Hybridization Reaction Research Articles

DNA Polymer Nanoparticles Programmed via Supersandwich Hybridization for Imaging and Therapy of Cancer Cells.

Spherical nucleic acid (SNA) constructs are promising new single entity materials, which possess significant advantages in biological applications. Current SNA-based drug delivery system typically employed single-layered ss- or ds-DNA as the drug carriers, resulting in limited drug payload capacity and disease treatment. To advance corresponding applications, we developed a novel DNA-programmed polymeric SNA, a long concatamer DNA polymer that is uniformly distributed on gold nanoparticles (AuNPs), by self-assembling from two short alternating DNA building blocks upon initiation of immobilized capture probes on AuNPs, through a supersandwich hybridization reaction. The long DNA concatamer of polymeric SNA enables to allow high-capacity loading of bioimaging and therapeutics agents. We demonstrated that both of the fluorescence signals and therapeutic efficacy were effectively inhibited in resultant polymeric SNA. By further modifying with the nucleolin-targeting aptamer AS1411, this polymeric SNA could be specifically internalized into the tumor cells through nucleolin-mediated endocytosis and then interact with endogenous ATP to cause the release of therapeutics agents from long DNA concatamer via a structure switching, leading to the activation of the fluorescence and selective synergistic chemotherapy and photodynamic therapy. This nanostructure can afford a promising targeted drug transport platform for activatable cancer theranostics.

Graphitic C3N4 nanosheet and hemin/G-quadruplex DNAzyme-based label-free chemiluminescence aptasensing for biomarkers

Here we first reported that graphitic carbon nitride nanosheet (g-C3N4 NS) could effectively quench the chemiluminescence (CL) of luminol-hydrogen peroxide (H2O2) system. According to the new discovery, a label-free and homogeneous CL aptasensing platform was designed for sensitive detecting of biomarkers. In the absence of target, DNA probe containing hemin/G-quadruplex DNAzyme structure was adsorbed on the surface of g-C3N4 NS, causing the CL quenching of luminol through an electron transfer process. However, in the presence of the target, a DNA-DNA duplex was formed due to DNA hybridization reaction and target recognition effect, which could not be adsorbed onto the g-C3N4 NS surface because of its weak affinity. Thus, the electron transfer was blocked and the CL emission of luminol could be enhanced. The proposed CL aptasensor could detect carcinoembryonic antigen (CEA) with a detection limit of 63.0 pg/mL and it can also be used as a general detecting strategy for adenosinetriphosphate (ATP) detection. This aptasensing platform exhibited high sensitivity toward biomarkers and the probe need not be labeled, showing great promise for disease diagnosis.

Detection of BCR/ABL Fusion Gene Based on MNAzyme‐mediated Target‐cycling and ssDNA‐assisted Cascade Hybridization Reaction

AbstractA novel and simple electrochemical strategy has been developed for ultrasensitive and specific detection of BCR/ABL fusion gene based on multi component nucleic acid enzyme (MNAzyme) and ssDNA‐assisted cascade hybridization reaction. In the presence of Mg2+, a stable active MNAzyme was formed by the homogeneous recognition and specific binding with the target, initiating shear reaction of hairpin probe (HP) to produce numerous triggers. These triggers could hybridize with the immobilized capture probes and the biotinylated P1 and P2, which caused the heterogeneous ssDNA‐assisted cascade hybridization reaction on the surface of the electrode. Binding with the biotin‐rich long concatamers structure, streptavidin‐alkaline phosphatase (ST‐AP) could catalyze α‐naphthyl phosphate (α‐NPP) to produce amplified electrochemical signals. The electrochemical DNA biosensor showed high sensitivity for target gene with detection limit of 2.19×10−14 M. In addition, the established biosensor was successfully applied for detecting target BCR/ABL gene in complex samples. Thus, the electrochemical biosensing strategy presents a simple and pragmatic platform toward ultrasensitive fusion gene detection in chronic myelogenous leukemia (CML) diagnosis.

