to fabricate a label free “signal-on” ECL biosensor for ultrasensitive detection of Pb 2+ . A dynamic range from 10 pmol·L −1 to 1 μmol·L −1 with a low limit of detection (1.2 pmol·L −1 ) is obtained. These porphyrin dots provide evidence for the use of organics-based dots as new NIR ECL luminophores for fundamental ECL research, and highlight the significance of small-sized organic nanomaterials in ECL sensing applications.

DNAzyme-Au nanoprobe coupled with graphene-oxide-loaded hybridization chain reaction signal amplification for fluorometric determination of alkaline phosphatase.

A sensing platform is presentedfor the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu2+ ions. In the presence of ALP, the released Cu2+ can promote the Cu2+-dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10UL-1, and the limit of detection (LOD) is about 0.14UL-1. The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n = 3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.

Ultrasensitive cloth-based microfluidic chemiluminescence detection of Listeria monocytogenes hlyA gene by hemin/G-quadruplex DNAzyme and hybridization chain reaction signal amplification.

In this work, a cloth-based chemiluminescence (CL) biosensor has been firstly presented for highlysensitive determination of long PCR amplicons. Under the action of a hybridization chain reaction, a good deal of hemin/G-quadruplex DNAzyme molecules are produced, which can effectively enhance the CL signal. Moreover, effective cloth-based DNA biosensors can be fabricated by sequential wax screen-printing and surface-modification processes. Especially, the integration of a desirable hydrophobic barrier and gravity/capillary flow onto the flow channel of the cloth-based device makes the biosensor easy to be fabricated and to be associated with a flow CL. For the luminol/H2O2-based CL system, the signals are triggered by the hemin/G-quadruplex DNAzyme and are recorded by a low-cost CCD. Under optimized conditions, the determination range of target DNA is 0.002-20,000pM and its limit of detection is calculated to be 1.1fM. The results show that the proposed CL biosensor has a good analytical performance, such as high detectability and specificity, wide linear range, and receivable reproducibility and stability. Finally, the proposed biosensor is proven by the fact that this method can successfully detect the target DNA prepared from the Listeria monocytogenes-spiked milk samples. Therefore, it is believed to have the potential application prospects in food safety and environmental monitoring. Graphical abstract.

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.

Electrochemical Sensing of Exosomal MicroRNA Based on Hybridization Chain Reaction Signal Amplification with Reduced False-Positive Signals.

MicroRNAs (miRNAs) in cancer cell-derived exosomes are important cancer biomarkers. Herein, a sensitive hybridization chain reaction (HCR) electrochemical assay was fabricated for the detection of exosomal microRNA-122 (miR-122). The hairpin DNA (hpDNA) probes were first immobilized on the surface of a gold electrode. In the presence of miR-122, the hairpin structure of the hpDNA could be opened and triggered the HCR through the cross-opening and hybridization of two helper DNA hairpins. Long nicked double helixes generated from HCR are used to capture more RuHex and increase the signal of differential pulse voltammetry (DPV). In this assay, the density of the hpDNA probes on the surface of the gold electrode was precisely controlled by the simultaneous immobilization of hpDNA and short 12 nucleotides single-stranded DNA (S-12), providing a very high amplification efficiency. More importantly, the false positive signal could be reduced or completely eliminated by applying exonuclease I (Exo I) before the introduction of target miR-122. Under optimal conditions, the assay offers very high sensitivity with an attomolar level detection limit, a linear range with 9 orders of magnitude, and specificity in single mismatch discrimination. This sensitive electrochemical assay could successfully evaluate the miR-122 concentration in different cancer-derived exosomes, indicating its potential use in cancer diagnostics.