Distance-dependent quenching and enhancing of electrochemiluminescence from tris(2, 2′-bipyridine) ruthenium (II)/tripropylamine system by gold nanoparticles and its sensing applications

Understanding the role of gold nanoparticles (AuNPs) in electrochemiluminescence (ECL) processes of the Ru(bpy)32+ (bpy= 2, 2′-bipyridine)/tripropylamine (TPA) system would be beneficial to develop novel ECL sensors for a variety of applications. In this work, we found that the AuNPs on the surface of indium tin oxide (ITO) electrode could catalyze the electrochemical oxidation of TPA, greatly enhancing the ECL signal of Ru(bpy)32+/TPA, present in the solution. If physical separation of AuNPs away from electrode surface after hybridization with target ssDNA, ECL signal decreased dramatically due to the loss of electrochemical activity of AuNPs, based on which a sensitive and specific DNA sensor in a “switch-off” mode was constructed with a detection limit of 0.2 pM. In addition, a suppressing effect of the AuNPs on the ECL of Ru(bpy)32+ was experimentally confirmed by decreasing the electrocatalytic effect to overall ECL emission, including selection of oxalate as a coreactant instead of TPA, or introduction of gold electrode as substrate. Furthermore, when Ru(bpy)32+ and AuNPs were both immobilized on the ITO electrode at close proximity, the ECL quenching induced by energy/electron transfer was predominant. ECL emission of the Ru(bpy)32+/TPA system resulted from a competition between electrocatalytic enhancement and quenching effect. However, the quenched ECL signal would return in case of the AuNPs moving far away from ECL emitters after a hybridization reaction as before, and a separation distance dependent surface enhancement was observed as well. Based on the role change for AuNPs from quenching to enhancing ECL intensity of Ru(bpy)32+/TPA system, a novel ECL DNA sensing strategy in a “turn-on” mode was developed, enabling quantitative analysis of target ssDNA over the range of 0.05 pM to 0.5 nM with a detection limit of 12 fM. Overall, we demonstrated the existence of three effects of AuNPs on the ECL of Ru(bpy)32+/TPA system, and which played the leading role was dependent on the placement of AuNPs, Ru(bpy)32+, and their separation distance. The ECL sensors based on the role change for AuNPs showed both high sensitivity and excellent selectivity.

Layered Double Hydroxide Nanomaterials Encapsulating Angelica gigas Nakai Extract for Potential Anticancer Nanomedicine.

We prepared hybrids consisting of Angelica gigas Nakai (AGN) root or flower extract and layered double hydroxide (LDH) for potential anticancer nanomedicine, as decursin species (DS) in AGN are known to have anticancer activity. Dimethylsulfoxide solvent was determined hybridization reaction media, as it has affinity to both AGN and LDH moiety. In order to develop inter-particle spaces in LDH, a reversible dehydration-rehydration, so-called reconstruction route, was applied in AGN-LDH hybridization. Quantitative analyses on AGN-LDH hybrids indicated that the content of DS was two times more concentrated in the hybrids than in extract itself. Using X-ray diffraction, FT-IR spectroscopy, scanning electron microscopy, and zeta-potential measurement, we found that AGN extract moiety was incorporated into inter-particle spaces of LDH nanoparticles during the reconstruction reaction. Time-dependent DS release from hybrids at pH 7.4 (physiological condition) and pH 4.5 (lysosomal condition) exhibited a pH-dependent release of extract-incorporated LDH hybrids. An anticancer activity test using HeLa, A549, and HEK293T cells showed that the AGN-LDH hybrid, regardless of extract type, showed enhanced anticancer activity compared with extract alone at an equivalent amount of DS, suggesting a nanomedicine effect of AGN-LDH hybrids.

A gold nanoparticle-based lateral flow biosensor for sensitive visual detection of the potato late blight pathogen, Phytophthora infestans

Phytophthora infestans, the causal agent of late blight in potatoes and tomatoes, is the most important and ongoing pathogenic threat to agricultural production worldwide. Rapid and early identification of P. infestans is an essential prerequisite for countering the further spread of infection. In this study, a novel method for visual detection of P. infestans has been developed by integrating universal primer mediated asymmetric PCR with gold nanoparticle (AuNP)-based lateral flow biosensor. We employed asymmetric PCR to generate large amounts of single-stranded DNA (ssDNA) by amplifying a region of P. infestans-specific repetitive DNA sequence. The ssDNA products were then applied to the lateral flow biosensor to perform a visual detection using sandwich-type hybridization assays. In the presence of target DNA, sandwich-type hybridization reactions among the AuNP–probe, target DNA and capture probe were performed on the test line of the biosensor, and then a characteristic red band was produced for the accumulation of AuNPs. Quantitative analysis obtained by recording the optical intensity of the red band demonstrated that this biosensor could detect as little as 0.1 pg μL−1 genomic DNA. Furthermore, the specificity of the biosensor was confirmed by detecting three other Phytophthora species and two pathogenic fungi. We believe this method has potential application in early prediction of potato late blight disease and instigation of management actions to reduce the risk of epidemic development.

DNA-Based Sensor for the Detection of an Organophosphorus Pesticide: Profenofos.

In this work, we propose an electrochemical DNA aptasensor for the detection of profenofos, an organophosphorus pesticide, based on a competitive format and disposable graphite screen-printed electrodes (GSPEs). A thiol-tethered DNA capture probe, which results to be complementary to the chosen aptamer sequence, was immobilised on gold nanoparticles/polyaniline composite film-modified electrodes (AuNPs/PANI/GSPE). Different profenofos solutions containing a fixed amount of the biotinylated DNA aptamer were dropped onto the realized aptasensors. The hybridisation reaction was measured using a streptavidin-alkaline phosphatase enzyme conjugate, which catalyses the hydrolysis of 1-naphthyl -phosphate. The 1-naphtol enzymatic product was detected by means of differential pulse voltammetry (DPV). The aptasensor showed itself to work as a signal off sensor, according to the competitive format used. A dose response curve was obtained between 0.10 μM and 10 μM with a detection limit of 0.27 μM.

A novel and sensitive electrogenerated chemiluminescence biosensor for detection of p16INK4a gene based on the functional paste-like nanofibers composites-modified screen-printed carbon electrode

In this work, we offered a novel and sensitive electrogenerated chemiluminescence (ECL) biosensing system for detection of p16INK4a gene using the functional paste-like nanofibers composites-modified screen-printed carbon electrode (SPCE). The paste-like nanofibers composites (PG/GR/CS) which were comprised of the electrospun nanofibers (PG, the graphene (GR) doped polycaprolactam 6 (PA6) were prepared via one-step electrospinning), graphene (GR) and chitosan (CS) were served as the nanosized backbones for pyrrole (Py) electropolymerization. The functional paste-like nanofibers composites (PG/GR/CS/PPy) used as a substrate for dsDNA (hybridization reaction of ssDNA1, p16INK4a gene and the Ru(bpy)32+/silver nanoparticles (AgNPs) doped gold (Au) core-shell luminescent composite nanoparticles labeled ssDNA2 (RuAg@AuNPs-ssDNA2)) immobilization. Under optimal conditions, a linear relationship between ECL intensity and p16INK4a gene concentration was found in a range of 0.1 pM–1 nM with the detection limit of 0.05 pM (S/N = 3). This ECL biosensor based on the PG/GR/CS/PPy-modified SPCE demonstrated excellent electrochemical performance for the detection of p16INK4a gene and this platform can be used for the determination of various analytes.

A molecularly imprinted electrochemiluminescence sensor for ultrasensitive HIV-1 gene detection using EuS nanocrystals as luminophore

Development of simple, sensitive and specific method for human immunodeficiency virus (HIV) assays are urgently demand. In this study, we developed a novel molecularly imprinted polymer (MIP) electrochemiluminescence (MIP-ECL) sensor for the highly sensitive and selective HIV-1 gene detection using Europium sulfide nanocrystals (EsNCs) as signal producing compound. Here, the HIV aptamer as the template and o-phenylenediamine as the functional monomer, were electropolymerized directly on the surface of ITO electrode. With the hybridization reaction between the assemblies of EuS NCs functionalized 5-amino-labeled oligonucleotides as capture probes and oligonucleotides as detection target (HIV gene), the ECL signal significantly increased using K2S2O8 as coreactant. Taking advantage of both MIP-ECL assays and the strong electrochemiluminescence emission of EuS NCs, the sensitive and selective HIV gene detection has been achieved in a linear range 3.0 fM to 0.3 nM with a detection limit of 0.3 fM. The present MIP-ECL biosensor showed good specificity for HIV DNA detection compared to non-complementary and two bases mismatched sequences. The proposed ECL biosensor was applied to detect of HIV DNA in real human serum samples and satisfactory results were obtained. Due to high sensitivity and selectivity, excellent reproducibility and stability of the proposed assay, EuS NCs can be used as novel luminophore for development of MIP-ECL sensors for detection of other DNA biomarkers.

A novel electrochemical biosensor for HIV-related DNA detection based on toehold strand displacement reaction and cruciform DNA crystal

A sensitive and specific electrochemical biosensor was developed for quantitative detection of HIV-related DNA by integrating toehold strand displacement reaction (TSDR) with cruciform DNA crystal. The TSDR system can be specifically triggered by HIV-related DNA generating plentiful double-stranded DNA products and releasing the target DNA to undergo an additional cycle of amplification. The hybridization reaction of double-stranded DNA products with capture probes on Au electrode surface makes the binding sites of cruciform DNA crystal available. After the attachment of cruciform DNA crystal and the employment of streptavidinylated alkaline phosphatase, a significantly amplified electrochemical signal is obtained. This strategy was applied to the highly sensitive detection of HIV-related DNA in a linear range from 1 pM to 100 nM and a detection limit as low as 0.21 pM was achieved. The proposed strategy can be accomplished without the employment of any enzymes or intensive purification. In addition, this method has good selectivity and anti-interference ability, which could be applied in complex samples with comparable analytical performance. For these advantages, the proposed strategy holds great potential for rapid and early clinical diagnosis of HIV infection.

Probing DNA Hybridization Equilibrium by Cationic Conjugated Polymer for Highly Selective Detection and Imaging of Single-Nucleotide Mutation.

Hybridization-based probes emerge as a promising tool for nucleic acid target detection and imaging. However, the single-nucleotide selectivity is still challenging because the specificity of hybridization reaction is typically low at room temperature. We disclose an effective and simple method for highly selective detection and in situ imaging of single-nucleotide mutation (SNM) by taking the advantages of the specific hybridization of short duplex and the signal amplifying effect of cationic conjugated polymer (CCP). Excellent discrimination of the nucleic acid strands only differing by single nucleotide was achieved enabling the sensitive detection of SNM at the abundance as low as 0.1%. Single-molecule fluorescence resonance energy transfer (smFRET) study reveals that the presence of CCP enhances the perfect matched duplex and the mismatched duplex to a different extent, which can be an explanation for the high single-nucleotide selectivity. Due to the simple design of the probe and the stable brightness of CCP, highly selective mRNA in situ imaging was achieved in fixed cells. Melanoma cell line A375 with BRAF V600E point mutation exhibits higher FRET efficiency than liver cancer cell line HegG2 that was not reported having the mutation at this point.

An electrochemiluminescence biosensor for detection of CdkN2A/p16 anti-oncogene based on functional electrospun nanofibers and core-shell luminescent composite nanoparticles

An electrochemiluminescence (ECL) biosensor based on functional electrospun nanofibers for hybridization detection of specific CdkN2A/p16 anti-oncogene at trace level via binding luminescent composite nanoparticles for signal amplification has been developed. The carboxylated multiwalled carbon nanotubes (MWCNTs) doped polycaprolactam 6 (PA6) electrospun nanofibers (PA6-MWCNTs) was prepared via electrospinning, which served as the nanosized backbones for silica nanoparticles (SiO2) electrodeposition. The functional electrospun nanofibers (PA6-MWCNTs-SiO2) used as supporting scaffolds for single-stranded DNA1 (ssDNA1) immobilization can dramatically increase the amount of DNA attachment and the sensitivity of hybridization. The sandwich construction of ssDNA1-CdkN2A/p16 anti-oncogene -tri(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+)/silver nanoparticles (AgNPs) doped gold (Au) core-shell luminescent composite nanoparticles (RuAg@AuNPs)-labeled ssDNA2 (RuAg@Au-ssDNA2) was fabricated through a hybridization reaction. It was observed that high amount of doped Ru(bpy)32+ in RuAg@AuNPs successfully amplify the recognition signal by adding tripropylamine (TPrA). The change of ECL intensity was found to have a linear relationship in respect to the logarithm of the CdkN2A/p16 anti-oncogene concentrations in the wide range of 1.0 × 10–15~1.0 × 10–12 M, with a detection limit of 0.5 fM (S/N = 3) which is comparable or better than that in reported anti-oncogene assays. Excellent sensitivity and selectivity make the developed biosensor a promising tool for the detection of tumor biomarkers.

Nano polythionine-based electrochemiluminescence biosensor for detection of the p16INK4a gene using RuAg@AuNPs core-shell nanocomposites as DNA labels

In this work, a nano polythionine (NPTh)-based universal sandwich-type electrochemiluminescence (ECL) biosensor for sensitive and specific detection of the p16INK4a gene using core-shell luminescent nanocomposites as excellent DNA labels was constructed. The NPTh electrode was prepared by in situ electropolymerization technique, which had a large surface area and was served as effective biosensing substrates for ssDNA1 immobilization by charge desorption. Meanwhile, tri(2,2′-bipyridyl)ruthenium(II) (Ru(bpy)32+)/silver nanoparticles (AgNPs) doped gold (Au) core-shell nanocomposites (RuAg@AuNPs) were utilized as excellent ssDNA2 labels. The construction of dsDNA was fabricated through a hybridization reaction of ssDNA1, p16INK4a gene and RuAg@Au-ssDNA2. The change of ECL intensity on the NPTh electrode was found to have a linear relationship in respect to the logarithm of the p16INK4a gene concentrations in the wide range of 1.0 × 10–13~1.0 × 10−1 M, with a detection limit of 0.05 p.M. (S/N = 3). The presented biosensing strategy may provide great potential in the assay of other clinically biomarkers.

Lateral flow test for visual detection of multiple MicroRNAs

The authors describe a rapid and low-cost approach for multiplex microRNA(miRNA) assay on lateral flow nucleic acid biosensor (LFNAB). The principle of assay is based on sandwich-type nucleic acid hybridization reactions to produce gold nanoparticle (GNP)-attached complexes (ssDNA-microRNA-ssDNA/GNPs), which are captured and visualized on the test zone of LFNAB. By designing three different test zones on LFNAB, simultaneous detection of microRNA-21, microRNA-155 and microRNA-210 was achieved with an adding-measuring model by using GNP as visual tag. The method was challenged by testing the microRNAs in spiked serum samples with satisfied results. In our perception, the test is a particularly valuable tool for clinical application and biomedical diagnosis, particularly in limited resource settings.

Specific colorimetric ELISA method based on DNA hybridization reaction and non–crosslinking gold nanoparticles aggregation for the detection of amantadine

Amantadine (AMD), a banned antiviral veterinary drug, is still being abused. This study developed a novel enzyme linked immunosorbent assay for the colorimetric detection of AMD involving DNA hybridization reaction and non–crosslinking gold nanoparticles (AuNPs) aggregation. Accordingly, the Primer 1–AuNPs–anti–AMD monoclonal antibody (mAb) could be captured by AMD artificial antigen on ELISA wells. Primer 2, which was complementary paired to Primer 1, was eventually added into the ELISA wells. After the hybridization reaction, the free Primer 2 in the supernatant was mixed with AuNPs and NaCl and induced a rapid color change of AuNPs. The lack of AMD in the sample resulted in capturing a substantial Primer 1–AuNPs–mAb complex and limited free Primer 2 in the supernatant. After adding NaCl, the color of AuNPs turned blue with limited Primer 2. This simple and visualized novel method had good sensitivity (0.033 μM) and exhibited a potential application for AMD screening on site.

A sandwiched electroanalysis method for probing Anthrax DNAs based on glucose-induced gold growth and catalytic coupling of tyramine using gold-mineralized glucose oxidase

Gold nanoparticles was doped into the protein matrix of glucose oxidase (GOx) by the in-site biomineralization route, yielding the GOx with mineralized gold (GOx-Gold) showing the double catalysis activities of GOx and peroxidase-like gold catalysis. A magnetic separation-based detection method was thus tailored for the sandwiched electroanalysis of Anthrax DNAs using GOx-Gold and tyramine linker as the probe labels. After the DNA hybridization reactions, the tyramine-mediated linking of DNA capture and detection probes was conducted through gold-catalytic oxidization of tyramines labelled at the probe terminuses, followed by the glucose-triggered gold growth catalyzed by GOx. Highly amplified electrochemical output of gold signals was thus achieved toward the ultrasensitive detection of DNAs in blood, with the detection limit down to ∼0.10 fM. Also, the discrimination of DNAs with single-base mutation could be expected. Importantly, such a biomimic gold mineralization route can be tailored for remolding various enzymes with improved intrinsic catalysis and electrocatalysis, thus promising the wide applications in the catalysis, biosensing, and biomedical fields.

Localized DNA Hybridization Chain Reactions on DNA Origami.

The field of DNA nanoscience has demonstrated many exquisite DNA nanostructures and intricate DNA nanodevices. However, the operation of each step of prior demonstrated DNA nanodevices requires the diffusion of DNA strands, and the speed of these devices is limited by diffusion kinetics. Here we demonstrate chains of localized DNA hybridization reactions on the surface of a self-assembled DNA origami rectangle. The localization design for our DNA nanodevices does not rely on the diffusion of DNA strands for each step, thus providing faster reaction kinetics. The locality also provides considerable increased scalability, since localized components of the devices can be reused in other locations. A variety of techniques, including atomic force microscopy, total internal reflection fluorescence, and ensemble fluorescence spectroscopy, are used to confirm the occurrence of localized DNA hybridization reactions on the surface of DNA origami. There are many potential biological applications for our localized DNA nanodevices, and the localization design is extensible to applications involving DNA nanodevices operating on other molecular surfaces, such as those of the cell.

ViroFind: A novel target-enrichment deep-sequencing platform reveals a complex JC virus population in the brain of PML patients.

Deep nucleotide sequencing enables the unbiased, broad-spectrum detection of viruses in clinical samples without requiring an a priori hypothesis for the source of infection. However, its use in clinical research applications is limited by low cost-effectiveness given that most of the sequencing information from clinical samples is related to the human genome, which renders the analysis of viral genomes challenging. To overcome this limitation we developed ViroFind, an in-solution target-enrichment platform for virus detection and discovery in clinical samples. ViroFind comprises 165,433 viral probes that cover the genomes of 535 selected DNA and RNA viruses that infect humans or could cause zoonosis. The ViroFind probes are used in a hybridization reaction to enrich viral sequences and therefore enhance the detection of viral genomes via deep sequencing. We used ViroFind to detect and analyze all viral populations in the brain of 5 patients with progressive multifocal leukoencephalopathy (PML) and of 18 control subjects with no known neurological disease. Compared to direct deep sequencing, by using ViroFind we enriched viral sequences present in the clinical samples up to 127-fold. We discovered highly complex polyoma virus JC populations in the PML brain samples with a remarkable degree of genetic divergence among the JC virus variants of each PML brain sample. Specifically for the viral capsid protein VP1 gene, we identified 24 single nucleotide substitutions, 12 of which were associated with amino acid changes. The most frequent (4 of 5 samples, 80%) amino acid change was D66H, which is associated with enhanced tissue tropism, and hence likely a viral fitness advantage, compared to other variants. Lastly, we also detected sparse JC virus sequences in 10 of 18 (55.5%) of control samples and sparse human herpes virus 6B (HHV6B) sequences in the brain of 11 of 18 (61.1%) control subjects. In sum, ViroFind enabled the in-depth analysis of all viral genomes in PML and control brain samples and allowed us to demonstrate a high degree of JC virus genetic divergence in vivo that has been previously underappreciated. ViroFind can be used to investigate the structure of the virome with unprecedented depth in health and disease state.

Molecular Localization of Latent Epstein – Barr Virus Early Repeats (EBERS) in Cervical Tissues with Adenocarcinoma by RNA-In Situ Hybridization

Epstein-Barr virus (EBV) has an etiological relevancy to the pathogenesis of an increasing number of cancers. Cervical carcinogenesis is a multifactorial stepwise process that has a possible link with many infective factors, including EBV infections where the standard procedures in their histopathological diagnosis rely on in situ hybridization. This retrospective research was designed to(1) investigate the frequency of EBV infections in correlation with the age of patients with cervical adenocarcinoma, (2)validate the impact this virus on the histopathological grade expression of those cancers; and(3)rating the EBV-infections by evaluating the signal scores and intensities of RNA -in situ hybridization (RISH) reactions. A total number of 49 patients, who had undergone hysterectomies or punch biopsies from their cervices were enrolled in this study. Twentyone tissues were collected from cervical adenocarcinoma where as the control groups comprised 28 blocks from cervical tissues either without any significant pathological changes or from chronic cervicitis. Molecular detection of Latent EpsteinBarr Virus Early Repeats (EBERS) was performed by using ultra-sensitive versions of RNA-in situ hybridization (RISH) technique. The mean age of this group of Iraqi patients with cervical adenocarcinoma was 46.7 ± 11.6 years and histopathologically, well and poor grades of cervical adenocarcinoma each constituted 14.3% whereas 71.4% was moderately differentiated. The EBV was detected in 38.1% (8 out of 21) of cervical adenocarcinoma tissues while EBERs were neither detected in chronic cervicitis tissues nor in those healthy cervical tissues. Seven out of eight cases (87.5%) with positive EBERs–ISH reactions have well and moderate differentiation grades while only one case (12.5%) has poor differentiation. It may conclude that EBV infections in cervical adenocarcinoma could point for an initiating and/or cofactor roles, along with other important oncogenic viruses, in cervical oncogenesis. The observed impact of EBV on the differentiation of cervical adenocarcinoma could possibly shade light on an early- eventual occurrence of such molecular attack in cervical carcinogenesis.

A cascade amplification strategy of catalytic hairpin assembly and hybridization chain reaction for the sensitive fluorescent assay ofthe model protein carcinoembryonic antigen.

A cascade nucleic acid amplification strategy is presented for fluorometric aptamer based determination of the model protein carcinoembryonic antigen (CEA). Amplification is accomplished by combining catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR). In this assay, a specially designed single-stranded DNA containing the aptamer sequence (AS) specific for CEA is hybridized with an inhibitor strand (IS) to form a double-stranded DNA (IS@AS). In the presence of CEA, it will recognize and bind to the AS strand which causes the release of IS. By introducing two DNA hairpins (H1 and H2; these containing complementary sequences) CHA will be activated via the hybridization reactions of H1 and H2. This is accompanying by the formation of a double-stranded DNA (H1-H2) and the release of CEA@AS. The liberated CEA@AS further drives successive recycling of the CHA, thereby generating further copies of H1-H2. The resultant H1-H2 hybrids act as primers and trigger HCR with the help of other two DNA hairpins (H3 and H4) containing G-rich toehold at the 5'-terminus and 3'-terminus of H3 and H4, respectively. The fluorescent probe N-methyl mesoporphyrin IX (NMM) is finally intercalated into the G-rich domains of the long DNA nanostructures due to formation of G-quadruplex structures. This generates a fluorescent signal (best measured at excitation/emission wavelengths of 399/610nm) that increases with the concentration of target (CEA). This aptamer-based fluorescence assay is highly sensitive and has a linear range that covers the 1pg·mL-1 to 2ng·mL-1 CEA concentration range, with a 0.3pg·mL-1 detection limit. Graphical abstract By integrating catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for effective signal enhancement, a novel cascade amplification strategy is presented to develop a sensitive and selective fluorescent method for the assay of the model protein carcinoembryonic antigen (CEA).